Upload
others
View
7
Download
0
Embed Size (px)
Citation preview
Overview of Lux Polysaccharide Columns,
General Method Development and
Optimization Practices in Analytical and
Preparative HPLC
Prof. Bezhan Chankvetadze
Department of Physical and Analytical Chemistry
Tbilisi State University, Tbilisi, Georgia
• History of Lux® chlorinated phases
• Lux polysaccharide based CSPs
• Column screening strategies
• Elution order reversal
• Chemo- and enantio-selectivity
• New developments
• Preparative scale separations
Outline
• Polysaccharide based materials are rather universal CSPs
• These materials can be used with normal, reversed, and polar organic modes
• Applicable for both pressure and voltage-driven separations
• Extremely high enantioselectivity can be achieved using these CSPs for various
group of analytes
• A large family of polysaccharide-based CSPs are available
• Polysaccharide-based CSPs are very useful for preparative and product scale
enantioseparations
Why shall we use polysaccharide derivatives as chiral
stationary phases (CSPs) in liquid phase separation techniques?
S
C
O
O
O
10 20
10 155
20 40 680 760
+c)
b) +
0
0
a)
0
Elution time /min
αααα=112
Enantioseparation of chiral sulphoxide with
CDCPC column
Chemistry of a chiral selector
Chemistry, morphology and nature of inert carrier
Coating or immobilization technology
Pretreatment of a packing material
Column hardware
Column packing technology
What can be optimized when developing a new
chiral column?
Chlorinated Chiral Selectors
O
O
OCONH
OCONH
CH3
Cl
CH3
Cl
CH3
Cl HNOCO
Lux® Cellulose- 4
Cellulose tris (4-chloro-3 methylphenylcarbamate)
O
O
OCONH
Cl
Me
Cl
MeOCONH
Cl
HNOCOMe
Lux® Cellulose-2
Cellulose tris(3-chloro-4 methylphenylcarbamate)
O
O
OCONH
OCONH
HNOCO
Cl
Cl
CH3
CH3
Cl
CH3
Lux® Amylose-2
Amylose tris(5-chloro-2-methylphenylcarbamate)
Substitution Pattern
Spectral Properties
Chiral Recognition Ability
(or Chromatographic Performance)
Comparative Chiral Recognition of Cellulose tris (3,5-Dimethylphenylcarbamate)
(Lux® Cellulose-1/CHIRALCEL®-OD®) and Cellulose tris (3,5-Dichlorophenylcarbamate)
Eluent: Hexane/2-Propanol (90:10). Flow rate: 0.5mL/min.
CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.
Intramolecular Hydrogen Bonding Between
the Adjacent Glucopyranose Units in Polysaccharide
Phenylcarbamates
23
2
3
C
Phenyl
Phenyl
CO
O
O
O
O
N H
O
O
H N
The Carbamate Moiety (-CONH-) Plays a Dual Role
in the Structure of Polysaccharide Phenylcarbamates:
1) It is most likely interaction site of the chiral analyte with the chiral
selector and thus responsible for chiral recognition ability
2) It facilitates a formation of intramolecular hydrogen bonding between
adjacent glucose unites in the structure of polysaccharide
phenylcarbamate and thus, it is responsible for its higher ordered
secondary structure and solubility in certain solvents
Designed Synthesis of Cellulose-based Chiral Stationary Phases
(Correlation between Spectroscopic Characteristics and
Chromatographic Performance)
IR Spectra in N-H range for
chloromethylphenyl-
carbamate derivatives of
cellulose (1a, 1c, 1d, 1e and 1f)
FT-IR
1H NMR spectra of cellulose phenyl
carbamate derivatives
(a) 1a, (b) 1b, (c) 1c, (d) 1d, (e) 1e
and (f) 1f
NMR
B. Chankvetadze et al. J. Chromatogr. A, 670, 1994, 39-49
1
Designed Synthesis of Cellulose-based Chiral Stationary Phases
(Correlation between Spectroscopic Characteristics and
Chromatographic Performance)
CD spectra of cellulose phenyl
carbamate derivatives (1a, 1c, 1d,
1e and 1f) in THF
CD
Dependence of Rs/N for 2,2’-
diamino-6,6’-dimethylbiphenyl on
the N-H chemical shift of cellulose
phenylcarbamate derivatives (1a, 1c,
1d, 1e, and 1f)
HPLC
B. Chankvetadze et al. J. Chromatogr. A, 670, 1994, 39-49
Review by: C. Yamamoto, Y. Okamoto Bull. Chem. Soc. Jpn., 2004, 227-257
a). The Sign in parentheses represents the optical rotation of the first-eluted enantiomer. Eluent: Hexane/2-Propanol (90:10); flow rate:
0.5mL/min. b). Eluent: Hexane/2-Propanol (98:2); 0.5mL/min.
