Microphytobenthos composition in Heron Reef, Australia, by High Performance Liquid Chromatography (HPLC) (July 2012)

We quantified pigment biomarkers by high performance liquid chromatography (HPLC) to obtain a broad taxonomic classification of microphytobenthos (MPB) (i.e. identification of dominant taxa). Three replicate sediment cores were collected at 0, 50 and 100 m along transects 5-9 in Heron Reef lagoon (n=15) (Fig. 1). Transects 1-4 could not be processed because the means to have the samples analysed by HPLC were not available at the time of field data collection. Cores were stored frozen and scrapes taken from the top of each one and placed in cryovials immersed in dry ice. Samples were sent to the laboratory (CSIRO Marine and Atmospheric Research, Hobart, Australia) where pigments were extracted with 100% acetone during fifteen hours at 4°C after vortex mixing (30 seconds) and sonication (15 minutes). Samples were then centrifuged and filtered prior to the analysis of pigment composition with a Waters - Alliance HPLC system equipped with a photo-diode array detector. Pigments were separated using a Zorbax Eclipse XDB-C8 stainless steel 150 mm x 4.6 mm ID column with 3.5 µm particle size (Agilent Technologies) and a binary gradient system with an elevated column temperature following a modified version of the Van Heukelem and Thomas (2001) method. The separated pigments were detected at 436 nm and identified against standard spectra using Waters Empower software. Standards for HPLC system calibration were obtained from Sigma (USA) and DHI (Denmark).本研究通过高效液相色谱（High Performance Liquid Chromatography，HPLC）对色素生物标志物进行定量分析，以实现底栖微藻（microphytobenthos，MPB）的宽泛分类学归类，即鉴定优势类群。研究于赫伦礁潟湖的5-9号样带沿线，分别在0 m、50 m和100 m处采集3份平行沉积物岩芯，总计15份样本（n=15），详见图1。由于野外数据采集阶段不具备HPLC分析样品的条件，1-4号样带的样品未能进行后续处理。岩芯样品经冷冻保存后，从其顶部刮取表层沉积物，置于干冰冷却的冻存管中。样品被送至澳大利亚霍巴特的联邦科学与工业研究组织海洋与大气研究部（CSIRO Marine and Atmospheric Research, Hobart, Australia）实验室，在4℃条件下经涡旋混匀（30秒）、超声处理（15分钟）后，用100%丙酮进行了15小时的色素萃取。随后对样品进行离心与过滤处理，再采用配备光电二极管阵列检测器的Waters Alliance高效液相色谱系统进行色素组成分析。色素分离采用安捷伦科技（Agilent Technologies）生产的Zorbax Eclipse XDB-C8不锈钢色谱柱（柱长150 mm、内径4.6 mm、粒径3.5 μm），并通过二元梯度洗脱系统结合升高柱温的方式，基于经改良的Van Heukelem与Thomas（2001）方法完成分离。分离后的色素在436 nm波长处被检测，并通过沃特世Empower软件对照标准光谱完成定性鉴定。高效液相色谱系统校准所用的标准品购自美国Sigma公司与丹麦DHI公司。