Embed Size (px)
Citation preview
Unusual retention mechanism and selectivity:CD-Screen column for analysis of cyclodextrin-derivatives and
-Select column for general purpose applications
Julianna Szemán1, Katalin Csabai1, Gábor Varga2
1 CYCLOLAB Cyclodextrin R&D Laboratory Ltd., Budapest, Hungary, e-mail: [email protected] 2 CHIROQUEST Chiral Technologies Development Ltd., Budapest, Hungary, e-mail: [email protected]
In our previous work novel stationary phase was prepared by bonding N-(4-nitrophenyl)-carbamide group to the silica gel matrix [1,2]. The new phase has primarily been developed for analysis of cyclodextrins (CDs) and their derivatives.
Taking into account the structure of this selector it seemed to be a promising tool for the separation of various families of compounds. Based on theoretical considerations several types of interactions can be expected with different types of molecules. The main characteristic property of this stationary phase is the essential role of π-π interactions in the retention mechanism [3, 4]. Due to the electron-withdrawing nitro-group in para position, retention forces become stronger and shape selectivity can be significantly better compared to other commercially available phenyl-bonded silica phases [5].
In general, hydrogen bonding was regarded as one of the reasons of poor peak symmetry and low efficiency of chromatographic media. However, the rationally planned, well-defined, non silanol type hydrogen bonding capability can play an important role in the selectivity and retention. Due to the balanced ratio of several types of interactions, it can be supposed that this stationary phase would be suitable both for reversed and normal phase applications, as well.
Although the numerous types of - active HPLC phases have been already designed and investigated [3, 4], columns having diverse and multiple interacting sites for selectivity tuning can still expect great interest to solve separation problems e.g. in orthogonal chromatographic systems.
The aim of this work was to study the separation potency of 4-nitrophenyl-carbamide stationary phases and to optimize its capability for different separation problems.
INTRODUCTIONINTRODUCTION
N+
O-
O
NH
OO
NHCH2CH2CH2
O
O Si δ+
H-acceptor
H-donors
STRUCTURE OF THE SELECTORSTRUCTURE OF THE SELECTOR
PRINCIPLE OF SEPARATIONPRINCIPLE OF SEPARATION
RESULTS AND DISCUSSIONRESULTS AND DISCUSSION
Illustration of the interaction between the apolar cavity of cyclodextrin and the nitrophenyl-carbamide selector
• higher surface coverage compared to CD-Screen stationary phase to obtain strong interaction
• fully endcapped to eliminate the silanol interactions, to obtain well defined H-bonding
• optimized surface coverage fitting to the size of cyclodextrin molecules
• secondary interactions with free silanols to increase the selectivity
-SELECT-SELECT CD-SCREENCD-SCREEN
Electrostatic potential surface of 4-nitrophenyl and phenyl-carbamide in complex with phenol
The steric possibilities of the interactions were examined by molecular modelling methods. Both selector and solute molecular models were geometry-optimized using HyperChem MM+ molecular mechanics computational method. The energy minimization of the system consisting of these molecules together was the next step using the same method. The resulted complexes show clearly the presence of one or more hydrogen-bridges, depending on the chemical structure of analyte. Examining the electrostatic potential surfaces of the molecules in these complexes the role of the electron- withdrawing nitro-group in the retention mechanism can be easily understood.
Illustration of hydrogen-bonding between 4-nitro-phenyl-
carbamide and phenol
min0 2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20 1
2
min0 2 4 6 8 10 12 14 16 18
mAU
0
5
10
15
20
25
12
π-Select
Conventional phenyl-column
k’=0.54
k’=0.25
Polar analytes
Separation of phenol and caffeine
• higher retention due to the - interactions and well defined hydrogen-bonding
• residual silanols are eliminated, good peak shape • in pH 2-8 interval the retention time of phenol
is relatively high and does not depend on the pH (on conventional phenyl column the retention time of phenol is near to t0 at higher pH)
Separation of apolar, aromatic analytes
• high retention on the -Select column, in spite of the fact that the carbon-content of this stationary phase is very low
• the shape-selectivity and CH2-selectivity are higher, even diethyl-phthalate and biphenyl are well resolved.
