Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92) 600–1
SECTION 600Pesticide Analytical Manual Vol. I
Table of Contents
page date
601: General Information
601 A: Principles 601-1 1/94
601 B: Modes of Operation 601-2 1/94
Liquid-Solid Chromatography 601-2 1/94
Liquid-Liquid Chromatography 601-3 1/94
Bonded Phase Chromatography 601-3 1/94
Ion Exchange Chromatography 601-4 1/94
Size Exclusion Chromatography 601-4 1/94
601 C: Instrumentation and Apparatus 601-5 1/94
Basic Components 601-5 1/94
HPLC System Plumbing 601-7 1/94
601 D: Solvents and Reagents 601-10 1/94
Potential Problems 601-10 1/94
Specific Solvents 601-12 1/94
Chapter 1
Regulatory Operations
Chapter 2
General Analytical
Operations and Information
Chapter 3
Multiclass
MRMs
Chapter 5
GLC
Chapter 4
Selective
MRMs
Chapter 6
HPLC
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92)600–2
Pesticide Analytical Manual Vol. ISECTION 600
page date
Reagent Blanks 601-14 1/94
Safety Precautions 601-14 1/94
601 E: Sample Preparation 601-14 1/94
Sample Cleanup 601-14 1/94
Sample Filtration 601-14 1/94
Sample Solvent Degassing 601-15 1/94
Choice of Sample Solvent 601-15 1/94
601 F: Reference Standards 601-15 1/94
Stock Solutions 601-15 1/94
Working Standard Solutions 601-16 1/94
Storage 601-16 1/94
References 601-16 1/94
602: Columns
602 A: Column Selection 602-1 1/94
602 B: Analytical Columns 602-1 1/94
Liquid-Solid Chromatography 602-2 1/94
Bonded Phases 602-2 1/94
Ion Exchange 602-3 1/94
Ion Pair 602-4 1/94
Size Exclusion 602-4 1/94
602 C: Column Evaluation 602-5 1/94
602 D: Column Specifications 602-6 1/94
602 E: Analytical Column Protection 602-8 1/94
Filters 602-8 1/94
Precolumns 602-9 1/94
Guard Columns 602-9 1/94
602 F: Column Maintenance and Troubleshooting 602-9 1/94
Column Care 602-9 1/94
Column Evaluation by Injection of 602-10 1/94
Test Mixtures
Column Storage 602-11 1/94
Column Regeneration 602-11 1/94
References 602-12 1/94
603: Mobile Phase Selection, Preparation, and Delivery
603 A: Mobile Phase Selection 603-1 1/94
Normal Phase Chromatography 603-1 1/94
Reverse Phase Chromatography 603-3 1/94
Ion Exchange Chromatography 603-4 1/94
Ion Pair Chromatography 603-5 1/94
Size Exclusion Chromatography 603-5 1/94
Gradient Elution in HPLC 603-5 1/94
603 B: Mobile Phase Preparation 603-6 1/94
Filtering Solvents 603-6 1/94
Degassing Solvents 603-6 1/94
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92) 600–3
SECTION 600Pesticide Analytical Manual Vol. I
page date
Preparation of Multisolvent Mobile Phases 603-7 1/94
Solvent Reservoirs 603-7 1/94
603 C: Mobile Phase Delivery Systems 603-8 1/94
Pumps 603-8 1/94
Gradient Programming Systems 603-10 1/94
603 D: Maintenance and Troubleshooting 603-10 1/94
Problems with Pumps 603-10 1/94
References 603-13 1/94
604: Injection Systems
604 A: Injection Valves 604-1 1/94
604 B: Automatic Injectors 604-3 1/94
604 C: Operation, Maintenance, Troubleshooting, 604-3 1/94
and Repair of Injection Valves
References 604-4 1/94
605: Detectors
605 A: UV/VIS Absorbance Detectors 605-2 1/94
Fixed Wavelength UV Detectors 605-3 1/94
Variable Wavelength UV Detectors 605-4 1/94
Solvents 605-4 1/94
Performance Characteristics 605-4 1/94
Multichannel or Photodiode Array Detectors 605-5 1/94
Applications 605-5 1/94
Problems, Maintenance, and 605-5 1/94
Troubleshooting
605 B: Fluorescence Detectors 605-6 1/94
Detector Design 605-6 1/94
Solvents 605-7 1/94
Performance Characteristics 605-7 1/94
Parameter Adjustments 605-8 1/94
Applications 605-8 1/94
Detector Maintenance 605-8 1/94
605 C: Electrochemical Detectors 605-8 1/94
Conductivity Detectors 605-9 1/94
Amperometric and Coulometric Detectors 605-9 1/94
Performance Characteristics 605-10 1/94
Applications 605-11 1/94
605 D: Photoconductivity Detectors 605-11 1/94
Apparatus 605-11 1/94
Performance Characteristics 605-11 1/94
Applications 605-13 1/94
605 E: Mass Spectrometric Detectors 605-13 1/94
605 F: Derivatization for Detection Enhancement 605-14 1/94
Comparison of Pre- and Post-Column 605-14 1/94
Derivatization
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92)600–4
Pesticide Analytical Manual Vol. ISECTION 600
page date
Post-Column Reactor Design 605-14 1/94
References 605-16 1/94
606: Residue Identification and Quantitation
606 A: Residue identification 606-1 1/94
Co-chromatography 606-1 1/94
Use of Alternative Columns 606-2 1/94
Spectrometric Confirmation 606-2 1/94
606 B: Quantitation 606-3 1/94
Reference 606-3 1/94
607: Quality Assurance and Troubleshooting
607 A: Liquid Chromatograph Monitoring and 607-1 1/94
Performance Testing
607 B: Troubleshooting from Chromatograms 607-2 1/94
608: Bibliography
General Texts 608-1 1/94
Columns 608-1 1/94
Detectors 608-2 1/94
Troubleshooting 608-2 1/94
Application to Pesticides 608-2 1/94
Figures
601-a Chromatographic Separation Techniques 601-1 1/94
601-b HPLC Modes of Operation 601-2 1/94
601-c Guide to Selection of HPLC Mode 601-5 1/94
601-d Block Diagram of HPLC System 601-6 1/94
601-e Column Outlet Fittings 601-7 1/94
601-f Low Dead Volume Fitting 601-7 1/94
601-g Standard Internal Fitting 601-8 1/94
601-h Internal Thread Low Dead Volume Fitting 601-8 1/94
602-a Calculation of Column Performance Parameters 602-5 1/94
603-a Reciprocating Pump 603-8 1/94
603-b Gradient System I 603-9 1/94
603-c Gradient System II 603-9 1/94
603-d Gradient System III 603-10 1/94
604-a External Loop Injector: Six-Port Injection Valve 604-1 1/94
604-b Internal Loop Injector 604-2 1/94
605-a UV/VIS Detector 605-3 1/94
605-b Variable Wavelength UV/VIS Detector 605-4 1/94
605-c Fluorescence HPLC Detector 605-6 9/96
605-d Three Electrode Electrochemical Detector 605-10 1/94
605-e Post-Column Reactors 605-15 1/94
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92) 600–5
SECTION 600Pesticide Analytical Manual Vol. I
page date
Tables
602-a: HPLC Column Specification Elements 602-7 1/94
602-b: Minimum Efficiency Values 602-8 1/94
603-a: Properties of Common HPLC Solvents with 603-2 1/94
Alumina Columns
603-b: Classification of Solvent Selectivity 603-2 1/94
Transmittal No. 96-E1 (9/96)
Form FDA 2905a (6/92)600–6
Pesticide Analytical Manual Vol. ISECTION 600
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–1
Pesticide Analytical Manual Vol. I
601: GENERAL INFORMATION
In recent years, high performance liquid chromatography (HPLC) has grown in
popularity as a determinative step for residue analysis, until today it is accepted as
complementary to the more traditional gas liquid chromatography (GLC). HPLC
provides capabilities not possible with GLC, most importantly the ability to sepa-
rate and quantitate residues of polar, nonvolatile, and heat-labile chemicals. These
characteristics make HPLC the determinative step of choice for many residues
previously beyond the applicability of multiresidue methodology.
601 A: PRINCIPLES
Chromatography comprises a family of sepa-
ration techniques (Figure 601-a), all of which
share common characteristics. A narrow ini-
tial zone of mixture is applied to a sorptive
stationary phase having a large surface area.
Development with mobile phase causes com-
ponents of a mixture to move through the
stationary phase at different rates and to
separate from one another. Differential mi-
gration occurs because of differences in dis-
tribution between the two phases. The mo-
bile phase can be a gas or a liquid. Liquid
chromatography is divided into two main
types, planar (thin layer and paper chroma-
tography) and column. Column liquid chro-
matography, both the classical (low pressure)
version and the high performance version
discussed here, is further subdivided accord-
ing to the mechanism of separation into five
major types: liquid-solid (adsorption) chromatography, LSC; liquid-liquid (parti-
tion) chromatography, LLC; bonded phase chromatography, BPC; ion exchange
chromatography, IEC; and size exclusion chromatography, SEC.
HPLC developed steadily during the late 1960s as high efficiency, small particle
packings and improved instrumentation were produced. In contrast to classical
column liquid chromatography, HPLC uses high pressure pumps; short, narrow
columns packed with microparticulate phases; and a detector that continuously
records the concentration of the sample.
HPLC systems use the principles of classical column chromatography in an analyti-
cal instrument. Development of HPLC has been directly related to availability of
suitable hardware (columns, pumps, inlet systems, low dead volume fittings, etc.)
that allows precise flow control under the elevated pressures needed, as well as the
ability to manufacture a wide variety of column packing materials in particle sizes
of exacting micron (μm) dimensions.
In contrast to GLC, where the gas mobile phase is inert and does not affect
separation of analytes from one another, the HPLC mobile phase is critical to this
Chapter 6 is revised from a chapter on HPLC written for FDA in 1989-90 by Joseph Sherma,
Ph.D., Lafayette College, Easton, PA.
Figure 601-a
Chromatographic Separation
Techniques
Chromatography
Gas
Liquid
Column
Planar
Classical HPLC
LSC LLC BPC IEC SEC
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–2
resolution. Choice of mobile phase is second only to the choice of operating
mode in determining the suitability of the system to produce the desired separa-
tions.
HPLC had limited use for routine trace multiresidue analysis in the absence of
sensitive element-selective detectors. Early development work relied primarily on
refractive index (RI) or fixed wavelength UV absorbance detectors. Neither detec-
tor demonstrated sufficient sensitivity or selectivity for use in trace residue analysis.
In the mid-1970s, the fluorescence detector was shown to provide the needed
sensitivity and specificity for pesticides that are naturally fluorescent or can
be chemically labeled with a fluorophore. This resulted in the first practical appli-
cation of HPLC to multiresidue pesticide determination (see method for
N-methylcarbamates, Section 401).
More recently, scientists have investigated photoconductivity and electrochemical
detectors and certain applications of the newer multiwavelength UV detectors.
This research indicates that these detectors can also fulfill the sensitivity and
selectivity requirements for determination of certain pesticides at residue levels.
601 B: MODES OF OPERATION
Separations by HPLC are achieved
using the five basic operational modes
(Figure 601-b). The mode chosen for
a particular application will depend on
the properties of the analyte(s) to be
separated and determined. For residue
determination, as for HPLC analyses
in general, BPC is the most widely
used.
There are two variations within the five
operational modes of HPLC operation;
these distinctions are based on the relative polarities of stationary and mobile
phases:
1) normal phase (NP) chromatography: stationary phase is more polar than
the mobile phase; the least polar analytes elute first; analyte retention is
increased by decreasing mobile phase polarity.
2) reverse phase (RP) chromatography: stationary phase is less polar than
the mobile phase; the most polar analytes elute first; analyte retention is
increased by increasing mobile phase polarity.
Liquid-Solid Chromatography
LSC, also called adsorption chromatography, uses an adsorbent, usually uncoated
silica gel. The basis for separation is the selective adsorption of polar compounds,
presumably by hydrogen bonding, to active silanol (SiOH) groups by orientation
and on the surface of the silica gel. Analytes that are more polar will be attracted
more strongly to the active silica gel sites. The solvent strength of the mobile phase
determines the rate at which adsorbed analytes are desorbed and eluted.
Figure 601-b
HPLC Modes of Operation
HPLC
LSC
LLC
BPC
IEC
SEC
Ion
suppression
Ion
pair
SAX SCX
GPC GFC
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–3
Pesticide Analytical Manual Vol. I
LSC is useful for separation of isomers and classes of compounds differing in polarity
and number of functional groups. It works best with compounds that have relatively
low or intermediate polarity. Highly polar compounds may irreversibly adsorb on the
column. Poor LSC separations are usually obtained for chemicals containing only
nonpolar aliphatic substituents.
Liquid-Liquid Chromatography
LLC, also called partition chromatography, involves a solid support, usually silica
gel or kieselguhr, mechanically coated with a film of an organic liquid. A typical
system for NP LLC is a column coated with ?,?'-oxy dipropionitrile and a nonpolar
solvent like hexane as the mobile phase. Analytes are separated by partitioning
between the two phases as in solvent extraction. Components more soluble in the
stationary liquid move more slowly and elute later. LLC has now been replaced by
BPC for most applications.
Bonded Phase Chromatography
BPC uses a stationary phase that is chemically bonded to silica gel by reaction of
silanol groups with a substituted organosilane. Unlike LLC, the stationary phase is
not altered by mobile phase development or temperature change. All solvents can
be used, presaturation of the mobile phase with the stationary phase is not re-
quired, and gradient elution can be used to improve resolution.
Specialized applications of BPC have been developed for ionized compounds,
which are highly water soluble and generally not well retained on RP BPC col-
umns. Retention and separation can be increased by adding an appropriate pH
buffer to suppress ionization (ion suppression chromatography) or by forming a
lipophilic ion pair (ion pair chromatography) between the analyte and a counter
ion of opposite charge. The resultant nonionic species are separated by the same
column techniques used for naturally nonionic organic molecules.
Ion suppression is the preferred method for separation of weak acids and bases,
for which the pH of the mobile phase can be adjusted to eliminate analyte ioniza-
tion while remaining within the pH 2-8 stability range of bonded silica phases. The
analyte is chromatographed by RP HPLC, usually on a C-18 column, using metha-
nol or acetonitrile plus a buffer as the mobile phase. The technique is often
preferred over IEC (see below) because C-18 columns have higher efficiency,
equilibrate faster, and are generally easier to use reproducibly compared to ion
exchange phases. Strong acids and bases are usually separated on an ion exchange
column or by ion pair chromatography.
Ion pair chromatography is used to separate weak or strong acids or bases as well
as other types of organic ionic compounds. The method involves use of a C-18
column and a mobile phase buffered to a pH value at which the analyte is com-
pletely ionized (acid pH for bases, basic pH for acids) and containing an appro-
priate ion pairing reagent of opposite charge. Trialkylammonium salts are com-
monly used for complex acidic analytes and alkylsulfonic acids for basic analytes.
The ion pairs separate as if they are neutral polar molecules, but the exact mecha-
nism of ion pair chromatography is unclear. Retention and selectivity are affected
by the chain length and concentration of the pairing reagent, the concentration
of organic solvent in the mobile phase, and its pH. Retention increases up to a
point as the chain length of the pairing reagent or its concentration increases,
then decreases or levels off [1].
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–4
Compounds not ionized at the operative pH will not pair with the reagent, but
they may still be strongly retained by a C-18 column depending on their alkyl
structure. In this case, however, retention will not increase with the addition of an
ion pairing reagent, and some decrease in retention may occur, probably due to
reagent competition for the stationary phase [1].
Ion Exchange Chromatography
IEC is used to separate ionic compounds. Microparticulate insoluble organic poly-
mer resin or silica gel is used as the support. Negatively charged sulfonic acid
groups chemically bound to the support produce strong acid cation exchange
(SCX) phases. Positively charged quaternary ammonium ions bound to the sup-
port produce strong base anion exchange (SAX) phases. The most widely used
resin support is cross-linked copolymer prepared from styrene and divinylbenzene.
Mobile phases are aqueous buffers.
Separations in IEC result from competition between the analytes and mobile phase
ions for sites of opposite charge on the stationary phase. Important factors control-
ling retention and selectivity include the size and charge of the analyte ions, the
type and concentration of other ions in the buffer system, pH, temperature, and
the presence of organic solvents.
Ion chromatography, a subcategory of IEC, has been used primarily for separa-
tions of inorganic cations or anions. Because a conductivity detector is usually
employed, some means is required to reduce the ionic concentration and, hence,
the background conductance of the mobile phase. A second ion exchange sup-
pressor column to convert mobile phase ions to a nonconducting compound may
be used. Alternatively, a stationary phase with very low exchange capacity may be
used with a dilute, low conductance mobile phase containing ions that interact
strongly with the column.
Size Exclusion Chromatography
SEC separates molecules based on differences in their size and shape in solution.
SEC cannot separate isomers. SEC is carried out on silica gel or polymer packings
having open structures with solvent-filled pores of limited size range. Small analyte
molecules can enter the pores and spend a longer amount of time passing through
the column than large molecules, which are excluded from the pores. Ideally,
there should be no interaction between the analytes and the surface of the station-
ary phase.
Two important subdivisions of SEC are gel permeation chromatography (GPC)
and gel filtration chromatography (GFC). GPC uses organic solvents for organic
polymers and other analytes in organic solvents. GFC uses aqueous systems to
separate and characterize biopolymers such as proteins and nucleic acids.
The chemist developing an HPLC method must first consider the properties of
the analytes of interest and choose an HPLC separation method that best takes
advantage of those properties. Many of the references in the bibliography (Section
608) offer guidance to making these choices. A general, simplified guide for
selecting an HPLC mode according to the properties of the analyte(s) is illustrated
in Figure 601-c; the guide is based on the principles of Snyder and Kirkland [2].
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–5
Pesticide Analytical Manual Vol. I
Figure 601-c
Guide to Selection of HPLC Mode
(based on analyte characteristics)
Molecular weight >2000?
Organic
soluble?
Molecular sizes
very different?
Ionizable?
Ion Suppression
Chromatography
Ion
Pairing
Anionic?
Anionic
Counter Ion
RP (C-8, C-18)
org-aq solvent
Cationic
Counter Ion
RP (C-8, C-18)
org-aq solvent
IEC SCX
aq solvent
Strongly lipophilic? SAX
aq solvent
BPC
RP, C-18, polar
org solvent
LSC
silica; polar
org solvent
BPC
normal phase
(CN, NH , diol)
org solvent
LSC
silica; polar
org solvent
BPC
RP, C-8
org-aq
solvent
BPC
RP
org-aq
solvent
SEC
Yes
No
No
Yes
No
Yes
Yes
Yes
No
No
No
Yes
No
Yes
GFC
GPC
Anionic?
2
This scheme categorizes analytes as either ionic/ionizable (and therefore water
soluble) or nonionic/nonionizable (not water soluble). Based on these distinc-
tions, and on the polarity of the analytes, the diagram provides general rules for
choosing an HPLC mode of operation likely to separate the analytes.
601 C: INSTRUMENTATION AND APPARATUS
Basic Components
The following basic components are typically included in an HPLC system (Fig-
ure 601-d): solvent reservoir(s); optional gradient-forming device; one or more
precision solvent delivery pumps; injector; analytical column and optional
precolumn and guard column; column oven; detector; recorder, integrator, or
computerized digital signal processing device; and associated plumbing and wir-
ing.
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–6
For analytical HPLC, typical flow rates of 0.5-5 mL/min are produced by pumps
operating at 300-6000 psi. Although pumps are capable of high pressure opera-
tion, state-of-the-art 25 cm × 4 mm id columns with 5 μm packings typically pro-
duce 1000-2000 psi at 1 mL/min. High pressures should be avoided because they
contribute to limited column life expectancies.
Sample extract is applied to the column from an injector valve containing a loop
that has been filled with sample solution from a syringe. After passing through the
column, the separated analytes are sensed by visible/UV absorption, fluorescence,
electrochemical, photoconductivity, or RI detectors. To minimize extra-column
peak spreading, the instrument components must be connected using low dead
volume (ldv) fittings and valves and tubing as short and narrow in bore as possible.
Analytical HPLC may use either isocratic or gradient elution methods. Isocratic
elution uses a mobile phase of constant composition, whereas the strength of the
mobile phase in gradient elution is made to increase continually in some prede-
termined manner during the separation. Gradient elution, which requires an
automatic electronic programmer that pumps solvent from two or more reservoirs,
reduces analysis time and increases resolution for complex mixtures in a manner
similar to temperature programming in GLC. Gradient elution capability is highly
recommended for systems to be used for residue determination. However, it is not
always possible to employ gradient elution because some HPLC column/solvent
systems and detectors are not amenable to the rapid solvent and pressure changes
involved.
Stationary phases are uniform, spherical, or irregular porous particles having
nominal diameters of 10, 5, or 3 μm. Bonded phases produced by chemically
bonding different functional groups to the surface of silica gel are most widely
used, along with unmodified silica gel and size exclusion gels. Columns are usually
stainless steel, 3-25 cm long and 4.6 mm id, prepacked by commercial manufac-
turers. There has been increasing use of microbore columns having diameters ≤2
mm. Although many HPLC separations can be carried out at ambient tempera-
ture, column operation in a thermostatted column oven is necessary for reproduc-
ible, quantitative results, because distribution coefficients and solubilities are tem-
perature dependent.
Depending on the nature of the analyte(s), certain additional equipment may be
required. For example, apparatus and reagents for performing post-column
Figure 601-d
Block Diagram of
HPLC System
[Reprinted with permission of
McGraw-Hill Book Company, from
West, C.D. (1987) Essentials of
Quantitative Analysis, Figure 14.1,
page 346.]
Pump A
Solvent A
Pump B
Solvent B
Sample
injection
system
Gradient
device
Thermostatted
column oven
Column
Thermostatted
detector oven
Detector
Amplifier
Recorder or
readout device
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–7
Pesticide Analytical Manual Vol. I
Stainless
steel frit,
2 μm
1/4"
stainless
steel
tubing
1/16"
tubing
(iii)(ii)(i)
derivatization, as used in Section 401 for N-methylcarbamates, may be needed to
convert analytes to compounds that can be detected with the required sensitivity
and/or selectivity.
HPLC System Plumbing
Band broadening can occur not only in the analytical and guard columns, but also
in dead volume in the injector, detector, or plumbing connecting the various
components of the HPLC system. This
effect, called extra-column dispersion,
must be minimized for high efficiency.
The proper choice and use of tubing
and fittings are critical in this regard.
Fittings. Figure 601-e illustrates three
types of column outlet fittings. The
conventional fitting (i) used in GLC
and general laboratory plumbing has
excessive dead volume. It has been
modified to produce a zero dead vol-
ume (zdv) fitting (ii) in which the
metal column and the tubing are
butted up directly against the stainless
steel frit. There is evidence that the
nature of the tubing connection in the
zdv fitting may lead to some loss in
efficiency, especially if the connection
is not made carefully. The ldv fitting
(iii) improves efficiency by use of a cone-shaped distributor connecting the gauze
or frit at the end of the column with the tubing. A typical dead volume for the ldv
fitting is 0.1 μL.
