Applications

Figure 6 shows the separation of the steroid flut-icasone propionate from two closely related impurities using micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate (SDS) as the micelle while Figure 7 shows the corresponding HPLC trace using 3 |im Spherisorb ODS-1 stationary phase with gradient elution (impurity 3 was not present in the MEKC experiment).

If the sample is analysed by CEC with the same batch of 3 |im Spherisorb ODS-1, then the chromato-gram shown in Figure 8 is obtained. Apart from a reversal in elution order from the MEKC/HPLC to the CEC the latter is extremely efficient with peak 3 in the CEC trace exhibiting an efficiency of almost 4 x 105 plates m"1 with a reduced plate height of 0.9.

If the electrolyte is changed from phosphate to borate and the organic content increased from 75 to 80%, then the chromatogram shown in Figure 9 is obtained. Although the capillary length has been increased, the analysis time has been reduced significantly. Also, a peak previously undetected has now shown up between peaks 1 and 2.

Increasing the organic content of the electrolyte can drastically reduce run times, but in order to maintain high efficiencies it is desirable to work at high buffer concentration. To meet this criterion it is necessary to work with so-called biological buffers since traditional inorganic buffers are insoluble in high concentrations of organic solvents. Figure 10 shows the fast, highly efficient analysis of the three impurity peaks from the parent steroid in less than 11 min using a mobile phase containing 0.1 mol L" 1 Tris buffer (2-amino-2-hydroxymethylpropane-1,3-diol).

Figure 11 shows the chromatogram of a crude sample of a prostaglandin, GR63779X, obtained by using an unadjusted phosphate buffer on a 3 |im Spherisorb ODS-1 phase, while Figure 12 is the same

Figure 5 Capillary packing procedure. 5000 psi + 34 000 kPa; 9000 psi + 62 000 kPa.

sample run under identical conditions on a 1.5 |m Zorbax C8 column.

The expected increase in efficiency on going from 3 |im to 1.5 |im particles has not been realized. The efficiency for the peak eluting at 20 min on the smaller-particle Zorbax column is about 300 000 plates m"1 with a reduced plate height of 1.6. This is not as good as the figures for the 3 |im column for two reasons: first, there is the difficulty in packing the smaller particles, and second, Zorbax itself is a difficult material to pack. However, for the two early

Figure 6 Separation of fluticasone from related compounds by MEKC. Capillary, 72 cm x 75 |m i.d.; detection at 238 nm with a range of 0.02 and rise time of 0.5 s; applied voltage, 30 kV; temperature, 60°C; carrier, 0.01 mol L"1 Na2HP04/0.006 mol L"1 Na2B407• 10H20/0.05 mol L"1 SDS in 20% methanol; injection, 1.0 s vacuum.

eluting peaks the 1.5 |im Zorbax column gives much better peak shapes and slightly different selectivity. Figure 13 shows a highly efficient separation of an angiotensin compound from 13 impurities. Apart from the two late emerging compounds, all the peaks are symmetrical with no apparent evidence of peak tailing. In contrast many of the compounds in this mixture gave tailing peaks when run on an HPLC column containing the same stationary phase. This may be due to the fact that the CEC separation was at high pH while the HPLC separation was at a pH less than 5. Experience has shown that it is possible to run columns under electrodrive conditions at a much higher pH than is possible in HPLC without any deterioration in performance and often with a considerable improvement in separation.

Figure 6 Separation of fluticasone from related compounds by MEKC. Capillary, 72 cm x 75 |m i.d.; detection at 238 nm with a range of 0.02 and rise time of 0.5 s; applied voltage, 30 kV; temperature, 60°C; carrier, 0.01 mol L"1 Na2HP04/0.006 mol L"1 Na2B407• 10H20/0.05 mol L"1 SDS in 20% methanol; injection, 1.0 s vacuum.

