2010-5-20 1
HPLC Column and System Troubleshooting
色谱柱和色谱系统的故障检修
? What Every HPLC
? User Should Know
?每个 HPLC使用者应该知道什么
2010-5-20 2
HPLC Components
HPLC 的组成
? Pump 泵
? Injector/Autosampler 进样器 /自动进样器
? Column 色谱柱
? Detector 检测器
? Data System/Integrator 数据系统 /积分仪表
? All of these components can have problems
and require troubleshooting.
? 所有这些部件可能出现故障而需要检修。
2010-5-20 3
Categories of Column Problems 色谱
柱问题的类型
?
? A,Pressure 压力
? B,Peak shape 峰形
? C,Retention 保留时间
2010-5-20 4
Pressure Issues
压力问题
? Column Observations Potential Problems
? 色谱柱观测 潜在的问题
? High pressure Plugged frit
? 压力高 堵塞滤头
? Column contamination
? 色谱柱污染
? Piugged packing
? 堵塞填料
2010-5-20 5
Determining the Cause and Correcting
High Back Pressure 查出原因 纠正压力
? Check pressure with/without column - many pressure problems
are due to blockages in the system or guard
? 有／无柱子时检查压力 － 许多压力问题是由于系统或保护柱的阻塞
? If Column Pressure is high 如果柱压高,
? Wash column - Eliminate column contamination and
? 冲洗柱子 plugged packing 排除柱污染和填料堵塞
? - high molecular weight/adsorbed
? compounds 高分子量／被吸附化合物
? - precipitate from sample or buffer
? 样品或缓冲液中的沉淀物
? Back flush column - Clear plugged frit 反冲柱子－清洁阻塞的滤头
? Change frit - Clear plugged frit 更换滤头
2010-5-20 6
Column Cleaning 柱子清洗
? F lush with stronger solvents than your mobile phase
? 用比你的流动相更强的溶剂冲洗
? Reversed-Phase Solvent Choices in Order of
? Increasing Strength 为了增加强度选择反相溶剂
? Use at least 25 ml of each solvent for analytical columns
? 对于分析柱每种溶剂至少用 25 ml 冲洗
? Mobile phase without buffer salts 没有缓冲盐的流动相
? 100% Methanol 甲醇
? 100% Acetonitrile 乙腈
? 75% Acetonitrile,25% Isopropanol 75乙腈, 25％异丙醇
? 100% Isopropanol 异丙醇
? 100% Methylene Chloride★ 二氯甲烷
? 100% Hexane ★ 已烷
? ★ When using either Hexane or Methylene Chloride the column must be flushed with
? Isopropanol before returning to your resvered-phase mobile phase.
? 当用已烷 蔌 二氯甲烷柱子时,必须先用 异丙醇 冲洗,然后再用你的反相流动相
2010-5-20 7
Column Cleaning柱子清洗
? Normal Phase Solvent Choices in
Order of Increasing Strength
? 为了增加强度选用正相溶剂
? Use at least 50 ml of each solvent
? 每种溶剂至少用 50 ml
? 50% Methanol, 50% Chloroform
? 50% 甲醇, 50% 氯仿
? 100% Ethyl Acetate 乙酸乙酯
2010-5-20 8
How to Change a Frit
怎样更换滤头
? Column Inlet Frit 柱进口滤头
? Column Body 柱身
? Compression Ferrule 压缩金属套圈
? Wear gloves 戴手套
? Do not allow bed to dry 不要让底座干燥
? Do not touch the column-body heat will extrude packing
不要使柱身受热,否则填料会喷出
? Do not over tighten 不要旋得太紧
? Female End Fitting 旋好尾端螺母
? Male End Fitting 旋好尾端螺头
2010-5-20 9
Preventing Back Pressure Problems
压力问题的预防
? Use column protection 柱保护
? - Guard columns 保护柱 /预柱
? - In-line filters 在线过滤器
? Sample Preparation 样品预处理
? Appropriate column flushing
? 适当的柱冲洗
? Filter buffered mobile phase
? 过滤缓冲流动相
2010-5-20 10
Preventing Back Pressure Problems:In-Line Devices
压力问题的预防,在线装置
? Mobile Phase Pre-Column Injector Filter Guard
? From Pump 预柱 进样器 滤头 Column
? 从泵来的流动相 保护柱
? Analytical
? Column
? Filter and Guard Column Act on Sample 分析柱
? 滤头和保护柱作用于样品
