TYPE-C Mechanisms

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A comparison of the selectivity with Silica-C™ and UDC-Cholesterol™ columns gives a valuable insight into the unique TYPE-C™ retention mechanisms

Both columns used in this study behaved very similarly to each other for the highly polar, basic compound, Metformin. Both exhibit an aqueous normal phase (ANP) response in an acidified (0.05% v/v H3PO4/acetonitrile) mobile phase. In creating a Retention Map for these columns and compounds, one would clearly see that there is an almost constant retention time near the solvent front as the acetonitrile percentage increases to approximately 55%.

As the percent concentration of acetonitrile increases, the retention time rapidly changed to being infinitely retained when the concentration was at and over 95% acetonitrile.

The considerable similarity in retention times of both Silica-C™ and the Cogent UDC-Cholesterol™ columns for Metformin suggests that the fundamental retention mechanism is related to the silica hydride surface common in all of the Cogent TYPE-C™ columns. This retention mechanism for Metformin was not retained much by the bonded ligand in the UDC-Cholesterol column.

Reviewing the Retention Maps for the mid polarity range compound Glyburide, it is clear that these two columns are quite different when using the same mobile phase (described above). Interestingly both columns demonstrate different effects suggesting differences in prime retention mechanisms.

On the Cogent Silica-C™ column, even though there is no bonded carbon phase, or carbon based end-capping, a reverse phase retention characteristic with Glyburide was observed while there is no retention on the column at high percentages of acetonitrile. A rapid increase in retention time occurs once acetonitrile concentration drops to less than 30%. The peaks, while symmetrical, are relative to their retention, very broad; a three-minute retention peak will have a peak width of well over a minute.

The Cogent UDC-Cholesterol™ column also exhibited a reverse phase retention characteristic for Glyburide, with retention at high percentages of Acetonitrile, but a rapid increase in retention times once the acetonitrile percentage became less than 70%.

The relative difference between the columns at 30% vs. 70% in acetonitrile concentration is extremely significant. At 70% ACN, the retention mechanism of the un-bonded Silica-C™ column had no retention effect.

It must therefore be assumed that on the Cogent UDC-Cholesterol column the Glyburide retention is almost wholly a bonded phase (UDC-Cholesterol) effect.

This fundamental difference in retention properties between these two columns was also suggested by the peak width characteristics; as the Cogent UDC-Cholesterol™ column’s Glyburide peak, (unlike the Silica-C™ column’s Glyburide peak) was very sharp with a width of less than 0.3 minute at a three minute retention time, as opposed to well over a minute peak width at a equivalent time with un-bonded Silica-C™ column.

Summarizing:

It would appear that when working in Aqueous (and possibly organic) Normal Phase (ANP) mode the silica-hydride surface of all TYPE-C Silica™ supports could be the dominant separation mechanism. But that when working in aqueous reverse phase mode, the silica hydride surface and the bonded phase can both have reverse phase characteristics and both can contribute differently to the separation mechanism depending on the sample.

In the case of Metformin and Glyburide the Cogent UDC-Cholesterol™ column is the preferred column, as the reverse phase characteristics of the silica-hydride surface for Glyburide do not come into effect at a stage when the useful normal phase characteristics for Metformin are effective. The situation with UDC-Cholesterol, however, results in both effects overlapping, thus chromatographers have a choice of having either compound elute first, with both solutes having significant retention so that neither overlaps with the solvent front.

This ability to have either the polar or non polar peak elute first, and to be able to manipulate one from near the solvent front and the other from co-elution to infinite retention is a characteristic of all TYPE-C™ products.

Click here to see actual chromatogram of Metformin and Glyburide Separated by the above.

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