Columns are usually received from
manufacturers with a 1/4–1/16" zdv or ldv
outlet fitting and a 1/4" nut and cap or
a reducing union at the inlet (i.e., not
1/4" in size, but suitable for 1/4" tubing).
Figure 601-f shows a complete ldv fitting
connection between a column and a
detector. The column fits snugly inside
the stainless steel end fitting and is sealed
by a high compression ferrule. A 2 μm
porous frit is firmly seated between the
column and end fitting. The column and
detector are connected by a short length
of stainless steel (or polymer) tubing.
The column is also connected to the
injection valve using a zdv or ldv fitting
and a short length of stainless steel tub-
ing.
(i) Conventional reducing union (dead volume is
shaded); (ii) zdv union; (iii) ldv union.
[Reprinted with permission of John Wiley and Sons, Inc., from
Lindsay, S. (1987) High Performance Liquid Chromatography,
Figure 2.3a, page 28.]
Figure 601-e
Column Outlet Fittings
Column
(1/4" od)
Ferrule
2 μm porous frit
1/4" end fitting
0.01" id
To detector
[Reprinted with permission of Howard Sloane, Savant,
from LC-102 audiovisual program.]
Figure 601-f
Low Dead Volume Fitting
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–8
External column end fittings (Figures 601-e and 601-
f), which were formerly popular, are not durable
during repeated attachments and removals. Thus,
the internal fitting is practically standard today. This
uses female threads in the fitting body and a male
nut (Figure 601-g).
Unions. Unions are fittings that connect two pieces
of tubing. The most commonly used type is the
internal thread ldv type (Figure 601-h). The union
is not drilled through completely, but a short (0.02")
web of metal is left between the two pieces of tub-
ing with a small diameter (approximately 0.02 or
0.01") hole drilled through. Even though the tub-
ing ends do not butt against each other as in early
zdv unions, there is essentially no dead volume added
to the system through their use. For this reason,
they are commonly classified as zdv unions. This
type of union has fewer assembly, re-assembly, and
tubing interchange problems than the early butt-
together zdv type.
Assembly of Fittings. Fittings consist of four parts:
the body, tubing, ferrule, and nut. The nut and
ferrule are slid onto the tube end, the tube is pushed all the way into the fitting
body and held there securely, the nut is finger-tightened, and then another three-
quarter turn is made with a wrench. This procedure should assure that the ferrule
is pressed (“swaged”) onto the tub-
ing. To replace the ferrule, the tub-
ing must be cut and the fitting re-
made. When using fittings to con-
nect system components, the nut
should be finger-tightened and then
tightened a one-half turn more with
a wrench. If leaking is observed,
slightly more tightening should be
sufficient to complete the seal. Over-
tightening of nuts can lead to fit-
ting distortion and leaks.
Fitting components from different
manufacturers have dissimilar de-
signs, sizes, and thread types and are usually not interchangeable. Ferrules from
different manufacturers have unique shapes, but they are usually interchangeable
because the front edge is deformed when pressed onto the tubing. However, as a
general rule, it is best to purchase all fittings and spare parts from one manufac-
turer. Even fittings from a given manufacturer differ slightly because of manufac-
turing tolerances. However, this is of concern only with microbore columns, for
which dead volume is a greater consideration. For these columns, it is best to not
even interchange fittings from the same manufacturer.
A variety of fittings are available that can be finger-tightened to the degree nec-
essary to seal stainless steel tubing at 2000-6000 psi. All of these are based on the
use of polymeric ferrules, but some have a steel nut, whereas others are all plastic.
[Reprinted with permission of Aster Publishing Corporation,
from Dolan, J.W., and Upchurch, P. (1988) LC-GC 6,
Figure 3, page 788.]
Column
Frit
Union
1/16" tubing
Figure 601-g
Standard Internal Fitting
[Reprinted with permission of John Wiley
and Sons, Inc., from Meyer, V.R. (1988)
Practical High Performance Liquid
Chromatography, Figure 6.18, page 80.]
Figure 601-h
Internal Thread Low Dead Volume Fitting
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–9
Pesticide Analytical Manual Vol. I
They are used mostly on frequently attached and detached high pressure connec-
tions, such as between the injector and column or column and detector, and for
polymer tubing waste lines from the injector or detector.
Fittings must be kept free of silica particles, which may scratch surfaces between
the ferrule and union and cause leaks.
Tubing. Stainless steel tubing is available commercially that is supposedly ready for
immediate use in HPLC systems. It is machine cut, polished, and deburred to
provide perfectly square ends. It is also cleaned by sonication, passivated, washed,
and rinsed with a solvent such as isopropanol to eliminate residual dirt or oils.
Despite this careful preparation, it is a wise precaution to rinse new tubing with
mobile phase under operating pressure before using it as part of the HPLC system.
The most commonly used tubing for connecting components of the chromato-
graph is 316 stainless steel, 1/16" od, with different inside diameters. Tubing with
0.01" (0.25 mm) id is commonly used in areas where dead volume must be mini-
mized to maximize efficiency, e.g., between the injector and column, precolumn
and column, columns in series, and column and detector, and for preparing pulse
damping spirals.
Typical lengths of tubing connections are 3-6 cm. Tubing with 0.005 or 0.007" id
is used to connect microbore or short 3 μm particle size columns to detectors and
injectors. Filtering of samples and solvents is especially critical to prevent clogging
of this narrow bore tubing. Tubing with 0.02-0.05" id is available when ldv is not
important and low resistance to flow and pressure drop is desirable. For example,
1 mm (0.04") tubing is often used between the pump and sample injector.
Tubing can be cut to any required length in the laboratory, but it is important not
to distort the interior or exterior during the process. The simplest method is to
score the tubing completely around the outside with a file and then bend it back
and forth while holding it on either side of the score with two smooth-jawed pliers.
The ends are filed smooth and deburred, and the tubing is thoroughly washed
with solvent. If the bore should become closed by the bending and filing, the tube
can be reamed out with an appropriate drill bit before final smoothing and wash-
ing. A number of types of manual and motorized tubing cutters are available from
chromatography accessory suppliers. Proper cutting of tubing to make leak-free
connections is an art that requires considerable practice.
Although stainless steel tubing and fittings are standard for systems using organic
and salt-free aqueous solvents, corrosion becomes a problem with buffers contain-
ing salts, particularly halide salts at low pH. HPLC companies have available a
variety of accessories that can solve this problem. These include titanium high
pressure system components, for use in the flow stream at all points of mobile
phase contact, and titanium or polymeric fluorocarbon tubing with id values simi-
lar to stainless steel. One such polymer is Tefzel (ethylene-tetrafluoroethylene
copolymer), which can withstand pressures of 5000 psi or higher. (Teflon is lim-
ited to pressures <1000 psi.) Titanium and polymeric plumbing components are
especially valuable for biochemical HPLC and ion chromatography.
Reference 3 is a valuable source of information to help avoid many tubing instal-
lation problems.
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–10
System Leaks. Leaks are relatively easy to detect in LC instruments because liquid
will be visible around a loose fitting. A loss of system pressure when using a
constant volume pump is a common sign that a leak may be present. If this occurs,
all fittings, especially sample valve and column fittings, should be checked and
tightened if necessary with two open-ended wrenches. Care must be taken not to
overtighten. If leaking does not stop, the faulty fitting must be replaced.
601 D: SOLVENTS AND REAGENTS
The mobile phase in HPLC is chosen for its ability, in combination with a particu-
lar column, to provide the required separation of the analyte(s). The solvents used
to prepare the mobile phase must be of high purity, most often HPLC grade,
spectrophotometric grade, or distilled from all-glass apparatus. Other factors of
importance include cost, viscosity, toxicity, boiling point, compressibility, UV trans-
parency (if a UV detector is used), RI (if an RI detector is used), vapor pressure,
flash point, odor, inertness with respect to sample compounds, and ability to cause
corrosion. Choices of solvents and reagents cannot be made without careful con-
sideration of the effect their presence can have on the entire HPLC system.
Solvents and reagents used in the HPLC determinative step and in sample prepa-
ration procedures preceding HPLC should not:
1) cause degradation or unintended reaction of the analyte(s);
2) cause the solvent delivery system to malfunction;
3) cause damage to the analytical column;
4) cause damage to the detector; or
5) contribute noise or increased or decreased detector response for the
analyte.
Potential Problems
Many of the problems with mobile phases arise because of the presence of impu-
rities, additives, dust or other particulate matter, or dissolved air. Examples of
some specific potential problems with solvents and reagents and suggested solu-
tions follow.
Degradation. Analytes can be degraded by solvents and reagents used in the ex-
traction and cleanup steps of the analysis, or in the HPLC step itself. Analyte
chemistry is usually known in advance, and reagents likely to cause degradation
can be avoided. Unexpected reaction of the analyte(s) will usually be demon-
strated by poor or no recovery of the compound(s) through the method, or by
detection of additional reaction products in the determinative step.
The presence of impurities in solvents or reagents is often the cause of such
unexpected reactions. For example, traces of oxidizing agents in solvents have
been found to degrade N-methylcarbamates prior to their determination by HPLC.
Purity of all reagents used in trace-level determinations should always be as high
as possible.
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–11
Pesticide Analytical Manual Vol. I
Dissolved Gases. The presence of dissolved gases in solvents composing the mo-
bile phase is a major cause of practical problems in HPLC. Gas bubbles can collect
in pumps, the detector cell, or other locations in the HPLC system. This can affect
the reproducibility of the volume delivered by the pump, or large bubbles may
completely stop the pump from working. Detection can be affected in various
ways. With the UV detector, air in the detector cell can cause seriously increased
detector noise or high absorbance. Dissolved oxygen can interfere with detection
at short wavelengths, as oxygen absorbs radiation at <200 nm. Solvents must be
“degassed,” a topic covered in Section 603 B, Mobile Phase Preparation.
Damage to Columns. HPLC columns are easily damaged and expensive to replace.
Bases can remove the functional groups from bonded HPLC phases. Therefore,
bases should not be used in analyses involving BPC unless their removal prior to
chromatography can be assured. Bonded phases are usually stable in the pH range
of approximately 2-8.
Microscopic particles and microorganisms can clog column frits or even the top
of the column itself. If this happens, the pressure drop across the column for a
given flow will gradually increase, and the column may eventually become com-
pletely blocked. Filtration of the sample solution and mobile phase to remove
particles ≥5 μm, and the use of an appropriate precolumn and guard column, are
recommended to protect the analytical column. Particles <5 μm may be of con-
cern with some columns and detectors.
Any mobile phase, especially one containing water or methanol, can dissolve silica
gel in unmodified and bonded silica gel columns. A precolumn containing silica
gel can be positioned between pump and injector to saturate the mobile phase
with silica gel so that the analytical column is not dissolved.
Both precolumns and guard columns are discussed in Section 602 E, Analytical
Column Protection.
The potential for damage to the column by reagents used in post-column
derivatization is unlikely but not impossible. If the flow of the mobile phase is
stopped, post-column reagents can diffuse back through the column effluent onto
the column. This can result in deterioration of the column packing.
Damage to Detectors. The potential for reagent damage varies with each detector.
As stated above, the compressibility of dissolved gases in solvents can cause bubbles
to appear in the detector cell and interfere with the analysis. Traces of oxygen are
incompatible with electrochemical detectors operating in the reductive mode;
oxygen can also cause quenching in fluorescence detectors, leading to reduced
sensitivity. Degassing of solvents is required. Porous flow-through coulometric
detectors can be clogged by the presence of particles ≥0.2 μm. Filtration of sol-
vents through a 0.22 μm filter is essential when using this type of detector.
Solvent Impurities. Many reagent grade solvents contain levels of impurities that
make them unsuitable for use in HPLC. Sometimes the impurities are added
deliberately by manufacturers as antioxidants, stabilizers, or denaturing agents.
For example, chloroform usually contains up to 1.0% methanol or ethanol, and
tetrahydrofuran may contain butylated hydroxytoluene or hydroquinone. These
impurities may cause increased or decreased detector response or change the
mobile phase strength and/or selectivity.
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–12
In some cases, incompatibility of a solvent or reagent with the HPLC system can
be determined in advance and avoided. In the case of unknown impurities, prob-
lems will be recognized only during use of the chemical; careful investigation will
be needed to determine the cause of the problem. Even microorganisms in inad-
equately purified water can cause a high background signal in some detectors (see
Water, below). Whenever possible, HPLC grade solvents should be used to pre-
pare mobile phases. Spectral or pesticide grade solvents may not be adequately
pure for HPLC use. Solvents should be adequately purified and tested before use.
Specific Solvents
Water. Water is probably the most commonly used solvent in HPLC because of its
role as the strength-adjusting solvent in RP mobile phases. It is also one of the
most difficult solvents to purify and maintain in the pure state. Purity of water is
especially critical in the determination of trace residues, when detectors are oper-
ated at high sensitivity.
Purification of water by distillation, even triple distillation, is inadequate because
volatile and codistilled organics will not be removed. Bonded RP columns will
collect these impurities over long term use, which can alter the properties of the
column or sometimes produce spurious peaks. Water can be purified by distilla-
tion from potassium permanganate, by passage through a coarse grained C-18
bonded phase column that is periodically regenerated with acetonitrile, or by
means of a commercial water purification system.
One widely used water purification system (Millipore Milli-Q) pumps distilled water
through a prefilter cartridge to eliminate particulates; then through sequential
cartridges of charcoal, ion exchange resin, and Organex-Q; and finally through a
0.22 μm filter. The activated charcoal cartridge removes organic impurities that
can interfere with spectroscopic detectors. The mixed bed ion exchange resin
cartridge(s) removes inorganics and ionized organics, as well as impurities leached
from the charcoal; this removal is essential for proper operation of electrochemi-
cal detectors. The Organex-Q cartridge eliminates any remaining organics, in
addition to traces of material leached from the ion exchange cartridge. The final
0.22 μm filter removes microscopic particles and microorganisms not eliminated
by the previous cartridges. This filtration step protects column frits, columns, and
porous flow-through detectors from particles that could clog them. It also mini-
mizes the possibility that microorganisms will grow sufficiently to cause a back-
ground detector signal. The quality of the feed water is improved and the life of
the purification system is extended if a reverse osmosis system is included between
the prefilter and carbon cartridges. This system lowers the base level of organics,
inorganics, and microorganisms.
Microorganisms such as bacteria and algae multiply rapidly in water. Therefore,
even when using water purified in the manner just described, it is wise to discard
all remaining water at the end of each week. The HPLC system should be flushed
with methanol to destroy any microorganisms that have entered it during the
week. At the beginning of a new work week, the water reservoir should be washed
with methanol prior to filling with newly purified water. Growth of microorgan-
isms can also be prevented by adding 0.02% sodium azide or acetonitrile (which
is present in many RP mobile phases) to the water.
Purified water is best stored in carefully cleaned glass containers. Plasticizers
can leach into water stored in plastic containers, interfering with RP systems or
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–13
Pesticide Analytical Manual Vol. I
contaminating the column. Leaching of metals from glass containers is also a
possibility, but this is usually less of a problem than introduction of organic impu-
rities.
HPLC grade water can be purchased from a number of commercial sources. This
water can be used successfully as received for most applications.
The following purity check can be used to test water for applicability in HPLC:
? Pump 100 mL water through C-18 column.
? With a UV detector in-line, run a linear gradient from 0 to 100% metha-
nol at 1 mL/min for 10 min and hold for 15 min.
? If the UV baseline shift at 0.08 AUFS is <10% and very few peaks of <3-
5% full scale deflection are observed, the water is pure enough for most
applications.
Acetonitrile. Acetonitrile is commonly used in RP HPLC mobile phases. Manufac-
turers’ specifications for HPLC solvent purity are usually based on acceptability for
UV detectors. Specifications for fluorescence and electrochemical detectors are
very difficult to define because of the complexity of instrumental parameters.
Methanol. Another of the more common solvents employed in RP HPLC is metha-
nol, which suffers from the same inadequacy of specifications as acetonitrile.
Methanol has the disadvantage of producing relatively viscous solutions when mixed
with water, giving rise to much higher pressures than with other mobile phases.
Chlorinated Solvents. Some chlorinated solvents are stabilized against oxidative
breakdown by addition of small amounts of methanol or ethanol. Alcohol will
increase polarity of mobile phases and shorten elution times in NP HPLC. Also,
reproducibility will be affected because the concentration of stabilizer will vary
slightly from batch to batch.
Chlorinated solvents can be purchased without stabilizer, or the stabilizer can be
removed by adsorption onto alumina, or by extraction with water followed by
drying. Unstabilized chlorinated solvents may slowly decompose, producing hydro-
chloric acid, which degrades columns and corrodes stainless steel. The rate of
decomposition may be accelerated by the presence of other solvents. Hydrochloric
acid can be removed by passing the solvent through activated silica or calcium
carbonate chips. Solvents can be stabilized with amylene to avoid these problems.
Gillespie et al. [4] noted problems such as increased detector response and discol-
oration of equipment when ethylene dichloride or methylene chloride was used
in HPLC mobile phases. The problems described were attributed to a reaction
between solvent impurities and stainless steel upon prolonged contact.
Ethers. Ethers contain additives to stabilize them against peroxide formation. For
example, tetrahydrofuran is often stabilized by addition of small amounts of hyd-
roquinone. This compound absorbs UV radiation and so interferes with UV ab-
sorption detection. It can be removed by distilling the solvent from potassium
hydroxide pellets. Inhibitor-free tetrahydrofuran should be stored in a dark bottle
and flushed with nitrogen after each use. Any peroxides that form should be
periodically removed by adsorption onto alumina.
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–14
Reagent Blanks
Blank samples should be analyzed to ascertain that no interferences from reagents
(or glassware) occur during analysis. Reagent blanks are especially important when
using nonspecific optical detectors such as UV or RI detectors.
Safety Precautions
Beyond the concern over damage to HPLC systems that can be caused by reagents
and solvents, it is important to protect the health of the analyst. An awareness of
the toxicity of the chemicals in use is essential. Care must be taken to minimize
exposure to toxic chemicals. See Reference 5 for more on laboratory safety for
HPLC analysis.
601 E: SAMPLE PREPARATION
Sample Cleanup
Extracts to be analyzed by HPLC must be cleaned up (i.e., interfering co-extrac-
tives removed) sufficiently to permit identification and quantitation of residues,
and to prevent contamination or harm to any part of the HPLC system. The
column and/or detector may be impaired by injection of dirty extracts, especially
when many samples are analyzed.
Cleanup procedures for trace residue determination by HPLC must be developed
to accommodate the selectivity of the detector. Dissolved interferences in the
sample solution that appear in the chromatogram as extra peaks must be re-
moved. Any materials that will be strongly adsorbed by the column must also be
removed to prevent their affecting chromatographic characteristics of the column,
causing baseline drift, or appearing as spurious peaks in later chromatograms.
A recent innovation combines cleanup of the sample extract in-line with the HPLC
determinative step [6]. A short column of SCX resin replaces the sample loop in
a six-port HPLC injection valve, where it effectively removes the analyte, formetanate
hydrochloride, from the extract. Solvent flushing of the column while the short
column is still off-line (disconnected from the analytical column) provides cleanup
and substitutes for traditional separatory funnel partitionings. Subsequent switch-
ing of the valve places the cleanup column in-line with the analytical SCX column
for elution and determination. This coupled column application and other mul-
tidimensional variations [7] provide simple, rapid analysis with minimum solvent
use.
Sample Filtration
Removal of particulate matter in the sample solution is critical for HPLC stability.
Both column frits and the top of the column packing can become clogged by
particles, leading to increased back pressure and adverse effects on chromato-
graphic results because of decreased column efficiency, production of split peaks,
etc.
At a minimum, samples should be passed through a commercial clarification
apparatus, such as a syringe and a 5 μm filter pad in a Swinny adapter, before
injection. In residue determination, passing samples through filters with <1 μm
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–15
Pesticide Analytical Manual Vol. I
pores is preferred. If the detector in use is of the porous flow-through type, the
sample should be filtered to remove particles >0.2 μm. In addition, in-line filters
placed ahead of the column can be used to prevent clogging of column frits. It
is important to ensure that the analyte is not lost on the filter medium, especially
for quantitative determination. This should be determined by analysis of samples
fortified with known concentrations.
Sample Solvent Degassing
Sample extracts should be prepared for injection using solvents that have been
degassed in the same manner as mobile phase solvents (see Section 603 B, Mobile
Phase Preparation). This will reduce the possibility of problems when the sample
solvent enters the detector cell. The sample solution itself should not be degassed
because evaporation will change its concentration.
Choice of Sample Solvent
Ideally, the sample should be dissolved in the mobile phase. This reduces the size
of the solvent peak, thereby aiding identification of early eluting sample peaks. It
also avoids sample precipitation on or before the column, which can result in the
loss of peaks for the analyzed sample and appearance of unknown, randomly
eluting peaks in chromatograms from subsequent injections. This could occur, for
example, if the mobile phase is methanol/water and the sample is dissolved in
neat methanol because of insolubility in the mobile phase. As a precaution after
using a different sample solvent, the column should be flushed with a strong
solvent that is compatible with the column, followed by equilibration with the
mobile phase before injection of the next sample. Ultrasonic mixing may aid in
dissolving the sample in the mobile phase or a similar solution.
If the sample must be prepared in a solvent different from the mobile phase, it
should be compatible with the column, as close as possible to the mobile phase in
composition, and of weaker elution strength if this is consistent with solubility
requirements. In addition to possible sample precipitation as described above,
injection in a stronger solvent can cause peak tailing. If a stronger solvent must be
used, the smallest possible volume should be injected.
601 F: REFERENCE STANDARDS
General procedures for storage, handling, and preparation of solutions of analyti-
cal reference standards for pesticide residue analysis are covered in Section 205.
Preparation, storage, and stability are described in greater detail in Reference 8.
The nature of HPLC makes it the preferred determinative step for many unstable,
reactive, or easily degraded pesticides. For this reason, the stability of the pesticide
in the solvent used to prepare standard solutions requires particular attention.
Stock Solutions
Considerations for the choice of a solvent for preparing stock standard solutions
are the same as for choosing a solvent in which to inject samples (see Section 601
E). If stability permits, standard solutions should be prepared in the mobile phase
to be used in the HPLC analysis. However, many pesticides have limited stability
in “reactive” solvents, such as methanol or water, often used for mobile phases. For
example, the fungicides thiophanate-methyl, captan, folpet, and captafol can be
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–16
stored indefinitely in benzene, acetone, or isooctane, but they quickly degrade in
methanol/water.
Alternatively, stock standard solutions can be prepared in a less reactive solvent
with a fairly high volatility (e.g., acetone). Working standard solutions can then be
prepared by evaporation of the volatile solvent from an aliquot and subsequent
dissolution in the HPLC mobile phase or other appropriate solvent.
