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Chiral Stationary-Phase Optimized Selectivity Liquid ... · Chiral Stationary-Phase Optimized...

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! !"#$% = ! ! ! ! + ! ! ! ! + ! ! ! ! + ! ! ! ! +! ! ! ! ! ! + ! ! + ! ! + ! ! +! ! The development of methods allowing improved chiral separation and purification of therapeutic agents is crucial as the pharmacokinetic and pharmacological behavior of both enantiomers and also of diastereomers can vary significantly from the active pharmaceutical ingredient. Recently Stationary-Phase Optimized Selectivity Liquid Chromatography (SOS-LC) has been successfully further developed and progressively used for isocratic and gradient liquid chromatography separations. Until now the potential of this methodology to facilitate the separation and purification of chiral isomers has not yet been investigated, which could speed up the purification process or allow for improved chiral screening of complex mixtures. In this work the possibilities of chiral Stationary-Phase Optimized Selectivity Liquid Chromatography (SOS-LC) are demonstrated on a limited set of commercial chiral columns. This approach allows for the prediction of the separation profiles through isocratic and gradient mode on any possible combination of the stationary phases based on a limited number of preliminary analyses. Isocratic and gradient predictions were performed with a commercially available and with an in-house developed Microsoft visual basic algorithm, respectively. The potential of this method is demonstrated via the prediction of the optimal chiral column combination for the baseline separation of 4 chiral pairs containing solutes of varying polarity. EXPERIMENTAL Chiral Stationary-Phase Optimized Selectivity Liquid Chromatography: a Novel Approach for the Separation of Mixtures of Enantiomers Ravindra Suryakant Hegade 1 , Maarten De Beer 2 , Frederic Lynen 1 * 1. Separation Science Group, Department of Organic And Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-Bis, 9000 Gent, Belgium 2. AmatsiSEPS, Technologiepark 4, B-9052 Ghent, Belgium INTRODUCTION 1. Chemicals and reagents Hexane, 2-Propanol, ethanol chiral mixture (trans-Stilbene oxide, 1,2,3,4 Tetrahydro-1- napthol, 4-phenyl-1,3-dioxane and hexobarbital). 2. Chromatographic conditions a. Instrument: Agilent 1260 HPLC system with (VWD); b . Mobile Phase: A. Hexane, B. IPA , c . Flow: 0.5 ml/min; d. Detector Wavelength: 210 nm; e . Column Oven: 25°C, f . Inj. Vol. : 2μL; g . Lux 3u Columns (50 mm x 4.6 mm, 3μm): Amylose 2, Cellulose 1, Cellulose 2, Cellulose 3 and Cellulose 4 (Phenomenax, USA) with Lux Amylose 2 (4 x 3.0 mm) pre- column; h. Software: Chemstation (Agilent), POPLC optimizer v 1.04.03 (Bischoff Chromatography) and Microsoft visual basic algorithm. 3. Separation of the chiral mixture on individual column segments. 4. Optimization of stationary phase CONCLUSION Stationary phase optimized selectivity approach is transferable to chiral separations both under isocratic and gradient conditions on a set of commercially available columns. Retention time predictions on combined polysaccharide phases are achievable with good predictive accuracies. Baseline separation of 4 chiral pairs could be achieved for a mixture of solutes of varying polarity through gradient analysis. The study also illustrated that the elution order of chiral pairs is not constant on all columns and that appears to be highly depending on the type of solute. Figure 2. Predicted (A) & experimental chromatogram (B) of the chiral mixture on 4 combined column segments (Amylose 2 + Cellulose 2 + Cellulose 3 + Cellulose 4). (For annotations refer Table 1) REFERENCES [1] M. De Beer, F. Lynen, K. Chen, P. Ferguson, M. Hanna-Brown, P. Sandra, Stationary-phase optimized selectivity liquid chromatography: Development of a linear gradient prediction algorithm, Anal. Chem. 82 (2010) 1733–1743 [2] P.J. Schoenmakers, H.A.H. Billiet, R. Tijssen ’, L. De Galan, Gradient selection in reversed-phase liquid chromatography, J. Chromatogr. 