Selection of appropriate chiral selectors for chiral analysis by regression modeling
of spectral data
Selorm Modzabi, Marianna A. Busch and Kenneth W. BuschBaylor University
One Bear Place #97348Waco, TX 76798
Need for chiral analysis*• Pharmaceutical industry
– Drug development– Process control
• Agro-chemical industry• Food and beverage industry• Fragrance industry• Basic research
*Chiral Analysis, K. W.Busch & M. A. Busch Eds., Elsevier, 2006
CHIRAL ANALYSIS BY REGRESSION MODELING OF SPECTRAL DATA
(CARMSD)
Modern chemical instrumentation allows us to combine—•Multivariate (multi-wavelength) data collectionwith•Multivariate modelingto give a powerful combination that can extract latent information from the multivariate data that would not be possible with univariate measurements
Basic Strategy of CARMSD1. Calibration Phase
1) Prepare a set of calibration samples
• Same total concentration of chiral analyte
• Different known enantiomeric compositions
• Fixed concentration of chiral auxiliary
Basic Strategy of CARMSD1. Calibration Phase
2) Collect spectral data on the calibration set
3) Perform PLS-1 regression modeling
Basic Strategy of CARMSD2. Validation Phase
1) Prepare a new set of validation samples
2) Collect spectral data3) Enter the spectral data into the
regression model and predict the enantiomeric compositions
4) Compare the predicted enantiomeric compositions with the known values
CARMSD•Clearly the chiral auxiliary is at the heart of the CARMSD method.•Regression modeling depends on changes in the spectral signature with enantiomeric composition of the sample. •The larger these spectral changes are, the easier it is to develop robust regression models.
CARMSD
• Chiral Selectors used to date– Cyclodextrins– Modified cyclodextrins– Surfactants & mixed cyclodextrins– Chiral Surfactants– Chiral Ionic Liquids
Enantiomer-CD transient inclusion complexes
C
CH3
ClH
BrC
CH3
H Cl
Br
Enantiomeric pair
Diastereomeric pair(hypothetical)
CDCD
Enantiomeric discrimination by transient noncovalent complex
formation with cyclodextrins— An Example
C
a
d bc
C
a
b dc
Chiral selectors used to date
Chiral selector Analyte concentration Prediction error (RMSEP) range
Cyclodextrins (CDs)
3.75 – 7.50 mM 0.02 – 0.07
Modified cyclodextrins (MCDs)
7.5 mM 0.05 – 0.6
Chiral surfactants (CSs) 1.5 – 6 % w/v
1.0 x 10-4 - 5.0 x 10-6
M0.02 – 0.05
Chiral ionic liquids (CILs) 30 and 150 mM
5 mM 0.05 – 0.09
Analyte to selector (CDs, MCDs) mole ratio: = 1 : 2RMSEP = xip – xi)2/n]1/2 : xi = known mole fraction, xpi = predicted mole fraction & n = total samplespredicted
Chiral analytes: amino acids, pharmaceuticals, other organics
Problems with CDs
•Limited Solubility•Extent of interaction depends on formation constant of inclusion complex•Possibility of more than one complex in solution (R—CD, CD—R—CD, etc.)•Inclusion complex formation depends on size of analyte in relation to cyclodextrin
What about other possible chiral auxiliaries?
