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Friday, December 30, 2011

Factors affecting efficiency of Column Chromatography

Factors affecting efficiency of Column Chromatography

Factors affecting efficiency of Column Chromatography

Efficiency of column chromatography or Resolution of column chromatography is dependent on various factors,  some of the few major factors includes

factors affecting chromatography

Column Chromatography 

Dimension of the column:
Columns of various dimensions are available for use, depending upon the process suitable column dimension need to be selected. Height or length / width ratio is important factor, increasing the length of the column shown to have improved separation / resolution. Mostly for desalting purposes long columns are shown to give satisfactory results. But optimization is needed for each process to show effective reproducible results.
Example of certain columns,XK columns are jacketed columns eg: XK 16/40,  XK 50/30, here 50 denotes the inner diameter of the column in millimeters (mm) and 30 is the length or height of the column in centimeters (cms).

Particle size of the matrix:
Particle size also plays important role in the improved separation, In gel filtration chromatography small medium and super fine range particles are available, the particle size and other details will be available on the specification sheet provided during the purchase of the chromatography matrix. Decreasing  the particle size shown to have improved separation.

Solvents used in the column chromatography should not affect the stability of the proteins or it should not cause any deleterious effects, solvents used should be compatible with the column and the matrix used. Generally a solvent with low viscosity is used since the viscosity can affect the flow rate and which in turn affects the separation.

Temperature is also an important factor in chromatography, since proteins can degrade at higher temperatures. Large scale protein purification processes are done mostly at 4oC.lab scale protein purification is usually done at room temperature. Some chromatographic columns can get damaged at higher temperatures of around 60oC. It is advisable to maintain lower temperatures for improving the yield and efficiency of column chromatography.

Flow Rate:
Flow rate can be maintained using a peristaltic pump or  AKTA systems ( Protein purification controller form GE Life Science). For each chromatographic media or matrix the maximum flow rate at which can be used during the process will be given in the specification sheet ( For flow rate conversions click here).
Running the column at Medium flow rate is recommended, because very slow flow rate can lead to zone spreadening and too fast flow rate can cause extensive tailing.

Column Packing (How to Pack a chromatography column) is yet another important factor in the resolution, proper packing of the column is very important to get better results. Few points to remember while column packing
  •  Degas the matrix and buffers, this will help to remove air bubble trapped inside, air bubbles in the column can lead to poor resolution.
  • Pour the matrix / media into the column in a single time attach reservoir to the top of the column.
  • Calculate the amount of matrix required for the column, if your swelling dry matrix calculate amount of dry powder required to get the matrix volume.(click here for matrix volume calculations).
  • Place the column properly, column need to be kept straight.
  • Check the filters (mesh) other tubing connections are proper before starting. Purging the lines (tubing and system lines if using AKTA) can remove air bubbles.
These are the major factor which can affect the resolution or separation in a column chromatography, equilibration, washing, suitable matrix selection etc also can influence the efficiency. process optimization need to be done to get better results. For long term storage, chromatographic matrix can be stored in 20% ethanol. while reusing  3-7 column volumes of water wash can be given to remove ethanol.

Analytical Biochemisrty, Dr. P Asokan
GE Lifescience Chromatography Literature
Tosoh Biosciences, Chromatography Literature
Personal Experience