Figure 7 Separation of fluticasone from related compounds by HPLC. Column, 15 cm x 4.6 mm i.d. packed with 3 ^m ODS-1; flow rate, 1.0mLmin~1; detection at 238 nm, 0.05 absorbance units full scale (aufs); gradient 40% acetonitrile/H2O p70% acetonitrile/H2O in 20 min. (By permission of the author.)

Figure 8 Separation of fluticasone from related compounds by capillary electrochromatography (CEC) with a phosphate buffer. Column, 40 cm x 50 ^m i.d. packed with 3 ^m ODS-1; detection at 238 nm, 0.05 aufs; voltage, 30 kV; temperature, 30°C; carrier, 75% acetonitrile/H2O; buffer 2 mmol Na2HPO4, pH 8.3; injection, 0.4 min at 20 kV.

Figure 8 Separation of fluticasone from related compounds by capillary electrochromatography (CEC) with a phosphate buffer. Column, 40 cm x 50 ^m i.d. packed with 3 ^m ODS-1; detection at 238 nm, 0.05 aufs; voltage, 30 kV; temperature, 30°C; carrier, 75% acetonitrile/H2O; buffer 2 mmol Na2HPO4, pH 8.3; injection, 0.4 min at 20 kV.

Figure 10 Rapid separation of fluticasone from related impurities by CEC with a Tris buffer. Column, 40 cm x 50 ^m i.d. packed with 3 ^m ODS-1; detection at 238 nm, 0.05 aufs; voltage, 30 kV; temperature, 30°C; carrier, 90% acetonitrile/10% 0.1 mol L~1 Tris; injection, 0.05 min at 10 kV.

Cephalosporins are a class of antibiotic compounds. As well as the chiral centre in the ^-lactam ring, the compound shown in Figure 14 possesses a chiral centre in the ester group on the adjacent six-membered ring, giving rise to a pair of dia-stereoisomers. In addition the oxime group can be in one of two positions - either the syn (E) or the anti (Z) - both of which give rise to isomers. Both pairs of diastereoisomers are readily resolved on the 3 |im Spherisorb ODS-1 column with a length of 40 cm. The use of such a column length for HPLC would be impractical because the pressure requirements would be beyond the range of current instruments. Since there is no pressure drop in CEC, the use of long columns presents no practical problems.

If the extremely high efRciencies obtained with CEC for achiral compounds could be matched with chiral compounds, then there would be less need to achieve high a values in order to separate the isomers. Figures 15 and 16 show the separation of bendro-flumethiazide and hexobarbital on an a-glycoprotein and a Cyclobond stationary phase, respectively. Although large a values are obtained with these columns, the overall efficiencies are extremely poor. This is possibly due to column overload, since these phases have a low sample capacity.

Nearly all CEC separations to date have been carried out on C18 or C8 phases. However, because EOF drops off substantially below pH 6, most studies have been carried out above pH 7 and as a result most CEC has been for neutral molecules. One way of promoting EOF at lower pH values is by the use of columns packed with a strong cation exchanger (SCX); such

Figure 9 Separation of fluticasone from related compounds by CEC with a borate buffer. Column, 60 cm x 50 ^m i.d. PC20 packed with 3 ^m ODS-1; detection at 238 nm, 0.05 aufs; voltage, 30 kV; temperature, 30°C; carrier, 80% acetonitrile/20% 5 mmol L~1 borate, pH 9; injection, 0.4 min at 30 kV.

Figure 11 Separation of a prostaglandin from related impurities by CEC. Column, 60 cm x 50 |im i.d. packed with 3 |im ODS-1; detection at 270 nm, 0.05 aufs; voltage, 30 kV; temperature, 35°C; carrier, 70% acetonitrile/30% 0.1 mol L_1 Na2HPO4; injection, 0.04 min at 30 kV. (By permission of the author.)