Formation of quaternary ammonium salts of carboxylic acids
Use of Chiral AminesIon-pair formation as a means of enantiomeric discrimination
R CO
OH+ RNH2
R CO
O- +NH3R
Chiral selector: (S)-1-phenylethylamine (S-PEA)
CARMSD with a Homochiral Amine
Determination of enantiomeric composition of Tyrosine with (S)-phenylethylamine using UV spectroscopy
Diastereomeric ion pairs
Zwitterion
+NH3
O
O-
HO
H2N CH3
H3N+ CH3
NH2
O
O-
HO
+D
S
SD
+NH3
O
O-
HO
H2N CH3
H3N+ CH3
NH2
O
O-
HO
+L
S
SL
Use of (S)-1-phenylethylamineDetermination of enantiomeric composition of Tyrosine
(Tyr to S-PEA ratio = 1 : 1)
Mean centered UV spectra for different compositions of D- and L-Tyrosine + (S )-PEA
-0.04
-0.03
-0.02
-0.01
0
0.01
0.02
0.03
0.04
285 305 325 345 365 385 405
Wavelength/nm
Mea
n ce
nter
ed A
bsor
banc
e
I sobestic point(339 nm)
-Two diff erent absorbing species: D- & L- Tyr quaternary salts-Solutions contain identical sum of Tyr species-Relative amounts determine by ratio of D- and L-Tyr -Tyr species have identical absorptivity at 339nm
-0.1
0.4
0.9
1.4
1.9
2.4
2.9
3.4
3.9
4.4
250 260 270 280 290 300 310 320 330 340 350
Wavelength/nm
Abs
orba
nce
Original spectra
Isosbestic point(343 nm)
Effect of pH on spectrum
Effect of varying PEA/Tyrmol ratios at neutral pH
Effect of varying PEA/Tyr mol ratios in acid solution
UV spectra: 2.5 mM L-Tyrosine + 2.5 mM PEA
-0.1
0.4
0.9
1.4
1.9
2.4
2.9
3.4
3.9
236 256 276 296 316 336 356
Wavelength/nm
Abs
orba
nce
2.5mM PEA
2.5mM Tyr
PEA1 : 8Tyr
PEA2 : 7Tyr
PEA3 : 6Tyr
PEA4 : 5Tyr
PEA4.5 : 4.5 Tyr
PEA5 : 4Tyr
PEA6 : 3Tyr
PEA7 : 2Tyr
PEA8 : 1Tyr
2.5 mM Tyr
PEA : Tyr 1 : 1
2.5 mM PEA
236 - 252 nm: hyperchromic effect
289 - 313 nm : hyperchromic effect
UV spectra: 2.5 mM L-Tyrosine + 2.5 mM PEA (Solution acidified with HCL)
-0.05
0.45
0.95
1.45
1.95
2.45
2.95
3.45
3.95
236 256 276 296 316 336 356
Wavelength/nmA
bsor
banc
e PEA1 : 9Tyr
PEA2 : 8Tyr
PEA3 : 7Tyr
PEA4 : 6Tyr
PEA5 : 5Tyr
PEA6 : 4Tyr
PEA7 : 3Tyr
PEA8 : 2Tyr
PEA9 : 1Tyr
236 - 252 nm : no hyperchromic effect
289 - 313 nm : no hyperchromic effect
PEA : Tyr 1 : 1
Job’s Plot
Job’s plot in neutral solution indicating 1:1 ion pair formation
Job’s plot in acid solution indicating lack of ion pair formation
Job's Plot: 2.5 mM Tyrosine + 2.5 mM (S)-PEA
0
0.5
1
1.5
2
2.5
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85
Volume of (S)-PEA/ml
Abs
orba
nce
at 2
43 n
m
Job's Plot: 2.5 mM L-Tyrosine + 2.5 mM PEA (solutions acidified with HCl)
0.49
0.5
0.51
0.52
0.53
0.54
0.55
0.56
0.57
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Volume of S-PEA/mlA
bsor
banc
e at
243
nm
Determination of enantiomeric composition of Tyrosine with (S)-
phenylethylamine using UV spectroscopy— PLS1 Calibration Model
PlotsRandomly Selected Calibration Samples: 0.0500, 0.150, 0.300, 0.400, 0.500, 0.650, 0.850, 0.950
-5
-4
-3
-2
-1
0
1
2
3
4
5
317 367 417 467 517
Wavelength/nm
Reg
ress
ion
coef
ficie
nt
D-TyrL-Tyr
Cross validation of calibration samples: 0.0500, 0.150, 0.300, 0.400, 0.500, 0.650, 0.850, 0.950
Actual D-Tyr mole fraction
Predicted D-Tyr mole fraction
Predicted L-Tyr mole fraction
0.100 0.101 0.899 (0.900)0.250 0.250 0.750 (0.750)0.300 0.303 0.697 (0.700)0.400 0.401 0.599 (0.600)0.450 0.462 0.538 (0.550)0.650 0.659 0.341 (0.350)0.750 0.758 0.242 (0.250)0.800 0.802 0.198 (0.200)0.900 0.901 0.099 (0.100) RMSEP: D-Tyr & L-Tyr = 0.006Values in bracket = Actual mole fraction of L-Tyr
