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Hydrophobic and Silanophilic Interaction Tests
How are the “Hydrophobic and Silanophilic Interaction” tests useful?

These tests help an HPLC column shopper compare column characteristics based on a set of standard conditions using the same test mixtures.

The hydrophobicity test is a measure of how well a column will retain non-polar compounds. The higher the value the greater the retention of these solutes. The silanophilic test measures strong polar/adsorptive interactions with the surface. The lower the value the less surface adsorption which results in better peak shape for polar analytes.

  1. The hydrophobicity of Cogent™ Bidentate C18™ columns is high and comparable to Luna® C18 and the Zorbax® Eclipse XDB.
  2. The hydrophobicity of Cogent™ UDC-Cholesterol™ columns is mid range and is similar to many of the C8 phases.
  3. Both Cogent TYPE-C™ phases tested below show low silanol (silanophilic) activity.

Actual Test Conditions:
Mobile phase: 35:65 20mM phosphate buffer, methanol, pH 7
Flow rate: 0.5 mL/min for Cogent™ columns and 1 mL/min for Phenomenex™ column
Injection volume: 1 µL
Detection: UV 214 nm
Sample I: uracil (16 mg/L), naphthalene (60 mg/L), acenaphthene (200 mg/L) in methanol
Sample II: amitryptiline hydrochloride (100 mg/L), acenaphthene (200 mg/L) in methanol

Columns Tested Catalog No. Dimension (mm)
Cogent Bidentate C18™, 4µm 40018-75P 4.6 x 75
Cogent UDC-Cholesterol™, 4µm 69069-7.5P 4.6 x 75
Phenomenex, Luna® C18(2) 5µm 00F-4252-E0 4.6 x 150

Test Parameters: From the Neue Test1
Hydrophobicity Parameter():
was calculated according to equation (1)1:

     ln(k) = x V – 3.068 (1)

where V is the molar volume of the analyte (acenaphthene or naphthalene).
Results obtained are presented in the Table below:

Silanophilic Interaction Parameter(S):
of amitriptyline was calculated according to equation (2)1
     S = ln (kamitriptyline) – 0.7124 x ln (kacenaphthene) + 1.9748 (2)

Results obtained are presented in the Table below:

Column * ** S (AS) ***
Cogent Bidentate C18™, 4µm 0.0397 - 1.429 1.02
Cogent UDC Cholesterol™, 4µm 0.0339 - 0.949 0.95
Phenomenex, Luna® C18(2) 5µm 0.0400 0.0397 2.28 1.73

* - actual measurement
** - value from Table 1 in reference 1
*** - asymmetry parameter for amitriptyline

Discussion and Rationale:

Two tests using the methods for HPLC packings by Neue, et. al. [1] were run in order to understand some of the retention and surface characteristics of the hydride stationary phases compared to typical commercially available reverse phase packings. The first test is for Hydrophobicity which compares the retention of naphthalene and acenaphthene under a standard set of conditions.

The hydrophobicity parameters () for the two TYPE-C silicon-hydride phases were as follows:
     Bidentate C18™ = 0.0397
     UDC-Cholesterol™ = 0.0339

In order to verify that the measurements were done correctly the same determination was made on a Phenomenex Luna C18 column. The experimentally determined value of was 0.0400 for this column, in comparison to the reported value of 0.0397 [1] in the literature.

The hydrophobicity of the Bidentate C18™ column with respect to the more than 100 commercial packings tested [1] is similar to the Luna® C18 and the Zorbax® Eclipse XDB C18. These stationary phases are at the higher end of the hydrophobicity values of the phases tested and reported.

The UDC-Cholesterol™ has lower hydrophobicity as expected and is similar to many of the C8 phases [1].

The other phase property tested was the silanophilic interaction parameter (S). The values determined were as follows:
     Bidentate C18™ = 1.429
     UDC-Cholesterol™ = 0.949

In comparison to the more than 100 columns tested [1], the Cogent Bidentate C18™ is at the low end of classical packings, particularly with respect to its silanol activity and assymetry. The UDC-Cholesterol™ phase has an even lower S parameter and is similar to many phases with polar embedded groups as well as some phenyl and cyano phases. The “S” test is based on the retention of the polar compound amitriptyline.

For both TYPE-C™ (hydride) phases the peak symmetry was excellent indicating that relatively few silanol groups were interacting with this basic solute, AS values were found:
     Bidentate C18™ = 1.02
     UDC-Cholesterol™ = 0.95

Reference: The Neue Test

  1. “Characterization of HPLC Packings” – U.D. Neue, K.V. Tran, P.C. Iraneta, B.A. Alden, J. Sep. Sci., Vol. 26, No 3-4, March (2003) 174-186.


Hydrophobic and Silanophilic Interaction Tests

How are the “Hydrophobic and Silanophilic Interaction” tests useful?