Chromatograms, from HPLC

Figure 4 shows the chromatograms from HPLC separations of fractions F2 of both chlorinated and unchlorinated samples. These... 

Figure 13.20 displays chromatograms from HPLC analysis of amino acids deriva-tized with phenyhsothiocyanate to yield derivatives with strong absorption at the common Hg-lamp line of 254 nm. The single letter component abbreviations are those of Fig. 13.19, with some additional peaks such as ammonia (NH3), phosphoserine (PH-S), phosphothreonine (PH-T), hydroxyproline (OH-P), galactosamine (Gal), and norleucine internal standard (NLE) and excess derivatizing reagent (Re). The fact that... 

A GC trace of Octa, under conditions similar to those described by Timmons and Brown (ref. 4), closely resembled the published chromatogram for trace impurities in Deca. Material isolated from HPLC peak 7 (Figure IB) eluted from the GC (Figure 2) in a position corresponding to one of the octabromo isomers in the published work. This agrees with the assignment of peak 7 as an octabromo isomer in this work. Material isolated from HPLC peaks 8 and 9 (Figure IB and... 

Extraction efficiency. The efficiency of extractions of imidacloprid from vegetables and crops decreases in the order acetonitrile > acetone > ethyl acetate. Acetonitrile is not a suitable extraction solvent because of the large number of co-eluting peaks on the chromatograms on HPLC at 210 or 270 nm. ... 

Fig. 2.114. RP-HPLC profiles of ACTs and SEC fractions (fr.) of ACTs. Each lyophilized sample was dissolved in water (1 mg/ml), and analysed by RP-HPLC. Upper chromatogram RP-HPLC profile of ACTs. Lower chromatograms with fraction numbers RP-HPLC profiles of SEC fractions of ACTs. The numbers of identified peaks in each chromatogram are (1) procyanidin B1 (PB1), (2) (+)-catechin, (3) procyanidin B2 (PB2), (4) procyanidin Cl (PCI), 5 (—)-epicatechin (EC). AU means relative absorbance units (at 280 nm). For details on the RP-HPLC conditions see text. Reprinted with permission from A. Yanagida et al. [253].

Fig. 3.81. HPLC analysis of reacted and unreacted RB4 (300 mg/1) total chromatogram at 598 nm (a and b), enlarged chromatogram from 27 to 28.5 min retention time (c), spectra of reacted RB4 componenets (d), and spectra of unreacted RB4 components (e) (DH, dihydrolysed MH, monohy-drolysed UH, unhydrolysed. Reprinted with permission from W. J. Epolito et al. [145].

In HPLC, a sample is separated into its components based on the interaction and partitioning of the different components of the sample between the liquid mobile phase and the stationary phase. In reversed phase HPLC, water is the primary solvent and a variety of organic solvents and modifiers are employed to change the selectivity of the separation. For ionizable components pH can play an important role in the separation. In addition, column temperature can effect the separation of some compounds. Quantitation of the interested components is achieved via comparison with an internal or external reference standard. Other standardization methods (normalization or 100% standardization) are of less importance in pharmaceutical quality control. External standards are analyzed on separate chromatograms from that of the sample while internal standards are added to the sample and thus appear on the same chromatogram. 

Fig. 7.6 Nonbound SCH 23390 in a competitive MS binding assay for dopamine Di receptors monitored at a transition from 288.1 91.2 m/z from binding samples without or with (+)-butaclamol. Intensity (/) is shown (a) without (+)-butaclamol, (b) with 30 nM (+)-butaclamol, (c) with 10 pM (+)-butaclamol. (a-c) Representative chromatograms after HPLC separation (RP8 column solvent CH3CN/0.1% HCOOH in H2O 1 1 300 pL min ).

Understandably, the impurity profiles of the same drug substance produced by different S3mthetic routes will differ qualitatively and quantitatively. This is commonly observed when a drug substance is provided by different suppliers. For example, the HPLC chromatograms from samples of fluoxetine hydrochloride obtained from four different suppliers show the differences in the impurities produced by the presumably different synthetic routes (Figure 1.1) [7]. Supplier A is the innovator company. Supplier B is in Italy, and Suppliers C and D are in India. 

FIGURE 1.3 HPLC profile for 28 different polyphenols on a Cis column. Classes of compound are shown in the upper part of the chromatogram. (From Sakakibara, H., Honda, Y., Nakagawa, S., Ashida, H., and Kanazawa, K., J. Agric. Food Chem., 51, 571, 2003. With permission.)... 

