New 3-Micron Polysaccharide-based Chiral Columns for Fast HPLC and SFC Geoffrey B. Cox, Norbert M. Maier Chiral Technologies, Inc Pilar Franco and Tong Zhang Chiral Technologies, Europe
Benefits of 3-Micron Columns High Speed separations Reduction in analysis time More samples per day Reduction in cost per sample High efficiency separations Higher resolution for difficult samples Higher precision from higher resolution
Why Stop at 3-Microns? Pressure 3-Micron columns give attainable pressure drops at high flow rate Pressure increases dramatically with smaller particles [ΔP = f(1/d p2 )] Equipment Conventional HPLC systems cannot reach the high pressures needed for very small particles Conventional HPLC systems need modification for very small particles
R s / Time (min) Pressure (bar) Pressure, R s and Particle Size 12 6 1 DP Time 5 8 4 6 3 4 2 2 R s 1 1 2 3 4 5 6 dp Calculated for methanol at the optimum flow velocity for each particle
Effect of Equipment on Performance CHIRALPAK Agilent 12 SL Agilent 11 Relative Loss AD -3 (ECV optimized) (standard) (%) Column Size N 1 N 2 N 1 N 2 N 1 N 2 25 x 4.6 mm I.D. 323 3969 3136 39 2.1.2 15 x 4.6 mm I.D. 158 1562 152 1516 3.9 2.9 5 x 4.6 mm I.D. 666 65 636 641 4.5 1.4 (Trifluorophenylethanol; n-hexane/2-propanol 9:1; 1 ml/min, 21 nm, 25 C) The 3-Micron columns may be used in conventional (but modern) HPLC units without significant loss in performance, even with the smaller columns. With older units, small volume flow cells and narrow diameter capillaries may be needed for optimum performance.
Reduction in Particle Size Gives Faster Mass Transfer 2.5-Microns Bandwidth due to pore diffusion 1.5-Microns This effect increases with flow velocity
HETP (mm) Van Deemter Plots: 3- vs 5-Microns.35.3.25.2.15.1 5-micron CHIRALPAK AD-H CHIRALCEL AD-3 CHIRALPAK IC -3 CHIRALPAK IA -3 9:1 Hexane : IPA trans-stilbene xide Higher optimum flow rate with smaller particles leads to faster analyses and higher efficiency Smaller HETP allows shorter columns and faster analyses.5 3-Micron...5 1. 1.5 2. 2.5 3. 3.5 4. 4.5 5. Flow Velocity (mm/sec)
HETP (mm) Van Deemter Plots Reversed Phase Mobile phase: Water-Acetonitrile 6:4 (v/v) Detection: 21 nm Temp.: 25 C Sample volume: 1. microl.25 1-Indanol, 1st Enantiomer.2.15 5-Micron CHIRALPAK AD-3R.1 CHIRALPAK AD-3R CHIRALPAK AD-RH.5 3-Micron...5 1. 1.5 2. 2.5 u (mm/s)
2.252 Reversed Phase Tests AD-3R CHIRALPAK AD-3R 15 x 4.6 mm 4% ACN.2M KH 2 P 4.5 ml/min; 25 C 5 1 N1 N2 4 2 3 Mephenesin (1) 29 254 1-Indanol (2) 2325 236 1-Naphthylethanol (3) 292 1961 3 2-Methyl-1-tetralone (4) 2191 228 2 4 1 2 4 6 8 1 12 14 16 18 min After 3 week pressure stability test
CHIRALPAK IA (5µm) CHIRALPAK IC (5µm) Selectivity: Transfer From 5-Micron 7. 6. y = 1.367x R 2 =.996 7. 6. y =.9948x R 2 =.999 5. 5. 4. 4. 3. 3. 2. 2. 1. 1. 2. 3. 4. 5. 6. 7. CHIRALPAK IA-3 (3µm) 1. 1. 2. 3. 4. 5. 6. 7. CHIRALPAK IC-3 (3µm) Effortless method transfer from 5- to 3-Micron columns
k CHIRALPAK IA (5µm) CHIRALPAK IA (5µm) Retention Factors: Transfer from 5-Micron 3.5 3. 4.5 4. 2.5 2. 1.5 1..5...5 1. 1.5 2. 2.5 3. 3.5 k CHIRALPAK IA-3 (3µm) 3.5 3. 2.5 2. 1.5 1..5.. 1. 2. 3. 4. 5. CHIRALPAK IA-3 (3µm) Effortless method transfer from 5- to 3-Micron columns
