7.9,Flash Column Chromatography Guide
Overview,
Flash column chromatography is a quick and (usually) easy way to separate complex
mixtures of compounds,We will be performing relatively large scale separations in 5.301,around
1.0 g of compound,Columns are often smaller in scale than this and some of you will experience
these once you move into the research lab,Column chromatography uses the same principles
discussed in the TLC Handout,but it can be used on a preparative scale,We will be running flash
columns since we will use compressed air to push the solvent through the column,This not only
helps provide better separation,but it also cuts down on the amount of time required to run a
column,
Reading,
For an excellent description,see LLP pages 205–214,
Preparing and Running a Flash Column,
1) Determine the dry,solvent-free weight of the mixture that you want to separate,
2) Determine the solvent system for the column by using TLC,You should aim for R
f
values between 0.2 and 0.3,If your mixture is complicated,this may not be possible,In
complex cases,you will probably have to resort to gradient elution,This simply means that
you increase the polarity of the solvent running through the column (eluent) throughout the
course of the purification,This technique will be described in more detail later in the
handout,but for the TLC analysis you should determine which solvent systems put each of
the different spots in the 0.2 to 0.3 R
f
range,
3) Determine the method of application to the column,You have three choices,neat,in
solution,or deposited on silica,
Neat,If your compound is a non-viscous oil,it is sometimes easiest to apply it
neat,You can use a long Pasteur pipet to apply the liquid to the column and then rinse any
traces of it into the column using the predetermined solvent system,
In Solution,Neat application can sometimes lead to column cracking,so a more
common method for liquids,as well as solids,is to dissolve the sample in a solvent and
apply it to the column in solution,In the best case,all of the components of your mixture
should have an R
f
of zero in this solvent - usually pentane or hexane,In many situations
63
WARNING NOTICE,The experiments described in these materials are potentially hazardous and require a high level
ofsafety training,special facilities and equipment,and supervision by appropriate individuals,You bear the sole
responsibility,liability,and risk for the implementation of such safety procedures and measures,MIT shall have no
responsibility,liability,or risk for the content or implementation of any of the material presented,Legal Notices
this is not possible,so a solvent that moves only one compound in the mixture can be used,
or you can simply use the chosen eluent,Keep in mind that these last two options are risky
for difficult purifications,
Adsorption onto Silica,The final technique that is useful for some liquids and all
solids is to deposit (adsorb) the compound on silica,Caution,silica gel is acidic,and this
procedure can destroy acid-sensitive compounds that normally survive on silica gel
columns,First,using a round-bottomed flask,dissolve the mixture in dichloromethane and
add silica gel (double the weight of your compound to determine the weight of silica),
Concentrate the solution on the rotary evaporator,Caution,Silica gel is a very fine powder
and can easily get sucked into the rotavap,Plug the opening of the adapter or bump guard
with glass wool to prevent "bumping" of the solid,Fast rotation also helps prevent this
problem,Once the solid is mostly dry (you will know it's dry when most of the solid has
fallen off the sides of the flask),remove the flask from the rotavap and finish removing the
solvent using the vacuum pump - assuming nothing in your mixture is volatile,Caution,
Use a glass wool plug in the vacuum adapter,or you will find silica gel (and your
compound) throughout the vacuum tubing and manifold,Once it is totally dry (no more
bubbling from the solid),remove the flask from the vacuum line and scrape the sides clean
with a spatula,The solid can now be added to the top of the column by simply using a
powder funnel followed by a few 1.5-mL rinses with the eluent,
4) Determine the appropriate silica gel to compound ratio,Easy separations require ratios
between 30-50:1 (by weight),while harder separations call for ratios of up to 120:1,The
reading in LLP and discussions with more experienced colleagues can help you make this
tough decision,
5) Pick the appropriate column,The amount of silica gel you are going to use determines
the size of your column,There is an ongoing debate about whether to use silica columns
that are short and wide or ones that are tall and skinny,In 5.301 we will argue that the
short,wide columns provide better separation,but this statement may be challenged by some
of your future co-workers,When you are first starting,the best way to select the correct
column for a given amount of silica gel is to ask other members of your lab which column
they would use and record this in your notebook,(This is much easier than measuring
column diameters.) In 5.301 we will only have one size to choose from,so the choice will
be fairly straightforward!
