A simple, robust orthogonal background correction method for two-dimensional liquid chromatography

Abstract

Background correction is a very important step that must be performed before peak detection or any quantification procedure. When successful, this step greatly simplifies such procedures and enhances the accuracy of quantification. In the past, much effort has been invested to correct drifting baseline in one-dimensional chromatography. In fast online comprehensive two-dimensional liquid chromatography (LC×LC) coupled with a diode array detector (DAD), the change in the refractive index (RI) of the mobile phase in very fast gradients causes extremely serious baseline disturbances. The method reported here is based on the use of various existing baseline correction methods of one-dimensional (1D) liquid chromatography to correct the two-dimensional (2D) background in LC×LC. When such methods are applied orthogonally to the second dimension ((2)D), background correction is dramatically improved. The method gives an almost zero mean background level and it provides better background correction than does simple subtraction of a blank. Indeed, the method proposed does not require running a blank sample.

Figure 1

Structure of the LC×LC background represented by three single ²D chromatograms of a dummy run sampled at different times of the gradient in the ¹D. The amount of acetonitrile in the sample solvent transferred from the ¹D to the ²D (region A) corresponds to 10 % for the black dotted curve, 30 % for the blue curve and 50 % for the red dashed curve. Region B is where most peaks elute and region C shows the system flush-out peak.

Figure 2

Comparison of the effect of two different baseline correction methods on a typical single ²D chromatogram. The chromatograms are intentionally offset by 7 mAU to help visualization. (a) Conventional baseline correction methods: the blue solid line chromatogram is the original single ²D chromatogram; the black dashed line is the corrected baseline using a moving-median filter and the red dot-dashed line is the corrected baseline by the asymmetric polynomial fitting method. (b) The two methods are applied in combination with the OBGC method; line format same as in (a).

Figure 3

(a) Contour plot of a LC×LC chromatogram of an indole standard sample, the blue dotted line shows a cut taken at 7.0 s in the ²D. (b) Blue curve: chromatogram taken as the ¹D cut represented by the blue dotted line in panel (a); black dotted curve: baseline generated by the moving-median filter; red dashed curve: baseline resulting from the asymmetric polynomial fitting.

Figure 4

3D plots of the OBGC method represented as (a) the original hybrid chromatogram of 20 simulated 2D peaks over a dummy LC×LC chromatogram, (b) the recreated background structure obtained applying the OBGC method in combination with the moving-median filter; (c) the result of subtracting the recreated background shown in (b) to the original hybrid chromatogram shown in (a).