Cation exchange chromatography

Strong acid cation exchange resins manufactured by the sulphonation of polystyrene or polydivinyl benzenes undergo the following reaction wi cations  

Weak acid cation exchange resins manufactured, eg by the polymerisation of methacrylic acid undergo the following reaction with cations  

Analysis is achieved by passing a large volume of water sample, suitably adjusted in pH and reagent composition down a small column of the resin. The adsorbed ions are then desorbed with a small volume of a suitable reagent in which the metals or metal complexes or anionic species dissolve. This extract can then be analysed by any suitable means. 

The exchange reaction with a cation M+ that occurs at the stationary phase of a cation exchanger can be represented as follows  

The separation of cations is determined by their different affinities toward the stationary phase. 

1) A crude mixture was charged onto the column, which had been filled with 

2) Impurities were eluted while 4 remained adsorbed on the H-form resin. 

3) Compound 4 was exchanged with sodium cations and eluted from the Na-form resin. 

In principle, an extraction technique involving pH adjustment of the aqueous phase can offer purification similar to ion exchange chromatography. Although the method uses a smaller volume of solvent, it has limited ability to remove low-level impurities. Therefore, the replacement of ion exchange chromatography with extraction requires some ingenuity. 

The earlier purification method involving CEC required a large volume of solvent (about 900 L/kg of 4), although the resin could be recycled. In contrast, the purification involving extraction in combination with salt crystallization required a smaller volume of solvent (about 25 L/kg of 4). Thus, extraction-based purification contributes significantly to the reduction of solvent waste. 

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Before polyacrylamides are sold, the amount of residual acrylamide is determined. In one method, the monomer is extracted from the polymer and the acrylamide content is determined by hplc (153). A second method is based on analysis by cationic exchange chromatography (154). For dry products the particle si2e distribution can be quickly determined by use of a shaker and a series of test sieves. Batches with small particles can present a dust ha2ard. The percentage of insoluble material is determined in both dry and emulsion products. 

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]...

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). 

Acetyl esterase (AE) has been purified to homogeneity from orange peels. The purification steps included cation exchange chromatography and gel filtration. The enzyme has affinity for triacetin and sugar beet pectin with K, of 39 mM and of 26 mg/ml, respectively. AE has a MW of 42 kD and is a monomer. The isoelectric point is at pH > 9. 

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. 

RG-lyase was purified from Pectinex Ultra SP-L, produced by Aspergillus aculeatus, using anion- and cation-exchange chromatography. The purified RG-lyase differed from RG-hydrolase in pi and pH optimum and stability (Table I). 

Breter, H.-J., Seibert, G., and Zahn, R. K., Single-step separation of major and rare ribonucleosides and deoxyribonucleosides by high-performance liquid cation-exchange chromatography for the determination of the purity of nucleic acid preparations, ]. Chromatogr., 140, 251, 1977. 

Callmer, K. and Davies, L., Separation and determination of vitamin Bj, B2, Bj, and nicotinamide in commercial vitamin preparations using high-performance cation-exchange chromatography, Chromatographia, 7, 644, 1974. 

Kwon, S.-M., Lee, K.-P., Tanaka, K., and Ohta, K., Simultaneous determination of anions and cations by ion-exclusion chromatography-cation-exchange chromatography with tartaric acid/18-crown-6 as eluent, /. Chromatogr. A, 850, 79, 1999. 

Weitzhandler, M., Farnan, D., Horvath, J., Rohrer, J. S., Slingsby, R. W., Avdal-ovic, N., and Pohl, C., Protein variant separations by cation-exchange chromatography on tentacle-type polymeric stationary phases, /. Chromatogr. A, 828, 365, 1998. 

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. 

Litowski, J. R., Semchuk, P. D., Mant, C. T., and Hodges, R. S., Hydrophilic interaction/cation-exchange chromatography for the purification of synthetic peptides from closely related impurities Serine side-chain acetylated peptides, /. Peptide Res., 54, 1, 1999. 

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. 

Essader, A.S., Cargile, B.J., Bundy, J.L., Stephenson, J.L., Jr. (2005). A comparison of immobilized pH gradient isoelectric focusing and strong-cation-exchange chromatography as a first dimension in shotgun proteomics. Proteomics 5, 24—34. 

Aebersold, R., Mann, M. (2003). Mass spectrometry-based proteomics. Nature 422,198-207. Alpert, A.J., Andrews, P.C. (1988). Cation-exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica. J. Chromatogr. 443, 85-96. 

Figure 10.2 Optimization of a purification protocol by LC-MS analysis. (1) LC-MS analysis of C-terminal fragment from reference molecule purified by cation-exchange chromatography (CEX) and hydroxyapatite chromatography (HA). (2) Incubation at 37°C under acidic conditions shows degradation of the purified molecule if CEX precedes HA. (3) The molecule is stabilized when the sequence of the two purification steps is swapped. 

O Neil JR (1986) Theoretical and experimental aspects of isotopic fractionation. Rev Mineral 16 1-40 Oi T, Kawada K, Hosoe M, Kakihana H (1991) Fractionation of lithium isotopes in cation-exchange chromatography. Sep Sci Tech 26 1353-1375... 

Purification by cation exchange chromatography (Dowex 50WX8, 200-400 mesh, H" " form) eluted with 1 m NH4OH afforded the amine as a white solid in 78 % yield (48 mg, 0.25 mmol). 

Rey, M., Pohl, C., Bordunov, A. and DeBorba, B., Cation Exchange Chromatography with Conductivity Detection Suppressed or Not Suppressed Poster B-6, International Ion Chromatography Symposium, 2003. 

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