64
6) Pick appropriate collection test tubes,This is also a good time to consult your more
experienced colleagues,but a simple guideline is to divide the volume of silica that you used
by four and pick test tubes that can accommodate this volume,(200 mL of silica = 50 mL
fractions)
7) Once you have selected a column,you need to plug the stopcock end so that the silica
will not drain out,This is normally done with a small piece of cotton or glass wool and a
long stick or glass rod,
8) Mount the column in the hood - due to the large volumes of volatile solvents used and
the health risks associated with dry silica gel,you should never run a column outside of the
hood,Check to see that the column is perfectly vertical - crooked columns make separation
more difficult,
9) Close the stopcock and add a few inches of eluent,
10) Add sand (dried and washed) to the column using a funnel,Your goal is to produce a
thin layer of sand (no more than 1 cm) above the plug that will help prevent the silica from
ending up in the collection flasks,
11) Measure out the correct volume of silica,The safest way to do this is by volume in the
hood,Silica gel has a density equal to 0.5 g/mL so you can use an Erlenmeyer flask to
measure it out (100 g = 200 mL),Don't fill the Erlenmeyer more than one third full of
silica since you will be adding solvent to the flask as well,
12) Make a slurry of the silica by adding at least 1.5 times the volume of solvent as silica
you just measured out,Mix it thoroughly by swirling or stirring vigorously to remove all
the air from the silica,(Air bubbles will compromise the effectiveness of the column.)
13) Using a powder funnel,carefully and slowly pour the slurry into the column making
sure not to disturb the layer of sand,Stop pouring frequently to swirl the slurry so that the
silica is evenly mixed,Once you've finished pouring,rinse the Erlenmeyer several times
with the eluent and add the remaining solvent/silica mixture to the column,
14) Using a pipet and eluent,rinse any silica stuck to the sides of the top of the column into
the solvent layer,
65
15) Once all of the silica is off the sides of the column,open the stopcock and use the
compressed air to pack the column,The silica level in the column will shrink to about half
of its original height,Check to make sure that the top of the column is flat,If not,it must
be stirred up and allowed to settle undisturbed,As the excess solvent elutes under the
applied pressure,tap the sides of the column gently with a rubber stopper or the end of a
pencil,This will improve the packing of the silica particles,Collect all the solvent that
elutes from the column and recycle it for use after your compound has been added,
Caution,Never let the solvent level drop below the top of the column,
16) Once the column is packed,add a protective layer of sand to the top of the silica,This
should be relatively level and about 2 cm thick,This will protect the column when you are
adding solvent - if you add solvent too fast,it can destroy a flat column (thus hurting
separation) unless it is protected by sand,
17) Using the compressed air,lower the solvent level until it is even with the height of the
sand,
18) Close the stopcock and put the first test tube under the column outlet,
19) Carefully add your compound to the column - when adding liquids be sure to drip them
down the sides of the glass,not directly onto the top of the column,When rinsing the
flasks that contained the mixture,carefully add a one pipet-full of the rinse solution to the
column at a time,Then open the stopcock and drain the liquid level down to the top of the
column and close the stopcock,Rinse the flask three times using this procedure,For
mixtures that were deposited on silica gel,an additional 2 cm layer of sand is now added,
20) Carefully fill the column with eluent,At first,add the solvent by Pasteur pipet,Once 1
cm of solvent has been added,the stopcock can be opened for good,Keep adding the
solvent by pipet until a few centimeters of solvent are above the column,Now add the
solvent from an Erlenmeyer through a powder funnel—slowly—letting it first run down the
side of the column,Be patient,you do not want to disturb the top of the column,
21) Once you have filled the column with eluent,you are ready to "run" the column,
Remember that a quick flow rate helps to give good separation,Adjust the air pressure to
66
give a swift flow rate—no fire-hoses,though! Keep the pressure on and change the test
tubes once they become filled,Remember to replenish the solvent in the column frequently,
22) Monitor the column's progress by TLC - this can get a little hectic,trying to run TLCs
and collect your fractions,so in the beginning you might want to decrease the air pressure
(or remove it entirely) when you are checking the progress of the column,
23) When running a gradient elution column,use the initial solvent system until the higher
R
f
compounds have come off the column,Once they are safely in collection flasks,
increase the polarity of the eluent,Caution,Increase the polarity gradually,Drastic
polarity changes can "crack" the silica gel - sending fissures through the column like in a
bad earthquake movie,This cannot help your separation! Instead,increase the polarity by
about 5% for every 100 mL (or more) until you reach the desired solvent system,Then,
stay with this eluent until your desired compound has eluted,At this point,you can either
change eluents again or proceed to the next step,
24) Once you have determined that all of the compounds you are interested in have eluted
from the column,you are ready to wrap everything up,First,put a large Erlenmeyer flask
underneath the column,and use a green Keck clip to attached the compressed air source to