Table 1. Separation Factors (α) in the Resolution of a-j on Cellulose and Amylose Phenylcarbamates
Amylose DerivativesCellulose DerivativesLux® Cellulose-1/CHIRALCEL® OD®
Chloro-methyl derivative (Lux Cellulose-4) is more universal
Comparative Chiral Recognition of Chlorinated and non-
Chlorinated Chiral Selectors
FT-IR and CD spectra of
tris(chloromethylphenylcarbamates) of amylose
Separation of enantiomers of cobalt
(III) acetylacetonate on (a) 1e, (b) 1b
and (c) 1d.
Separation of enantiomers of 2,2’-
dihydroxy-1,1’-binaphthyl on (a) 1e, (b)
1b and ( c ) 1d. Flow rate 1.0mL/min.
B. Chankvetadze et al. J. Chromatogr. A, 694, 1995, 101-111
Designed Synthesis of Amylose-based Chiral Stationary Phases
(Correlation between Spectroscopic Characteristics and
Chromatographic Performance)
Lux® Cellulose-1/CHIRALCEL® OD® Cellulose Derivatives
a). The Sign in parentheses represents the optical rotation of the first-eluted enantiomer. Eluent: Hexane/2-Propanol (90:10); flow rate:
0.5mL/min. b). Eluent: Hexane/2-Propanol (98:2); 0.5mL/min.
Separation Factors (α) in the Resolution of a-j on Cellulose and Amylose Phenylcarbamates
Amylose DerivativesCHIRALPAK® AD®
Lux Amylose-2
Review by: C. Yamamoto, Y. Okamoto Bull. Chem. Soc. Jpn., 2004, 227-257
Comparative Chiral Recognition of Chlorinated
and non Chlorinated Chiral Selector
CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.
Chemistry of a chiral selector
Chemistry, morphology and nature of inert carrier
Coating or immobilization technology
Pretreatment of a packing material
Column hardware
Column packing technology
What can be optimized when developing a new
chiral column?
The pressure stability at least up to 300 bar
pH stability in the range of 2.0-9.0 pH units(the applications up to pH 11.0 are described in the literature)
Applicability under normal-, reversed- and polar organic mobile phase conditions
High plate number
Applicability for SFC separations
Easy conversion of analytical to prep-scale separations
Plus all common advantages of polysaccharide-based chiral columns.
Due to careful optimization of all above mentioned parameters all
chiral columns in the Lux series provide:
Portfolio Overview:
Lux® Phases Chiral Selectors
Commercial name
Polysaccharide Type of chiral selector
Substituent
Lux Cellulose-1 Cellulose Carbamate Me
Lux Cellulose-2 Cellulose Carbamate Me, Cl
Lux Cellulose-3 Cellulose Benzoate Me
Lux Cellulose-4 Cellulose Carbamate Me, Cl
Lux Amylose-2 Amylose Carbamate Me, Cl
Lux® Polysaccharide-Based CSPs
Cellulose triscarbamate Amylose triscarbamate
Lux® Cellulose-1 vs. CHIRALCEL® OD-H®
CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.
Polar organic mode
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mv
-100
0
100
200
300
400
500
600
700
800
mv
-100
0
100
200
300
400
500
600
700
800
1: 220 nm, 4 nm
BIFONAZOLE_LUX1_1
In SFC with Bifonazole
Method Information:
Column size: 250 x 4.6 mm
Flow Rate: 2.5mL/min
15% MeOH:0.1%DEA
85% CO2
Detection: UV @ 220 nm
Temperature: 35°C
Minutes
0 2 4 6 8 10 12 14 16 18 20 22 24
mv
-100
0
100
200
300
400
500
600
700
800
900
mv
-100
0
100
200
300
400
500
600
700
800
9001: 220 nm, 4 nmBIFONAZOLE_102308_1
2
Chiralcel-OD-H
Lux-Cellulose-1
N
N
QC test Chromatogram n=5 Symmetry Efficiency
CHIRALCEL® OD-H® 0.62 15312
Lux 5u Cellulose- 1 0.74 17768
Lux shows a 17% increase in peak symmetryand a 14% increase in peak efficiency
Symmetry vs. Efficiency
Lux® Cellulose-1 vs. CHIRALCEL® OD-H®
CHIRALCEL, CHIRALPAK, AD, AS, OD, OD-H, and OJ are registered trademarks of DAICEL Chemical Industries, Ltd.
Lux® Cellulose-2 vs. CHIRALCEL® OZ-H®
CHIRALCEL OZ-H is a registered trademarks of DAICEL Chemical Industries, Ltd.
Lux 5µ Cellulose-2
Rs: 2.43
Chiralcel OZ-H
Rs: 0.92
Bupivacaine (0.1%DEA in Hexane/IPA (90:10) )
Lux 5µ Cellulose-2
Rs: 2.30
Chiralcel OZ-H
Rs: 1.84
Doxylamine (0.1%DEA in Hexane/IPA (80:20) )
Lux® Cellulose-3 vs. CHIRALCEL® OJ-H®
CHIRALCEL OJ-H is a registered trademarks of DAICEL Chemical Industries, Ltd.
