**9. Sample preparation**

338 Ion Exchange Technologies

samples.

starting buffer.

**7. Column chromatography** 

realising the full separation potential of any gel.

clogged, are difficult to clean and cause artifacts (10).

achieved by increasing the column diameter.

**8. Quantity and preparation of ion exchanger** 

illustrated by the frequency with which phosphate buffers are cited in the literature in connection with anion exchangers. In those instances when a buffering ion which interacts with the ionic groups on the matrixnis used, extra care must be taken to ensure that the

Good results in column chromatography are not solely dependent on the correct choice of gel media. The design of the column and good packing technique are also important in

The material used in the construction of the column should be chosen to prevent destruction of labile biological substances and minimize non-specific binding to exposed surfaces. The bed support should be designed so it is easily exchangeable to restore column performance whenever contamination and/or blockage in the column occur. Bed supports made from coarse sintered glass or glass wool cannot be recommended because they soon become

The pressure specifications of the column have to match the back-pressure generated in the packed bed when run at optimal flow rate. This is particularly important when using high performance media with small bead size. All are easy to dismantle and reassemble to allow thorough cleaning, which is a particularly important aspect when handling biological

As for most adsorptive, high selectivity techniques, ion exchange chromatography is normally carried out in short columns. A typical ion exchange column is packed to a bed height of 5-15 cm. Once the separation parameters have been determined, scale-up is easily

The amount of ion exchanger required for a given experiment depends on the amount of sample to be chromatographed and on the available or dynamic capacity of the ion exchanger for the sample substances. For the best resolution in ion exchange chromatography, it is not usually advisable to use more than 10-20% of this capacity, although this value can be exceeded if resolution is adequate. Preparation of the ion exchanger Having chosen the appropriate ion exchanger and starting buffer it is essential that the exchanger is brought to equilibrium with start buffer before sample application.

To prepare the gel, the supernatant is decanted and replaced with starting buffer to a ratio of approximately 75% settled gel to 25% buffer. If large amounts of ion exchangers are to be equilibrated with a weak buffer, the ion exchanger should first be equilibrated with a 10 times concentrated buffer solution at the correct pH, and then with a few volumes of

system has come to equilibrium before application of sample.

The amount of sample which can be applied to a column depends on the dynamic capacity of the ion exchanger and the degree of resolution required. For the best resolution it is not usually advisable to use more than 10-20% of this capacity(11). Information on the available capacities for the different exchangers is given in the relevant product sections.

The ionic composition should be the same as that of the starting buffer. If it is not, it can be changed by gel filtration on Sephadex G-25 using.Desalting Columns, dialysis, diafiltration or possibly by addition of concentrated start buffer.

If the ion exchanger is to be developed with the starting buffer (isocratic elution), the sample volume is important and should be limited to between 1 and 5% of the bed volume. If however, the ion exchanger is to be developed with a gradient, starting conditions are normally chosen so that all important substances are adsorbed at the top of the bed. In this case, the sample mass applied is of far greater importance than the sample volume. This means that large volumes of dilute solutions, such as pooled fractions from a preceding gel filtration step or a cell culture supernatant can be applied directly to the ion exchanger without prior concentration.Ion exchange thus serves as a useful means of concentrating a sample in addition to fractionating it. If contaminants are to be adsorbed, and the component of interest is allowed to pass straight through, then the sample volume is less important than the amount of contaminant which is present. Under these conditions there will be no concentration of the purified component, rather some degree of dilution due to diffusion.

The viscosity may limit the quantity of sample that can be applied to a column. A high sample viscosity causes instability of the zone and an irregular flow pattern. The critical variable is the viscosity of the sample relative to the eluent. This corresponds to a protein concentration of approximately 5%. Approximate relative viscosities can be quickly estimated by comparing emptying times from a pipette.
