(1Chiral Technologies, Inc. 2DAICEL Corporation)

Stephen G. SWARTZ1, Joseph M. BARENDT1, Kanji NAGAI2, Satoshi SHINKURA2, Tohru SHIBATA2, Atsushi OHNISHI2

1. Introduction

Poly(4-vinylpyridine) Based Novel Stationary Phase

Investigated under Supercritical Fluid Chromatography

DAICEL DCpak® is registered trademarks of DAICEL Corporation. All chromatograms were collected by SHIMADZU instrument (Nexera UC).

Chiral Technologies, Inc, 800 North Five Points Road, West Chester, PA19380, USA URL: http//www.chiraltech.com E-mail: jbarendt@chiraltech.com

2. Previous Work: Poly(butylene terephthalate) (PBT) Column Investigated under SFC Condition

■ Commercially Available Achiral Stationary Phase

3. Poly(4-vinylpyridine) (P4VP) Column Investigated under SFC Condition

2018/07/28–08/02

HPLC2018

P-T-1309

Most achiral stationary phases are based on low-molecular-weight selectors.

We focus our attention on novel polymer based stationary phase.

Multiple dipole interaction High durability Cooperative interaction

DAICEL DCpak P4VP

(Particle size: 5 mm and 3 mm)

Commercially

available 2EP (5 mm)

n = 1

n = 20

n = 41

n = 60

n = 80

B3

B12

B6 Cycles

UV 230 nm DAICEL DCpak

P4VP (5 mm)

n = 1

n = 20

n = 40

n = 60

n = 80

B3

B12 B6 Cycles

UV 230 nm Cyanocobalamin (B12) Nicotinamide (B3) Pyridoxal HCl (B6)

■ Cycle vs Retention Factor (k)

P4VP

2-EP

P4VP

2-EP P4VP

2-EP • We used newly purchased columns and CO2 cylinders.

Flow rate: 3.0 ml/min, CO2/MeOH = 75/25, 40 oC,

BPR: 15 MPa, Time: 15 min.

Run over same analysis for 80 cycles.

Cyanocobalamin

(Vitamin B12)

0 2 4 6 8 10

(min)

Column: 4.6 x 150 mm, 5 mm, Flow: 3 ml/min, Temp.: 40 oC, BPR: 15.0 MPa. Eluent: MeOH +

20 mM ammonium formate (10% to 60% gradient in 10 min).

260 nm

266 nm

284 nm

260 nm

361 nm

268 nm

Nicotinamide

(Vitamin B3)

Thiamine

(Vitamin B1)

Pyridoxal HCl

(Vitamin B6)

Nicotinic acid

(Vitamin B3)

Riboflavin

(Vitamin B2)

■ Water Soluble Vitamins

o-Terphenyl (1)

m-Terphenyl (2)

p-Terphenyl (3)

■ Terphenyl isomers

1

2, 3

Commercially

available 2-EP

(5 mm)

Nicotinamide (B3) Cyanocobalamin (B12) Pyridoxal HCl (B6)

UV 254 nm

2

1

3

DAICEL DCpak

PBT (5 mm)

1

2,3

4 5

6

7

8 Commercially

available 2-EP

(5 mm)

2

3

4 5

6

7

8 1 DAICEL DCpak

P4VP (5 mm)

2 3

DAICEL DCpak

P4VP (3 mm)

2

3

4 5

6

7

8 1

2

3

Column: 4.6 x 150 mm, Eluent: MeOH gradent (3 to 38%), Flow: 3 ml/min, Temp.: 40 oC, BPR: 15.0 MPa

Naphthalene (1)

Anthracene (2)

Phenanthrene (3)

Pyrene (4)

Naphthacene (5)

Chrysene (6)

Triphenylene (7)

Peryene (8)

1 2, 3, 5

4 6

Column: 4.6 x 150 mm, Eluent: MeOH (30% isocratic), Flow: 3 ml/min, Temp.: 40 oC, BPR: 15.0 MPa

2 3

4 5

6

1

DAICEL DCpak

P4VP (5 mm)

■ Steroids

N1 = 9238

N2 = 9840

N3 = 8808

N4 = 9396

N5 = 8782

N6 = 8694

■ Polyaromatic hydrocarbons

UV 220 nm

1

2

3

4

Commercially

available 2EP

(5 mm)

without additive

Alprenolol (1)

Propranolol (2)

Atenolol (3)

Pindolol (4)

DAICEL DCpak

P4VP (5 mm)

1

2

3

4 UV 220 nm

UV 254 nm UV 225 nm

without additive

High Planarity Recognition

P4VP Column shows excellent planarity recognition and molecular shape recognition.

