Typical set up

for manual
column
chromatography.
A chemist in the 1950s using
column chromatography. The
Erlenmeyer receptacles are on the
floor.
From Wikipedia, the free encyclopedia
(Redirected from Flash chromatography)
Column chromatography in chemistry is a method
used to purify individual chemical compounds from
mixtures of compounds. It is often used for
preparative applications on scales from micrograms
up to kilograms.
The classical preparative chromatography column, is
a glass tube with a diameter from 50 mm and a
height of 50 cm to 1 m with a tap at the bottom. Two
methods are generally used to prepare a column; the
dry method, and the wet method. For the dry
method, the column is first filled with dry stationary
phase powder, followed by the addition of mobile
phase, which is flushed through the column until it is
completely wet, and from this point is never allowed
to run dry. For the wet method, a slurry is prepared
of the eluent with the stationary phase powder and
then carefully poured into the column. Care must be
taken to avoid air bubbles. A solution of the organic
material is pipetted on top of the stationary phase. This layer is usually topped with a small
layer of sand or with cotton or glass wool to protect the shape of the organic layer from the
velocity of newly added eluent. Eluent is slowly passed through the column to advance the
organic material. Often a spherical eluent reservoir or an eluent-filled and stoppered
separating funnel is put on top of the column.
The individual components are retained by the stationary phase differently and separate
from each other while they are running at different speeds through the column with the
eluent. At the end of the column they elute one at a time. During the entire chromatography
process the eluent is collected in a series of fractions. The composition of the eluent flow
can be monitored and each fraction is analyzed for dissolved compounds, e.g. by analytical
chromatography, UV absorption, or fluorescence. Colored compounds (or fluorescent
compounds with the aid of an UV lamp) can be seen through the glass wall as moving
bands.
1 Overview
2 Stationary phase (adsorbent)
3 Mobile phase (eluent)
4 Automated Systems
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Column chromatography proceeds by a series of steps.
5 Column Chromatogram Resolution Calculation
6 Column Adsorption Equilibrium
7 See also
8 References
9 External links
The stationary phase or adsorbent in column chromatography is a solid. The most common stationary phase for
column chromatography is silica gel, followed by alumina. Cellulose powder has often been used in the past.
Also possible are ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography
or expanded bed adsorption (EBA). The stationary phases are usually finely ground powders or gels and/or are
microporous for an increased surface, though in EBA a fluidized bed is used.
The mobile phase or eluent is either a pure solvent or a mixture of different solvents. It is chosen so that the
retention factor value of the compound of interest is roughly around 0.2 - 0.3 in order to minimize the time and
the amount of eluent to run the chromatography. The eluent has also been chosen so that the different
compounds can be separated effectively. The eluent is optimized in small scale pretests, often using thin layer
chromatography (TLC) with the same stationary phase.
A faster flow rate of the eluent minimizes the time required to run a column and thereby minimizes diffusion,
resulting in a better separation, see Van Deemter's equation. A simple laboratory column runs by gravity flow.
The flow rate of such a column can be increased by extending the fresh eluent filled column above the top of
the stationary phase or decreased by the tap controls. Better flow rates can be achieved by using a pump or by
using compressed gas (e.g. air, nitrogen, or argon) to push the solvent through the column (flash column
chromatography).
[1][2]
The particle size of the stationary phase is generally finer in flash column chromatography than in gravity
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An automated ion chromatography
system.
column chromatography. For example, one of the most widely used silica gel grades in the former technique is
mesh 230 400 (40 63 m), while the latter technique typically requires mesh 70 230 (63 200 m) silica
gel.
[3]
A spreadsheet that assists in the successful development of flash columns has been developed. The spreadsheet
estimates the retention volume and band volume of analytes, the fraction numbers expected to contain each
analyte, and the resolution between adjacent peaks. This information allows users to select optimal parameters
for preparative-scale separations before the flash column itself is attempted.
[4]
Column chromatography is an extremely time consuming stage in any
lab and can quickly become the bottleneck for any process lab.
