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2000
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April
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In the Laboratory
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The Microscale Laboratory
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Complete Analysis of a Biologically Active Tetrapeptide: A Project Utilizing Thin-Layer Chromatography and Tandem Quadrupole Mass Spectrometry
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Joseph W. LeFevre
Chemistry Department, SUNY Oswego, Oswego, NY 13126
David W. Dodsworth
Bristol-Myers Squibb, Syracuse, NY 13221-4755
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April 2000
Vol. 77 No. 4
p. 503
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| Abstract |
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The biologically active tetrapeptide d-Ala-Gly-l-Phe-d-Leu ([des-Tyr1-d-Ala2-d-Leu5]enkephalin) was analyzed for its amino acid content and stereochemistry by normal and reversed-phase thin-layer chromatography (TLC), and its sequence was determined by tandem quadrupole mass spectrometry. The project involved sequential N-dansylation of a portion of the tetrapeptide, hydrolysis, isolation, and identification of the N-terminal amino acid as dansyl-alanine by comparison with standards using normal-phase TLC. A second portion of the tetrapeptide was hydrolyzed and the resulting four free amino acids were converted to their corresponding dansyl derivatives and purified by preparative normal-phase TLC. The three dansyl amino acids not identified previously were identified by TLC. The stereochemistry of each was determined by comparison with dansyl-dl-amino acid standards using reversed-phase TLC in the presence of ß-cyclodextrin, a chiral mobile phase additive. Finally, the correct amino acid sequence was determined by tandem quadrupole mass spectrometry. This project gives students valuable experience in microscale synthesis, both normal and reversed-phase TLC, stereochemical analysis, and mass spectrometry.
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| Supplement |
Detailed student procedures, questions, and Instructors' Notes are available. The Instructors' Notes include comments on this experiment, tables of TLC retention factors (Rf values), MS/MS fragmentation pattern data, answers to questions, equipment and reagent lists, detailed procedures for preparing reagents, and hazard alerts.
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| More Information |
Citation |
LeFevre, Joseph W. ; Dodsworth, David W. J. Chem. Educ. 2000 77 503.
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Keywords |
Laboratory Instruction; Organic Chemistry; Biochemistry; Proteins / Peptides; Amino Acids; Stereochemistry; Mass Spectrometry; Microscale
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History |
Created:
Last Updated: |
March 2, 2000
August 31, 2005
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| Home > JCE Print > Journal of Chemical Education > Issues >
2000
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April
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503
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