Reversed-phase retention

Fig. 2-15. Reversed-phase retention of the first eluted and the seeond eluted enantiomers of 5-methyl-5-phenylhydantoin as a funetion of mobile phase eomposition. The eolumn was a 250 x 4.6 mm vaneomyein CSR The buffer was triethylammonium aeetate at pH 7.0. The flow rate was 1.0 mL min at ambient temperature (23 °C).

Ion suppression is a technique used to suppress the ionization of compounds (such as carboxylic acids) so they will be retained exclusively by the reversed-phase retention mechanism and chromatographed as the neutral species. 

The pH dependence of the tailing of Dil was investigated in separate experiments. The experimental conditions were the same but the pH of the mobile phase was adjusted to different values by HC1. The effect of pH on the retention behaviour of the dye is illustrated by chromatograms in Fig. 3.113. The pH dependence of tailing was tentatively explained by the marked contribution of free silanol groups to the reversed-phase retention of the dye... 

T. Cserhati, E. Forgacs and S. Balogh, Relationship between the reversed-phase retention of monotetrazolium and ditetrazolium salts and their physicochemical parameters. J. Planar Chromatogr.-Mod. TLC, 12 (1999) 446 451. 

T. Cserhati and G. Oros, Impact of molecular surface characteristics on the reversed-phase retention behaviour of synthetic dyes. Biomed. Chromatogr., 13 (1999) 525-530. 

Carbon-based material on a silica template has been pioneered by Knox (34). It can be used at any pH. However, the mechanism of retention on this support is quite different from that for the average alkyl-bonded silica (35). Further information on reversed-phase retention can be found in Ref. 36. 

SL Abidi, TL Mounts. Reversed-phase retention behavior of fluorescence labeled phospholipids in ammonium acetate buffers. J Liq Chrom 17 105-122, 1994. 

Ion suppression is a technique used to suppress the ionisation of compounds (such as carboxylic acids) so they will be retained exclusively by the reversed phase retention mechanism and chromatographed as the neutral species. Column packings with an extended pH range are needed for this application as strong acids or alkalis are used to suppress ionisation. In addition to carboxylic acids, the ionisation of amines can be suppressed by the addition of a base to the mobile phase, thus allowing chromatography of the neutral amine. 

Dinitrobenzoic acid proved capable of improving reversed phase retentions of chemical warfare agent derivatives [137] and [S-(R,R)]-(-)-bis(-a-methylbenzyl) amine hydrochloride was an effective IPR for zwitterionic analytes [138]. 

FIGURE 9.1 Theoretical curves showing basic analyte reversed phase retention as a func-amonds p/... 

Coym, J.W. and Dorsey, J.G. Reversed-phase retention thermodynamics of pure-water mobile phases at ambient and elevated temperature. J. Chromatogr. A. 2004, 1035, 23-29. 

The use of pennanently coated columns was not the only strategy to eliminate IPRs in eluents. The possibility of adding an IPR only to the solution of sample to be injected was successfully explored. It was suggested that ion-pairing in a sample is sufficient to provide improved reversed phase retention. The effectiveness of this stratagem has both theoretical and practical value. 

Alkyl-Type Phases (C1-C18, C30). Probably 90% of all reversed-phase columns are alkyl-type bonded phases. An enormous amount of publications are devoted to the classiflcation, standardization, and comparison of these phases. In their book Practical HPLC Method Development, Snyder and Kirkland [57] indicate that reversed-phase retention for nonpolar and nonionic compounds generally follows the retention pattern Cl < C4 < C8 = Cl 8. At the same time, they refer to the comparison of Cl 8-type columns from different manufacturers and find dramatic variation in the retention of both polar and nonpolar compounds at the same conditions on different columns. 

U. D. Neue, C. H. J. Phoebe, K. Tran, Y. Cheng, and Z. Lu, Dependence of reversed-phase retention of ionizable analytes on pH, concentration of organic solvent and silanol activity, /. Chromatogr. A 925 (2001), 49-67. 

