Mobile Phase Design and Parameter Optimization

Different investigators have suggested that the mechanism of chiral recognition on cellulose and amylose derivatives is based on:

1. Hydrogen bonding

2. Dipole-dipole interaction

3. Charge transfer complex formation (n-n interactions)

4. Possible inclusion into chiral cavities or channels of the chiral stationary phase

Whatever the type of interaction involved, the mobile phase must be considered as a dynamic part of the system, capable of interacting with both the enan-tiomeric solute and the chiral stationary phase. For solutes where hydrogen bonding plays an important role in the selective chiral interaction process, proton-donating polar modifiers can compete with the solute for the hydrogen-bonding sites of the stationary phase. In other cases, n-n interactions between an aromatic moiety on the solute and the chiral stationary phase seems to be the most important interaction force.

Most of the separations on physically coated cellulose and amylose columns have been performed with mobile phases consisting of «-hexane or «-heptane as the major component, mixed with various aliphatic alcohols. The choice of solvent combination is principally based on recommendations for use by the manufacturer. Concerns about the stability of these columns combined with the relatively high cost discouraged the use of other solvent combinations than those recommended by the manufacturer. However, in the last few years, other aprotic solvents, e.g., acetonitrile, methyl-tertiary-butyl ether and ethyl acetate have been applied on certain types of the derivatized polysaccharide columns. Certainly in the field of preparative chromatographic enan-tiomer separations. These alternative solvents widen the application range of this type of stationary phase.

For method development on this type of phase screening experiments on 50 x 4.6 mm ID pre-col-umns filled with respectively Chiralcel OJ, Chiralcel OD, Chiralpak AD and Chiralpak AS are convenient. In use such column are rapidly equilibrated, give fast separations and have in many cases more than sufficient separation power for initial method development work. As a general rule, pure ethanol at a flow rate of 0.5 mL min"1 is used as the eluent of choice to perform the first experiments.

Pure ethanol is chosen based on the experience that, for a lot of products, below a certain amount of polar modifier in an «-hexane or «-heptane based mobile phase, the retention factors strongly increase. In general, the effect of the polar modifier on the retention factor decreases upon increasing modifier content. An indication that the competition between the solute and the polar modifier in the eluent for the hydrogen-bonding sites of the stationary phase is a saturable process and a maximum effect on the retention factor will be reached within a certain range of polar modifier concentration (for our type of compounds, mostly situated between 15 and 20%). If, in the screening experiments, the analytes are insufficiently retained or no separation is observed, ethanol is mixed with «-heptane or «-hexane in different ratios. Once a suitable «-heptane-ethanol ratio has been found to reach a k' value between 3 and 6, ethanol is replaced by the same molar amount of one of the other lower aliphatic alcohols (1- or 2-pro-panol, primary, secondary or tertiary butanol). In many cases, large effects on the resolution values can be observed when ethanol as polar modifier is replaced by another alcohol.

Experience shows that pure methanol or mixtures of ethanol and methanol or ethanol and 2-propanol are often very useful to improve a separation.

On these phases it has been observed that the retention factors of a range of 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid esters increased with increased chain length of the alcohol used for chromatography on a Chiracel OB column. This effect might be based on a reduced capability of the larger alcohols to compete with the solute for hydrogen-binding sites on the stationary phase.

The higher retention values measured for the branched alcohols compared to their corresponding linear analogues may be due to steric influences, which result in a reduced tendency of these alcohols to interact by hydrogen bonding with the polysacchar-ide phase.

The observed decrease in retention factor with increasing chain length of the ester group could be an indication that hydrophobic effects also contribute to the interaction mechanism between the solute and the chiral stationary phase.

For the lower members of the homologue series of the 2,3-dihydro-1,4-benzodioxin-2-carboxylic acid esters, practically no difference in resolution values can be observed between the different polar modifiers. Depending on the type of alcohol used, the resolution rapidly decreases for the pentyl ester and the higher homologues. Only for n-butanol as polar modifier, is the decrease in resolution value rather limited. For ethanol the lowest resolution values are observed for the whole product series. There are indications that, for the investigated solutes, hydrogen-bonding forces certainly play an important role in the chiral recognition process.

This type of experiment clearly demonstrates that, when n-hexane or n-heptane-alcohol mixtures are used, testing different alcohols as polar modifier during method development should be performed, because in many cases, large differences in enantioselec-tivity may be observed.

