[Reader Insight] A Guide to Selective Columns for Isomer Separation
This article is submitted by expert chromatographer SHI Hang. Welch Materials, Inc. is authorized to translate this article to English and publish it on behalf of the author.
Table of content
Introduction
In liquid chromatography method development, isomers are one of the most challenging types of compounds to separate, which include positional isomers, diastereomers, and cis-trans isomers. These compounds are generally difficult to separate due to similar logD values, thus requiring specialized columns.
In this article, we will review some "unique columns" commonly used in daily analysis that involves separation of isomers.
Phenyl and Pentafluorophenyl (PFP) Columns
When dealing with benzene ring positional isomers, Phenyl and PFP columns are often the go-to choices due to their unique ability to offer hydrophobic interactions, π-π interactions, dipole-dipole interactions, hydrogen bonding, and spatial selectivity.
The main types of Phenyl and PFP columns are:
1. Phenyl/PFP-Alkyl Columns
Here, phenyl groups are bonded to silica through alkyl chains, typically hexyl or propyl, where hexyl offers stronger hydrophobicity. Replacing phenyl with pentafluorophenyl results in PFP columns. Due to the rigid structure of the benzene ring, the bonding density isn’t very high, meaning these columns generally lack alkali resistance.
While the labeled pH range is usually 2–8, lower column temperatures are recommended at pH >7 to prevent silica breakdown and stationary phase loss (exceptions include hybrid silica bases).
Such columns are common in the markets. Examples include:
- Phenyl columns: Xtimate Phenyl-Hexyl, Waters XBridge BEH Phenyl (both with hybrid silica bases and high pH tolerance)
- PFP columns: Ultisil XB-PFP, YMC-Triart PFP
Application Example: Separation of six isomers
Compounds: 4-Hydroxydiphenylamine, 2-Phenoxyaniline, 4-Phenoxyaniline, 4-Amino-4'-Hydroxybiphenyl, 3--Hydroxydiphenylamine, 3--Phenoxyaniline
Column: Boltimate EXT-PFP
2. C18-Phenyl/PFP Columns
With alternating C18 and phenyl groups (sometimes labeled as phenyl at the C18 terminus due to patent restrictions), these columns offer the selectivity of both traditional C18 and phenyl columns, making them ideal for complex sample analysis where regular impurities and positional isomers need simultaneous control.
Examples include: Aurashell C18/AR, ACE Excel 3 C18-PFP, etc.
3. Biphenyl/Pentafluorobiphenyl Columns
Here, two phenyl or PFP groups are bonded in tandem to silica, enhancing π-π interactions for separating isomers that phenyl or PFP columns can’t resolve.
Examples include: Comixsep Polar BiPFP, Shim-pack Velox Biphenyl, etc.
4. Other Types
Other varieties include ether-phenyl columns (e.g., Ultisil Phenyl-Ether, ChromCore Phenyl-Ether) and positively charged phenyl columns (e.g., Waters XSelect CSH Phenyl-Hexyl).
Application Example: Separation of alcohol derivatives and their ortho and meta isomers
Column: Ultisil Phenyl-Ether
Embedded Amide Columns
As the name suggests, these columns use amide linkages (patented by different brands, often comprising amide or carbamate groups). Initially developed for analyzing basic compounds under acidic conditions, these columns have shown excellent separation for diastereomers during method development, likely due to the C=O group providing some shape selectivity through spatial hindrance.
Examples: Ultisil Polar-RP, ShimNex HE C18-EP, etc.
Application Example:
Samples structures:
Column: Ultisil Polar-RP
C8 Columns
Known for weaker hydrophobicity than C18, C8 columns also demonstrate spatial selectivity. Their shorter carbon chains lead to controlled bonding density, allowing compounds to more easily fit into spaces between bonded phases, making C8 suitable for separating conformational isomers with differing spatial structures.
Examples: Xtimate C8, ZORBAX SB-C8
Adamantyl Columns
Adamantyl columns feature a unique cage-like structure that provides spatial selectivity. This technology, patented by Sanyo Fine, includes the model CAPCELL PAK ADME HR.
Specialized C18 Columns
1. Polymeric-Bonded PAH Columns
Designed specifically for polycyclic aromatic hydrocarbons (PAHs), these columns have high spatial selectivity. They are often labeled after PAH.
Examples: Ultisil PAH, ChromCore PAH
2. Multi-layer Bonded Columns
Using multi-layer bonding to increase bonding density, these columns offer improved spatial selectivity.
Chiral Columns
Chiral columns are diverse, including polysaccharide derivatives, macrocyclic antibiotics, cyclodextrins, and Pirkle-type columns. Since they are chiral selective, these columns can separate diastereomers and positional isomers. However, due to their high cost, chiral columns are typically not recommended for non-diastereomer separations.
Conclusion
In conclusion, columns with spatial selectivity are plentiful. Choosing the most cost-effective column depends on the specific type of isomer. Phenyl/PFP, amide, and C8 columns cover the majority of separation needs: for benzene ring positional isomers, phenyl/PFP columns are ideal; for cis-trans and diastereomers, amide and C8 columns are preferred.