? Pre-Column Acts on Mobile Phase
? 预柱作用于流动相 To Detector
? 到检测器
2010-5-20 11
Preventing Back Pressure Problems,Sample Preparation
压力问题的预防,样品制备
? Solvent/Chemical Environment
? 溶剂 /化学环境
? Particulate/Aggregate Remove
? 微粒 /凝聚物的除去
? Filter samples 过滤样品
? Centrifugation 离心分离
? Solid Phase Extraction(S.P.E.) 固相萃取
? Cartridges or Plates 薄膜或薄层板
? Disks or Membranes 圆盘或生物膜
2010-5-20 12
Peak Shape Issues
峰形问题
? Split peaks 裂峰
? Peak tailing 峰拖尾
? Broad peaks 宽峰
? Poor efficiency 低柱效
? Many peak shape issues also combinations-I.e,Broad
and tailing or tailing with increased retention许多峰形问题
是结合在一起的 -例如,
? 展宽和拖尾或拖尾和保留时间增加
2010-5-20 13
Split Peaks 裂峰
?Can be caused by,
?可能的原因,
? Column contamination 柱污染
? Partially plugged frit 部分阻塞滤片
? Column void 柱头塌陷
? Injection solvent effects 溶剂效应
2010-5-20 14
Split Peaks 裂峰
Column Contamination 柱污染
? Column,StableBond SB-C8,4.6 × 250mm,5μm
? Mobile Phase:60%25mM Na2HPO4,pH3.0, 40%MeOH
? Flow Rate,1.0 mL/min Temperature,35oC
? Detection,UV 254 nm
? Sample,Filtered OTC Cold Medication,
? 1,Pseudoephedrine 2,APAP 3,Unknown
? 4,Chlorpheniramine
? Injection 1 峰 3 峰形较好
? Injection 30 峰 3 裂峰
? Injection 1 After Column Wash with 100% CAN
? 经 100%乙腈冲洗后 峰 3 峰形极好
2010-5-20 15
Split Peaks 裂峰
Injection Solvent Effects 溶剂化效应
? Column,StableBond SB-C8,4.6 × 250mm,5μm
? Mobile Phase,82%H2O, 18%ACN
? Injection Volume,30 μL
? Sample,
? 1,Caffeine 咖啡因 2,Salicylamide 水杨酰胺
? A,Injection Solvent B,Injection Solvent
? 100% Acetonitrile Mobile Phase
? 峰形宽拖尾 峰形尖锐对称
?样品溶剂尽可能与流动相匹配
2010-5-20 16
Determining the Cause of Split Peaks
裂峰原因的确定
? 1,Complex sample matrix or many samples analyzed- likely
column contamination or partially plugged frit
? 复杂样品的基质或分析许多样品 - 柱污染或部分阻塞滤片
? 2,Mobile phase pH>=7 - likely column void due to silica
dissolution (unless specialty column used)
? 流动相 pH>=7 - 由于硅胶溶解使柱塌陷 (除非使用专门的柱子 )
? 3,Injection solvent stronger than mobile phase - likely split
and broad peaks,dependent on volume
? 溶剂比流动相的可能性大 - 裂峰和宽峰,由样品量来决定
2010-5-20 17
Peak Tailing,Broadening and Loss of Efficiency
峰拖尾、变宽和柱效降低
? Can be caused by,可能的原因
?
? Column,secondary interactions”
? 柱“次级效应”
? Column void 柱塌陷
? Column contamination 柱污染
? Column aging 柱老化
? Column loading 柱负荷超载
? Extra-column effects 柱外效应
2010-5-20 18
Peak Tailing Column,Secondary Interactions”
峰拖尾柱“次级效应”
? Column,Alkyl-C8,4.6 × 150mm,5μm
? Mobile Phase:85%25mM Na2HPO4,pH7.0, 15%ACN
? Flow Rate,1.0 mL/min Temperature,35oC
? Sample,1,Phenylpropanolamine 苯丙醇胺／去甲麻黄碱
? 2,Ephedrine 麻黄碱 3,Amphetamine 安非他明／苯丙胺
? 4,Methamphetamine 脱氧麻黄碱 5,Phenteramine 苯三胺
? No TEA 无三乙胺 10 mM TEA 10 mM 三乙胺
? USP TF(5%) 美国药典拖尾因子 USP TF(5%) 美国药典拖尾因子
? 1,1.29 1,1.19
? 2,1.91 2,1.18
? 3,1.63 3,1.20
? 4,2.35 4,1.26
? 5,1.57 5,1.14
? Peak tailing eliminated with mobile phase modifier (TEA) at pH 7
? 用流动相减尾剂 (三乙胺 )在 pH 7 消除峰拖尾
2010-5-20 19
Peak Tailing Column,Secondary Interactions”
峰拖尾柱“次级效应”
? Column,Alkyl-C8,4.6 × 150mm,5μm
? Mobile Phase:85%25mM Na2HPO4,pH7.0, 15%ACN
