Size Exclusion Chromatographic Columns

Size Exclusion Chromatographic Columns

Size Exclusion Chromatographic columns separates molecules based upon their size, not molecular weight. A common packing material for these columns is molecular sieves. Zeolites are a common molecular sieve that is used. The molecular sieves have pores that small molecules can go into, but large molecules cannot. This allows the larger molecules to pass through the column faster than the smaller ones. Other packing materials for size exclusion chromatographic columns are polysaccharides and other polymers, and silica. The pore size for size exclusion separations varies between 4 and 200 nm. 

Figure 3: Schematic of a size exclusion column. The larger particles will elute first because they are too big to fit inside the pores. The smallest particles will elute last because they fit very well inside the pores. This figure was created with Microsoft Paint.

Chiral Columns

Chiral columns are used to separate enantiomers. Separation of chiral molecules is based upon steriochemistry. These columns have a stationary phase that selectively interacts with one enantiomer over the other. These types of columns are very useful for separating racemic mixtures.

Some Stationary Phases Used to Separate Enantiomers

Stationary Phase	Method(s) Used
Metal Chelates	GC, LC
Amino Acid Derivatives	GC, LC
Proteins	LC
Helical Polymers	LC
Cyclodextrin Derivatives	GC, LC

Table 5: This table shows some stationary phases that are used to separate enantiomers and the corresponding chromatographic methods that they are applied to.

Column Efficiency

Peak or band broadening causes the column to be less efficient. The ideal situation would to have sharp peaks that are resolved. The longer a substance stays in the column it will cause the peaks to widen. Lengthening the column is a way to improve the separation of different species in the column. A column usually needs to remain at a constant temperature to remain efficient. Plate height and number of theoretical plates determines the efficiency of the column. Improving the efficiency would be to increase the number of plates and decrease the plate height.

The number of plates can be determined from the equation:

N=L/H

where L is the length of the column and H is the height of each plate. N can also be determined from the equation:

N=16(t_R/W)² or N=5.54(t_R/W_1/2)²

where t_R is the retention time, W is the width of the peak and W_1/2 is half the width of the peak.
Height equivalent to a theoretical plate (HETP) is determined from the equation:

H=L/N

or HETP can also be determined by the equation:

H=A+B/u+Cu

where H equals HETP, A is the term for eddy diffusion, B is the term for longitudinal diffusion, C is the coefficient for mass-transfer between the stationary and mobile phases, and u is the linear velocity. The equation for HETP is often used to describe the efficiency of the column. An efficient column would have a minimum HETP value.

Gas chromatographic columns have plate heights that are at least one order of magnitude greater than liquid chromatographic column plates. However GC columns are longer, which causes them to be more efficient. LC columns have a maximum length of 25 cm whereas GC columns can be  100 meters long.