It is also interesting to note that, by changing the polar modifier phase, an inversion of the elution order can occur, as illustrated in the following example. On a Chiralcel OD column, it was possible to separate the investigated compound (R89439) in its two enan-tiomers using a mixture of n-hexane-ethanol in an 80 : 20 volume ratio. The desired enantiomer eluted as the first peak under these experimental conditions, as illustrated in Figure 2A. When the ethanol as polar modifier was partially replaced by methanol, a reversal of elution order was observed, as illustrated in Figure 2B.

Some investigators have suggested that binding a polar modifier to sites near the chiral cavities might alter the steric environment of these cavities. If the environment of these cavities changes, it can certainly have an influence on the steric fit of the chiral solutes in these cavities, which may in part be responsible for the observed phenomenon of reversal in elution order. It is furthermore interesting to note that a reversal of elution order can often be achieved by the changeover of a carbamate-type phase towards a ben-zoate-type phase. This knowledge can be very useful when a small amount of one enantiomer has to be determined besides a large excess of the other enan-

tiomer, because it is easier to quantify a small peak in front of a large one than in the opposite situation. Based on experience, it is also important to mention that sometimes even small variations in experimental conditions can have a tremendous effect on the resolution of enantiomers on this type of stationary phases.

The often observed strong influence of small differences in experimental conditions on the resolution, certainly has to be considered when robust and reproducible methods have to be developed on this type of phases, for regulatory (Good Laboratory (GLP), Good Manufacturing (GMP)) purposes.

An example of the use of aprotic modifiers, which can also affect the separation on these phases, is as follows. About 40 g of a diastereomeric mixture had to be separated into its four enantiomers. Very good separation was obtained on an amylose 3,5-dimethyl-phenyl carbamate (Chiralpak AD) column using pure ethanol, pure methanol or a mixture of ethanol and 2-propanol in a 90 : 10 volume ratio. Unfortunately, the solubility of the diastereomeric mixture in the alcohol used was very poor. We therefore decided to study the behaviour of ethanol-acetonitrile mixtures, because the solubility of the product was a lot better in acetonitrile.

The results of these experiments are summarized in Table 2 and graphically represented in Figure 3.

As illustrated in Figure 3, the addition of an aprotic modifier to the polar organic solvent has a clear effect on the retention behaviour and the enantioselectivity, especially for the most retained enantiomers. The investigated solute molecule contains different hydrogen-bonding groups, which can strongly interact with the carbamate functionality of the stationary phase. Combined with poor solubility of the substance in the lower molecular weight alcohols, this makes these solvents less suitable to displace the solute from the stationary phase and probably explains the high retention values observed for pure methanol or ethanol.

The addition of acetonitrile improves the solubility of the product. This at first simplifies the transfer of the solute into the mobile phase and furthermore gives the protic solvent a better chance to compete for the hydrogen-bonding sides of the stationary phase, which may explain the observed systematic decrease in retention factors with increasing acetonitrile concentration. Below a certain concentration of protic solvent in the eluent, the strong hydrogen-bonding properties of the solute predominate again, resulting in an increase in retention factor. Using a mixture of ethanol and acetonitrile in a 30: 70 volume ratio allowed the separation of the diastereomeric mixture in its four enantiomers without difficulty.

Figure 2 Effect of mobile-phase composition on the elution order. Column: 250 x 4.6 mm. Chiralcel OD (cellulose 3,5 dimethylphenyl carbamate). Mobile phase: (A) n-hexane-ethanol (80:20; v/v); (B) n-hexane-ethanol-methanol (80:5:15; v/v). Flow rate: 1 mL min-1. Sample: R89439 (racemate plus pure enantiomer).

Table 2 Effect of the addition of acetonitrile on retention behaviour and enatioselectivity

Acetonitrile

(%)

k'1

k'2

k'3

k'4

a (1-2)

a (3-4)

0

2.94

6.43

11.97

28.24

2.187

2.359

5

1.80

3.6

8.67

14.30

2

1.649

10

1.25

2.7

7.55

10.25

2.16

1.358

15

0.95

2.08

6.34

8.03

2.189

1.267

20

0.66

1.59

5.47

6.62

2.409

1.210

30

0.50

1.25

5.26

5.26

2.50

1.0

40

0.46

1.1

5.05

5.05

2.391

1.0

50

0.45

1.04

5.72

5.72

2.311

1.0

70

0.90

1.73

9.38

12.05

1.922

1.285

80

1.31

2.67

16.86

27.0

2.038

1.601

Basic or acidic additives can also have major effects on the separations. Thus, for some basic or acidic substances, it is often necessary to add respectively a base or an acid to improve the peak shape. Based on the manufacturer's recommendations, it is possible to use up to 1% of di- or triethylamine to reduce the tailing of basic substances. Acetic or trifluoroacetic acid can be used for the analysis of acidic substances.

