ABSTRACT
Initial attempts to monitor ascorbic acid and sulfite, in a beer matrix, by combining ion-exclusion chromatography with a pulsed amperometric detector using a single applied voltage to the platinum working electrode, were unsuccessful. Alternatively, good chromatograms for the separation of the two antioxidants were achieved utilizing a standard, amperometric cell. However, remarkably superior results were observed when this standard cell was operated in a pulsed mode and cleaning cycles were continually applied throughout the analysis. The working electrode stability and precision have been examined. Preliminary spike recovery data indicate acceptable accuracy for the method. Comparisons of this method to standard reference methods are currently ongoing.
Subject(s)
Ascorbic Acid/analysis , Chromatography, Gel/methods , Sulfites/analysis , Beer/analysis , Chromatography, Gel/instrumentation , ElectrodesABSTRACT
Six fractions (I-VI) of pyridoxal 5'-phosphate (PLP) hemoglobin (Hb), prepared by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205] have been isolated and purified by anion-exchange high-performance liquid chromatography (HPLC). Total phosphate analyses indicate that I is HbA, II and III are double-labelled, IV and V are tetra-labelled and VI contains 6 mol of phosphorus per mol of hemoglobin. The purified components have been resolved into their alpha and beta chains by preparative reversed-phase HPLC using a macroporous C4 support. Phosphate analyses indicate that the beta chains of II, III and IV each contain one phosphate per chain while the beta chains of V and VI each contain two phosphates. The alpha chains of IV and VI were found to be monophosphate-labelled. Reversed phase HPLC analysis of the tryptic peptides of the beta chains indicates that the label is bound exclusively to the 1-valine residue in II, III and IV while both the 1-valine and the 82-lysine are labelled in V and VI. Similarly, modification of the 1-valine residue of the alpha chains of IV and VI was detected. Components II and III have the same molecular formula. Evidence is presented which shows that they are interconvertible and that they correspond to the PLP2-Hb species and component V is Benesch's PLP4-Hb [J. Biol. Chem., 257 (1983) 1320 and references cited therein]. Component IV is III with one additional PLP per alpha chain and similarly VI is V with monolabelled alpha chains.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Blood Substitutes/analysis , Hemoglobins/analysis , Pyridoxal Phosphate/blood , Chromatography, High Pressure Liquid , Globins/analysis , Humans , Hydrolysis , Oxygen/analysis , Peptides/analysis , Phosphates/analysis , TrypsinSubject(s)
Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/analysis , Blood Substitutes/analysis , Hemoglobins/analysis , Oxygen/analysis , Chemical Phenomena , Chemistry, Physical , Chromatography, High Pressure Liquid , Humans , Indicators and Reagents , Phosphates/analysis , Protein Binding , Spectrophotometry, UltravioletABSTRACT
Pyridoxal 5'-phosphate hemoglobin (PLP-Hb), prepared from hemoglobin and a four-fold excess of pyridoxal 5'-phosphate by the method of De Venuto and Zegna [J. Surg. Res., 34 (1983) 205], has been chromatographically resolved into six components via a quaternary ammonium monobead support. On an analytical scale, the separations have been found to be rapid (ca. 50 min) and highly reproducible. The results also indicate that the preparation of PLP-Hb yields a reproducible product ratio. The potential of the analytical method for the routine quality control of blood substitutes derived from PLP-Hb is discussed. All five of the PLP derivatives (components II-VI), isolated and purified via a combination of conventional and preparative monobead anion-exchange chromatography, gave single peaks when analyzed by high-performance liquid chromatography. Total phosphate analyses indicated that components II and III each contain two PLPs per Hb, IV and V four and VI six.