? Flow Rate,1.0 mL/min Temperature,35oC
? Sample,1,Phenylpropanolamine 苯丙醇胺／去甲麻黄碱
? 2,Ephedrine 麻黄碱 3,Amphetamine 安非他明／苯丙胺
? 4,Methamphetamine 脱氧麻黄碱 5,Phenteramine 苯三胺
? pH 3.0 pH 7.0
? USP TF(5%) 美国药典拖尾因子 USP TF(5%) 美国药典拖尾因子
? 4,1.33 4,2.35
? Reducing the mobile phase pH reduces interactions with silanols that
cause peak tailing
? 降低流动相的 pH,减小与硅醇基作用引起的峰拖尾
2010-5-20 20
Peak Tailing 峰拖尾
Column Contamination 柱污染
? Column,StableBond SB-C8,4.6 × 250mm,5μm
? Mobile Phase,20% H2O, 80% MeOH Flow Rate,1.0 mL/min
Temperature,R.T,Detection,UV 254 nm
? Sample,1,Uracil 尿嘧啶 2,Phenol 苯酚
? 3,4-Chloronitrobenzene 4-氯硝基苯 4,Toluene 甲苯
? Plates TF Plates TF Plates TF
? 理论板数 拖尾因子 理论板数 拖尾因子 理论板数 拖尾因子
? 1,7629 2.08 1,7906 1.43 1,7448 1.06
? 2,12043 1.64 2,12443 1.21 2,12237 1.21
? 3,13727 1.69 3,17999 1.19 3,15366 1.11
? 4,13355 1.32 4,17098 1.25 4,19067 1.17
? QC test forward direction QC test reverse direction QC test after cleaning
? 100% IPA,35oC
? 向前方向 相反方向 100%异丙醇冲洗后
2010-5-20 21
Peak Tailing/Broadening
Sample Load Effects
峰拖尾 /变宽 样品过载效应
? Column,4.6 × 150mm,5μm
? Mobile Phase:40%25mM Na2HPO4,pH7.0, 60%ACN
? Flow Rate,1.5 mL/min Temperature,40oC
? Sample,
? 1,Desipramine 去郁敏 /脱甲基丙米嗪 2,Nortriptyline 3,Doxepin
4,Imipramine 丙米嗪 5,Amitriptyline 阿米替林 6,Trimipramine
? Tailing Eclipse XDB-C8 Broadening / Competitive C8
? USP TF 拖尾因子 Plates 理论板数
? High Load / 10 Low Load High Load / 10 Low Load
? 高载负 /10倍 低载负 高载负 /10倍 低载负
? 1,1.60 1.70 850 5941
? 2,2.00 1.90 815 7842
? 3,1.56 1.56 2776 6231
? 4,2.13 1.70 2539 8359
? 5,2.15 1.86 2735 10022
? 6,1.25 1.25 5189 10725
2010-5-20 22
Peak Broadening,Splitting Column Void
峰变宽,裂开 柱塌陷
? Mobile Phase:流动相：
? 50%CAN, 50%Water, 0.2%TEA (~pH 11)
? 50％ 乙腈, 50％水, 0。 2％三乙胺 (~pH 11)
? After 30 injections Initial 开始时
? 进样 30次后
? Multiple peak shape changes can be caused by the
same column problem,In the case a void resulted from
silica dissolved at high pH.
? 多数峰形改变可能是同一柱问题引起的。在这个例子中，
高 pH溶解硅胶导致塌陷
2010-5-20 23
Broad Peaks Unknown
“Phantom” Peaks
? Column,Extend-C18,4.6 × 150 mm,5 μm
? Mobile Phase,40% 10 mM TEA,pH 11, 60% MeOH
? Flow Rate,1.0 mL/min Temperature,R.T,Detection,UV 254
? Sample,1,Maleat 顺丁烯二酸 2,Pseudeophedrine 伪麻黄碱
3,Chlorphenniramine 氯非尼拉明
? Plates 理论板数
? 1,5922
? 2,9879
? 3,779, Phantom” peak from first injection 前次进样未出完的峰
? The extremely low plates are an indication of an extremely late
eluting peak from the preceding run.
? 极低的理论板数表明这是一个前面进样极迟洗脱出来的峰
2010-5-20 24
Peak Tailing峰拖尾
Injector Seal Failure进样器密封性差
? Column,Bonus-RP,4.6 × 75mm,3.5μm
? Mobile Phase,30% H2O, 70% MeOH Flow Rate,1.0 mL/min
Temperature,R.T,Detection,UV 254 nm
? Sample,1,Uracil 尿嘧啶 2,Phenol 苯酚
? 3,N,N-Dimethylaniline N,N-二甲苯胺
? Plates TF Plates TF
? 理论板数 拖尾因子 理论板数 拖尾因子
? 1,2235 1.72 1,3670 1.45
? 2,3491 1.48 2,10457 1.09
? 3,5432 1.15 3,10085 1.00
? Before After replacing rotor seal and isolation seal
? 之前 转动杆和隔离密封垫检修后
? Overdue instrument maintenance can cause peak shape problems.