Lux® Cellulose-3 vs. CHIRALCEL® OJ-H®
(Separation of stereoisomers of difenoconazole)
Hex:EtOH 90:10 V/V
Flow rate: 1 ml/min
Lux® Cellulose-3
CHIRALCEL® OJ-H®
CHIRALCEL OJ-H is a registered trademarks of DAICEL Chemical Industries, Ltd.
Coated vs. covalently immobilized polysaccharide-based
chiral columns
Coated vs. Immobilized CSPs
CHIRALPAK® AD- H® CHIRALPAK® IA™
Ghanem, L. Naim, J. Chromatogr. A 1101, 2006, 171-178
CHIRALCEL, CHIRALPAK, AD, IA are registered trademarks of DAICEL Chemical Industries, Ltd.
Ghanem, L. Naim, J. Chromatogr. A 1101, 2006, 171-178
Amylose triscarbamate
Coated vs. Immobilized CSPsLux® Cellulose-1 vs. CHIRALPAK® IB™
CHIRALPA IB is a trademarks of DAICEL Chemical Industries, Ltd.
Coated vs. Immobilized CSPsLux® Cellulose-1 vs. CHIRALPAK® IB™
Percentage of Compounds Resolved on Coated and Immobilized CSPs using Hex:IPA
CHIRALPA IB is a trademarks of DAICEL Chemical Industries, Ltd.
Method Development
•Column complementarity
•Mobile phase complementarity
•Mobile phase additives
•Temperature
•Elution order reversal
•Chemo- and enantioselectivity
Separation of Flavanone Using
Lux® Amylose-2 with NP and PO
0 10 20 30 t, min
CH3CN
Hexane/2-Propanol (9:1)
Complementary Chiral
Recognition in NP and RP
NP (Hexane/IPA)
Columns Rs
Lux Cellulose-1 0
Lux Cellulose-2 0
Lux Amylose-2 0
OH
F
O
Flurbiprofen
min0 2 4 6 8 10
mAU0
50
100
150
200
250
300
DAD1 D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0209\LP206091.D)
7.585
8.510
Lux Amylose-2Rs: 3.15 (CH3CN/aq* (6:4)
min0 2 4 6 8 10
mAU0
50
100
150
200
DAD1 D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0109\LP901103.D)
6.603
Lux Cellulose-2 Rs: 0 (CH3CN/aq* (6:4)
min0 2 4 6 8 10
mAU0
50
100
150
200
250
300
DAD1 D, Sig=220,8 Ref=off (F:\PHEN4157\HPCHEM\1\DATA\LP0109\LP901027.D)
6.769
Lux® Cellulose-1Rs: 0 (CH3CN/aq* (6:4)
* aq: 0.1% HAc
Separation of Bifonazole by SFC
M i n u t e s
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4
mv
- 5 0
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
5 0 0
5 5 0
6 0 0
6 5 0
mv
- 5 0
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
3 0 0
3 5 0
4 0 0
4 5 0
5 0 0
5 5 0
6 0 0
6 5 01 : 2 2 0 n m , 4 n m
B I F O N A Z O L E _ L U X 2 _ 1 0 2 2 0 8 _ 2
MP:MeOH+0.1%DEA/CO2(15:85)
Lux 5 µm Cellulose-2
M i n u t e s
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0 2 2 2 4
mv
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
mv
- 1 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
6 0 0
7 0 0
8 0 0
1 : 2 2 0 n m , 4 n m
B I F O N A Z O L E _ L U X 1 _ 1
MP: MeOH+0.1%DEA/CO2(15:85)
Lux® 5 µm Cellulose-1 N
N
Oct 2010-Oct 2012
244 Accepted projects
161 Successful projects
Chiral Screening Summary
244 Accepted projects
161 Successful projects
Chiral Separation Mode Statistics
244 Accepted projects
161 Successful projects
Reverse Phase Conditions
69% Success Rate
(111 successful projects)
244 Accepted projects
161 Successful projects
Normal Phase Conditions
55% Success Rate
(88 successful projects)
244 Accepted projects
161 Successful projects
Polar Organic Phase
20% Success Rate
(32 successful projects)
244 Accepted projects
161 Successful projects
Chiral Screening Summary
Method Development
Purpose:
- chemo- + enantioselectivity
- desired enantiomer elution order
Means:
- Chiral stationary phase (CSP)
- Mobile phase
- Separation temperature
Justification
Chemo- + enantioselectivity:
- resolve chiral drugs and impurities
- chiral drugs and metabolites
Desired enantiomer elution order:
- analysis
- purification
Chemo + enantioselectivity
cis-Diltiazem
Compound A
13
.29
10
.52 Lux 5µm Cellulose-1
Rs: 3.92
MP: 0.1 %DEA in Hexane/Ethanol (95:5)
Lux 5µm Amylose-2
Rs: 1.21
MP: 0.1 %DEA in Hexane/Ethanol (8:2)
Separation of Stereoisomers of cis-
Diltiazem on Lux® Chiral Columns
Unpublished result
N-desmethyl cis-
diltiazem
Compound BB
A
Lux 5µm Cellulose-2
Rs: 0.83, 0.63
MP: 0.1 % FA Hexane/Ethanol (8:2)
Lux 5µm Cellulose-1
Rs: 3.80
MP: 0.1 % FA Hexane/Ethanol (8:2)
Cyclothiazide
Lux 5µm Amylose-2
Rs: 1.22, 4.09
MP: 0.1 % FA Hexane/Ethanol (8:2)
Nadolol
Separation of Stereoisomers of Cyclothiazide
and Nadolol on Lux® Chiral Columns
Unpublished result
Unpublished result
Mobile phase: n-Hex/EtOH99/1 (v/v), 1 ml/min.
