Reverse phase separation

In reverse-phase chromatography, which is the more commonly encountered form of HPLC, the stationary phase is nonpolar and the mobile phase is polar. The most common nonpolar stationary phases use an organochlorosilane for which the R group is an -octyl (Cg) or -octyldecyl (Cig) hydrocarbon chain. Most reverse-phase separations are carried out using a buffered aqueous solution as a polar mobile phase. Because the silica substrate is subject to hydrolysis in basic solutions, the pH of the mobile phase must be less than 7.5. 

A useful guide when using the polarity index is that a change in its value of 2 units corresponds to an approximate tenfold change in a solute s capacity factor. Thus, if k is 22 for the reverse-phase separation of a solute when using a mobile phase of water (P = 10.2), then switching to a 60 40 water-methanol mobile phase (P = 8.2) will decrease k to approximately 2.2. Note that the capacity factor decreases because we are switching from a more polar to a less polar mobile phase in a reverse-phase separation. 

Haddad and associates report the following capacity factors for the reverse-phase separation of salicylamide (k i) and caffeine... 

This reversed-phase chromatography method was successfully used in a production-scale system to purify recombinant insulin. The insulin purified by reversed-phase chromatography has a biological potency equal to that obtained from a conventional system employing ion-exchange and size-exclusion chromatographies (14). The reversed-phase separation was, however, followed by a size-exclusion step to remove the acetonitrile eluent from the final product (12,14). 

Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

FIGURE 13.57 NOTE The importance of Solvent/column interaction using Jordi DVB columns cannot be over emphasized. We have found that a SOySO mbc of MeOH/ACN for the strong solvent Is adequate for many reverse phase separations and is better than either alone. We have now observed that the use of THF/ACN as strong solvent is often better than MeOH/ACN. In general Lewis bases (electron donor solvents) deactivate the aromatic rings and often dramatically increase column efficiencies. 

Reverse phase chromatography is finding increasing use in modern LC. For example, steroids (42) and fat soluble vitamins (43) are appropriately separated by this mode. Reverse phase with a chemically bonded stationary phase is popular because mobile phase conditions can be quickly found which produce reasonable retention. (In reverse phase LC the mobile phase is typically a water-organic solvent mixture.) Rapid solvent changeover also allows easy operation in gradient elution. Many examples of reverse phase separations can be found in the literature of the various instrument companies. 

Reversible Phase Separation Driven by Photodimerization of Anthracene A Novel Method for Processing and Recycling Polymer Blends... 

Flgvire 4.5 The influence of endcapping on peak shape and retention of soee PTH-anino acids using a reversed-phase separation system. Peak identification 1 PTH-histidine, 2 PTH-arginine and 3 PTH-valine. (Reproduced with permission from ref. 71. Copyright Preston Publications, Inc.)... 

Martin, O. C. and Pagano, R. E., Normal- and reverse-phase separation of fluorescent (NBD) lipids, Anal. Biochem., 159, 101, 1986. 

Membranes offer a format for interaction of an analyte with a stationary phase alternative to the familiar column. For certain kinds of separations, particularly preparative separations involving strong adsorption, the membrane format is extremely useful. A 5 x 4 mm hollow-fiber membrane layered with the protein bovine serum albumin was used for the chiral separation of the amino acid tryptophan, with a separation factor of up to 6.6.62 Diethey-laminoethyl-derivatized membrane disks were used for high-speed ion exchange separations of oligonucleotides.63 Sulfonated membranes were used for peptide separations, and reversed-phase separations of peptides, steroids, and aromatic hydrocarbons were accomplished on C18-derivatized membranes. 

A review of the properties of silica as applied to reversed phase separations summarizes a number of issues that have been debated for many years.71 The review categorizes unmodified silanols as free, geminal, vicinal, and internal. The pK, values of silanols average about 7.1, but some silanols may have pK, values as low as 3. As reported in Chapter 4, heavy metal... 

A number of recommendations have been made in the development of quantitative chromatographic methods. The American Society for Testing Materials — using as a benchmark the reversed phase separation of benzyl alcohol, acetophenone, benzaldehyde, benzene, and dimethylterephthalate — discovered substantial laboratory-to-laboratory differences in quantitative analysis.53 These compounds are routinely used to test column performance or for system suitability testing. A followup study, using benzyl alcohol, acetophenone, p-tolualdehyde, and anisole, showed that measurement of... 

Zhu, Y. Lubman, D. M. Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations. Electrophoresis 2004, 25, 949-958. 

Jt is not possible to bond all of the surface silanol groups. Unreacted silanols are capable of adsorbing polar molecules, and will thus affect the chromatographic properties of the bonded phase. Usually, the unreacted silanols produce undesirable effects, such as tailing and excessive retention in reverse phase separations, although there have been cases reported where the unreacted silanols improve such... 

This will follow the rules for reverse phase separations (Fig. 3.1c). Increasing the amount of organic solvent will decrease the polarity of the mobile phase and will decrease retention. 

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