Cation Exchange-Size Exclusion

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. 

FIGURE 8.1 Schematic diagram of a 2D CEX x SEC instrument. Reprinted from Evans, C. R. and Jorgenson, J. W. (2004) Anal. Bioanal. Chem. 378, 1952-1961, with kind permission of Springer Science and Business Media. 

---

Schenk et al. reported the quantification of cytokines in cell extracts using micro-HPLC packed microcolumns coupled with immunochemical detection. Various types of chromatographies were associated with the immunochemical detection system in order to perform the separation of cytokines. These included cation-exchange, size exclusion, and re-versed-phase chromatography, with the latter being the most successful. 

Another example of vims clearance is for IgM human antibodies derived from human B lymphocyte cell lines where the steps are precipitation, size exclusion using nucleases, and anion-exchange chromatography (24). A second sequence consists of cation-exchange, hydroxylapatite, and immunoaffinity chromatographies. Each three-step sequence utilizes steps based on different properties. The first sequence employs solubiUty, size, and anion selectivity the second sequence is based on cation selectivity, adsorption, and selective recognition based on an anti-u chain IgG (24). 

The size-exclusion and ion-exchange properties of zeoHtes have been exploited to cause electroactive species to align at a zeoHte—water interface (233—235). The zeoHte thus acts as a template for the self-organization of electron transfer (ET) chains that may find function as biomimetic photosynthetic systems, current rectifiers, and photodiodes. An example is the three subunit ET chain comprising Fe(CN)g anion (which is charge-excluded from the anionic zeoHte pore stmcture), Os(bipyridine)3 (which is an interfacial cation due to size exclusion of the bipyridine ligand), and an intrazeoHte cation (trimethylamino)methylferrocene (F J ). A cationic polymer bound to the (CN) anion holds the self-assembled stmcture at an... 

Ion-exchange chromatography (both cation and anion) can also be undertaken in HPLC format. Though not as extensively employed as reverse-phase or size-exclusion systems, ion-exchange-based systems are of use in analysing for impurities unrelated to the product, as well as detecting and quantifying deamidated forms. 

---