? 仪器保养超期可能引起峰形问题 /进样器划痕
2010-5-20 25
Peak Tailing 峰拖尾
Extra-Column Volume 柱外死体积效应
? Column,StableBond SB-C18,4.6 × 30mm,3.5μm
? Mobile Phase,85% H2Owith 0.1% TFA, 15% CAN
? Flow Rate,1.0 mL/min Temperature,35oC
? Sample,1,Phenylalanine 2,5-benzyl-3,6-dioxo-2-piperazine
? 3,Asp-phe 4,Aspartame
? 10 μl extra-column volume 50 μl extra-column volume
2010-5-20 26
Determining the Cause of Peak Tailing
峰拖尾原因的确定
? Evaluate mobile phase effects - alter mobile phase pH
and additives to eliminate secondary interactions
评价流动相效果 - 改变流动相 pH和添加剂消除次级效应 /流动相中组份
与柱子相互作用
? Evaluate column choice - try column with high purity
silica or different bonding technology
评价柱选择 - 试用高纯度硅胶柱或不同键合技术柱
? Reduce sample load 减小进样量
? Eliminate extra-column effects 消除柱外效应
? Flush column and check for aging/void
冲洗柱子检查柱子老化 /塌陷
2010-5-20 27
Retention Issues
保留时间问题
? Retention time changes(tr)
保留时间改变
? Capacity factor (retention) changes (k′)
容量因子改变
? Selectivity changes (?)
选择性改变
2010-5-20 28
Changes in Retention Same Column,Over Time
相同柱子上保留时间改变,超时
? May be cause by,
可能的原因,
? Column aging 柱老化
? Column contamination 柱污染
? Insufficient equilibration 不够平衡
? Poor column/mobile phase combination
柱 /流动相组成差
? Change in mobile phase 改变流动相
? Change in flow rate 改变流速
? other instrument issues 其他仪器问题
2010-5-20 29
Mobile phase Change Causes Change in Retention
流动相改变引起保留时间改变
? 60%MeOH:40% 0.1% TFA Fresh TFA Added to Mobile Phase
? Volatile TFA evaporated/degassed from mobile phase,
易挥发的三氟乙酸从流动相中蒸发 /挥发。
? Replacing it solved problem,
换用新的流动相就解决问题。
?使用有机挥发性酸时，流动相应新配。
2010-5-20 30
Column Aging/Equilibration Causes
Retention/Selectivity Changes
柱老化／平衡不够引起保留时间／选择性改变
? Column 1 - Initial 1 号柱 开始时
? Column 1 - Next Day 1 号柱 第二天
? Column 1 - After Cleaning with 1% H3PO4
1 号柱用 1% H3PO4 清洗
? The primary analyte was sensitive to mobile phase
aging of the column,最初的分析对流动相老化柱子是灵敏的
? The peak shape was a secondary issue resolved by
flushing the column,冲洗柱子第二要解决的问题是峰形
? Retention and peak shape were as expected after
cleaning,清洗后保留时间和峰形都达到了预期效果
2010-5-20 31
Determining the Cause of Retention Changes on the
same Column
同一柱上保留时间改变原因的确定
? 1,Determine K′,?,and tr for suspect peaks 对
怀疑峰 K′,?和 tr的确定
? 2,Wash column 冲洗柱子
? 3,Test new column - note lot number
试验新柱 - 记录一批数据
? 4,Review column equilibration procedures
观察柱平衡过程
? 5,Make up fresh mobile phase4 and test
配制新鲜流动相再试验
? 6,Check instrument performance 检查仪器性能
2010-5-20 32
Change in Retention/Selectivity Column-to Column
柱与柱之间保留时间 /选择性的改变
? Different column histories (aging)
不同的柱历史 (老化 )
? Insufficient/inconsistent equilibration
? 平衡不够 /不一致
? Poor column/mobile phase combination
柱 /流动相差
? Change in mobile phase流动相改变
? Change in flow rate 流速改变
? Other instrument issues其他仪器问题
? Slight changes in column bed volume (tr only)
柱内体积轻微改变
2010-5-20 33
Lot-to-Lot Selectivity Change (pH)
批与批之间选择性改变 (pH)
? pH 4.5 - Lot 1 pH 3.0 - Lot 1
? pH 4.5 - Lot 2 pH 3.0 - Lot 2
? pH 4.5 shows selectivity change from lot-to-lot
for basic compounds
pH 4.5 显示批与批之间对碱性化合物的选择性改变
? pH 3.0 shows no selectivity change from lot-to-
lot,indicating silanol sensitivity at pH 4.5 pH
3.0显示批与批之间对碱性化合物的无选择性改变,表明在
pH 4.5硅醇基的灵敏性
2010-5-20 34
Conclusions 结论
? HPLC column problem are evident as,
高效液相色谱柱问题明显的为,
? High pressure 高压
? Undesirable peak shape 不希望得到的峰形
? Changes in retention/selectivity 保留时间 /选择性的改变
? Often these problems are not associated with
the column and may be caused by instrument
and experimental condition issues.