The usefulness of these mobile-phase additives, is shown in the following example. For the enantiomer separation of [( $ ) 2,6-dichloro-a-(4-chlorophenyl)-4-(4,5-dihydro-3,5-dioxo-1,2,4-triazin-2-(3H)-yl) benzene acetonitrile] (diclazuril) initially a method was used in which the acidic NH-group in the 3,5-dioxo-1,2,4 triazin part of the molecule was methylated with diazomethane. The derivatized product

Figure 3 Effect of acetonitrile on (A) the retention factor; (B) enantioselectivity. Column: 250 x 4.6 mm Chiralcel AD (amylose 3,5 dimethylphenyl carbamate). Mobile phase: ethanol-acetonitrile in different ratios. Flow rate: 1 mL min"1. Filled circles, k'1; triangles, k'2; open circles, k'3; squares, k'4.

Figure 3 Effect of acetonitrile on (A) the retention factor; (B) enantioselectivity. Column: 250 x 4.6 mm Chiralcel AD (amylose 3,5 dimethylphenyl carbamate). Mobile phase: ethanol-acetonitrile in different ratios. Flow rate: 1 mL min"1. Filled circles, k'1; triangles, k'2; open circles, k'3; squares, k'4.

could easily be analysed on an amylose 3,5 dimethylphenyl carbamate (Chiralpak AD) column, using ethanol-w-hexane in an 80 : 20 volume ratio.

A chromatogram of this separation is illustrated in Figure 4A.

As can be seen from Figure 4A, reaction with dia-zomethane results in different reaction products, with both nitrogen alkylated and oxygen alkylated compounds observed. Because direct analysis of this product on the cellulose- or amylose-based stationary phases, using the classical eluents, was not possible, due to the presence of the acidic NH-group in the 3,5-dioxo-1,2,4-triazin part of the molecule (which resulted in a retardation of the substance on the stationary phase), the effect of the addition of triflu-oroacetic acid was examined. The result of such an experiment on a micro-LC column is shown in Figure 4B.

The use of an amine as tailing reducer is illustrated in the following example. A few grams of a chiral amino alcohol had to be separated in its two enantio-mers. Only partial separation could be obtained on a Chiralcel OD (cellulose 3,5-dimethylphenyl carbamate) column using a mixture of «-hexane and 2-propanol in a 70 : 30 volume ratio. Because a severe tailing was observed, we investigated the effect on the addition of triethylamine as mobile-phase additive. A small quantity of 0.1 vol% of triethylamine improved the peak shape and the resolution. However, a much better result was observed when the triethylamine content was increased to 0.5 vol%. Thereafter, triethylamine was replaced for the same

Figure 4 (A) Separation of diclazuril after derivatization with diazomethane. Column: 250 x 4.6 mm i.d. Chiralpak AD (amylose 3,5 dimethylphenyl carbamate). Mobile phase: ethanol-n-hexane(80 : 20; v/v). Flow rate: 0.5 mL min~1. Detection: UV(290 mm). Injection volume: 10 ^L. Temperature: ambient. Peak 1: Enantiomers of A/-methylated product. Peaks 2 and 3: Enantiomers of O-methylated product. (B) Separation of diclazuril using trifluoroacetic acid as mobile-phase additive. Column: 150x0.32 mm i.d. Chiralpak AD. Mobile phase: ethanol-1 % trifluoroacetic acid. Flow rate: 5 ^L min~1. Detection: UV (280 nm). Injection volume: 60 nL. Temperature: ambient.

amount of diethylamine. The addition of 0.5% die-thylamine resulted in the highest resolution value.

Therefore, the first experiment on the preparative column was performed with a mobile phase containing 0.5 vol% of diethylamine. The obtained result was rather poor. The enantiomers eluted as relatively broad peaks, which were only partially resolved. Because for preparative chromatographic application we prefer to work with triethylamine instead of di-ethylamide, the method was therefore further optimized using triethylamine as tailing reducer. To reach maximum resolution on the preparative column, the amount of 2-propanol had to be reduced from 30% to 10 vol%, while the triethylamine concentration had to be increased to 2 vol%.

