Separation of Hydrophobic Compounds with HPLC & UHPLC

This AppNote shows separation of analytes within a range of hydrophobicity. A simple gradient is used to elute all the compounds and baseline separation is obtained for the critical pair (peaks 4 and 5) and the least hydrophobic compound is adequately retained.

A comparison is shown in the figure below with a 4μm Cogent Bidentate C18 Column and a similar 2.o (2.2µm) Column. The retention profiles are quite comparable, meaning Method Transfer from one Column to the other will be easy to achieve.

Separation of Hydrophobic Compounds ChromatogramsSeparation of Hydrophobic Compounds Chemical Structures

1. Acetanilide, 2. Benzonitrile, 3. Methyl Benzoate, 4. Ethyl Benzoate,
5. Phenyl Benzoate, 6. Benzyl Benzoate, 7. Bibenzyl 8. Triphenylamine

Method Conditions

Columns: Cogent Bidentate C18 2.o™, 2.2μm, 120Å; Cogent Bidentate C18™, 4um, 100Å
Catalog Nos.: 40218-05P-2; 40018-05P-2
Dimensions: 2.1 x 50mm for both Columns
Mobile Phase:
A: DI Water / 0.1% Formic Acid (v/v)
B: Acetonitrile / 0.1% Formic Acid (v/v)

Time (minutes) %B
0 20
1 20
25 80
26 80
27 20

Injection vol.: 1 µL
Flow rate: 0.3mL / minute
Detection: UV @ 254nm
Sample Preparation: Mixture of solutes in 80:20:0.1 Acetonitrile / DI Water / Formic Acid Diluent. Peak identities were confirmed with individual standards.
t0: 0.7 minutes


No 274 Transfer HPLC Method to UHPLC with Hydrophobic Compounds pdf 1 Mb  Download File