? 经常遇到的这些问题不一定与柱有关,而与仪器和
实验条件方面的问题有关
HPLC Column and System Troubleshooting
色谱柱和色谱系统的故障检修
? What Every HPLC
? User Should Know
?每个 HPLC使用者应该知道什么
2010-5-20 2
HPLC Components
HPLC 的组成
? Pump 泵
? Injector/Autosampler 进样器 /自动进样器
? Column 色谱柱
? Detector 检测器
? Data System/Integrator 数据系统 /积分仪表
? All of these components can have problems
and require troubleshooting.
? 所有这些部件可能出现故障而需要检修。
2010-5-20 3
Categories of Column Problems 色谱
柱问题的类型
?
? A,Pressure 压力
? B,Peak shape 峰形
? C,Retention 保留时间
2010-5-20 4
Pressure Issues
压力问题
? Column Observations Potential Problems
? 色谱柱观测 潜在的问题
? High pressure Plugged frit
? 压力高 堵塞滤头
? Column contamination
? 色谱柱污染
? Piugged packing
? 堵塞填料
2010-5-20 5
Determining the Cause and Correcting
High Back Pressure 查出原因 纠正压力
? Check pressure with/without column - many pressure problems
are due to blockages in the system or guard
? 有／无柱子时检查压力 － 许多压力问题是由于系统或保护柱的阻塞
? If Column Pressure is high 如果柱压高,
? Wash column - Eliminate column contamination and
? 冲洗柱子 plugged packing 排除柱污染和填料堵塞
? - high molecular weight/adsorbed
? compounds 高分子量／被吸附化合物
? - precipitate from sample or buffer
? 样品或缓冲液中的沉淀物
? Back flush column - Clear plugged frit 反冲柱子－清洁阻塞的滤头
? Change frit - Clear plugged frit 更换滤头
2010-5-20 6
Column Cleaning 柱子清洗
? F lush with stronger solvents than your mobile phase
? 用比你的流动相更强的溶剂冲洗
? Reversed-Phase Solvent Choices in Order of
? Increasing Strength 为了增加强度选择反相溶剂
? Use at least 25 ml of each solvent for analytical columns
? 对于分析柱每种溶剂至少用 25 ml 冲洗
? Mobile phase without buffer salts 没有缓冲盐的流动相
? 100% Methanol 甲醇
? 100% Acetonitrile 乙腈
? 75% Acetonitrile,25% Isopropanol 75乙腈, 25％异丙醇
? 100% Isopropanol 异丙醇
? 100% Methylene Chloride★ 二氯甲烷
? 100% Hexane ★ 已烷
? ★ When using either Hexane or Methylene Chloride the column must be flushed with
? Isopropanol before returning to your resvered-phase mobile phase.
? 当用已烷 蔌 二氯甲烷柱子时,必须先用 异丙醇 冲洗,然后再用你的反相流动相
2010-5-20 7
Column Cleaning柱子清洗
? Normal Phase Solvent Choices in
Order of Increasing Strength
? 为了增加强度选用正相溶剂
? Use at least 50 ml of each solvent
? 每种溶剂至少用 50 ml
? 50% Methanol, 50% Chloroform
? 50% 甲醇, 50% 氯仿
? 100% Ethyl Acetate 乙酸乙酯
2010-5-20 8
How to Change a Frit
怎样更换滤头
? Column Inlet Frit 柱进口滤头
? Column Body 柱身
? Compression Ferrule 压缩金属套圈
? Wear gloves 戴手套
? Do not allow bed to dry 不要让底座干燥
? Do not touch the column-body heat will extrude packing
不要使柱身受热,否则填料会喷出
? Do not over tighten 不要旋得太紧
? Female End Fitting 旋好尾端螺母
? Male End Fitting 旋好尾端螺头
2010-5-20 9
Preventing Back Pressure Problems
压力问题的预防
? Use column protection 柱保护
? - Guard columns 保护柱 /预柱
? - In-line filters 在线过滤器
? Sample Preparation 样品预处理
? Appropriate column flushing
? 适当的柱冲洗
? Filter buffered mobile phase
? 过滤缓冲流动相
2010-5-20 10
Preventing Back Pressure Problems:In-Line Devices
压力问题的预防,在线装置
? Mobile Phase Pre-Column Injector Filter Guard
? From Pump 预柱 进样器 滤头 Column
? 从泵来的流动相 保护柱
? Analytical
? Column
? Filter and Guard Column Act on Sample 分析柱
? 滤头和保护柱作用于样品