Using this method, it was possible to inject 250 mg of the product. The optimized method enabled sufficient amount of the pure enantiomers to be prepared in a reasonable amount of time.

The solute structure will also clearly have a direct effect on the separation. Thus, the chiral recognition process on polysaccharide phases results from differences in the summation of binding energies originating from:

1. hydrogen bonding

2. dipole-dipole interactions

3. charge transfer (n-n) complex formation

4. steric interactions

It is not possible to generalize which type of interaction forces plays the key role in the solute-chiral stationary phase complex formation. Hydrogen bonding certainly has a strong role in the selective chiral interaction process.

Based on the knowledge that on polysaccharide phases different mechanisms play a role in the chiral recognition process and small variations in experimental conditions or solute structure can strongly affect the enantioselectivity, it is clear that small changes in the molecular structure of a specific type of compounds that are in general well separated can often be a challenge to find an acceptable separation method for some of the members of such a product series.

The effect of small structural changes on the retention behaviour and the enantioselectivity is illustrated in the following examples.

In the first example, a few imidazole derivatives bearing an ester function on the imidazole ring were investigated on a Chiralcel OC (cellulose phenyl carbamate) as well as on a Chiralcel OD (cellulose 3,5-dimethylphenyl carbamate) column, using n-hexane-2-propanol as the mobile phase. On the Chiralcel OC column a mixture of n-hexane-2-pro-panol in a 90 : 10 volume ratio was used. Because with this mobile-phase composition the products were not sufficiently retained on the Chiralcel OD column, the amount of polar modifier had to be reduced to 5 vol% on this column type. On both column types the retention factor strongly decreased with increasing chain length of the eater group attached to the imidazole ring. Also steric effects seem to play a role, because on both columns the smallest retention value is measured for the 2-propyl ester.

When the resolution values are considered, a clear difference was to be observed between both column types. For the 1-propyl and 2-propyl esters, baseline resolution is obtained on both stationary phases. However, the observed differences for the ethyl and methyl ester are striking. On the Chiralcel OC column the ethyl ester is still baseline-resolved, while the methyl ester is only partially separated, whereas on the Chiralcel OD column, the methyl ester is very well separated and the ethyl ester did not display any separation at all. Certainly there is an indication that small but nevertheless influential contributions play an important role in the chiral recognition process, which of course makes it not always that easy to predict whether a new product in a series of comparable structures will be separated or not on a particular type of stationary phase.

The next example also demonstrates that it is not always easy to predict whether a product within a series will be separated on a certain type of polysac-charide phase.

Some tetramisol derivatives were investigated on three different carbamate-type phases (Chiralcel OC, Chiralcel OF and Chiralcel OD) using «-hexane-2-propanol in a 70 : 30 volume ratio. For the investigated compounds, the highest retention is measured on the Chiralcel OF (p-chlorophenyl carbamate) column, while the smallest values are observed on the Chiralcel OD column. However, on the three different column types the meta-substituted derivative has always the most strongly retained.

Although the products were most strongly retained on the Chiralcel OF column, not one of the 10 investigated substances was fully baseline-resolved. Two of the ort^o-substituted compounds and the unsubstituted tetramisol were best resolved on the Chiralcel OC column, while for the meta- and para-substituted derivatives the highest resolution value was in general measured on the Chiralcel OD column.

It is quite clear therefore from the different examples that small changes in solute structure or experimental conditions can have a strong influence on the chromatographic behaviour. This phenomenon makes it often difficult to predict whether a product will be separated on a certain type of polysaccharide phase or not. Therefore, even when a good mobilephase composition has been found on a particular column, it is always advisable to test this solvent mixture on another column of the same type (carba-mate or benzoate).

Achiral derivatization can be exploited to improve resolution as it is well known from experience that compounds bearing an ionizable group, e.g. a car-boxyl, hydroxyl or amino group, are often poorly resolved on the polysaccharide type of phases. De-rivatization of these functional groups to the corresponding ester, carbamate or amide derivatives frequently improves the separation.

