Use in protein separations

The nomenclature of the RP is not consequent. The RP most often used contains octyl (RP C8) or octadecyl (RP C18) groups. There is no differentiation even when two methyl groups are introduced additionally with the silane (as with monofunctional silanes) or only one (difunctional) or none (trifunctional silane). Some manufacturer use silanes with bulky side groups (e.g., isopropyl groups) to improve the hydrolytic stability of the bonded phases, but here also, only the longest alkyl group is used in nomenclature. RP C8 and RP C18 are the work horses in HPLC. Shorter chains (RP4) are used in protein separations, and special selectivity can be obtained with bonded phenyl, cyano, amino or fluoro groups. 

Hydrophobic chromatography has not been used in protein separation as intensive as ion-exchange or affinity chromatography since sharp separations are not achieved. 

Isoelectric Focusing. Isoelectric focusing is a technique used for protein separation, by driving proteins to a pH where they have no mobiUty. Resolution depends on the slope of a pH gradient that can be achieved in a gel. 

Nevertheless, despite the inherent disadvantages of exclusion chromatography, there are instances where it is the only practical method of choice. The technique is widely used in the separation of macro-molecules of biological origin, e.g. polypeptides, proteins, enzymes, etc. In fact, it is in this area of biotechnology where the major growth in HPLC techniques appears to be taking place. 

Table 6.2 Chromatographic techniques most commonly used in protein purification protocols. The basis of separation is listed in each case...

Instead of ion-exclusion, size exclusion has been used in the separation of NH4S04 from a protein [41]. In that case, the adsorption isotherms were found to be simply linear. A hydrophobic interaction separation has been used for desalting in the case of phenylalanine and NaCl [41]. NaCl shows almost no interaction with the packing and consequently has a linear adsorption isotherm. The phenylalanine, on the other hand, showed a classical Langmuir-type adsorption isotherm. 

Proteins are frequently powerful immunogens and the availability of specific antibodies, particularly monoclonal antibodies, makes the technique of affinity chromatography very useful in the separation and purification of individual proteins. The technique has been used to purify a wide range of proteins such as hormones, membrane receptors and complement proteins. However, it is not restricted to proteins and is potentially applicable to any immunogenic substance. The availability of suitable antibodies is essential and these may be raised by whole animal polyclonal techniques or by monoclonal cell culture. The former antibodies may need some prior purification before being immobilized. 

One of the major advantages of CE as a separation technique is the wide variety of separation modes available. Analytes can be separated on the basis of charge, molecular size or shape, pi, or hydrophobicity. The same CE instrument can be used for zone electrophoresis, IEF, sieving separations, isotachophoresis, and chromatographic techniques such as MEKC and capillary electrokinetic chromatography. This section provides a brief description of each separation mode. Zone electrophoresis, IEF, and sieving are the primary modes used for protein separations, and these will be discussed in detail in the following sections. 

The most effective supports used in the separation of proteins all have certain common characteristics. They should be hydrophilic as separations are almost always carried out in aqueous buffers. Supports must be inert in that nonspecific binding is minimized. It is also desirable that the support does not contribute to the separation in ways different from the active groups attached to it. This helps to insure predictability and reproducibility of the separations among different manufactured lots of chromatographic media. 

In CGE the capillary is filled with a gel containing cross-linked or linear polymers. The gel thereby acts as molecular sieve. Traditionally, cross-linked polyacrylamides and agarose have been utilized in the slab and tube format. Polyacrylamides when cross-linked have smaller pore sizes and are used for protein separations. The larger pore sizes of agarose gels are more suitable for DNA separation. Polyacrylamides yield very viscous gels. Therefore, polymerization is usually done on column, which has a lot of practical problems. 

Salt (ionic strength) gradients in lEC discussed in Section 5.4.3.3 are frequently used in the separation of complex peptides, proteins, and other biopolymer samples as a complementary technique to RP solvent gradient separations, often in a 2D setup [99,100]. The gradients usually start at a low salt (chloride, sulfate, etc.) concentration and typically run from 0.005 to 0.5 M. A buffer is used to control the pH acetonitrile and methanol may be added to improve the resolution and urea to improve the solubility of proteins that are difficult to dissolve. Ion exchangers with not strongly hydrophobic matrices usually prevent protein denaturation in aqueous mobile phases. 

The transfer protocols are the same for DNA and RNA. In opposition to the methods given in Protocol 2.5.3, the forces driving the biomolecules from the separating gel to the receiving membrane are diffusion and capillary flow. This type of transfer is also applicable for proteins, but because the pores of polyacrylamide gels used for protein separation are mostly smaller, transfer times are longer and transfer efficiencies are lower than by electrotransfer. 

In gas or liquid column chromatography, the adsorbent particles are packed into a column, after which a small amount of fluid containing several solutes to be separated is applied to the top ofthe column. Each solute in the applied fluid moves down the column at a rate determined by the distribution coefficient between the adsorbent and the fluid and emerges at the outlet of the column as a separated band. Liquid column chromatography is the most common method used in the separation of proteins and other bioproducts. 

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