Separation of Stereoisomers
of Ethyl-3-methyl-3-phenylglycidate
and Ethyl-3-phenylglycidate on Lux® Cellulose-4
Separation of stereoisomers of difenoconazole
n-Hex/EtOH 90:10 v/v
Flow rate: 1 ml/min
Lux® Cellulose-3
2S,4R
2R,4S
2R,4R
2S,SR
F. Dong, J. Li, B. Chankvetadze, Y. Cheng, X. Liu, J. Xu, X. Chen, Y. Li, C. Bertucci,
D. Tedesco, R. Zanasi, Y. Zheng, The chiral triazole fungicide difenoconazole:
absolute stereochemistry, stereoisomer bioactivity, aquatic toxicity and environmental
behavior in vegetable and soil, Environmental Science & Technology, 47, 2013, 3386-3394.
Lux Cellulose-4 0.01% 2-propanol
Proprietary agrochemical product-1
RC
E
RT
Z
SC
ES
TE
ST
ZS
CZ
RT
E
RC
Z
Lux Cellulose-1
Enantiomer elution order
Chiral stationary phase (CSP)
Mobile phase
Separation temperature
Column Complementarity
Enantiomer Elution Order
L
DLux Cellulose-4
D
L
Lux Cellulose-3
D
L
Lux Cellulose-1
L
DLux Cellulose-2
FMOC-IsoLeucine in normal phase
300
300
150
150
30
0
10 20
0
mAU
min
Cellulose tris (3,5-dimethylphenylcarbamate)
Lux Cellulose-1
Amylose tris (3,5-dimethylphenylcarbamate)
R
S
Naproxen
Mobile phase: n-hexane/ethanol/formic acid
92/8/0.1 (v/v/v)
The effect of polysaccharide backbone on the
enantiomer recognition pattern
10 20
030
min
200
100
200
100
0
mAU
Cellulose Carbamate vs. Cellulose Benzoate
Naproxen
Mobile phase: n-hexane/ethanol/formic acid
92/8/0.1 (v/v/v)
Cellulose tris (4-methylbenzoate)
Lux Cellulose-3
5 10
0
20
min15
200
100
200
100
0
mAU
The effect of substituent position
n-hexane/2-propanol/formic acid
(99.5/0.5/0.1, v/v/v)
Ibuprofen
Enantiomer elution order
Chiral stationary phase (CSP)
Mobile phase
Separation temperature
(-)
(+)
(+)
(+)(-)
(-)
b
c
a
Analyte: Bifonazole
Column: Lux Amylose-2
Methanol
Ethanol
Acetonitrile
The Effect of Mobile Phase on
Enantiomer Elution Order
The effect of a type of
alcohol/polar modifier
200
100
300
150
0
mAU
10 20
030
min
n-hexane/2-propanol/formic acid (95/5/0.1, v/v/v)
n-hexane/ethanol/formic acid (95/5/0.1, v/v/v)
Chiral column: Lux Amylose-2
n-Hex/IPA/FA=60/40/0.1
n-Hex/IPA/FA=85/15/0.1
D
L
D,L
L
D
n-Hex/IPA/FA=95/5/0.1
The effect of IPA concentration on
enantiomer elution order in HPLC
Lux Cellulose-1FMOC-
isoleucine
0.1 %FA in Hexane/IPA(85:15)D
L
0.3 %FA in Hexane/IPA(85:15)
0.5 %FA in Hexane/IPA(85:15)
1.0 %FA in Hexane/IPA(85:15)
The Effect of FA Concentration
on Enantiomer Elution Order in HPLC
D,L
L
D
LD
CH3
OHO
CH3
H O
H
C
N
O
Enantioseparation
of Fmoc-(D,L)-Isoleucine
*
L. Chankvetadze et al. Journal of Chromatography A, 2011, 6554-6560
K.S.S. Dossou et al. Journal of Separation Science, 34 (15), 2011, 1772-1780
Enantiomeric Impurity
Determination in S-Amlodipine
0 5 10 15 20 25 30 35 40
0
50
100
150
200
250
mA
U
Time (min)
S
S
R
R
ACN/0.1% DEA/0.02% FA
0 5 10 15 20
0
50
100
150
200
250
mA
U
Time (min)
R
S
S
R
ACN/0.1% DEA/0.01% FA
The Effect of Addition of Acidic Additive
to Basic Mobile Phase on the Elution Order of Amlodipine
R
S
R
S
R
S
Time (min.)
