Determination of Formation Constant of CSEnantiomer Complex

The present technique has an advantage over conventional HPLC by allowing the determination of the formation constant (Kf) for the CS-enantiomer complex which is useful for developing the CS for chiral chromatography.

In a schematic view of the portion of the separation column in Figure 5, enantiomers (A+ and A_) are partitioned between the organic stationary phase (upper half) and the aqueous mobile phase (lower half). In the organic stationary phase enantiomers form a CS complex [CSA+] according to their formation constants (Kf ). In this situation:

Figure 3 Preparative separation of DNB-leucine racemate by high speed CCC. Experimental conditions: solvent system: hexane/ethyl acetate/methanol/10 mM HCl (6 : 4 :5 : 5) where the organic stationary phase containing CS at 10 to 60 mM as indicated; samples: ( + )-DNB-leucine 125-1000 mg dissolved in 10-45 mL of solvent. (For other conditions, see the Figure 1 caption.)

Figure 3 Preparative separation of DNB-leucine racemate by high speed CCC. Experimental conditions: solvent system: hexane/ethyl acetate/methanol/10 mM HCl (6 : 4 :5 : 5) where the organic stationary phase containing CS at 10 to 60 mM as indicated; samples: ( + )-DNB-leucine 125-1000 mg dissolved in 10-45 mL of solvent. (For other conditions, see the Figure 1 caption.)

where D0 is the partition coefficient of the analyte in a CS-free solvent system (this is also called the parti tion ratio) and D ± is the partition coefficient in a CS-containing solvent system. From these equations, the partition coefficient of the analyte is given by the following equation:

Figure 4 Separation of DNB-leucine racemate by pH-zone-refining CCC. Experimental conditions: apparatus: see the Figure 1 caption; solvent system: methyl t-butyl ether/water; stationary phase: upper organic phase to which trifluoroacetic acid (40 mM) and CS (40 mM) were added; mobile phase: lower aqueous phase to which aqueous ammonia was added to 20 mM; sample: (+)-DNB-leucine 2 g; flow rate: 3.3 mL min~1; revolution: 800 rpm; analysis: chirality by analytical HSCCC (see Figure 1) and pH by a portable pH meter. Note that the analysis of the fraction from the mixing zone (middle chromatogram) shows three peaks corresponding to (-)-DNB-leucine, impurity and ( + )-DNB-leucine from left to right.

Figure 4 Separation of DNB-leucine racemate by pH-zone-refining CCC. Experimental conditions: apparatus: see the Figure 1 caption; solvent system: methyl t-butyl ether/water; stationary phase: upper organic phase to which trifluoroacetic acid (40 mM) and CS (40 mM) were added; mobile phase: lower aqueous phase to which aqueous ammonia was added to 20 mM; sample: (+)-DNB-leucine 2 g; flow rate: 3.3 mL min~1; revolution: 800 rpm; analysis: chirality by analytical HSCCC (see Figure 1) and pH by a portable pH meter. Note that the analysis of the fraction from the mixing zone (middle chromatogram) shows three peaks corresponding to (-)-DNB-leucine, impurity and ( + )-DNB-leucine from left to right.

Figure 5 Schematic diagram of simple chemohydrodynamic equilibrium between the racemates (A±) and chiral selector (CS) in the separation column.

Here, [CS]org is the difference between the initial concentration of the CS and the concentration of the CSA± complex, i.e.:

In Figure 5, which shows the chemodynamic equilibrium in a portion of the separation column, if the analytes are ionizable (e.g. acids) they will be partially dissociated to form anions [A±]aq which are almost insoluble in the organic phase.

In this equilibrium state, the following set of equations is given for each racemate:

D± = ([A±H]org + [CSA±H]org)/([A±H]aq # [A±]aq)

When [A±]org«[CS]initiai, [CS]org approaches [CS^iai hence, eqn [4] may be rewritten:

The validity of eqn [6] has been examined by a series of experiments, the results of which indicated that the separation factor (D + /D_) is increased as expected by increasing the concentration of chiral selector in the stationary phase (Figure 2).

For computation of Kf, eqn [6] can be modified into a more convenient form:

where D±, D0, Ka, and Kf± represent the partition coefficient, the partition ratio, the dissociation constant, and the CS-complex formation constant for each racemate, respectively. From these equations, we obtain:

In pH-zone-refining CCC, the peak resolution is mainly determined by the difference in pH between where D± and D0 can be computed from the chromatograms obtained with and without the chiral selector in the stationary phase.

Using eqn [7] the formation constant (Kf±) has been determined by a series of experiments where small amounts (0.1-0.2 mg) of enantiomers were separated at various CS concentrations in the stationary phase. Figure 6 is drawn by plotting the (D ± — D0)/D0 values from each enantiomer against the initial CS concentration in the stationary phase where the formation constant is computed from the slope of the straight line. These results indicate that the method is useful for computing the formation constants of various analyte-CS pairs.

General Chemodynamic Model in Chiral CCC

This second model deals with more generalized condition where the ionic analytes are dissociated in the aqueous mobile phase. This approach can be useful for predicting the feasibility of chiral resolution by pH-zone-refining CCC.

Figure 6 Determination of formation constant of CS-DNB-amino acids by the standard HSCCC technique. The diagram was produced by plotting (D± — D0)/D0 (eqn [7]) against the initial CS concentration in the organic stationary phase. The slope of each line indicates the formation constant (Kf) of the corresponding enantiomer.

Figure 6 Determination of formation constant of CS-DNB-amino acids by the standard HSCCC technique. The diagram was produced by plotting (D± — D0)/D0 (eqn [7]) against the initial CS concentration in the organic stationary phase. The slope of each line indicates the formation constant (Kf) of the corresponding enantiomer.

where [CS]org/Z+ and [CS]org/Z- are the free CS concentrations in the A+ and A_ zones, respectively. When K# > Kf_, [CS]org/Z+ < [CS]org/Z- < [CS]initial. Eqn [13] indicates that chiral resolution can be improved by increasing D0/Kr and/or choosing the CS with a large Kf+/Kf_ value. It also implies that increasing the CS concentration will yield higher peak resolution.

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  • haris
    What is formation constant in chiral chromatography?
    2 years ago

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