Benzene is a good solvent for most pesticide standards, but its toxicity makes its
use inadvisable. Isooctane and hexane dissolve most organochlorine pesticides;
isooctane’s low volatility minimizes evaporative loss during storage, but also pre-
cludes its use in cases where it is desirable to evaporate the original solvent prior
to dissolution in the mobile phase. Chloroform is useful for triazines, methylene
chloride or methanol for carbamates, acetone for benzimidazole-related fungi-
cides, and methanol for phenylurea herbicides.
Because of possible deterioration due to evaporation and/or instability, it may be
necessary to remake stock standard solutions frequently. Because standard refer-
ence materials are often supplied in limited quantities (<100 mg), use of a mi-
crobalance is preferred for accurate weighing of low mg quantities of standard for
preparation of stock solutions. Direct preparation of dilute solutions in this way
can also reduce the number of dilutions required to make the working standard
solution.
Working Standard Solutions
These solutions are prepared at concentrations suitable to the detector in use and
the expected levels of pesticides in sample extracts. Concentrations of working
standard solutions should closely match those in sample extracts for the most
reliable comparison of peak heights or areas. For general screening purposes or
multiresidue analysis, working standard solutions can be made up as mixtures of
pesticides resolvable by the method.
Stability of working standard solutions should be confirmed by periodic compari-
son against newly prepared solutions or fresh dilutions of stock solutions. Solvents
used to prepare working standard solutions should be compatible with the sample
solvent and the HPLC system (see Section 601 E) and should be checked for
contaminants that could possibly interfere with the analysis.
Storage
Stock standard solutions should be stored in an explosion-proof refrigerator at
≤4° C. Benzene solutions can freeze at these temperatures and may crack contain-
ers. Organochlorine pesticide stock solutions can be stored for at least 6 months
without deterioration. Organophosphorus and carbamate solutions are less stable
and should be discarded 3-4 months after preparation. Some standard solutions
degrade quickly and must be made fresh at least daily.
References
[1] Gilvydis, D.M., and Walters, S.M. (1990) J. Assoc. Off. Anal. Chem. 73, 753-761
SECTION 601
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 601–17
Pesticide Analytical Manual Vol. I
[2] Snyder, L.R., and Kirkland, J.J. (1979) Modern Liquid Chromatography, 2nd ed.,
Wiley, New York
[3] Callahan, F.J. (1985) Swagelok Tube Fitting and Installation Manual, Markad
Service Co.; available from Supelco, Bellefonte, PA 16823-0048
[4] Gillespie, A.M., et al. (Feb. 1986) “Cautionary Note on Use of Ethylene Dichlo-
ride and Methylene Chloride in HPLC Mobile Phases,” LIB 3011, FDA,
Rockville, MD
[5] Runser, D.J. (1981) Maintaining and Troubleshooting HPLC Systems — A User’s
Guide, Wiley, New York
[6] Niemann, R.A. (1993) J. AOAC Int. 76, 1362-1368
[7] Wojtowicz, E.J. (Feb. 1992) “HPLC Fluorometric Analysis of Benomyl and
Thiabendazole in Various Agricultural Commodities,” LIB 3650, FDA,
Rockville, MD
[8] EPA Manual of Quality Control for Pesticides and Related Compounds in Human
and Environmental Samples (2nd rev., 1981) Section 3-O, Environmental Pro-
tection Agency, Washington, DC
SECTION 601 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)601–18
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 602–1
SECTION 602Pesticide Analytical Manual Vol. I
602: COLUMNS
The nature and dimensions of the column packing, together with the nature of the
mobile phase, largely determine the selectivity and efficiency of the separation that
is achieved. HPLC columns are packed with small particles (usually 3-10 μm) hav-
ing a narrow size distribution (approximately ± 20%). The use of microparticulate
materials requires that the mobile phase be pumped through the column at high
pressure. Columns can be prepared in the laboratory, but most analysts purchase
commercial prepacked, pretested columns.
An HPLC column is a highly efficient filter, and any particulate matter or strongly
retained impurity that is injected will remain on the top. To prevent deterioration
of the analytical column, a guard column should be installed between it and the
injection device. The guard column is discarded or repacked after a certain num-
ber of sample injections. A saturation precolumn situated between the pump and
injector device may be used to ensure equilibrium between the two phases in a
liquid-liquid chromatography system, or to prevent dissolution of silica from an
unmodified or bonded silica analytical column. Although columns of different
sizes have been used, 25 cm × 3-5 mm id columns packed with 5 or 10 μm station-
ary phase material have provided adequate separation in a reasonable time for
many applications.
602 A: COLUMN SELECTION
Column selection is not a straightforward process. The best approach is to search
the literature for work published on a separation that is the same as, or similar to,
the one that needs to be accomplished. Many of the references in Section 608
discuss column selection techniques for different sample types, and most column
manufacturers have published guides and technical data sheets that will aid in
column selection.
A knowledge of the chemistry of the sample, often determined by some simple wet
chemistry experiments, combined with a systematic trial and error approach, is
probably the method used most often in column selection. If the molecular weight,
range of solubility, and molecular or ionic structure of the analyte are known, a
mode of separation can be selected as discussed previously (see Figure 601-c). The
most appropriate column for that mode is then chosen, based on the experience
of the analyst, column manufacturers’ recommendations, or a search of the litera-
ture.
602 B: ANALYTICAL COLUMNS
Factors important in producing efficient columns include narrow particle size
distribution in the packing and minimal dead volume in the tubing, fittings, cells,
and other components of the HPLC instrument.
Most packed columns are made from stainless steel. In addition, glass cartridge
columns are common and radial compression columns prepared from heavy wall
polyethylene cartridges are available. The latter columns are radially compressed
in a hydraulic press during use to minimize void volumes and wall effects and
thereby increase column efficiency.
Pesticide Analytical Manual Vol. ISECTION 602
602–2
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Recent advances in column technology include use of 3-10 cm columns packed with
3-5 μm particles. The major advantages of these shorter columns over conventional
25 cm columns are faster separations and improved sensitivity of detection. Another
trend is the use of microbore columns, 0.2-1 mm id columns packed with conventional
bonded phases. Microbore columns can be made very long, providing up to one
million theoretical plates for difficult separations. They require only small volumes of
mobile phases and allow novel detection possibilities, including flame ionization,
chemical ionization mass spectrometry, and IR spectrometry.
Normal phase (NP) HPLC is carried out on adsorbent (silica gel, alumina) col-
umns or polar bonded (cyano, amino, diol) columns. Liquid solid chromatog-
raphy and polar bonded phase chromatography are suitable for separation of
nonionic multifunctional compounds and isomers. Silica gel is by far the most
used column for NP separations. However, because NP columns have not been
used widely for analytical work, most discussion of columns in this section refers
to various types of reverse phase (RP) chromatography used for pesticide determi-
nation.
Liquid-Solid Chromatography
Until recently, little use had been made of liquid-solid chromatography (LSC) for
pesticide analysis. Now, however, a column of porous graphitic carbon, a nonpolar
RP adsorbent, has been successfully applied to the determination of
ethylenethiourea (ETU) using a strongly acid mobile phase [1]. Such columns
offer stability for applications requiring pH extremes and are complementary to
silica-based columns.
Bonded Phases
Most analytical HPLC systems use RP chromatography on silica-based C-18 or C-8
bonded phases. Other RP bonded packings include those having C-1, C-2, C-4, C-
12, cyano, phenyl, diol, or cyclohexyl groups. In RP mode, the stationary phase is
hydrophobic and nonpolar, and mobile phases are relatively polar (usually water
with methanol or acetonitrile). Nonpolar sample components are strongly re-
tained, and polar components are less retained. Bonded columns are stable and
reproducible compared to nonbonded columns with physically adsorbed coatings,
which they have almost completely replaced. The major limitation is the narrow
pH range for column stability.
Most commercially available bonded phases are of the siloxane type, Si-O-Si-R.
They are prepared by reacting surface silanol groups on silica with an
organochlorosilane reagent, the organic portion of which is the moiety to be
bonded (octyl, octadecyl, phenyl, aminopropyl, cyanopropyl, etc.). Packings can be
prepared, for example, by using mono-, di-, or trichloroorganosilanes to produce
products having different chromatographic properties. Monochloroorganosilanes
react with silica to form a monomolecular layer of bonded organic groups. Di- or
trichloroorganosilanes react with silica in the presence of a protic reagent to form
a linear or cross-linked polymeric layer, the structures and properties of which are
not as well defined as with monomeric phases. Polymer bonded phases have poorer
mass transfer characteristics but higher loadability. Some of the accessible unreacted
silanols on the silica surface after the primary bonding reaction may be removed
by end-capping, which involves reaction with a less bulky reagent such as
trimethylchlorosilane.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 602–3
SECTION 602Pesticide Analytical Manual Vol. I
Most of the current bonded RP columns have 5 μm spherical silica as the base
material. Pore size ranges from 60-300 nm, with 80-120 nm most common.
To increase the range of pH stability, bonded columns having polystyrene-
divinylbenzene (DVB) polymer as the base material have been developed.
Another approach is a base material composed of alumina coated with a
polybutadiene polymer layer to protect the bonded surface from attack by hydrox-
ide. Stability up to pH 13 is possible for such columns because alumina is stable at
this pH.
Short chain phases such as C-2 and C-4 are used to reduce hydrophobic interac-
tions in separating high molecular weight analytes, such as proteins and peptides.
Cyanopropyl phases can be used in NP work by selective interactions with the
cyano functional group or as a short chain RP material for separation of polar
analytes. Diol phases, whose structures involve two hydroxy groups on adjacent
carbon atoms in an aliphatic chain, are less polar than silica and are used in both
NP and RP chromatography. Phenyl phases are prepared by the reaction of
dimethylphenylchlorosilane with silica gel. They are nonpolar and have special
affinity for aromatic compounds. Cyclohexyl phases have selectivity for alicyclic
compounds compared to straight chain compounds. Some RP columns are base-
deactivated to optimize separation of basic compounds without tailing or need for
mobile phase modifiers for ion pairing or ion suppression.
The determinative steps of Sections 401, 403, and 404, methods for N-methyl-
carbamates, substituted ureas, and benzimidazoles, respectively, provide examples
of applications of bonded RP HPLC to pesticide residue analysis.
Ion Exchange
Four types of microparticulate packings are available for high performance ion
exchange chromatography (IEC). Polystyrene-DVB polymeric gel resin particles of
5-10 μm diameter substituted with ionogenic groups were the earliest of these
packings. The amount of DVB added for the polymerization reaction determines
the degree of cross-linking and, hence, the pore structure. Resins with <6% DVB
are not pressure stable and cannot be considered HPLC packings. Slow diffusion
of analytes within the polymer matrix and the resulting poor efficiency led to
development of pellicular ion exchange materials, consisting of a glass core, an
intermediate coating of silica, and an outer ion exchanger polymer film. These
materials suffer from low efficiency due to their relatively large particle size and
low sample capacity.
Silica-based ion exchange packings are prepared in a manner similar to other
bonded phases. Controlled porosity glass column packings with attached hydro-
philic polymeric groups can be used for high speed separations of large ionic
molecules such as proteins and nucleic acids.
Virtually all commercial ion exchange materials contain sulfonate (strong cation
exchange), carboxylate (weak cation exchange), tetraalkylammonium ion (strong
anion exchange), or an amine (weak anion exchange) functional group. The
capacity of exchangers is a function of the pH of the mobile phase. Full exchange
capacity is exhibited by different exchangers at the following pH values: strong
cation, above 3; weak cation, above 8; strong anion, below 9; and weak anion,
below 6. The wide exchange range of strong exchangers makes them most useful
for general analytical work. The pH of the mobile phase controls retention by its
effect on the ionic nature of both the sample and the exchange sites.
Pesticide Analytical Manual Vol. ISECTION 602
602–4
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
IEC has been applied to determination of residues of formetanate hydrochlo-
ride [2]. A strong cation exchange mechanism is used for the chromatography of
this ionic residue.
Ion Pair
RP ion pair chromatography is an alternative to IEC. It is an extension of ion
suppression chromatography, in which weak acids or bases are separated on an
RP bonded column by addition of a pH modifier to the mobile phase to ensure
that analytes are in their undissociated forms.
In ion pair chromatography, a charged organic compound is added to the mobile
phase to form a neutral ion pair with an analyte of opposite charge. For example,
an alkylsulfonate can be added to cationic samples and tetrabutylammonium
phosphate to anionic substances. Ion pair chromatography is suitable for separat-
ing mixtures of anions, cations, and neutral substances; the pH of the mobile
phase will suppress the ionic character of one of the types of ions, while the
counter ion will react with the other type to form ion pairs. For example,
tetrabutylammonium phosphate buffered to pH 7.5 can form ion pairs with strong
and weak acids, and the buffering suppresses weak base ions. Amphoteric mol-
ecules can be chromatographed with either quaternary amine or sulfonate counter
ions at an appropriate pH value.
Selectivity can be affected by the concentration and choice of the ion pair reagent.
The k values (see Section 602 C) of analytes are proportional to the counter ion
concentration. The longer the alkyl chain length, the greater are k values. Reten-
tion times can also be adjusted by changing the composition of the mobile phase,
which is usually a mixture of water with either methanol or acetonitrile.
Quaternary ammonium salts in alkaline medium are damaging to silica gel. Col-
umns should never be stored in such solutions. A precolumn placed in front of the
injector, to saturate the mobile phase with silica gel, is highly recommended in
these systems.
An example of pesticide determination using ion pair chromatography (on a
bonded phase) is the determination of benzimidazole residues (Section 404). In
addition, two methods for determining residues of paraquat and diquat use the
ion pair mechanism, one with a polymeric (PRP-1) column [3], and the second
with a silica column using NP mode chromatography [4].
Size Exclusion
Separations in the size exclusion (SEC) mode are based on molecular size and are
controlled by the pore size of the packing material. Particle sizes in the 5-20 μm
range are used to provide good column efficiency. Packings for SEC include semi-
rigid organic gels, porous silica, and controlled pore glasses.
The major use of SEC in pesticide determination is for cleanup of residues from
fatty samples by gel permeation chromatography, rather than as a determinative
step. The most used packing for this purpose has been styrene-DVB copolymer
such as Bio-Beads S-X3 (Section 304 C5, Section 402). The Bio-Beads S-X series
offers exclusion limits from 400-14,000 molecular weight; S-X3 has a 2000 exclu-
sion limit. The exclusion limit is determined by the amount of DVB cross-linking
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 602–5
SECTION 602Pesticide Analytical Manual Vol. I
of the gel, as well as by the degree of swelling that can occur in different solvents.
Maximum expansion of the gel occurs in relatively nonpolar solvents. Typical
solvents used include benzene, toluene, xylene, carbon tetrachloride, methylene
chloride, and mixtures such as methylene chloride/hexane. The sample should
not interact with the stationary phase in any way, e.g., by adsorption. Stationary
phase with a smaller particle size will provide greater peak capacity, and better
and faster separations.
602 C: COLUMN EVALUATION
An HPLC column can be evaluated by measuring certain performance characteris-
tics or parameters, many of which can be visualized or measured on the chromato-
grams produced by the column. Column efficiency and peak symmetry reflect the
quality of the column, whereas the capacity factor and selectivity indicate its
capability to retain and separate compounds of interest.
Several terms must be measured in order to calculate the parameters of a column.
Figure 602-a provides a visual representation of these terms:
The time from injection to the peak maximum is known as the retention time, t
r
.
The retention time consists of two parts, t
o
and t'
r
. t
o
is the time from injection to
emergence of the solvent front, which may be noted as a small shift or disturbance
in the baseline or a solvent peak if the sample solvent is different from the mobile
phase and is sensed by the detector. t'
r
, the adjusted retention time, equals t
r
minus t
o
. t'
r
represents the time that the analyte is retained in the stationary phase.
?t is the time between the maxima of two peaks, and W is the peak width deter-
mined between the intersections of tangents drawn on the sides of the peaks with
the baseline. All of these time values can be measured in mm directly on the
Terms
tr = retention time, mm
to = elution distance of unretained component, mm
t'r =tr-to (adjusted retention time)
a,b = peak half-width at 10% peak height, mm
?t = time between peak maxima, mm
W = peak width at base, mm
h = peak height, mm
Wh = peak width at half height, mm
Figure 602-a
Calculation of Column Performance Parameters
tr
D t
2
1
h
Wh
ab
10% h
W
to
Injection
1
tr
2
Capacity factor, k = t'r/to
Selectivity, α =k
2
/k
1
or t'r
2
/t'r
1
Efficiency, n theoretical plates = 16(tr/W)
2
or 5.54(tr/Wh)
2
Efficiency, HETP = column length (cm)/n
Resolution, Rs =2(?t)/(W
1
+W
2
) or (1/4)(α?1) n(k/1+k)
Peak asymmetry, As = b/a
References: Walters, M.J., et al. (Nov.1980) "Recommendations for HPLC Columns," LIB 2447, FDA, Rockville, MD;
ASTM Standards on Chromatography (1981) E682.
Pesticide Analytical Manual Vol. ISECTION 602
602–6
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
recorder trace of the chromatogram. These terms are used to calculate the follow-
ing parameters for evaluation of columns: capacity factor, selectivity, efficiency,
resolution, and peak asymmetry.
The capacity factor, k, measures retention of an analyte by the column in terms of
column volumes. It is affected by the strength (e.g., polarity) of the mobile phase
and the strength (retentivity) of the column packing. A k value of 2-10 for the
most retained component is generally optimal for good resolutions but may be
higher for difficult separations.
Selectivity is a thermodynamic factor that measures the ability of a particular
column/mobile phase combination to provide different distribution constants for
two substances, thereby causing a different degree of retention for the two sub-
stances, as indicated by the separation of their peak maxima. It is symbolized by α
and calculated as the ratio of t'
r
values or k values for two peaks, with the largest
value placed in the numerator. Selectivity is affected by the chemistry of the entire
system, including the functionality of the sample components.
Efficiency is a kinetic factor that indicates the ability of the column/mobile phase
combination to produce narrow peaks. Efficiency is dependent on particle size,
column dimensions, and packing technique. It is determined by the number of
theoretical plates, n, and height equivalent to a theoretical plate, HETP.
Resolution is the ability of the column/mobile phase combination to separate the
peaks representing two substances. It is a function of efficiency, selectivity, and
retention and is improved by increasing the separation of the peaks (selectivity)
and/or by decreasing their width (increasing efficiency). Resolution should be >1
to minimize error in quantitative analysis. A retention, k, of 2-10 is usually as-
sumed.
Peak asymmetry describes the shape of a chromatographic peak. Theory assumes
a symmetrical, Gaussian shape for peaks, but asymmetry can be caused by extra-
column effects, poorly packed columns, deterioration of packing, incompatability
between analyte and packing, etc. The peak asymmetry factor is the ratio, at 10%
peak height, of the distance between the peak apex and the back side of the
chromatographic curve to the distance between the peak apex and the front side
of the chromatographic curve. A value of 1 indicates a symmetrical peak, a value
>1 is a tailing peak, and a value <1 is a fronting peak.
Higher efficiency, which leads to sharper peaks, is achieved by using columns with
small, uniform, tightly packed particles and optimized column flow rates. High
selectivity, which is manifested by well separated peak maxima, is influenced mostly
by the nature of the stationary and mobile phases.
602 D: COLUMN SPECIFICATIONS
The parameters described above can be used to evaluate column quality. Columns
that produce the desired separation should be defined for future reference by
the measured parameters. A “system suitability test” that specifies acceptable op-
eration of the HPLC determinative step should be included with any method
description; this may require the use of specific compounds involved in the proce-
dure. System suitability test elements that relate to column specifications are listed
in Table 602-a.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 602–7
SECTION 602Pesticide Analytical Manual Vol. I
At a minimum, a new analytical column should be checked for efficiency by calcu-
lating and recording the number of theoretical plates using an appropriate test
solution. This value is compared with the manufacturer’s specifications and used
in later column quality control evaluations.
Expected minimum efficiency values are shown in Table 602-b. In general, effi-
ciency (plates per meter) decreases with larger or less uniform size column pack-
ing, lower temperature, increased extra-column volume in the system, and larger
samples. Efficiency improves when k = <2 unless extra-column effects are domi-
nant.
Specifications and test systems for six satisfactory HPLC bonded phase silica col-
umns were recommended at an early stage of HPLC application [5]. These recom-
mendations are useful as a guideline for comparing and defining columns, but the
specifications themselves are no longer applicable because of subsequent improve-
ments in HPLC column technology. Other protocols for column testing and evalu-
ation have been suggested. For example, Poole and Schuette [6] described test
conditions and specifications for a 10 μm C-18 RP column using a mixture of
resorcinol, naphthalene, and anthracene and a UV detector.
Commercial bonded silica RP columns from different manufacturers are not equiva-
lent, and information on the degree of hydrocarbon coverage in a column is not
usually provided. In addition, the free (unreacted) silanol sites vary among
Table 602-a: HPLC Column Specification Elements
Physical Description
Packing material
? particle type: size, shape, pore size
? bonded surface type: functionality, mono or polymeric
? surface coverage: (% concentration or μmoles/m
2
)
? additional silylation
Column dimensions
Performance Characteristics
[Requires that the test system be defined by specifying mobile phase solvent and
flow rate, test solution compounds, and solvent. Characteristics must be related
to peak(s) that were used to measure each.]
? Minimum theoretical plates, n
? Resolution, Rs
? Selectivity, α
? Capacity factor, k
? Asymmetry, As
Pesticide Analytical Manual Vol. ISECTION 602
602–8
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
columns and manufacturers, and these can have significant effects on the chroma-
tography of polar analytes. A test scheme developed for classifying and selecting
C-18 bonded columns was used to classify 12 brands of columns into three
major groups based on a hydrophobicity index, free silanol index, and column
efficiency [7].
602 E: ANALYTICAL COLUMN PROTECTION
HPLC analytical columns are expensive and subject to damage during use. The
following items must be used to protect the column and prolong its useful life:
Filters
A major cause of column deterioration and damage is the buildup of particulate
and chemical contamination at the head of the column. This can lead to increased
back pressure and anomalous chromatographic results. Particle buildup is mini-
mized by proper filtering of mobile phase solvents (see Section 603 B) and by
choosing a sample solvent that will not cause precipitation (Section 601 E). In
addition, in-line column filters help to eliminate particulate impurities.
Columns normally contain stainless steel inlet and outlet filters or frits to retain
the column packing. The pore size of the frit must be smaller than the particle
diameter of the packing, e.g., a 2 μm frit for 5 μm packing. Frits are either incorpo-
rated into the ends of the column itself or made an integral part of the column
end fittings.
Periodic cleaning of end fittings and frits in an ultrasonic bath in a solution such
as 6 M nitric acid is recommended, especially when column back pressure in-
creases. Before removing column end fittings, the manufacturer’s literature should
be read carefully for procedural instructions or notification of any loss of warranty
if the column is taken apart. Some companies seal end fittings onto the column
with epoxy and do not guarantee the column if the seal is broken.
