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Comparison of buffers and detection systems for high-pressure liquid chromatography of peptide mixtures.

ABSTRACT: The chromatographic properties of peptides ranging in length from 2 to 65 residues have been compared on reverse-phase packings in three buffer systems at low pH. Of the buffers examined, two are widely used in connection with u.v. detection [(i) phosphate/acetonitrile or (ii) phosphate/propan-2-ol] and the third for fluorescence detection [(iii) pyridine/formate-pyridine/acetate/propan-1-ol]. The addition of a chaotropic salt, NaClO4, to the phosphate buffers, as first described by Meek (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1632-1636, is shown to significantly improve the chromatographic behaviour of more hydrophobic peptides. The two most commonly used detection systems, u.v. and fluorescence, are compared in terms of ease of use and sensitivity.

SUBMITTER: Wilson KJ

PROVIDER: S-EPMC1163332 | biostudies-other | 1981 Oct

REPOSITORIES: biostudies-other

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Project description:Hemoglobin High-performance liquid chromatography (Hb HPLC) is a standard first-line technique for diagnosis of thalassemia and hemoglobinopathies. We compared two HPLC systems for detection and quantification of normal and abnormal Hb fractions. EDTA samples from 100 normal healthy subjects and 107 subjects affected with hemoglobinopathies or carriers were analysed using HPLC systems Tosoh HLC-723G11 and Bio-Rad Variant-II. Retention time (RT) and area of peaks for HbA2, HbF and other structural variants were compared. In discrepant cases samples were run on Sebia Capillary zone electrophoresis (CZE) for confirmation of results (39 out of 107 cases with HbE, HbD Iran, Hb Lepore and HbQ). Measurement of HbA2 and HbF in normal samples and HbF in those with variant Hbs showed good correlation by both analyzers (R 2 = 0.83, 0.9 and 0.99 respectively). HbE co-elutes with HbA2 in Bio-Rad. Correlation done using the apparent HbA2 concentration from Bio-Rad with (HbE + HbA2) from Tosoh G11 showed good correlation (R 2 = 0.97). Correlation of HbS (Eluting at S-window at RT 3.11 min in Tosoh G11 and 4.33 min in Bio-Rad) as well as HbD Punjab (Eluting at D-window at RT 2.82 min in Tosoh G11 and 4.06 min in Bio-Rad) by both instruments was good. HbD Iran (Eluting at E-window at RT 2.69 min in Tosoh G11 and with HbA2 at 3.53 min in Bio-Rad); HbQ (Eluting at C-window at RT 3.78 min in Tosoh G11 and unknown window at 4.7 min in Bio-Rad), HbH (Eluting at P00 window at RT 0.13 min in Tosoh G11 and giving pre-integration peak in Bio-Rad), Hb Lepore (Eluting at P08 window at RT 2.67 min in Tosoh G11 and with HbA2 at 3.46 min in Bio-Rad) gave comparable results. Correlation with findings of CZE was done in few cases when needed. Two automated HPLC instruments demonstrated similar usefulness for screening patients for hemoglobinopathies. However, complex elution patterns as well as co-elution of variants like HbA2, HbE, Hb Lepore, HbD Iran (in Bio-Rad); HbD Iran and HbE (Tosoh G11) pose difficulty in interpretation. A complementary second method like CZE may be required.

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Comparison of buffers and detection systems for high-pressure liquid chromatography of peptide mixtures.

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