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Making Conductivity Curves

Often times during a protein purification one wants to determine the salt concentration in a sample. For example, if a protein is eluted off an ion exchange column with a salt gradient it is useful to measure the salt concentration in the fractions in order to determine the ionic conditions at which the protein eluted. This is done by comparing the conductivity of the chromatographic fractions with that of a standard curve.

To make the standard conductivity curve a series of buffers that only differ in the concentration of one of their components is employed. These buffers can be conveniently generated by mixing, in different proportions, two buffers that differ only in their salt concentrations. For example, a buffer series for 25 mM Tris-HCl, pH 7.4, X mM KCl can be generated in the following manner:

ml of 25 mM TrisHCl ml of 25 mM TrisHCl, 1M KCl Final mM KCl

100
0
0

80
20 20

60
40
40

45
60
60

20
80
80

0
100
100

To measure the conductivity of these buffers they are diluted 1:100 in deionized water (10 ml + 990 ml of water in 1.5 ml Eppendorf tubes). The conductivity of the 1 ml dilutions is measured by using a conductivity meter.

A graph of conductivity (µS/ cm) vs. salt concentration (mM KCl) is then generated and a linear regression is performed to obtain the equation for the line.

To determine the salt concentration of an unknown, add 10 ml of sample to 990 ml of deionized water, record the conductivity and convert it into salt concentration using the equation from the standard curve.

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revised 6/19/1998 cla