Determination of enantiomeric composition of Tyrosine with (S)-
phenylethylamine using UV spectroscopy— Results of CARMSD
Cross validation plot
Determination of enantiomeric composition of Phenylalanine with (S)-
phenylethylamine using UV spectroscopy- Results of CARMSD
Cross validation of calibration samples: 0.0500, 0.100, 0.200, 0.392, 0.500, 0.527, 0.700, 0.950
Actual D-Phe mole fraction
Predicted D-Phe mole fraction
Predicted L-Phe mole fraction
0.150 0.185 0.823 (0.850)0.267 0.264 0.738 (0.733)0.352 0.348 0.649 (0.648)0.468 0.464 0.529 (0.532)0.486 0.483 0.515 (0.514)0.527 0.524 0.465 (0.473)0.600 0.597 0.397 (0.400)0.650 0.640 0.346 (0.350)0.819 0.814 0.161 (0.181)RMSEP: D-Phe = 0.013 & L-Phe = 0.011Values in bracket = Actual mole fraction of L-Phe
Determination of enantiomeric composition of Alanine with(S)-
phenylethylamine using UV spectroscopy- Results of CARMSD
Cross validation of calibration samples: 0.0500, 0.100, 0.250, 0.350, 0.500, 0.650, 0.750, 0.850
Actual L-Ala mole fraction
Predicted L-Ala mole fraction
Predicted D-Ala mole fraction
0.200 0.211 0.789 (0.800)
0.300 0.279 0.721 (0.700)
0.400 0.404 0.596 (0.600)
0.600 0.631 0.369 (0.400)
0.700 0.692 0.308 (0.300)
RMSEP: D-Ala & L-Ala = 0.018
Values in bracket = Actual mole fraction of D-Ala
Fischer Esterification
Use of Chiral Alcohols
Esterification results in the formation of true covalent diastereomers
+ ROH + HOHH+
heatC
O
R OHC
O
R OR
CARMSD with Homochiral (S)-(+)-1,2-propanediol
Determination of enantiomeric composition of Phenylalanine with (S)-1,2-propanediol using UV spectroscopy
NH2
O
OH
OH
OH
+NH3-Cl
O
O+NH3
-Cl
O
O+NH3
-Cl
O
O
70oC , HCL
OH
D or L-Phe
D- or L hydroxy monoester compound D- or L diester compound
excess
Possible products
Determination of enantiomeric composition of Phenylalanine with
1,2-propanediol using UV spectroscopy
Original UV spectra (15 samples)
Mean centered UV spectra (15 samples)
-0.01
-0.008
-0.006
-0.004
-0.002
0
0.002
0.004
0.006
0.008
0.01
222 242 262 282 302 322 342 362 382
Wavelength/nm
Mea
n C
ente
red
Abs
orba
nce
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
225 245 265 285 305 325 345
Wavelength/nm
Abs
orba
nce
Randomly selected calibration samples: 0.050, 0.150, 0.250, 0.300, 0.500, 0.750, and 0.950
Determination of enantiomeric composition of Phenylalanine with 1,2-propanediol using UV spectroscopy- PLS1 Calibration Model Plots
Randomly selected calibration samples: 0.050, 0.150, 0.250, 0.300, 0.500, 0.750, and 0.950
Determination of enantiomeric composition of Phenylalanine with
1,2-propanediol using UV spectroscopy- Results of CARMSD
Actual D-Phe mole fraction
Predicted D-Phe mole fraction
Predicted L-Phe mole fraction
0.103 0.0848 0.915 (0.897)0.400 0.407 0.593 (0.600)0.451 0.425 0.575 (0.549)0.597 0.596 0.404 (0.403)missing 0.773 0.227
(missing)0.801 0.801 0.208 (0.199)0.851 0.859 0.141 (0.149)missing 0.877 0.123
(missing) RMSEP: D-Phe & L-Phe = 0.014Values in bracket = Actual mole fraction of L-Phe
CARMSD with noncovalent diastereomers vs. CARMSD with covalent diastereomers
RMSEP figure of merit analysis of chiral discrimination strategies
LEL = lower RMSEP limit and UEL = upper RMSEP limit
0
0.1
0.2
0.3
0.4
0.5
0.6
Cyclodextrins ModifiedCyclodextrins
Chiral Surfactants Chiral Ionic Liquids Covalent/ionic Diatereomers
LEL
UEL
UEL - LEL
