Cation-Exchange Chromatography of Proteins

The last two examples show that, in a number of cases, cation-exchange chromatography can be regarded as an alternative to reversed-phase chromatography on chemically bonded silica phases, especially because the characteristic interactions between the basic compounds and the free silanol groups of silica are not observed with polymer-based cation exchangers. 

Proteins are isolated and characterized with different chromatographic techniques. Depending on the protein, ion-exchange (lEX), size-exclusion (SEC), affinity (IMAC), hydrophobic interaction (HIC), and reversed-phase chromatography (RPLC) may be applied. Traditionally, separation materials with low mechanical stability and limited resolution were used. In recent years, HPLC materials were developed that offered the separation power required by the protein chemist. Now, pellicular ion-exchange resins as well as organic polymer monoliths exhibit high resolution at relatively short retention times, while 

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Fig. 3. Cation-exchange chromatography of protein standards. Column poly(aspartic acid) Vydac (10 pm), 20 x 0.46 cm. Sample 25 pi containing 12.5 pg of ovalbumin and 25 pg each of the other proteins in the weak buffer. Flow rate 1 ml/min. Weak buffer 0.05 mol/1 potassium phosphate, pH 6.0. Strong buffer same +0.6 mol/1 sodium chloride Elution 80-min linear gradient, 0-100% strong buffer. Peaks a = ovalbumin, b = bacitracin, c = myoglobin, d = chymotrypsinogen A, e = cytochrom C (reduced), / = ribonuclease A, g = cytochrome C (oxidised), h = lysozyme. The cytochrome C peaks were identified by oxidation with potassium ferricyanide and reduction with sodium dithionite [47]...

Xu, W. and Regnier, F. E., Electrokinetically driven cation-exchange chromatography of proteins and its comparison with pressure-driven high-performance liquid chromatography, /. Chromatogr. A, 853, 243, 1999. 

Simultaneous optimizatiou of the steepuess of a salt gradient, column length, and mobile phase flow rate using iso-resolution plots was recently applied to cation-exchange chromatography of proteins on short columns [129,130]. 

In 1958, Sober and Peterson [I] described the use of diethylaminoethyl (DEAE)-derivatized cellulose in the anion-exchange separation of proteins. This hydrophilic matrix overcame many of the problems of the PSDVB-based materials. Shortly thereafter, carboxymethyl-derivatized cellulose was introduced for the cation-exchange chromatography of proteins. Separations achieved using... 

Polymethacrylate Weak cation exchanger (carboxylic acid) ProSwift WCX Dionex Corporation 50x4.6mm 50x 1 mm Ion exchange chromatography of proteins... 

Saufi SM, Fee CJ. Simultaneous aiuon and cation exchange chromatography of whey proteins using a customizable mixed matrix membrane. J. Chromatogr. A 2011 1218 9003-9009. 

The enz3rme activity was adsorbed to the cation exchange chromatography. Three peaks of activities were found (fraction no. 78-81, 83-85 and 86-89). The major peak (no.83-85) was coUected (Figure 2. The enz3rme was purified to approx. 100 fold with the higher specific activity, 534.7 unit/mg protein (Table 1). 

The dialysed sample was fractionated by cation exchange chromatography. A 40-50 ml sample was applied to a CM-Sepharose CL-6B column (1.5 x 15 cm). Unbound proteins were removed with 50 mM MES pH 6.8, 1 mM DTT, and the bound proteins were eluted with an increasing NaCl gradient from 0 - 0.4 M NaCl in a total volume of 500 ml. The flow was 25 ml/h and fractions of 8.33 ml were collected. The protein profile was measured at 280 nm. 

Gerstner, J. A. and Cramer, S. M., Cation-exchange displacement chromatography of proteins with protamine displacers effect of salt-induced gradients, Biotechnol. Prog., 8, 540, 1992. 

Alpert, A. J., Cation-exchange high-performance liquid chromatography of proteins on poly(aspartic acid)-silica, /. Chromatogr, 266, 23, 1983. 

Figure 2.6. LC-tandem mass spectrometry to examine complex mixtures. The mixture of many different proteins is digested to yield peptides and the peptides are resolved into fractions hy cation exchange chromatography followed by reverse phase chromatography. The fractionation steps resolve the peptides into fractions that he processed hy tandem mass spectrometry to yield sequence information suitable for database searching.

It was in this context that the first true comprehensive online LC x LC separation was reported (Bushey and Jorgenson, 1990). Mixtures of intact proteins were analyzed using cation-exchange chromatography (CEX) as the first dimension and size exclusion chromatography (SEC) as the second. This research demonstrated that the practical difficulties of coupling two dissimilar LC modes for a comprehensive 2D separation are relatively easy to overcome when instrumentation is properly configured. 

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