Küpper H, Spiller M, Küpper F C
Mathematisch-Naturwissenschaftliche Sektion, Universität Konstanz, Konstanz, D-78457, Germany.
Anal Biochem. 2000 Nov 15;286(2):247-56. doi: 10.1006/abio.2000.4794.
Accurate quantification of pigments in mixtures is essential in all cases in which separation of pigments by chromatography is impracticable for one reason or another. An example is the analysis of in vivo formation of heavy metal-substituted chlorophylls in heavy metal-stressed plants. We describe here a novel, accurate UV/VIS spectrophotometric method for the quantification of individual chlorophyll derivatives in complex mixtures, which has the potential for universal applicability for mixtures difficult to separate. The method is based on the description of each pigment spectrum by a series of Gaussian peaks. A sample spectrum is then fitted by a linear combination of these "Gauss-peak spectra" including an automatic correction of wavelength inaccuracy and baseline instability of the spectrometer as well as a correction of the widening of absorbance peaks in more concentrated pigment solutions. The automatic correction of peak shifts can also partially correct shifts caused by processes like allomerization. In this paper, we present the Gauss-peak spectra for Mg-chlorophyll a, b, c, pheophytin a, b, c, Cu-chlorophyll a, b, c, and Zn-chlorophyll a in acetone; Mg-chlorophyll a, b, pheophytin a, b, Cu-chlorophyll a, b, allomerized Cu-chlorophyll a, b, and Zn-chlorophyll a, b in cyclohexane; Mg-chlorophyll a, b, pheophytin a, b, and Cu-chlorophyll a, b in diethyl ether.
在因某种原因无法通过色谱法分离色素的所有情况下，准确定量混合物中的色素至关重要。一个例子是分析重金属胁迫植物中重金属取代叶绿素的体内形成情况。我们在此描述一种新颖、准确的紫外/可见分光光度法，用于定量复杂混合物中的单个叶绿素衍生物，该方法有可能普遍适用于难以分离的混合物。该方法基于用一系列高斯峰描述每种色素的光谱。然后，通过这些“高斯峰光谱”的线性组合拟合样品光谱，包括自动校正光谱仪的波长不准确和基线不稳定，以及校正更浓缩色素溶液中吸光度峰的展宽。峰位移的自动校正也可以部分校正由异构化等过程引起的位移。在本文中，我们给出了丙酮中镁叶绿素a、b、c、脱镁叶绿素a、b、c、铜叶绿素a、b、c和锌叶绿素a的高斯峰光谱；环己烷中镁叶绿素a、b、脱镁叶绿素a、b、铜叶绿素a、b、异构化铜叶绿素a、b和锌叶绿素a、b的高斯峰光谱；乙醚中镁叶绿素a、b、脱镁叶绿素a、b和铜叶绿素a、b的高斯峰光谱。