Table 602-b: Minimum Efficiency Values
(in thousands of theoretical plates per meter)
Particle Size
Column Type 10 μm5 μm3 μm
porous RP bonded 12-20 35-40 80-100
porous silica gel
adsorbent 24 40
porous ion
exchangers 10-15
semirigid organic
size exclusion gels 9-12
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 602–9
SECTION 602Pesticide Analytical Manual Vol. I
Precolumns
The terms “precolumn” and “guard column” are often used interchangeably, but
the two types of columns are different and serve separate primary functions.
Precolumns are positioned in the HPLC system prior to the sample injector. Their
purpose is to saturate the mobile phase with silica so that the silica or bonded
silica analytical column packing is not dissolved during use. Precolumns packed
with inexpensive, coarse silica are suitable for this mobile phase conditioning
function.
Guard Columns
A guard column is inserted between the injector and analytical column to protect
the latter from damage or loss of efficiency due to the presence of particulate
matter or strongly adsorbed impurities from analytical samples. It can also serve as
a saturator column to prevent dissolution of the stationary phase, in addition to,
or instead of, a precolumn as described above. The use of a guard column is
especially important when injecting relatively crude sample extracts or biological
fluids.
Guard columns are short (2-6 cm) disposable columns containing the same pack-
ing as the analytical column. The guard column must be changed frequently, as
dictated by the contamination level of the samples, to ensure that the lifetime of
the analytical column, which should be several hundred hours running time, is
not shortened.
The use of a commercial guard column having the same particle diameter packing
as the analytical column, in combination with low dead volume fittings and short
lengths of connection tubing, should cause essentially no loss in efficiency. Guard
columns containing 5 or 10 μm particles can be purchased in the form of prepacked
disposable cartridges (often 2 cm). They must be slurry packed if prepared in the
laboratory. When a greater loss of efficiency is not critical, guard columns contain-
ing larger particle (20-40 μm) microporous or pellicular packings can be dry-
packed in the laboratory using the tap and fill method, but the pellicular packings
do not provide as much protection because of their lower surface area.
602 F: COLUMN MAINTENANCE AND TROUBLESHOOTING
Column Care
Chromatography companies usually supply a booklet describing recommended
care and use of their columns. Topics covered typically include column descrip-
tion; directions for initial inspection, connection, equilibration, operation, regen-
eration, repair, and storage; mobile phase requirements; and information about
solvent purification, protector columns, and replacement of frits. Any such litera-
ture should be read carefully and the suggestions followed as closely as possible.
The following items describe routine handling and maintenance:
? Do not jar, drop, or vibrate columns.
? Pass solvent through the column in the direction specified by the manu-
facturer. If a flow direction is indicated, operation in the opposite direc-
tion may disturb the packing and reduce column efficiency.
Pesticide Analytical Manual Vol. ISECTION 602
602–10
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
? When starting up the HPLC system, gradually increase column flow rate
and pressure to avoid pressure shock and formation of voids in the pack-
ing.
? Operate the column at a constant temperature using a column oven. If
temperature significantly above ambient is used, raise the temperature
slowly with solvent flowing. Elevated temperature improves column effi-
ciency and reduces operating pressure by lowering solvent viscosity. How-
ever, thermal expansion of the column wall can lead to sinking or chan-
neling of the column packing and loss of efficiency. Do not operate ana-
lytical columns at >60? C.
? Allow the column to equilibrate with the mobile phase, as indicated by a
stable detector baseline, before injecting samples. Be sure that back pres-
sure is acceptable for the required flow rate.
? Check fittings visually and by feel to be sure there are no mobile phase
leaks. Leaks too small to see may be detected by the coolness of fittings to
the touch.
? Particulate matter can become caught in the inlet frit, causing high back
pressure. Replace the frit to return the column to the lowest possible
operating pressure. Alternatively, clean the frit by washing with dilute
nitric acid in an ultrasonic bath, dry, and replace. Never remove the
bottom frit from the column. Use a precolumn filter to avoid the need to
change the inlet frit and possibly disturb the column packing. Filter
samples that may contain particulate matter to prevent contamination of
the sample valve or column inlet frit. A commercial clarification kit that
attaches to a syringe is a convenient way to filter samples.
? Do not overtighten column end fittings, or threads may be stripped, caus-
ing a leak.
? Flush the column with a solvent stronger than the mobile phase at the
end of the day if dirty samples were injected.
? Handle columns gently to avoid shock and the formation of voids.
? Label columns with complete information on their source, identity, his-
tory and conditions of use, and regeneration and storage solvents. Keep a
log notebook for each column from time of installation.
? Do not subject columns to operating conditions that may destroy their
structure; be aware of the appropriate solvents and conditions that are
compatible with the particular column.
Column Evaluation by Injection of Test Mixtures
Inject a test mixture to evaluate efficiency, selectivity, k, peak shape, etc., according
to the laboratory’s instrument quality assurance requirements (see Section 602 C).
Choose the test mixture according to the purpose of the test:
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 602–11
SECTION 602Pesticide Analytical Manual Vol. I
? To compare a chromatogram to the one supplied with a prepacked
column by the manufacturer, use the same compounds and conditions
specified by the manufacturer for comparable results.
? If an in-house test mixture for column assessment is needed, prepare it
to contain the following types of components:
1) an unretained (but not excluded) component for assessment of the
volume between the particles and in the pores;
2) a minimally retained component (k = about 0.2) to assess zone broad-
ening caused mainly by the injector, column, and detector. Because
the peak volume of this component will be small, it will be a critical
test of the effect of these system components on performance;
3) a moderately retained component (k = 1-3);
4) a well retained component (k = 7-20). This component is optional
because zone broadening will not be obvious because of the large
peak volume; and
5) a totally excluded component for determination of column void vol-
ume.
Column Storage
When no longer in use, columns should be equilibrated with an appropriate
storage solvent, disconnected from the HPLC system, and the ends capped se-
curely for storage.
Buffer solutions and halogen salts can easily damage column packings and stain-
less steel columns. Columns should be flushed with water after the use of buffers
and should never be stored in such a solution. LSC columns are best stored in a
dry organic solvent; RP columns in methanol, acetonitrile, or a water/acetonitrile
or water/methanol mixture (use of water-free organic solvent reduces silica disso-
lution); IEC columns in a compatible solvent with the same ion as the form of the
exchanger; and SEC columns in a solvent compatible with the swelling properties
of the packing. Columns are not normally stored under pressure. The tempera-
ture and humidity of the storage area should be moderate and consistent.
Column Regeneration
Columns should not be operated with excessive pressure as this can create a void
at the column head, resulting in a significant loss of efficiency. The cause of
increased back pressure should be determined and steps taken to remedy
the situation. Pressure buildup due to particulates can sometimes be relieved by
back flushing the column or changing the frit at the head of the column. The
simplest method of removing strongly retained material is washing with a solvent
stronger than the mobile phase. If an appropriate guard column is in use, rejuve-
nation of the system should be possible in most cases by merely replacing the
guard column.
Pesticide Analytical Manual Vol. ISECTION 602
602–12
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
A void at the head of the column can be observed after removing the inlet fitting.
Voids can be filled with either glass beads or the same or similar packing as
originally in the column.
Flushing with pure organic solvents such as methanol, tetrahydrofuran, chloro-
form, or acetonitrile is useful for regenerating bonded polar phase columns. When
using any series of washes, the order of solvents should be weak to strong (nonpo-
lar to polar for NP, and polar to nonpolar for RP), with consideration of mutual
solubility at each stage. Basic impurities may be washed out with 1-5% aqueous
phosphoric or acetic acid and acidic impurities with 1-5% aqueous pyridine. Bio-
logical materials and fats are removed from RP columns by washing with methyl-
ene chloride and making several 0.2-1 mL injections of dimethylsulfoxide during
elution. Typically, 75 mL of each wash solvent is used at a flow rate of 0.5-3 mL/
min. If washing does not remove adsorbed impurities from the top of the column
bed, the upper, contaminated layers of packing must be removed with a spatula
(exercising great care to avoid scratching the internal column wall), and the col-
umn repacked as described above for the case of a void.
In all cases, the last wash in the regeneration process should be with a solvent that
is miscible with the mobile phase, and the column should be finally re-equilibrated
with the mobile phase. After regeneration (or between washing stages to check
progress), a test mixture should be chromatographed to evaluate plate number,
k, and peak shape. Regeneration and return to equilibrium with the mobile phase
can also be monitored by keeping the column connected to the detector and
observing baseline drift. This should not be done if eluted impurities might con-
taminate the detector cell.
References
[1] Krause, R.T. (1989) J. Liq. Chromatogr. 12, 1635-1644
[2] Niemann, R.A. (1993) J. AOAC Int. 76, 1362-1368
[3] Worobey, B.L. (1987) Pestic. Sci. 18, 245-257
[4] Chichila, T.M., and Walters, S.M. (1991) J. Assoc. Off. Anal. Chem. 74, 961-967
[5] Walters, M.J., et al. (Nov. 1980) “Recommendations for HPLC Columns,” LIB
2447, FDA, Rockville, MD
[6] Poole, C.F., and Schuette, S.A. (1984) Contemporary Practice of Chromatography, pp.
241-252, Elsevier, New York
[7] Walters, M.J. (1987) J. Assoc. Off. Anal. Chem. 70, 465-469
Pesticide Analytical Manual Vol. I SECTION 603
603–1
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
603: MOBILE PHASE SELECTION,
PREPARATION, AND DELIVERY
Mobile phases for different HPLC modes were described briefly under Modes of
Operation (Section 601 B). Solvents used to prepare mobile phases were discussed
under Solvents and Reagents (Section 601 D). This section presents additional
considerations related to the preparation and delivery of mobile phases.
603 A: MOBILE PHASE SELECTION
Mobile phases in HPLC are usually mixtures of two or more individual solvents
with or without additional additives or modifiers. The mobile phase is an active
partner with the column in obtaining the required separation. The usual approach
is to choose what appears to be the most appropriate column, and then to design a
mobile phase that will optimize the retention and selectivity of the system.
The two most critical parameters for nonionic mobile phases are strength and
selectivity. Mobile phase strength is related directly to polarity and ability to dis-
solve polar analytes in normal phase (NP) chromatography, while the opposite
relationship exists for reverse phase (RP) chromatography. The general strategy
for choosing a mobile phase is to find a solvent or solvent mixture with the correct
strength to give k values in the optimum 2-10 range, and then to alter the phase to
give the needed selectivity while maintaining the same strength. Solvents have
been classified according to strength and selectivity to allow the selection process
to be at least somewhat systematic.
Solvent strength for any solvent is dependent on the stationary phase adsorbent.
An eluotropic series is a ranking of solvent strengths on a given adsorbent. Table
603-a lists solvent strengths for common solvents when used with different station-
ary phases.
Table 603-b shows solvents that are members of the different selectivity groups; the
solvents most preferred for HPLC are underlined. Compounds in different groups
interact in different ways with the analytes to be separated, e.g., dispersion interac-
tions, dipole forces, and hydrogen bonding. To optimize selectivity and improve
separations, mobile phases are prepared from solvents in different selectivity groups.
Normal Phase Chromatography
The eight groups of solvents shown in Table 603-b emerged from Snyder’s plot of
solvents within a triangle of selectivity coordinates representing relative proton
donor, proton acceptor, and dipole parameters [1, 2]. Maximum selectivity is ob-
tained if one solvent is chosen from each group closest to the corners of the
triangle. Based on other factors, such as viscosity and UV absorption properties, the
three solvents chosen are usually diethyl ether or methyl tert-butyl ether (MTBE),
chloroform, and methylene chloride. Hexane is used as the base solvent to adjust
polarity (solvent strength). A binary mixture of hexane with one of these three
solvents can be used to determine the appropriate solvent strength, and other
binary, tertiary, and quaternary mixtures with the same strength can be tested in
a systematic trial and error fashion for the required selectivity. The overall strength
(P′) of a mixture is the sum of the product of the individual P′ values times
the volume fraction for each component solvent. Other useful combinations of
603–2
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. ISECTION 603
Table 603-a: Properties of Common HPLC Solvents with Alumina Columns
Solvent Solvent
UV Polarity Strength
Cut-off, Refractive Boiling Viscosity Parameter, Parameter,
Solvent nm Index Point, ?C cP, 25?C P' ε? Group
isooctane 197 1.389 99 0.47 0.1 0.01 —
n-hexane 190 1.372 69 0.30 0.1 0.01 —
methyl t-butyl ether 210 1.369 56 0.27 2.5 0.35 —
benzene 278 1.501 81 0.65 2.7 0.32 VII
methylene chloride 233 1.421 40 0.41 3.1 0.42 V
n-propanol 240 1.385 97 1.9 4.0 0.82 II
tetrahydrofuran 212 1.405 66 0.46 4.0 0.82 II
ethyl acetate 256 1.370 77 0.43 4.4 0.58 VIa
chloroform 245 1.443 61 0.53 4.1 0.40 VIII
dioxane 215 1.420 101 1.2 4.8 0.56 VIa
acetone 330 1.356 56 0.3 5.1 0.56 VIa
ethanol 210 1.359 78 1.08 4.3 0.88 II
acetic acid 1.370 118 1.1 6.0 Large IV
acetonitrile 190 1.341 82 0.34 5.8 0.65 VIb
methanol 205 1.326 65 0.54 5.1 0.95 II
water 1.333 100 0.89 10.2 Very Large VIII
Table 603-b: Classification of Solvent Selectivity
Group Solvents
I aliphatic ethers, methyl t-butyl ether
1
, tetramethylguanidine, hexamethylphosphoric acid
amide, alkyl amines
II aliphatic alcohols, methanol
III pyridine derivatives, tetrahydrofuran, amides (except formamide), glycol ethers, sulfoxides
IV glycols, benzyl alcohol, acetic acid, formamide
V methylene chloride, ethylene chloride
VI a) tricresyl phosphate, aliphatic ketones and esters, polyesters, dioxane
b) sulfones, nitriles, acetonitrile, propylene carbonate
VII aromatic hydrocarbons, toluene, halosubstituted aromatic hydrocarbons, nitro compounds,
aromatic ethers
VIII fluoroalcohols, m-cresol, water, chloroform
1
Underlined solvents are those generally preferred.
[Both tables reprinted with permission of Elsevier Science Publishers, from Poole, C.F., and Schuette, S.A.
(1984) Contemporary Practice of Chromatography, Table 4.16, page 260.]
Pesticide Analytical Manual Vol. I SECTION 603
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Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
solvents with different selectivity characteristics plus miscibility over the entire range
of mixture composition include methylene chloride, MTBE, and acetonitrile in
Freon FC-113 (1,1,2-trifluoro,1,2,2-trichloroethane), and methylene chloride, MTBE,
and ethyl acetate in hexane.
Solvents for liquid-solid chromatography (LSC) HPLC should contain at least a
small concentration (e.g., 0.01-1%) of a polar modifier (water, alcohol, acetoni-
trile) to de-activate highly adsorptive sites that can cause tailing of chromato-
graphic peaks. Water is the most important de-activator, and it can have a pro-
found effect on chromatographic results. It is very difficult to control exactly the
amount of water dissolved in nonpolar solvents such as pentane, hexane, heptane,
and methylene chloride; this is one of the major causes of slow column equilibra-
tion with mobile phases and poor reproducibility in LSC. The following are useful
precautions when using alumina and silica columns:
? Use 50% water-saturated solvents for silica gel and 25% water-saturated
solvents for alumina, except for pentane, hexane, and heptane, which
should contain 0.05% acetonitrile. (50% water-saturated means that the
solvent has 50% of the water it would have if it were totally saturated.
50% water-saturated solvents are prepared by mixing equal volumes of
dry solvent and saturated solvent or by passing dry solvents through a
special moisture control column for specified time periods.)
? Change from one solvent to another in small steps along the eluotropic
series. Do not attempt to follow a very polar solvent with a very nonpolar
one directly, or vice versa.
? Chromatograph a test mixture repeatedly to test column equilibrium
each time a column is used after being shut down or when changing
mobile phases.
? If possible, use a separate column for each mobile phase to avoid prob-
lems associated with slow equilibrium. Avoid gradient elution with silica
gel or alumina columns.
Reverse Phase Chromatography
This section will only consider mobile phases for bonded polar phase columns
such as C-8 and C-18, which predominate in pesticide determinations. Classical
liquid-liquid chromatography (LLC) will not be covered because it has been al-
most completely superseded by bonded phase chromatography.
In RP chromatography, the mobile phase is more polar than the stationary phase,
and the most polar compounds elute first from the column. Mobile phases gener-
ally consist of mixtures of water, the weakest solvent for RP HPLC, or aqueous
buffers with water-soluble organic solvents. Typically used solvents include, in or-
der of decreasing polarity and increasing elution strength: methanol, acetonitrile,
ethanol, isopropanol, 1-propanol, dioxane, and tetrahydrofuran (THF). Acid and
basic buffers are used in the ion suppression mode to convert, respectively, weak
acid and weak base analytes to their nonionic, hydrophobic forms, which are
selectively retained on RP phases. Totally nonaqueous mobile phases are being
increasingly used for the “nonaqueous RP” HPLC of polar substances.
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The selectivity triangle approach described above for NP HPLC is applied equally
well to RP HPLC. The solvents nearest to the corners of the triangle and having
the requisite water solubility are acetonitrile (dipole interactions), methanol (pro-
ton acceptor), and THF (proton donor properties). It is most common to start
with a water/methanol mixture to find the optimum solvent strength (P'), and
then add one or both of the other solvents to maintain the strength but increase
selectivity for the required separation. Each mobile phase modifier imparts a spe-
cial selectivity by causing lower k values (faster elution) for compounds with a
particular type of functional group.
A systematic four-solvent, seven-mixture mobile phase optimization strategy based
on the approach described above is widely used for isocratic NP and RP HPLC [3].
In the case of acids or bases, an additional modifier to adjust pH may be necessary,
e.g., 1% acetic acid in water for the chromatography of phenols. One of the seven
mixtures tested by this protocol should provide the required separation. If not, a
different column, temperature, pH, or solvent modifier is required. An HPLC
system with four solvent reservoirs and computerized solvent mixing capability
makes this optimization routine a simple matter.
Ion Exchange Chromatography
Solvents for the separation of ionic compounds (e.g., pesticides with acidic or basic
groups) by ion exchange chromatography (IEC) include aqueous acids, bases, or
buffers that allow the analytes to possess full or partial electronic charges and to be
more or less attracted to the ionic groups of the stationary phase.
Ion exchange separations usually depend on several equilibria, the positions of
which are a function of the following factors: the relative affinities of the analyte
and the mobile phase counter ions, the ionic strengths of the analyte and counter
ions, the acid or base strengths of the analyte and the stationary phase functional
groups, and the mobile phase pH.
The general approach to designing a mobile phase is to first adjust the ionic
strength to give analyte k values between 2 and 10, and then adjust the pH to
control selectivity. Low ionic strength facilitates retention and high ionic strength
elution. A change in pH affects both the character of the functional groups of the
stationary phase and the ionization of the analyte. Retention is favored by a mobile
phase/exchanger pH between the pKa values of the exchanger and the analyte
(both must be charged). Elution is facilitated by mobile phase/exchanger pH
above the pKa of a cation or below the pKa of an anion. Efficiency is improved by
elevated temperatures and lower flow rates. An increase in counter ion concentra-
tion increases mobile phase strength. The proper choice of counter ion can im-
prove selectivity. In general, exchangers prefer ions with higher charge, smaller
hydrated diameter, and greater polarizability. Retention of analytes is favored if
the exchanger is equilibrated with counter ions that are weakly held, and elution if
the mobile phase/exchanger contain strongly held counter ions. Addition of an
organic modifier generally increases solvent strength (especially if analytes are
interacting with the mobile phase by a hydrophobic mechanism) and increases
efficiency by lowering viscosity.
Pesticide Analytical Manual Vol. I SECTION 603
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Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Ion Pair Chromatography
The mobile phase for ion pair chromatography is at a pH where the analyte is in
its ionic form, and it also contains a pairing agent that conjugates with the analyte
to form a hydrophobic, uncharged species that is selectively retained by a C-18 or
C-8 bonded column. Typical pairing agents are a quaternary amine for weak acids
and an alkyl sulfate or sulfonate for weak bases.
The choice of a mobile phase is aided by the following guidelines:
1) Methanol/water mixtures are preferred as the mobile phase to mini-
mize counter ion solubility problems.
2) Short chain counter ions are recommended for analytes with little dif-
ference in molecular structure, and longer chain, hydrophobic counter
ions for greater retention.
3) If silica-based bonded columns are used, the pH of the mobile phase
must be maintained within the column’s stability range. Use of porous
polymer packings avoids this concern.
4) The mobile phase should be degassed before adding the counter ion to
prevent possible foaming.
5) Typical concentrations for counter ions are 0.005-0.01 M, and 0.0005-
0.001 M for buffer components.
6) Counter ions should not absorb UV light if a UV detector is in use.
7) To prevent salt precipitation, the pump should not be turned off until
mobile phase is washed out of the system. Alternatively, a slow flow of
mobile phase can be maintained overnight. It is best to have a dedicated
column only for ion pair HPLC.
Size Exclusion Chromatography
Because of the nature of size exclusion chromatography, there are only two basic
requirements for a mobile phase: it must readily dissolve the analyte and not
damage the stationary phase. Solvents that are not compatible with polystyrene-
divinylbenzene (DVB) phases include water, alcohols, acetone, methyl ethyl ke-
tone, and dimethylsulfoxide. If the analyte does not dissolve well in the mobile
phase, tailing and/or delayed elution due to interaction of the analyte with the
stationary phase can occur. Adsorption effects are reduced by using a mobile
phase chemically related to the stationary phase, e.g., toluene for polystyrene-DVB
columns.
Gradient Elution in HPLC
The preceding discussions relate principally to mobile phases for isocratic HPLC.
Isocratic elution is widely used because of its convenience and reproducibility. It is
not adequate, however, for separation of analytes containing components with
greatly different retention times. Gradient elution improves resolution of early
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Pesticide Analytical Manual Vol. ISECTION 603
eluting peaks while causing later eluting peaks to elute sooner and in a narrower
band. Alternative approaches to the general elution problem include column cou-
pling and flow and temperature gradient, but these will not be discussed here.
Solvent gradients are usually composed of a binary mixture of a weak solvent to
which continuously increasing amounts of stronger solvent are added in a linear,
convex, or concave relationship over time. Isocratic elution periods are often in-
cluded at the beginning and/or end of the gradient sequence. Important consid-
erations include the solvents chosen, the initial and final composition, and the
gradient shape and steepness. Stepwise gradients are also possible. Methods are
available for predicting and optimizing gradients for the different HPLC modes
[4, 5], but the most suitable gradient for a particular separation is usually deter-
mined empirically.
Gradient elution is used widely in NP and RP bonded phase and IEC. It is not
recommended for LSC and cannot be used with LLC or with refractive index (RI)
or conductivity detectors. Regeneration at the end of the gradient must return the
column to equilibrium with the initial solvent. Solubility considerations may re-
quire purging the system with an intermediate strength solvent, or it may be
possible to simply pass 5-10 column volumes of the first solvent through the col-
umn.
pH and ionic strength gradients are common in IEC to control mobile phase
strength and selectivity. Gradients for ion pair chromatography must be checked
to be sure that the counter ion and buffer components are soluble in all solvent
compositions used. Ion pair gradients may involve a solvent gradient with constant
pH and counter ion concentration, or these may be changed along with, or in-
stead of, the methanol/water (or other solvent) composition.