ACN/H2O/DEA/FA (95:5:0.1:0.04)
ACN/H2O/DEA (95:5:0.1)
ACN/H2O/DEA/FA (95:5:0.1:0.06)
The Reversal of Elution Order
COOCH2CH3
CH2OCH2CH2NH2H3C
H3COOC
ACN/H2O/DEA/FA (90:10:0.1:0.03)
ACN/H2O/DEA/FA (90:10:0.1:0.3)
S
R
ACN/H2O/DEA (90:10:0.1)
S
R
R+S
No Reversal of Elution Order
COOCH2CH3
CH2OCH2CH2NH2H3C
H3COOC
Aqueous mobile phase:
Does this always mean
a reversed-phase behaviour?
R
R
R
R
R
R
R
R
S
S
S
S
S
S
S
S
ACN/DEA = 100/0.1
ACN/H2O/DEA = 90/10/0.1
ACN/H2O/DEA = 80/20/0.1
ACN/H2O/DEA = 70/30/0.1
ACN/H2O/DEA = 60/40/0.1
ACN/H2O/DEA = 50/50/0.1
ACN/H2O/DEA = 40/60/0.1
ACN/H2O/DEA = 95/5/0.1
t, min
Aqueous mobile phase:
Does this always mean
a reversed-phase behaviour?
Amlodipine
Column: Lux Cellulose-4
Effects observed:
Decrease of enantioselectivity
Disappearance of enantioselectivity
Increase of enantioselectivity
Appearance of enantioselectivity
Reversal of enantiomer recognition pattern
Effects observed:
Decrease of enantioselectivity
Disappearance of enantioselectivity
Increase of enantioselectivity
Appearance of enantioselectivity
Reversal of enantiomer recognition pattern
ACN/DEA=100/0.1
ACN/DEA/FA=100/0.1/0.1
R
S
R
S
0
20
40
0
20
40
20 40
20 40mAU
mAU
min
min
Carvedilol
Lux Cellulose-1
Decrease of enantioselectivity
RS
R
S
0
20
40
60
0
20
40
10 20
10 20mAU
mAU
min
min
IPA/DEA=100/0.1
IPA/DEA/FA=100/0.1/0.1
Amylose tris(3,5-dimethylphenylcarbamate)
Effects observed:
Decrease of enantioselectivity
Disappearance of enantioselectivity
Increase of enantioselectivity
Appearance of enantioselectivity
Reversal of enantiomer recognition pattern
a)
b)
0
40
80
120
0
200
400
600
10
10
20
20
R+S
R
S
mAU
mAU
ACN/DEA=100/0.1
ACN/DEA/FA=100/0.1/0.1
min
min
Lux Cellulose-2
Disappearance of enantioselectivity
Pindolol
Effects observed:
Decrease of enantioselectivity
Disappearance of enantioselectivity
Increase of enantioselectivity
Appearance of enantioselectivity
Reversal of enantiomer recognition pattern
IPA/DEA=100/0.1
IPA/DEA/FA=100/0.1/0.1
R
S
R
S
5 15 25
5 15 25
40
80
0
0
20
40
mAU
mAU
min
min
35
35
The effect of acidic additive on separation of basic compound
Propranolol
Column: Lux-Cellulose-1
R
S
R
S
20 40
20 40
0
40
80
40
80
mAU
mAU min
min
IPA/DEA=100/0.1
IPA/DEA/FA=100/0.1/0.1
The effect of acidic additive on separation of basic compound
Column: Lux-Cellulose-1
Pindolol
ACN=100%
ACN/DEA=100/0.1
ACN/ACA=100/0.1
R
S
R
S
R
S
0
100
200
50
100
0
0
100
200
10 20
10 20
10 20
mAU
mAU
mAU
min
min
min
Lux Amylose-2
Increase of enantioselectivity
Pindolol
Effects observed:
Decrease of enantioselectivity
Disappearance of enantioselectivity
Increase of enantioselectivity
Appearance of enantioselectivity
Reversal of enantiomer recognition pattern
R+S
RS
IPA/DEA=100/0.1
IPA/DEA/FA=100/0.1/0.1
10 20
10 20
0
100
200
0
200
400
600
mAU
mAUmin
min
The effect of acidic additive on separation of basic compound
Propranolol
Column: Lux-Cellulose-4
ACN/DEA=100/0.1
ACN/DEA/FA=100/0.1/0.1
0
0
40
80
50
100
150
10 20 30
10 20 30
mAU
mAUmin
min
Carazolol
Lux Cellulose-4
Appearance of Enantioselectivity
R+S
RS
RS
ACN/DEA=100/0.1
ACN/FA=100/0.1
ACN/DEA/FA=100/0.1/0.1
10 20
10
10
20
20
0
0
0
50
100
50
100
200
400
mAU
mAU
mAU
min
min
min
