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The reduction of carbon in analytical HPLC fractions to graphite for AMS
Author(s) -
Kim SeungHyun,
de Moura Charlene C Ho Fabiana F,
Chuang Jennifer,
Haack Kurt W,
Ognibene Ted J,
Buchholz Bruce A,
Vogel John S,
Clifford Andrew J
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a135
Subject(s) - high performance liquid chromatography , chromatography , chemistry , accelerator mass spectrometry , graphite , radiochemistry , mass spectrometry , organic chemistry
Accelerator mass spectrometry (AMS) is an isotope ratio measurement method for long‐lived radioisotopes at attomole levels. It is a powerful technique to conduct true tracer experiment of naturally occurring micronutrients and their metabolites once isolated by analytical HPLC. Typically, a healthy subject is administered a small tracer dose (5–100 nCi) of 14 C‐labeled micronutrients (e.g. β‐carotene, vitamin E, or lutein) and serial blood is collected by time since dose. The metabolites of interest are isolated on a single HPLC run. HPLC fractions are collected at 20 s intervals into quartz tubes and dried under reduced pressure. To avoid contamination, inner most tube containing the sample should not be touched. A 50μℓ aliquot of sub‐modern tributyrin in methanol is added to contain 1mg carbon and dried. Copper oxide (CuO) is prepared by heating at 500°C for 2 h and cooled. About 40 mg CuO is added to the HPLC fraction and heated at 900°C for 2h to convert carbon to CO 2 . The CO 2 is transferred to a septa‐sealed vial containing zinc catalyst and iron. This is heated at 525°C for 6h to convert the CO 2 to graphite that coats the iron particles to graphite suitable for AMS. We demonstrate a detailed and simplified sample preparation protocol on graphitizing of low labeled metabolites of HPLC fractions for AMS in nutritional studies to novice scientists. Work performed under the auspices of the US DOE by the University of California, LLNL under Contract No. W‐7405‐Eng‐48, NIH NCRR P41 RR 13461, and NIDDK DK48307.