Application Note F2.

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Quick Overview of Column Chromatography

SiliaFlash®
1-Methodology

Before performing a separation on a chromatographic column, it is highly recommended to make different

2-Theory of separation

For technique such as thin layer chromatography, it is not reliable to measure the retention of a compound
as a function of the time but rather as a function of the distance. For this matter, the “retention factor”, R f, is
defined by the ratio of the distance travelled by the analyte over the distance travelled by the solvent front.
The difference between two R f, namely ∆R f, is a measure of the distance between analytes.

R f = Distance travelled by the analyte

∆R f = R f1 – R f2

Typically, the analyte of interest must have an R f of 0.15-0.35 and a ∆R f of at least 0.15 with the nearest
compound, to obtain the best separation.

In flash chromatography separation is govern by column volumes (CV). This corresponds to the volume of
solvent necessary to fill all the void volume in a packed column, i.e., sorbent pores and interstitial spaces
between sorbent particles. The number of CV for a particular compound is the number of column volumes
necessary to elute this compound. The correlation between the Rf and the CV is defined by:

CV = 1/R f

The separation between two peaks, ∆CV is defined by:

∆CV = 1/R f1 – 1/R f2

Ideally, the analyte of interest should have a CV between 3-6 and a ∆CV greater than 1 with the nearest
compound.

3-Choice of solvent

Choice of the solvent, the mobile phase, is of crucial importance. The choice is guided by the polarity of the
solvent depending of whether normal or reversed phase is used (low or high polarity respectively). Here is a
list of different solvents and their relative strength:

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Solvent Solvent strength

SiliaFlash®
Methanol 0.95
Ethanol 0.88
2-propanol 0.82
acetonitrile 0.65
ethyl acetate 0.58
Tetrahydrofuran 0.57
Acetone 0.56
Dichloromethane 0.42
Chloroform 0.40
Diethyl ether 0.38
Toluene 0.29
Hexane 0.01

Combination of different solvents may be necessary to obtain a ∆R f greater than 0.2. Also, mixtures of sol-
vent of equivalent overall strength do not necessarily have the same selectivity. Acetonitrile does not have
the same selectivity as methanol or ethanol. A mixture of 1:1 hexane/Ethyl acetate and 1:2 Hexane/dichlo-
romethane may provide similar strength but may demonstrate different selectivity. Hence the trick is to try
different combinations of solvents and solvent ratios.

4-Loading the column

The best method to fill a column with a sorbent is by the slurry method. In this technique, a container is filled
with the least eluting solvent that is going to be used during the separation, non-polar or polar for normal
or reversed phase respectively. The sorbent is then added to the solvent (and not the reverse) in order to
make a slurry fluid enough so that it can be poured easily. The container is swirled to obtain an homogeneous
solution and is rapidly poured into the column through a funnel. Quantities of sorbent added in this fashion
should not exceed a layer of about 2 cm at the time. It is also important to tap gently the side of the column
to improve packing. Drain excess solvent and make sure that the column never runs dry.

5-Solvent equilibration

Before adding the sample, it is important to condition the column with the starting elution mixture, in order
to obtain optimum and reproducible results. It can be done by washing the column with a volume of solvent 2
times that of the column volume. See the following table for details.

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Approximate

SiliaFlash®
Column Size (g) Typical Solvent Volume (mL)
Column Volume (mL)
2 2.5 5
5 6.5 13
10 12.5 25
20 25 50
25 31.3 62.5
50 62.5 125
70 88 176
100 125 250
150 188 376

6-Loading of the sample

Two different ways may be adopted: wet loading and dry loading.

Wet loading: This technique requires dissolving the sample to be purified in a minimum amount of solvent
that has the least affinity with the sorbent. This means that non-polar solvent, such as hexane, should be
used to load the sample in normal phase chromatography and that polar solvent, such as methanol, should
be used to load the sample in reversed phase chromatography. The level of solvent in the column is brought
down just to that of the sorbent. The dissolved sample is then added directly to the top of the column, dis-
pensed evenly on the surface. Let the sample enter completely the column by lowering the level of liquid to
the line of the sorbent. Rinse the side of the column with the same solvent and lower the level again to make
sure that the entire sample is in contact with the sorbent.

Dry loading: This technique is recommended when the sample is not soluble enough in the prescribed sol-
vent. Small quantities of a better solvent can be added to ensure complete dissolution. The sample is pre-ad-
sorbed on a small quantity of sorbent, varying from a ratio sample/sorbent of 1:1 to 1:3 by volume. Solvent
can be evaporated off on a rotary evaporator and the sample added above the top frit of the column. Finally,
another frit may be placed atop. Press down this new surface to secure tight packing and prevent movement
of the bed.

7-Sample capacity

Typically, 5 to 10% of mass sample relative to the column bed mass may be used for purification. Different
factors such as analytes, concentration of reaction products, elution solvent used and sample matrix may
affect the capacity of the column.

[email protected], www.SiliCycle.com
Tel.: 1 418.874.0054, Toll-free: 1 877.SILICYCLE (North America only)