Desalting and Gel Filtration Chromatography

In desalting and buffer exchange, the macromolecular components of a sample are recovered in the buffer used to pre-equilibrate the gel-filtration matrix through which the sample is processed. The method is commonly referred to as desalting when the goal is to remove buffer salts from a sample in exchange for water (with water used to pre-equilibrate the gel-filtration resin). Buffer exchange is the term used when one set of buffer salt in a sample is exchanged for another set.

Applications for desalting include:

• Removing salts from protein solutions
• Removing phenol or unincorporated nucleotides from nucleic acid preparations
• Separating excess crosslinking, labeling or derivatization reagents from conjugated proteins

Buffer exchange is used to place a protein solution into a more appropriate buffer before subsequent applications such as:

• Electrophoresis
• Ion exchange (IEX)
• Affinity chromatography (AC)

Gel filtration chromatography is useful for many of the same purposes as dialysis, because both methods are based on similar ranges of molecular weight cut-off (MWCO) limits that exclude molecules based on size. Compared to dialysis, gel filtration has the advantage of speed (a few minutes vs. hours for dialysis), which is necessary in certain experimental situations. For example, reduced peptides must be desalted quickly to remove reductant and initiate subsequent sulfhydryl-reaction procedures before oxidation back to disulfides occurs. Also, gel filtration is compatible with organic and other solvents that dissolve or otherwise compromise the integrity of dialysis membranes. Finally, in contrast to dialysis, gel filtration chromatography allows the contaminating material to be removed in a relatively small volume (and is left on the column), an important feature when working with toxic or radioactive substances.

Gel filtration separates molecules of different dimensions based on their relative abilities to penetrate into a suitable stationary phase or chromatographic resin. The resin has size-exclusion properties and usually consists of very small, uncharged porous particles in an aqueous solution, which are packed into a column and then used for the separation. The resin particles have a range of pore sizes that determine the size of molecules that can be separated. The average or maximum effective pore size defines what is called the fractionation range or exclusion limit of the resin. Molecules smaller than the fractionation range can enter the pores of the resin, while molecules larger than the fractionation range are excluded from entering the pores.

When a sample solution is passed through a column of packed gel filtration resin, small molecules in the sample (buffer salts, small molecules, etc.) enter the pores of resin beads that they encounter and are forced to follow a circuitous path before later exiting the beads. By contrast, large molecules flow around the resin beads, taking a relatively direct path through the column. In effect, small molecules experience a much larger column volume than large molecules. (Keep in mind that resin beads are extremely porous; most of their total volume is water). Thus, the difference in the flow rates of small molecules and excluded molecules allows the faster-flowing macromolecules to become separated from the slower small molecules as the sample travels the distance of the resin bed packed in the column.

When an appropriate gel filtration resin is used, many different classes of macromolecules can be separated from buffer salts, unconjugated labeling reagents and other molecules to achieve rapid purification before downstream applications. This is done by passing samples through a column whose resin-bed is sufficiently tall and voluminous to fully separate the emergence from the end of the column of macromolecules compared to the small molecules. By collecting small fractions, the macromolecules are easily separated from the small molecules emerging in the later fractions.

As mentioned above, gel filtration can be effectively utilized for protein desalting and is accomplished by first equilibrating the gel filtration column with water. However, buffer exchange is accomplished by first equilibrating the column resin with the target buffer. In both desalting and buffer-exchange modes of gel filtration, the buffer constituents carrying the sample into the column will be replaced by the solution in which the resin bed was originally saturated (i.e., pre-equilibrated). As a loaded sample enters into a resin bed, it displaces an identical volume of water or buffer already present in the column. As sample is pushed through the column (usually by addition of more buffer at the top of the column), the equilibration solution is pushed out through the end of the column. Because the macromolecules emerge from the column before the buffer they were originally carried in, they end up emerging from the column in the equilibration solution. As described in more detail in the section below, there are three methods for performing gel filtration that employ the use of gravity-flow columns (drip columns), centrifuge (spin) columns or chromatography cartridges.