Good Peak Shape without Additive

P4VP column can afford symmetrical peaks even in the absence of any additives for APIs.

■ Experiment

Column: 4.6 x 150 mm, Eluent: MeOH (3% isocratic), Flow: 3 ml/min, Temp.: 40 oC, BPR: 15.0 MPa

2 3

4 5

6

1 N1 = 18414

N2 = 19693

N3 = 17813

N4 = 18698

N5 = 17572

N6 = 17090

Prednisone (1)

Estrone (2)

Prednisolone (3)

Estradiol (4)

Dexa- methasone (5)

Estriol (6)

■ Cycle Dependent SFC Chromatogram

VB12

VB3 (Nicotinamide) VB1

VB6 VB3 (Nicotinic acid)

VB2

Column: 4.6 x 150 mm, 5 mm, Eluent: MeOH + 20 or 0 mM ammonium formate gradent (10 to 35% in 10 min), Flow: 3 ml/min, Temp.: 40 oC, BPR: 15.0 MPa

1 3

DAICEL DCpak

P4VP (5 mm)

2 4 UV 280 nm

1

2

3

4 UV 280 nm

Commercially

available 2EP

(5 mm)

with HCO2H•NH3

DAICEL DCpak

P4VP (3 mm)

5. Conclusion

A novel P4VP based column was designed and its performance

was evaluated under SFC conditions.

This column showed unique molecular shape recognition for

planar molecules.

P4VPcolumn afforded symmetric peaks for APIs probably due to

the effective shield of residual silanols by polymeric pyridine

selector.

Stability towards cycle durability was confirmed.

Application in HPLC mode was attained.

We would like to appreciate Mr. Jun Zhu and Ms Yanlin Wang in

DAICEL Chiral Technologies (China) for their help in HPLC analysis.

DAICEL DCpak PBT

(Particle size: 5 mm and 3 mm)

Unique planarity recognition of isomeric and closely similar

samples.

Orthogonal retention relationship of 2-ethylpyridine (2-EP)

and this columns was attained.

Column robustness was confirmed by using a variety of

modifiers and by cycle durability testing.

K. Nagai, T. Shibata, S. Shinkura, A. Ohnishi, J. Chromatogr. A 2018, 1549, 85–92.

Durability Testing

Column robustness of P4VP column was confirmed by cycle durability testing.

4. P4VP Column Investigated under HPLC Modes

K. Nagai, T. Shibata, S. Shinkura, A. Ohnishi, submitted.

■ b-Blockers (without additive) ■ b-Blockers (with 20 mM ammonium formate)

■ NAD, NADH

Column: 2.1 x 150 mm, 3 mm, Flow: 0.3 ml/min, Temp.: 40 oC, Eluent: CH3CN/ammonium acetate

buffer (pH 4.8) = 40/60 (isocratic).

■ AMP, ADP, ATP

DAICEL DCpak

P4VP (3 mm)

NAD NADH

AMP

NA

D

NA

DH

ADP

ATP

Column: 2.1 x 150 mm, 3 mm, Flow: 0.3 ml/min, Temp.: 40 oC, Eluent: CH3CN/10 mM

phosphate buffer (pH 7.0) = 10/90 (isocratic).

AM

P A

DP

AT

P

Versatile Column

P4VP column can be applied from non-polar to polar

samples.

HPLC application dealing with hydrophilic samples was conducted.

logP = –0.45

logP = –2.11

logP = –0.57

logP = 0.29

logP = 0.67

logP = –1.05

UV 210 nm DAICEL DCpak

P4VP (3 mm) UV 254 nm

Commercially

available 2-EP

(5 mm)

with HCO2H•NH3