the column,Allow the air to push all of the remaining solvent out of the column and then to
dry the silica gel,(It's difficult to remove the silica from the column until it is completely
dry.) This will take at least one hour for large columns,
25) While the column is drying,start to combine fractions,Using TLC,determine which
test tubes contain your pure compound(s),Combine fractions of similar purity in large
round-bottomed flasks and concentrate them on the rotavap,For longer duration columns,
combine fractions while the column is still going to expedite the process,
26) Once the solvent is completely removed,analyze the compounds by NMR,
67
Overview,
Flash column chromatography is a quick and (usually) easy way to separate complex
mixtures of compounds,We will be performing relatively large scale separations in 5.301,around
1.0 g of compound,Columns are often smaller in scale than this and some of you will experience
these once you move into the research lab,Column chromatography uses the same principles
discussed in the TLC Handout,but it can be used on a preparative scale,We will be running flash
columns since we will use compressed air to push the solvent through the column,This not only
helps provide better separation,but it also cuts down on the amount of time required to run a
column,
Reading,
For an excellent description,see LLP pages 205–214,
Preparing and Running a Flash Column,
1) Determine the dry,solvent-free weight of the mixture that you want to separate,
2) Determine the solvent system for the column by using TLC,You should aim for R
f
values between 0.2 and 0.3,If your mixture is complicated,this may not be possible,In
complex cases,you will probably have to resort to gradient elution,This simply means that
you increase the polarity of the solvent running through the column (eluent) throughout the
course of the purification,This technique will be described in more detail later in the
handout,but for the TLC analysis you should determine which solvent systems put each of
the different spots in the 0.2 to 0.3 R
f
range,
3) Determine the method of application to the column,You have three choices,neat,in
solution,or deposited on silica,
Neat,If your compound is a non-viscous oil,it is sometimes easiest to apply it
neat,You can use a long Pasteur pipet to apply the liquid to the column and then rinse any
traces of it into the column using the predetermined solvent system,
In Solution,Neat application can sometimes lead to column cracking,so a more
common method for liquids,as well as solids,is to dissolve the sample in a solvent and
apply it to the column in solution,In the best case,all of the components of your mixture
should have an R
f
of zero in this solvent - usually pentane or hexane,In many situations
63
WARNING NOTICE,The experiments described in these materials are potentially hazardous and require a high level
ofsafety training,special facilities and equipment,and supervision by appropriate individuals,You bear the sole
responsibility,liability,and risk for the implementation of such safety procedures and measures,MIT shall have no
responsibility,liability,or risk for the content or implementation of any of the material presented,Legal Notices
this is not possible,so a solvent that moves only one compound in the mixture can be used,
or you can simply use the chosen eluent,Keep in mind that these last two options are risky
for difficult purifications,
Adsorption onto Silica,The final technique that is useful for some liquids and all
solids is to deposit (adsorb) the compound on silica,Caution,silica gel is acidic,and this
procedure can destroy acid-sensitive compounds that normally survive on silica gel
columns,First,using a round-bottomed flask,dissolve the mixture in dichloromethane and
add silica gel (double the weight of your compound to determine the weight of silica),
Concentrate the solution on the rotary evaporator,Caution,Silica gel is a very fine powder
and can easily get sucked into the rotavap,Plug the opening of the adapter or bump guard
with glass wool to prevent "bumping" of the solid,Fast rotation also helps prevent this
problem,Once the solid is mostly dry (you will know it's dry when most of the solid has
fallen off the sides of the flask),remove the flask from the rotavap and finish removing the
solvent using the vacuum pump - assuming nothing in your mixture is volatile,Caution,
Use a glass wool plug in the vacuum adapter,or you will find silica gel (and your
compound) throughout the vacuum tubing and manifold,Once it is totally dry (no more
bubbling from the solid),remove the flask from the vacuum line and scrape the sides clean
with a spatula,The solid can now be added to the top of the column by simply using a
powder funnel followed by a few 1.5-mL rinses with the eluent,
4) Determine the appropriate silica gel to compound ratio,Easy separations require ratios
between 30-50:1 (by weight),while harder separations call for ratios of up to 120:1,The
reading in LLP and discussions with more experienced colleagues can help you make this
tough decision,
5) Pick the appropriate column,The amount of silica gel you are going to use determines
the size of your column,There is an ongoing debate about whether to use silica columns
that are short and wide or ones that are tall and skinny,In 5.301 we will argue that the
short,wide columns provide better separation,but this statement may be challenged by some
of your future co-workers,When you are first starting,the best way to select the correct
column for a given amount of silica gel is to ask other members of your lab which column
they would use and record this in your notebook,(This is much easier than measuring
column diameters.) In 5.301 we will only have one size to choose from,so the choice will
be fairly straightforward!
