HPLC profiles

Reversed-phase high performance Hquid chromatography has come into use for estimating the purity of proteins and peptides as weU. However, before employed, a high performance Hquid chromatographic (hplc) profile of a given protein must be completely vaHdated (43). 

Fig. 3-2. Semipreparative RP-HPLC profile of eyelo(Arg-Lys-X-Pro-X-Ala). The erude sublibrary (160 mol) was dissolved in 0.1 % (v/v) TFA and applied to a Whatman Partisil 10 pm ODS-2 (1 x 50 em) eolumn. The peaks were eluted using a 40-min linear gradient of 0-25 % aeetonitrile in water at a flowrate of 7 mL min . Fractions were collected every 2 min and pooled in three fractions as indicated by arrows 130 pmol of peptides was reeovered (yield 81 %). (Reprinted with permission from ref. [75]. Copyright 1998, Ameriean Chemieal Soeiety.)...

Guyomarc h, R, Binet, A., and Dufosse, L., Production of carotenoids by Brevibac-terium linens variation among strains, kinetic aspects and HPLC profiles, J. Ind. Microbiol. BiotechnoL, 24, 64, 2000. 

Amino acid derivatives can be examined for enantiomeric purity by the same procedures after removal of the protecting groups. Another approach is to couple them directly with another derivative to give protected dipeptides whose diastereomeric forms are usually easy to separate by HPLC (see Section 4.11). An A-protected amino acid is coupled with an amino acid ester, and vice versa. Use of soluble carbodiimide as reagent (see Section 1.16), followed by aqueous washes, gives clean HPLC profiles. It is understood that the derivative that serves as reagent must have been demonstrated to be enantiomerically pure.43 84-89... 

Fig. 2.16. HPLC profile of carotenoids in an extract of vegetable soup. An expansion of the profile from 30 to 39 is shown in the inset (A). Monitored wavelengths were 436, 440, 464, and 409 nm for peaks 9,10,11,12, and 14, respectively, in the inset (A). Peak identification 1 + 1" = all-trans-lutein and cw-lutein 2 = 5,6-dihydroxy-5,6-dihydrolycopene (lycopene-5,6-diol) 3 = j3-apo-8 -carotenal (internal standard) 4 = lycopene 1,2-epoxide 5 = lycopene 5,6-epoxide 6 = 1,2-dimethoxyproly-copene (tentative identification) 7 = 5,6-dimethoxy-5,6-dihydrolycopene 8 = lycopene 9 = pheo-phytin b 10 = neurosporene 11 = (-carotene 12 = pheophytin a 13 = (-carotene 14 = pheophytin a isomer and (-carotene 15 = a-carotene 16 and 16" = all-trans-/fcarotene, cis-/J-carotene 17 and 17" = all-trans- or cA-phytofluene 18 and 18" = all-trans- or cw-phytoene. Reprinted with permisson from L. H. Tonucci et al. [40].

Fig. 2.23. Reversed-phase gradient HPLC profiles of carotenoids in human plasma. A human volunteer was given an oral dose of 5,6-epoxy-/l-carotene (9.1 /imol). Plasma was analysed for carotenoids before (a) and 6h after (b) the oral dose. Peak identification 1, bilirubin 2, lutein 3, zeaxanthin 4, /1-cryptoxanthin 5, 5,6-epoxy-/l-carotene 6, lycopene 7, /1-carotene. The detection wavelength was 445 nm. AU, absorbance unit. Reprinted with permission from A. B. Barua [50],...

Fig. 2.36. Representative HPLC profiles of the extract of microcapsules, initially and after eight weeks of storage (a) and pure astaxanthin after eight weeks of storage (b). Chromatographic conditions are described in the text. Reprinted with permission from I. Higuera-Ciapara et al. [79].

Fig. 2.96. HPLC profiles of Starking Delicious juice, rose cider, and GPC fractions from rose cider recorded at 520 nm. Each lyophilized sample was dissolved in 10 per cent EtOH (20 mg/ml). Peak 1 with retention time of 27.5min is Cyn-3-gal, which is the main anthocyanin in apple, and peaks 5-9 are unidentified anthocyanins, which may be newly produced during the vinification process. Reprinted with permission from T. Shoji et al. [231],...

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. 2.116. The HPLC profile of the Sephadex LH-20 fraction containing the carboxypyranocyani-dins l i. Reprinted with permission from T. Fossen et al. [257].

Fig. 3.26. Typical elution HPLC profiles of MG residues extracted from (a) salmon spiked at 20 pg/kg LMG and MG each (b) residue-incurred salmon fillet (2.9 pg/kg). Analysis of the residue-incurred salmon was repeated using the LC-MS-MS system as shown in (c) the profile shows the monitoring of the m/z = 329.5 to m/z 313.3 fragmentation. The elution positions of MG, LMG and the internal standard brilliant green (BG) are indicated. Note BG is not detected in the m/z = 329.5 to m/z 313.3 trace (c) and its position is therefore depicted as an under broken arrow. Reprinted with permission from A. A. Bergwerft et al. [105],...