min0 2 4 6 8
mAU
0
2.5
5
7.5
10
12.5
15
17.5
20
2
3 4
1
min0 2 4 6 8
mAU
0
100
200
300
400
500
600
700
800
1
2+3
4
π-Select
Conventional phenyl-column
C: 4.7%
C: 7.0%
Eluent: MeOH-water 30:701.: Phenol 2.: Caffeine
Eluent: MeOH-water 60:401.: Dimethyl-phtalate; 2.: Diethyl-phtalate3.: Biphenyl 4.: o-Terphenyl
Apolar, aromatic analytes
• The higher surface coverage of conventional phenyl columns causes steric hindrance, complex formation is restricted [5, 6].
On CD-Screen column• The retention mechanism is based on the complex
forming capability of CD rings with the functional groups of columns
• Due to the strong retention wider range of eluent composition can be used.
• Higher efficiency - lower LOQ of unsubstituded cyclodextrins.
• The separation of component groups gives the possibility to follow batch-to batch reproducibility.
Shape selectivity of -Select columnPolyaromatic hydrocarbons
[min.]Time
0 1 2 3 4 5 6 7
[mV]
Voltage
0
200
400
600
800
TOH_MeOH_85_ism
1
2 3
6 7
[min.]Time
0 2 4 6 8
[mV]
Voltage
0
100
200
300
400
500
600
ToL_MeOH_70
12
4
35
S
S
1. Benzothiophene
4. Dibenzothiophene
7. Benzo[g,h,i]perylene
6. Benzo[a]pyrene
3. Fluoranthene
2. Fluorene 5. Pyrene
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
0
50
100
150
200
min0 2.5 5 7.5 10 12.5 15 17.5 20
mAU
-10
0
10
20
30
40
Plate number 16685
asymmetry: 1,189
Plate number: 5649
asymmetry: 1,261
π-Select 250x4 mm
LiChrosphere Si 60 250x4 mm
Prostaglandine intermediate product
Eluent: MeOH-water 85:15 Eluent: MeOH-water 75:25
O
O O Si
COOCH3
Z and E
R and S*
Diastereomer peptides
[min.]Time
0 1 2 3 4 5 6 7
[V]
Voltage
0.0
0.2
0.4
0.6
0.8
1.0
D:\Doc\Cég\Marketing\Balaton2005\aminoacids\aminoacids
1.: 4-OH-Phenylglycine2.: Phenylglycine3.: Phenylalanine4.: Tryptophane
Eluent: MeOH-0.1% H3PO4 30:70 0.8 ml/min pH: 2.6
Aromatic amino-acids Basic drugs
[min.]Time
0 2 4 6 8 10
[mV]
Voltage
0
20
40
60
80
100
összes
1.:N-methyl-ephedrine2.: Athenolol3.: Pindolol4.: Sertraline
[min.]Time
0 2 4 6 8 10 12
[mV]
Voltage
0
20
40
60
összes
1.: Trasicor2.: Propranolol
[min.]Time
0 10 20 30 40 50
[µV]
Voltage
0
100
200
300
400
piszelebmtpppn
*Tyr-Pro-Phe-Atc-NH2* racemic amino-acidAtc-NH2: 2-amino-decaline-2-carboxylic acid
Eluent: MeOH-0.1% H3PO4 60:40 0.8 ml/min pH: 2.6
Eluent: MeOH-0.1% H3PO4 30:70 0.8 ml/min pH: 2.6
min2 4 6 8 10 12 14 16
mV
50
100
150
200
250
300
350
400
α-CD
min2 4 6 8 10 12 14
V
20
40
60
80
100
120
min 0 2 4 6 8 10 12 14
V
0
50
100
150
200
250
300
350
400 Batch 1
Batch 2
Batch 3
Batch 4
min 0 2 4 6 8 10 12 14
V
0
50
100
150
200
250
300
RAMEA synthesis 3
RAMEA synthesis 1
DIMEA synthesis 2
CD-Screen Phenyl column
min5 10 15 20 25 30
Norm.