? Pre-Column Acts on Mobile Phase
? 预柱作用于流动相 To Detector
? 到检测器
2010-5-20 11
Preventing Back Pressure Problems,Sample Preparation
压力问题的预防,样品制备
? Solvent/Chemical Environment
? 溶剂 /化学环境
? Particulate/Aggregate Remove
? 微粒 /凝聚物的除去
? Filter samples 过滤样品
? Centrifugation 离心分离
? Solid Phase Extraction(S.P.E.) 固相萃取
? Cartridges or Plates 薄膜或薄层板
? Disks or Membranes 圆盘或生物膜
2010-5-20 12
Peak Shape Issues
峰形问题
? Split peaks 裂峰
? Peak tailing 峰拖尾
? Broad peaks 宽峰
? Poor efficiency 低柱效
? Many peak shape issues also combinations-I.e,Broad
and tailing or tailing with increased retention许多峰形问题
是结合在一起的 -例如,
? 展宽和拖尾或拖尾和保留时间增加
2010-5-20 13
Split Peaks 裂峰
?Can be caused by,
?可能的原因,
? Column contamination 柱污染
? Partially plugged frit 部分阻塞滤片
? Column void 柱头塌陷
? Injection solvent effects 溶剂效应
2010-5-20 14
Split Peaks 裂峰
Column Contamination 柱污染
? Column,StableBond SB-C8,4.6 × 250mm,5μm
? Mobile Phase:60%25mM Na2HPO4,pH3.0, 40%MeOH
? Flow Rate,1.0 mL/min Temperature,35oC
? Detection,UV 254 nm
? Sample,Filtered OTC Cold Medication,
? 1,Pseudoephedrine 2,APAP 3,Unknown
? 4,Chlorpheniramine
? Injection 1 峰 3 峰形较好
? Injection 30 峰 3 裂峰
? Injection 1 After Column Wash with 100% CAN
? 经 100%乙腈冲洗后 峰 3 峰形极好
2010-5-20 15
Split Peaks 裂峰
Injection Solvent Effects 溶剂化效应
? Column,StableBond SB-C8,4.6 × 250mm,5μm
? Mobile Phase,82%H2O, 18%ACN
? Injection Volume,30 μL
? Sample,
? 1,Caffeine 咖啡因 2,Salicylamide 水杨酰胺
? A,Injection Solvent B,Injection Solvent
? 100% Acetonitrile Mobile Phase
? 峰形宽拖尾 峰形尖锐对称
?样品溶剂尽可能与流动相匹配
2010-5-20 16
Determining the Cause of Split Peaks
裂峰原因的确定
? 1,Complex sample matrix or many samples analyzed- likely
column contamination or partially plugged frit
? 复杂样品的基质或分析许多样品 - 柱污染或部分阻塞滤片
? 2,Mobile phase pH>=7 - likely column void due to silica
dissolution (unless specialty column used)
? 流动相 pH>=7 - 由于硅胶溶解使柱塌陷 (除非使用专门的柱子 )
? 3,Injection solvent stronger than mobile phase - likely split
and broad peaks,dependent on volume
? 溶剂比流动相的可能性大 - 裂峰和宽峰,由样品量来决定
2010-5-20 17
Peak Tailing,Broadening and Loss of Efficiency
峰拖尾、变宽和柱效降低
? Can be caused by,可能的原因
?
? Column,secondary interactions”
? 柱“次级效应”
? Column void 柱塌陷
? Column contamination 柱污染
? Column aging 柱老化
? Column loading 柱负荷超载
? Extra-column effects 柱外效应
2010-5-20 18
Peak Tailing Column,Secondary Interactions”
峰拖尾柱“次级效应”
? Column,Alkyl-C8,4.6 × 150mm,5μm
? Mobile Phase:85%25mM Na2HPO4,pH7.0, 15%ACN
? Flow Rate,1.0 mL/min Temperature,35oC
? Sample,1,Phenylpropanolamine 苯丙醇胺／去甲麻黄碱
? 2,Ephedrine 麻黄碱 3,Amphetamine 安非他明／苯丙胺
? 4,Methamphetamine 脱氧麻黄碱 5,Phenteramine 苯三胺
? No TEA 无三乙胺 10 mM TEA 10 mM 三乙胺
? USP TF(5%) 美国药典拖尾因子 USP TF(5%) 美国药典拖尾因子
? 1,1.29 1,1.19
? 2,1.91 2,1.18
? 3,1.63 3,1.20
? 4,2.35 4,1.26
? 5,1.57 5,1.14
? Peak tailing eliminated with mobile phase modifier (TEA) at pH 7
? 用流动相减尾剂 (三乙胺 )在 pH 7 消除峰拖尾
2010-5-20 19
Peak Tailing Column,Secondary Interactions”
峰拖尾柱“次级效应”
? Column,Alkyl-C8,4.6 × 150mm,5μm
? Mobile Phase:85%25mM Na2HPO4,pH7.0, 15%ACN