149 (1978). [3] S. Nyiredy, Z. Szucs, L. Szepesy, Stationary phase optimized selectivity liquid chromatography: Basic possibilities of serially connected columns using the “PRISMA” principle, J. Chromatogr. A. 1157 (2007) 122–130. [4] De Beer, Maarten; Lynen, Frederic; Hanna-Brown, Melissa; et al. Chromatographia (2009) Volume: 69 Issue: 7-8 Pages: 609 - 614 [5] Chen, Kai; Lynen, Frederic; De Beer, Maarten; et al. J. Chromatogr. A (2010) Volume 1217 Issue: 46 Pages 7222-7230 Min mAU mAU mAU mAU Min Min C D Min A Min B Figure 1. Isocratic chromatograms obtained on the Amylose 2 (A, B) and on the cellulose 3 column (C, D) obtained with 90/10 (A, C) and 95/5 (B, D) hexane/IPA (% v/v) mobile phases. (For annotations refer Table 1) On none of the individual stationary phase, baseline separation could be obtained for the chiral mixture even when mixing 5% or lower IPA concentration. Void Time Enan,omer R Enan,omer S Predic'on of Reten'on Time (RT) On Amylose 2 + Cellulose 2 + Cellulose 3 + Cellulose 4 AMYLOSE 2 CELLULOSE 3 CELLULOSE 2 CELLULOSE 4 CELLULOSE 1 TSO TSO TSO TSO TSO trans-S'lbene oxide (TSO) RT k RT k RT k RT k RT k 1.5 1.57 1.41 1.47 1.44 2.61 0.74 3.26 1.08 1.95 0.38 1.98 0.35 2.85 0.98 Enan'omer 1 (R) 9.74 3.66 1.44 3.89 1.48 3.5 1.48 3.56 1.42 5.06 2.51 Enan'omer 2 (S) 14.61 THN THN THN THN THN 1,2,3,4-Tetrahydrol-1-napthol RT k RT k RT k RT k RT k (THN) 1.5 1.56 1.41 1.47 1.45 3.01 1.01 2.8 0.79 2.58 0.83 2.63 0.79 3.1 1.14 Enan'omer 3 (S) 11.02 3.03 1.02 3.1 0.99 2.84 1.01 2.79 0.9 3.1 1.14 Enan'omer 4 (R) 11.76 HXL HXL HXL HXL HXL hexobarbital (HXL) RT k RT k RT k RT k RT k 1.49 1.56 1.41 1.47 1.45 5.95 2.99 11.05 6.08 17.37 11.32 27.94 18.01 8.8 5.07 Enan'omer 5 (R) 92.79 12.18 7.17 12.78 7.19 31.68 21.47 42.71 28.05 9.92 5.84 Enan'omer 6 (S) 70.7 4PD 4PD 4PD 4PD 4PD 4-Phenyl-1,3-dioxane (4PD) RT k RT k RT k RT k RT k 1.5 1.54 1.41 1.48 1.44 3.1 1.07 4.1 1.66 2.57 0.82 2.54 0.72 2.99 1.08 Enan'omer 7 (R) 13.21 3.44 1.29 4.74 2.08 2.76 0.96 2.68 0.81 4.79 2.33 Enan'omer 8 (S) 12.6 Table 1. Retention times (RT) and retention factors (k) as measured for each solute when using 90/10 hexane/ IPA (% v/v) as mobile phase. 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 Min 1 3 4 8 7 2 6 5 1 3 4 8 2 6 5 7 A B 1 3 4 8 2 7 Preliminary measurements were performed to obtain void time, retention time (RT) and retention factor ( k) for every individual enantiomer from chiral mixture on all 5 column segments by using the identical chromatographic conditions. Thus, the measured k’ on the individual column segments are then used to predict the RT’s on a possible linear column combination of the segments using equation 1. Where k A , k B , k C , k D and k E correspond to the retention factors of a respective compounds while ϕ A , ϕ B , ϕ C , ϕ D and ϕ E represent the lengths of the respective segments in a combined column. RESULTS & DISCUSSION The possible column combinations were simulated with the straightforward isocratic algorithm (based on equation 1) and ranked in order of decreasing selectivity of the critical pair, which provides the optimal column combination composed of Amylose 2, Cellulose 2, Cellulose 3 and Cellulose 4 columns (assembled in random order). A. Isocratic Chiral SOS-LC B. Gradient Chiral SOS-LC -0.8 -0.3 0.2 0.7 1.2 1.7 2.2 0 10 20 30 40 -1.5 -0.5 0.5 1.5 2.5 3.5 0 10 20 30 40 C D ln(k) ln(k) ϕ ϕ A B ln(k) ln(k) -1.5 -0.5 0.5 1.5 2.5 3.5 0 10 20 30 40 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 0 10 20 30 40 ϕ ϕ A B 1. Concept of the prediction algorithm for retention times under