Reversed-phase chromatography chromatogram

The LC/ESI-MS analysis of PAs is usually performed by reverse-phase chromatography, chromatograms relative to analysis of a grape seed extract are reported in Fig. 6.10. 

The reaction of amines and amino acids with orthophthaldehyde has been widely used in postcolumn and precolumn derivatization in analyses of foods (99-104) and in analyses of peptides from biological samples. Figure 2 (87) presents a chromatogram for OPA derivatives of tryptic peptides from two proteins. The sensitivity of the method was on the order of picomoles. The authors have themselves performed postcolumn OPA derivatization of low-molecular-weight peptides from blue cheeses separated by reversed-phase chromatography (86). 

Figure 1. Reversed-phase HPLC chromatograms of human relaxin side fraction before and after reduction with dithiothreitol. The chromatography was performed on a Vydac C4 column using TFA-containing mobile phases, and eluted with an acetonitrile linear gradient from 18 to 50%.

Fig. 3.11 shows chromatograms of 2,3-dihydroxybenzoate and 2,5-dihyroxybenzoate separated by HPLC reversed-phase chromatography fitted with electrochemical detection (Halliwell et al., 1988). HPLC is carried out on a Spherisorb 50DS column (25 cm x 4.6 mm) applying 97.2% (v/v) 30 mM sodium citrate/2.7 mM acetate... 

Fig. 10. Chromatograms illustrating the effect of pairing ions on the separation of the dipeptides Ala-Tyr (1), Pro-Tyr (2), and Leu-Tyr (3) by reversed-phase chromatography. Column /x-Bondapak C flow rate 2 ml/min temperature 20 C eluents A, 5% methanol-95% water-50 mJVf KHjP04-5 mM HjPOi, pH 3.0 B, same as A plus 5 laM n-hexyl sulfonate C, 50% methanol-50% water-50 mW KH1PO4-5 mW HjP04-5 mA/ do-decyl sulfate, pH 3.0. Reprinted with permission from Hearn el al. (29). Copyright by Elsevier Scientific Publishing Co., Amsterdam.

When some of the components of the mobile phase are adsorbed by the stationary phase, the chromatograms of even small samples are not predicted correctly by linear chromatography. Then the sample injection causes a perturbation of their equilibrium, which is not properly described by linear chromatography im-less the equilibrium isotherms of these adsorbed components of the mobile phase are linear. There are basically two types of situations that may arise. They are typically exemplified by normal and reversed phase chromatography. In the for-... 

The best mixture (with respect to retention) for separating ten phenols by reversed-phase chromatography was found to be water-methanol (60 40), represented by chromatogram (J). The mobile phase polarity can be calculated as foUows ... 

Figure 7.5. HPLC analysis of sweetener, flavors, and preservative in a soft drink sample using gradient reversed-phase chromatography and UV detection. Chromatogram courtesy of PerkinElmer, Inc.

Figure 7.30. HPLC analysis of pre-column derivatized amino acids (50 pmoles) using Waters AccQ.Tag reagents with reversed-phase chromatography separation and fluorescence detection. Chromatogram courtesy of Waters Corporation.

Figure 7.34. HPLC analysis of synthetic oligonucleotides using ion-pair gradient reversed-phase chromatography on a column packed with 2.5-pm nonporous hybrid particles with UV detection at 260nm. Chromatogram courtesy of Waters Corporation.

Two different sample preparation methods by size-exclusion and reverse-phase chromatography were proposed for analysis of PCs and PAs in wine. In the former, a volume of 5 mL of dealcoholized wine is passed through a Fractogel Toyopearl TSK gel HW-50 (F) (12 x 120 mm) column. The stationary phase is washed with 25 mL of water and the simple polyphenols are eluted with 50 mL of an ethanol/water/trifluo-roacetic acid 55 45 0.005 (v/v/v) solution. The polymeric fraction is recovered with 50mL of acetone/H20 60 40 (v/v). Figure 6.19 shows the LC/ESI-MS extracted ion chromatograms of dimers and trimers in a wine (Fulcrand et al., 1999). 

A. flos-aquae strains they obtained an excellent linearity in their calibration curve in the range, 20-100 ng (on-column) (Harada et al., 1989). Wong and Hindin studied the applicability of LC-UV for the isolation, identification, and quantification of AN. Both normal phase and reversed phase chromatography were examined, but both phases showed poor chromatograms with broad peaks with extensive band tailing (Wong and Hindin, 1982). 

Fig. 8. Administration of le-Ci -all-cis-AS.n.i -Cig.a (D- Gas chromatograms of Cjo-PFA (F2, F3, and Fg) from liver after reversed phase chromatography.

TIC chromatogram (bottom chart) and contour plot for the LC-ESI-MS analysis of Triton X-100, by using gradient reversed-phase chromatography [2 mm i.d. octadecylsrUca column, 20/80 to 50/50 acetonitrile/10 mM ammonium acetate in 20 min, 200 /iL/min flow rate, 3 1 effluent split, nebulizer-assisted electrospray]. See Figure 4.9 for the oligomer series (I-IV) separated. (Courtesy of PE Sciex, Foster City, CA)... 

Figure 3 Chromatogram of a plasma extract showing separation of the anticonvulsants primidone, phenobarbitone, phenytoin, and carbamazepine by isocratic reversed-phase chromatography. Solid-phase extraction with a CIS column was used for sample preparation. Methanol (45%) in dilute acid is used as the mobile phase. Flow rate 1.0 ml min"h Detection UV absorption at 225 nm. Column CDS Hypersil 5 pm, 15 cm x 4.6 mm. Two internal standards are used IS 1 is 4-methyl primidone and IS 2 is 5-ethyl-5-p-tolybarbituric acid.

Figure 22-27 Chromatogram showing 20 ppb BPA leached into water from a polycarbonate hiking bottle. Reversed-phase chromatography on 150 x 3.9-mm column of 5-p.m RP18 with gradient elution over 15 min going from 40% to 100% solvent B. Solvent A = 0.1 wt% formic acid in HjO. Solvent B = acetonitrile. Mass spectral detection of negative ions with electrospray ionization and selected reaction monitoring of m/z 227 - 133 transition. (From M. Swartz, LCGC, January 2010, p. 42.]...

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