603 B: MOBILE PHASE PREPARATION
Mobile phases must be prepared from high purity solvents, including water that
must be highly purified (see Section 601 D). Mobile phases must be filtered through
≤1 μm pore size filters and be degassed before use.
Filtering Solvents
Particulate matter in solvents can damage pumps, block flow in tubing, and de-
grade column performance. Filtering of all HPLC solvents should be a routine
laboratory procedure. Filtering is especially important for removal of particles
when solvents are stored over molecular sieves. Commercial units that attach to
any vacuum line are available for simultaneous filtration and degassing of solvents,
or similar apparatus can be assembled in the laboratory. Commercial nylon mem-
brane filters with 0.22-1.2 μm pore size are compatible with all solvents commonly
used in HPLC. Most commercial HPLC grade solvents are prefiltered through a
0.2 μm filter and should not require additional filtration.
Degassing Solvents
Degassing of solvents is necessary to avoid problems with columns, pumps, and
detectors caused by gas bubbles in the system. The filtering step, if carried out with
an aspirator or vacuum pump, can also provide degassing. Degassing of volatile
Pesticide Analytical Manual Vol. I SECTION 603
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Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
solvent mixtures with a vacuum can change the composition of the solvent; vacuum
degassing should never be used for such mixtures.
Other degassing methods include boiling, use of an in-line degassing unit with a
gas-permeable membrane, or by agitation in an ultrasonic bath. However, the
most effective and convenient degassing method is helium sparging. A commercial
unit can be used, or a setup can be made in the laboratory from Teflon tubing and
an inlet line frit attached to a helium supply. The frit is immersed in the solvent
reservoir, and helium is bubbled for a few minutes with about 3-4 psi pressure at
the tank. The helium flow is reduced to a trickle during operation of the system. If
solvent mixtures are made manually, individual solvents are degassed prior to
preparation, and the mixture is kept under helium during use.
Preparation of Multisolvent Mobile Phases
Mobile phase mixtures can be prepared either by manual blending or by in-line
mixing using the HPLC solvent blending and delivery apparatus. Laboratory glass-
ware used for preparing mobile phases should be exceptionally clean so it does
not introduce particles or impurities.
Two different approaches to manual preparation of solvents are possible. Either is
valid, as long as the preparation method is clearly recorded so others can repro-
duce the results. In the first method, volumes of solvents A and B measured in
graduated cylinders or pipets are mixed together in the mobile phase reservoir. In
the second method, a measured volume of solvent A is placed in a volumetric
flask, and the solution is diluted to the line with solvent B and transferred to the
mobile phase reservoir. Solutions prepared by these methods will be slightly differ-
ent, especially for water/alcohol mixtures, because of the nonexact additivity of
volumes upon mixing. It is good practice to prepare the mobile phase fresh each
day, especially if a volatile solvent is involved. If the mobile phase will be used for
longer periods, it should be definitely proven, e.g., by measuring RI or
chromatographing a test mixture, that there is no change in composition with
time.
If an error in composition is suspected for a mobile phase prepared in-line, a new
batch of the mobile phase should be carefully prepared manually and the separa-
tion repeated.
Solvent Reservoirs
The solvent container should be made of a material from which the solvent cannot
leach significant impurities, and should have a cover with a small opening through
which the Teflon or stainless steel delivery tubing fits snugly but without constriction.
The reservoir is placed away from sunlight or drafts to avoid temperature gradients,
and above the solvent delivery system to provide siphon feed to the pump. The
reservoir should be labeled with the composition and date of preparation of the
mobile phase, and a solvent reservoir filter (sinker frit) should be attached to the end
of the delivery tube.
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3) The flow should be constant, repro-
ducible within at least 1%, and
pulseless or have a damping system
to minimize detector noise generated
by the pulses. It should be easy to set,
measure, and change the flow rate.
4) It should be easy to change from one
mobile phase to another.
5) The internal volume of the pump and
all of the plumbing between the pump
and the injector should be as small as
possible.
6) The pump should be useful for
isocratic or gradient operation.
7) The pump should be adaptable to the
use of small volumes of mobile phase,
a high volume mobile phase reservoir,
or a heated reservoir.
603 C: MOBILE PHASE DELIVERY SYSTEMS
Pumps
The function of the pump in HPLC is to deliver the mobile phase through the
column at high pressure with a controlled flow rate. Two major categories of
pumps are constant flow or volume and constant pressure. Constant pressure
pumps apply a constant pressure to the mobile phase; flow through the column is
determined by the flow resistance of the column and any other restrictions in the
system. Constant flow pumps generate a certain flow rate of mobile phase; the
pressure depends on the flow resistance.
Constant flow pumps are recommended for HPLC because flow resistance may
change with time due to swelling or settling of the column, small temperature
variations, or buildup of particulate matter. These effects will cause flow rate
changes with a constant pressure pump and result in nonreproducible retention
data and erratic baselines.
A suitable pump should have the following characteristics:
1) The interior of the pump should be made of inert materials that resist
corrosion by any solvents being used.
2) Pressures up to 6000 psi and a wide range of flow rates (0.1-≥10 mL/min)
should be available, and the flow rate should be easy to change. High flow
rate capability is especially important for preparative work.
8) The pump should be easy to maintain and repair. Even with the best of
care, seals, rings, and gaskets will require occasional replacement, and it
will help if these are easy to access.
Figure 603-a
Reciprocating Pump
[Reprinted with permission of John Wiley and Sons,
Inc., from Lindsay, S. (1987) High Performance
Liquid Chromatography, Figure 2.2d, page 21.]
Mobile phase outlet
Mobile phase inlet
Seals
Piston
Solvent
chamber
Eccentric cam
Check
valves
Pesticide Analytical Manual Vol. I SECTION 603
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Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Neither constant pressure pumps nor screw-driven syringe constant flow pumps
are described here because the reciprocating pump (Figure 603-a) is used in most
HPLC instruments. In this pump, a small piston is driven in and out of a solvent
chamber by a motor-driven eccentric cam and gear arrangement. On the forward
stroke, the inlet check valve closes, the outlet valve opens, and the mobile phase is
pumped to the column. On the return stroke, the outlet valve closes and the
chamber is refilled.
Because the displaced volume is small, the pump must cycle frequently. Abrasion
is minimized by using hard, smooth piston material such as borosilicate glass,
sapphire, or chrome-plated steel. Solvent capacity of
a reciprocating pump is unlimited if the external
reservoir is filled as required. The internal chamber
volume can be very small (e.g., 10-100 μL), allowing
rapid change of mobile phases. The flow rate, 0.01-
50 mL/min, is changed by varying the length of the
piston stroke or the speed of the motor. Piston seals
and check valves must remain leak free. This requires
regular maintenance and periodic replacement of
parts. Access to valves and seals for maintenance is
usually quite easy.
Figure 603-a shows a single-head reciprocating pump,
in which solvent is delivered to the column for only
one-half of the pumping cycle. Flow pulsations arise
from the piston action, which may produce noise
with some detectors during high sensitivity analyses.
Flow noise is reduced if the pump is designed with a
rapid stroke rate so the detector cannot respond rap-
idly enough to sense the flow changes. Other ways to
obtain constant flow rate are the incorporation of
dampeners or a feedback control system.
Twin- or dual-piston reciprocating pumps have two
heads operated 180 degrees out of phase by the ac-
tion of a single cam so that one pumps while the
others refills, producing a constant, pulseless flow
and reduced noise.
The diaphragm or membrane reciprocating pump is
a variation of the piston pumps described above. A
piston is driven back and forth by an eccentric disc.
The movement is conveyed hydraulically to a flexible
steel membrane, which flexes and displaces the sol-
vent out to the column, and then pulls in mobile
phase from the reservoir when the diaphragm re-
turns. Check valves at the inlet and outlet ensure
flow in the proper direction. The piston does not
contact the solvent directly, so seals are not needed.
Pulsations caused by discontinuous pumping and suc-
tion cycles are stabilized by incorporation of a damp-
ing system or two pistons synchronized to minimize
pulse lag. Back pressure changes in the column and
the elasticity of the diaphragm can cause flow rate
[Reprinted with permission of John Wiley
and Sons, Inc., adapted from Lindsay, S.
(1987) High Performance Liquid
Chromatography, Figure 2.2f, page 24.]
Figure 603-b
Gradient System I
[Reprinted with permission of John Wiley
and Sons, Inc., adapted from Lindsay, S.
(1987) High Performance Liquid
Chromatography, Figure 2.2f, page 24.]
Solvent
A
Solvent
B
Metering
pump
Low
pressure
mixing
chamber
High
pressure
pump
To column
To column
Proportioning
valves
Low
pressure
mixing
chamber
High
pressure
pump
Solvent
A
Solvent
B
Figure 603-c
Gradient System II
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Figure 603-d
Gradient System III
Solvent
A
Solvent
B
High
pressure
mixing
chamber
High
pressure
pumps
To column
Programmer
Flow
controller
High
pressure
pumps
[Reprinted with permission of John Wiley
and Sons, Inc., adapted from Lindsay, S.
(1987) High Performance Liquid
Chromatography, Figure 2.2f, page 24.]
deviations. A feedback flow controller should
be incorporated to ensure constant flow rate.
Gradient Programming Systems
Figures 603-b, -c, and -d show the arrangements
for gradient-forming systems involving two sol-
vents.
In Figure 603-b, the gradient is formed at low
pressure by metering controlled amounts of
solvents A and B from low pressure pumps into
a mixing chamber (volume <1 mL) fitted with a
magnetic stirrer, from which it is drawn into a
high pressure pump for delivery to the column.
The arrangement in Figure 603-c is similar, but
composition of solvent in the low pressure mix-
ing chamber is regulated using microprocessor-
controlled, solenoid-operated time-proportion-
ing valves. Low pressure systems with no mixing
chamber are also available. Such systems, which
ensure that the gradient is not retarded, involve highly precise valve control and a
great deal of mechanical and electronic equipment to mix extreme volume ratios.
In Figure 603-d, the separate solvents are pumped with two high pressure pumps
into a high pressure mixing chamber. The type of gradient formed is controlled by
programming the delivery rate of each pump. Electronic control must ensure that
the total volume is always constant and that compensation is made for changes in
viscosity. The post-pump chamber must provide rapid and complete mixing and
have a small volume and no “dead” areas. This method is more expensive than low
pressure mixing because a separate pump is required for each solvent and thus is
decreasingly favored.
Technically sophisticated systems, usually involving low pressure, controlled mix-
ing and delivery to the column with a high pressure pump, are now available for
gradients involving three and four different solvents, which are becoming more
widely used for separation of complex mixtures.
Errors in gradient formation can be caused by restricted lines and loose tubing
connections, which can be corrected by the operator. Problems with valve control-
lers or software usually must be handled by a manufacturer’s service technician.
603 D: MAINTENANCE AND TROUBLESHOOTING
Problems with Pumps
The following considerations are important in the maintenance and troubleshoot-
ing of all types of HPLC pumps:
? Have pumps set up by a manufacturer’s service technician, who should
explain proper operation, maintenance, troubleshooting procedures, and
precautions to all users and in-house service personnel.
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Transmittal No. 94-1 (1/94)
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? Obtain all available operation manuals and require that they are read
carefully by users. Many manufacturers provide extensive maintenance
and troubleshooting information with their pumps.
? Stock an adequate supply of parts that require routine replacement or
may be damaged or broken during routine maintenance, such as seals,
plungers, fittings, cams, O-rings, heads, check valves, springs, clamps, etc.
? Maintain a log notebook for each pump. List maintenance and repair
dates and procedures, and use and storage history.
? Do not store corrosive solvents or buffers in the pump overnight.
? Periodically lubricate pump motors with the proper grade of oil.
? Do not attempt to replace one solvent with another unless both are
completely miscible.
? Degas solvents to avoid bubbles in the pump head(s) and filter all sol-
vents. These are the two primary precautions for preventing pump prob-
lems.
? If possible, avoid highly volatile solvents (e.g., pentane), which with the
pumping action can cause volatilization and bubbles.
? Avoid pump overheating by working in a well-ventilated area.
? Confirm that the pressure limit switch, if available, is set properly.
? Inspect pump heads and fittings for leaks on a daily basis. Leaks can be
caused by dirty pistons and worn piston seals. Gentle tightening of fit-
tings usually eliminates leaks. Overtightening of fittings can cause leaks
and permanently damage the part. This can be an expensive matter if
the affected fitting threads into a pump check valve or head.
? Dirty, sticking, or malfunctioning check valves can cause irregular or
inaccurate flow and drifting baselines, or stop flow altogether. Check
valves can be replaced or cleaned. In either case, follow the
manufacturer’s instructions.
? Determine the useful lifetime of pump seals under the operating condi-
tions in each laboratory. Replace the seals on a regular basis before the
useful lifetime is over, or at least on a yearly basis. At the same time,
inspect the piston for scratches.
? Verify the flow rate of pumps on a periodic basis, to an accuracy of 10%,
by delivery into a graduated cylinder while timing with a stop watch.
When an accuracy of ±1% is desired, use a stop watch and buret. Mea-
sure the time interval as the meniscus passes two marks on the buret a
known volume apart. At least a 2 min period is desirable for this degree
of accuracy. If the delivered flow rate is not within specifications, check
for leaks and/or make adjustments or repairs as outlined in the operat-
ing manual.
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? If the pump starts up but does not move the solvent, it is probably in need
of priming. Pump-priming procedures vary from one instrument to an-
other; check the correct procedure in the manufacturer’s instrument
manual.
Problems Caused by Air. Most problems with HPLC pumps are caused by air
bubbles. These arise when air is drawn into the pump when the solvent reservoir
runs dry or the solvent inlet line is lifted out of the reservoir; from leaks at the
fittings that connect the inlet tubing to the pump; from bubbles generated when
the mobile phase components are mixed; or from cavitation of the mobile phase
in the inlet line or pump head. The symptom in each case is stoppage of flow or
fluctuating pressure.
A sinker frit on the end of the inlet tubing or tight connection through a cap at
the mouth of the reservoir will keep the tubing submerged in the bottom of the
reservoir and prevent air in the reservoir from reaching the pump.
If an air leak on the inlet side of the pump is suspected, carefully tighten each of
the fittings, including the check valve. Do not overtighten plastic fittings to the
point of distortion. If the leak persists, disassemble the fittings and examine them
for damage.
Recut suspect tube ends and re-assemble or replace suspect low pressure or com-
pression fittings until the problem is solved. If buffers have been used, flush the
fitting with nonbuffered solvent before re-assembly.
When RP solvents (e.g., water and methanol) are mixed, the mixture has a lower
capacity for dissolved gases than the pure component solvents. This is why bubbles
often are seen evolving from freshly mixed mobile phase. With manual mixing,
excess gas bubbles from the solution, but the mixture remains saturated with air.
Therefore, when the pump begins to fill, pressure is reduced and gas bubbles
form in the pump head. With low pressure mixing, solvents are combined just
prior to the pump. Mobile phase entering the pump is supersaturated with air,
which bubbles out in the pump. With high pressure mixing, solvents are mixed
after the pump, so bubble problems should not occur in the pump. Proper degas-
sing of solvents (Section 603 B) is essential.
Cavitation occurs when the pump draws solvent through a line with restricted flow
and creates a partial vacuum in the line. This vacuum can cause dissolved air to
expel, forming bubbles in the inlet line or pump head. Blockage of the inlet filter
in the mobile phase reservoir is a common cause of cavitation that can be cor-
rected by replacing the restricted filter. Another cause is a tightly fitting reservoir
cap that is not properly vented. Drilling a very small (<1 mm) hole in the cap or
loosening it can remedy this problem.
Problems Caused by Dirt. The most damaging pumping system problems are
caused by dirt, a term that encompasses particulate matter introduced by the
mobile phase, buffer evaporation, or wearing of seals. The main problems caused
by the presence of dirt are malfunctioning check valves and premature pump seal
wear.
Particulate matter can prevent proper sealing of check valves, resulting in pres-
sure fluctuations and poor pump delivery. With high pressure mixing, a dirty
check valve can cause proportioning problems. If simple flushing does not cure a
Pesticide Analytical Manual Vol. I SECTION 603
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Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
suspected check valve problem, the valve should be replaced. If the problem is
eliminated, the dirty check valve should be cleaned, if possible, and later re-used.
If it cannot be cleaned, it can be returned to the manufacturer for rebuilding. A
dirty check valve is cleaned by rinsing with HPLC grade solvent or sonicating in
10% nitric acid followed by rinsing with HPLC grade water.
If a pump containing buffered mobile phase is shut off and allowed to sit over-
night or longer without washing out the buffer, mobile phase behind the pump
seal will evaporate and abrasive solid crystals will form. When the pump is re-
started, these crystals will abrade the seal and cause accelerated wear. Abraded seal
particles can also cause check valve problems and block the top column frit.
Flushing with 10-20 column volumes of nonbuffered solvent at the end of each day
is recommended. Some pumps are designed to allow direct flushing of buffer
from behind the seal. The pump operation manual should be consulted.
Proportioning Problems. To prevent proportioning problems, solvents must flow
freely with no restrictions. Change inlet (sinker) frits in solvent reservoirs before
they become blocked. Make buffers fresh daily to extend frit lifetime by retarding
microbial contamination. Make sure that low pressure fittings are sealed properly
so that air cannot leak in and solvent out. Thoroughly degas solvents to prevent
bubble problems. Elevate solvent reservoirs above the proportioning manifold to
apply slight head pressure and improve the reliability of solvent delivery.
Run reference tests routinely to recognize mechanical problems with the propor-
tioning valves and problems with the controlling software. For example, set the
programmer so the solvent does not flow through the column. Use any convenient
(miscible) solvents of HPLC quality in the pumps. Attach a recorder to the pro-
gram monitor terminal jacks so the pen traces the gradient. Operate the program-
mer as outlined in the instrument operating manual. Compare the trace on the
recorder to determine whether the correct programs are actually being produced.
Test each program that is regularly used for actual analyses.
Another test may involve a series of 10% isocratic steps from 0 to 100% of solvent
B (containing a UV absorber to allow detection), changed every 5 min. The result-
ing trace of absorption vs time should yield rising steps that have the same height
and are fairly square. A third test is a 20 min blank gradient run at 4 mL/min
using the same spiked B solvent. This trace should be essentially straight (espe-
cially between 5 and 95% B), with angular intersections at the 0% baseline and
100% plateau.
Bubble problems can be caused by air leaks, which can occur when a proportion-
ing valve diaphragm becomes perforated or other damage to the solvent propor-
tioning manifold occurs. The proportioning manifold can be tested by connecting
the manifold inlet and outlet tubing with a union. If the bubble problem disap-
pears, the manifold is bad and must be replaced.
References
[1] Snyder, L.R. (1974) J. Chromatogr. 92, 223-230
[2] Snyder, L.R. (1978) J. Chromatogr. Sci. 16, 223-234
[3] Glajch, J.L., et al. (1980) J. Chromatogr. 199, 57-79
603–14
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. ISECTION 603
[4] Snyder, L.R., et al. (1988) Chapter 6, Practical HPLC Method Development, Wiley-
Interscience, New York
[5] Poole, C.F., and Schuette, S.A. (1984) Contemporary Practice of Chromatography, pp. 265-
271, Elsevier, New York
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 604–1
Pesticide Analytical Manual Vol. I SECTION 604
604: INJECTION SYSTEMS
The purpose of the injection system is to apply the sample extract onto the column
in a narrow band. Three techniques are available: direct syringe injection, stop-
flow syringe injection, and use of an injection valve.
In direct syringe injection, the extract is injected into the flowing mobile phase
through a septum using a high pressure syringe in a manner similar to GLC. A
septumless injector is also available for direct syringe injection without interrupt-
ing mobile phase flow. In stop-flow injection, injection is made at ambient pressure
after depressurizing the injection port by use of a sliding seal and shut-off valve, or
by turning off the solvent delivery pump. The direct syringe injection and stop-flow
injection techniques are now obsolete and rarely used.
604 A: INJECTION VALVES
The injection valve is at present the most widely used injection device for repro-
ducibly introducing sample extracts into pressurized columns without flow inter-
ruption. Valves can be made with external or internal loops. Virtually all commer-
cial external loop valves are variations of the six-port design shown in Figure 604-a.
A fixed volume loop is connected across two of the ports. The extract is introduced
through the injection port, and excess extract flows out through the waste port.
The other two ports provide a path for the mobile phase as it is pumped into
the column. External loops are available in sizes ranging from 5 μL to 2 mL.
The 10 and 20 μL sizes are probably most widely used. Loops are usually made
from standard 1/16" stainless steel, but other materials are used in biocompatible
injectors.
When the valve is rotated to the load position on the left in Figure 604-a, mobile phase
flows directly from the pump to the column and the loop connects the injection and
waste ports. The loop is at ambient pressure and is filled with extract from a regular
[Reprinted with permission of Valco Instruments Inc.]
Sample
syringe
To column
Mobile
phase
Waste
Sample
loop
Sample
syringe
To column
Mobile
phase
Waste
Sample
loop
Load position Inject position
Figure 604-a
External Loop Injector: Six-Port Injection Valve
SECTION 604 Pesticide Analytical Manual Vol. I
604–2
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
microliter syringe. When the valve is rotated to the inject position, shown on the
right, mobile phase flows through the loop, sweeping the extract rapidly onto the
column.
One common technique is to fill the loop completely with extract (the “filled
loop” technique). In this case, the loop volume fixes the injection volume, and the
loop must be changed to vary the extract volume. To achieve good reproducibility,
an extract volume equal to twice the loop volume should be used to flush and fill
the loop. If a Teflon waste tube is used, air and excess solvent will be seen going to
waste. All air should be expelled when filling the loop. For applications in which
limited extract is available, some valves have a special loop-filler port that permits
loading of the loop with minimal waste.
Other methods for varying the volume of extract injected include filling a fixed
volume loop completely with a combination of extract and solvent; partially filling
the loop (the “partial loop” technique), following the manufacturer’s procedural
guidelines; or using special variable volume valves. For best results with the partial
loop technique, the volume of extract injected should be <50% of the nominal
loop volume, e.g., 10 μL extract in a 25 μL loop. If a tiny air bubble is injected just
before the extract to isolate it from previous solution in the loop, >50% of the
nominal loop volume can be injected. The partial loop technique allows flexibility
of injection volume, but precision is dependent on the analyst’s skill in reproduc-
ibly injecting specific extract aliquots from a syringe.
Recently, the six-port valve has been adapted to place cleanup and analyte concen-
tration steps in-line with the determinative step. A short cleanup column chosen
for its ability to retain the analyte replaces the loop shown in Figure 604-a [1].
