Mobile-phase modifier concentration

Nine different commercially available ceramides (Ceramide III, IIIB, and VI N-palmitoyl-, V-stearoyl-, V-oleoyl- and V-nervonoyl-D-sphingosine, and V-palmi-toyl- and V-lignoceroyl-DL-dihydrosphingosine) were analyzed on a 30°C Cj colunm (ELSD), drift tube 35 C, N2 at 1 bar) using a 15-tnin 95/5/0 — 35/5/60 acetonitrile/THF/n-propyl alcohol (all phases made lOmM in both triethylamine and formic acid) gradient. The authors studied the effects of mobile phase components (methanol and dichloromethane offered no advantages). Peak area was shown to be a strong positive function of mobile phase modifier concentration from 1 to 10 mM, whereas the overall retention was unaffected [1037]. 

FIGURE 13.4 Total ion chromatograms from the ID LC/MS analysis of a yeast ribosomal protein fraction separated using 0.1% TFA (Panel a) and 0.1% formic acid (Panel b) as mobile phase modifiers. TFA produced narrower, more concentrated, peaks for mass analysis that did not overcome the significant electrospray ionization suppression associated with using this modifier for LC/MS studies, resulting in an overall reduction in component intensities. 

Straub and Voyksner" developed a schematic approach for ionization optimization at a variety of pH values and organic modifiers. As a general rule, for every 5-10% increase of organic modifiers in the mobile phase, the mass response will double. A compromise approach must be adopted in choosing the type and ratio of organic modifier so that desirable separation and ionization can be achieved simultaneously. As a general rule, in either methanol- or acetonitrile-based mobile phases, a concentration range of 20% to 80% of either methanol or acetonitrile is optimal. 

When acetonitrile/methanol was used as the mobile phase, the concentration of methanol had to be increased to 50% before the TGs could be eluted in a reasonable length of time when ethanol was used as the polar organic modifier with ACN, resolution and detection were improved and only 20% ethanol was required. 

Buffer—Mobile phase modifier used to control pH. Usually salts of weak acids or bases, most effective at their pKa, where concentrations of ionized and unionized form are equal. 

Type IV includes chiral phases that usually interact with the enantiomeric analytes through the formation of metal complexes. There are usually used to separate amino acid enantiomers. These types of phases are also called ligand exchange phases. The transient diastereomeric complexes are ternary metal complexes between a transitional metal (usually Cu +), an amino acid enantiomeric analyte, and another compound immobilized on the CSP which is able to undergo complexation with the transitional metal (see also the ligand exchange section. Section 22.5). The two enantiomers are separated based on the difference in the stability constant of the two diastereomeric species. The mobile phases used to separate such enantiomeric analytes are usually aqueous solutions of copper (II) salts such as copper sulfate or copper acetate. To modulate the retention, several parameters—such as the pH of the mobile phase, the concentration of the copper ion, or the addition of an organic modifier such as acetonitrile or methanol in the mobile phase—can be varied. 

The effect of the structure, type, and concentration of the polar mobile phase modifier (mpm) on retention (k ) and stereoselectivity (ot) has been studied by a number of investigators (28-30). In general, an increase in the steric bulk of the mpm results in an increase in the observed enantioselec-tivity. An example of these studies is the work reported by Zief et al. (28) in which the solute was 2-trifIuoro-l-(9-anthryl)ethanol and the mobile phases were composed of hexane and either ethanol, 2-propanol, or f-butanol as the mpm. Of the three alcohols tested, the use of t-butanol as... 

The basic mobile phase used with the AGP CSP is composed of phosphate buffer and one or more modifiers. The type and concentration of the mobile phase modifier are extremely important and both k and a. can be altered by changing the mobile phase (9-11). 

