Derivatized Cyclodextrins

Native cyclodextrin columns cannot be used effectively for the separation of enantiomers under normal phase chromatographic conditions. On the other hand, different naturally occurring chiral molecules that have been derivatized are extensively and very successfully used in the normal phase mode of operation.

Triacetylcellulose, obtained by heterogeneous acylation of cellulose, was one of the first commercially available derivatives. However, the later developed and commercialized aromatic cellulose and amylose derivatives (benzoates, carbamates), compared with triacetylcellulose, are much more universally applicable. Owing to the broad applicability of the cellulose and amylose derivatives similar cyclodextrin-based stationary phases have been developed.

In our laboratories we investigated the possibilities of the derivatized cyclodextrin columns under normal as well as reversed-phase chromatographic conditions. We also compared these phases with the corresponding cellulose derivatives.

Our first experiments on the functionalized cyclo-dextrin phases were performed under normal phase conditions. A series of 42 products covering a broad range of organic chemistry were investigated on the S-naphthylethyl carbamate, the para-toluoyl and 3,5-dimethylphenyl carbamate cyclodextrin derivative, using n-hexane-2-propanol in different ratios as the mobile phase.

The results obtained were rather poor. Of the whole test series only six products were partially or completely resolved on the 3,5-dimethylphenyl car-bamate column. The situation was even worse on the two other columns tested. Therefore, we decided to switch immediately to the reversed-phase mode of operation. The initially used test series of 42 products was first investigated on the three above-mentioned cyclodextrin columns with a mobile phase consisting of 0.5% ammonium acetate in water and methanol in a 30-70 volume ratio. This mobile phase composition was chosen after some preliminary experiments, which demonstrated that higher water content caused a tremendous increase in retention times. Compared with the results under normal phase conditions, the number of products separated and the degree of separation were much better on all the investigated column types. Because earlier experiments on the native cylcodextrin phases have demonstrated that an acidic pH generally results in better separations, a 20 mM tetrabutylammonium hydrogensulfate solution (pH 2.3) was thereafter used as tailing reducer.

Owing to the well-known reversed-phase effect of retention decrease with lowering pH values, the methanol content in the mobile phase had to be reduced to 40 vol% instead of the 80 vol% used in the experiments with ammonium acetate as tailing reducer. Under these experimental conditions, the largest number of products was separated on the S-naphthylethyl carbamate column, although the results on the other two columns only differed slightly. It was also interesting to observe that some products, which could not be separated on native cyc-lodextrin, were completely resolved on one of the derivatized phases. In the next set of experiments, a 20 mM solution of respectively tetramethyl-, tetraethyl- and tetrabutylammonium hydroxide was adjusted with sulfuric acid to a pH value of 2.5 and used in combination with 70 vol% of methanol as the mobile phase. The effect of the cationic part of the tailing reducer is not clear. With tetraethylam-monium hydroxide the largest number of products are partially or completely resolved, but the resolution values are in general somewhat higher with tetramethylammonium hydroxide, although the differences with the two other alkylammonium hydroxides are minimal. Only for one member of the test series was tetramethylammonium hydroxide required to obtain partial resolution.

On native cyclodextrin columns, we could clearly demonstrate the influence of the anionic part of the tailing reducer. Therefore, a similar test was done on the 3,5-dimethylphenyl carbamate cyclodextrin derivative, using 20 mM tetramethylammonium hydroxide adjusted to pH 2.5 with respectively triflu-oroacetic, hydrochloric, phosphoric, (d)-camphor-sulfonic and sulfuric acids, (d)-Camphorsulfonic acid has been deliberately chosen to investigate eventual additional effects by introducing chirality in the mobile phase. After pH adjustment, the aqueous phase was mixed with methanol in a 30-70 volume ratio. Some products were also tested with a 50-50 mixture of aqueous phase and methanol. Comparable with the observations on the native cylcodextrin column, and also on the functionalized cyclodextrin, pH adjustment with sulfuric acid resulted in the largest number of separations. For all the other acids, the number of partially or completely resolved products dropped to about 50% or less of the value observed for sulfuric acid. However, three products which could not be separated with one of the different acids tested were partially resolved when (^)-camphorsul-fonic acid was used to adjust the pH of the tetramethylammonium hydroxide solution.