The effect of acidic additive on
separation of basic compound
Propranolol
Column: Lux-Amylose-2
ACN=100%
ACN/DEA=100/0.1
ACN/ACA=100/0.1
R+S
R+S
RS
10 20 30
10 20 30
10 20 30
0
50
100
0
20
40
60
0
100200
300
mAU
mAU
mAU
min
min
min
Propranolol
Lux Cellulose-1
Appearance of Enantioselectivity
Effects observed:
Decrease of enantioselectivity
Disappearance of enantioselectivity
Increase of enantioselectivity
Appearance of enantioselectivity
Reversal of enantiomer recognition pattern
R
S
R
S
0
100
200
300
0
50
100
150
5 15 25
5 15 25
mAU
mAU
IPA/DEA=100/0.1
IPA/DEA/FA=100/0.1/0.1
min
min
Lux Cellulose-4
Carvedilol
Reversal of Enantiomer Elution Order
ACN/DEA=100/0.1
ACN/FA=100/0.1
ACN/DEA/FA=100/0.1/0.1
R
S
R
S
S
R
0
20
40
040
80
120
2000
400600
a)
b)
c)
mAU
mAU
mAU
min
min
min10 20 30
10 20 30
10 20 30
The effect of acidic additive on
separation of basic compound
Carvedilol
Column: Lux Cellulose-4
R
S
R
S
0
100
200
300
0
100
200
300
400
IPA/DEA=100/0.1
IPA/DEA/FA=100/0.1/0.1
10
10 20
20
mAU
mAU
min
min
Reversal of Enantiomer Elution Order
Lux Cellulose-4
Pindolol
R
S
S
R
0
10
20
30
0
40
80
10 20
10 20
ACN/DEA=100/0.1
ACN/DEA/FA=100/0.1/0.1
mAU
mAU
min
min
Reversal of Enantiomer Elution Order
Carvedilol
Lux Cellulose-4
1S,2S
1R,2R
1R,2R
1S,2S
ACN/DEA=100/0.1
ACN/DEA/FA=100/0.1/0.1
0
20
40
40
0
80
10 20
10 20
mAU
mAU
min
min
Reversal of Enantiomer Elution Order
cis-Tramadol
Lux-Cellulose-4
2-Propanol=100%
2-Propanol/DEA=100/0.1
2-Propanol/FA=100/0.1
IPA/DEA/FA=100/0.1/0.1
040
80
120
050
100
150
0
100
200
0
100
200
300
10 20 30
10 20 30
10 20 30
10 20 30
R+S
R
S
S
R
SR
mAU
mAU
mAU
mAU
min
min
min
min
Reversal of Enantiomer Elution Order
Carvedilol
Lux-Cellulose-2
Enantiomer elution order
Chiral stationary phase (CSP)
Mobile phase
Separation temperature
The Effect of Temperature on
Enantiomer Elution Order
5°C
20°C
50°C
D
L
D,L
L
D
Analyte: FMOC-Isoleucine
Column: Lux Cellulose-1
Mobile phase: Hex/IPA/FA 90/10/0.1 (v/v/v)
10 20
030
800
400
800
400
800
400
mAU
5°C
55°C
75°C
R
R+S
R
S
S
t, min
Analyte: Ketoprofen
Column: Lux Cellulose-2
Mobile phase: nHex/IPA/FA 95/5/0.1 (V/V/V)
The Effect of Temperature on
Enantiomer Elution Order
(+)
(-)
(+)
(+)
(-)
(-)
(±)
55 °C
65 °C
35 °C
75 °C
Analyte: Bifonazole
Column: Lux Amylose-2
Mobile phase: Acetonitrile
The Effect of Temperature on
Enantiomer Elution Order
10 200
30
50
25
50
25
50
25
mAU R+S
S
R
0°C
25°C
75°C
S
R
t, min
Analyte: Ibuprofen
Column: Lux Cellulose-4
Mobile phase: n-Hex/IPA/FA 99.5/0.5/0.1 (v/v/v)
The Effect of Temperature on
Enantiomer Elution Order
Preparative Scale Separations
Loadability of Chiral
Stationary Phases
0.1
0.5
1.0
5.0
10
50
mg/g CSP100 Protein-based CSPs
Vancomycin CSP
Cyclodextrin-based CSPs
Tetraamide CSPs (Kromasil
Polyacrylamide (Chiraspher)
Brush-type CSPs (Pirkle)
Polysacchiride-based CSPs
Historical Preparative Chiral
Column Performance
Performance decreases as column diameter increases
• Chiral Columns do not perform as well as achiral columns
– Analytical columns offer always higher performance
• High efficiency
• Good peak symmetry
– Preparative columns have lower performance
• Lower efficiency for same particle size
• Peak tailing is prevalent
Limitations of Conventional