Figure 14 Separation of diastereoisomers by CEC. Column, 40 cm x 50 |m i.d. packed with 3 |m ODS-1; detection at 276 nm, 0.03 aufs; voltage, 30 kV; temperature, 30°C; carrier, 50% acetonitrile/50% 0.01 molL"1 Na2HPO4; injection 0.2 min at 20 kV.

Figure 12 Separationofa prostaglandin from related impurities by CEC with a column packed with 1.5 |m C8 silica. Column, 40 cm x 50 |m i.d. packed with 1.5 |m Zorbax; detection at 270 nm, 0.05 aufs; voltage, 30 kV; temperature, 30°C; carrier, 70% acetonitrile/30% 0.1 molL"1 Na2HPO4; injection, 0.04min at 20 kV.

a material has been used for the CEC of neutral and charged analytes. The SCX used (manufactured by Phase Separations Ltd, Deeside, UK) had a sulfonic acid group linked to 3 | m silica via a propyl group to give a loading of 0.031 mEq g"1.

Figure 17 shows the structures of a group of tricyclic antidepressants. Because of the highly basic nature of these compounds, with typical pKa values of around 8, severe tailing is encountered with HPLC and even the new base-deactivated phases made from ultrapure silica still give some tailing.

When a test mixture containing bendrofiumethi-azide and four of the antidepressants was analysed on a Spherisorb ODS-1 capillary, only the neutral ben-drofiumethiazide eluted (Figure 18). However, CEC

Figure 14 Separation of diastereoisomers by CEC. Column, 40 cm x 50 |m i.d. packed with 3 |m ODS-1; detection at 276 nm, 0.03 aufs; voltage, 30 kV; temperature, 30°C; carrier, 50% acetonitrile/50% 0.01 molL"1 Na2HPO4; injection 0.2 min at 20 kV.

analysis at low pH on the 3 | m SCX phase gave unexpectedly high efficiencies. Figure 19 shows the separation of three tricyclic antidepressants on the cation exchange phase at pH 3.5. It is noticeable in this chromatogram that the neutral compound bendrofumethiazide, despite eluting first, gives a significantly broader peak than the later eluting compounds. This suggests that some form of focusing effect is taking place. Plate numbers on this phase have been measured in excess of 8 x 107, which is many orders of magnitude above that explicable by current theory.

Attempts to use a mixed-mode column with a C18 ligand and a -SO3H functional group attached to the same silica particle were not successful. When the antidepressant test mixture is injected onto the mixed-mode column at both pH 9.8 and pH 5.7, only

Figure 15 Chiral separation of bendroflumethiazide by CEC with an a-glycoprotein stationary phase. Column, 40 cm x50 |m i.d.; detection at 225 nm, 0.05 aufs; voltage, 30 kV; temperature, 30°C; carrier, 10% IPA/0.01 mol L"1 Na2HPO4 buffer, pH 7; injection, 0.4 min at 30 kV. (By permission of the author.)

Figure 13 Separation of an angiotensin compound from impurities by CEC at a relatively high pH. Column, 30 cm x 50 |m i.d. packed with 3 |m ODS-1; detection at 220 nm, 0.03 aufs; voltage, 30 kV; temperature, 28°C; carrier, 76% acetonitrile/H2O in 2 mmol L"1 Na2HPO4 buffer, pH 7.3; injection, 0.4 min at 20 kV.

Figure 15 Chiral separation of bendroflumethiazide by CEC with an a-glycoprotein stationary phase. Column, 40 cm x50 |m i.d.; detection at 225 nm, 0.05 aufs; voltage, 30 kV; temperature, 30°C; carrier, 10% IPA/0.01 mol L"1 Na2HPO4 buffer, pH 7; injection, 0.4 min at 30 kV. (By permission of the author.)

