Reversed-phase HPLC column selectivity

An on-line concentration, isolation, and Hquid chromatographic separation method for the analysis of trace organics in natural waters has been described (63). Concentration and isolation are accompHshed with two precolumns connected in series the first acts as a filter for removal of interferences the second actually concentrates target solutes. The technique is appHcable even if no selective sorbent is available for the specific analyte of interest. Detection limits of less than 0.1 ppb were achieved for polar herbicides (qv) in the chlorotriazine and phenylurea classes. A novel method for deterrnination of tetracyclines in animal tissues and fluids was developed with sample extraction and cleanup based on tendency of tetracyclines to chelate with divalent metal ions (64). The metal chelate affinity precolumn was connected on-line to reversed-phase hplc column, and detection limits for several different tetracyclines in a variety of matrices were in the 10—50 ppb range. 

In reversed-phase HPLC, column temperature is a strong determinant of retention time and also affects column selectivity. A column oven is therefore required for most automated pharmaceutical assays to improve retention time precision, typically at temperatures of 30-50°C. Temperatures >60°C are atypical due to concerns about thermal degradation of the analytes and column lifetimes. Exceptions are found in high-throughput screening where higher temperatures are used to increase flow and efficiency. Ambient or snb-ambient operation is sometimes found in chiral separations to enhance selectivity. Column ovens... 

Fig. 3 Two-dimensional nonlinear selectivity map of reversed-phase HPLC columns. Number of iterations 377. Maximum error 2.1 x 10 (A) Ci silica (B) C2 silica (C) Ce silica (D)...

Reverse-phase HPLC is the standard analytical tool used for the control of the purity of synthetic peptides. Analysis is most commonly performed on reversed-phase HPLC columns, consisting of 3-5 pm, 100-300 A pore size C8 or C18 silica the larger pore material is generally favoured for the analysis of larger peptides (> 30 residues). Samples are eluted with a gradient formed between water and acetonitrile. An ion-pairing reagent is added to both solvents to improve resolution and selectivity. The most popular buffer systems are listed in Table 3. 

Cohen K A, Grillo S A, Dolan J W 1985 A comparison of protein retention and selectivity on large-pore reversed-phase HPLC columns. LC Mag 3 37-40... 

Although some normal phase methods have been used, the majority of carotenoid separations reported in the literature were carried out by reversed phase HPLC. Among the Cjg columns employed for determination of complete carotenoid compositions in foods, the polymeric Vydac brand is preferably used for separation of cis isomers. Several examples of different C,g columns and mobile phases are cited in the literature, but not aU carotenoids are baseline separated in most systems. Table 6.2.1 shows some examples employing different brands of Cjg columns." Acetonitrile did not improve selectivity toward separation of carotene isomers in a Vydac 201TP column and resolution was strongly dependent on the Vydac column lot. ... 

Discussed below are various modes of separations in HPLC. Included here is brief coverage of mobile-phase selection for various modes of chromatography and elementary information on mechanism, choice of solvents and columns, and other practical considerations. It should come as no surprise that reversed-phase HPLC is discussed at greater length in this section because it is the most commonly used technique in HPLC (more detailed discussion is provided in Section 15.8). Clearly,... 

Variations in retention and selectivity have been studied in cyano, phenyl, and octyl reversed bonded phase HPLC columns. The retention of toluene, phenol, aniline, and nitrobenzene in these columns has been measured using binary mixtures of water and methanol, acetonitrile, or tetrahydrofuran mobile phases in order to determine the relative contributions of proton donor-proton acceptor and dipole-dipole interactions in the retention process. Retention and selectivity in these columns were correlated with polar group selectivities of mobile-phase organic modifiers and the polarity of the bonded stationary phases. In spite of the prominent role of bonded phase volume and residual silanols in the retention process, each column exhibited some unique selectivities when used with different organic modifiers [84],... 

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

Colistin (COL) is a multicomponent antibiotic (polymyxins E) that is produced by strains of inverse Bacillus polymyxa. It consists of a mixture of several closely related decapeptides with a general structure composed of a cyclic heptapeptide moiety and a side chain acetylated at the N-terminus by a fatty acid. Up to 13 different components have been identified. The two main components of colistin are polymyxins El and E2 they include the same amino acids but a different fatty acid (216). A selective and sensitive HPLC method was developed for the determination of COL residues in milk and four bovine tissues (muscle, liver, kidney, and fat). The sample pretreatment consists of protein precipitation with trichloracetic acid (TCA), solid-phase purification on Cl 8 SPE cartridges, and precolumn derivatization of colistin with o-phthalaldehyde and 2-mercaptoethanol in borate buffer (pH 10.5). The last step was performed automatically, and the resulting reaction mixture was injected into a switching HPLC system including a precolumn and the reversed-phase analytical column. Fluorescence detection was used. The structural study of El and E2 derivatives was carried out by HPLC coupled with an electrospray MS. Recoveries from the preseparation procedure were higher than 60%. 

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