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RECEIVED for review May 21,1990. Accepted October 8,1990. This work was supported by the Italian Ministry of the University and Scientific, Technological Research (MURST) and the Italian Research Council (CNR).
Isolation and Purification of Chlorophylls a and b for the Determination of Stable Carbon and Nitrogen Isotope Compositions Robert R. Bidigare,' Mahlon C. Kennicutt II,* and Wendy L. Keeney-Kennicutt Geochemical & Environmental Research Group, 833 G r a h a m Road, T e x a s A & M University, College S t a t i o n , T e x a s 77845 Stephen A. MackoZ D e p a r t m e n t of E a r t h Sciences, Memorial University, St. J o h n s , Newfoundland, Canada A l B 3 x 9
A method is described to Isolate and purify chlorophyll pigments from plant tissues for the determination of stable carbon and nltrogen isotope composltlons. Chlorophylls are initially isolated by solvent preclpltation, followed by preparative C18 reverse-phase high-performance liquid chromatography (HPLC) for final purlflcation. The purity of the isolated pigments (chlorophyll a and chlorophyll b ) was assessed by analytical HPLC, absorption spectroscopy, elemental analysis, and 'H NMR spectroscopy. A step-by-step evaluation of the separation indicates that molecular and isotopic integrity of the pigments are preserved during purification. Measurement of stable Isotope composition across the HPLC peak illustrated the necessity to collect the entire peak in order to maintain Isotopic integrity. Multiple stable Isotope compositions (613C, 615N) of Individual molecular markers (Le., pigments) are a powerful new tool to identify the sources of complex organic matter mixtures in the bio- and geospheres.
INTRODUCTION Organic matter in the bio- and geospheres is a complex mixture of living organisms and detrital remains. This complexity results from the multitude of source organisms, variable biosynthetic strategies available to organisms, and transfor-
* T o whom correspondence should be addressed. Present address: Department of Oceanography, 1000 Pope Rd, Universitv of Hawaii, Honolulu, HI 96822. *Present address: Department of Environmental Sciences, University of Virginia, Charlottesville, VA 22903.
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0003-2700/91/0363-0130$02.50/0
mations that occur during diagenesis and catagenesis. Chemical and stable isotopic characterization of organic matter can provide insight into its origin. However, the existing analytical techniques lack the specificity required to adequately characterize most natural mixtures. The stable isotope composition of bulk organic matter and molecular marker distributions are often difficult to interpret due to indistinct boundaries between sources and an inadequate understanding of source compositions (1-4). To resolve this complexity, the determination of the stable isotope composition of individual compounds has been investigated including, amino acids ( 5 ) ,photosynthetic pigments (6, 7 ) , monosaccharides (8), aliphatic and aromatic hydrocarbons (9-12), and geoporphyrins (10, 13-15). Chlorophyll-related compounds are ideal candidates for use in molecular stable isotope studies because (a) chlorophylls are the basis of the photosynthetic process that produces biomass, (b) tetrapyrroles contain two elements amenable to stable isotope analysis, (c) early diagenetic reactions are well defined, and (d) porphyrins and chlorins are abundant in nature. Tetrapyrrole-based diagenetic alteration products, geoporphyrins, are ubiquitous and stable in the geosphere as atested by their presence in coal and petroleum. The molecular and stable isotope compositions of individual geoporphyrins have been demonstrated to be useful in delineating precursor-product relationships for this class of compounds in geological samples (10, 14, 15). In contrast to the expanding data base on geoporphyrins, little or no information is available on the stable isotopic composition of pigments of extant plant species. This basic information is needed to model the formation and cycling of 0 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 2, JANUARY 15, 1991
Residue
Isotopes I C:N
1
1 . Dioxane ppl,
lH.0H:DioYn.:P2
0
= 10 :1:1 )
2 . Tilter
* Filtrate
Residue
+
metone 1 2 d )
Discard
2.
+
1. Acetone
2.
-
+
Meon Acetone
Figure 1. Anatytlcal protocol used for the purification of plant pigments.
organic matter in the biosphere as well as to interpret geoporphyrin data. Toward this goal, we present a method for the isolation of milligram quantities of chlorophylls a and b for the determination of stable carbon (b13C) and nitrogen (b15N)isotope ratios. Carbon and nitrogen are two abundant and essential building blocks for organic matter synthesis, and thus, their distributions and isotopic compositions are uniquely associated with organic matter production. Data are presented for bulk organic matter, lipid extracts, and individual pigments derived from representative plants to illustrate the efficacy of this approach.
EXPERIMENTAL SECTION Pigment Standards and Plant Materials. Commercially available pigment standards (chlorophylla a and b; Sigma Chemical Company) were used to evaluate stable isotope fractionation effects during purification. Naturally occurring photosynthetic pigments were isolated from six different species of terrestrial plants for stable isotope analysis including ragweed (Ambrosia artomisifolia), parsley (Petroselinum crispum), sorghum (Sorghum halepense), Bermuda grass (Cynodon dactylon), Johnson grass (Sorghum bicolor), and brussel sprouts (Brassica gemmifera). Pigment Isolation. Pigments were purified according to the analytical protocol outlined in Figure 1. Plant tissues were freeze-dried ("bulk organic matter"), homogenized (15 g) in 75 mL of MeOH using a Tekmar Tissumizer, and filtered through a 47-mm polyester membrane filter (0.4 pm, Nuclepore Corporation). These procedures were carried out in the dark to minimize photolytic degradation. The filter was rinsed three times with 5 mL of methanol. Pigments were precipitated from the filtrate ("lipid extract") by the sequential addition of dioxane and high-performance liquid chromatography (HPLC) grade water (Me0H:dioxane:water (v/v): 1O:l:l) following the methods of Watanabe et al. (26). The suspension was placed in the freezer (-20 "C) for 1 h and then filtered through a 47-mm polyester membrane filter (0.4 pm), rinsing the filter three times with cold Me0H:dioxane:water (1Ol:l). The pigment-containing residue was dissolved in 2 mL of acetone, and 0.5-2.0 mL were injected onto a Spectra-Physics SP8100 liquid chromatograph. Base-line separations of chlorophylls a and b are achieved by using a Dynamax preparative-scale C18column (21.4 X 250 mm; 5-pm Mi-
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crosorb particles; Rainin Instrument Co.). Pigments were eluted with MeOH at a flow rate of 10 mL min-' are detected with a Waters Model 440 absorbance detector (254 nm) with a total analysis time of 45 min. Under these conditions, the retention times of chlorophylls a and b are 22 and 37 min, respectively. The high-performanceliquid chromatography step can be shortened to 15 min without a loss of resolution if a "high-flow" head attachment (30 mL min-') is used. The entire peak, corresponding to each pigment of interest, was collected and evaporated to dryness with a purified stream of nitrogen. The pigment residues were redissolved in acetone and subsampled for the analyses described below. Following each run, the remaining nonpolar compounds are eluted from the C18 column with acetone. The entire isolation procedure can be completed in