I = 21 y = 0.394 r = 0.9476 F = 49.8 where log P is the hydrophobicity, bondrefr is the molecular refractivity, delta is the submolecular polarity parameter, ind indicator variable (0 for heterocyclics and 1 for benzene derivatives). Calculations indicated that PBD-coated alumina behaves as an RP stationary phase, the bulkiness and the polarity of the solute significantly influencing the retention. The separation efficiency of PBD-coated alumina was compared with those of other stationary phases for the analysis of Catharanthus alkaloids. It was established that the pH of the mobile phase, the concentration and type of the organic modifier, and the presence of salt simultaneously influence the retention. In this special case, the efficiency of PBD-coated alumina was inferior to that of ODS. The retention characteristics of polyethylene-coated alumina (PE-Alu) have been studied in detail using various nonionic surfactants as model compounds.It was found that PE-Alu behaves as an RP stationary phase and separates the surfactants according to the character of the hydrophobic moiety. The relationship between the physicochemical descriptors of 25 aromatic solutes and their retention on PE-coated silica (PE-Si) and PE-Alu was elucidated by stepwise regression analysis. 

Indeed, one usually deals with retention of analytes on the stationary phase, which reduces the concentration of the analyte in the moving zone of the mobile phase and requires additional amounts of the mobile phase to elute the retained portion of the analyte from the stationary phase. Cases of peak compression in chromatography are mostly coupled with the displacement of the adsorbed portion of the analyte (or analytes) by an auxiliary component of the mobile phase (a displacer or mobile phase modifier). In order to act like this, the latter must be adsorbed on the stationary phase even stronger than the displaced analytes. Only in frontal analysis can several weaker retained components of a mixture be obtained with an enhanced concentration, at the expense of the stronger retained component that functions as a displacer and remains in the column [175]. 

The enclosed CD-ROM contains everything needed to run MICHROM. This software is able to take the results obtained with a set of compounds and several compositions of hydro-alcoholic micellar phases, and calculate the affinity constants to predict the results for compositions of mobile phase (surfactant concentration, modifier concentration and pH). 

This is not the case when low levels of volatile modifiers are used. The greatest problems arise with the use of extremely volatile low-level mobile phase modifiers (MPMs), such as trifluoroacetic acid and triethylamine in reversed-phase solvent systems and ethyl acetate or dichloromethane components in normal-phase solvent systems. These low-level MPMs are typically used at the 0.1-1.0% v/v level. A continuous sparge over the course of the day will greatly reduce their concentrations via volatilization, and dramatic changes in peak retention and peak shape can result (see Fig. 1.13),... 

The robustness of a method is typically determined during the method development stage and is a measure of how consistently a method generates the same analytical result when small deliberate changes in operating parameters are made. Many times it is part of the intralaboratory development/validation process. For example, changeable parameters could include organic level in mobile phase, pH of mobile phase, concentration of mobile phase modifiers, and colutim. 

Yang and Thyrion [1072] studied peak shape and retention on a wide range of basic amine-containing compounds procaine, adiphenine, drofenine, nafronyl, tetracaine, meclofenoxate, 4-aminobenzoic acid, and caffeine. The basic mobile phase was 65/35 acetonitrile/water (20 mM acetate buffer at pH 4.5) and the column a Ci8 (A =260 and 280 nm). The effect of the identity (i.e., di-n-butylamine and triethylamine) and concentration of mobile phase modifier from 0 to 0.3% was studied. Retention times from 2 to 20 min resulted and asymmetry factors in general were <2.5. 

Casamenti et al. [1399] developed a method for screening 11 central nervous system drugs (phenobarbital, olanzapine, clozapine, risperidone, loxapine, haloperidol, imipramine, amitriptyline, fluoxetine, chlorpromazine, paroxetine) on a Cjg column (A = 230 nm) using a 20/11.7 water (0.4g tetramethylammonium perchlorate with 0.2 mL of 7% (m/m) HCIO4 to pH 2.8 with ammonia)/acetonitrile mobile phase. Keep in mind that perchlorates, when concentrated with some metals, are hazardous. Elution was complete in 35 min with good resolution for most compounds. Plots of the effects of mobile phase modifier level and percent acetonitrile on overall retention are presented. Linear ranges of 25-5000 ng/mL with detection limits of 10-250 ng/mL (analyte dependent) are reported. 

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