LONG-LIVED AFTERGLOW OF PHOTOSYNTHETIC PIGMENTS IN SOLUTION: PHOTOCHEMILUMINESCENCE

Abstract —Our recent research on photochemiluminescence (PCL) of pigments in solutions is reviewed. PCL was observed in the course of photooxidation by oxygen of chlorophyll a , bacteriochlorophyll, protochlorophyll, their analogs, synthetic dyes and aromatic hydrocarbons. The PCL of chlorophyll was studied in detail. It depends on oxygen concentration, intensity of exciting light, pH, nature of pigments, solvents etc. The thermochemiluminescence was observed after illumination of liquid and solid pigment solutions at low temperature (down to - 170C). The excitation spectra of PCL coincide with the pigment absorption spectra. The PCL emission spectra in most cases differ from those of pigment fluorescence. Electron acceptors, electron donors, radical inhibitors and β-carotene quench PCL. The quenching efficiency of electron acceptors is similar to their action on the chlorophyll triplet state. The quenching effect of radical inhibitors and β-carotene correlates with their activity in reaction with singlet oxygen. The effect of quenchers on the chlorophyll fluorescence, photobleaching and pigment sensitized oxygenation was studied. Analysis of experimental data allowed the assumption that chemiluminescence accompanies the decomposition of labile pigment peroxides. The accumulation of peroxides is probably due to the reaction in the complex of pigment and singlet oxygen, formed as a result of energy transfer from photoexcited (triplet) pigment molecules to oxygen. The terminal chemiluminescence emission proceeds from the singlet excited states of molecules of pigments and products of their oxidation.     

OPTICAL SPECTROSCOPY OF BILAYER MEMBRANES

Abstract— The absorption and fluorescence spectroscopy of natural and model bilayer lipid membranes is reviewed. Basic structural features of biological membranes and the relative advantages of black lipid membranes (BLM) and of liposomes are discussed. Theoretical considerations show that the wavelengths of absorption maxima are affected by the refractive index and dielectric constant of the medium surrounding the chromophore. Techniques of obtaining photoelectric action spectra, direct absorption spectra, and reflection spectra of BLM are described. Polarized spectra can give information about the orientation of membrane constituents and show, for example, that the porphyrin ring of chlorophyll in BLM is tilted at 45 ± 5° to the membrane surface. Absorption maxima of chlorophyll in BLM are compared with solution spectra of various chlorophyll adducts and aggregates. It is concluded that chlorophyll in BLM exists largely as solvated monomer and dimer (or oligomer), depending on concentration, and is not coordinated with water. From the theory of fluorescence spectroscopy it follows that aggregation and the polarity of the environment affect the fluorescence yield and lifetime of a membrane component, and also the wavelength of its emission maximum. The microviscosity of the membrane matrix affects the anisotropy of fluorescence. Techniques of steady-state fluorescence spectroscopy and of fluorescence lifetime measurements are reviewed. Examples of the use of fluorescent probes in membrane studies are given. Certain probes such as anilinonaphthalene sulfonate (ANS) preferentially bind to membrane proteins. The location of a probe in a particular membrane region can be pinpointed from its fluorescence yield and emission maximum. The orientation of the hydrocarbon chains of membrane lipids has been found, from fluorescence polarization of certain probes, to be normal to the membrane surface as postulated a priori on the basis of the lipid bilayer model. Anisotropy of fluorescence shows that elongated probe molecules rotate rapidly about their long axes when surrounded by phospholipids but become immobilized when bound to proteins. Changes in intensity and anisotropy of fluorescence as function of temperature have demonstrated the existence of phase transitions and phase equilibria of membrane lipids. Excimer fluorescence has been used as a measure of the available lipid core volume of membranes. Mechanisms of energy transfer between membrane components are reviewed. The theoretical dependence of energy transfer on distance and orientation for several rigid and fluid membrane models is discussed in terms of the structural information it can provide. Fluorescence sensitization resulting from energy transfer within and across bilayer membranes has been demonstrated in various systems. Quantitative measurement of energy transfer efficiency in BLM has shown that such transfer is about five times more efficient than in solutions at comparable donor-acceptor distances. Lipid membranes can be viewed as structures which maintain their components at high concentrations, in a reactive state, and at favourable orientations.     

