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An example of how to use HPLC:
A range of pigments were identified by reverse phase HPLC. For the full methodology see Wiltshire & Schroeder (1994).
Extraction involved adding either 1 ml of 100% acetone or 90% DMF to the sediment sample (0.04-0.1 g) and pigments were extracted for a minimum of 24 hours at -70 0C or 40C in the dark.
Extractant and sediment were separated by filtration through a 0.2 m m pore syringe filter (WhatmanTM). Chl a standards (derived from Anacystis nidulans, SigmaTM) were analysed with every sample batch to obtain a standard curve.
The HPLC system consisted of a quaternary high pressure pump (Perkin Elmer 410), an autosampler (Waters WISP 417) and a diode-array detector (Waters 910).
The column was a Nucleosil C18 (Capital HPLC Ltd) kept in a column oven at 25 ¡C. Extractions were injected onto a binary gradient.
The flow rate was 1 ml min-1 and the two solvents used were: eluant A:- 80% methanol, 10% water, 10% buffer (1.5 g acetate, 7.7 g ammonium acetate in 100 ml of distilled water), eluant B:- 90% methanol, 10% acetone. Chl a eluted at 25 minutes, and analysis of peak area was used to quantify the pigment (Wiltshire & Schroeder, 1994).
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