Slurry Packing
High pressure solvent forces
sedimentation of the slurry
After sedimentation, column is disassembled from slurry chamber and capped (as quickly as possible)
During disassembly the
bed “relaxes” and
extrudes from column
• Packed bed disturbed
• Packing density reduced
• Non-uniform density
- Lower density at the “uncapped” end
• Inherent in all slurry packed columns
• Revolution in preparative column design and
manufacturing – R&D 100 Award in 2006
• Patented Hydraulic Piston Compression
technology, adapting Dynamic Axial Compression
to high-throughput disposable column
• Removes bed collapse/voiding as a source of
column failure and greatly improved performance
• Packing is 100% micro-processor controlled
leading to vast improvements in column-to-
column consistency and overall process control
Axia™ Patented Technology
AXIA is a trademark of Phenomenex.Axia is patented by Phenomenex. U.S. Patent No. 7,674,383
Axia™ Patented Technology
Integrated axial compression technology into pre-packed preparative columns
http://www.phenomenex.com/Info/WebDocumentServe/demo9.swf
Axia™-packed Lux® Cellulose-1 250 x 21.2 mm
Media Asymmetry Plates/Meter
Lux Cellulose-1 1.37 77435
High efficiencies are maintained when scaling from analytical to
preparative dimensions
up to 50mm IDs
Column Performance
N = 70,664
αααα = 1.19
SN 461603-13
N = 75,156
αααα = 1.19
SN 456633-1Analytes: TSO
Column: Lux® Cellulose-2
Mobile Phase: Hexane:IPA 9:1 (V/V)
Ab
so
rba
nc
eA
bs
orb
an
ce
0
0 Minutes 18
Column 250 x 50 mm
Column 250 x 4.6 mm
Columns tested:
• Lux® Cellulose-1 250 x 4.6 mm, 5 µm
• Lux Cellulose-1 250 x 4.6 mm, 10 µm
• Lux Cellulose-1 250 x 4.6 mm, 20 µm
Analytical testing conditions:
• Mobile Phase: 60:40 Hex:IPA with 0.1% formic acid
• Method Type: Isocratic
• Flow Rate: 1mL/min
• Concentration of sample: ~0.5mg/mL
• Injection Volume: 10 uL
• Temperature: Ambient
• Detection: UV at 254nm
• Runtime: 15 minutes
Chiral Separation of Warfarin
2 4 6 8 10 12
mAU0
5
10
15
20
25
30
35
40
DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...IRAL PS 120831A 2012-08-31 14-59-46\WARFARIN LUX1-20U_1MG_ML_2.D)
4.6
84
7.4
60
Part α Rs
5 µm 2.4 8.7
10 µm 2.5 4.6
20 µm 2.4 2.2
10 µm
5 µm
20 µm
Chiral Separation of Warfarin
Lux® Cellulose-1
Columns tested:
• Lux® Cellulose-1 250 x 4.6 mm, 5 µm
• Lux Cellulose-1 250 x 4.6 mm, 10 µm
• Lux Cellulose-1 250 x 4.6 mm, 20 µm
Analytical testing conditions:
• Mobile Phase: 60:40 n-Hexane:Isopropanol with 0.1% formic acid
• Method Type: Isocratic
• Flow Rate: 1mL/min
• Concentration of sample: ~0.5mg/mL
• Injection Volume: 10 uL
• Temperature: Ambient
• Detection: UV at 220nm
• Runtime: 15 minutes
Chiral Separation of Lansoprazole
Part α Rs
5 µm 1.3 2.3
10 µm 1.4 2.1
20 µm 1.4 0.9
min2 4 6 8 10 12 14
mAU-50
0
50
100
150
200
DAD1 A, Sig=220,4 Ref=off (C:\CHEM32\...24 2012-08-28 14-52-09\LANSOPRAZOLE LUX1-5U 60_40 HEXANE_IPA_1.D)
2.9
31
6.3
89
7.4
47
Chiral Separation of Lansoprazole
10 µm
5 µm
20 µm
Lux® Cellulose-1
Summary for Lux® Cellulose-1
• Selectivity (α): Constant across all
particle sizes (5 µm, 10 µm, 20 µm)
• Efficiency (N): As particle size increases,
efficiency of column decreases.
