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/dp2)]

Equipment

Conventional HPLC systems cannot reach the high

pressures needed for very small particles

Conventional HPLC systems need modification for very

small particles

Pressure, Rs and Particle Size

0

2

4

6

8

10

12

0 1 2 3 4 5 6

dp

Rs /

Tim

e (

min

)

0

100

200

300

400

500

600

Pre

ss

ure

(b

ar)

Rs

Time DP

Calculated for methanol at the optimum flow velocity for each particle

Effect of Equipment on Performance

CHIRALPAK®

AD®-3

Agilent 1200 SL

(ECV optimized)

Agilent 1100

(standard)

Relative Loss

(%)

Column Size N1 N2 N1 N2 N1 N2

250 x 4.6 mm I.D. 32030 30969 31360 30900 2.1 0.2

150 x 4.6 mm I.D. 15800 15620 15200 15160 3.9 2.9

50 x 4.6 mm I.D. 6660 6500 6360 6410 4.5 1.4

(Trifluorophenylethanol; n-hexane/2-propanol 90:10; 1 mL/min, 210 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

1.5-Microns

2.5-Microns Bandwidth due to pore diffusion

This effect increases with flow velocity

Van Deemter Plots: 3- vs 5-Microns

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Flow Velocity (mm/sec)

HE

TP

(m

m)

CHIRALPAK AD-H

CHIRALCEL® AD-3

CHIRALPAK IC™-3

CHIRALPAK IA™-3

9:1 Hexane : IPA

trans-Stilbene Oxide

5-micron

3-Micron

• Higher optimum flow rate

with smaller particles leads

to faster analyses and

higher efficiency

• Smaller HETP allows

shorter columns – and

faster analyses

Van Deemter Plots – Reversed Phase

1-Indanol, 1st Enantiomer

0.0 0.5 1.0 1.5 2.0 2.5 0.000

0.005

0.010

0.015

0.020

0.025

CHIRALPAK AD-3R

CHIRALPAK AD-3R

CHIRALPAK AD-RH

u (mm/s)

HE

TP

(m

m)

Mobile phase: Water-Acetonitrile 60:40 (v/v)

Detection: 210 nm Temp.: 25°C

Sample volume: 1.0 microL

5-Micron

3-Micron

min 0 2 4 6 8 10 12 14 16 18

0

100

200

300

400

500

2.2

52

1

2 3

4

CHIRALPAK AD-3R 150 x 4.6 mm

40% ACN – 0.2M KH2PO4 0.5 ml/min; 25°C

After 3 week pressure stability test

Reversed Phase Tests – AD-3R

N1 N2

Mephenesin (1) 20900 20540

1-Indanol (2) 23250 23060

1-Naphthylethanol (3) 20920 19610

2-Methyl-1-tetralone (4) 21910 20280

Selectivity: Transfer From 5-Micron C

HIR

AL

PA

K IA

(5

µm

)

CHIRALPAK IA-3 (3µm)

y = 1.0367x

R2 = 0.996

1.0

2.0

3.0

4.0

5.0

6.0

7.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0

CH

IRA

LP

AK

IC

(5

µm

)

CHIRALPAK IC-3 (3µm)

y = 0.9948x

R2 = 0.999

1.0

2.0

3.0

4.0

5.0

6.0

7.0

1.0 2.0 3.0 4.0 5.0 6.0 7.0

Effortless method transfer from 5- to 3-Micron columns

Retention Factors: Transfer from 5-Micron

0.0 1.0 2.0 3.0 4.0 5.0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

k’ C

HIR

AL

PA

K IA

(5

µm

)

k’ CHIRALPAK IA-3 (3µm)

CH

IRA

LP

AK

IA

(5

µm

)

CHIRALPAK IA-3 (3µm)

Effortless method transfer from 5- to 3-Micron columns

Transfer to 3-Micron: CHIRALPAK IA-3

CHIRALPAK IA

(150 x 5.6 mm I.D., 5 mm)

Hexane-Isopropanol 90:10 (v/v) UV 210 nm, 25° C

flow rate: 0.6 mL/min

min 0 1 2 3 4 5 6 7 8 9

mAU

0

100

200

300

400 1 2 3

4 5

6 8

7

min

CHIRALPAK IA-3

(150 x 5.6 mm I.D., 3 mm)

Hexane-Isopropanol 90:10 (v/v) UV 210 nm, 25° C

flow rate: 1.75 mL/min

Mecoprop methyl ester: 1,2

trans-Stilbene oxide: 3,7

Benzoin ethyl ether: 4,5

Ruelene: 6,8

3-fold faster

0 1 2 3 4

mAU

0

100

200

300

400

1 2

3

4 5

6 8

7

N = 18,000

N = 23,000

* ~ Equal plate numbers

Transfer to 3-Micron: CHIRALPAK IC-3

0 1 2

mAU

0

100

200

300

400

500 min 0 1 2 3 4 5 6 7

mAU

0

100

200

300

400

500

CHIRALPAK IC-3

(150 x 5.6 mm I.D., 3 mm)

