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  • Title: Two dimensional assisted liquid chromatography - a chemometric approach to improve accuracy and precision of quantitation in liquid chromatography using 2D separation, dual detectors, and multivariate curve resolution.
    Author: Cook DW, Rutan SC, Stoll DR, Carr PW.
    Journal: Anal Chim Acta; 2015 Feb 15; 859():87-95. PubMed ID: 25622610.
    Abstract:
    Comprehensive two-dimensional liquid chromatography (LC×LC) is rapidly evolving as the preferred method for the analysis of complex biological samples owing to its much greater resolving power compared to conventional one-dimensional (1D-LC). While its enhanced resolving power makes this method appealing, it has been shown that the precision of quantitation in LC×LC is generally not as good as that obtained with 1D-LC. The poorer quantitative performance of LC×LC is due to several factors including but not limited to the undersampling of the first dimension and the dilution of analytes during transit from the first dimension ((1)D) column to the second dimension ((2)D) column, and the larger relative background signals. A new strategy, 2D assisted liquid chromatography (2DALC), is presented here. 2DALC makes use of a diode array detector placed at the end of each column, producing both multivariate (1)D and two-dimensional (2D) chromatograms. The increased resolution of the analytes provided by the addition of a second dimension of separation enables the determination of analyte absorbance spectra from the (2)D detector signal that are relatively pure and can be used to initiate the treatment of data from the first dimension detector using multivariate curve resolution-alternating least squares (MCR-ALS). In this way, the approach leverages the strengths of both separation methods in a single analysis: the (2)D detector data is used to provide relatively pure analyte spectra to the MCR-ALS algorithm, and the final quantitative results are obtained from the resolved (1)D chromatograms, which has a much higher sampling rate and lower background signal than obtained in conventional single detector LC×LC, to obtain accurate and precise quantitative results. It is shown that 2DALC is superior to both single detector selective or comprehensive LC×LC and 1D-LC for quantitation of compounds that appear as severely overlapped peaks in the (1)D chromatogram - this is especially true in the case of untargeted analyses. We also anticipate that 2DALC will provide superior quantitation in targeted analyses in which unknown interfering compounds overlap with the targeted compound(s). When peaks are significantly overlapped in the first dimension, 2DALC can decrease the error of quantitation (i.e., improve the accuracy by up to 14-fold compared to 1D-LC and up to 3.8-fold compared to LC×LC with a single multivariate detector). The degree of improvement in performance varies depending upon the degree of peak overlap in each dimension and the selectivities of the spectra with respect to one another and the background, as well as the extent of analyte dilution prior to the (2)D column.
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