Effect of Mobile Phase on Retention

TABLE 5-1. NPC Solvent Strength (e ) and Selectivity of Varions Solvents Employed in HPLC 

Binary mixtures, however, have only limited abilities for controlling mobile-phase selectivity. Therefore, ternary and even quaternary mobile phases that contain two or more different polar solvents along with a nonpolar solvent are often used to achieve the required selectivity. If the ratio of the concentration of two polar solvents is constant but the sum of the their concentration is being changed with respect to that of the nonpolar solvent, the effect on retention is much the same as when the concentration of the single strong solvent 

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DS RP/polymer/IPAJV Various mobile phases Effect of mobile phase on retention of amoxicillin and clavulanate 126... 

C. Effects of Mobile Phase on Purine and Pyrimidine Retention Characteristics... 

Concentrations of moderator at or above that which causes the surface of a stationary phase to be completely covered can only govern the interactions that take place in the mobile phase. It follows that retention can be modified by using different mixtures of solvents as the mobile phase, or in GC by using mixed stationary phases. The theory behind solute retention by mixed stationary phases was first examined by Purnell and, at the time, his discoveries were met with considerable criticism and disbelief. Purnell et al. [5], Laub and Purnell [6] and Laub [7], examined the effect of mixed phases on solute retention and concluded that, for a wide range of binary mixtures, the corrected retention volume of a solute was linearly related to the volume fraction of either one of the two phases. This was quite an unexpected relationship, as at that time it was tentatively (although not rationally) assumed that the retention volume would be some form of the exponent of the stationary phase composition. It was also found that certain mixtures did not obey this rule and these will be discussed later. In terms of an expression for solute retention, the results of Purnell and his co-workers can be given as follows,... 

From a practical point of view the change in retention with solvent concentration will be greater at the lower concentrations of chloroform where interactions in both phases are being changed. At concentrations above 50%, however, interactions are only changing in the mobile phase and so the effect of solvent concentration on retention will be less significant. 

Also in this case the calculated (predicted) retention values showed good agreement with the experimental results. It has been concluded that pH gradient elution may enhance the separation efficacy of RP-HPLC systems when one or more analyses contain dissociable molecular parts [81]. As numerous natural pigments and synthetic dyes contain ioniz-able groups, the calculations and theories presented in [80] and [81] and discussed above may facilitate the prediction of the effect of mobile phase pH on their retention, and consequently may promote the rapid selection of optimal chromatographic conditions for their separation. 

A study of the effects of mobile phase composition on retention and selectivity of some carboxylic acids and amino acids was performed on a commercially available teicoplanin CSP, under analytical conditions, on the profile of the adsorption isotherms of the enantiomers and on the overloaded separation [87]. 

The results indicate that there is little effect of mobile phase composition on the retention volume of the solutes employed, or the silica dispersion. It should be pointed out, however, that there are no values Included for methanol between 0% and 10% methanol where adsorption of the methanol on the reverse phase surface significantly changes the value of Its retention volume. 

To determine the effect of mobile-phase composition on the sorption behavior of TGs on reverse-phase columns, two mixtures were employed acetonitrile/ethanol (80 20) and aceto-nitrile/methanol (80 20). A very rapid analysis resulted, with excellent peak shape and adequate resolution, when ethanol was used as the secondary solvent. Substituting an equal amount of methanol for ethanol resulted in increased solute retention, poor detector response, and asymmetrical peaks. Methanol forms a monomolecular layer on octadecyl-derived silica, which may explain the increase in solute retention caused by methanol. Also, the use of methanol would... 

FIGURE 5-51. Effect of mobile phase polarity on retention of phthalate plasticizers. Column juPorasil (silica, 10 p,m), 3.9 mm ID x 30 cm flow rate 2 mL/min detector UV at 254 nm. Mobile Phase (a) is 5% ethyl acetate in isooctane, (b) is 5% butyl acetate in isooctane. 

As reported by Zakaria and Brown (Zl), changing the mobile-phase ionic strength has little effect on the retention behavior of nucleosides and bases. The effect of mobile-phase ionic strength is much more pronounced in the case of ribonucleotides, where the compounds are ionized under the mobile-phase conditions. 