EXCITATION TRANSFER BY CHLOROPHYLL a IN MONOLAYERS AND THE INTERACTION WITH CHLOROPLAST GLYCOLIPIDS*

In mixed monolayers with purified chloroplast glycolipids and other colorless lipids, chlorophyll a fluorescence exhibits a decrease in quantum efficiency with increasing chlorophyll concentration. The fluorescence, which is strongly polarized in dilute films, becomes progressively depolarized as the area fraction of chlorophyll increases, and it is completely depolarized in a pure chlorophyll a monolayer. The observed behavior is consistent with an inductive resonance mechanism of energy transfer among the chlorophyll molecules with a critical transfer distance of 20–90 Å, depending on the model chosen for the energy transfer mechanism. The purified glycolipids–mono-and digalactosyl diglycerides and sulfoquinovodiglyceride–separately form stable, compressible monolayers of the liquid-expanded type on an aqueous subphase and in an atompshere of nitrogen. At maximum compression the three glycolipids occupy areas of 55, 80 and 47 A²-molecule^-1, respectively, in the monolayer. Mixed monolayers of chlorophyll a with, separately, the monogalactolipid and the sulfolipid behave upon compression as two-dimensional solutions. The fluorescence polarization at high chlorophyll concentrations in mixed monolayers indicates that several of the lipid diluents facilitate local ordering of the pigment molecules.     

SPECTROSCOPIC STUDY OF CHLOROPHYLL a IN ORGANIC SOLVENTS AND POLYMERIZED ANHYDROUS POLYVINYL MATRIX

In this paper we present a spectroscopic study of chlorophyll a in solutions and in anhydrous polyvinyl alcohol films. Absorption, excitation and emission spectra, combined with fluorescence lifetime and time-resolved anisotropy measurements show that chlorophyll a in anhydrous polyvinyl alcohol films exists in a purely monomeric state. Furthermore, it appears that the monomeric chlorophyll a exhibits an efficient excitation energy transfer in this polyvinyl alcohol matrix. These results are rationalized in terms of a model in which the chlorophyll a molecules are located within pockets, formed by the polymer chains. It is concluded that the chlorophyll a-anhydrous polyvinyl alcohol film is a suitable system for studying energy transfer processes, especially because the factors governing energy transfer such as mutual orientation and separation of the molecules can easily be controlled.     

CHLOROPHYLL a AND CAROTENOID AGGREGATES AND ENERGY MIGRATION IN MONOLAYERS AND THIN FILMS

Abstract— The spectra of absorption, fluorescence and excitation of monolayers and thin films containing chlorophyll a together with a carotenoid (cis-β-carotene, trans-β-carotene, fucoxanthin, or zeaxanthin), were measured at — 196°C. The concentration ratios used, (Chl)/(Car), were 6:1, 4:1, 3:1, 2:1, 1:1 and 1:3, and the area densities, 3·70, 2·55, 1·76, 0·71, 0·37 and 0·17 nm²/pigment molecule. In dilute monolayers, (3·70 nm²/molecule), with a constant concentration ratio (Chl)/(Car) = 3:1, evidence of three β-carotene forms, with absorption bands at 460, 500 and 520 nm (C₄₆₀, C₅₀₀ and C₅₂₀), and of a chlorophyll a form with an absorption band at 669–672 (Chl_669–672) was found. On increasing the density to 0·2–0·3 nm²/molecule, a conversion of C₄₆₀ and C₅₂₀ into C₅₀₀, was observed, and several more additional (probably more strongly aggregated) chlorophyll a forms appeared, with absorption bands at 672–733 nm. With excess carotene [(Chi)/(Car) = 1:3] the forms C₄₆₀, C₅₀₀, C₅₂₀ and Chl_669–672 were present even in the most dense films (0·2–0·3 nm²/molecule). The same was found with other carotenoids: if one of the pigments was in excess, aggregated forms of the other tended to disappear. In the transfer of energy from carotenoids to chlorophyll a, C₅₀₀ was found to be the main donor. In layers with a concentration ratio (Chl)/(Car) = 3:1, the efficiency of transfer was less than 10 per cent at the lowest density used (3·70 nm²/molecule); it increased to 50 per cent, as the density was increased to 0·20 nm²/molecule. When the relative concentration of the carotenoid was increased to (Chl)/(Car) = 1:1, the efficiency of energy transfer dropped to 25 per cent even at 0·20 nm²/molecule. It seems that the efficiency of energy transfer between carotene molecules (prior to its transfer to chlorophyll a) is low, and effective transfer occurs only between β-carotene and immediately adjacent chlorophyll a molecules.     

