Simultaneous determination of chlorophyll a and chlorophyll b by derivative spectrophotometry

Chlorophyll a (Chl a) and chlorophyll b (Chl b) plant pigments, which are important in the food industry and are beneficial as environmental pollution indicators, have been extracted with a novel solvent mixture (1:1 v/v acetone–propanol) not containing chloroform and simultaneously determined by first-derivative spectrophotometry. The results were statistically compared to those obtained by the ordinary absorption spectrophotometric reference utilizing the principle of additivity of absorbances. The testing of the developed method in synthetic mixtures of Chl a and Chl b and in real plant material samples (grass, spinach, chard, purslane, black cabbage, crisp lettuce, rocket, dill and seaweed) proved successful in that the developed extractive derivative spectrophotometric method was both rapid and precise, and was not dependent on the Chl a/b ratio in contrast to the reference method which was adversely affected by the latter parameter.     

Loop currents in chlorophyll a

Loop currents involve the circulation of electrons (or holes) around closed conjugated-bond loops. From the length of the orbital perimeter the kinetic energy per electron can be calculated. In chlorophyll a, four electronic excitations have been analyzed, including: (1) a single positive charge (hole) circling a 19-bond orbit with a Möbius twist (this is proposed as the effective ground state); (2) nine electrons, unevenly spaced, circling the same 19-bond orbit but without the Möbius twist (proposed as the red excited state); (3) nine electrons in the same orbit but evenly spaced and vibrationless (the hopping exciton); (4) ten electrons, unevenly spaced, in a 16-bond orbit (the blue excited state). The fit of theory to experiment is examined.     

Photostabilized chlorophyll a in mesoporous silica: adsorption properties and photoreduction activity of chlorophyll a

Chlorophyll a was adsorbed to mesoporous silica (FSM, folded-sheet mesoporous material) to form a chlorophyll-FSM conjugate, in which a nanometer-scale interaction between chlorophyll a molecules resembles a living plant leaf. The mesopores of FSM acted as nanoscale spaces not only for an interaction between chlorophyll molecules and the silica support but also for a nanoscale interaction between the absorbed chlorophyll molecules. These interactions contribute to photostability. An increase in the amount of chlorophyll adsorbed to the pores of FSM leads to an enhancement of the photostability accompanied by a shift in the absorbance maximum to a longer wavelength. The physiological function of the chlorophyll-FSM conjugate was explored as chlorophyll-FSM exhibited the photoinduced ability to catalyze the reduction of methyl viologen (an electron carrier). The evolution of hydrogen gas was observed for 14 h without deterioration when an aqueous suspension containing chlorophyll-FSM, methyl viologen, 2-mercaptoethanol (an electron donor), and platinum was illuminated with visible light.     

Micellar enhanced spectrofluorometric determination of chlorophyll a and chlorophyll b in fresh waters

The enhancement of the fluorescence of chlorophyll a and chlorophyll b in a non-ionic micellar media is used for the establishment of a new analytical method for the determination of these pigments. After optimization of the variables, including concentration of surfactant, pH, temperature and percentage of organic solvent, the analytical figures of merit are determined. High recovery percentages are found when the method is applied to the simultaneous determination of chlorophyll a and b in synthetic mixtures. Finally, the method is used for the determination of both chlorophylls in local fresh waters, finding excellent agreement with the results obtained by an HPLC/fluorescence method.     

Carbon-13 NMR spectra of chlorophyll a,chlorophyll a′, pyrochlorophyll a and the corresponding pheophytins

