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Column Chromatography

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Column chromatography is one of the most widely researched and studied topics in chromatography. Chromatography falls into two major types namely 2-dimensional/planar chromatography and 3-dimensional/ column chromatography. There are two main sub-types of planar chromatography i.e., paper chromatography and thin-layer chromatography. But there are numerous types and multiple applications of column chromatography. We have already learned about the former in our previous articles, this article thus specifically introduces column chromatography to you.

What is column chromatography

Column chromatography is a type of chromatography in which the stationary phase is contained into a glass tube called a chromatographic column. The stationary phase could either be packed into the column or it could be coated along the inner walls of the column. Accordingly, the chromatographic column could either be a ‘packed column’’ and/or an open tubular/capillary column.

High-performance liquid chromatography (HPLC) and gas chromatography (GC) are two principal types of column chromatographies. HPLC along with other interesting column chromatographic techniques such as affinity chromatography, hydrophobic interaction chromatography, size/molecular exclusion chromatography and ion exchange chromatography all utilize a stationary phase packed column. While gas chromatography stands prominent in using an open tubular column.

Types of columns in chromatography

1. Stationary phase packed columns

If the column tube is filled with stationary phase as a packing material, then such a chromatographic column is known as a packed column. The stationary phase may constitute a support surface with different functional groups attached. In that case, the support must be thermally and mechanically stable as well as it should be non-reactive with low affinity towards the targeted analyte.   

Smaller the stationary phase particle size, more compactly packed the column hence offering a high surface area for analyte retention. The mobile phase sweeps pass the stationary phase by travelling through the interstitial spaces present in the packing material.

Pressure needs to be applied to support the mobile phase flow in case of extremely close packing such as that present in HPLC columns. The composition of the stationary phase packed column may vary as per requirement in different chromatographic techniques. It can act as a normal phase or a reverse phase for analyte retention.

The commonly used stationary phase material for column packing in each technique is as shown in the table below.

Type of Chromatography Stationary phase for column packing
High-performance liquid chromatography (HPLC)Silica, octylsilane, octadecylsilane, alumina, polymers
(polymethyl siloxane, squalene, diatomaceous earth) etc.
Affinity chromatographySilica as support with covalently bonded ligands having -CO, -SH, -NH2, -COOH groups exposed to the analyte as per requirement
Molecular/Size exclusion chromatographyDextran, agarose, polyacrylamide, polystyrene etc.
Ion exchange chromatographyResins, ion-exchange gels (made of cellulose, dextrose, polyacrylamide etc., with labile, charged functional groups as side chains) and inorganic exchangers
 
Hydrophobic interaction chromatography (HIC)
Hydrophobic stationary phase such as agarose gel with phenyl or alkyl groups attached

The modern scientific world makes use of commercially packed columns for chromatographic applications such as the stainless-steel columns available for HPLC (as shown below).

Stainless-steel HPLC Columns  
Image by YMC.EU

But in order to perform chromatography at a smaller scale/laboratory level, the following step-by-step guide can be used for column packing in a glass tube.

Step I:  Pre-requisites

The right stationary phase is selected. In case of silica, you must know the pore size and dimensions of silica gel/beads you are using for your column packing.

Step II: Pre-treatment

i) Silica Activation: The silica gel is activated by treatment with a concentrated acid such as a 30% solution of conc. hydrochloric acid (HCl) by soaking the gel in this acidic solution overnight. Treatment with acid, activates the hydroxyl functional groups (OH) present on the surface of silica.

ii) Agarose Gel: In case of another stationary phase material such as the agarose gel employed in size exclusion chromatography, the gel is soaked in its respective mobile phase such as a buffer solution. This soaking practice allows swelling of the gel, flexible bead formation occurs which prevents stationary phase cracking later on in the column.

Step III: Column Packing

A slurry of the stationary phase is then prepared in the prospective mobile phase. A  cotton plug is placed at the bottom of the column and the stationary phase slurry is consequently loaded from the top of the column. It is allowed to settle down the column, under gravitational action. Once settled, the solvent is allowed to drain off from the bottom end prior to loading the analyte mixture and reloading a layer of mobile phase from the top of the column for analytical separation.

Stationary phase packing, thickness of the stationary phase, column length and the internal diameter of the column, all these factors strongly influence the overall chromatographic separation. Usually, a narrow bore column with a long length offers better column chromatographic efficiency and separation.

An HPLC column can last for many years, if maintained in good health. If should be properly flushed with and stored in a 100% organic solvent such as acetonitrile after every use. A chromatographic column performance can be and should be monitored periodically by assessing critical parameters such as number of peaks obtained, peak symmetry and retention factor for a standard etc. Any abnormal changes in these parameters may indicate column degradation.

2. Open tubular/capillary columns

Instead of packing the stationary phase into the internal cavity, capillary columns have stationary phase coatings along the inner walls of the column. This type of columns is preferably used for performing gas chromatography. The capillary columns in GC are much longer (15-60) m as opposed to packed columns of HPLC (3-25) cm long. Henceforth, capillary columns of GC are situated into the system in a coiled form. These columns are installed in an oven to meet the high temperature requirements of a gas chromatographic procedure.

Coiled capillary columns for GC. Image by labcompare.com

Porous polymers such as polyimide, solid particles, organic and inorganic materials are among the most common stationary phase choices for capillary columns. A thin film of stationary phase coating is always preferred over a thicker coating. The different coating options can be read in further detail on our article what is stationary phase in chromatography.

Capillary columns are usually considered more reliable, offering better chromatographic analysis as opposed to packed columns. Some of the merits of capillary columns over packed columns are as follows.

Capillary columns offer:

  • Higher sensitivity
  • Shorter analysis time
  • Less chances of peak broadening by eddy diffusion thus greater resolution
  • Requires small sample size

Packed columns on the other hand offers a greater sample loading capacity but comes with longer analyte retention times, a high chance of peak broadening thus inferior chromatographic resolution.

Conclusion

Overall, it depends on the specific chromatographic applications that determines which type of column will be more valuable for you to perform column chromatography.

You may like the following video tutorials on column chromatography:-

  1. Column chromatography
  2. Learn about how a column is constructed

References

1. G. Guiochon and C. L. Guillemin (1988). Methodology Open Tubular Columns Chapter 8. Journal of Chromatography Library., Elsevier. 42: 247-317.

2. Cazes, J. (2009). Encyclopedia of Chromatography.

3. D.F., M. and M. B.B. (2016). “Column Chromatography “: 218.

Column Chromatography

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