? Flow Rate,1.0 mL/min Temperature,35oC
? Sample,1,Phenylpropanolamine 苯丙醇胺／去甲麻黄碱
? 2,Ephedrine 麻黄碱 3,Amphetamine 安非他明／苯丙胺
? 4,Methamphetamine 脱氧麻黄碱 5,Phenteramine 苯三胺
? pH 3.0 pH 7.0
? USP TF(5%) 美国药典拖尾因子 USP TF(5%) 美国药典拖尾因子
? 4,1.33 4,2.35
? Reducing the mobile phase pH reduces interactions with silanols that
cause peak tailing
? 降低流动相的 pH,减小与硅醇基作用引起的峰拖尾
2010-5-20 20
Peak Tailing 峰拖尾
Column Contamination 柱污染
? Column,StableBond SB-C8,4.6 × 250mm,5μm
? Mobile Phase,20% H2O, 80% MeOH Flow Rate,1.0 mL/min
Temperature,R.T,Detection,UV 254 nm
? Sample,1,Uracil 尿嘧啶 2,Phenol 苯酚
? 3,4-Chloronitrobenzene 4-氯硝基苯 4,Toluene 甲苯
? Plates TF Plates TF Plates TF
? 理论板数 拖尾因子 理论板数 拖尾因子 理论板数 拖尾因子
? 1,7629 2.08 1,7906 1.43 1,7448 1.06
? 2,12043 1.64 2,12443 1.21 2,12237 1.21
? 3,13727 1.69 3,17999 1.19 3,15366 1.11
? 4,13355 1.32 4,17098 1.25 4,19067 1.17
? QC test forward direction QC test reverse direction QC test after cleaning
? 100% IPA,35oC
? 向前方向 相反方向 100%异丙醇冲洗后
2010-5-20 21
Peak Tailing/Broadening
Sample Load Effects
峰拖尾 /变宽 样品过载效应
? Column,4.6 × 150mm,5μm
? Mobile Phase:40%25mM Na2HPO4,pH7.0, 60%ACN
? Flow Rate,1.5 mL/min Temperature,40oC
? Sample,
? 1,Desipramine 去郁敏 /脱甲基丙米嗪 2,Nortriptyline 3,Doxepin
4,Imipramine 丙米嗪 5,Amitriptyline 阿米替林 6,Trimipramine
? Tailing Eclipse XDB-C8 Broadening / Competitive C8
? USP TF 拖尾因子 Plates 理论板数
? High Load / 10 Low Load High Load / 10 Low Load
? 高载负 /10倍 低载负 高载负 /10倍 低载负
? 1,1.60 1.70 850 5941
? 2,2.00 1.90 815 7842
? 3,1.56 1.56 2776 6231
? 4,2.13 1.70 2539 8359
? 5,2.15 1.86 2735 10022
? 6,1.25 1.25 5189 10725
2010-5-20 22
Peak Broadening,Splitting Column Void
峰变宽,裂开 柱塌陷
? Mobile Phase:流动相：
? 50%CAN, 50%Water, 0.2%TEA (~pH 11)
? 50％ 乙腈, 50％水, 0。 2％三乙胺 (~pH 11)
? After 30 injections Initial 开始时
? 进样 30次后
? Multiple peak shape changes can be caused by the
same column problem,In the case a void resulted from
silica dissolved at high pH.
? 多数峰形改变可能是同一柱问题引起的。在这个例子中，
高 pH溶解硅胶导致塌陷
2010-5-20 23
Broad Peaks Unknown
“Phantom” Peaks
? Column,Extend-C18,4.6 × 150 mm,5 μm
? Mobile Phase,40% 10 mM TEA,pH 11, 60% MeOH
? Flow Rate,1.0 mL/min Temperature,R.T,Detection,UV 254
? Sample,1,Maleat 顺丁烯二酸 2,Pseudeophedrine 伪麻黄碱
3,Chlorphenniramine 氯非尼拉明
? Plates 理论板数
? 1,5922
? 2,9879
? 3,779, Phantom” peak from first injection 前次进样未出完的峰
? The extremely low plates are an indication of an extremely late
eluting peak from the preceding run.
? 极低的理论板数表明这是一个前面进样极迟洗脱出来的峰
2010-5-20 24
Peak Tailing峰拖尾
Injector Seal Failure进样器密封性差
? Column,Bonus-RP,4.6 × 75mm,3.5μm
? Mobile Phase,30% H2O, 70% MeOH Flow Rate,1.0 mL/min
Temperature,R.T,Detection,UV 254 nm
? Sample,1,Uracil 尿嘧啶 2,Phenol 苯酚
? 3,N,N-Dimethylaniline N,N-二甲苯胺
? Plates TF Plates TF
? 理论板数 拖尾因子 理论板数 拖尾因子
? 1,2235 1.72 1,3670 1.45
? 2,3491 1.48 2,10457 1.09
? 3,5432 1.15 3,10085 1.00
? Before After replacing rotor seal and isolation seal
? 之前 转动杆和隔离密封垫检修后
? Overdue instrument maintenance can cause peak shape problems.