A FLASH PHOTOLYSIS STUDY OF INTERMEDIATES IN PHOTOCHEMICAL REACTIONS OF CHLOROPHYLL

Abstract— The photochemical reactions of chlorophyll intermediates in vitro have been studied by the flash photolysis method. The flash excitation of pigment solutions has been shown to involve the population of a chlorophyll triplet state where the oxidation-reduction processes occur. The mechanism and kinetics of pigment triplet decay have been investigated from 20°to — 50°C and the ability of chlorophyll molecules to carry out triplet-triplet energy transfer has been established. The latter phenomenon has been used to show up the role of chlorophyll triplets in the reversible photooxidation reaction with P -quinone. There have been studied initial products of pigment photoreduction with ascorbic acid and phenylhydrazine. Experimental data of the mechanism of the initial oxidation and reduction in chlorophyll photosensitized reactions have been analysed. There have been also obtained the differential spectra of chlorophyll triplets and radicals. A calculation has been made of rate constants for a few elementary reactions.     

ENERGY TRANSFER AND ENERGY LOSSES IN BILAYER MEMBRANE VESICLES (LIPOSOMES)

Abstract. The efficiency of singlet-singlet energy transfer was studied in bilayer lipid membrane vesicles (liposomes) for the following donor-acceptor systems: (1) p -terphenyl (TP) and diphenyloctatetraene (DPO); (2) DPO and chlorophyll a (Chl a ); and (3) β-carotene and Chl a. The energy transfer efficiency φ_DA was measured by sensitized fluorescence of the acceptor. Fractional quenching of the donor φ_Q was found from the donor fluorescence in absence and presence of the acceptor. For TP-DPO and for DPO-Chl a , the transfer efficiency increased with increasing acceptor concentration but was essentially independent of the donor concentration. No energy transfer from β-carotene to Chl a could be detected. In liposomes, φ_DA differed only slightly from φ_Q at all donor and acceptor concentrations, thus demonstrating the absence of any appreciable energy losses. For solutions of the same donor-acceptor pairs in cyclohexane φ_Q was considerably larger than φ_DA. The difference represents energy lost, principally by internal conversion, due to collisional quenching. The principal function of the lipid membrane appears to be the suppression of such losses. In addition, the rate of energy transfer in lipid membranes is about double that in solutions (at the same intermolecular distance) due to more favorable orientation.     

MIGRATION OF EXCITATION ENERGY FROM THIONINE TO METHYLENE BLUE IN MICELLES

Abstract— The main absorption bands of thionine (Th⁺) and methylene blue (MB⁺) in aqueous solution lie at 598 nm and 664 nm, respectively. This position permits excitation energy transfer from Th⁺ to MB⁺, but not vice versa. We describe here studies of such transfer between these molecules adsorbed on micelles of sodium lauryl sulfate (SLS), imitating, at least to some extent, the state of pigments in chloroplasts.
The SLS concentration was varied from 3.0 to 11 × 10^-3 M. In the presence of dye, aggregation to micelles, each containing 70–100 detergent molecules, begins at about 3.0 × 10^-3 M SLS. Practically all dye ions are adsorbed on these micelles as soon as their formation begins.
Energy transfer from adsorbed Th⁺ ions to adsorbed MB⁺ ions can be demonstrated by observing the quenching of the fluorescence of thionine and the sensitization of that of methylene blue.
At [Th⁺] = [MB⁺] = 1 × 10^-5 M , the most efficient energy transfer (82 per cent efficiency, as derived from measurements of the quenching of Th⁺ fluorescence, or 90 per cent, as derived from sensitization of MB⁺ fluorescence) is observed at the lowest SLS-concentration (3.0 × 10^-3 M ), when the only micelles present are those formed by aggregation of dye-carrying low molecular complexes of SLS with dye cations. Each micelle carries, under these conditions, 10–14 molecules of the two dyes, and the distance between two closest dye ions is about 16 A. Transfer becomes less efficient as the SLS-concentration increases, causing pigment molecules to distribute themselves among a greater number of micelles.     

