Reversed Phase Applications

As mentioned before, two types of polysaccharide phases which are specifically designed for reversed-phase applications are currently on the market. This type of phase is extremely interesting for the direct injection of aqueous solutions (ionic products, plasma samples, etc.). Furthermore, the columns are useful in column-coupling techniques (for example, an octadecylsilica column followed by a polysacchar-ide column) to solve difficult separation problems. The manufacturer recommends on this type of column to use a mobile phase consisting of an aqueous solution of a sodium, ammonium or potassium salt in combination with methanol, ethanol or acetonitrile as the organic modifier. The choice of the cationic part of the salt seems not to have a significant effect on the separation. However, a significant difference in separation has been observed with various anions. Anions, such as ClO^ and PF^ in general show good separation. Also the salt concentration can strongly affect the retention time and the resolution.


Figure 5 Diastereomer separation. Column: 50x4.6 mm i.d. Chiralpak AD (amylose 3,5 dimethylphenyl carbamate). Flow rate: 1 mL min~1. Mobile phase: 1, ethanol; 2, ethanol-acetonitrile (95 : 5; v/v); 3, ethanol-acetonitrile (90 : 10; v/v); 4, acetonitrile.

Mobile-phase design and parameter optimization

Triethylamine salts are very popular as tailing reducers in reversed-phase applications. We have therefore investigated the usefulness of these salts on the poly-saccharide-type phases. Experience with cyclodextrin and derivatized cyclodextrin columns taught us that a salt concentration of 50 mmol in general is high enough to obtain good peak shapes for basic substances. Experimental work was started with this salt concentration. Afterwards, specific experiments performed to investigate the effect of the tailing reducer concentration on the chromatographic parameters confirmed that 50mmolL~1 was a good choice. In a concentration range between 10 and 50 mmol L_1 the highest resolution values were always measured for the highest salt concentration. However, this concentration also resulted in the strongest retardation of the compounds.

In a first set of experiments, the difference in chromatographic behaviour of a few products under normal-phase and reversed-phase conditions was investigated on a Chiralcel OJ-R and Chiralcel OD-R column. The columns were first equilibrated with ethanol. Thereafter, a mixture of n-hexane-2-pro-panol in a 70 : 30 volume ratio was pumped through the column until equilibrium was reached and the different products were analysed. After rinsing the columns with ethanol, the column was equilibrated with a mixture consisting of 70 vol% methanol and 30 vol% of a 50 mmol L_1 triethylamine solution in water adjusted with sulfuric acid to a pH value of 2.5.

In general the products are better retained when reversed-phase conditions are applied. It was also striking that for most of the investigated products there was a large difference in resolution values between the two modes of operation.

Type of tailing reducer Experiments on cyclodex-trin and derivatized cyclodextrin columns have shown us that, for the analysis of basic compounds, the anionic part of the tailing reducer has a clear effect on enantioselectivity. To investigate this effect, we selected some imidazole derivatives.

To perform the experiments, a 55 mmol L_1 solution of triethylamine in analytical grade water was prepared and 2 L portions of this solution were adjusted to a pH value of 2.5 with respectively hydrochloric, trifluoroacetic, methanesulfonic, camphor-sulfonic, sulfuric and phosphoric acid. After pH adjustment the solution was further diluted to obtain a solution which contains exactly 50mmolL~1 triethylamine.

As eluent a mixture composed of 70 vol% of methanol and 30 vol% of the triethylamine solution was used. On the Chiralcel OJ-R and on the Chiralcel

OD-R column, the retention values for phosphate or sulfate anion are markedly higher than the values measured for the other anions. The lowest values were in most cases measured for chloride as the counterion.

On both column types, the anionic part of the tailing reducer has a clear effect on enantioselectivity (Figure 6). In general, the best results are obtained for phosphate or sulfate as the anion, although for some products better results were obtained using another type of anion.

The Chiralcel OJ-R column is much more suitable for the separation of the investigated compounds than the Chiralcel OD-R column.

Type of polar modifier In experiments using a mixture of methanol and triethylamine in a 70 : 30 volume ratio, high retention times were observed for some compounds. Therefore, it was interesting to investigate the effect of the addition of acetonitrile on retention time and enantioselectivity. To perform these experiments, a mixture of 70 vol% methanol and 30 vol% triethylamine adjusted to pH 2.5 with phosphoric acid was prepared and thereafter mixed with different amounts of acetonitrile. The different chromatograms obtained during these experiments are shown in Figure 7.

As Figure 7 clearly illustrates, acetonitrile strongly affects retention of the investigated compound. Although the resolution steadily decreases with increasing acetonitrile content, the k' value of the second eluting peak could be reduced by a factor of 38 with a full baseline resolution of the enantiomers as a result.

Because the elution power of acetonitrile is significantly higher than for methanol, we generally prefer to start new experiments with a methanol-water-tail-ing reducer mixture. If the compounds of interest are too strongly retained with this eluent, methanol is systematically replaced by acetonitrile until an acceptable compromise between the retention factor and the resolution value is found.

pH In the analysis of ionizable compounds, pH can certainly have a strong effect on the chromatographic behaviour. We therefore investigated for the product series summarized in Table 2 the effect of pH variations in the range between 2.5 and 4.5 on a Chiralcel OJ-R using a mobile phase composed of meth-anol-acetonitrile and a 50mmolL~1 triethylamine solution in water adjusted to the desired pH value with phosphoric acid. For one of the products investigated, the different chromatograms obtained during these experiments are depicted in Figure 8.

Figure 8 shows that the retention factors steadily increase with increasing pH value, reaching

Figure 6 Effect of the anionic part of the tailing reducer on the resolution. Column: 250 x4.6 mm i.d. Chiralcel OJ-R (p-methyl-benzoyl cellulose). Flow rate: 1 mLmin~1. Mobile phase: 50 mmol L~1 triethylamine adjusted to a pH value of 2.5 with different acids-methanol (30 : 70; v/v).

maximum at pH 4 and decreasing again above this pH value. A similar pattern was also observed for the resolution value.

The different experiments that have been performed clearly indicate that, for ionizable compounds, pH can have an important effect on the enantioselectivity, which of course makes it a parameter to be investigated thoroughly during method development and optimization.

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