The ¹³C NMR spectra of C-10 epimeric chlorophylls a and a′, pheophytins a and a′, pyrochlorophyll a and pyropheophytin a have been recorded and assigned by chemical shift comparison, by long-range selective ¹H decoupling experiments and by the examination of the fully coupled spectra. Various factors influencing the ¹³C chemical shifts of the chlorophyll derivatives, e.g. the coordination of magnesium to the chlorin nucleus, the effect of solvent and the steric strain at the periphery of the macrocycle, have been examined. The ¹³C NMR spectra of chlorophyll a measured in acetone-d₆ and tetrahydrofuran-d₈ (THF) were compared, and remarkable solvent effects on the ¹³C chemical shifts were observed. These effects were interpreted mostly in terms of specific chlorophyll-solvent interactions. Different electron donor and steric properties of acetone and THF were considered to cause conformational alterations in the macrocycle, induced by the ligation of the solvent molecule(s) to the axial position(s) of the central magnesium atom of chlorophyll a. These results show that ¹³C NMR spectroscopy is a method of high information value for investigations of the unique electron donor acceptor (EDA) properties of the chlorophylls. The structural differences between the C-10 epimeric chlorophylls and pheophytins were examined in terms of the substituent chemical shift (SCS) parameters for the C-10 methoxycarbonyl group. The analysis showed that the change from the (10R) to the 10(S) configuration induces conformational alterations in the whole macrocycle which are, however, most prominent in rings IV and V. Owing to the increased steric interaction (repulsion) between the bulky substituents at C-7 and C-10, the peripheral strain is larger in the (10S) form, and is relieved by more pronounced deviations of rings IV and V from the macrocyclic plane compared with the (10R) form. The examination of the SCS parameters also showed that the peripheral steric strain is dissipated to a larger extent over the entire macrocycle in the Mg-free derivatives. These results confirm the previous conclusions based on ¹H NMR and CD data. The possible function of chlorophyll a′ in photosynthesis is briefly discussed.     

The primary electron donor of photosystem II of the cyanobacterium Acaryochloris marina is a chlorophyll d and the water oxidation is driven by a chlorophyll a/chlorophyll d heterodimer

We present a theoretical analysis of the flash-induced absorbance difference spectrum assigned to the formation of the secondary radical pair P(+)QA(-) in photosystem II of the chlorophyll d-containing cyanobacterium Acaryochloris marina. An exciton Hamiltonian determined previously for chlorophyll a-containing photosystem II complexes is modified to take into account the occupancy of certain binding sites by chlorophyll d instead of chlorophyll a. Different assignments of the reaction center pigments to chlorophyll a or d from the literature are investigated in the calculation of the absorbance difference spectrum. A quantitative explanation of the experimental data requires one chlorophyll a molecule per reaction center, located at the site of P(D1). The remaining sites are occupied by chlorophyll d and pheophytin a. By far, the lowest site energy is found for the accessory chlorophyll of the D1 branch, Chl(D1), which represents the sink of excitation energy and therefore the primary electron donor. The cationic state is stabilized at the chlorophyll a, which drives the oxidation of water.     

Synthesis of chlorophyll a glycoconjugates using olefin cross-metathesis

Chlorophyll a glycoconjugates were obtained by cross-metathesis between olefin moieties of chlorophyll derivatives and allylβ-galactopyranoside tetraacetate.     

Highly efficient electrogenerated chemiluminescence of natural chlorophyll a

Highly efficient electrogenerated chemiluminescence (ECL) of natural chlorophyll a (Chl a) was observed in acetonitrile with 1-butyl-3-methylimidazolium hexafluorophosphate as electrolyte and tri-n-propylamine (TPrA) as a coreactant. The collected ECL spectrum displayed a maximum emission peak at ca. 670 nm, suggesting that the same excited states with the photo-excitation processes were generated. The possible ECL reaction mechanism of this Chl a–TPrA system was discussed and established. ECL intensity of Chl a was proportional to its concentration over the range of 0.1–11 μM, and a high ECL efficiency (Φ_ecl) of 0.86 was calculated using Ru(bpy)₃^2 + as the standard (Φ_ecl = 1). Herein, an important property of natural Chl a was expanded and a new kind of ECL luminophores was developed. Moreover, it is expected that this high ECL efficiency of natural porphyrin complex has great potential to expand its ECL sensing application.     

Cyclodextrin/chlorophyll a complexes as supramolecular photosensitizers

The interactions between chlorophyll a, and three cyclodextrins, hydroxypropyl-beta-cyclodextrin heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin and hydroxypropyl-gamma-cyclodextrin, were studied in aqueous solutions by means of absorption, emission and circular dichroism spectroscopy. Nanosecond laser flash photolysis and steady-state singlet oxygen generation experiments were performed to clarify the photoactivity of chlorophyll a in these systems. Moreover the photosensitizing activity of these complexes towards human leukemia T-lymphocytes (Jurkat cells) was tested and compared with that of the free sensitizer, chlorophyll a. The results obtained indicate that each cyclodextrin is able to carry the pigment in monomeric form inside of cells producing singlet oxygen.     