The present model has so f2ir assumed that interactions between molecules of solvent or solute in either phase can be ignored. Now we will examine the effects of these interactions on retention in various LSC systems. Equation (4) for the retention of a solute X in a mobile phase M recognizes intermolecular interactions in the mobile phase (n), but assumes that adsorbed-phase free energies ( ia) are not a function of intermolecular interactions within the adsorbed phase. We can recognize these adsorbed-phase intermolecular interactions by adding an energy term Eja" to Eq. (4) for each adsorbed species i ... 

Figure 4-18. Effect of mobile-phase pH on the retention of ionizable compounds. (Reprinted from reference 55, with permission.)...

S. Heinisch and J. L. Rocca, Effect of mobile phase composition, pH and buffer type on the retention of ionizable compounds in reversed-phase liquid chromatography Application to method development,/. Chromatogr. A 1048 (2004), 183-193. 

Both the solvent-interaction model (Scott and Kucera, 1979) and the solvent-competition model (Snyder, 1968, 1983) have been used to describe the effects of mobile-phase composition on retention in normal-phase liquid chromatography. The solvent interaction model on the one hand provides a convenient mathematical model for describing the relationship between retention and mobile phase composition. The solvent competition model on the other hand provides a more complete, quantitative description of the relative strengths of adsorbents and organic solvents used in normal-phase chromatography. 

The effect of methanol percentage in the mobile phase on retention times was studied by using a mobile phase consisting of 25.0 mM citrate buffer, pH=3.25, r=30.0°C, and 4.00 mM octane sulphonate and methanol at 42.0%, 45.0%, and 50.0%. Figure 5 shows that, with increasing percentages of methanol, retention times drop considerably and resolution decreases, but up to 50.0% methanol, this remains within acceptable limits. 

Figure 10.7 Effect of chain length on retention [reproduced with permission from G.E. Berendsen and L. de Galan, J. Chromatogr., 196,21 (1980)]. Conditions mobile phase, methanol-water (60 40). Peaks T— acetone 2 = p-methoxy-phenol 3 — phenol 4-— m-cresol 5 = 3,5-xylenol 6 = anisole 7 = p-phenylphenol.

Ion-pairing with alkyl sulfonates or sulfates enhances the retention of cationic amine moieties on the lipophilic stationary phase. The effects of mobile phase composition (ion-pairing agents, ionic strength or organic components) on the chromatographic and electrochemical behavior of catecholamines and their metabolites has been reviewed previously." ... 

Tugcu, N., Song, M., Breneman, C.M., Sukumar, N., Bennett, K.P. and Cramer, S.M. (2003) Prediction of the effect of mobile-phase salt type on protein retention and selectivity in anion exchange systems. Anal. Chem., 75, 3563-3572. 

Figure 2.10. Six RPLC chromatograms illustrating the effect of mobile phase solvent strength on solute retention and resolution. LC conditions were column Waters Symmetry C18, 3pm, 75x4.6 mm, 1 mL/min, 40°C, Detection at258nm. Mobile phase is mixture of acetonitrile (ACN) and water. Solutes were nitrobenzene (NB) and propylparaben (PP).

Xiao, Y.Z., Freed, A.S., Jones, T.T., et al. (2006) Protein instability during HIC Describing the effects of mobile phase conditions on instability and chromatographic retention. Biotechnol Bioeng, 93 (6), 1177-1189. 

Figure 7 Schematic representations of the effects of mobile phase composition, pressure, and temperature on retention in packed column SFC using binary fluids. Adjusting composition has the most effect on retention, but, since few of the lines cross, selectivity is only modestly affected. Temperature has little effect on retention but sometimes has a strong influence on selectivity. With binary fluids, pressure is a secondary control variable with modest influence on both retention and selectivity.

The effect of methanol percentage in the mobile phase on retention times was studied by using a mobile phase consisting of 25.0 mM citrate buffer, pH = 3.25,... 

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