linear gradient conditions on combined column segments. For chiral gradient SOS-LC a discontinuous algorithm based on numerical integration was developed allowing the prediction of retention times on columns with serially combined chiral stationary phases. A linear gradient was thereby considered as the sequence of small isocratic stages, which correspond to small time intervals t. This discontinuous approach allows for the determination of the intermediate migrated distance through the column at each time point during the analysis. This is a useful approach when serially coupled columns with different stationary phases are considered. Sta$onary phase A Sta$onary phase B Δt φ d c Σ d c Δt 1 φ 1 d c,1 d c,1 Δt 2 φ 2 d c,2 d c,1 +d c,2 Δt 3 φ 3 d c,3 d c,1 +d c,2 +d c,3 d c,1 Δt 1 d c,2 Δt 2 Δt 3 d c,3 d c,4 Δt 4 Δt 4 φ 4 d c,4 d c,1 +d c,2 +d c,3 +d c,4 d c,5 Δt 5 Δt 5 φ 5 d c,5 d c,1 +d c,2 +d c,3 +d c,4 +d c,5 t = 0me φ i = frac0on organic modifier near the migra0ng analyte band d c = covered distance L = column length If Σd c becomes equal to L, then the component elutes from the column t r = Δt 1 + Δt 2 + … + Δt 5 Necessary informa.on - ln(k) = aφ 2 + bφ + c - gradient profile - knowledge of φ near the migra0ng analyte band - dwell 0me - dwell 0me in column - linear velocity of the mobile phase 2. Calculations of nonlinear regressions based on the quadratic relationship between ln(k) and ϕ [ln(k) = aφ 2 + bφ + c]. The obtained relationships allowed for utilization of the visual basic algorithms in Microsoft Excel for retention time predictions and subsequent ranking of the optimal column combinations. Figure 3. Acquisition of the ln(k) = aϕ 2 + bϕ + c curves, based on all preliminary isocratic measurements for the chiral mixture on a Cellulose 2 (A), Cellulose 3 (B), Cellulose 1 (C), and Cellulose 4 (D) column 3. Figures of merit of the chiral gradient approach The effectiveness of the above methodology for the prediction of retention time on coupled chiral columns has been proved for trans-Stilbene oxide on a number of random column combinations represented in Figure (left). gradient from 1 to 15 % (v/v) IPA in 60 min was thereby used on all column combinations. (For annotations refer Table 1) 0 2 4 6 8 10 12 14 16 18 20 22 24 Min 2 C 2 B 1 2 1 1 A Amylose 2 (A3) Cellulose 2 (C2) Cellulose 3 (C3) Cellulose 4 (C4) A2 + C3 C3 + C2 + A2 C3 + C4 + A2 + C2 OPTIMAL PHASE COMBINATION The complete chiral SOSLC methodology was subsequently performed on all 8 solutes on all possible column combinations for a fixed gradient of 1 to 20% IPA in 60 min. From the 325 possible solutions (columns combinations) the optimal combination consisting and of the Cellulose 3 column at the inlet, followed by the Cellulose 2 phase and ending with the Amylose 2 column. This resulted in a total column length of 15 cm and corresponded to the highest value for the retention time difference of the critical peak pair of all combinations, while enabling analysis within a stipulated maximum time limit of 60 min. (Figure 4) A 0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20 mAU mAU ϕ ϕ B 1 8 0 5 10 15 20 25 30 35 40 45 50 55 60 7 2 3 4 6 5 1 8 7 2 3 4 6 5 Min 0 5 10 15 20 0 5 10 15 20 Op#mal Phase Combina#on Cellulose 3 + Cellulose 2 + Amylose 2 Figure 4. Predicted (A) & experimental chromatogram (B) of the chiral mixture. (For annotations refer Table 1) Optimal Phase Combination Amylose 2 + Cellulose 2 + Cellulose 3 + Cellulose 4 Ravindra Suryakant Hegade gratefully acknowledges Social Justice and Special Assistance Department, Government of Maharashtra (India) for co-funding this research.
Transcript
Page 1: Chiral Stationary-Phase Optimized Selectivity Liquid ... · Chiral Stationary-Phase Optimized Selectivity Liquid Chromatography: a Novel Approach for the Separation of Mixtures of