CHLOROPHYLLS IN POLYMERS. I. STATE OF CHLOROPHYLL a IN UNSTRETCHED POLYMER SYSTEMS

Abstract— Model systems for the study of energy transfer processes are useful for the elucidation of the various factors governing the mechanism of energy transfer in photosynthetic systems. Here we describe the characterization of two systems, consisting of chlorophyll a incorporated in anhydrous nitrocellulose and polyvinylalcohol films. First, optical spectroscopy and time-resolved fluorescence techniques are used to characterize the state of the chlorophyll molecules in the films. We find that in nitrocellulose films the state of chlorophyll a depends strongly on the ratio of nitrocellulose to dimeth-ylsulfoxide in the solutions from which the films are cast. The state of chlorophyll a in polyvinylalcohol films does not depend on the amount of polymer originally dissolved in dimethylsulfoxide. In these films the pigment is monomeric at low concentrations of chlorophyll a, but aggregates are formed at much lower concentrations than in nitrocellulose. The latter fact is explained by the existence of pockets in polyvinylalcohol, leading to high local concentrations.
To further test the suitability of the nitrocellulose polymer films as model systems for energy transfer processes, time-resolved fluorescence anisotropy profiles are measured in dependence of the concentration of pigments in the matrix. Fits of the observed decay profiles to the predicted decay show good correspondence, as long as no traps are present. Furthermore, the fitted decay times yield the correct value of the Forster radius R₀ as compared to the value obtained spectroscopically. We thus conclude that the chlorophyll a-nitrocellulose system can be very appropriate for the study of energy transfer processes between photosynthetic pigment, since the pigments are uniformally distributed in the matrix.     

ROOM TEMPERATURE TIME-RESOLVED IN μs RANGE DELAYED LUMINESCENCE OF CHLOROPHYLL a AND CHLOROPHYLL-LUTEIN MIXTURE SOLUTIONS

Using time-resolved in μS range luminescence spectroscopy, we observed at 20°C the emission of chlorophyll a, pheophytin a and chlorophyll a-lutein mixture solutions. This delayed emission exhibits several maxima in the650–750 nm region. The positions and kinetics of decay of delayed emission bands depend on chlorophyll concentration, and vary as a result of pheophytinization and addition of lutein. Our results can be explained by supposition that upon excitation, charge transfer species are formed in various pigment complexes. The back electron transfer reactions yield chlorophyll excited singlet states contributing to observed delayed emission. Delay in emission seems to be due also to the trapping of excitation on the triplet states of various forms of pigment and its detrapping with the participation of thermal energy followed by energy transfer to the forms of pigment characterized by different decay times.     

Energy transfer between surface-immobilized light-harvesting chlorophyll a/b complex (LHCII) studied by surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that combines rapid and efficient excitation energy transfer with effective protection of its pigments from photobleaching. These properties make LHCII potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Most of such applications would require the LHCII to be immobilized on a solid surface. In a previous study we showed the immobilization of recombinant LHCII on functionalized gold surfaces via a 6-histidine tag (His tag) in the protein moiety. In this work the occurrence and efficiency of Fo?rster energy transfer between immobilized LHCII on a functionalized surface have been analyzed by surface plasmon field-enhanced fluorescence spectroscopy (SPFS). A near-infrared dye was attached to some but not all of the LHC complexes, serving as an energy acceptor to chlorophylls. Analysis of the energy transfer from chlorophylls to this acceptor dye yielded information about the extent of intercomplex energy transfer between immobilized LHCII.     

ENERGY TRANSFER IN A LIGHT-HARVESTING CAROTENOID-CHLOROPHYLL c-CHLOROPHYLL a-PROTEIN OF PHAEODACTYLUM TRICORNUTUM

Abstract— The marine diatom Phaeodactylum tricornutum was readily disrupted in 0.1 N Tris-HCl buffer, pH 7.8, in a Braun Model MSK cell homogenizer at 0-5°C. Treatment of the suspension with sodium lauryol sarcosinate (3 molecules per 10000 daltons of protein) at 5°C in the dark and subsequent centrifugations produced a pigmented, protein fraction whose excitation spectrum exhibited energy transfer from carotenoids to chlorophyll a (Chi a ). Disruption of the pigment-protein complex by heating in 1% sodium dodecylsulfate resulted in loss of energy transfer. For each Chi a molecule this fraction had 1 Chi c , 4 fucoxanthin, and 6.7 accessory pigment molecules. Presence of the accessory complex of Photosystem II in this preparation is suggested by the high xanthophyll content. Further, based on Chl a concentrations, this fraction had about 18 times more apparent fluorescence emission at 680 nm when excited at 470 nm than the intact cells.     