Comparison of derivatized cyclodextrins and the corresponding cellulose derivatives Because derivatized cellulose and amylose columns are extensively used in our laboratories for both analytical and preparative chromatographic applications, it seemed worthwhile to compare these phases with the corresponding cyclodextrin derivatives. At present only two cellulose phases are commercially available which can be used equally well under reversed-phase and normal phase conditions, namely Chiralcel® OD-R (3,5-dimethlyphenyl carbamate and Chiralcel® OJ-R (para-methylbenzoate) (Daicel, Japan). We compared these phases with Cyclobond®-DMP and Cyclobond®-PT columns (Advanced Separation Technologies), respectively.

The first experiments were performed under normal phase conditions, using n-hexane-2-propanol in a 70-30 ratio as the mobile phase. If products eluted too fast with this mobile phase composition, the amount of 2-propanol was reduced to respectively 20 or 10 vol%. A total of 21 different products were examined. The results of these experiments are summarized in Table 3.

From this data it is clear that for the investigated product classes the cellulose derivatives are far superior compared to the corresponding cyclodextrin phases when normal phase conditions are applied. We thereafter examined the same product series under reversed-phase conditions using a mixture of 70 vol% of methanol and 30% of a 50 mM triethylamine solution adjusted to pH 2.5 with sulfuric acid. The results of these experiments are summarized in Table 4.

When we compare this data with the results obtained under normal phase conditions, we have to conclude that in the reversed phase mode of

Table 3 Derivatized cyclodextrins versus the corresponding cellulose derivatives under normal phase conditons

Column type

Good separa

Partially

Total

tion 3 > 0.90

resolveda

Number %

Number %

Number %

Chiralcel OD-R

9 42.9

6 28.6

15 71.4

Cyclobond I-DMP

2 9.5

3 14.3

5 23.8

Chiralcel OJ-R

13 61.9

5 23.8

18 85.7

Cyclobond I-PT

- -

2 9.5

2 9.5

aFor the partially resolved peaks, the resolution on the cellulose derivatives is always much higher than on the cyclodextrin derivatives.

aFor the partially resolved peaks, the resolution on the cellulose derivatives is always much higher than on the cyclodextrin derivatives.

Table 4 Derivatized cyclodextrins versus the corresponding cellulose derivatives under reversed phase conditons

Column type

Good separation 9 > 0.90

Partially resolved

a

Total

Number %

Number

%

Number

%

Chiralcel OD-R

2 9.5

10

47.6

12

57.1

Cyclobond I-DMP

2 9.5

5

23.8

7

33.3

Chiralcel OJ-R

14b 66.7

-

-

14

66.7

Cyclobond I-PT

- -

3

14.3

3

14.3

aFor the partially resolved peaks, the resolution on the cellulose derivatives is always much higher than on the cyclodextrin derivatives.

bAll products are fully baseline resolved (3 = 1.00).

aFor the partially resolved peaks, the resolution on the cellulose derivatives is always much higher than on the cyclodextrin derivatives.

bAll products are fully baseline resolved (3 = 1.00).

operation fewer products are separated on the cellulose derivatives, although on the Chiralcel® OJ-R column all separated products are fully baseline resolved. The smallest resolution value equals 2.5 while the largest value is greater than 12.5, while under normal phase conditions the highest resolution value observed equals 6.3.

On the cyclodextrin derivatives a few more products are separated but the increase in number is certainly not spectacular. Furthermore, in many cases where partial resolution has been indicated in the table the chromatograms only showed a small deviation in the peak shape, indicating the early beginning of separation.

For a series of products which under comparable experimental conditions are all very well separated on the native cyclodextrin column, the results on the different cyclodextrin and cellulose derivatives using 50 mM triethylamine adjusted to pH 2.5 with sulfuric acid and methanol in a 30-70 volume ratio as mobile phase are illustrated in Figure 10.

Because only one substance of the test series is separated on the Cyclobond-PT column, and most of the products are only partially resolved on the Cyclobond-DMP column, while all these products are perfectly baseline resolved on native cyclodex-trine, it is clear that other parameters must play a role in the chiral recognition process on the derivatized phases.