50
100
150
200
250
CRISMEB
DS=1.5
RAMEB
DS=14
min2 4 6 8 10 12 14 16 18
mV
150
200
250
300
350
400
Degradation products
Examination of cyclodextrin derivatives on CD-Screen column
Determination of the remnant non-substituted αCD in the randomly methylated αCD
Different batches of randomly methylated αCD, same
production method and DS
Randomly methylated αCD prepared by different production
methods, DS~12
m2 4 6 8 1 1 1 1 1
V
30
35
40
45
50
55
60
65
70
BCD
Eluent: 45% methanol
min2 3 4
mV
50
60
70
80
90
100
110
120
130
Eluent: 18% methanol
The commercially available cyclodextrin derivatives are statistically substituted having different degree of substitution (DS) and substitution patterns. Detailed “fingerprint” chromatograms give the possibility to compare the identity or similarity of materials.
Component distribution of randomly methylated βCD
cyclodextrins having different DSDecomposition of randomly methylated β-cyclodextrins
CONCLUSIONSCONCLUSIONSNormal phase applicationEluent: n-hexane - t-buthyl-methyl-ether 97:3
Apparatus: Agilent 1050 HPLC system with UV-VIS Detector at 205 or 254 nm. For detection of cyclodextrins Evaporative Light Scattering Detector PL-ELS 1000, (Polymer Laboratories) was used (Evaporation: 110°C, Nebulization: 90 °C, Gas flow: 1.2 l/min) Columns: The stationary phases (Hungarian Patent Application Pending 2004) were prepared by ChiroQuest Ltd. Column size: 250 mm x 4.0 mm I.D; Mobile phases: methanol – water or acetonitrile – water; Column temperature: 30 °C; Flow rate: 1.0 ml/min.Samples: CDs and CD derivatives of Cyclolab Ltd., Hungary and Wacker Chemie, Germany were analysed.
EXPERIMENTAL
Separation of various families of compounds have been investigated on 4-nitrophenyl-urea bonded stationary phase in both reversed and normal phase systems.The new chromatographic media kept its retentive capability even in extremely polar or apolar conditions and proved to be suitable for the separation of different type of substances:-Select:
separation of polyaromatic hydrocarbons separation of diastereomers in reversed and normal phase conditions, as well analysis of aromatic amino-acids and basic drugs in acidic conditions
CD-Screen: characterisation of component distribution of statistically substituted CD derivatives,
batch-to-batch reproducibility, control of degree of substitution or synthesis method determination of parent CDs, and degradation products in substituted CD derivatives quality control of single isomer 6-mono-amino-β-cyclodextrin
[1] PCT Application Number PCT/HU 05/00043, May 30, 2005[2] J. Szemán, K. Csabai, K. Kékesi, L. Szente, G. Varga; J. Chromatogr. A, submitted for publication[3] J. Horak, W. Lindner ; J. Chromatogr. A, 1043, 177-194 (2004)[4] J. Horak, N. M. Maier, W. Lindner; J. Chromatogr. A, 1045, 43-58 (2004)[5] I. Caron, C. Elafkir, M. Dreux; Chromatographia 47, 383-390 (1998)[6] A. Salvador, B. Herbretau, M. Dreux; J. Chromatogr. A, 855, 645-656 (1999)
REFERENCES
Quality controll of single isomer 6-monoamino β-cyclodextrin
Eluent: 0.1% TFA in water-0.1% TFA in MeOH 75:25
Chromatographic profile of hydroxypropyl--cyclodextrinComparison to phenyl column
The authors are grateful to Ms. Zs. Zachár and Ms. E. Erdei to their valuable technical assistance. The work was supplied by the National R&D programme (NKFP-1/A-04104).
ACKNOWLEDGEMENT
