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52 May / June 2021


Chromatography Today Help Desk Diluting the challenges with 2D LC


There are typically two questions that a separation scientist will be asked.


• What compounds are in my sample? • How much of those compounds are in my sample?


Liquid chromatography can help with answering both of these questions, although it should be noted that strictly speaking chromatography is blind [1] and that to be able to answer these questions a suitable detector is required. Ideally, the detector will be able to determine/confirm the identity of the compound and quantify the amount of compound present. A variety of detectors are available in liquid chromatography, each having advantages and sometimes disadvantages, with financial considerations often a major factor in the acquisition decision-making process. Therefore, although it might be argued that mass spectrometry is a better detection technique in terms of quantification and identification, it is not the most populous detection system due to financial considerations.


The demands placed on separation scientists to answer these two fundamental questions are becoming increasingly more challenging, and as a consequence, liquid chromatography has seen continuous developments. One of these developments addresses the need for a greater understanding of complex samples, by using two or higher dimension liquid chromatography. The theory behind this is well understood and has been the subject of previous Chromatography Today articles [2], and in essence, states that if two modes of chromatography are different then it is feasible to improve the resolution up to a maximum of the square of that obtained using a single dimension. The practicalities of liquid chromatography mean that in reality this is not achieved, however, significant improvements have been shown to occur when coupling columns that are orthogonal.


One of the challenges associated with the use of 2D LC is transferring the sample from the first-dimension column to the second-dimension


column, and the Help Desk will focus on this in the present article. Interestingly many of the challenges, which are not always identified, also exist in one-dimensional chromatography and are associated with the injection of the sample into the mobile phase.


In transferring between the first and second dimension, as has previously been stated, it is preferred to use an orthogonal separation mechanism, which presents the challenge of ensuring that the transfer solute is compatible with the mobile phase in the second dimension. There are typically two aspects that can occur and relate to either a solubility/mixing issue or the solute plug being transferred in a strong eluotropic mobile phase.


Figure 1 demonstrates what can happen if there is a solubility issue during the injection. It can be seen that in the example shown that the transfer solute is kept the same and that the mobile phase is altered, in this case from acetonitrile to methanol. It can be seen that the detector response for the system using acetonitrile resulted in a non-linear response curve and that obtained for methanol resulted in a linear response. If only the initial data was taken, it may have been assumed that the detector had gone outside of its linearity range, and the Help Desk suggests that this may be occurring in one or two examples in a one-dimensional system and that solubility has not been a consideration. The same observation can also be seen if the transfer solvent is not fully miscible with the second-dimension mobile phase.


The other scenario that can occur is when the eluotropic strength of the transfer solvent is too strong and this results in the premature early elution of weakly retained analytes, Figure 2. It is also possible that the peak shape may become distorted as the analyte peak effectively starts to surf on the transfer solvent plug. Some examples of the different observed peak shapes are also shown in Figure 2.


Figure 2 shows a range of different observed peaks obtained for weakly retained (on the second dimension LC column) compounds. In one scenario the analyte peak can be seen effectively eluting with the transfer solvent. In another scenario, the analyte peak is split between components that are eluting with the transfer solvent elution time and the retention time that would be expected to be seen if there were no adverse effects from the transfer solvent. The final figure in this series shows a fronting peak. It should be noted that all of these observations can also be seen in one- dimensional chromatography and that the volume of solvent, physiochemical properties of the analyte used in the transfer can also affect the outcome.


Figure 1


The two scenarios that have been explained in the previous sections are


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