Transfer to 3-Micron: CHIRALPAK IA-3 mau 4 3 1 2 3 CHIRALPAK IA (15 x 5.6 mm I.D., 5 mm) Hexane-Isopropanol 9:1 (v/v) UV 21 nm, 25 C flow rate:.6 ml/min * ~ Equal plate numbers 2 N = 18, 7 4 5 1 6 8 1 2 3 4 5 6 7 8 9 min mau 4 3 1 2 3 CHIRALPAK IA-3 (15 x 5.6 mm I.D., 3 mm) Hexane-Isopropanol 9:1 (v/v) UV 21 nm, 25 C flow rate: 1.75 ml/min N = 23, 7 Mecoprop methyl ester: 1,2 trans-stilbene oxide: 3,7 Benzoin ethyl ether: 4,5 Ruelene: 6,8 2 4 5 1 6 8 1 2 3 min 4 3-fold faster
Transfer to 3-Micron: CHIRALPAK IC-3 mau 5 4 3 CHIRALPAK IC-5 (15 x 5.6 mm I.D., 3 mm) hexane/2-propanol 9:1 (v/v) UV 21 nm, 25 C flow rate:.6 ml/min 3 4 * ~ Equal plate numbers 2 1 N = 19,2 1 2 1 2 3 4 5 6 7 5 6 min mau 5 4 CHIRALPAK IC-3 (15 x 5.6 mm I.D., 3 mm) Hexane-Isopropanol 9:1 (v/v) UV 21 nm, 25 C flow rate: 1.75 ml/min 3 4 1-Phenyl-2,2,2-trifluoroethanol: 1,2 Tetrahydronaphthol: 3,4 Mecoprop methyl ester: 5,6 3 2 N = 21,8 1 2 1 1 2 5 6 3-fold faster
High Efficiency Separations - Metolochlor N CH 3 Cl 2 x CHIRALCEL D-3 (15 x 4.6 mm I.D.) R s1,2 2.2 R s3,4 1.92 N1 1998 N2 2129 N3 196 N4 18435 1 2 3 4 min Serial coupled columns, n-hexane/2-propanol 97.5:2.5 (v/v);.5 ml/min; 235 nm; 15 C
CHIRALPAK IA-3 (4.6 x 15 mm) High Speed Separations Cl Cl N N Furoin H Cl.2.4.6.8 1. 1.2 1.4 min.2.4.6.8 1. 1.2 1.4 min MtBE-MeH 8:2 (+.1% Ethanolamine) Flow rate: 3.5 ml/min MeH 1% Flow rate: 3. ml/min
Pressure Stability For high speed separations, the columns must be pressure stable Extensive pressure testing of the 3-Micron columns shows no practical limit with conventional HPLC systems
operational pressure (bar) Pressure Stability 5 4 Column: CHIRALPAK AD-3 (15 x 4.6 mm I.D.) pressurizing solvent: ethanol; stress test performed without guard cartridge. 3 2 1 2.3 ml/min EtH 43 bar operation pressure for 1 week total volume of mobile phase passing through column 23 L (= 14,5 column volumes) 1 2 3 4 5 6 7 exposure time (days) Test EtH 1 Test EtH 2 Test EtH 3 Test EtH 4 Test EtH 5 Test Hex 1 Test Hex 2
3.359 3.373 18.862 18.742 7.223 7.344 Column Tests Pressure Stability mau 3 25 2 15 1 5 TS Ethanol:.5 ml/min Before 2.5 5 7.5 1 12.5 15 17.5 2 min Column: CHIRALPAK AD-3 (15 x 4.6 mm I.D.) mau 35 3 25 2 15 1 5 After 2.5 5 7.5 1 12.5 15 17.5 min 2 Compound QC-Test N unchallenged N after stress N Diff (%) N1 N2 N1 N2 N1 N2 TS/Ethanol Test EtH 2227 1713 2243 179 +.7 -.2 Troeger base Hexane / IPA Test EtH 1844 1394 1847 1375 +.2-1.4 TS Hexane/IPA Test Hex 2123 1553 2224 1599 + 4.8 + 2.9
Fast Separations N H H N Cl CH xprenolol.hcl Cl Dichlorprop Cl Carbinoxamine N 6 12 18 24 3 seconds 6 12 18 seconds 24 6 12 18 24 3 seconds CHIRALPAK AD-3 (5 x 4.6 mm I.D.); for oxprenolol and carbinoxamine: n-hexane/ 2-propanol/diethylamine 9/1/.1 (v/v/v); for dichlorprop: n-hexane/2-propanol/trifluoroacetic acid 9/1/.1 (v/v/v); flow rate: 5. ml/min; UV-detection: 235 nm; temperature: 25 C.
Reversed Phase CHIRALPAK AD-3R (4.6 x 15 mm) Tolperisone.2M NH 4 HC 3 aq. (ph9. -DEA) ACN 4:6 Methyl 2-phenylsulfinylacetate H 2 -ACN 6:4 N S 1 2 3 4 5 1 2 3 4 5
mv mv SFC 16 Devrinol CHIRALPAK IC-3 15 x 4.6 mm 15% methanol / C 2 14 3 ml/min 12 bar bp 12 1 8 6 4 2 N 1 Vince lactam 8 6 4 2 CHIRALPAK IA-3 15 x 4.6 mm 3% methanol / C 2 3 ml/min 12 bar bp N H 1 2 3 4 5 6 7 Minutes.5 1. 1.5 2. 2.5 3. 3.5 Minutes
3-Micron Columns Column Normal Reversed SFC Fast High Phase Phase Separations efficiency CHIRALPAK IA-3 CHIRALPAK IC-3 CHIRALPAK AD-3 CHIRALPAK AD-3R CHIRALCEL D-3 CHIRALCEL D-3R
Conclusions High efficiency at high flow rates Robust Pressure limit > 4 bar (Determined by HPLC unit) Improves productivity by reduction of analysis time High efficiency allows difficult separations Can be used in conventional HPLC units