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An automated orthogonal two-dimensional liquid chromatograph.
Cadapakam J Venkatramani1, Yury Zelechonok
1Pharmacia Corporation, Global Chemical Process R & D, 4901 Searle Parkway, Skokie, Illinois 60077, USA. cvenkatramani@yahoo.com
Analytical Chemistry
|October 23, 2003
Summary
A novel two-dimensional liquid chromatography (2D-LC) system enhances separation efficiency by coupling columns with different selectivities. This method minimizes correlation between dimensions, leading to orthogonal separations and improved peak capacity for complex mixtures.
Area of Science:
- Analytical Chemistry
- Chromatography
Background:
- Conventional liquid chromatography (LC) can struggle with complex mixtures, leading to co-elution and limited peak capacity.
- Two-dimensional LC (2D-LC) offers enhanced separation power but often suffers from column correlation, reducing overall efficiency.
Purpose of the Study:
- To develop a simple and efficient 2D-LC system using a standard HPLC setup and a multi-port valve.
- To achieve orthogonal separations by minimizing correlation between the two chromatographic dimensions.
- To improve peak capacity and enable positive identification of components in complex mixtures.
Main Methods:
- Coupling two LC columns with different selectivities using a 12-port, dual-position valve.
- Implementing continuous, periodic sampling from the primary column eluent onto the secondary column.
- Utilizing a solvent gradient in the primary dimension and incremental solvent strength in the secondary dimension ('tuning') to minimize column correlation.
- Evaluating the system with a test mixture of aromatic amines and non-amines using various secondary columns (ODS-AQ/ODS monolith, ODS/amino, ODS/cyano).
Main Results:
- The developed 2D-LC system achieved separations comparable to conventional HPLC in the primary dimension, with rapid (<10 seconds) separations in the secondary dimension.
- Optimized tuning minimized cross-correlation between dimensions, resulting in an orthogonal separation plane.
- Peak capacity was significantly enhanced, theoretically reaching around 450 in 30 minutes.
- The choice of secondary column (amino and cyano columns showing high complementarity) greatly influenced the separation pattern, with reversed elution orders observed for several components.
- A 2D-LC system with amino and cyano columns rapidly resolved most components of the test mixture within minutes.
Conclusions:
- The developed 2D-LC system provides a simple yet powerful approach for analyzing complex mixtures.
- Orthogonal separations achieved through column tuning significantly enhance resolution and peak capacity.
- The system allows for positive identification of components based on unique retention time pairs and provides two independent measures of molecular properties.