LCGC The Peak - April 2008 - (Page 10)

10 THE PEAK APRIL 2008 20 16 13,14 15 18,19 20 23 24 26 3 7 2 5 12 10 6 15 21 11 25 17 10 1 4 8 9 22 5 0 0 5 10 15 20 25 30 First dimension retention time (min) Figure 2: LC LC separation of an indolic metabolite standard mixture. First dimension: Gradient-elution conditions from 5 to 40% B in 20 min; column: 50 mm 2.1 mm Discovery HS-F5; solvent A: 20 mM sodium phosphate, 20 mM sodium perchlorate, pH 5.7; solvent B: acetonitrile; temperature: 40 °C; flow rate: 0.10 mL/min; injection volume: 10 mL; detection by absorbance at 220 nm. Second dimension: column: 50 mm 2.1 mm ZirChrom-CARB; flow rate: 3.00 mL/min; gradient elution from 0 to 70% B in 17.4 s, where A: 20 mM perchloric acid in water, and B: acetonitrile; temperature: 110 °C; injection volume: 34 mL; detection by UV absorbance at 220 nm. Reprinted from reference 2 with permission. resolution obtained in the first dimension be maintained, despite cutting the chromatogram in a limited number of fractions. The first criterion is met by using a valveswitching system like that shown in Figure 1 (5). The second criterion is more difficult to meet. It has long been assumed (6) that about four “cuts” would need to be taken along every first-dimension peak. Recent results from Tanaka’s group (7) suggest that two cuts per peak can be optimal. The questions that will be addressed in this communication involve the value of LC LC in practice. Why would one want to employ the technique, given that the equipment is more complicated (an additional LC pumping system is required) and that a valve-switching system and dedicated software are needed? The answer is twofold, and it will be provided in the next two sections. One deals with the peak capacity that can be obtained from comprehensive twodimensional chromatographic separations. In the second one, the concept of sample dimensionality will be discussed and we will see how structured, readily interpretable two-dimensional chromatograms can be obtained. Peak capacity in LC LC truly comprehensive separations, such a two-dimensional chromatogram is representative for the entire sample. There are two criteria for calling a twodimensional separation “comprehensive” (4). One is that every bit of the sample is being analyzed in both dimensions, without anything going to waste. The second is that the Second dimension retention time (s) The peak capacity of any separation can be defined as the number of peaks that can be separated with a specific resolution. The assumption is that the peaks are spread equally throughout the chromatogram. While this is quite feasible (for example, the separation of a homol-

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LCGC The Peak - April 2008 Contents LC3LC: Comprehensive Two-Dimensional Liquid Chromatography Split Injections in Gas Chromatography: How to Reduce Inlet Discrimination A Simple and Solvent-Free Method for Determining Tetracyclines in Prawns Industry News Events Education

LCGC The Peak - April 2008

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