Therefore, several manufacturers have developed automated flash
chromatography systems (typically referred to as LPLC, low pressure
liquid chromatography, around 50-75 psi) that minimize human
involvement in the purification process. Automated systems will include
components normally found on more expensive HPLC systems such as a
gradient pump, sample injection ports, a UV detector and a fraction
collector to collect the eluent. Typically these automated systems can
separate samples from a few milligrams up to an industrial kg scale and
offer a much cheaper and quicker solution to doing multiple injections on
prep-HPLC systems.
The resolution (or the ability to separate a mixture) on an LPLC system will always be lower compared to
HPLC, as the packing material in an HPLC column can be much smaller, typically only 5 micrometre thus
increasing stationary phase surface area, increasing surface interactions and giving better separation. However,
the use of this small packing media causes the high back pressure and is why it is termed high pressure liquid
chromatography. The LPLC columns are typically packed with silica of around 50 micrometres, thus reducing
back pressure and resolution, but it also removes the need for expensive high pressure pumps. Manufacturers
are now starting to move into higher pressure flash chromatography systems and have termed these as medium
pressure liquid chromatography (MPLC) systems which operate above 150 psi.
The software controlling an automated system will coordinate the components, allow a user to only collect the
factions that contain their target compound (assuming they are detectable on the system's detector) and help the
user to find the resulting purified material within the fraction collector. The software will also save the resulting
chromatograph from the process for archival and/or later recall purposes.
A representative example of column chromatography as part of an undergraduate laboratory exercise is the
separation of three components (out of 28) in the oil of spearmint: carvone, limonene and dehydrocarveol
[5]
. A
microscale setup consisting of a Pasteur pipette as column with silica gel stationary phase can suffice. The
starting eluent is hexane and solvent polarity is increased during the process by adding ethyl acetate.
Typically, column chromatography is set up with peristaltic pumps flowing buffers and the solution sample
through the top of the column. The solutions and buffers pass through the column where a fraction collector at
the end of the column setup collects the eluted samples. Prior to the fraction collection, the samples that are
eluted from the column pass through a detector such as a spectrophotometer or mass spectrometer so that the
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concentration of the separated samples in the sample solution mixture can be determined.
For example, if you were to separate two different proteins with different binding capacities to the column from
a solution sample, a good type of detector would be a spectrophotometer using a wavelength of 280 nm. The
higher the concentration of protein that passes through the eluted solution through the column, the higher the
absorbance of that wavelength.
Because the column chromatography has a constant flow of eluted solution passing through the detector at
varying concentrations, the detector must plot the concentration of the eluted sample over a course of time. This
plot of sample concentration versus time is called a chromatogram.
The ultimate goal of chromatography is to separate different components from a solution mixture. The resolution
expresses the extent of separation between the components from the mixture. The higher the resolution of the
chromatogram, the better the extent of separation of the samples the column gives. This data is a good way of
determining the columns separation properties of that particular sample. The resolution can be calculated from
the chromatogram.
The separate curves in the diagram represent different sample elution concentration profiles over time based on
their affinity to the column resin. To calculate resolution, the retention time and curve width are required.
Retention Time: The time from the start of signal detection by the detector to the peak height of the elution
concentration profile of each different sample.
Curve Width: The width of the concentration profile curve of the different samples in the chromatogram in
units of time.
A simplified method of calculating chromatogram resolution is to use the plate model
[6]
. The plate model
assumes that the column can be divided into a certain number of sections, or plates and the mass balance can be
calculated for each individual plate. This approach approximates a typical chromatogram curve as a Gaussian
distribution curve. By doing this, the curve width is estimated as 4 times the standard deviation of the curve, 4.
The retention time is the time from the start of signal detection to the time of the peak height of the Gaussian
curve.
From the variables in the figure above, the resolution, plate number, and plate height of the column plate model
can be calculated using the equations:
Resolution (R
s
):
R
s
= 2(t
RB
t
RA
)/(w
B
+ w
A
)
Where:
t
RB
= retention time of solute B
t
RA
= retention time of solute A
w
B
= Gaussian curve width of solute B
w
A
= Gaussian curve width of solute A
Plate Number (N):
N = (t
R2
)/(w/4)
2
Plate Height (H):
H = L/N
Where L is the length of the column.