Figure 3. Normal-phase HPLC chromatograms from XAD-2/ethyl ether extracts of chlorinated and unchlorinated water. A 25-min linear gradient was used from 1% to 50% isopropyl alcohol in n-hexane. (Reproduced with permission from reference 16. Copyright 1986 Water Research Centre.)...

Figure 6 shows chromatograms from reverse-phase HPLC separations of the extracts of chlorinated tyrosine and chlorinated phenylalanine. Authentic standards were used to locate the compounds that had previously been identified by GC-MS. The chromatograms show the presence of several compounds not observed by GC-MS. This result indicates the presence of products of chlorination, which are nonvolatile. Other techniques are currently being employed to identify such compounds, for example, HPLC fractionation techniques combined with mutagenicity testing of the fractions and soft-ionization mass spectrometric analyses of the mutagenic fractions. 

Figure 6. Reverse-phase HPLC chromatograms from XAD-2/ethyl ether extracts of (a) chlorinated phenylalanine and (b) chlorinated tyrosine. An eluent of 55% methanol in water was used in (a), and an eluent of 35% methanol in water was used in (b).

Ruiz-Sala et al. (129) described a reversed-phase HPLC method with a light-scattering detector for the analysis of TGs in milk fat. The identification of TGs was carried out by a combination of HPLC and gas-liquid chromatography (GLC), and was based on the equivalent carbon numbers and retention times of different standard TGs. Finally, quantitation of peak areas from HPLC chromatograms was carried out after applying a deconvolution program to the parts of chromatograms with poor resolution. 

Figure 1.18. Comparison of MRM chromatograms following HPLC-MS/MS and UPLC-MS/ MS analysis of a mixture containing alprazolam, ibuprofen, d5-alprazolam, diphenhydramine, naproxen, and prednisolone. Each set of chromatograms was obtained from a single 100-ng/ mL injection of rat plasma. d5-Alprazolam was used as the internal standard for quantification of alprazolam. (Reprinted with pemnission from Yu et al., 2006.)...

Figure 2. Radioactivity chromatogram of sulfur compounds derivatized with monobromobimane. The reversed-phase HPLC separation is based on the hydrophobic properties of the bimane-sulfur adducts but peak area is based on "S-radioactivity of the compounds. At time 0 sulfite and thiosulfate impurities are present before addition of the hepatopancrease tissue homogenate. This was a 60 min experiment to determine the sulfide detoxifying functions of the hepatopancrease of the hydrothermal vent crab Bythograea thermydron. During this time the proportion of radioactivity in sulfide rapidly decreases and thiosulfate and sulfate accumulate as end products. Two intermediates, pi and p2 accumulate then decrease during the experiment. The two intermediates are believed to be polysulfides based on similar elution times of polysulfide standards. (Figure is the unpublished chromatograms from the data in Vetter et al. (24)-) continued on next page.

A capillary electrophoresis system is comparatively simple. The basic components (Fig. 6.1) include the power supply which provides the high voltage necessary for the separation, the capillary in which the separation takes place, the detector which determines the sensitivity of the separation, and the data acquisition system which records the electropherogram. Some instruments also perform fraction collection. The final electropherogram looks similar to a chromatogram obtained from HPLC. 

An external standard method is used when the standard is analyzed on a separate chromatogram from the sample. Quantitation is based on a comparison of the peak area/height (HPLC or GC) of the sample to that of the reference standard for the analyte of interest. The external standard method is more appropriate for samples with a single target analyte and narrow concentration range, where there is a simple sampling procedure, and for the analysis of hydrocarbon fractions. The calculation requires an accurate extract final volume and constant injection size. The peak area of an analyte is compared with that from a standard or standard curve and corrected for volume ... 

Figure 3. Chromatogram of HPLC analysis of an air sample from an aluminum reduction plant sample was collected on a glass fiber/silver membrane filter combination.

Vanilla extract is used to flavor a wide variety of foods and 1s very expensive. Herrman and StockH (48) developed an HPLC method to determine the major components of vanilla extracts. This method can be used to help differentiate natural vanilla extracts from cheaper substitutes. Shown in Figure 2a 1s a separation of standards of the major components 1n vanilla. Shown in Figure 2b is the chromatogram from a typical vanilla extract. It should be noted in Figure 2b that natural vanilla extracts apparently lack ethyl vanillin, a commonly used flavor substitute. 

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