Retention factor phosphate buffers

PARTITION COEFFICIENTS (P ), RETENTION FACTOR (K) AND MOBILITY DATA OF DYE INTERMEDIATES MEASURED WITH A PHOSPHATE-(TTAB) BUFFER AT PH 5.0 ... 

FIGURE 1.4 Dependencies of retention factors k on counterion (i.e., phosphate) concentration [X]. Experimental conditions Mobile phase, methanol-sodium dihydrogenphosphate buffer (50 50 v/v) (pHa 6.5 adjusted in the mixture with sodium hydroxide) flow rate, 1 mLmin temperature, 25°C CSP, 0-9-[3-(triethoxysilyl)propylcarbamoyl]-quinine bonded to silica [30] column dimension, 150 x 4 mm ID. 

Fio. 47. Retention factors of pteroyl-oliso-yL-glutamates as a ftinction of the eluent pH. The solid lines were calculated as diKussed in Bush et at. (204). Data were obtained for folic acid (FA) and oligo yglutainates containing one (Ft Glu,), three (Ft GIU ). hve (Ft GtU ), and seven (Ft Glur) utamyl residues. The data were obtained on 5 Partisil ODS-2 with 0.1 M phosphate buffers containing 696 (v/v) acetonitrile as eluent, 4S C, with detection at 254 nra. Reprinted with permission from Bush rf a/. (204). 

Fio. 38. Plot of the algorithm of the retention factor, k, and log P, the water-/i>octanol partition coefficient of eight amino acids. The chromatographic data were obtained on 3 ftm LiChrosorb kP-8, 230 x 4.6 mm i.d. eluierit 0.1 M aqueous phosphate buffer, pH 6.7, T 70 C. Eluites Trp, tryptophan Phe, phenylalanine Leu, leudne Val. valine Tyr, tyrosine Lys, lysine Ala, alanine Gly, glycine. Reprinted with permission from Molnar and Horvith QOS). 

FIGURE 15.7 Retention factor for naphthalenesulfonate as a function of the volume fraction of methanol in the mobile phase (10%, 25%, and 40%) and the pairing ion (octylsulfonate) concentration. Mobile phase phosphate buffer at pH = 2.1 and constant ionic strength (175 mM Na+). The theoretical line is obtained by combining Equations 15.38 and 15.39. Experimental data from Ref. [11]. (Reproduced with permission from Bartha, A. et al., J. Chromatogr., 506, 85, 1990.)... 

Inject 20 /d of solution (5). When the chromatograms are recorded under the prescribed conditions, the retention times are 5-vinyl-2-pyrro-lidone, about 18 min and vigabatrin, about 21 min. The test is not valid unless the resolution factor between the peaks corresponding to 5-vinyl-2-pyrrolidone and vigabatrin is at least 1.5 if necessary, adjust the concentration of the mobile phase (reduce the concentration of the phosphate buffer solution to increase the retention time of vigabatrin and increase the concentration of acetonitrile to decrease the retention time of 5-vinyl-2-pyrrolidone). 

Figure S-IS. Comparison of retention factors for 4-ethylpyridine as a function of pH using three different acidic modifiers. Column 150 x 4.6 mm Zorbax XDB-C18. Mobile phase acetonitrile - 10 mM. sodium phosphate buffer adjusted with trifluoroacetic acid, (10 90) flow rate 1.0 ml/min 25 C, UV 2.54 nm, sample 1 pi injection.

This equation predicts a linear relationship between the logarithm of the retention factor and the molal salt concentration, which is indeed commonly observed. Figure 13.1 shows the retortion behavior of several proteins on a silica-based polar bonded phase, propylacetamide. The eluents were various concentrations of ammonium sulfate in 0.1 M phosphate buffer, pH 7. The slope of the relationship is 2-2.S (LAnoI) for this stationary phase. For n-butyl-and phenyl-derivatized silica-based phases, values of between 1 and 2 (LAnoO were observed (5). 

Figare 13.1 Dependence of the retention factor on the molal salt concentration. The stationary phase is propylacetamide bonded to a silica with an average pore size of 25 nm. Mobile phase ammonium sulfate in 0.1 M phosphate buffer, pH 7. (Reprinted from Ref. 10, p. 3232, by courtesy of Marcel Dekker, New York, 1990.)... 

Although the target function of neutral CDs is to exert chiral selection in the EKC separation of optical isomers, they have often been used as auxiliary complex ligands as a means of improving resolution of closely eluting achiral positional and structural related compounds or to reduce significantly apparent retention factors. The separation of seven positional and structural naphthalenesulfonate isomers (pH 3.0 phosphate buffer/p-CD) " and five 2,4-dinitrophenylhydrazine (DNPH)-aldehyde derivatives in vehicular emission (pH 9.0 borate buffer/SDS/p-CD) are examples of neutral CD-mediated separations. 

This has been repeatedly confirmed in several studies see, for example. Figure 4.4. Here, the retention (bars) and separation factors (lines) of tricyclic antidepressants in acidic acetonitrile/phosphate buffer are shown. In the case of quite different retention factors depending on the hydrophobic character of the phases (differently strong interactions), apart from a few exceptions very similar separation factors result. Although the analytes are retained for different lengths of time on the stationary phases, they show a similar selectivity behavior. This is observed especially with neutral or - by means of the pH-neutralized molecules. In this case, mainly hydrophobic interactions between the analytes and the surface of the material dominate. These are not especially specific the individual differences of the phases with respect to selectivity do not come into effect. 

Fig. 1 Dependence of the retention factor, fe, on temperature, T. The solutes are (1) resorcinol, (2) phenol, (3) p-nitroaniline, (4) o-cresol, (5) nitrobenzene, (6) 2,6-xylenol, (7) 2,4-xylenol, (8) toluene, (9) 2-naphthol, (10) p-propylphenol, (11) p-butylphe-nol, and (12) p-amylphenol. Capillary 50 p,m I.D. X 570 mm (effective length, 500 mm) separation solution 50 mM SDS in 100 mM borate-50 roM phosphate buffer (pH 7.0) applied voltage 15 kV detection wavelength 214 nm.

An RP mechanism can be enforced by the pH value. If a uniform state of ionization of the analytes that are to be separated is generated by a given pH value (here, suppression of ionization), then their individual polar character recedes into the background, and the separation runs purely according to their different organic character. As mentioned above, apolar interactions result in a narrow range of selectivities, and the a values are small and similar. Figure 7 shows the retention and separation factors for the separation of tricyclic anti-depressants in an acidic phosphate buffer on several different phases. 

Fig. 8. On the bandwith of retention and separation factors by the separation of metabolites of tricyclic anti-depressants in acetonitrile/acidic phosphate buffer.

Chem. 1 Comparison according to all retention factors in acidic/alkaline methanol/acetonitrile phosphate buffers, S2. 

Chem. 6 Comparison according to retention and separation factors in neutral and alkaline methanoi/acetonitriie phosphate buffers, S2 (scores plot). 

In addition to the solvent, additives are often used in HPLC in low amounts (0.01-1%) to optimize performance and minimize undesired side effects, such as peak broadening. One of the prime factors determining retention is the charge state of the analyte that strongly depends on the pH. For this reason buffers (traditionally potassium phosphate buffers) are typically used to adjust the pH accurately. Note that in the case of HPLC-MS, nonvolatiles cannot be used, so typically ammonium acetate or formate buffer is preferred. Various other additives may also be used, such as trimethyl amine or trifluoroacetic acid, to suppress the interaction of analytes with the residual silanol groups of the stationary phase, thereby improving the resolution. 

---