• Resolution (Rs): As particle sizes
increases, resolution of peaks
decreases.
Part α N Rs
5 µm 2.4 103472 8.7
10 µm 2.5 31000 4.6
20 µm 2.4 9660 2.2
Warfarin
Part α N Rs
5 µm 1.3 66208 2.3
10 µm 1.4 25212 2.1
20 µm 1.4 7024 0.9
Lansoprazole
Columns tested:
• Lux® Cellulose-1 250x4.6 mm, 10 µm
• Lux Cellulose-1 250x4.6 mm, 20 µm
Preparative testing conditions:
• Mobile Phase: 60:40 Hex:IPA with 0.1% formic acid
• Method Type: Isocratic
• Flow Rate: 1mL/min
• Concentration of sample: 20mg/mL
• Temperature: Ambient
• Detection: UV at 254nm
• Runtime: 15 minutes
Loadability Experiments
with Warfarin
2 4 6 8 10 12
mAU0
500
1000
1500
2000
2500
3000
DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...T TEST\CHIRAL PS 120904A 2012-09-04 17-50-03\WARFARIN 10U_6MG_.D)
2 4 6 8 10 12
mAU0
500
1000
1500
2000
2500
3000
3500
DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...T TEST\CHIRAL PS 120904A 2012-09-04 16-36-45\WARFARIN 20U_6MG_.D)
Loadability 10 µm vs. 20 µm
Part α Rs
10 µm 2 1.1
20 µm 1.9 0.6
“touching bands”
10 µm
300 µL injection: 6mg of compound at 0.24% specific load
20 µm
2 4 6 8 10 12
mAU0
500
1000
1500
2000
2500
3000
DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...TEST2\CHIRAL PS 120905A 2012-09-05 14-20-08\WARFARIN 10U_14MG_.D)
2 4 6 8 10 12
mAU0
500
1000
1500
2000
2500
DAD1 B, Sig=254,4 Ref=off (C:\CHEM32\...TEST2\CHIRAL PS 120905A 2012-09-06 11-10-52\WARFARIN 20U_14MG_.D)
“Touching bands”
Part α Rs
10 µm 2.1 0.6
20 µm <1.1 0
700 µL injection: 14 mg of compound at 0.55% specific load
Loadability 10 µm vs. 20 µm
No resolution
10 µm
20 µm
Axia™ Column Family
Axia Packing Process now scaled to provide three diameters
and three lengths for Lux® products
21.2 mm
50 mm
30 mm
New Developments
Enantioseparation with Chromolith Si (R) modified by coating
of polysaccharide derivative on the surface of silica monoliths
Column size: 50 x 4.6 mm
Mobile phase: n-Hex/2-Propanol=9/1
Flow rate: 20 ml/min
Backpressure: 163 bar
t1=12.8 sec
t2 =23.0 secC CH
2OH
CF3
H
CH
OH
CF3
0 2 4 t, min.
a)
CH C
OOH
t, min.0 2 4
CH3CH3
OH OH
t, min.0 2 4
b) c)
(+)
(-)
(+)
(-) (-)
(+)
Enantioseparation of some chiral chemicals on the
monolithic capillary column coated with cellulose
tris(3,5-dimethylphenylcarbamate)
Capillary dimension: 100 mm x 15 cm; Mobile phase: n-Hex/2-Prop=9/1
CH
OH
CF3
0 15 30 45 t, sec.
(-)
(+)
Fast LC enantioseparation using monolithic capillary column
modified with cellulose tris(3,5-dimethylphenylcarbamate)
Capillary dimension: 100 mm x 15 cm
Mobile phase: n-Hex/2-Prop=9/1
t1~9 sec
t2~22 sec
Fused-core silica based CSPs
Enantioselective peak focusing in HPLC
N=
13
44
0
N=
14
67
5
N=
17
44
5
N=
53
80
5
Run 1
Run 3
Run 7
N=
16
67
0
N=
16
36
0
N=
19
23
4
N=
51
33
8N
=5
15
63
N=
18
81
6
N=
16
43
5
N=
15
52
2
Enantioselective peak focusing
Column: Amylose
tris (3,5-dimethylphenylcarbamate)
Sample diluted with/dissolved in:
n-hexane/etanol 95/5 (v/v)
Mobile Phase:
n-hexane/etanol 99.9/0.1 (v/v)
N=
12
06
8
N=
11
86
7N
=1
71
70
N=
81
66
6
Column: Lux Cellulose-3
Sample diluted with/dissolved in:
n-hexane/etanol 95/5 (v/v)
Mobile Phase:
n-hexane/etanol 99.9/0.1 (v/v)
I would like to thank for a fruitful co-operation over several years
my colleagues from Phenomenex Inc., especially:
Dr. Tivadar Farkas
Dr. Ismail Rustamov
as well as my co-workers and students at Tbilisi State University
(Tbilisi, Georgia).
Acknowledgments