As a general conclusion it can be stated that for the type of substances investigated the derivatized cyclo-dextrin columns are, in both modes of operation (normal as well as reversed phase), less universally applicable than the corresponding cellulose derivatives.

Hydroxypropyl-/-Cyclodextrin Derivative

A hydroxypropyl-/-cyclodextrin column (experimental phase of the Chromatography Research group of Merck Darmstadt) in the reversed-phase mode of operation has been extensively investigated. The result on this type of material were completely comparable with the data obtained on the native cyclodextrin columns. However, for the whole range of products the degree of separation was in general

Figure 10 Derivatized cylcodextrin columns versus the corresponding cellulose derivatives. Experimental conditions: column: 250 mm x 4.6 mm ID (Cyclobond®-DMP, Cyclobond®-PT, Chiralcel® OD-R, Chiralcel® OJ-R); mobile phase: 50 mM triethylamine adjusted to pH 2.5 with sulfuric acid-methanol (30-70, v/v); flow rate: 1 mL min~1.

Figure 10 Derivatized cylcodextrin columns versus the corresponding cellulose derivatives. Experimental conditions: column: 250 mm x 4.6 mm ID (Cyclobond®-DMP, Cyclobond®-PT, Chiralcel® OD-R, Chiralcel® OJ-R); mobile phase: 50 mM triethylamine adjusted to pH 2.5 with sulfuric acid-methanol (30-70, v/v); flow rate: 1 mL min~1.

better on the hydroxypropyl column. To investigate new products, we therefore always start our experiments on a hydroxypropyl-^-cyclodextrin column instead of using the classical native ^-cyclodextrin type of material.

y-Cyclodextrin

A test series of 28 products, which also have been investigated on ^-cylcodextrin, have been examined on a y-cyclodextrin column using a mobile phase consisting of 80 vol% of a 50 mM triethylamine solution in water adjusted to pH 2.5 with sulfuric acid and 20 vol% of methanol. On the ^-cyclodextrin column, 22 products were partially or completely resolved. On the y-derivative only nine products could be resolved.

Compared with the results obtained on the ^-cyclo-dextrin column, it is clear that from a general point of view the y derivative is less suitable for the separation of the investigated product series. Nevertheless, it remains an additional tool that might help to solve a separation problem when experiments on other types of cyclodextrin columns fail.

Dynamically Generated Cyclodextrin Phases

Instead of using the commercially available chemically bonded cyclodextrin phases, it is also possible to perform enantiomer separations on a reversed-phase column after addition of cyclodextrin or cyclodextrin derivates to the mobile phase. A number of experiments have been performed with hydroxypropyl-^-cyclodextrin as mobile phase additive. This derivative was initially chosen because it is readily soluble in water.

To investigate the possibilities of a dynamically generated cyclodextrin phase, a test series of 22 products was examined on three different types of reversed-phase packing material. RP Select B (Merck), Hypersil BDS (Shandon) and Aluspher RP Select B (Merck) were selected as stationary phases. An aqueous solution containing 50 mM triethylamine and 50 mM of hydroxypropyl-^-cyclodextrin adjusted to pH 3 with sulfuric acid combined with methanol in an 80-20 volume ratio was used as the mobile phase.

Compared with the data obtained on a chemically bonded hydroxypropyl-^-cyclodextrin column using the same eluent, the chemically bonded column gives, in general, better results than the dynamically coated reversed-phase materials. Furthermore, alumina as stationary phase matrix seems to be less effective. However, in a few exceptional cases the Aluspher Select B column gives as good or even better results than the silica-based materials.

Cyclodextrin Phases in Preparative Chromatographic Applications

The importance of preparative chromatographic enantiomer separations in industry is continuously growing. Therefore it was very interesting to investigate the usefulness of cyclodextrin phases in preparative chromatographic applications.

On an experimental hydroxypropyl-^-cyclodextrin phase (Merck Darmstadt), several products which showed a good resolution on the corresponding analytical material were investigated on a preparative scale. An example of a preparative chromatographic separation is illustrated in Figure 11.

On the hydroxypropyl-^-cyclodextrin phase, in general a loading capacity of 2mgg~1 of packing material was used. However, in some other cases we were able to load up to 4 mg of product per gram of stationary phase, which from a preparative chromatographic point of view certainly can be considered as a reasonable value for this type of application.

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