While the valve is in the load position, sample extract is injected onto the cleanup
column; subsequent injection of solvent removes co-extractives to waste without
removing the analyte. When the valve is rotated to the inject position, mobile
phase flows through the cleanup column and elutes the analyte onto and through
the analytical column to the detector. Variations of this technique can involve
cleanup and analytical columns of the same or different HPLC modes [2]. When
the injection valve is used this way, no loop is available, so the extract volume
injected must be measured accu-
rately in a syringe.
Internal loop injectors are valves
with four ports, with the loop in
the form of an engraved groove
or slot in the body of the valve.
These are used for injecting ex-
tracts in the 0.05-5 μL range. Fig-
ure 604-b illustrates the internal
loop injector. These injection
valves are also available for
microbore HPLC. This type of
valve, which is designed for nar-
row bore (1-2 mm) columns, has
a 0.2-1 μL interchangeable cham-
ber and built-in needle port with
0.3 μL holdup volume to reduce
extract loss.
Figure 604-b
Internal Loop Injector
Load
position 1
Load
position 2
Sample
Sample
Pump Column Pump Column
[Reprinted with permission of John Wiley and Sons, Inc., adapted
from Lindsay, S. (1987) High Performance Liquid Chromatography,
Figure 2.2g, page 26.]
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 604–3
Pesticide Analytical Manual Vol. I SECTION 604
Ten-port multifunctional valves are now available for HPLC. These can perform as
a standard six-port injection valve and in addition allow extract injection followed
by back flush, injection into two columns simultaneously, injection into either of
two columns (random access), extract injection followed by precolumn back flush,
trace enrichment, alternate extract injection from two streams, two-column selec-
tion with flow maintained in both, heart-cutting operations, and fast, sequential
injections of a single extract.
Injection valves used with the filled loop technique are easy to use, provide the
best precision for quantitative HPLC, typically <1% relative standard deviation, are
easily adapted for automatic injection, and allow high pressure operation (up to
7000 psi). The partial loop injection technique is not as reproducible as the filled
loop technique for manual injections. It is best used only for preliminary experi-
ments when determining the optimum injection volume, or with automatic injec-
tors that provide high precision automatic syringe delivery. To maximize column
efficiency, the smallest convenient extract volume should be injected using an
extract solvent that is weaker (i.e., more polar for reverse phase HPLC) than the
mobile phase.
The method used to insert the valve in the HPLC system is critical for minimizing
loss of efficiency. A 5 cm length of 0.15 or 0.50 mm id tubing between the valve
and column with connection via a low or zero dead volume fitting is recom-
mended. When using the partial fill technique, the valve should be connected as
shown in Figure 604-a. This plumbing arrangement delivers the extract to the
column before the solution previously in the loop, preventing band spreading due
to dilution of the extract.
604 B: AUTOMATIC INJECTORS
Automatic injectors are valve injectors whose rotation is controlled by pneumatic
or electric actuators. Most use a mechanized syringe to dispense the extract into
the loop. Systems with both fixed and adjustable volumes are available for unat-
tended operation. A typical adjustable volume model allows injection of 1-100 μL,
accurate measurement by a motor-driven microsyringe, a positive displacement
mechanism to minimize extract waste, microprocessor-controlled injection sequenc-
ing, and a multivial sample turntable. The microprocessor controls movement of
the turntable, sampling needle, microsyringe, and injection valve. All injection
parameters, including sampling interval, sample number, and injections per sample,
are entered from a keyboard. Injection precisions are typically quoted as 0.5-1%.
604 C: OPERATION, MAINTENANCE, TROUBLESHOOTING,
AND REPAIR OF INJECTION VALVES
The following considerations are important for trouble-free operation, mainte-
nance, and repair of injection valves:
? Read carefully the literature packed with the valve for information on
installation, use, maintenance, and repair.
? The major cause of injector problems is particulate matter entering the
valve. Particles can lodge in moving parts, scratch the rotor surface, and
cause leakage. They can also block the connecting tubing or sample
loop. To avoid formation of particles, dissolve the sample extract in the
SECTION 604 Pesticide Analytical Manual Vol. I
604–4
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
mobile phase itself. If a solvent is used in which the analyte is more
soluble, components of the extract may precipitate when contact is made
with the mobile phase. To eliminate particulate matter, install an in-line
5 μm filter between the pump and the valve, and filter any extracts that
have visible particulate material or are cloudy or opalescent.
? If blockage occurs, locate it and back flush the blocked passage; disas-
semble the valve and sonicate the blocked part in soapy water, rinse in
clear water, and blow the passage clear with compressed air; or replace
the blocked tubing. Return blocked valves that cannot be cleared in the
laboratory for reconditioning by the manufacturer.
? To minimize rotor seal wear, prevent abrasive particles from entering the
valve as described above, and do not allow buffered or corrosive mobile
phases to remain in the valve for extended periods of time without flush-
ing.
? Do not operate above the pressure limit of the valve (usually 1500-7000
psi) or leakage may occur. Operate at the lowest possible pressure to
reduce rotor seal wear.
? Use valves that are constructed of materials compatible with extract and
mobile phase components. In addition to the usual valves constructed
from stainless steel with a polymeric rotor, specialty valves made from
more inert materials are commercially available.
? Maintain a good supply of spare parts, e.g., dead volume fittings, ferrules,
and rotors. It is best to stock backup valves in case repair cannot be done
in the laboratory and return of a malfunctioning valve to the manufac-
turer is necessary.
? Engrave an identification number on each valve, and keep a log notebook
to monitor the history of use and repairs.
? Do not overtighten valve fittings. Overtightening can cause leaks or dam-
age to the valve body.
? To minimize dead volume and peak broadening, use connecting tubing
between the injection valve and the column, and the column and the
detector, that is as short as possible and has a small id. See that valve
tubing is straight and has a perpendicularly flat end that is sealed tightly
inside the port in the valve body. Do not allow metal pieces formed in the
tube-cutting process to enter the valve body.
? Identify crossport leaks by observing mobile phase emerging from a Teflon
exit line when the valve is in the load or run position.
References
[1] Niemann, R.A. (1993) J. AOAC Int, 76, 1362-1368
[2] Wojtowicz, E.J. (Feb. 1992) “HPLC Fluorometric Analysis of Benomyl and
Thiabendazole in Various Agricultural Commodities,” LIB 3650, FDA, Rockville,
MD
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 605–1
SECTION 605Pesticide Analytical Manual Vol. I
605: DETECTORS
The function of an HPLC detector is to continuously and instantaneously monitor
the mobile phase emerging from the column. The output of the detector is an
electrical signal that results from measuring some property of the mobile phase
and/or the analytes. Application of HPLC to trace residue determination is depen-
dent on the availability of sufficiently specific and sensitive detectors. The common
HPLC refractive index (RI) detectors are nonselective and require microgram
quantities of analyte, so were never adequate.
The UV/VIS absorbance and fluorescence detectors are now commonly applied to
pesticide residue determination, because methods research has resulted in schemes
that utilize their capabilities while overcoming their limitations. Other detectors
available for HPLC include photoconductivity, electrochemical, and mass spectro-
metric; published applications to residue determination increase each year. Use of
combined detection systems is also increasing, e.g., the combination of UV absor-
bance and electrochemical detection.
The following are important characteristics of HPLC detectors:
Sensitivity. Detector sensitivity is a gauge of the detector’s response (signal) to the
presence of an analyte. In HPLC applications, the usual quantitative measurement
is called minimum detectability, defined as concentration of analyte that causes
the detector to produce a response twice that of instrument noise.
Minimum detectability refers to detector response to an analyte in pure solution.
“Limit of detection” or “limit of determination,” however, takes into account the
amount of sample extract that can be introduced to the detector and is partially
dependent on the degree of cleanup provided by the analytical method (see Sec-
tion 105). Injection volume is an important parameter in determining the limit of
detection of an HPLC analysis, because aqueous extracts prepared for HPLC de-
termination are not easily concentrated. However, HPLC can tolerate large vol-
umes of solvent without loss of column performance, thus improving detection.
Injection volumes up to 100 μL are not unusual for analytical HPLC.
Linearity. The linear range is the concentration range over which response is
directly related to analyte concentration (i.e., a plot of response vs concentration
has a constant slope). Linearity is commonly expressed in units such as 1:10,000 or
10
4
, which might indicate a range of 10
-10
(the minimum detectable quantity) to
10
-6
g/mL for the UV detector.
Selectivity of Response. Universal detectors respond to all components in the
extract, while specific (selective) detectors respond to only certain compounds
depending on their structure. Selective detectors are usually more sensitive and
less affected by variations in the mobile phase.
Effect of Changing Conditions. Ideally, the detector should not be affected by
changes in temperature or mobile phase composition.
Time Response. The detector-recorder combination should react rapidly so that
quickly eluting, narrow peaks can be measured accurately. The time constant of the
detector-recorder, which is defined as the time required to reach 63 or 98% of full
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–2
scale deflection, should be at least in the 0.1-0.3 sec range for high speed HPLC
and narrow peaks. Modern instruments are capable of response in the millisecond
range.
Cell Volume. Detector cell volume should be minimized to limit peak broadening
and maximize efficiency. A cell volume of 8 μL is typical, with smaller values for
micro-HPLC. The volume of associated tubing should also be minimized. How-
ever, as the volume of the detector cell decreases, sensitivity of detection is poorer.
Detector design should eliminate dead corners in the cell from which the analyte
is not quickly washed by the mobile phase.
Noise and Drift. Baseline noise, as indicated by variations in the recorder signal
with no sample in the detector cell, is caused mostly by the electronics of the
detector, recorder, or amplifier. Additional sources include the mobile phase
(bubbles, changes in flow rate or pressure, leaks, impurities) and temperature
fluctuations. To reduce the chance for bubble formation from depressurized mo-
bile phase in the detector cell, modern detectors include a back pressure restrictor.
Baseline drift may occur when the HPLC system is first turned on. If drift persists
after warmup, it is most likely due to changes in mobile phase composition, leaks,
temperature variations, column bleed, or a gas bubble in the detector cell. Baseline
drift can also be caused by slow elution of highly retained components left on the
column from previous samples.
Nondestructiveness. An analyte can be collected for further characterization if its
chemical form is not changed by the detector. UV and fluorescence detectors are
examples of nondestructive detectors, while the electrochemical and photocon-
ductivity detectors are destructive.
Ruggedness, reliability, and ease of use, maintenance, and repair are also impor-
tant qualities to seek in HPLC detectors.
605 A: UV/VIS ABSORBANCE DETECTORS
The most popular HPLC detectors are the fixed and variable wavelength UV/VIS
types. Fixed wavelength UV detectors most often operate at 254 nm, which is
useful for molecules with aromatic rings, carbonyl, conjugated double bonds, and
other suitable chromophores. Variable wavelength detectors usually operate in the
190-380 nm range with a deuterium source or 190-900 nm with a supplemental
tungsten source. Other supplementary lamps that have been used are the 229 nm
cadmium lamp and 214 nm zinc lamp.
Variable wavelength detectors provide several advantages. A wider applicability
and significant increase in sensitivity often result from operating at wavelengths in
the 190-230 nm range. This is due to the large molar absorptivities of many pesti-
cides in this region. Typically, aromatic systems have a 10-50 times greater absor-
bance at 214 and 229 nm than at 254 nm. Different peaks in the chromatogram
can be detected at different optimum wavelengths.
Very pure solvents, including water, must be used to avoid noise and unstable
baselines at the lower wavelengths. Increased selectivity for pesticides with aro-
matic and certain other functional groups can be obtained by measuring at longer
wavelengths such as 280 or 295 nm. This gives the analyst the ability to “edit out”
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 605–3
SECTION 605Pesticide Analytical Manual Vol. I
Figure 605-a
UV/VIS Detector
[Reprinted with permission of John Wiley and Sons, Inc., from
Meyer, V.R. (1988) Practical High Performance Liquid
Chromatography, Figure 5.5, page 69.]
Lamp
Lens
Measuring
cell
Eluate in
Filter
Photodiode
Window
Reference cell
Mask
unwanted peaks. This approach, however, may lead to some loss in sensitivity if
measurement of the analyte cannot be made at its wavelength of maximum absor-
bance. An additional advantage of a spectrophotometric-type detector is that un-
known components can be identified by stopping the mobile phase flow and
scanning the full UV/V IS spectrum of the component trapped in the sample cell
(stop-flow scanning).
Fixed Wavelength UV Detectors
There are several types of fixed wavelength UV detectors that differ mainly in the
output of the source. One type has a low pressure mercury lamp source that emits
a sharp line spectrum. A filter passes the principal 254 nm line and removes other
weaker lines. A second type has
a modified mercury lamp with a
phosphor and provides output at
254 or 280 nm. A third type em-
ploys a medium pressure mer-
cury lamp and band pass filters
to isolate emission lines at 220,
254, 280, 313, 334, or 365 nm.
Figure 605-a shows a diagram of
the light path and liquid flow
path for a double-beam fixed
wavelength UV/VIS detector.
Light from the source is focused
by a quartz lens onto sample and
reference cells. Column effluent
flows continuously through the
sample side (top), while the ref-
erence side (bottom) is filled
with pure mobile phase or air. A
wavelength of absorption is cho-
sen by the filter. After passing
through the filter, the radiation
is chopped by a rotating sector
so that alternating pulses fall on the detector (double beam in time). The pres-
ence of absorbing analyte in the sample cell decreases the intensity of the sample
beam relative to the reference beam. The difference in signal between the two
beams, which represents the absorbance of the analyte, is amplified and recorded.
In an alternative detector design, the sample and reference radiation fall on two
photodetectors whose outputs pass through pre-amplifiers to a log comparator,
which produces the absorbance signal. Double-beam design compensates for
changes in source output and photomultiplier tube response.
Detector cells are typically 1 mm id with a 10 mm optical path, or approximately 8
μL volume. The most common designs are the H-cell, Z-cell, and the tapered cell.
The latter design minimizes the effects of changing mobile phase RI, as can occur
during gradient elution.
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–4
Figure 605-b
Variable Wavelength UV/VIS Detector
[Reprinted with permission of Elsevier Science Publishers, from Poole, C.F., and Schuette, S.A. (1984) Contemporary
Practice of Chromatography, Figure 5.6A, page 377.]
Variable Wavelength UV Detectors
The second major UV/VIS detector is the continuously variable wavelength type
(Figure 605-b). Light from a continuous deuterium source (or tungsten source for
the visible region) is focused on the entrance slit of a grating monochromator,
which disperses it into its component wavelengths. The monochromatic light emerg-
ing through the exit slit is divided into sample and reference beams by a beam
splitter or chopper. The detector measures the difference in absorbance between
the sample and reference.
Solvents
Many solvents absorb strongly in the UV and cannot be used in certain spectral
regions. It is important to choose solvents that are transparent at the wavelength(s)
being used. For example, carbon tetrachloride, benzene, and acetone cannot be
used at 254 nm because they absorb too strongly. Hexane, chloroform, methanol,
and water are transparent at 254 nm and provide a wide range of solvent strength
for preparing mobile phases. The choice of solvents with UV cutoffs <220 nm is
quite limited.
Performance Characteristics
The UV detector is sensitive to 10
-6
to 10
-10
g/mL of many compounds. It has a
wide linear range and is relatively insensitive to small changes in flow or back
pressure, although at the detection limit the detector is very sensitive to changes in
temperature. Detection is limited to compounds that absorb at the chosen wave-
length.
Chopper
and
Fan Drive
Motor
Monochromator
Scan Assembly
Monochromator
UV Lamp Vis Lamp
Lamp Selection Mirror
Filter Wheel
(Mechanical Coupling
to Wavelength Drive)
Sample
Compartment
Light Path
Reference
Cell
Sample
Cell
Read
Out
Electronic Unit
VIS Lamp
Supply
UV Lamp
Supply
Synchronizing
Pulses
Multiple
Trigger
Assembly
EHT
Pre-amplifier
Input
(Mechanically
Synchronized
to Beam
Choppers)
Rotating
Chopper
Mirror (2 off)
PM
Tube
Transmittal No. 96-1 (9/96)
Form FDA 2905a (6/92) 605–5
SECTION 605Pesticide Analytical Manual Vol. I
Gradient elution is possible provided the solvents do not absorb. At very sensitive
settings, changes in RI, as caused by gradient elution or pressure and flow changes,
can produce baseline shifts with some types of detector cells.
The fixed wavelength detector is less versatile but is much less expensive and
often gives less noise than the continuously variable wavelength spectrophotomet-
ric detector. As mentioned above, the great advantage of the variable wavelength
detector is the ability to optimize sensitivity and/or selectivity for each analyte by
detection at the most favorable wavelength.
Multichannel or Photodiode Array Detectors
In a photodiode array detector, polychromatic radiation is passed through the
detector flow cell, and emerging radiation is diffracted by a grating so that it falls
on an array of photodiodes. Each diode receives a different narrow wavelength
band. The complete array of diodes is scanned by a microprocessor many times a
second. The resulting spectra may be displayed on a cathode ray tube monitor
and/or stored in the instrument for transfer to a recorder or printer. The detector
is best used in conjunction with a computerized data station, which allows various
post-run manipulations, such as identity confirmation by comparison of spectra
with a library of standard spectra recalled from disk storage. Detection can be
made at a single wavelength or at a number of wavelengths simultaneously, or
wavelength changes can be programmed to occur at specified points during the
run. Absorbance ratios at selected wavelengths (e.g., 254 and 280 nm) can be
displayed for each peak, which aids in determining identity and the presence of
unresolved components.
Applications
The UV detector has been the most widely used for pesticide residue determina-
tion. Section 404 uses UV and fluorescence detectors to determine benzimidazole
residues, whereas other references describe combinations of UV and photocon-
ductivity [1-3]; the photodiode array is applicable to determining paraquat and
diquat [4].
Problems, Maintenance, and Troubleshooting
Air bubbles in UV flow cells can produce a series of very fast noise spikes on the
chromatogram, or pronounced baseline drift. Falsely high absorbance readings
can be caused by impure or improperly prepared mobile phase, large air bubbles
in the flow cell, a misaligned flow cell, or dirty end windows. Gas bubbles develop
in the detector cell because they are pumped through the system or the solvent is
degassed in the detector. Prevent bubbles from being pumped through the system
by eliminating system leaks, expelling air from the pumping system, avoiding very
volatile solvents, and not stirring the mobile phase reservoir too vigorously. Pre-
vent solvent degassing in the sample cell by degassing the mobile phase prior to
use. If the cell has no back pressure valve, raise cell pressure above atmospheric by
attaching ≥10' spiral steel or Teflon tubing to the detector outlet to act as a flow
restrictor, and placing the tubing outlet above the detector. The tubing must not
shut off flow completely, as too great a pressure increase could shatter the cell
windows.
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 96-1 (9/96)
Form FDA 2905a (6/92)605–6
To dissolve gas bubbles lodged in the cell, briefly increase cell back pressure by
holding a piece of rubber septum over the detector outlet or by connecting a
syringe to the outlet. With aqueous systems, it may be necessary to fill the cell with
methanol and repeat application of back pressure.
Protect the detector from temperature fluctuations by placing the system away
from direct sunlight and drafts, and regularly monitor flow rate and pressure for
change.
Detector response can drop because dirt in the cell or a bad source lamp reduces
the level of radiation reaching the photocell. Some detectors have a meter that
allows easy determination of light level. If it is low, clean the detector or change
the source lamp. (Avoid eye damage by not viewing the light directly.) Consult
the detector manual for the proper procedure for changing the lamp and clean-
ing the cell. The average life of a 254 nm lamp is approximately 5000 hr, but it
should be replaced as soon as aging begins to cause significant intensity changes.
Some cells can be taken apart, the optical components cleaned with a suitable
solvent and dried, and the cell re-assembled. Others cannot be taken apart and are
cleaned by flushing the cells with a series of solvents delivered from a 50 mL glass
syringe, e.g., acetone, 6 M nitric acid, distilled water, and acetone, then drying with
a flow of clean, dry nitrogen before reconnection to the column. If necessary,
allow 6 M nitric acid to stand in the cell overnight. To remove particles most
effectively, draw nitric acid through the cell with a syringe in a direction opposite
to the normal flow.
605 B: FLUORESCENCE DETECTORS
Fluorescence detectors provide two to three orders of magnitude more sensitivity
than UV detection. Selectivity is also excellent because of the choice of excitation
and emission wavelengths and the fact that only a small fraction of all compounds
naturally fluoresce.
The simplest type of instrumentation is a
fixed wavelength fluorometer with bandpass
filters for both excitation and emission.
More convenient and versatile fluoromet-
ric detectors can operate at variable wave-
lengths. These are equipped with mono-
chromators to select excitation and emis-
sion wavelengths. Most compounds that
fluoresce naturally have a rigid, planar con-
jugated cyclic structure. Nonfluorescent
compounds can be detected if they are first
converted to fluorescent compounds by
pre- and post-column derivatization.
Detector Design
Figure 605-c is a schematic diagram of a
simple filter fluorometer detector. Light
from a mercury lamp passes through a fil-
ter that selects the excitation wavelength.
An interference filter providing a 10-20 nm
Figure 605-c
Fluorescence HPLC Detector
[Reprinted with permission of John Wiley and
Sons, Inc., from Meyer, V.R. (1988) Practical
High Performance Liquid Chromatography, Figure
5.10, page 74.]
Photodiode
Cell
Lamp
Emission
filter
Excitation
filter
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 605–7
SECTION 605Pesticide Analytical Manual Vol. I
bandpass is commonly used. Lenses focus the radiation on the sample cell, which
contains the flowing column effluent. If fluorescent compounds are present, they
absorb the incident radiation and re-emit at a longer wavelength.
Although it is emitted in all directions, the re-emitted or fluorescent light is usually
measured at a right angle to the direction of the incident light. A second filter
isolates a suitable wavelength from the fluorescence spectrum and rejects any
scattered exciting radiation from the source. A lens focuses the emitted light on a
photomultiplier tube.
The second filter can be a bandpass filter for selectivity, or a cutoff filter for
greater sensitivity.
The right angle measurement design allows monitoring of the incident beam as
well as the emitted light, so that dual UV absorption and fluorescence detection is
possible with some commercial detectors. A more common arrangement is to
place a separate UV detector in tandem with a fluorescence detector to check the
reliability of the fluorescence measurements, especially at low levels of quantita-
tion.
Other more sophisticated fluorescence detectors use grating monochromators in-
stead of filters and are termed continuous wavelength spectrofluorometric detec-
tors. These usually have either a deuterium (190-400 nm) or xenon (200-850 nm)
arc source. Because these sources are more unstable than a mercury discharge
source, detector design is often modified to correct for fluctuations in source
intensity by splitting off a portion of the exciting light to a reference detector.
Variable wavelength detectors have the advantage of allowing low wavelength exci-
tation, which is necessary for some naturally fluorescent compounds such as in-
doles and catecholamines.
Fluorescence detectors that use a laser as the excitation source are being studied.
