new, 3-micron polysaccharide-based chiral columns for fast ... · 6/8/2014 · equipment...
TRANSCRIPT
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