About Comprehensive Two-Dimensional LC

What is Multidimensional Chromatography?

Multidimensional chromatography uses a combination of several chromatography techniques, separation modes, and columns to separate multiple components. It achieves significantly higher separation than normal one-dimensional chromatography. Various separation modes and the corresponding mobile phases can be selected for HPLC, and the diverse permutations available suggest the possibility of achieving a degree of selectivity not possible using one-dimensional separation alone.

• Comprehensive Two-Dimensional Liquid Chromatograph : Nexera-e
• Trap-Free 2D LC/MS system for Quadrupole LCMS
• Comprehensive GCxGC System

What is Comprehensive Two-Dimensional LC?

Two-dimensional LC is a commonly used HPLC multidimensional chromatography technique. It is sometimes called "LC×LC". One of the two-dimensional LC techniques is comprehensive two-dimensional LC, in which all the eluent from the first-dimension column is introduced online into the second-dimension column, such that the entire sample is subjected to two-dimensional separation.

Fig.1 Differences between a conventional mono-dimensional separation and a multi-dimensional separation

System Configuration for Comprehensive Two-Dimensional LC

Fig. 2 shows the system configuration for comprehensive two-dimensional LC. This system permits gradient elution at both the first- and second-dimension stages. The sample solution is injected by the autosampler and separated in the first-dimension column. The sample flow rate is generally low in the first-dimension column (such as 50 μL/min) to allow separation over a long period of time. The eluent from the first column is sent to the loop of the flow-switching valve.

When the loop becomes full (after two minutes at a 50 μL/min flow rate with the 100 μL loop used in this example), the valve operates to send the eluent for separation in the second-dimension column. As the flow-switching valve operates every two minutes in the example shown here, analysis in the second-dimension column (including column equilibration time for gradient elution) must be completed within two minutes.

A UHPLC (ultra high performance liquid chromatography) column has been adopted in recent years as the second-dimension column in order to achieve a sufficiently high degree of separation in about one minute.

Comprehensive Two-Dimensional LC Analysis Example

Fig. 3 shows the analysis of a Kakkonto extract granule using a comprehensive two-dimensional LC system. 
The sample was separated using a semi-micro reversed-phase column (100 mm × 1.5 mm I.D., 2.2 µm) and neutral pH mobile phase for the first-dimension and an ultra-high speed reversed-phase column (50 mm × 2.1 mm I.D., 2.7 µm) and acidic pH mobile phase for the second-dimension. 

The alignment of peaks in the vertical-axis (second-dimension) direction indicates separation of components in the second-dimension column that could not be separated in the first-dimension column. Therefore, it can be said that comprehensive two-dimensional LC is able to separate components at the second-dimension that could not be separated at the first-dimension.