Figure 16 Chiral separation of hexobarbital by CEC with a Cyclobond 1 stationary phase. Detection at 210 nm, 0.05 aufs; carrier, 5% acetonitrile/95% Na2HPO4, pH 7.1; injection, 0.3 min at 10 kV. (By permission of the author.)

the neutral bendroflumethiazide is eluted. Similar results were obtained with a mixed-mode phase with a C6 chain. It is believed that the hydrophobic ligands

Figure 17 Structures of some tricyclic antidepressants separated by CEC.

Figure 18 CEC of bendroflumethiazide and four tricyclic antidepressants by CEC on a 3 |im ODS-1 column. Detection at 210 nm, 0.05 aufs root temperature 0.2; voltage, 30 kV; temperature, 30°C; carrier, 70% acetonitrile/30% 0.01 mol L"1 Na2HPO4, pH 9.8 unadjusted; injection, 0.3 min at 3 kV. Peak 1 represents bendroflumethiazide. Nortriptyline, clomipramine, methdilazine and imipramine were not eluted.

Figure 18 CEC of bendroflumethiazide and four tricyclic antidepressants by CEC on a 3 |im ODS-1 column. Detection at 210 nm, 0.05 aufs root temperature 0.2; voltage, 30 kV; temperature, 30°C; carrier, 70% acetonitrile/30% 0.01 mol L"1 Na2HPO4, pH 9.8 unadjusted; injection, 0.3 min at 3 kV. Peak 1 represents bendroflumethiazide. Nortriptyline, clomipramine, methdilazine and imipramine were not eluted.

collapse onto the -SO3H groups and shield them from participation in the focusing process. If a strong cation exchanger is produced, using a different link from the propyl group used for the original material, then the focusing effect returns. Figure 20 shows the electrochromatogram of methdilazine, clomipramine and imipramine on a column containing a stationary phase where the sulfonic acid group is attached to the silica via a phenyl group. Once again, highly efficient separations are obtained. Methdilazine now elutes before clomipramine, whereas on the propyl -SO3H phase elution was the other way round.

Nonporous materials are used in HPLC for the analysis of highly hydrophobic compounds that

Figure 19 CEC of bendroflumethiazide and three tricyclic antidepressants on a 3 |im Spherisorb SCX column. Detection at 220 nm, 0.045 aufs; voltage, 30 kV; temperature, 30°C; carrier, 70% acetonitrile/30% 0.05 mol L"1 Na2HPO4, pH 3.5; injection, 0.5 min at 2 kV.

Figure 17 Structures of some tricyclic antidepressants separated by CEC.

Figure 19 CEC of bendroflumethiazide and three tricyclic antidepressants on a 3 |im Spherisorb SCX column. Detection at 220 nm, 0.045 aufs; voltage, 30 kV; temperature, 30°C; carrier, 70% acetonitrile/30% 0.05 mol L"1 Na2HPO4, pH 3.5; injection, 0.5 min at 2 kV.

Figure 20 CEC separation of three tricyclic antidepressants on an SCX column with the S03H group attached to the silica via a phenyl group. Packed length =40 cm, total length =56 cm; voltage, 30 kV; detection at 210 nm; 0.02 aufs; carrier, 70% acetonitrile/30% 0.01 mol L"1 NaH2P04, pH 2.3; injection, 0.4 min at 2 kV.

Figure 20 CEC separation of three tricyclic antidepressants on an SCX column with the S03H group attached to the silica via a phenyl group. Packed length =40 cm, total length =56 cm; voltage, 30 kV; detection at 210 nm; 0.02 aufs; carrier, 70% acetonitrile/30% 0.01 mol L"1 NaH2P04, pH 2.3; injection, 0.4 min at 2 kV.

would normally be too strongly retained on conventional porous reversed-phase packings. Micropell C18, made by Horv^th (Yale, USA), has been used successfully for CEC with relatively small molecules even though there is little retention because of the very small surface area. Figure 21 shows the separation of the neutral diol, GR5788X, from the diben-zyl compound, GR57994X, in less than 4 min on a 40 cm capillary packed with this 2 |m nonporous phase.

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