HPLC

TM

UDC Cholesterol

TM

Molecular Shape and Polarity Recognition by HPLC One Column Can Achieve Orthogonal, Purity Confirmation Method Conditions Mobile Phase

Acetonitrile Chromatogram A: 2 1

2

Chromatogram D: 2

20° C

2

8

2

10 12

Chromatogram E: 20° C 2

SOLVENT FRONT

1

1

5

3

1

5

8

10 12

Chromatogram C:

Chromatogram F:

45° C

45° C

2

SOLVENT FRONT

3

Steroid Compound Type Peak 1: Ethinyl Estradiol Peak 2: Norgestrel

2+1

5

8

15

Column

Cogent UDC-Cholesterol™

Cat. No.

69069-75R

Dimensions

75 x 4.6mm id

Flow Rate

1.0 ml/min

Detection:

UV, 240nm

Discussion of Chromatograms Chromatograms A, B & C Using a Cogent UDC-Cholesterol™ column at 3 different temperatures and with Acetonitrile as an organic modifier, produced similar results (shown) as achieved on 17 different, available C18 columns. Little effect on selectivity and retention time occurred.

Chromatograms D, E & F When using Methanol as the modifier, the relative retention times differ so much that the elution order is reversed to: Peak 2 eluting first, then Peak 1. A 15 temperature change between 15°C and 45°C has a profound effect on both selectivity and retention times. Implications The Cogent UDC-Cholesterol™ column, when used with methanol, has a fundamental selectivity mechanism based on shape recognition. The same column when used with Acetonitrile has the fundamental selectivity mechanism based on polarity recognition (ARP).

1

2

Chromatograms A, B & C 50% Aqueous (0.1% TFA) 50% Acetonitrile (ACN) Chromatograms D, E & F 45% Aqueous (0.1% TFA) 55% Methanol

15° C

3

Chromatogram B:

SOLVENT FRONT

Methanol

SOLVENT FRONT

SOLVENT FRONT

15° C

SOLVENT FRONT

APPLICATION NOTES

No. 1 of Series C

10 12

Advantages Using different selectivity mechanisms with the same 15 column can achieve both shape and polarity recognition of some analytes. Uses One column can be used to confirm compound purity and identify degradation products.

MTC • MicroSolv Technology Corporation 1 Industrial Way West, Bldg E, Eatontown, NJ 07724 USA Ph. 1-732-578-1777 • Fx. 1-732-578-9777 www.mtc-usa.com Email: [email protected] 03-H-09-C1