PHOTOREDUCTION OF PROTOCHLOROPHYLL AND ITS DERIVATIVES

Abstract —Photoreduction of protochlorophyll and a series of its derivatives (with gradual simplification of the structure) was studied in ascorbic acid-propanol-pyridine mixtures. Additions were introduced into the solutions to suppress some side processes. Conditions were found for the photoreduction of pigment on the 7,8-linkage, with the formation of corresponding chlorins. Chlorin yields depended on the nature of the metal in the centre of the pigment molecule. The yield of chlorophyll upon photoreduction of protochlorophyll was 30 per cent, and the yields of Zn-derivatives of protochphyll was about 35–80 per cent. Photochemically prepared chlorins were isolated by chromatography. Some differences were discovered between their electronic, infrared, and nuclear magnetic resonance spectra and those of natural chlorins. These differences result from chlorins with a cis arrangement of hydrogen atoms on the 7,8-linkage being selectively formed upon photoreduction, while natural chlorins have the trans arrangement. The mechanism of the photoreduction reaction is discussed.     

LOCAL ELECTRIC FIELD EFFECT ON THE CHLOROPHYLL FLUORESCENCE

Abstract. Polarized absorption and fluorescence of chlorophyll a and bacteriochlorophyll solutions in nematic liquid crystal mixtures have been studied in the presence of an external electric field.
The electric field caused a reorientation of the pigment molecules as concluded from changes in the polarized absorption. However, no correlation was found between the change in the polarized absorption and the change in the polarized fluorescence as a function of the field strength. The field influence was much stronger than expected only from the molecular orientation. The fluorescence polarized parallel to the direction of the liquid crystal was found to increase, whereas the similarly polarized absorption decreases. As a whole, the fluorescence yield significantly increased. It is suggested that the additional electric field is reinforced by a local field, most probably due to a protonation of the liquid crystal molecules.
Charged solvent molecules are reoriented in an external electric field which changes the local electric field acting on chlorophylls. Similar changes in CHI fluorescence yield due to local electric field can be created in vivo by the shift of charged molecules present in surrounding pigments. Such effects can be at least partially responsible for slow induction of fluorescence phenomena.     

EXCITATION ENERGY TRANSFER IN THE LIGHT HARVESTING ANTENNA SYSTEM OF THE RED ALGA Porphyridium cruentum AND THE BLUE-GREEN ALGA Anacystis nidulans: ANALYSIS OF TIME-RESOLVED FLUORESCENCE SPECTRA

Abstract— Time-resolved fluorescence spectra of intact cells of red and blue-green algae Porphyridium cruentum and Anacystis nidulans were measured by means of a ps laser and a time-correlated photon counting system. Fluorescence spectra were observed successively from various pigments in the light harvesting system in the order of phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC) and chlorophyll a (Chl a ). The spectrum changes with time in the range of0–400 ps in P. cruentum and of0–1000 ps in A. nidulans . The time-resolved spectra were analyzed into components to obtain the rise and decay curve of each fluorescence component. Overall time behaviors of the sequential fluorescence emissions from various pigments can be interpreted with a decay kinetics ofexp(–2 kt ^½). The rate constants of the energy transfer show that the energy transfer takes place much faster in the red alga P. cruentum than in the blue-green alga A. nidulans , particularly in the step PCAPC. Results also indicated that a special form of APC, far-emitting APC, exists in the pigment system of A. nidulans , but it does not mediate a main energy transfer from phycobilisome to Chl a.     

IS ZEAXANTHIN CAPABLE OF ENERGY TRANSFER TO CHLOROPHYLL a IN PARTIALLY GREENED LEAVES? A STUDY OF FLUORESCENCE EXCITATION SPECTRA DURING VIOLAXANTHIN DEEPOXIDATION

Absorbance spectra and excitation spectra of chlorophyll a fluoresence were recorded during the light-induced deepoxidation of violaxauthin to zeaxanthin in bean leaves (Phaseolus coccineus) greened under intermittent light. Light minus dark fluorescence excitation difference spectra showed distinct minima at 440, 465, and 500 nm corresponding to maxima in the absorbance difference spectra. Both difference spectra were prevented by the deepoxidase inhibitor dithiothreitol and were inverted when zeaxanthin was epoxidized. The fluorescence excitation difference spectra were successfully modeled by considering the absorbance differences between violaxanthin and zeaxanthin, assuming no energy transfer from the two pigments to chlorophyll a, and accounting for light-induced scattering changes. The pigment stoichiometry and the scattering changes of the simulation were in accordance with experimental data. The results indicate that, in the early stage of leaf development, light absorbed by the cycle pigments violaxanthin and zeaxanthin is not transferred to chlorophyll.     