Metabolites formed during the decolourization of the azo dye Reactive red 22 by Pseudomonas luteola were separated and identified by HPLC-DAD and HPLC-MS. The chemical structures of Reactive red 22 (3-amino-4-methoxyphcnyl-/fhydroxyl-sulphonc sulphonic acid ester) and its decomposition products are shown in Fig. 3.92. RP-HPLC measurements were carried out in an ODS column using an isocratic elution of 50 per cent methanol, 0.4 per cent Na2HP04 and 49.6 per cent water. The flow rate was 0.5 ml/min, and intermediates were detected at 254 nm. The analytes of interest were collected and submitted to MS. RP-HPLC profiles of metabolites after various incubation periods are shown in Fig. 3.93. It was concluded from the chromatographic data that the decomposition process involves the breakdown of the azo bond resulting in two aromatic amines [154],... 

Collier et al. (10) demonstrated that HPLC was an effective technique for the separation of aromatic hydrocarbon metabolites in exposed marine organisms. Radioactive bioconversion products were studied in liver and gall bladder of coho salmon dosed with H-naphthalene. Quantitative identifications of glucuronide, sulphate, dihydrodiol, glycoside, and 1-naphthol derivatives were obtained. Three additional polar compounds of unknown structure were found. A typical HPLC profile is shown in Figure 2. 

Figure 2. HPLC profile of extracts of liver of 3H-naphthalene-exposed coho salmon. Data were obtained by single analysis of pooled tissue from four fish, each fed 74.6 fid 3H-naphthalene 16 hr earlier (lO).

Figure 3. HPLC profile of extracts of 3H-naphthalene-exposed herring larvae...

Figure 5. HPLC profiles of products from reactions catalyzed by secreted pectate lyase activities from Erwinia chrysanthemi after 66 min and 192 min. Reaction mixtures contained 4 units of enzyme activity per ml and 0.1% PGA in 0.05 M Tris-HCl, pH 8.5, 0.2 mM CaClg. Injections of 0.05 ml were made from reaction mixtures with a WISP automatic sample injector and eluted at 2.0 ml/min with a run time of 60 min. Compounds eluting with retention times of 5.36, 6.40, 7.76, and 9.56 min corresponded to unsaturated oligogalacturonate reference standards with DP values of 2, 3, 4, and 5, respectively.

Figure 3. HPLC profile of Aphantoxin most toxic peak (left corresponds to saxitoxin) and the major toxic component (second from left corresponds to neosaxitoxin). A total of 150 mouse units were loaded. A mouse unit is equal to the amount of toxin needed to kill a 20 g mouse in 15 min.

Figure 4. HPLC profile of Aphantoxin major peak (top-left 500 mouse units) and neosaxatoxin standard (2A) (bottom-left 200 mouse units).

Figure 5. HPLC profile of Anatoxin-a(s) toxic peak (far-left ...

FIGURE 19.5 HPLC profile of PITC derivatives of amino acids from a red seaweed (UV 254nm). (From Sanchez-Machado, D.I. et al., Chromatographia, 58, 159, 2003. With permission from Springer Science + Business Media.)... 

Figure 1 HPLC Profile of Synthetic Dendrotoxin-I Intermediates in the Deprotection and Folding Process, (a) Crude Product Obtained by the HF Deprotection Reaction, (b) Purified Peptide (6 Acm). (c) Completely Deprotected Peptide, (d) After Folding Reaction, (e) Final, Purified Product. Column YMC-Pac ODS A-302 (4.6 x 150 mm) Eluent 20-40% MeCN/0.1% TFA 25 min at 40 °C Flow Rate 1.0 mL min-1 Detection 220 nm. Individual Chromatographs from this Figure have Previously Appeared in Ref 01...

Figure 1 HPLC Profiles of Crude (a) and Purified (b) Fully Protected Segment 1. Column Vydac Diphenyl (250 x 4.6 mm) Eluant 80-90% Solution B in Solution A Linear Gradient for 25 min. HPLC Profiles of H-Asn103-Val124-OH Cys(Acm)110 (c) and (d) Obtained from Deprotection of Crude (a) and Purified (b) Fully Protected Segment 1, Respectively. Column Vydac C8(250 x 4.6 mm) Eluant 20 10% Solution B in Solution A Linear Gradient 25 min (Solution A 0.1% aq TFA and Solution B 90% MeCN/10% Solution A) Detection 215 nm...

Figure 1 HPLC Profile of Oxidative Folding of Elafin (a) After Folding Reaction in NH4OAc Buffer Containing 2M Gn-Cl, without Redox Reagents, (b) After Folding Reaction in NH4OAc Buffer Containing 2M Gn-Cl with Redox Reagents (100 10).,78 a...

Figure 2 HPLC Profile of Oxidative Folding of Reduced r-Conotoxin GIIIB Peak Labelled was Identified as the Cys10/Cys15 Disulfide Bonded Intermediate1861 ab...

Figure 3 HPLC Profile of the Effects of Reaction Temperature and NH4OAc Concentration on the Folding of Reduced co-Conotoxin MVIIC in the Presence of Redox Reagents Buffer NH4OAc pH 7.7 Peptide/GSH/ GSSG 1 100 10 Time 24hl la b...

Figure 4 HPLC Profile of the Progress of the Oxidative Folding of Reduced co-Conotoxin MVIIC and Stability of Isomer 21 in the Presence of Redox Reagents in 50 mM NH4OAc Buffer Containing 2M (NH4)2S04at pH 7.7 (a, c) and in 50 mM NH4OAc Buffer at pH 7.7 (b, d) at 5 °C Aliquots were Analyzed at the Time Shown in Each Paneli891a b...

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