!!"#$% = !!!! + !!!! + !!!! + !!!!+!!!!!! + !! + !! + !!+!!

The development of methods allowing improved chiral separation and purification of therapeutic agents is crucial as the pharmacokinetic and pharmacological behavior of both enantiomers and also of diastereomers can vary significantly from the active pharmaceutical ingredient. Recently Stationary-Phase Optimized Selectivity Liquid Chromatography (SOS-LC) has been successfully further developed and progressively used for isocratic and gradient liquid chromatography separations. Until now the potential of this methodology to facilitate the separation and purification of chiral isomers has not yet been investigated, which could speed up the purification process or allow for improved chiral screening of complex mixtures. In this work the possibilities of chiral Stationary-Phase Optimized Selectivity Liquid Chromatography (SOS-LC) are demonstrated on a limited set of commercial chiral columns. This approach allows for the prediction of the separation profiles through isocratic and gradient mode on any possible combination of the stationary phases based on a limited number of preliminary analyses. Isocratic and gradient predictions were performed with a commercially available and with an in-house developed Microsoft visual basic algorithm, respectively. The potential of this method is demonstrated via the prediction of the optimal chiral column combination for the baseline separation of 4 chiral pairs containing solutes of varying polarity. EXPERIMENTAL

Chiral Stationary-Phase Optimized Selectivity Liquid Chromatography: a Novel Approach for the Separation of Mixtures of Enantiomers

Ravindra Suryakant Hegade1, Maarten De Beer2, Frederic Lynen1*

1. Separation Science Group, Department of Organic And Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-Bis, 9000 Gent, Belgium

2. AmatsiSEPS, Technologiepark 4, B-9052 Ghent, Belgium

INTRODUCTION

1. Chemicals and reagents Hexane, 2-Propanol, ethanol chiral mixture (trans-Stilbene oxide, 1,2,3,4 Tetrahydro-1-napthol, 4-phenyl-1,3-dioxane and hexobarbital).

2. Chromatographic conditions a. Instrument: Agilent 1260 HPLC system with (VWD); b. Mobile Phase: A. Hexane, B. IPA , c. Flow: 0.5 ml/min; d. Detector Wavelength: 210 nm; e. Column Oven: 25°C, f. Inj. Vol. : 2µL; g. Lux 3u Columns (50 mm x 4.6 mm, 3µm): Amylose 2, Cellulose 1, Cellulose 2, Cellulose 3 and Cellulose 4 (Phenomenax, USA) with Lux Amylose 2 (4 x 3.0 mm) pre- column; h. Software: Chemstation (Agilent), POPLC optimizer v 1.04.03 (Bischoff Chromatography) and Microsoft visual basic algorithm.

3. Separation of the chiral mixture on individual column segments.

4. Optimization of stationary phase

REFERENCES

CONCLUSION •  Stationary phase optimized selectivity approach is transferable to chiral separations both

under isocratic and gradient conditions on a set of commercially available columns. •  Retention time predictions on combined  polysaccharide phases are achievable with good

predictive accuracies. •  Baseline separation of 4 chiral pairs could be achieved for a mixture of solutes of varying

polarity through gradient analysis. •  The study also illustrated that the elution order of chiral pairs is not constant on all columns

and that appears to be highly depending on the type of solute.

Figure 2. Predicted (A) & experimental chromatogram (B) of the chiral mixture on 4 combined column segments (Amylose 2 + Cellulose 2 + Cellulose 3 + Cellulose 4). (For annotations refer Table 1)

REFERENCES [1] M. De Beer, F. Lynen, K. Chen, P. Ferguson, M. Hanna-Brown, P. Sandra, Stationary-phase optimized selectivity liquid chromatography: Development of a linear gradient prediction algorithm, Anal. Chem. 82 (2010) 1733–1743 [2] P.J. Schoenmakers, H.A.H. Billiet, R. Tijssen ’, L. De Galan, Gradient selection in reversed-phase liquid chromatography, J. Chromatogr. 149 (1978). [3] S. Nyiredy, Z. Szucs, L. Szepesy, Stationary phase optimized selectivity liquid chromatography: Basic possibilities of serially connected columns using the “PRISMA” principle, J. Chromatogr. A. 1157 (2007) 122–130. [4] De Beer, Maarten; Lynen, Frederic; Hanna-Brown, Melissa; et al. Chromatographia (2009) Volume: 69 Issue: 7-8 Pages: 609 - 614 [5] Chen, Kai; Lynen, Frederic; De Beer, Maarten; et al. J. Chromatogr. A (2010) Volume 1217 Issue: 46 Pages 7222-7230

Min

mAU

mAU

mAU

mAU

Min Min

C D

Min

A

Min

B Figure 1. Isocratic chromatograms obtained on the Amylose 2 (A, B) and on the cellulose 3 column (C, D) obtained with 90/10 (A, C) and 95/5 (B, D) hexane/IPA (% v/v) mobile phases. (For annotations refer Table 1) On none of the individual stationary phase, baseline separation could be obtained for the chiral mixture even when mixing 5% or lower IPA concentration.