Hexane-Isopropanol 90:10 (v/v) UV 210 nm, 25° C

flow rate: 1.75 mL/min

CHIRALPAK IC-5 (150 x 5.6 mm I.D., 3 mm)

hexane/2-propanol 90:10 (v/v) UV 210 nm, 25° C

flow rate: 0.6 mL/min

1-Phenyl-2,2,2-trifluoroethanol: 1,2

Tetrahydronaphthol: 3,4

Mecoprop methyl ester: 5,6

1 2

3 4

5 6

1 2

3 4

5 6

3-fold faster

N = 21,800

N = 19,200

* ~ Equal plate numbers

High Efficiency Separations - Metolochlor

N

OCH3

O

Cl

Serial coupled columns, n-hexane/2-propanol 97.5:2.5 (v/v); 0.5 mL/min; 235 nm; 15°C

2 x CHIRALCEL OD-3

(150 x 4.6 mm I.D.)

Rs1,2 2.02

Rs3,4 1.92

N1 19098

N2 21029

N3 19006

N4 18435

min 0 10 20 30 40

High Speed Separations

CH

IRA

LP

AK

IA

-3

(4.6

x 1

50 m

m)

min 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

N

N

O

Cl

Cl

Cl

MeOH 100%

Flow rate: 3.0 ml/min

MtBE-MeOH 80:20 (+0.1% Ethanolamine)

Flow rate: 3.5 ml/min

min 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

O O

OH

O

Furoin

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

Pressure Stability

Column: CHIRALPAK® AD-3 (150 x 4.6 mm I.D.) pressurizing solvent: ethanol; stress test performed without guard cartridge.

TestEtOH 1

2.30 mL/min EtOH 430 bar operation pressure

for 1 week

0

100

200

300

400

500

exposure time (days) 0 1 5 7

op

era

tio

nal

pre

ssu

re (

bar

)

TestHex 1

TestEtOH 5

TestHex 2

TestEtOH 2 TestEtOH 3

total volume of mobile phase passing through

column 23 L (= 14,500 column volumes)

2 3 4 6

TestEtOH 4

Column Tests – Pressure Stability

Compound QC-Test N unchallenged N after stress N Diff (%)

N1 N2 N1 N2 N1 N2

TSO/Ethanol TestEtOH 22270 17130 22430 17090 + 0.7 - 0.2

Troeger base

Hexane / IPA

TestEtOH 18440 13940 18470 13750 + 0.2 - 1.4

TSO Hexane/IPA TestHex 21230 15530 22240 15990 + 4.8 + 2.9

Column: CHIRALPAK® AD-3 (150 x 4.6 mm I.D.)

min 2.5 5 7.5 10 12.5 15 17.5 20

mAU

0

50

100

150

200

250

300

3.3

73

7.3

44

18.7

42

mAU

0

50

100

150

200

250

300

350

min 0 2.5 5 7.5 10 12.5 15 17.5 20

3.3

59

7.2

23

18.8

62

Before After

TSO

Ethanol: 0.5 ml/min

Fast Separations

Oxprenolol.HCl

seconds

0 6 12 18 24 30

CHIRALPAK® AD-3 (50 x 4.6 mm I.D.); for oxprenolol and carbinoxamine: n-hexane/

2-propanol/diethylamine 90/10/0.1 (v/v/v); for dichlorprop: n-hexane/2-propanol/trifluoroacetic acid 90/10/0.1 (v/v/v); flow rate: 5.0 mL/min; UV-detection: 235 nm; temperature: 25°C.

OHN

OH

O

Dichlorprop

24 0 12 18 6

seconds

O COOH

Cl

Cl

Carbinoxamine

0 6 12 18 24 30

seconds

NCl

O

N

Reversed Phase

CHIRALPAK AD-3R (4.6 x 150 mm)

0 1 2 3 4 5

Tolperisone

0.02M NH4HCO3 aq. (pH9.0 -DEA)

ACN 40:60

N

O

0 1 2 3 4 5

Methyl 2-phenylsulfinylacetate

H2O-ACN 60:40

SO

OO

SFC

Minutes 0 1 2 3 4 5 6 7

mv

0

200

400

600

800

1000

1200

1400

1600 Devrinol CHIRALPAK IC-3 150 x 4.6 mm

15% methanol / CO2

3 ml/min 120 bar bp

ON

O

mv

0

20

40

60

80

100 Vince lactam

CHIRALPAK IA-3 150 x 4.6 mm

30% methanol / CO2

3 ml/min 120 bar bp

Minutes

0.5 1.0 1.5 2.0 2.5 3.0 3.5 0

NH

O

3-Micron Columns

Column Normal

Phase

Reversed

Phase

SFC Fast

Separations

High

efficiency

CHIRALPAK IA-3

CHIRALPAK IC-3

CHIRALPAK AD-3

CHIRALPAK AD-3R

CHIRALCEL OD-3

CHIRALCEL OD-3R

Conclusions

High efficiency at high flow rates

Robust

Pressure limit > 400 bar (Determined by HPLC unit)

Improves productivity by reduction of analysis time

High efficiency allows difficult separations

Can be used in conventional HPLC units