The intensity of lasers is about 10
4
higher than that of conventional sources,
providing a 10-100 fold improvement in detection sensitivity. The use of lasers can
reduce stray light, because their radiation is entirely monochromatic and coherent
and the beam has a small cross section and is nondiverging. At present, the use of
lasers as detector sources is limited by their high cost and relatively narrow range
of available excitation wavelengths.
Solvents
The intensity of fluorescence is affected by the composition of the mobile phase
and the presence of impurities. Quenching can occur with halide ions, water and
other strong hydrogen-bonding solvents, and buffers. High temperature and oxy-
gen in the mobile phase can also induce quenching. Compounds that fluoresce in
organic solvents may show a shift in intensity and fluorescence maximum wave-
length with change in solvent polarity.
Performance Characteristics
Sensitivity is greater for fluorescence because the signal is measured directly against
a dark background, and signal intensity can be increased by an increase in source
intensity. The sensitivity of fluorescence detectors is in the range of 1-100 pg/mL
for favorable compounds. Because both the excitation and detected wavelengths
can be varied, selectivity is high for fluorescence detection. The linear range is
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–8
generally two or three orders of magnitude at low concentrations where absor-
bance is <0.05. At higher concentration levels, the linear range can be very small.
Parameter Adjustments
Optimum wavelengths for fluorescence detection are chosen by scanning analyte
excitation and emission spectra with a fluorescence spectrofluorometer. For most
compounds, excitation and emission spectra are mirror images that more or less
overlap at longer excitation and shorter emission wavelengths. When excitation
and emission maxima are close together, optimization of fluorescence detector
monochromator settings is critical for achieving maximal emission output while
avoiding light-scattering effects due to the overlap. Careful choice of emission
wavelength and slit width based on spectral characteristics is necessary for maxi-
mum sensitivity, as well as for controlling background noise if fluorescent impuri-
ties are present in the injected extract [5].
Applications
Section 401, method for N-methylcarbamates, uses post-column hydrolysis and
derivatization to produce a chemical detectable by a fluorescence detector; a varia-
tion of that determinative step detects naturally fluorescent residues without the
post-column reactions. Section 403 uses photolysis to degrade substituted ureas for
subsequent fluorometric labeling and determination. Section 404 uses UV and
fluorescence detectors for benzimidazole residues.
Detector Maintenance
Clean the cell compartment with chromic acid (or equivalent) cleaning solution,
followed by thorough rinsing with dilute nitric acid and water. An overnight
soaking in chromic acid cleaning solution may be necessary to remove stubborn
impurities.
605 C: ELECTROCHEMICAL DETECTORS
Electrochemical (ECh) detectors include the conductivity detector for the deter-
mination of ionic analytes and amperometric, coulometric, and polarographic
detectors for analytes with oxidizable or reducible functional groups. The first
ECh detector for HPLC used polarography, but because use of this mode today is
infrequent, it will not be discussed in this chapter. The most widely used type is the
thin layer amperometric detector using a glassy carbon electrode, or, less fre-
quently, a gold amalgam or carbon paste electrode, which can provide sensitive
and selective determination of compounds with appropriate structures.
ECh detection can be used with reverse phase (RP) columns because of the high
polarity of RP mobile phases. For the detector to function properly, the mobile
phase must possess good electrical conductivity. Salt or buffer at a concentration
of 0.05 M is often used to provide the required ionic strength. The detector is
impractical for normal phase (NP) HPLC, and RP systems with high modifier
concentrations may also cause problems.
ECh detection has been applied to the HPLC determination of phenols, amines,
mercaptans, halogen compounds, ketones, aldehydes, and nitroaromatics. It is
suitable for quantitation of various pesticide classes, such as dinitroaniline,
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 605–9
SECTION 605Pesticide Analytical Manual Vol. I
bipyridinium, triazine, and phenylurea herbicides; nitrophenyl, dinitrophenol, car-
bamate, and organophosphorus insecticides; and azomethine insecticides and fun-
gicides. Any pesticide that produces an electroactive compound (phenol, aromatic
amine, aromatic nitro compounds) on metabolism or decomposition, such as
carbamates, ureas, anilides, etc., can potentially be determined using the ECh
detector.
Conductivity Detectors
Conductivity detectors measure the conductance (reciprocal of resistance) of the
effluent, which is proportional to ionic analyte concentration if the cell is suitably
designed. They are usually used to detect inorganic or organic ions after separa-
tion by ion exchange or ion chromatography. Since these modes of HPLC employ
mobile phases with high conductances, it is necessary to incorporate a chemical or
electronic means of eliminating the conductance of the mobile phase before the
analyte can be measured sensitively and accurately with a conductivity detector.
Typical detectors have a cell with a small (2 μL) active volume, composed of
insulating material, into which graphite or noble metal electrodes are implanted.
A constant alternating voltage is applied to the electrodes, and conductance is
measured with an appropriate circuit, such as a Wheatstone bridge. Since conduc-
tivity is highly temperature dependent, a means for automatic temperature com-
pensation is usually included.
Amperometric and Coulometric Detectors
ECh detectors are most often amperometric or coulometric detectors that mea-
sure current associated with the oxidation or reduction of analytes. Oxidation is
carried out at an anode bearing a positive potential, and reduction at a cathode
having a negative potential. Different compounds require unique potentials for
these electrochemical reactions to occur. The current produced by the electrode
reaction is measured in a flow cell at the column outlet. Detector selectivity and
sensitivity are changed by varying the potential of the electrode.
The use of electrochemical reduction as an HPLC detection method is impractical
because oxygen is easily reduced, and it is difficult to remove oxygen completely
from the mobile phase. Most ECh detector applications are, therefore, based on
oxidation. Mobile phases that work best are aqueous/organic mixtures with added
salts or buffers.
The coulometric type of ECh detector reacts all of the electroactive analyte passing
through it, yielding a higher current for the electroactive species than the
amperometric detector. However, background noise is also greater, so it is not
more sensitive. The coulometric detector is insensitive to flow rate and tempera-
ture changes, and, like the GLC microcoulometric detector, it responds in an
absolute manner, eliminating the need for calibration. However, it is more prone
to electrode contamination and must be designed to provide strict potential
control over the entire electrode area. The coulometric type of ECh detector is
much less popular than the amperometric type.
The amperometric ECh detector uses a smaller electrode surface and reacts only
about 1-10% of the electroactive analyte, so that most of the analyte leaves the
detector cell unchanged. Small currents in the nanoampere range are produced.
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–10
Silver/
silver
chloride
reference
electrode
Glass
carbon
working
electrode
Solvent
inlet
Stainless
steel
auxiliary
electrode
[Reprinted with permission of John Wiley and Sons, Inc.,
from Lindsay, S. (1987) High Performance Liquid
Chromatography, Figure 2.4j(i), page 68.]
Figure 605-d
Three Electrode Electrochemical
Detector
These currents can be amplified and
measured accurately, leading to
sensitivities as low as 0.1 pmol in fa-
vorable cases. Amperometric ECh
detectors are also simple in design
and relatively inexpensive and can
be made with a very small internal
volume (0.1-5 μL), thereby minimiz-
ing band broadening, but they are
difficult to use.
Figure 605-d is a simplified sche-
matic diagram of an amperometric
ECh detector. Three electrodes are
used: the working electrode at which
the current due to the analyte is
measured, a silver/silver chloride
reference electrode against which
the potential at the working elec-
trode is selected, and a stainless steel auxiliary electrode to carry the current
arising from the electrochemical reaction. The working electrode is most com-
monly glassy carbon, which is a highly polished, inert, and electrically conducting
form of carbon. To maintain reproducibility, the solid carbon electrode requires
regular maintenance in the form of polishing and cleaning, so detectors must be
relatively simple to take apart and re-assemble.
The chromatogram is obtained by measuring the current at the working electrode,
which is maintained at a fixed potential relative to the reference electrode, as the
electroactive analyte elutes from the column. The working electrode potential is
usually at or near the limiting current plateau of the analyte. The background
current, which is constant for a given mobile phase flow rate and composition, is
subtracted from the analytical signal to give a detector current that is proportional
to the concentration of the analyte according to Faraday’s law. Cyclic voltammetry
is often used to obtain preliminary electrochemical data that determine the opti-
mum applied potential and the effect of variables such as solution pH, mobile
phase composition and concentration, and analyte structure.
Performance Characteristics
In general, ECh detection offers better sensitivity and selectivity than the UV
detector for pesticide residue determination. Detection limits are generally at
picogram levels, whereas for the UV detector, detection limits are, at best, low
nanogram levels. On the other hand, the UV detector has greater long term
stability and is easier to use on an everyday basis.
Detector operation and sensitivity are critically dependent on flow rate constancy,
solution pH, ionic strength, temperature, cell geometry, condition of the elec-
trode surface, and the presence of electroactive impurities (e.g., dissolved oxygen,
halides, trace metals). The detector cannot be used with flow or solvent program-
ming if these changes affect the baseline, and waiting periods of ≥10 min are
required for variations in conditions such as flow rate, applied voltage, or mobile
phase, or for initial startup each day. Both increased flow rate and an increase in
the volume of injected extract decrease detection sensitivity.
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Form FDA 2905a (6/92) 605–11
SECTION 605Pesticide Analytical Manual Vol. I
Applications
An ECh detector was used for oxidative detection of coulometrically reduced
organonitro pesticides separated by RP HPLC [6]. Pesticides were separated on a
C-8 bonded column and monitored indirectly by means of a porous graphite
coulometric detector. The organonitro functional groups were reduced in the
guard cell of the detector, and the reduction products were then detected by
electrochemical oxidation. A 20-90% acetonitrile/water gradient with constant
electrolyte concentration could be used without occurrence of a significant baseline
change. The cell required periodic cleaning with dilute nitric acid and sodium
hydroxide solutions to eliminate negative peaks.
HPLC with ECh detection was also used to determine 0.01-0.02 ppm ethylene-
thiourea (ETU) in foods by a revised official AOAC method [7, 8]. The prepared
extract was chromatographed on a graphitized carbon column with acetonitrile/
aqueous 0.1 M phosphoric acid/water (5:25:70) mobile phase, and the eluted
ETU was detected by using an amperometric ECh detector having a gold/mercury
working electrode.
605 D: PHOTOCONDUCTIVITY DETECTORS
The photoconductivity detector (PCD) is sensitive and selective for organic halo-
gen, sulfur, and nitrogen compounds that form strong, stable ions upon photoly-
sis. The effluent is split as it leaves the column; one-half is passed through the
reference cell of a conductivity detector and the other half is irradiated with 214
or 254 nm UV light. Suitable analytes become ionized, and the resulting conduc-
tance is measured by the detector. Operation of the PCD requires an ion ex-
change resin to purify the mobile phase and lower background conductivity. Both
RP and NP (nonaqueous) systems have been used with the PCD, but the former
are more commonly used for pesticide determination and will be emphasized in
this section.
Apparatus
Publications [9, 10] describing applications of a PCD to pesticide residue determi-
nation employed a system with a reciprocating pump, loop injector or autosampler,
forced draft column oven, variable wavelength UV detector in tandem ahead of
the PCD, dual recorders, a data system for peak integration, and C-18 and cyano
bonded columns. A flow splitter was adjusted to give equal flow rate of column
effluent through the analytical and reference cells of the detector. Balance of
flows through the reference and analytical loops is facilitated by a metering valve
in the solvent line exiting from the reference compartment of the conductivity
cell. This apparatus, or an equivalent system, will be assumed in this section.
Performance Characteristics
The following performance characteristics have been determined by studies of a
PCD-UV detector system for residue determination.
Mobile Phase Preparation. Optimum sensitivity and stable baselines are achieved
when the mobile phase has a minimal ionic background concentration. De-ioniza-
tion of the mobile phase solvents, either individually or as a mixture, is carried out
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–12
by circulation through a mixed bed cartridge (1:1 mixture of anion and cation
exchange resins). Ion exchange treatment of aqueous mobile phases shortly be-
fore use with the PCD is recommended. A 24 hr period of resin circulation at 2.5
mL/min was chosen arbitrarily for “complete purification” of the solvent. Resin-
treated acetonitrile was found to be incompatible with the PCD-UV detector sys-
tem, and the use of resin-treated methanol rather than treated or untreated aceto-
nitrile is recommended.
Temperature Control. More sensitive and consistent detection was obtained when
the column, photolysis reaction chamber, conductivity cells, and associated plumb-
ing were all maintained at a constant, elevated temperature (35-40? C) within a
column oven.
Mobile Phase Flow Rate. The use of low flow rates improves detector response and
reduces the expenditure of purified mobile phase. However, lower flow rates lead
to longer analysis time unless the strength of the mobile phase can be increased
without losing the required resolution. A compromise among speed, resolution,
and detection sensitivity is necessary, depending on the requirements of a particu-
lar analysis.
Pressure. The PCD is very sensitive to pressure fluctuations. Thorough mobile
phase degassing and subsequent gentle sparging with helium help maintain stable
pumping pressure. Use of gradient elution is limited by pressure variations that
occur as the mobile phase composition changes, leading to excessive baseline
shift, especially in high sensitivity applications.
Reproducibility of Response. Improved reproducibility was shown to result from
complete purification of the mobile phase by ion exchange resin treatment and,
to a lesser degree, temperature control.
NP (Nonaqueous) Solvent Operation. The practical application of the PCD to NP
HPLC is limited by the low polarity of the mobile phases used. This results in poor
ion mobility and poor charge transfer in the conductivity cell and thus adversely
affects peak shape and response. Some workers have attained adequate polarity for
good response by adding acetic acid or other polar or ionic modifiers to nonaque-
ous mobile phases, but this approach is limited by the increase in background
noise the added compounds can cause. Reduced background conductivity and
diminished need for purification by de-ionization are advantages of the use of
certain nonaqueous solvents.
Choice of Irradiation Wavelength. In general, the 254 nm mercury lamp provides
greater detection sensitivity than the 214 nm zinc lamp. However, response can be
improved for certain compounds if the zinc lamp is substituted for the mercury
lamp. Greater stability is ensured if the detector, including the lamp, is left on at
all times.
Sensitivity, Selectivity, and Linearity. The PCD can detect low ng levels of many
pesticides and was found to be linear from 1-100 ng injected. Injection aliquots are
typically 5 μL containing 1-20 ng pesticide. UV detection typically shows more
background interferences from crop extracts than the PCD, indicating superior
selectivity for the PCD. Because the PCD is more complex and sensitive to varia-
tions in system conditions (e.g., de-ionization and degassing of mobile phases,
temperature, pumping fluctuations), it should be operated with a tandem UV
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 605–13
SECTION 605Pesticide Analytical Manual Vol. I
detector to monitor the chromatographic system and aid in the diagnosis of anoma-
lies.
Applications
The PCD has been included in several methods for pesticide residues [1-3, 10].
605 E: MASS SPECTROMETRIC DETECTORS
Mass spectrometric (MS) determination can be definitive, providing information
on analyte retention and concentration while simultaneously confirming its iden-
tity. Interpretation of mass spectra permits determination of molecular mass,
empirical formula, arrangement of molecular constituents, and, ultimately, mo-
lecular identity.
Successful use of MS as a chromatographic detector requires introduction of col-
umn effluent to the MS without breaking the vacuum in which the detector oper-
ates. This task is now easily accomplished for GLC-MS, but systems for vaporizing
or otherwise eliminating the HPLC mobile phase before introduction to the MS
are still being developed. Numerous interfaces and ionization techniques for HPLC-
MS have been made available, but so far no one system serves all needs.
An HPLC-MS interface involves two stages: effluent introduction and analyte
ionization. Available effluent introduction techniques include spray techniques, in
which the analyte is introduced as an aerosol; direct liquid introduction, and
mechanical transfer, such as the moving belt interface. Spray techniques are most
common; the aerosol may be produced through nebulization of the effluent using
thermal (e.g., Thermospray), pneumatic (heated nebulizer, particle beam,
Thermabeam), or electrostatic (electrospray, Ion Spray) processes.
The particle beam interface actually combines several processes to remove solvent
from the effluent. After nebulization, the resulting aerosol loses its more volatile
components in a desolvation chamber. Subsequently, the remaining aerosol is
pumped through a momentum separator, a series of skimmers and pumps that
divert most of the solvent vapors. The heavier analyte molecules then pass into the
MS.
Several approaches to analyte ionization exist; the combination of effluent intro-
duction and ionization must be compatible. Direct ionization of the effluent oc-
curs in Thermospray or electrospray interfaces; vaporization and ionization occur
simultaneously. Another direct ionization technique, called fast atom bombard-
ment, ionizes the analyte by bombarding the effluent with fast moving argon
atoms. Electron impact ionization can be achieved if the solvent is removed first,
such as with the particle beam interface. Chemical ionization (CI) can be used
without removal of solvent, using “filament-on” Thermospray, direct liquid intro-
duction, or one of several atmospheric pressure chemical ionization (APCI) sys-
tems that now exist.
APCI is a recent and significant improvement in HPLC-MS interfacing technology.
APCI is a process of ion formation that occurs at atmospheric pressure outside the
MS. APCI ion sources are unique and versatile because they can be utilized with
most of the interfaces described above. One unique advantage of APCI systems is
that interfaces (e.g., electrospray, heated nebulizer) can be readily interchanged
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–14
without venting the MS. APCI most commonly provides CI spectra, but with tan-
dem MS instruments (MS/MS), more complex spectral information is obtainable.
Selection of an interface for a particular HPLC-MS application requires consider-
ation of many factors. Among these, HPLC mobile phase flow rate may limit the
choice of interface to those capable of handling the volume; e.g., Ion Spray inter-
face is limited to 200 μL/min, while Thermospray can accept 1 mL/min. Thermal
stability of the analyte(s) may also restrict choice of interface. The interface must
also be compatible with mobile phase composition; e.g., nonvolatile salts or buffers
may clog some interfaces, flammable solvents are generally unsuitable, and high
aqueous content may inhibit volatilization.
Numerous applications of HPLC-MS have been published, and reviews of the
applications are available [11, 12]. References to the use of particle beam interface
[13-15] and to APCI [16, 17] provide information about application to pesticide
residue determination.
605 F: DERIVATIZATION FOR DETECTION ENHANCEMENT
The detection properties of an analyte can in many cases be enhanced by pre- or
post-column derivatization. Pre-column derivatization is usually carried out inde-
pendent of the instrument and post-column derivatization is usually performed in-
line. Derivatization reactions have been carried out mostly in conjunction with
fluorescence detectors, but visible absorption and ECh detectors are also widely
applied.
Comparison of Pre- and Post-Column Derivatization
Pre-column derivatization procedures have the advantages that long reaction times
and extreme reaction conditions can be used, and reagents can be employed that
have the same detection properties as the derivatives. This is not possible with
post-column reactions, because excess reagent is fed with the effluent to the detec-
tor. Pre-column derivatization may serve as a purification step, and the derivatives
may chromatograph more favorably than the parent compounds. However, the
derivatives are usually more similar structurally than the parent compounds, re-
ducing the chromatographic selectivity. Pre-column derivatization requires a quan-
titative reaction resulting in a stable and well defined product; by-products formed
in the reaction may interfere with the analyte in the chromatogram, necessitating
extensive cleanup after the derivatization reaction. No instrumental modifications
are required for pre-column derivatization, unless it is carried out in-line. In gen-
eral, post-column procedures allow for a higher degree of automation. Reactions
for post-column derivatization should be rapid to avoid extra-column band broad-
ening. An upper practical limit for high efficiency HPLC is about 20 min.
Post-Column Reactor Design
Post-column in-line derivatization is carried out in a reactor located between the
column and detector. The mobile phase flow is not interrupted, although it may
be augmented by addition of a secondary solvent to aid the reaction or meet
detector requirements. This is especially important for ECh detectors, for which
the mobile phase and derivatization reagent are seldom fully compatible. Since
the reactor is located after the column, products of derivatization will not interfere
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Form FDA 2905a (6/92) 605–15
SECTION 605Pesticide Analytical Manual Vol. I
with chromatography. The de-
rivatization reaction does not have to
go to completion or be well defined
if it is reproducible. The reaction
should take place in a reasonable
time, and the reagent should not be
detectable under the same conditions
as the derivative. A high concentra-
tion of reagent is usually used to mini-
mize dilution effects, and a moder-
ately elevated temperature to speed
the reaction.
Figure 605-e shows schematic repre-
sentations of three types of post-col-
umn detectors, the designs of which
are strongly influenced by the time
required for the reaction. In all of
these designs, controlled volumes of
one or more reagents are added to
the column effluent, followed by mix-
ing and incubation for a certain time
period with controlled temperature.
Reagents are added at low pressure
using pulse-free peristaltic pumps.
In the open tubular or open capillary
reactor (i), reagent is pumped via a
mixing tee into the column effluent
containing the separated analytes. The
reactor, which is a coil of stainless steel
or Teflon capillary tubing (typically
0.3 mm id, 150-600 μL volume), provides the necessary time for the reaction
without significantly contributing to band broadening. The combined streams are
finally passed on to the detector. This type of reactor is suitable for fast (≤ about
1 min) derivatization reactions, e.g., for determination of amines with
o-phthalaldehyde reagent.
The segmented stream tubular reactor (ii) is used for slower reactions (5-30 min).
Bubbles of air or a nonmiscible liquid are introduced into the stream at fixed time
intervals. This segments the column effluent into a series of reaction volumes
whose size is governed by the dimensions of the reaction tube and the frequency
of the bubble introduction. Optimal conditions include small liquid segments
introduced at high frequency; short, small id reaction tubes; and a high flow rate.
This type of reactor reduces analyte diffusion and band broadening. The segmen-
tation agent is generally removed by a phase separator prior to the detector, but
noise can also be suppressed electronically.
For intermediate speed reactions (0.3-5 min), packed bed reactors (iii) consisting
of a column containing a nonporous material such as glass beads have been used.
Reagent is pumped into the flowing effluent stream, and the mixture enters the
reactor column. Improper packing of the reaction column can lead to band broad-
ening in the same way as for the analytical column.
Figure 605-e
Post-Column Reactors
(i) Open tubular reactor; (ii) Segmented
reactor; (iii) Packed bed reactor
[Reprinted with permission of John Wiley and Sons, Inc.,
from Lindsay, S. (1987) High Performance Liquid
Chromatography, Figure 2.4p, page 79.]
Reagent
Pump
Pump
Air
Mixing tee
Reactor
Heating bath
Reactor
Detector
Debubbler
Detector
From column
From column
Pump
From column
Reactor
Heating bath
Detector
Reagent
(i)
(ii)
(iii)
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–16
A second detector can be placed ahead of the reaction detector to gather addi-
tional information about the analyte. For example, a UV detector can be utilized
prior to a derivatization/fluorescence detector.
