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HPLC Application Sheet
Using Shape Recognition Selectivity of Steriods for Peak Identity Confirmation


This complete method for peak identity was developed in less than 25 laboratory hours.

View stationary phase specifications

Peaks:

  1. Prednisolone
  2. Corticosterone
  3. Estradiol
  4. Ethinyl Estradiol
  5. Estrone
  6. Norgesterel
  7. Progesterone
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printable Application Sheet

 Method Conditions

  Chromatogram A Chromatogram B
Mobile Phase 60% Aqueous (0.1%TFA)
40% Acetonitrile		 56% Aqueous (0.1% TFA)
24% MeOH / 20% Acetonitrile
Column Cogent Bidentate-C18™ Cogent UDC-Cholesterol™
Cat. No. 40018-75R 69069-75R
Dimensions 75 x 4.6mm id 75 x 4.6mm id
Flow Rate 1.0 ml/min 1.0 ml/min
Detection: UV 240nm 240nm
Temperature 15°C 15°C


Discussion of Chromatograms:
Chromatogram A: Near baseline resolution was rapidly achieved for these steroids.
Chromatogram B: A near optimal resolution is shown. The selectivity shown here employs shape recognition as the predominant separation mechanism.

Subsequent baseline resolution of both methods were easily predicted and achieved using 250x4.6mm columns.

Implications
Selectivity based on hydrophobicity exhibitied in Chromatogram A is similar to other standard C18 polar embedded columns when used with similar conditions. Many variations of the relative elution orders of peaks 3,4,5 & 6 were possible by controlling temperature and or the relative percent concentrations of MeOH and Acetonitrile with the UDC-Cholesterol™ column shown in Chromatogram B.

Peak identity of very similar steroidal compounds could be assessed and confirmed with the two different selectivity mechanisms of these columns.

Advantages
Faster flow rates can be employed during experimentation resulting in short test runs for a very rapid evaluation of test conditions. Improvement of data and information in a shorter period of time results.

Uses
These two columns can be used to confirm other laboratory data or used to trouble shoot problems with closely eluting impurities or degradation products in a very short time period.


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