HPLC fractionation

HPLC fractionation is the first step (dimension) in the two-dimensional analysis of complex peptide mixtures. This step is generally carried out by gradient elution 

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Figure 2.8 (a) HPLC fractionation of orange oil on Lichrosorb 100 diol. (b) LC-GC-NPD analysis of peel orange oil (from Florida), contaminated with etliion. Reprinted from Proceedings of the 20tlr International Symposium on Capillary Chromatography, F. David et ai, On-line LC-PTV-CGC determination of pesticides in essential oils , 1998, with permission from Sandra P. 

In addition to the development of the powerful chiral additive, this study also demonstrated that the often tedious deconvolution process can be accelerated using HPLC separation. As a result, only 15 libraries had to be synthesized instead of 64 libraries that would be required for the full-scale deconvolution. A somewhat similar approach also involving HPLC fractionations has recently been demonstrated by Griffey for the deconvolution of libraries screened for biological activity [76]. Although demonstrated only for CE, the cyclic hexapeptides might also be useful selectors for the preparation of chiral stationary phases for HPLC. However, this would require the development of non-trivial additional chemistry to appropriately link the peptide to a porous solid support. 

HPLC fraction Measured" Mr (Da) Calculated " Mr (Da) Sequence position... 

Figure 10. Reversed-phase HPLC analysis of PAHs extracted from SRM 1649, urban dust/organics, with UV detection, preceded by normal-phase HPLC fractionation based on ring carbon number. (Reprinted from reference 72. Copyright 1984 American Chemical Society.) Continued on next page.

The effects of various enzymes on the activity of HPLC fractions that inhibited 3H-PCP binding were investigated. As shown in table 1, pronase (0.5 pg/ml), carboxypeptidase A (0.1 unit/ml), and trypsin (3.0 g/ml ) markedly decreased the potency of 10 n units of PCP-like activity. No significant change in activity was. seen when fractions were incubated with alpha-chymotrypsin. 

Panel B Activity profiles of bombesin, neuropeptide Y, and substance p from the same HPLC fractions. 

Activity has been detected In the Lemna bloassay with HPLC fractions of unknown allelochemlcals from sunflower using quantities as low as 5 pi of a 28 ppm w/v solution (see Saggese et al. this publication). 

Ultimately, it is important to develop explant bioassays which can help quantify allelochemicals. This will require greater availability of purified active products. However, at this time, the explant systems can be used to show qualitative effects whether in crude extracts, leachates or from HPLC fractionation (17). Once target species effects are characterized, other bioassays which may be more sensitive could be used as well. 

Dai, Y. Li, L. Roser, D. C. Long, S. R. Detection and identification of low-mass peptides and proteins from solvent suspensions of Escherichia coli by HPLC fractionation and MALDI mass spectrometry. Rapid Comm. Mass Spectrom. 1999,13, 73-78. 

He et al. (2002) used an off-line HPLC/CE method to map cancer cell extracts. Frozen ovarian cancer cells (containing 107 cells) were reconstituted in 300 pL of deionized water and placed in an ultrasonic bath to lyse the cells. Then the suspension was centrifuged and the solubilized proteins were collected for HPLC fractionation. The HPLC separation was carried out on an instrument equipped with a RP C-4 column, 250 mm x 4.6 mm, packed with 5-pm spherical silica particles. Extracted proteins were dissolved in 300 pL of DI water, and lOOpL was injected onto the column at a flow rate of 1 mL/min. Buffer A was 0.1% TEA in water and buffer B was 0.1% TFA in acetonitrile. A two-step gradient, 15-30% B in 15 min followed by 30-70% B in 105 min, was used. The column effluent was sampled every minute into a 96-well microtiter plate with the aid of an automatic fraction collector. After collection, the fractions were dried at room temperature under vacuum. The sample in each well was reconstituted before the CE analysis with 10 pL deionized water. The... 

FIGURE 16.12 CZE analysis of four HPLC fractions of cancer cell extracts (reprinted with... 

Feltz, K.P., D.E. Tillitt, R.W. Gale, and PH. Peterman. 1995. Automated HPLC fractionation of PCDDs and PCDFs and planar and nonplanar PCBs on C18-dispersed PX-21 carbon. Environ. Sci. Technol. 29 709-718. 

E2, EE, El Solvent extraction with sonification, HPLC fractioning, derivatization GC-MS/MS 0.4-1 [42]... 

The major components are series of homologous trimers, tetramers, and pentamers of the three acids 44-46, along with smaller quantities of dimers, hexamers, and heptamers. Furthermore, the secretion contains several isomers of each oligomer, furnishing a combinatorial library of several hundred macro-cyclic polyamines [51, 52]. Using repeated preparative HPLC fractionation, the most abundant trimeric, tetrameric and pentameric earliest-eluting compounds were isolated. One and two-dimensional H NMR spectroscopic analyses showed that these molecules were the symmetric macrocyclic lactones 48, 49, and 50 (m, n, o, p, q=7) derived from three, four or five units, respectively, of acid 46. Moreover, using preparative HPLC and NMR methods, various amide isomers, such as 53,54, and 55 (Fig. 9) were also isolated and characterized [51,52]. 

Figure 6.3 YES assay of HPLC fractions of extracts from SPMDs exposed to the Elizabeth River, VA, USA. Reproduced courtesy of Andrew Rastall, University of Heidelberg, Heidelberg, Germany.

Figure 6.8 UV spectra of standard chlorogenic acid (a) trans-cinnamic acid (b) cafFeic acid (c) p-coumaric acid (d) and femlic acid (e). The spectra of peaks 1 (chlorogenic acid) (f), peak 2 (chlorogenic acid isomer) (g), and peak 3 (caffeic acid) (h) were determined with HPLC fractions isolated from extracts of Superior potato peel.

Fig. 17.6 Stereodifferentiation of Furaneol (J) and mesifuran (2) from strawberries a HPLC chromatogram of strawberry extract mesifuran (fraction ft), Furaneol (fraction/2) b HPLC fractions, analysed by enantioselective capillary GC [23]...

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