Solvent Games in 2D-LC
How can we avoid band broadening and distortion of high performance liquid chromatographic peaks in the second dimension?
It is well known that pouring cold water into a hot glass causes a thermal contraction that will form a crack, if not shatter the vessel. The temperature of the water is not to blame, rather it is the change in temperature happening in the glass. If warm water is added to the glass it will survive to live another day.
Similarly, an easy way to lose efficiency from a HPLC separation is to pay little attention to sample preparation, in particular the injection solvent in which the analyte is dissolved. Indeed, if the solvent within the sample vial is only a little stronger than that of the mobile phase (at the initial composition for a gradient separation), peak broadening will start to erode the baseline between closely resolved peaks. If the solvent strength miss-match is even higher, significant band distortion can occur.
In a two dimensional (2D)-HPLC separation, small aliquots of a chromatographic separation (the first dimension) are sequentially transferred to a different separation environment (the second dimension) to enhance the overall capacity to resolve chemical compounds and enhance the resolution of structurally similar molecules. However, trying to complete a 2D-HPLC separation using a gradient in the first dimension is inherently challenging because the concentration of the aliquot transferred between dimensions (i.e., the second dimension injection solvent) is continually increasing. The solvent strength miss-match can deform the peak and remove any ability to extract meaningful data from a separation.
We recently incorporated a third pump into the 2D instrumental configuration to address the problem (1). The aim was to introduce a “counter gradient” to offset the changing mobile phase produced by the first dimension. I found that the second dimension injection solvent could be artificially manipulated so that a consistent composition was sent to the second dimension. The counter gradient has significance in reversed phase (RP) × RP, but also HILIC × RP separations, where the mobile phases are at the opposite ends of the solvent strength spectrum. We also created a free-to-use interactive application that calculates the required counter method for a single step gradient to produce a required transfer solvent strength.
You may be thinking that it would be useful for me to describe the metric that I used to assess the improvement; however, the state of peak shape was so terrible without the counter gradient that a suitable value could not be measured. I can say that the shape of chromatographic peaks in the second dimension were enhanced markedly with the inclusion of a counter gradient, restoring them to a typical Gaussian profile, which allowed for a quantitative 2D-HPLC determination of antioxidant limit of detection (LOD) for the first time.
Unfortunately, the counter gradient approach has its limitations. We found that the mobile phase concentration after the counter gradient was limited to approximately 20 percent solvent component. Below that threshold, several experimental design issues arose, including very slow first dimension times; counter gradient flow rates above instrument max; analytical scale sample loops of insufficient size; and first dimension dilution leading to limited LOD.
We have not used the counter gradient method with reverse and normal phases of chromatography, but I anticipate that the inclusion of a solvent that is miscible in both of these mobile phase environments will improve their compatibility. If successful, it will dramatically enhance the time and space efficiency of 2D-HPLC analyses.
- P. G. Stevenson et al., “Improving Peak Shapes with Counter Gradients in Two-dimensional High Performance Liquid Chromatography”, J. Chromatogr. A, 1337, 147–54 (2014).
“Many curious children like to pull things apart to see how they work, now I get to do this on a molecular scale” says Paul Stevenson of his career as an analytical scientist. After completing his PhD at the University of Western Sydney in September 2010 he completed a post-doctoral appointment at the University of Tennessee and now works at Deakin University, Australia. Paul is an early career researcher and has investigated the separation of a number of food products while developing novel approaches. “The separation of natural product extractions is fascinating because the small changes in concentration profiles can say so much about the underlying biological processes,” he says.