In the past, several theoretical models were proposed for the description of the reversed-phase retention process. Some theories based on the detailed consideration of the analyte retention mechanism give a realistic physicochemical description of the chromatographic system, but are practically inapplicable for routine computer-assisted optimization or prediction due to then-complexity [9,10]. Others allow retention optimization and prediction within a narrow range of conditions and require extensive experimental data for the retention of model compounds at specified conditions [11]. 

QSRR Eqs. (11.15) and (11.16) clearly demonstrate that the organic modifier of binary aqueous eluents used in reversed-phase liquid chromatography also modifies the stationary phase. The hydrocarbon brush on the silica matrix adsorbs the modifier and gets to some extent its properties 117]. QSRR enables differences in the mechanism of reversed-phase retention in individual HPLC. systems employing the. same stationary phase material, are characterized in a numerical manner. 

In addition to a wide range of polar and nonpolar hydrocarbons that can be analyzed by RP-HPLC, it is also possible to separate ionic species. Because water is used as part of almost all mobile phases, those species which are acids and bases can be neutralized by control of pH. In cases where neutralization is not possible, then the addition of a counterion into the mobile phase so that the analyte will form a neutral complex can be used to enhance RP retention. The same principle can be applied to inorganic species by forming a neutral complex that results in reversed-phase retention. 

Only one research group has illustrated the reversed phase retention behavior of solutes on zirconia-silica modified surfaces. In the study by Melo et al. on PMOS gamma-irradiated modified surfaces, the reversed phase retention behavior of a test mixture containing acetone, benzonitrile, benzene, toluene, and naphthalene was evaluated. The authors illustrated good resolution of the five-component text mixture as shown in Fig. 7. Retention, however, decreased after the stationary phase was washed with 5000 column volumes of base (pH 10). Despite the base washing, uniform peak shape was maintained, with only a slight reduction in resolution as discussed in Alkaline Stability above. 

It is widely recognized that sensitivity in either ESI or APCI is improved as the percentage of organic modifier is increased, due to the improved facility for desolvation. Adequate reverse-phase retention (/< = 1 to 5) is therefore important when LC-MS is performed. Temesi and Law studied the effect of LC mobile-phase composition on ESI response for a series of 35 compounds [18]. In their investigation, the signal response for positive-ion ESI was on average 10-20% higher in MeOH than ACN. 

The second general approach to overcome poor reverse-phase retention is to employ NP-LC. By comparison to RP-LC, the historic use of NP-LC for bioanalysis is negligible because of several limitations. The most notable limitation is the inability to perform reproducible gradient elution. Nevertheless, NP-LC can be a viable option for analytes too polar for RP-LC and produces far less back pressure. Perhaps, the most visible application of NP-LC is for the bioanalysis of chiral drugs [96]. This critical niche is largely the result of the frequent use of NP mobile phases with chiral stationary phases. 

While the driving force of reversed-phase retention is the interaction of the hydrophobic part of the analyte with the hydrophobic stationary phase, the interaction of the polar functional groups of the analyte with the mobile phase and with the residual silanols on a silka-based packing ate reqwnsible for the selectivity of a separatioiL This has two important consequences ... 

An extremely fruitful area of research and publication in the field of preformulation testing by LC is that of partition coefficient or HpophiHcity determination. Several standard procedures are available, including the relationship between partition coefficient and reversed-phase retention behavior (logP versus log ). Many variations in method and calculations have been proposed, such as comparison with an internal standard partitioning, variation in organic content of the mobile phase for the retention model, and rudimentary assay of organic and aqueous phase in the shake-flask experiment by LC. 

Heam and co-workers [460,461] have published a series of results relating amino acid hydrophobicity to reversed-phase retention of amino acids and peptides. The authors used wide-pore C4 or C]g columns and a gradient of IPA/acetonitrile/water (0.1% TEA) in generating results. A total of 1738 peptides covering 12 previously generated amino acid hydrophobicity scales were part of the study. Predicted and actual retention times of overlapping heptamers in myohemerythrin were presented. This study and previous studies cited therein offer excellent theoretieal and experimental bases for the predictive chromatography of amino acids and peptides. 

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