HPLC analysis of algal pigments: a comparison exercise among laboratories and recommendations for improved analytical performance
HPLC analysis of algal pigments: a comparison exercise among laboratories and recommendations for improved analytical performance
复制标题
藻类色素的 HPLC 分析：实验室之间的比较练习和改进分析性能的建议
DOI：
10.1016/0304-4203(95)00056-9
复制
发表时间：
1996
期刊：
影响因子：
3
通讯作者：
M. C. Kennicutt
中科院分区：
文献类型：
--
作者：
M. Latasa;R. Bidigare;M. Ondrusek;M. C. Kennicutt
在联合全球海洋通量研究（JGOFS）颜料分析师之间分配纯的单个和混合颜料标准品，以估计其分光光度和色谱系统的可变性。为了在比较过程中监测色素的完整性，将叶绿素和类胡萝卜素标准品存档并通过高效液相色谱法（HPLC）定期分析。在-20 °C的氮气下，在黑暗中储存的颜料标准品被发现至少稳定一年。从三个独立的相互校准练习的结果文件比HPLC分光光度分析更好的协议。对于分光光度比较，参与实验室分析的90%的颜料在平均“一致”值的± 6%范围内。相比之下，当比较色谱分析时，分别有65%和85%的实验室同意在± 10%和± 20%以内。用二极管阵列型分光光度计测得的叶绿素吸收比用单色器型分光光度计测得的低6-9%。这些低估可能是由于叶绿素荧光污染与二极管阵列分光光度计的光学配置。还确定了不能将单乙烯基叶绿素a与二乙烯基叶绿素a分离的HPLC方法可在原绿球藻属占优势的海洋沃茨中产生15-25%的总叶绿素a浓度高估。一个简单的二色性的方法，用于消除这种可变的误差源所造成的这些结构相关的颜料的共洗脱。强烈建议使用内标物和外标物进行定期校准检查，以提高分析性能。
Pure individual and mixed pigment standards were distributed among Joint Global Ocean Flux Study (JGOFS) pigment analysts to estimate the variability of their spectrophotometric and chromatographic systems. To monitor the integrity of the pigments during the comparison exercise, chlorophyll and carotenoid standards were archived and periodically analyzed by high-performance liquid chromatography (HPLC). Pigment standards stored in the dark under nitrogen at − 20 °C were found to be stable for periods of at least one year. Results from three separate intercalibration exercises document a better agreement for spectrophotometric analyses than for HPLC. For the spectrophotometric comparisons, 90% of the pigments analyzed by participant laboratories were within ± 6% of the mean “consensus” values. By contrast, 65 and 85% of the laboratories agreed to within ± 10 and ± 20%, respectively, when chromatographic analyses were compared. Chlorophyll absorption measurements obtained with a diode array-type spectrophotometer were 6–9% lower than those obtained with monochromator-type spectrophotometers. These underestimates probably result from chlorophyll fluorescence contamination associated with the optical configuration of the diode array spectrophotometer. It was also determined that HPLC methods which are not capable of separating monovinyl chlorophyll a from divinyl chlorophyll a can produce 15–25% overestimates of total chlorophyll a concentration in Prochlorococcus-dominated oceanic waters. A simple dichromatic approach is described for eliminating this variable source of error caused by co-elution of these structurally-related pigments. The use of internal standards and periodic calibration checks with external standards is highly recommended for improving analytical performance.