? 仪器保养超期可能引起峰形问题 /进样器划痕
2010-5-20 25
Peak Tailing 峰拖尾
Extra-Column Volume 柱外死体积效应
? Column,StableBond SB-C18,4.6 × 30mm,3.5μm
? Mobile Phase,85% H2Owith 0.1% TFA, 15% CAN
? Flow Rate,1.0 mL/min Temperature,35oC
? Sample,1,Phenylalanine 2,5-benzyl-3,6-dioxo-2-piperazine
? 3,Asp-phe 4,Aspartame
? 10 μl extra-column volume 50 μl extra-column volume
2010-5-20 26
Determining the Cause of Peak Tailing
峰拖尾原因的确定
? Evaluate mobile phase effects - alter mobile phase pH
and additives to eliminate secondary interactions
评价流动相效果 - 改变流动相 pH和添加剂消除次级效应 /流动相中组份
与柱子相互作用
? Evaluate column choice - try column with high purity
silica or different bonding technology
评价柱选择 - 试用高纯度硅胶柱或不同键合技术柱
? Reduce sample load 减小进样量
? Eliminate extra-column effects 消除柱外效应
? Flush column and check for aging/void
冲洗柱子检查柱子老化 /塌陷
2010-5-20 27
Retention Issues
保留时间问题
? Retention time changes(tr)
保留时间改变
? Capacity factor (retention) changes (k′)
容量因子改变
? Selectivity changes (?)
选择性改变
2010-5-20 28
Changes in Retention Same Column,Over Time
相同柱子上保留时间改变,超时
? May be cause by,
可能的原因,
? Column aging 柱老化
? Column contamination 柱污染
? Insufficient equilibration 不够平衡
? Poor column/mobile phase combination
柱 /流动相组成差
? Change in mobile phase 改变流动相
? Change in flow rate 改变流速
? other instrument issues 其他仪器问题
2010-5-20 29
Mobile phase Change Causes Change in Retention
流动相改变引起保留时间改变
? 60%MeOH:40% 0.1% TFA Fresh TFA Added to Mobile Phase
? Volatile TFA evaporated/degassed from mobile phase,
易挥发的三氟乙酸从流动相中蒸发 /挥发。
? Replacing it solved problem,
换用新的流动相就解决问题。
?使用有机挥发性酸时，流动相应新配。
2010-5-20 30
Column Aging/Equilibration Causes
Retention/Selectivity Changes
柱老化／平衡不够引起保留时间／选择性改变
? Column 1 - Initial 1 号柱 开始时
? Column 1 - Next Day 1 号柱 第二天
? Column 1 - After Cleaning with 1% H3PO4
1 号柱用 1% H3PO4 清洗
? The primary analyte was sensitive to mobile phase
aging of the column,最初的分析对流动相老化柱子是灵敏的
? The peak shape was a secondary issue resolved by
flushing the column,冲洗柱子第二要解决的问题是峰形
? Retention and peak shape were as expected after
cleaning,清洗后保留时间和峰形都达到了预期效果
2010-5-20 31
Determining the Cause of Retention Changes on the
same Column
同一柱上保留时间改变原因的确定
? 1,Determine K′,?,and tr for suspect peaks 对
怀疑峰 K′,?和 tr的确定
? 2,Wash column 冲洗柱子
? 3,Test new column - note lot number
试验新柱 - 记录一批数据
? 4,Review column equilibration procedures
观察柱平衡过程
? 5,Make up fresh mobile phase4 and test
配制新鲜流动相再试验
? 6,Check instrument performance 检查仪器性能
2010-5-20 32
Change in Retention/Selectivity Column-to Column
柱与柱之间保留时间 /选择性的改变
? Different column histories (aging)
不同的柱历史 (老化 )
? Insufficient/inconsistent equilibration
? 平衡不够 /不一致
? Poor column/mobile phase combination
柱 /流动相差
? Change in mobile phase流动相改变
? Change in flow rate 流速改变
? Other instrument issues其他仪器问题
? Slight changes in column bed volume (tr only)
柱内体积轻微改变
2010-5-20 33
Lot-to-Lot Selectivity Change (pH)
批与批之间选择性改变 (pH)
? pH 4.5 - Lot 1 pH 3.0 - Lot 1
? pH 4.5 - Lot 2 pH 3.0 - Lot 2
? pH 4.5 shows selectivity change from lot-to-lot
for basic compounds
pH 4.5 显示批与批之间对碱性化合物的选择性改变
? pH 3.0 shows no selectivity change from lot-to-
lot,indicating silanol sensitivity at pH 4.5 pH
3.0显示批与批之间对碱性化合物的无选择性改变,表明在
pH 4.5硅醇基的灵敏性
2010-5-20 34
Conclusions 结论
? HPLC column problem are evident as,
高效液相色谱柱问题明显的为,
? High pressure 高压
? Undesirable peak shape 不希望得到的峰形
? Changes in retention/selectivity 保留时间 /选择性的改变
? Often these problems are not associated with
the column and may be caused by instrument
and experimental condition issues.
? 经常遇到的这些问题不一定与柱有关,而与仪器和
实验条件方面的问题有关