EXCITATION ENERGY TRANSFER IN BILIRUBIN-CHLOROPHYLL AGGREGATES

Abstract— Solutions of chlorophyll a (chl a ) with different concentrations of bilirubin (blr) were used as a model system for studying the mechanism of energy transfer between bili-proteins and chl in blue-green algae. Several optical properties such as the absorption spectra, emission spectra for different wavelengths of excitation, degree of polarization and lifetime of fluorescence were measured. The measurements were carried out in several solvents (of different hydrogen bond-forming capacities) and at various dye concentrations.
The results indicate that aggregates of chl with blr are formed. Energy absorbed by blr in the aggregates is emitted as chlorophyll fluorescence, indicating energy transfer from blr to chl within the aggregate. The fluorescence yield and fluorescence polarization of aggregates are lower than those of isolated chl.     

Immobilization of light-harvesting chlorophyll a/b complex (LHCIIb) studied by surface plasmon field-enhanced fluorescence spectroscopy

The major light-harvesting chlorophyll a/ b complex (LHCIIb) of the photosynthetic apparatus in green plants can be viewed as a protein scaffold binding and positioning a large number of pigment molecules that engage in rapid excitation energy transfer. This property makes LHCIIb potentially interesting as a light harvester (or a model thereof) in photoelectronic applications. Such applications would require the immobilization of LHCIIb (or similar dye-protein complexes) on a solid surface. In this work, the immobilization of recombinant LHCIIb is tested and optimized on functionalized gold surfaces via a histidine 6 tag (His tag) in the protein moiety. Immobilization efficiency and kinetics are analyzed by using surface plasmon resonance (SPR) and surface plasmon field-enhanced fluorescence spectroscopy (SPFS). The latter was also used to assess the integrity of immobilized LHCIIb by recording Chl b-sensitized Chl a emission spectra. Since His tags have been included in a substantial number of recombinant proteins, the immobilization technique developed here for LHCIIb presumably can be extended to a large range of other membrane and water-soluble proteins.     

多变鱼腥藻藻胆蛋白共价复合物的合成及其分子内能量传递

利用双功能基偶联剂3-(2-吡啶联巯基)丙酸N-羟基琥珀酰亚胺酯(SPDP)合成了两个藻胆蛋白复合物,藻红蓝蛋白-变藻蓝蛋白复合物PEC-APC和藻红蓝蛋白-藻蓝蛋白复合物PEC-PC.利用吸收光谱和荧光光谱证明了藻胆蛋白构型与构象在反应后得到保持。通过荧光光谱观察到能量传递现象。计算出复合物PEC-APC的分子内能量传递效率约为90%.复合物PEC-PC中藻红蓝蛋白PEC的荧光寿命比PEC本身的寿命大大缩短,证明存在分子内能量传递。二硫苏糖醇(DTT)还原二硫桥键后能量传递被阻断。这进一步证明复合物合成成功及分子内能量传递。     

ENERGY TRANSFER FROM CHLOROPHYLL b TO CHLOROPHYLL a IN A HYDROPHOBIC MODEL SYSTEM

Abstract— Chlorophyll a and chlorophyll b purified by high-performance liquid chromatography (HPLC) were subsequently adsorbed on the surface of a pellicular reverse phase packing normally used in HPLC. The granule surface is reacted with octadecyl groups and furnishes an hydrophobic substrate for pigment adsorption. Reflectance spectra of chlorophyll a and chlorophyll b , each adsorbed at average spacings of about 11 nm² per molecule, had red region maxima at 664 and 643nm respectively. Fluorescence excitation spectra for 740nm emission from these surfaces peaked at about 420nm for chlorophyll a and 460nm for chlorophyll b. Adsorbed pigments excited at either of the two wave lengths had a single fluorescence emission peak at 683nm for chlorophyll a and at 664nm for chlorophyll b. A surface having both pigments adsorbed in approximately equal amounts with an overall average spacing of about 5.6nm² per molecule also had peaks at 420 and 460nm in the excitation spectrum. However, excitation of adsorbed molecules on this (latter) surface, at either 420 or 460nm, produced emission with the single chlorophyll a peak at 683nm. It is concluded that, under the conditions of our experiment, exciting adsorbed chlorophyll b contributes strongly to emission from adsorbed chlorophyll a.