doi:10.1016/j.chroma.2006.04.058 
Copyright © 2006 Elsevier B.V. All rights reserved.
Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography
Dwight R. Stolla, Jerry D. Cohenb and Peter W. Carra, 
, 
aUniversity of Minnesota, Department of Chemistry, Smith and Kolthoff Halls, 207 Pleasant Street SE, Minneapolis, MN 55455-0431, United States
bUniversity of Minnesota, Department of Horticultural Science, Alderman Hall, 1970 Folwell Hall, St. Paul, MN 55108, United States
Received 22 February 2006;
revised 11 April 2006;
accepted 19 April 2006.
Available online 23 May 2006.
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Abstract
A new approach to high speed, comprehensive online dual gradient elution 2DLC (LC × LC) based on the use of ultra-fast, high temperature gradient elution reversed phase chromatography is described. Entirely conventional gradient elution instrumentation and columns are assembled in a system which develops a total peak capacity of about 900 in 25 min; this is equivalent to roughly one peak/2 s. Each second dimension gradient is done in a cycle time of 21 s and the peak retention times measured for a set of twenty six indole-3-acetic acid (IAA) derivatives are reproducible to 0.2 s. Each peak eluting from the first dimension column is sampled at least twice across its width, as the corresponding peaks on the second dimension column appear in two or three consecutive second dimension chromatograms, clearly indicating that there is little loss in the resolution gained in the first dimension separation. Application to the separation of the low molecular weight components of wild-type and mutant maize seedlings indicates the presence of about 100 peaks on a timescale of 25 min. Compelling illustrations of the analytical potential of fast, high temperature 2DLC are evident in the clear presence of nine distinct peaks in a single second dimension chromatogram from a single quite narrow first dimension peak, and the great power of 2DLC to solve the “analytic dynamic range” problem inherent in the measurement of small peaks that are neighbors to a gigantic peak.
Keywords: Two-dimensional HPLC; High temperature HPLC; Ultra-fast gradient elution HPLC; Peak capacity; Metabolomics
Fig. 1. Block diagram of 2DLC instrument.
Fig. 2. Hypothetical separation of five components with 
to illustrate the effect of the first dimension sampling time (ts) on the effective first dimension peak capacity when ts is greater than or equal to the first dimension peak width. In this situation, decreasing the sampling time will increase the effective first dimension peak capacity (i.e., the number of peaks that can possibly be observed in the first dimension.
Fig. 3. Plot of the second dimension (- - -) and total two-dimensional (—) peak capacities as a function of the second dimension cycle time. The optimum total peak capacity is obtained with very short second dimension cycle times. 2nc and nc,2D were calculated using Eqs. (3) and (4), respectively.
Fig. 4. Structures of the twenty six indolic metabolites studied in this work. Compounds 6,10,11, and 14 are all structural isomers of indole-3-acetyl-myo-inositol.
Fig. 5. One-dimensional separation of twenty six indolic metabolites using the discovery HS-F5 column showing the high degree of saturation of the separation space when the separation is carried out in one dimension. Chromatographic conditions are given in Table 1.
Fig. 6. 2DLC separation of the indolic metabolite standard mixture. The two-dimensional separation space is much less saturated than the corresponding one-dimensional separation of the same sample shown in Fig. 5. Chromatographic conditions are the same as those in Table 1 and Table 2 for the first and second dimension separations, respectively.
Fig. 7. One-dimensional separations of maize extract samples using the Discovery HF-F5 column only. Panel A is representative of a wild-type sample, and panel B is representative of an orp mutant sample. Chromatographic conditions are the same as those described in Table 1.
Fig. 8. 2DLC chromatogram (absorbance at 220 nm) obtained from the separation of mutant maize extract. Other chromatographic conditions are the same as those described in Fig. 6.
Fig. 9. Second dimension chromatogram obtained from 10.15 to 10.50 min in the 2DLC separation of orp mutant maize extract (same sample as panel B in Fig. 7). At least nine components are observed in this second dimension separation of the fraction eluted from the first dimension column from 9.80 to 10.15 min (indicated by the first arrow in Fig. 7B).
Fig. 10. Second dimension chromatogram obtained from 11.55 to 11.90 min in the 2DLC separation of orp mutant maize extract (indicated by the second arrow in Fig. 7B). At least three low abundance components are observed in addition to the dominant peak at 10 s; it is impossible to observe these low level components in the one-dimensional separation in Fig. 7 because of the presence of the dominant peak.
Table 1.
First dimension gradient programa
a Column: 50 mm × 2.1 mm i.d. 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 μL; detection by absorbance at 220 nm unless otherwise noted.
Table 2.
Gradient elution performance characteristics of the second dimensiona of the 2DLC system
a Column: 50 mm × 2.1 mm i.d. 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 μL; detection by UV absorbance at 220 nm unless otherwise noted.
Table 3.
Firsta and second dimensionb gradient elution retention time for five randomly selected components of the indolic metabolite standard mixture
a First dimension conditions were the same as in
Table 1.
b Second dimension conditions were the same as in
Table 2.
Table 4.
Comparison of absolute peak capacities and peak capacity production rates achieved in this work and other published reports of comprehensive online 2DLC
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