Reversed-phase chromatography resolving power

Each column type has its own place of use. Column variety is what gives HPLC its versatility. It really depends on your compound and application. Approximately 80% of all separations are done on 5-10-jUm reverse phase Ci8 silica columns. Much of this is tradition. Reverse phase columns offer high-resolution separations for a wide variety of compounds and can be run in aqueous mobile phases. Ion exchange separations require salt solutions for separations, and these are not compatible with mass spectrometers. Size separations have lower resolving power and longer run times, but may be the only way to separate proteins solutions that will irreversibly stick to reverse phase columns. Use small pore size separation columns to remove salt from effluent from other chromatography separations. Zirconium and polymeric column are newer and offer possibilities for unique separations. 

CE, with its high resolving power, rapid method development, easy sample preparation, and low operational cost, is reported to be an excellent technique for resolving caseins (including different genetic variants), peptides derived from them, and whey proteins (16-19). Peptide profiles obtained by CE supplement the information obtained by reversed-phase high performance liquid chromatography (RP-HPLC) (17, 20). The application of CE to the assessment of proteolysis in milk and different cheese types has acquired an enormous importance in recent years. Reviews on the application of CE to this field can be found in papers by Otte et al. (21) and Redo et al. (22). 

There are a variety of HPLC columns and most of them are based on silica particles. The most popular column used today is the reversed-phase Cl8 column that uses a silica support with a bonded organic surface layer. Traditional HPLC analytical columns are usually 4.6 mm x 250 mm with a 5 pm particle size, as used in the USP official method. Modern columns for HPLC-mass spectrometry (LC-MS) applications are more efficient and offer the same or better resolving power in a much smaller package (2.1 mm x 150 mm with a 3.5 pm particle size or smaller). Ultra high-performance liquid chromatography (UHPLC) columns (sub 2pm particle size, 1-2 mm in diameter and 30-100 mm in length) are becoming more and more popular. 

---