Two-dimensional HPLC

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

Figure 9.8 Schematic illustration of the two-dimensional HPLC/fast-CZE instmmental setup. The argon laser beam was set at 488 nm, which was used to photodegrade and detect the fluorescein isothiocyanate tag.

J. S. Pati ick and A. L. Lagu, Determination of recombinant human proinsulin fusion protein produced in Escherichia coli using oxidative sulfitolysis and two-dimensional HPLC, Chem. 64 507-511 (1992). 

In early work, while compositional heterogeneity was recognized and could be predicted, it was difficult to measure. Now, methods such as GPC combined with NMR and/or MALDI,238 239 GPC coupled with FTIR240 and two dimensional HPLC or GPC241"245 can provide a direct measure of the composition distribution. 

Coldham, N. G. Woodward, M. J. Characterization of the Salmonella typhimurium proteome by semi-automated two dimensional HPLC-mass spectrometry Detection of proteins implicated in multiple antibiotic resistance. J. Proteome Res. 2004, 3,595-603. 

Horie, K., Kimura, H., Ikegami, T., Iwatsuka, A., Saad, N., Fiehn, O., Tanaka, N. (2007). Calculating the optimal modulation periods to maximize the peak capacity in two-dimensional HPLC. Anal. Chem. 79, 3764-3770. 

Haefliger, O.P. (2003). Universal two-dimensional HPLC technique for the chemical analysis of complex surfactant mixtures. Anal. Chem. 75, 371-378. 

Stoll, D.R., Carr, P.W. (2005). Fast, comprehensive two-dimensional HPLC separation of tryptic peptides based on high temperature HPLC. J. Am. Chem. Soc. 127, 5034-5035. 

Hu, L., Chen, X., Kong, L., Su, X., Ye, M., Zou, H. (2005). Improved performance of comprehensive two-dimensional HPLC separation of traditional Chinese medicines by using a silica monolithic column and normalization of peak heights. J. Chromatogr. A 1092, 191 198. 

FIGURE 9.4 Schematic diagram of the on-line comprehensive two-dimensional HPLC system including an integrated sample preparation step. 

TWO-DIMENSIONAL HPLC-CE METHODS FOR PROTEIN/PEPTIDE SEPARATION... 

Two-dimensional HPLC/CE separations are not limited to column HPLC/ column CE, as reported by Yang et al. (2003), who were able to couple capillary... 

FIGURE 18.8 Two-dimensional HPLC (NPLC/RPLC) chromatogram of Novel II 1412-70 with the corresponding chemical structure and average EO distribution as supplied by the manufacturer. Reprinted from Murphy et al. (1998b), with permission of the American Chemical Society. 

Svec F, Frechet JMJ. Pore-size specific modification as an approach to separation media for single-column, two-dimensional HPLC. Am Lab 1996 28 25. 

Semipreparative HPLC has been employed to obtain a vitamin D-rich fraction of the unsaponifiable matter for subsequent quantitative HPLC. Combinations of chromatographic modes used for offline semipreparative and quantitative analysis have included polar bonded-phase/adsorption (211,212), reversed-phase/adsorption (194,213), and adsorption/reversed-phase (70,125). An online two-dimensional HPLC technique using two polar bonded-phase columns has also been described (214). 

Normal-phase/reversed-phase chromatography is the ideal combination for semipreparative and quantitative separations in two-dimensional HPLC. Vitamins D2 and D3 coelute during the semipreparative stage, allowing a narrow retention window to be collected for analysis using internal standardization. By this means, Johnsson et al. (201) obtained a vitamin D3 detection limit of 0.1 yug/kg for milk and milk products. 

Mattila et al. (205) described a two-dimensional HPLC procedure for determining vitamin D3 and 25-hydroxyvitamin D3 in meat and milk products. Samples were saponified in the presence of vitamin D2 and 25-hydroxyvitamin D2 as internal standards, and the extracted un-saponifiable matter was subjected to normal-phase semipreparative HPLC to obtain a fraction containing 25-hydroxyvitamin D2 + 25-hydroxyvitamin D3 and a fraction containing vitamin D2 + vitamin D3. The collected fractions were evaporated and purified by reversed-phase HPLC. Fractions were again collected, after which vitamin D3 was quantified by tandem-column reversed-phase HPLC and 25-hydroxyvitamin D3 by tandem-column normal-phase HPLC. Analytical chromatograms of a purified extract of chicken are shown in Fig. 12. 

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