64
6) Pick appropriate collection test tubes,This is also a good time to consult your more
experienced colleagues,but a simple guideline is to divide the volume of silica that you used
by four and pick test tubes that can accommodate this volume,(200 mL of silica = 50 mL
fractions)
7) Once you have selected a column,you need to plug the stopcock end so that the silica
will not drain out,This is normally done with a small piece of cotton or glass wool and a
long stick or glass rod,
8) Mount the column in the hood - due to the large volumes of volatile solvents used and
the health risks associated with dry silica gel,you should never run a column outside of the
hood,Check to see that the column is perfectly vertical - crooked columns make separation
more difficult,
9) Close the stopcock and add a few inches of eluent,
10) Add sand (dried and washed) to the column using a funnel,Your goal is to produce a
thin layer of sand (no more than 1 cm) above the plug that will help prevent the silica from
ending up in the collection flasks,
11) Measure out the correct volume of silica,The safest way to do this is by volume in the
hood,Silica gel has a density equal to 0.5 g/mL so you can use an Erlenmeyer flask to
measure it out (100 g = 200 mL),Don't fill the Erlenmeyer more than one third full of
silica since you will be adding solvent to the flask as well,
12) Make a slurry of the silica by adding at least 1.5 times the volume of solvent as silica
you just measured out,Mix it thoroughly by swirling or stirring vigorously to remove all
the air from the silica,(Air bubbles will compromise the effectiveness of the column.)
13) Using a powder funnel,carefully and slowly pour the slurry into the column making
sure not to disturb the layer of sand,Stop pouring frequently to swirl the slurry so that the
silica is evenly mixed,Once you've finished pouring,rinse the Erlenmeyer several times
with the eluent and add the remaining solvent/silica mixture to the column,
14) Using a pipet and eluent,rinse any silica stuck to the sides of the top of the column into
the solvent layer,
65
15) Once all of the silica is off the sides of the column,open the stopcock and use the
compressed air to pack the column,The silica level in the column will shrink to about half
of its original height,Check to make sure that the top of the column is flat,If not,it must
be stirred up and allowed to settle undisturbed,As the excess solvent elutes under the
applied pressure,tap the sides of the column gently with a rubber stopper or the end of a
pencil,This will improve the packing of the silica particles,Collect all the solvent that
elutes from the column and recycle it for use after your compound has been added,
Caution,Never let the solvent level drop below the top of the column,
16) Once the column is packed,add a protective layer of sand to the top of the silica,This
should be relatively level and about 2 cm thick,This will protect the column when you are
adding solvent - if you add solvent too fast,it can destroy a flat column (thus hurting
separation) unless it is protected by sand,
17) Using the compressed air,lower the solvent level until it is even with the height of the
sand,
18) Close the stopcock and put the first test tube under the column outlet,
19) Carefully add your compound to the column - when adding liquids be sure to drip them
down the sides of the glass,not directly onto the top of the column,When rinsing the
flasks that contained the mixture,carefully add a one pipet-full of the rinse solution to the
column at a time,Then open the stopcock and drain the liquid level down to the top of the
column and close the stopcock,Rinse the flask three times using this procedure,For
mixtures that were deposited on silica gel,an additional 2 cm layer of sand is now added,
20) Carefully fill the column with eluent,At first,add the solvent by Pasteur pipet,Once 1
cm of solvent has been added,the stopcock can be opened for good,Keep adding the
solvent by pipet until a few centimeters of solvent are above the column,Now add the
solvent from an Erlenmeyer through a powder funnel—slowly—letting it first run down the
side of the column,Be patient,you do not want to disturb the top of the column,
21) Once you have filled the column with eluent,you are ready to "run" the column,
Remember that a quick flow rate helps to give good separation,Adjust the air pressure to
66
give a swift flow rate—no fire-hoses,though! Keep the pressure on and change the test
tubes once they become filled,Remember to replenish the solvent in the column frequently,
22) Monitor the column's progress by TLC - this can get a little hectic,trying to run TLCs
and collect your fractions,so in the beginning you might want to decrease the air pressure
(or remove it entirely) when you are checking the progress of the column,
23) When running a gradient elution column,use the initial solvent system until the higher
R
f
compounds have come off the column,Once they are safely in collection flasks,
increase the polarity of the eluent,Caution,Increase the polarity gradually,Drastic
polarity changes can "crack" the silica gel - sending fissures through the column like in a
bad earthquake movie,This cannot help your separation! Instead,increase the polarity by
about 5% for every 100 mL (or more) until you reach the desired solvent system,Then,
stay with this eluent until your desired compound has eluted,At this point,you can either
change eluents again or proceed to the next step,
24) Once you have determined that all of the compounds you are interested in have eluted
from the column,you are ready to wrap everything up,First,put a large Erlenmeyer flask
underneath the column,and use a green Keck clip to attached the compressed air source to
the column,Allow the air to push all of the remaining solvent out of the column and then to
dry the silica gel,(It's difficult to remove the silica from the column until it is completely
dry.) This will take at least one hour for large columns,
25) While the column is drying,start to combine fractions,Using TLC,determine which
test tubes contain your pure compound(s),Combine fractions of similar purity in large
round-bottomed flasks and concentrate them on the rotavap,For longer duration columns,
combine fractions while the column is still going to expedite the process,
26) Once the solvent is completely removed,analyze the compounds by NMR,
67