Thermo Fisher Scientific has developed various strategies for protein sample desalting. For example, Thermo Scientific Zeba desalting spin plates, spin columns, and cartridges contain unique gel filtration resins and were specifically designed to provide consistent performance over a wide range of protein concentrations and sample sizes. High protein recovery can be achieved even for dilute protein samples.

The three common formats for performing gel filtration include gravity-flow columns, centrifuge columns or chromatography cartridges. Gravity-flow-or drip-columns and chromatography cartridges use head-pressure from a buffer-chase to push the sample through a gel filtration matrix. Centrifuge columns use centrifugal force to move a sample through the matrix.

In the case of gravity-flow columns, sample is loaded through the top of an upright column and allowed to sink into the resin bed. The sample is then chased through the column by adding additional buffer or water to the top of the column. During this process, small fractions are collected and each is tested for the protein or other macromolecules of interest. In some cases, several fractions might contain the protein and may have to be pooled to improve yield.

Chromatography cartridges work in much the same way as gravity-flow columns except that the system is closed and liquid is forced into the device with the aid of fluid pressure generated from a syringe-plunger, pump or other device.

Thermo Scientific offers a variety of desalting columns as well as chromatography cartridges for efficient salt removal and protein recovery. The Thermo Scientific Pierce Zeba Desalting Chromatography Cartridges (7K MWCO, 5 mL) contain the Zeba High-Performance Resin for desalting or buffer exchange applications. This resin is ideal for removing low molecular weight compounds including salts, fluorescent dyes, biotin and other small labeling reagents. Protein samples can be processed with a high degree of recovery and ≥95% retention of salts and other small molecular contaminants (< 1,000 Da).

Centrifuge- or spin-columns are unique in that the force generated by a centrifuge is sufficient to push the sample through the resin bed without any buffer-chase. The centrifugal force causes the gel matrix to collapse to some extent, helping to squeeze the sample through and out the bottom of the column and to trap small molecules that enter the pores of the resin beads. Spin desalting is not only faster than gravity-flow and cartridge formats, it also reduces the amount of sample dilution.

It is important to select a column size that is suitable for the volume of sample to be desalted. A column that is too large will result in dilution of the protein sample. If the column is too small, the low molecular weight contaminants will not be adequately separated from the macromolecule of interest. Selecting a column size appropriate for the sample volume will minimize dilution and allow for complete and efficient separation. Generally, a column with a bed volume that is 4 to 20 times larger than the sample volume is adequate. The typically excluded volume (i.e., the column volume available to large molecules) is about 35% of the resin volume, while the total volume available to small molecules is nearly equal to the resin-bed volume.

The size-exclusion limit of the gel filtration resin bed is also important. For typical desalting and buffer exchange applications (as opposed to other types of size-exclusion chromatography), choosing a resin with size-exclusion limits (MWCO) between 2000 and 7000 is usually best. In practice, the small molecules one wishes to remove must be several times smaller than the MWCO; the macromolecules (e.g., proteins) one wishes to separate must be at least as large as the MWCO. For other applications, such as separating peptides from full-sized proteins, resins with larger exclusion limits (e.g., 40K) may be necessary. Be aware that resins with large exclusion limits are not always suitable for buffer exchange and desalting because the small molecules flow almost uninhibited (i.e., without a circuitous path) through the pores of the matrix.

Commercially available gel filtration resins are generally durable, chemically resistant and inert and have minimal nonspecific binding properties. Consequently, nearly any buffer system can be used effectively for desalting and buffer exchange. When desalting proteins into a water-equilibrated column, peak-broadening can result causing more sample dilution and poorer separation than when a buffered solution is used. Generally, better results are obtained using buffered solutions having some ionic character. As an alternative, volatile electrolytes (such as pyridinium acetate, ammonium bicarbonate and ethylenediamine acetate) can be added to the desalting buffer to increase the ionic strength and diminish the tailing effect when separation precision is critical for experimental success. These additives can be easily removed later by lyophilization.

• Porath, J. and Flodin, P. (1959(Gel filtration: a method for desalting and group separation. Nature 183: 1657-1659.
• Walker JM. 2009. The Protein Protocols Handbook. Third Edition. Springer-Verlag New York, LLC.