Post-column derivatization techniques can involve simple modification of solution
pH. For example, post-column conversion from slightly acid to a pH above 8
increases the fluorescence of coumarin anticoagulant rodenticides and allows their
sensitive determination with a fluorescence detector. More commonly, reagents
are used that produce fluorescent, UV-absorbing, colored, or electroactive deriva-
tives. Formation of a UV-absorbing derivative is difficult because most suitable
reagents are also strongly absorbing. Reagents can be directly reacted with the
analyte, or an initial hydrolysis or oxidation reaction is sometimes carried out,
followed by derivatization of the product. Fluorescamine, dansyl chloride, and o-
phthalaldehyde are examples of fluorogenic reagents, and ninhydrin is a common
chromogenic reagent for amino acids. Derivatives containing nitroaromatic chro-
mophores can be used for UV or ECh detection based on reduction. p-Aminophenol
derivatives of carboxylic acids and p-dimethylaminophenyl isocyanate derivatives
of arylhydroxyamines are also suitable for ECh detection.
The most important applications of post-column derivatization to pesticide deter-
mination have involved detection of amines. One example is the detection of N-
methylcarbamate insecticides and metabolites as described in Section 401.
Photochemical reactors have been employed to convert compounds to a more
readily detectable fluorescent species, or to a fragment that can be coupled with a
detection- enhancing reagent. The reactor often consists of a Teflon or quartz coil
wrapped around a high power UV lamp in a reflective housing. The length of the
coil is optimized in relation to the desired irradiation time. An example of this
technique applied to residue determination is the method for substituted urea
herbicides, Section 403.
References
[1] Gilvydis, D.M., and Walters, S.M. (1988) J. Agric. Food Chem. 36, 957-961
[2] Walters, S.M., and Gilvydis, D.M. (1983) LC Mag. 1, 302-304
[3] Walters, S.M., et al. (1984) J. Chromatogr. 317, 533-544
[4] Chichila, T.M., and Walters, S.M. (1991) J. Assoc. Off. Anal. Chem. 74, 961-967
[5] Walters, S.M., and Gilvydis, D.M. (May 1987) “HPLC-UV-Fluorescence Determi-
nation of Benomyl as its Hydrolysis Product MBC in Bananas,” LIB 3143, FDA,
Rockville, MD
[6] Krause, R.T., and Wang, Y. (1988) J. Chromatogr. 459, 151-162
[7] Krause, R.T. (1989) J. Assoc. Off. Anal. Chem. 72, 975-979
[8] Krause, R.T., and Wang, Y. (1988) J. Liq. Chromatogr. 11, 349-362
[9] Walters, S.M., and Gilvydis, D.M. (June 1986) “Studies on the Operation of
Photolysis-Conductivity Detection with HPLC,” LIB 3054, FDA, Rockville, MD
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 605–17
SECTION 605Pesticide Analytical Manual Vol. I
[10] Walters, S.M. (1983) J. Chromatogr. 259, 227-242
[11] Voyksner, R.D., and Cairns, T. (1989) in Analytical Methods for Pesticides and Plant
Growth Regulators, Volume XVII, Academic Press, NY
[12] Sherma, J. (1993) Anal. Chem. 65, 40R-54R, and previous biennial reviews
[13] Behymer, T.D., et al. (1990) Anal. Chem. 62, 1686-1690
[14] Doerge, D.R., and Miles, C.J. (1991) Anal. Chem. 63, 1999-2001
[15] Kim, I.S., et al. (1991) Anal. Chem. 63, 819-823
[16] Kawasaki, S., et al. (1992) J. Chromatogr. 595, 193-202
[17] Pleasance, S., et al. (1992) J. Am. Soc. Mass Spectrom. 3, 378-397
SECTION 605 Pesticide Analytical Manual Vol. I
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)605–18
Pesticide Analytical Manual Vol. I
SECTION 606
606–1
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
606: RESIDUE IDENTIFICATION AND QUANTITATION
606 A: RESIDUE IDENTIFICATION
The first step in determining residues in a cleaned up, concentrated extract is to run
a preliminary chromatogram. Tentative peak identification is made by comparing
retention data with data for standards measured under identical conditions. If these
data indicate the presence of one or more probable pesticide peaks, proper standard
solutions are prepared for qualitative confirmation and quantitation.
Identical retention characteristics of an analyte and reference substance in a single
determination do not assure accurate identification, because several compounds
may have the same retention time under any given set of conditions. Confirmation
of peak identity must be obtained by use of additional determinations (Section
103). Co-chromatography and HPLC using alternative (dissimilar) columns and/
or selective detectors are most appropriate for residues determined with HPLC.
Other chromatographic methods such as GLC or thin layer chromatography (TLC),
or UV, IR, nuclear magnetic resonance (NMR), or mass spectrometry (MS) may
also be useful for confirming residue identity.
Relative retention time, the ratio of the absolute retention of the compound of
interest to that of a selected reference standard (“marker compound”), is more
reproducible than the absolute retention time, so it is used to compare residue
and standard peaks. Only the composition of the mobile phase influences the
relative retention time, whereas absolute retentions can vary slightly from day to
day or even from hour to hour if instrumental parameters, such as mobile phase
flow rate, recorder chart speed, or injection technique vary. The marker com-
pound may be chromatographed just before or after the sample, but it is best to
include a portion of the marker compound in the injection of the sample extract;
in this way, both residue and marker peaks are chromatographed at the same
conditions and appear in the same chromatogram.
In addition to relative retention time, peak shape is often another useful aid in
comparing sample and standard chromatograms. Residue identity can be confirmed
by observing changes in absolute or relative retention time upon derivatization of
both the analyte and the appropriate reference standard.
Co-chromatography
Co-chromatography provides an alternative means of qualitative analysis based on
retention times. An amount of pure standard compound, thought to be the analyte,
is added to a portion of the sample extract at approximately double the amount
present, and an aliquot is re-injected. If the tentative identification of the residue
was correct, only the peak due to the analyte will be intensified, and peak shape
will not be distorted (i.e., no shoulders or broadening will be produced). This
method has the same limitation as comparison of retention times, in that it is
possible that another compound with chromatographic characteristics correspond-
ing to the added compound might be present. Compound identification using this
technique is enhanced by high column efficiency and resolution and optimized
operating parameters; when pesticides are well resolved from one another and
from nonpesticide artifacts co-extracted from the sample substrate, there will be
the greatest chance for the analyte and the co-injected standard to separate if they
SECTION 606
606–2
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
are not the same compound. If the HPLC system has recycling capability, the co-
injected mixture can be recycled several times to try to separate the analyte from the
added standard.
Use of Alternative Columns
Concurrence of retention times between analyte and standard peaks, or absence
of separation of peaks in a co-injected sample plus standard, on two or more
different HPLC columns (each with a suitable mobile phase) gives greater assur-
ance that the two peaks represent the same compound. However, the columns
must be judiciously chosen so that their separations are governed by distinctly
different mechanisms that produce different elution patterns. Reverse phase (RP)
and normal phase (NP) partition columns and silica gel adsorption columns have
been shown to be independent, complementary columns for confirmation of peak
identity for many compound types.
Spectrometric Confirmation
It is possible to confirm identification spectrometrically by collecting the analyte as
it elutes from the HPLC instrument, either manually or with an automatic fraction
collector, and analyzing it with UV, MS, IR, or NMR. However, the practical appli-
cation of this approach for trace pesticide determination is limited. Because the
sensitivity of such instruments is limited, eluates from HPLC are often difficult to
concentrate, and buffers and salts from RP mobile phases can interfere.
Spectrometric confirmation can be performed in-line. The absorbance (peak
height) ratio at two different UV wavelengths, e.g., 254 and 280 nm, can be charac-
teristic for a particular compound, and comparison of the ratio for the analyte and
a standard can be helpful for peak confirmation. The presence or absence of
peaks or the signal ratio when using different selective detectors provides addi-
tional confirmational information. Combinations that have been employed for
pesticide determination include the UV detector followed by a photoconductivity,
fluorescence, or electrochemical detector. Specificity is obtained by the position of
the absorption wavelength with the UV detector, the excitation and emission wave-
lengths with the fluorescence detector, and the reduction or oxidation potential
with the electrochemical detector.
Identification can be made by use of a scanning UV/VIS detector. The spectrom-
eter is initially set to a wavelength that produces a strong signal for the chromato-
graphic peak to be identified. When the absorbance signal is at its maximum, the
flow of mobile phase is stopped by means of a stop-flow valve and the full spectrum
of the trapped component is scanned. The flow of mobile phase is then restarted
and the analysis continued. A limitation of this approach is that UV and visible
absorption spectra are not as characteristic as IR, NMR, and MS for compound
identification.
Scanning of fluorescence spectra provides somewhat better characterization. If
full spectrum detection is used for identification confirmation, the following crite-
ria have been suggested [1]: the maximum absorption wavelength in the spectrum
of the analyte should be the same as that of the standard material to which it is
compared within a margin determined by the resolution of the detection system.
For diode array detectors, this is typically ±2 nm. The spectrum of the analyte
should not be visually different from the spectrum of the standard material
Pesticide Analytical Manual Vol. I
SECTION 606
606–3
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
for the parts of the two spectra with a relative absorbance >10%. This criterion is
met when the same maxima are present and when the difference between the two
spectra is never >10% of the absorbance of the standard material at any point.
606 B: QUANTITATION
Techniques for quantitating detector response by measurement of chromatographic
peaks are the same for HPLC as for GLC. Section 504 provides directions for both
manual peak measurement and use of electronic integrators; these directions should
be followed in HPLC quantitation.
Pesticide residues are quantitated by comparing the size (height or area) of the
peak for each analyte and the size of a peak from a similar, known amount of each
reference standard injected under the same HPLC conditions just before and/or
after the sample injection. Only one standard concentration is required for each
analyte if injections are made at concentration levels providing linear detector
response. This procedure, which is the most widely used, is known as the external
standardization method. Other quantitation methods, such as internal standard-
ization and standard additions, have not been widely used for pesticide residue
determination.
The exploratory chromatogram of the sample extract used to obtain qualitative
analysis will indicate to the analyst the proper standard solution to be used. The
solution should contain the pesticides to be quantitated at proper concentration
levels to fall within the linearity range of the detector and also to produce peaks
comparable in size (usually ±25%) to those obtained from the chromatogram of
the sample extract. Injection of the standard mixture may show that additional
dilution of the sample extract is required to produce peaks of the higher concen-
tration pesticides that are within the linear detector range and similar in size to
those from the standard mixture. If several standard mixtures are available at
different concentration levels, selection of one closely approximating the unknown
will facilitate the analysis. It cannot be emphasized too strongly that accurate
quantitation is not possible unless standards are prepared and maintained prop-
erly and replaced on schedule.
Peak height linearity in HPLC can be lost due to band spreading when the sample
solvent is significantly stronger than the mobile phase, e.g., the sample is dissolved
in methanol and injected into methanol/water (1:1) mobile phase in an RP col-
umn. If possible, the sample should be dissolved in the mobile phase to minimize
this problem. Otherwise, the injection volumes must be carefully considered; the
amounts of sample and standard injected should be equal, or the sample volume
must be kept small and the volume causing the onset of band spreading deter-
mined and not exceeded.
Reference
[1] de Ruig, W., et al. (1989) J. Assoc. Off. Anal. Chem. 72, 487-490
SECTION 606
606–4
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I
607–1
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I SECTION 607
607: QUALITY ASSURANCE AND TROUBLESHOOTING
Previous sections of this chapter included troubleshooting advice related to the
specific system components (Sections 601 D, 601 E, 602 F, 603 B, 603 D, 604 C, 605 A,
and 605 B). This section provides testing, maintenance, and troubleshooting proce-
dures for the HPLC system as a whole. These procedures are recommended to
minimize poor performance, damage, and downtime of the HPLC system and to help
ensure that results of HPLC analyses are accurate and precise.
607 A: LIQUID CHROMATOGRAPH MONITORING
AND PERFORMANCE TESTING
Consult the instruction manual supplied with each instrument component for
specific installation, operation, maintenance, and performance check procedures.
The following tests are general in nature and should be appropriate for most
HPLC systems and analyses.
Time of use:
? Check visually for solvent leaks at all fittings.
? Operate pump(s) at 1 mL/min, at the detector sensitivity anticipated,
and note baseline noise. If noise problems exist, consult the instrument
manual.
? Check detector sensitivity by chromatographing a reference standard
(or mixture) appropriate for the particular detector being used; note
detector response.
? Pretested columns, with which a test chromatogram is supplied by the
manufacturer, are preferred. Verify the adequacy of new columns by
repeating the manufacturer’s test. At time of use, test the column by
repeating the performance test specified when the column was pur-
chased, or use an alternative in-house test. Verify the performance of
columns packed in the laboratory in the same way and with the same
reference material as for commercially packed columns.
? With the column in place and pumps, detector, and recorder or integra-
tor in operation, inject several identical amounts of standard to check
that reproducibility is within laboratory specifications (typically ±3%).
? Before using the system for a new application, determine that the detec-
tor response is linear and reproducible by construction of a standard
curve. Using standards of different concentrations, occasionally spot-
check that linearity and response factors are within laboratory specifica-
tions.
? Check that the mobile phase components (solvents, salts) are of ad-
equate purity grade and are properly filtered and degassed. If there is
indication of contamination or significant concentration change, pre-
pare new mobile phase.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)607–2
SECTION 607 Pesticide Analytical Manual Vol. I
? Do not leave water or aqueous solutions of salts, acids, or bases in the
pump(s). Flush the system with an appropriate pure organic or aqueous/
organic solvent. Methanol is preferred for long term shutdown.
607 B: TROUBLESHOOTING FROM CHROMATOGRAMS
Efficiency is a measure of the ability of the column to produce narrow peaks
(Section 602 C). It is expressed by the plate number (number of theoretical
plates) of the column. The ability of the column to separate two components is
termed resolution. Resolution is a function of the peak widths (efficiency), the
separation between peak centers (selectivity), and the degree of retention for the
components by the column (capacity). This section reviews some problems that
can be detected by inspection of chromatograms and offers possible causes and
solutions for them. Specific procedures for solving most of the problems will be
found in the individual sections covering various instrument components.
? Peaks that elute too quickly with poor resolution are usually caused by a
flow rate that is too high or a capacity factor that is too low. Increase the
capacity factor (and affinity for the column) by using a mobile phase that
is weaker, i.e., less polar for adsorption chromatography and more polar
for reverse phase (RP) chromatography.
? If peaks of a mixture are not well separated but capacity and efficiency are
adequate (i.e., k' = 2-10 and narrow peaks), selectivity is too low. Vary
selectivity by changing to a second column operating with a completely
different mechanism (e.g., adsorption rather than RP), or try a different
mobile phase with both of the columns. If the column is old, its resolution
may have deteriorated; try a new column of the same type with the origi-
nal mobile phase.
? Poor resolution can also be due to low column efficiency. Improve effi-
ciency by lowering the flow rate to increase the number of theoretical
plates and improve resolution. Alternatively, improve efficiency by increas-
ing the column length (the analysis will take longer), by using a column
with smaller diameter packing, or by increasing the operating tempera-
ture (mass transfer is improved). A void at the top of the column can also
cause poor efficiency and resolution.
? Loss of retention from one chromatogram to the next can be caused by
incomplete column equilibration after gradient elution, adsorption of
sample impurities, or loss of column activity. Solve the first problem by
proper column regeneration after each gradient elution. Remove adsorbed
impurities by washing the column or replacing the top 2-3 mm column
packing. Maintain constant column activity by using properly dried sol-
vents (for adsorption chromatography). Use a guard column to help pre-
vent adsorption of high molecular weight and polar impurities by the
analytical column. Replace the guard column as required.
? An increase in retention times can be caused by too low flow rate, incor-
rectly prepared mobile phase mixture, the wrong solvent in one of the
pump reservoirs, or too slow rate of change of gradient. If silica gel is
being used, the activity of the column may have increased because of the
use of more completely dried solvents.
607–3
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. I SECTION 607
? Additional causes of drifting retention times include differences among
solvent batches, changes in the composition of a batch of mobile phase
upon standing, changes in temperature, a nonconstant recorder drive or
slipping chart paper, or changing mobile phase flow rate caused by
nonreproducible pump delivery or a leak in the system.
? Tailing peaks in adsorption chromatography can result from column
sites with too much activity. To solve this problem, add an optimum
amount of a deactivator (e.g., water) to the mobile phase.
? In RP HPLC, tailing can result if the sample is nearly insoluble in the
mobile phase or if the sample is partly or completely ionic. Bonded RP
columns have a high proportion of unreacted silanol groups (SiOH)
available for secondary reaction with analytes. Some of these silanol
groups cause bases to tail, and others affect acidic compounds. Different
commercial columns are better or worse in terms of their ability to
produce peaks with good peak shapes, but none will be completely free
from tailing problems.
? Add appropriate mobile phase additives to ensure neutrality of analytes,
thereby minimizing unwanted silanol interactions and the resultant peak
tailing. The best additives are usually 10-50 mM triethylamine or
dimethylhexylamine for suppressing base tailing, and approximately 1%
acetic acid for eliminating the tailing of acids. If both acidic and basic
sample components are present, combine the additives to give a cumula-
tive effect.
? In ion exchange chromatography, the cause of tailing peaks can be
mobile phase with too low an ionic strength or the wrong pH, or adsorp-
tion on the resin. Optimum buffer concentrations are sample depen-
dent, generally ranging from 10-100 mM. Ideally, the pH of the mobile
phase should ensure that the solute is completely ionized. Adsorption to
the resin can often be eliminated by an increase in temperature or
addition of a small percentage of organic modifier to the mobile phase.
? For all types of packings, tailing can be caused by a void at the top of the
column or excessively long or wide connection tubing between the in-
jection valve and column or between the column and detector. The
latter type of cause is indicated if the tailing of early peaks is greater
than that of later peaks, and if tailing is greater for faster flow rates.
? A peak exhibiting fronting (a slowly rising leading edge) is usually caused
by overloading. Remedy this by injecting less sample.
? A peak exhibiting a doublet or a shoulder (or tailing) results from a
dirty, channeled, or defective column. Regenerate dirty columns by wash-
ing or repacking the top of the bed. If the inlet frit rather than the
packing is dirty, clean or replace the frit.
? Peaks with a staircase shape that never reach true maximum height
result from an incorrect recorder damping control setting.
? A noisy recorder baseline can be caused by incorrect recorder damping
control setting, or by incorrect grounding of the recorder or the HPLC
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)607–4
SECTION 607 Pesticide Analytical Manual Vol. I
instrument, a defective source lamp or dirty cell windows (UV detector),
or contaminated solvent. Baseline noise in the form of successive sharp
spikes is most likely due to formation of bubbles in the detector cell.
Baseline drift is caused by contamination of the detector cell or column,
elution of adsorbed impurities, or a change in detector temperature.
? A negative recorder trace is usually caused by a leak between the sample
and reference cell compartments; locate and repair.
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92) 608–1
SECTION 608Pesticide Analytical Manual Vol. I
608: BIBLIOGRAPHY
GENERAL TEXTS
Brown, P.R., and Hartwick, R. A. (1989) High Performance Liquid Chromatogra-
phy, Wiley, New York
Engelhardt, H., ed. (1989) Practice of High Performance Liquid Chromatography,
Springer-Verlag, New York
Engelhardt, H., and Hupe, K.P. (1985) Coupling Methods in HPLC, GIT Verlag
GMBH, Darmstadt, Germany
Katz, E., ed. (1987) Quantitative Analysis Using Chromatographic Techniques, Wiley,
New York
Lawrence, J.F. (1981) Organic Trace Analysis by Liquid Chromatography, Aca-
demic Press, New York
Lindsay, S. (1987) High Performance Liquid Chromatography (Analytical Chemistry
by Open Learning), Wiley, New York
Meyer, V.R. (1988) Practical High Performance Liquid Chromatography, Wiley,
New York
Miller, J.M. (1988) Chromatography: Concepts and Contrasts, Wiley, New York
Parris, N.A. (1984) Journal of Chromatography Library, Volume 27: Instrumental
Liquid Chromatography. A Practical Manual on High Performance Liquid Chroma-
tography Methods, 2nd ed, Elsevier, New York
Poole, C.F., and Poole, S.K. (1991) Chromatography Today, Elsevier, New York
Poole, C.F., and Schuette, S.A. (1984) Contemporary Practice of Chromatography,
Elsevier, New York
Schoenmakers, P. (1986) Journal of Chromatography Library, Volume 35: Optimi-
zation of Chromatographic Selectivity. A Guide to Method Development, Elsevier, New
York
Snyder, L.R., et al. (1988) Practical HPLC Method Development, Wiley-Interscience,
New York
Snyder, L.R., and Dolan, J.W. LC Video Course: Getting Started in HPLC, LC
Resources, Inc., San Jose, CA (consists of five 45-60 min videotapes for begin-
ners in HPLC)
Yau, W.W., et al. (1979) Modern Size-Exclusion Liquid Chromatography, Wiley-
Interscience, New York
COLUMNS
Ishii, D., ed. (1988) Introduction to Microscale High Performance Liquid Chroma-
tography, VCH, Weinheim, Germany
608–2
Transmittal No. 94-1 (1/94)
Form FDA 2905a (6/92)
Pesticide Analytical Manual Vol. ISECTION 608
Kucera, P. (1984) Journal of Chromatography Library, Volume 28: Microcolumn
High Performance Liquid Chromatography, Elsevier, New York
Scott, R.P.W., ed. (1984) Small Bore Liquid Chromatography Columns: Their Prop-
erties and Uses, Wiley, New York
DETECTORS
Krull, I.S., ed. (1987) Chromatographic Science Series, Volume 34: Reaction Detec-
tion in High Performance Liquid Chromatography; Part A, Elsevier, New York
Ryan, T.H., ed. (1984) Electrochemical Detectors: Fundamental Aspects and Analyti-
cal Applications, Plenum Press, New York
Scott, R.P.W. (1986) Journal of Chromatography Library, Volume 33: Liquid Chro-
matography Detectors, 2nd ed, Elsevier, New York
Yang, E.S., ed. (1986) Detectors for Liquid Chromatography, Wiley, New York
TROUBLESHOOTING
Dolan, J.W., and Snyder, L.R. (1989) Troubleshooting LC Systems: A Practical
Approach to Troubleshooting LC Equipment and Separations, Humana Press, Clifton,
NJ
Dolan, J.W., and Snyder, L.R. Trouble Shooting HPLC Systems, Elsevier Science
Publishers, New York (consists of three 55 min training tapes)
Runser, D.J. (1981) Maintaining and Troubleshooting HPLC Systems — A User’s
Guide, Wiley, New York
Walker, J.Q., ed. (1984) Chromatographic Systems: Problems and Solutions, Preston
Publishers, Niles, IL
APPLICATION TO PESTICIDES
Follweiler, J.M., and Sherma, J. (1984) CRC Handbook of Chromatography: Pesti-
cides and Related Organic Compounds, Volume 1, CRC Press, Boca Raton, FL
Lawrence, J.F. (1982) High Performance Liquid Chromatography of Pesticides,
Volume XII of Analytical Methods for Pesticides and Plant Growth Regulators,
Zweig, G. and Sherma, J., eds., Academic Press, New York
Lawrence, J.F., ed. (1984) Food Constituents and Food Residues: Their Chromato-
graphic Determination, Marcel Dekker, New York
Lawrence, J.F., ed. (1984) Liquid Chromatography in Environmental Analysis,
Humana Press, Clifton, NJ