8


August/September 2010


Fundamental issues in the implementation of a two-dimensional LCxLC separation.


by Candice Grivel, Amélie Dechenaux, Sabine Heinisch*


Laboratoire des Sciences Analytiques – UMR CNRS 5180 – Université de Lyon – 43, Boulevard du 11 Novembre 1918 – 69622 Villeurbanne Cedex Tel.: 04 72 44 82 96 – Fax : 04 72 44 83 19 – e-Mail : sabine.heinisch@univ-lyon1.fr * Corresponding author


This article aims at presenting the fundamental steps when implementing a two-dimensional on-line separation (2D-LC) of complex samples. Firstly, an approach is proposed to make the search for orthogonal conditions easy. It is based on the treatment of retention data acquired from generic gradient separations of test mixtures in various systems (stationary phase / mobile phase / temperature). The regression coefficient of 2D retention plots and the practical peak capacity are both determined. The instrumental design and the choice of analytical conditions are also discussed. This article demonstrates that an ultra-fast second dimension combining high temperature and ultra high pressure (HT-UHPLC) is very attractive. Finally, relevant choices are illustrated by a 2D separation of a protein digest.


Key-words Two-dimensional on-line liquid chromatography (2D-LC); HT-UHPLC ultra-fast second dimension; orthogonal systems; instrumental design


Introduction In conventional liquid chromatography, the theoretical peak capacity is limited to about 1500. In addition to the use of very long columns, hours or even daysmay be required to attain such high values [1]


. In two-


dimensional liquid chromatography (2D-LC), the total peak capacity is theoretically given


as the product of the peak capacities in each dimension[2]


(Equation 1), thereby leading to impressive peak capacities.


nc,total = nc,1 x nc,2 (Equation 1) On the other hand, 2D-LC can be a powerful method for checking the peak purity. An example is given in Figure 1 for the 2D separation of aromatic compounds using two different RPLC systems.


2D-LC techniques include two different modes [3]


: the “heart-


cutting” (LC-LC) where only a few fractions of the first separation are sent to the second separation and the “comprehensive” (LCxLC), where the whole sample is subjected to both separations. The latter is intended for the screening or the analysis of complex samples such as pharmaceutical, environmental or biological ones.


Figure 1. 2D-separation of aromatic compounds. First Dimension, 150 mm×2.1 mm i.d. BetaBasic; 0.08 mL/min; 0–70% Methanol in 87 min; 30 ◦C; Second Dimension, 50 mm × 2.1mm i.d. Acquity BEH C18; 1.45 mL/min; 12–65% Acetonitrile in 0.27 min; 90◦C; detection at 220 nm.


The transfer of fractions between the two columns can be operated either on-line or off-line. In on-line transfer,


the two separations occur concurrently. Although the latter approach usually generates lower peak capacities, it offers many advantages: (1) it prevents sample


contamination or sample loss; (2) it allows automatable analysis; (3) it leads tomore reproducible and faster separations. However more complex instrumentation is required. Moreover, data handling and optimization of operating conditions become critical issues.


Themultiplicative rule (Eq.1) implies two criteria to be fulfilled [4]


.On the one hand,


selectivities in each dimensionmust be different in order to reach a sufficient degree of orthogonality.Orthogonality hasmainly been studied by comparing retention data of two different separations and by assessing their degree of orthogonality with the regression coefficient value r², which should be as small as possible [5]


. Chemometric techniques have also been investigated [6] .On


the other hand, the sampling rate of the first dimension peaksmust be suitable in order not to lose the resolution in the second dimension.Murphy et al. [7]


determined an adequate sampling rate as 3-4 cuts per peak.


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