HPLC Analysis of Tetrapyrrole Extracts

Tetrapyrrole extracts were analyzed using a JASCO HPLC 2000 series system (Jasco, Gross-Umstadt, Germany) as previously described.7 (link) The chromatographic separation of samples (20 μL injection volume) containing SAM and 5′-deoxyadenosine (DOA) was performed on a HYPERCARB column (Thermo Scientific, Dreieich, Germany) with 5 μm particle size and 100 mm × 2.1 mm column dimensions at 25 °C. The substances were eluted at a flow rate of 0.2 mL min^–1 using a gradient system. Solvent A consisted of 0.1% trifluoroacetic acid (for UV-spectroscopy, Fluka) and solvent B of acetonitrile (HPLC grade, Sigma-Aldrich) containing 0.08% trifluoroacetic acid (HPLC grade, Sigma-Aldrich). At the time of sample injection the mobile phase consisted of 100% solvent A. Within 25 min the content of solvent B was increased to 100% with concomitant decrease of solvent A. Then, the content of solvent B was held for 5 min before returning to the initial conditions. SAM and DOA were detected by photometric diode array analysis at 200–650 nm.

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Kühner M., Schweyen P., Hoffmann M., Ramos J.V., Reijerse E.J., Lubitz W., Bröring M, & Layer G. (2016). The auxiliary [4Fe–4S] cluster of the Radical SAM heme synthase from Methanosarcina barkeri is involved in electron transfer †Electronic supplementary information (ESI) available: The cyclic voltammetry experiments, the binding assays with the substrate analogs, the determination of Kd values, the SAM cleavage assays with the substrate analogs, the decarboxylase activity assays with the substrate analogs and the synthesis and characterization of Zn-COPRO III. See DOI: 10.1039/c6sc01140c. Chemical Science, 7(7), 4633-4643.

Publication 2016

HYPERCARB column HPLC grade

5 deoxyadenosine Acetonitrile Chromatographic Deoxyadenosine Held Hplc Photometric Solvent Spectroscopy Tetrapyrrole Trifluoroacetic acid

Corresponding Organization : Technische Universität Braunschweig

Other organizations : Leipzig University, Max Planck Institute for Chemical Energy Conversion, Ruhr West University of Applied Sciences

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Variable analysis

independent variables

Gradient system of mobile phase solvents (Solvent A: 0.1% trifluoroacetic acid, Solvent B: acetonitrile containing 0.08% trifluoroacetic acid)

dependent variables

Separation and detection of SAM and 5'-deoxyadenosine (DOA)

control variables

Injection volume (20 μL)
Column type (HYPERCARB column, Thermo Scientific, 5 μm particle size, 100 mm × 2.1 mm)
Column temperature (25 °C)
Flow rate (0.2 mL min-1)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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