For alcohol, for example, an esterification reaction with a para-substituted benzoic acid chloride has proven to be effective in solving difficult separation problems. The effect of an esterification reaction on the retention behaviour and the enantioselectivity is illustrated by means of the next examples.

The hydroxyl function of two completely different compounds was derivatized to yield the following

types of esters:

1. Benzoyl

2. p-Fluorine

3. p-Chlorine

4. p-Bromine

5. p-Trifluoromethyl

Benzoyl

6. p-Methyl

7. p-Methoxy

8. p-Nitro

9. p-Cyano

10. 1-Naphthoyl

11. 2-Naphthoyl

12. 9-Antracoyl

The different esters were respectively investigated on a Chiralcel OD, Chiralcel OJ, Chiralpak AD and Chiralpak AS column using pure ethanol as the elu-ent.

These compounds were retained more on the ben-zoate type of phase than on the carbamate type of stationary phase. Also, the resolution is significantly higher on the Chiralcel OJ column than on the other columns. The favourable results on the benzoate type of column inspired us to investigate the difference between ethanol and methanol as the mobile phase for this type of compound. For methanol much higher retention factors are measured than when ethanol is the eluent. In general, about twice as high resolution values are measured for methanol.

In the foregoing example, the best results for the different esters were obtained on a benzoate type of column. This is certainly not a general rule. Another alcohol [4,4-dimethoxy-1-(phenylmethyl)-3 piperidinol] was derivatized to yield a similar series of esters. This homologue series was also investigated on the four different types of polysaccharide phases, using ethanol as the eluent.

On the Chiralcel OJ column only the 2-naphthoyl derivative was partially separated. On the Chiralcel OD column only the 2-naphthoyl and the 9-an-thracoyl ester were partially resolved, while on the Chiralpak AS column only the 9-anthracoyl ester showed partial resolution. However, on the Chiral-pak AD column most of the products were separated. What is striking in this particular example is the partial separation of the native alcohol, while the benzoyl- and p-fluorobenzoyl esters do not show any separation at all.

For further synthesis applications, alcohols are frequently converted to the mesylated and tosylated derivative. When we are confronted with the preparative chromatographic separation of a racemic alcohol, we always investigate the mesylated or tosylated alcohol before carrying out other derivatization work, because we have experienced that in many cases, these compounds are easier to separate than the native alcohol. For very difficult separations we eventually had to synthesize the naphthylsulfonyl derivative.

Temperature is well known to affect chiral isolation. Thus, on chiral stationary phases, specific interaction mechanisms are involved in the separation process. Therefore, this type of phase often displays slow mass transfer characteristics.

Temperature, together with the mobile-phase velocity, certainly has to be considered as a factor that can be used to influence this mass transfer process.

In the next example, we examined the combined effects of temperature and flow velocity variations on enantioselectivity, column efficiency and resolution. The enantiomers of the investigated racemate were difficult to separate. Only partial separation could be achieved with w-hexane-ethanol in a 90 : 10 volume ratio on a Chiralcel OJ column, using our standard experimental conditions of flow rate and temperature. To investigate this separation problem further, the temperature was varied between 5 and 40°C and flow rates between 0.25 and 2 mL min-1 were tested.

A good linear relationship between the logarithm of the a value and the temperature has been observed. The a value steadily increased with decreasing temperature. However, the most significant parameter to indicate the separation between two products is the resolution value. This parameter is determined by both thermodynamic and kinetic contributions.

Although two completely different measuring principles have been used, comparable patterns are observed for the two resolution values. Both figures indicate that only for temperatures below 15°C and a flow rate of 0.25 mL min"1 can a nearly baseline separation be obtained. Although it was not possible to obtain a full baseline separation of the enantio-mers, the optimized analysis method was sufficiently accurate to follow up the investigations that were performed to develop a stereospecific synthesis method.

As this example indicates, during method development and optimization experiments, the factored temperature certainly has to be investigated. Besides essential information for analytical purposes, it also gives chromatographers performing preparative chro-matographic separations a good idea of whether it is possible to work at higher temperatures without losing efficiency. Due to the limited choice of possible mobile-phase compositions, the major problem one has to deal with in preparative chromatographic work on polysaccharide phases is the solubility of the product to be separated in the eluent used. Because solubility in most cases increases with increasing temperature, the ability to work at higher temperatures often improves throughput in preparative chromatographic separations.

The experiments performed also demonstrate that the flow velocity is a parameter that can be used to optimize a separation process.

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