VoidTime Enan,omerR Enan,omerSPredic'onofReten'onTime

(RT)OnAmylose2+Cellulose2+

Cellulose3+Cellulose4AMYLOSE2 CELLULOSE3 CELLULOSE2 CELLULOSE4 CELLULOSE1

TSO TSO TSO TSO TSOtrans-S'lbeneoxide

(TSO)RT k RT k RT k RT k RT k1.5 1.57 1.41 1.47 1.44 2.61 0.74 3.26 1.08 1.95 0.38 1.98 0.35 2.85 0.98 Enan'omer1(R) 9.743.66 1.44 3.89 1.48 3.5 1.48 3.56 1.42 5.06 2.51 Enan'omer2(S) 14.61

THN THN THN THN THN 1,2,3,4-Tetrahydrol-1-napthol

RT k RT k RT k RT k RT k(THN)

1.5 1.56 1.41 1.47 1.45 3.01 1.01 2.8 0.79 2.58 0.83 2.63 0.79 3.1 1.14 Enan'omer3(S) 11.023.03 1.02 3.1 0.99 2.84 1.01 2.79 0.9 3.1 1.14 Enan'omer4(R) 11.76

HXL HXL HXL HXL HXL

hexobarbital(HXL)RT k RT k RT k RT k RT k

1.49 1.56 1.41 1.47 1.45 5.95 2.99 11.05 6.08 17.37 11.32 27.94 18.01 8.8 5.07 Enan'omer5(R) 92.7912.18 7.17 12.78 7.19 31.68 21.47 42.71 28.05 9.92 5.84 Enan'omer6(S) 70.7

4PD 4PD 4PD 4PD 4PD

4-Phenyl-1,3-dioxane(4PD)RT k RT k RT k RT k RT k

1.5 1.54 1.41 1.48 1.44 3.1 1.07 4.1 1.66 2.57 0.82 2.54 0.72 2.99 1.08 Enan'omer7(R) 13.213.44 1.29 4.74 2.08 2.76 0.96 2.68 0.81 4.79 2.33 Enan'omer8(S) 12.6

Table1.Reten,on,mesandreten,onfactors(k)asmeasuredforeachsolutewhenusing90/10hexane/IPA(%v/v)asmobilephase.

Table 1. Retention times (RT) and retention factors (k) as measured for each solute when using 90/10 hexane/IPA (% v/v) as mobile phase.

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110Min

1

3 487 2

6 5

1 34

8

2

6 5

7

A

B1 3 4

8

2

7

Preliminary measurements were performed to obtain void time, retention time (RT) and retention factor (k) for every individual enantiomer from chiral mixture on all 5 column segments by using the identical chromatographic conditions. Thus, the measured ‘k’ on the individual column segments are then used to predict the RT’s on a possible linear column combination of the segments using equation 1.

Where kA, kB, kC, kD and kE correspond to the retention factors of a respective compounds while ϕA, ϕB, ϕC, ϕD and ϕE represent the lengths of the respective segments in a combined column.

RESULTS & DISCUSSION

The possible column combinations were simulated with the straightforward isocratic algorithm (based on equation 1) and ranked in order of decreasing selectivity of the critical pair, which provides the optimal column combination composed of Amylose 2, Cellulose 2, Cellulose 3 and Cellulose 4 columns (assembled in random order).

