Acidic analyte retention dependence

In order for proper description of the acidic analyte retention versus the mobile-phase pH, the pH shift of the aqueous portion of the mobile phase must be taken into account. Plot of the retention factor of 2-4dihydroxyben-zoic acid versus two different pH scales [ pH (Figure 4-34, line A) and pH (Figure 4-34, line B)] is shown in Figure 4-34. A theoretical curve (Figure 4-34, line C) of the retention dependence versus pH of the mobile phase was constructed for 2,4-dihydroxy benzoic acid, based on its potentiometric pKa of... 

S.4.2 Acidic Compounds. Similar retention curves can be obtained for acidic components, but obviously their retention dependence will be the mirror image of that for basic analytes (Figure 4-17). 

Figure 4-17. General retention dependence of acidic analyte on the mobile-phase pH. The inflection point of the curve corresponds to the component pAi. ...

Figure 4-23. Effect of analyte pH on the retention dependencies of acidic, basic, and zwitterionic analytes.

The analysis of Dorzolamide HCl at pH 2 with phosphoric acid shows early elution. The addition of hexafluorophosphate to the mobile phase leads to an enhancement of the retention. Figure 4-59 is an overlay of Dorzolamide HCl chromatograms at four increasing PFe concentrations. As the concentration increased, peak tailing decreased, and peak efficiency and analyte retention increased. Figure 4-60 shows the effect of different counteranions on basic analyte retention and peak efficiency. Depending upon the desired selectivity between a neutral component and a charged basic analyte, a particular chaotropic counteranion could be employed. 

In NPC, analytes retentions generally increase in the following sequence alkane < alkenes < aromatic hydrocarbons = chloroalkanes < sulfides < ethers < ketones = aldehyde = esters < alcohols < amides carboxylic acids [16]. The retention also depends to some extent on the... 

Figure 2 demonstrates the effects of adding octane sulfonic acid to the injection solvent for a reverse-phase separation of pyridinium and deoxypyri-dinium, components of collagen, in rat urine. These polyamine containing compounds are protonated and poorly retained under the usual acidic or neutral mobile-phase conditions. The sample preparation method is simple dilution and does not afford the removal of salts from the sample. Therefore if the analytes were inadequately retained the sensitivity, as well as the method accuracy, would suffer. As the concentration of octane sulfonic acid is increased to 50 mM (Fig. 2a), both the retention time and the peak response for the analytes improve significantly. Litde improvement is obtained at higher concentrations of octane sulfonic acid and retention is not strongly dependent on the injection volume (Fig. 2b). To protect the ion source from the fouling effects of octane sulfonic acid in the injection solvent, a timed divert valve was inserted before the ion source to shunt the excess ion pair reagents to waste during the first few minutes of each injection.

These were all done with perchloric acid as the modifier. It is considered to be a strong chaotropic agent. Weak chaotropic counteranions will produce the same type of retention dependence, but the overall effect of weak chaotropes on the analyte retention is much less pronounced. 

The dependence of the retention on the degree of ionization is shown in Fig. 1 for both an acidic and a basic analyte. For both compounds, the retention changes by more than an order of magnitude. This is typical for most compounds, the change in retention between the ionized and the non-ionized form is of the order of 10- to 30-fold. The ionized form always has lower retention in RP-chromato-graphy. Therefore, the retention is lowest imder acidic conditions for a basic analyte and under basic conditions for an acidic analyte. On the other hand, high retention is observed when the analyte is in its neutral form, i.e. imder acidic conditions for acidic analytes and under basic conditions for basic analytes. There is a transition... 

In Fig. 1, we have shown that the retention of an analyte may depend strongly on the pH of the mobile phase. In order to maintain reproducible pH values, one needs to use buffers in all pH ranges except at very acidic or strongly alkaline pH values. Buffers are solutions of ionogenic compounds that contain a conjugated pair of a proton donor and a proton acceptor. Thus, they stabilize the pH against the addition of small amounts of acid or base [6]. Let us discuss an acetate buffer as an example. This buffer contains an equimolar amount of acetic acid, the proton donor, and acetate, the proton acceptor. The pH of such a solution in water is 4.75. If one adds small amounts of acid or base (below the total buffer concentra-... 

The retention of an analyte strongly depends on its ionization. As mentioned above, the non-ionized form of the sample has a much higher retention, up to a factor of 30 higher, than the ionized form. However, the difference is not the same for all analytes. For some analytes, the retention changes more, for others less. This is shown in Fig. 8, where we have plotted the retention times imder gradient conditions for more than 70 samples under basic versus acidic conditions. 

This strategy consists in the initial modification of the silica surface with organosilanes having suitable anchoring groups, which are either reactive themselves or can be additionally activated for the final attachment of the chiral selector. The choice of the proper silane will depend on the presence of suitable functional groups on the chiral entity to be fixed to the matrix. As macrocyclic antibiotics contain hydroxyl, amine, and carboxylic acid functionalities, they can be linked to the silica surface in a variety of different ways [7, 55]. The obvious drawback of the stepwise assemblage of chiral selectors on the silica surface is the eventual formation of additional polar or ionizable sites on the matrix, which may cause unselective retention of chiral analytes. 

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