[6]
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For an adsorption column, the column resin (the stationary phase) is composed of microbeads. Even smaller
particles such as proteins, carbohydrates, metal ions, or other chemical compounds are conjugated onto the
microbeads. Each binding particle that is attached to the microbead can be assumed to bind in a 1:1 ratio with
the solute sample sent through the column that needs to be purified or separated.
Binding between the target molecule to be separated and the binding molecule on the column beads can be
modeled using a simple equilibrium reaction K
eq
= [CS]/([C][S]) where K
eq
is the equilibrium constant, [C] and
[S] are the concentrations of the target molecule and the binding molecule on the column resin, respectively.
[CS] is the concentration of the complex of the target molecule bound to the column resin.
[6]
Using this as a basis, three different isotherms can be used to describe the binding dynamics of a column
chromatography: linear, Langmuir, and Freundlich.
The linear isotherm occurs when the solute concentration needed to be purified is very small relative to the
binding molecule of the. Thus, the equilibrium can be defined as:
[CS] = K
eq
[C].
For industrial scale uses, the total binding molecules on the column resin beads must be factored in because
unoccupied sites must be taken into account. The Langmuir isotherm and Freundlich isotherm are useful in
describing this equilibrium. Langmuir Isotherm:
[CS] = (K
eq
S
tot
[C])/(1 + K
eq
[C]), where S
tot
is the total binding molecules on the beads.
Freundlich Isotherm:
[CS] = K
eq
[C]
1/n
The Freundlich isotherm is used when the column can bind to many different samples in the solution that needs
to be purified. Because the many different samples have different binding constants to the beads, there are many
different Keqs. Therefore, the Langmuir isotherm is not a good model for binding in this case.
[6]
Chromatography, an overview article covering all chromatographic techniques.
High performance liquid chromatography (HPLC) for column chromatography using high pressure.
Fast protein liquid chromatography (FPLC) for separation of proteins using column chromatography.
^ Still, W. C.; Kahn, M.; Mitra, A. J. Org. Chem. 1978, 43(14), 2923-2925. (doi:10.1021/jo00408a041
(http://dx.doi.org/10.1021%2Fjo00408a041) )
1.
^ Laurence M. Harwood, Christopher J. Moody. Experimental organic chemistry: Principles and Practice
(Illustrated edition ed.). pp. 180185. ISBN 978-0632020171.
2.
^ Normal phase column chromatography, Material Harvest (http://www.materialharvest.com/welcome
/silica_products/silica_gels_chromatography.html)
3.
^ Fair, J. D.; Kormos, C. M. J. Chromatogr. A 2008, 1211(1-2), 49-54. (doi:10.1016/j.chroma.2008.09.085
(http://dx.doi.org/10.1016%2Fj.chroma.2008.09.085) )
4.
^ Isolation of Three Components from Spearmint Oil: An Exercise in Column and Thin-Layer Chromatography 5.
Column chromatography - Wikipedia, the free encyclopedia http://en.wikipedia.org/wiki/Flash_chromatography
5 of 6 4/21/2010 3:36 PM
Davies, Don R.; Johnson, Todd M. J. Chem. Educ. 2007 84Abstract (http://jchemed.chem.wisc.edu/Journal/Issues
/2007/Feb/abs318.html)
^
a

b

c

d
Harrison et al. Bioseparations Science and Engineering. Oxford University Press. New York, New York.
2003
6.
Column chromatography howto (http://www.chem.ubc.ca/courseware/121/tutorials/exp3A
/columnchrom/)
Flash Column Chromatography Guide (http://ocw.mit.edu/NR/rdonlyres/Chemistry/5-301January--
IAP-2004/768356CA-FD2C-4CB7-B87A-27D12504BBA8/0/8_9_flash_column.pdf) (pdf)
Retrieved from "http://en.wikipedia.org/wiki/Column_chromatography"
Categories: Chromatography
This page was last modified on 13 April 2010 at 06:57.
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apply. See Terms of Use for details.
Wikipedia is a registered trademark of the Wikimedia Foundation, Inc., a non-profit organization.
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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 205214.
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
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 funnelslowlyletting 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 rateno 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