A. Isocratic Chiral SOS-LC

B. Gradient Chiral SOS-LC

A

B

ln(k)

ln(k)

ϕ

ϕ

-0.8

-0.3

0.2

0.7

1.2

1.7

2.2

0 10 20 30 40

1

2

3

4

5

6

7

8

-1.5

-0.5

0.5

1.5

2.5

3.5

0 10 20 30 40

12345678

C

D

ln(k)

ln(k)

ϕ

ϕ

A

B

ln(k)

ln(k)

-1.5

-0.5

0.5

1.5

2.5

3.5

0 10 20 30 40

1

2

3

4

5

6

7

8

-1.5

-1

-0.5

0

0.5

1

1.5

2

2.5

0 10 20 30 40

1

2

3

4

5

6

7

8

ϕ

ϕ

A

B

1. Concept of the prediction algorithm for retention times under linear gradient conditions on combined column segments.

For chiral gradient SOS-LC a discontinuous algorithm based on numerical integration was developed allowing the prediction of retention times on columns with serially combined chiral stationary phases. A linear gradient was thereby considered as the sequence of small isocratic stages, which correspond to small time intervals ∆t. This discontinuous approach allows for the determination of the intermediate migrated distance through the column at each time point during the analysis. This is a useful approach when serially coupled columns with different stationary phases are considered.

1

SOSLC Linear gradient

Sta$onaryphaseA Sta$onaryphaseB

Δt φ dc ΣdcΔt1 φ1 dc,1 dc,1Δt2 φ2 dc,2 dc,1+dc,2Δt3 φ3 dc,3 dc,1+dc,2+dc,3

dc,1

Δt1

dc,2

Δt2 Δt3

dc,3 dc,4

Δt4

Δt4 φ4 dc,4 dc,1+dc,2+dc,3+dc,4

dc,5

Δt5

Δt5 φ5 dc,5 dc,1+dc,2+dc,3+dc,4+dc,5

t=0meφi=frac0onorganicmodifiernearthemigra0nganalytebanddc=covereddistanceL=columnlength

IfΣdcbecomesequaltoL,thenthecomponentelutesfromthecolumntr=Δt1+Δt2+…+Δt5 Necessaryinforma.on

- ln(k)=aφ2+bφ+c- gradientprofile- knowledgeofφnearthemigra0nganalyteband-dwell0me-dwell0meincolumn-linearvelocityofthemobilephase

SOSLC Linear gradient

2. Calculations of nonlinear regressions based on the quadratic relationship between ln(k) and ϕ [ln(k) = aφ2 + bφ + c]. The obtained relationships allowed for utilization of the visual basic algorithms in Microsoft Excel for retention time predictions and subsequent ranking of the optimal column combinations.

Figure 3. Acquisition of the ln(k) = aϕ2 + bϕ + c curves, based on all preliminary isocratic measurements for the chiral mixture on a Cellulose 2 (A), Cellulose 3 (B), Cellulose 1 (C), and Cellulose 4 (D) column

3. Figures of merit of the chiral gradient approach The effectiveness of the above methodology for the prediction of retention time on coupled chiral columns has been proved for trans-Stilbene oxide on a number of random column combinations represented in Figure (left). gradient from 1 to 15 % (v/v) IPA in 60 min was thereby used on all column combinations. (For annotations refer Table 1)

0 2 4 6 8 10 12 14 16 18 20 22 24Min

2

C

2B1

21

1

AAmylose2(A3)Cellulose2(C2)Cellulose3(C3)Cellulose4(C4)

A2+C3

C3+C2+A2

C3+C4+A2+C2

OPTIMALPHASECOMBINATION

The complete chiral SOSLC methodology was subsequently performed on all 8 solutes on all possible column combinations for a fixed gradient of 1 to 20% IPA in 60 min. From the 325 possible solutions (columns combinations) the optimal combination consisting and of the Cellulose 3 column at the inlet, followed by the Cellulose 2 phase and ending with the

Amylose 2 column. This resulted in a total column length of 15 cm and corresponded to the highest value for the retention time difference of the critical peak pair of all combinations, while enabling analysis within a stipulated maximum time limit of 60 min. (Figure 4)

A

02468101214161820

02468101214161820

mAU

mAU

ϕ

ϕ

B1

8

0 5 10 15 20 25 30 35 40 45 50 55 60

7

23

4

6 5

1

8

7

23

4

6 5

Min0

5

10

15

20

0

5

10

15

20 Op#malPhaseCombina#on

Cellulose3+Cellulose2+Amylose2

Figure 4. Predicted (A) & experimental chromatogram (B) of the chiral mixture. (For annotations refer Table 1)

Optimal Phase Combination

Amylose 2 + Cellulose 2 + Cellulose 3 + Cellulose 4

Ravindra Suryakant Hegade gratefully acknowledges Social Justice and Special Assistance Department, Government of Maharashtra (India) for co-funding this research.

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