Written Report in Chemistry 52

NATURAL PRODUCTS CHEMISTRY, Lab

PREPARATIVE COLUMN CHROMATOGRAPHY

Submitted by:

VAL JASON G. LAGRADA

Submitted to:

MS. AILEEN MAY G. ANG

Column Chromatography is 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. Like thin layer chromatography, silica gel is used as a stationary phase and an

organic solvent, which is less polar than the silica gel, is used as the mobile phase.

Column chromatography is carried out in a glass tube clamped vertically with the initial

mixture placed at the top. Organic solvents run past the mixture on their way down the column.

Because of silica gel’s stronger affinity for the more polar components, the components with the

lower polarity will descend first through the column. During the chromatographic process, the

polarity of the mobile phase can be slowly increased by varying the solvent mixture used. As a

result, increasingly polar components will elute. Up to present, different types of columns are

used in the chromatographic process. These include the gravity column, flash column, vacuum

column, low pressure, medium pressure and high pressure columns. They are discussed further

below.

TYPES OF COLUMNS
A. Gravity Columns

The conventional gravity-driven, open-column chromatography method is still widely

used in both rapid filtration and true separation modes. In gravity column chromatography, the

mobile phase is allowed to move through the stationary phase with the help alone of the gravity

force.

Various stationary phases of different particle size (10–200 µm) and porosity (50 nm) are

available. Silica is the most widely used phase, but several bonded silica phases (cyano, amino,
hydroxyl, nitro) are also used, although they are more expensive. Styrene-divinylbenzene

polymers (XAD, HP, SP resins) can be used to advantage in desalting and extraction of organic

compounds from aqueous media. Alumina (Al2O3), initially much used in open-column

chromatography, should be used only with highly stable compounds because in both the acidic or

basic condition, it can become an excellent catalyst for undesired reactions.

Open-column chromatography is a slow method with the consequence that material is

lost by irreversible adsorption, particularly with silica and alumina, has low reproducibility (the

ease with which columns can be packed often leads to carelessness) and requires large amounts

of solvents, which, in step gradient solvent elution, are not easily recoverable.

B. Flash Columns

Flash Chromatography is a very convenient and simple technique that finds application in

both synthetic and natural products chemistry. Briefly, a column is preferably dry-filled with

adsorbent, the sample is introduced, and the solvent is forced through the column under pressure

from compressed air or nitrogen (1 bar above atmospheric pressure).

A solution of the sample is deposited on the column normally, and elution with different

solvents is achieved by pressure applied with a syringe containing the solvent. This method is

useful not only for sample preparation but also for separation and purification of compounds.

C. Vacuum Columns

Vacuum Liquid Chromatography rivals the foregoing Flash Column Chromatography

method for simplicity, but the flow of the solvent is maintained by vacuum. The column is

prepared in a sintered glass funnel using TLC grade packing (aluminium oxide, silica gel, or
reverse-phase supports). Uniform packing is achieved by initially tapping the funnel on the

bench and then by application of a vacuum from below the funnel. The sample is applied

uniformly at the top of the support. Step gradient elution is used and the column can be allowed

to run dry after collection of each fraction, approximating multiple-development PTLC. All the

usual stationary-phase adsorbents can be used, and the technique is applicable to large-scale

separations. Sample sizes from a few milligrams to 50 g can be accommodated by choosing the

appropriately sized funnel. The advantages over PTLC is that it reduced cost, is time saving, and

have a better resolution.

D. Preparative Pressure Liquid Chromatography

This term covers those techniques of column chromatography in which pressure is

applied by a pump operating above 2 bar pressure. Preparative, in this context, refers to amounts

ranging from micrograms to kilograms. The division between low (up to 5 bar), medium (5–20

bar), and high- pressure (>20 bar) liquid chromatography is not simply arbitrary but reflects the

use of different columns with different size packing material and size of sample that can be

fractionated.

i. Low-Pressure LC

This can be conducted with homemade glass or stainless steel columns. Ready-filled

glass columns (240 ×10 mm to 440 ×37 mm) packed with silica or RP-8 support (40–60 µm) are

commercially available. The system requires a pump capable of reaching 6 bar pressures and an

injection system. Smaller columns are suitable for sample loads up to 200 mg, whereas the larger

size will tolerate up to 3 gm. The selection of solvent can be extrapolated from TLC, and
normally a single solvent combination (isocratic) is used. Some representative examples have

been discussed. This technique is most useful for processing crude extracts into discreet smaller

fractions and, while it may not necessarily provide pure compounds, the individual fractions can

be submitted to chromatography with higher resolution potential.

ii. Medium-Pressure Columns

This mode uses larger columns and higher pressures delivered by a reciprocating pump. It

is a useful substitute for open column chromatography or FC in terms of sample load with the

advantage of higher resolution and shorter separation times. Compared to these other two

techniques, packings with smaller particle size (25–40 µm) are used. A flow rate of 100 mL/min

is usual and high loading capacity can be achieved.

ii. High-Pressure Columns

In this column, very fine silica gel is used so it has a greater separation power. However,

the flow rate of the mobile phase is severely decreased. High pressure pumps are used to push

the solvent through the column which in the case must be made of stainless steel.

In column, the preparation and application of the adsorbent used is crucial and is very

important. The adsorbent is applied to the Column in two ways, slurry packing or wet method

and dry method.

A. Slurry Packing (Wet method)

The adsorbent is suspended in the mobile phase and stirred very well to drive off all air

bubbles. The resulted slurry is then poured into the column. At the tap end of the column a
piece of glass wool or cotton must be added before the slurry application. Sand may be added

after the slurry. After slurry application the column must be allowed to settle overnight.

B. Dry Packing

In this method the dry adsorbent is poured to the column directly. Vibration is the applied

to get rid of air bubbles then the mobile phase as passed through the adsorbent. This method

cannot be applied gel Chromatography.

On the other hand, there are two types of the application of the sample. We have the wet

application and dry loading.

a. Wet application

The sample is dissolved in the initial mobile phase and apply by pipette to the top of the

column. This is very good method but in most of cases the samples are not soluble in the initial

mobile phase.

b. Dry loading

The sample is dissolved in any volatile solvent. The sample solution is then adsorbed on

small weight of adsorbent and the solvent is allowed to evaporate. The dry adsorbent loaded with

the sample is then applied to the column.

REFERENCES
COLEGATE, S. M. AND R. J. MOLYNEUX. 2008. Bioactive Natural Products: Detection,
Isolation, and Structural Determination. Taylor & Francis Group, LLC: NW.

CSEKE, L. J., KIRAKOSYAN, A., KAUFMAN, P. B., WARBER, S. L., DUKE, J. A. AND H.
L. BRIELMANN. 2006. Natural Products from Plants. Taylor & Francis Group,
LLC: NW.

MAYO, D. W., PIKE, R. M. AND P. K. TRUMPER. 2001. Microscale Techniques for the
Organic Laboratory: 2nd edition. John Wiley and Sons: New York, USA.