Cyanobacterial chlorophyll as a sensitizer for colloidal TiO2

Chlorophyll has been extracted from cyanobacteria. The adsorption of chlorophyll on the surface of colloidal TiO(2) through electrostatic interaction was observed. The apparent association constant (K(app)) of chlorophyll-TiO(2) obtained from absorption spectra is 3.78x10(4)M(-1). The K(app) value of chlorophyll-TiO(2) as determined from fluorescence spectra is 1.81x10(4)M(-1), which matches well with that determined from the absorption spectra changes. These data indicate that there is an interaction between chlorophyll and colloidal TiO(2) nanoparticle surface. The dynamics of photoinduced electron transfer from chlorophyll to the conduction band of colloidal TiO(2) nanoparticle has been observed and the mechanism of electron transfer has been confirmed by the calculation of free energy change (DeltaG(et)) by applying Rehm-Weller equation as well as energy level diagram. Lifetime measurements gave the rate constant (k(et)) for electron injection from the excited state chlorophyll into the conduction band of TiO(2) is 4.2x10(8)s(-1).     

Photoinduced electron transfer between chlorophyll a and gold nanoparticles

Excited-state interactions between chlorophyll a (Chla) and gold nanoparticles have been studied. The emission intensity of Chla is quenched by gold nanoparticles. The dominant process for this quenching has been attributed to the process of photoinduced electron transfer from excited Chla to gold nanoparticles, although because of a small overlap between fluorescence of Chla and absorption of gold nanoparticles, the energy-transfer process cannot be ruled out. Photoinduced electron-transfer mechanism is supported by the electrochemical modulation of fluorescence of Chla. In absence of an applied bias, Chla cast on gold film, as a result of electron transfer, exhibits a very weak fluorescence. However, upon negatively charging the gold nanocore by external bias, an increase in fluorescence intensity is observed. The negatively charged gold nanoparticles create a barrier and suppress the electron-transfer process from excited Chla to gold nanoparticles, resulting in an increase in radiative process. Nanosecond laser flash experiments of Chla in the presence of gold nanoparticles and fullerene (C60) have demonstrated that Au nanoparticles, besides accepting electrons, can also mediate or shuttle electrons to another acceptor. Taking advantage of these properties of gold nanoparticles, a photoelectrochemical cell based on Chla and gold nanoparticles is constructed. A superior performance of this cell compared to that without the gold film is due to the beneficial role of gold nanoparticles in accepting and shuttling the photogenerated electrons in Chla to the collecting electrode, leading to an enhancement in charge separation efficiency.     

Stimulated fluorescence and fluorescence quenching in chlorophyll a and bacteriochlorophyll a

This communication deals with the photophysical processes that take place in chlorophyll solutions under intense nitrogen laser irradiation. The effect of the pump photon density on the fluorescence yield depends strongly on the geometry of the irradiation and the sampling set-up. If the fluorescence cell and sampling probe are placed close to the transverse arrangement used for obtaining laser output, line narrowing and gain, which are processes associated with high population inversions and stimulated fluorescence, are observed. A normal fluorescence spectrum and a decrease in fluorescence quantum yield with increasing pump power are observed in the fluorescence cells in oriented at an angle of 20–40° with respect to the transverse axis of the exciting beam. The decrease in quantum yield appears to result from absorption of the pump photons by the excited singlet of the chlorophylls, and it is suggested that an analogous mechanism may be responsible for the anomalous fluorescence quantum yield reported for in in vivo Chlorella vulgaris algae.     

Non-linear absorption and fluorescence of chlorophyll a excited by a ruby laser

Excitation of chlorophyll a in solution by a Q-switched ruby laser permits the determination of the two-photon and excited singlet state cross sections from fluorescence and transmission measurements.     

Identification of novel sulfur-containing derivatives of chlorophyll a in a Recent sediment

Novel transformation products of chlorophyll a incorporating a methyl sulfide group in the substituent at the C-3 position have been identified in Recent sediments from an Antarctic lake.     

Characterisation of chlorophyll a solubilised in sodium lauryl sulphate micelles

Poisson statistics has been applied to the problem of solubilisation of chlorophyll a in sodium lauryl sulphate micelles. Dilution experiments have been carried out to support the finding that each unit of chlorophyll a contributing to the 740 nm band contains just one chlorophyll a molecule.     

Determination of chlorophyll a and b by simultaneous multi-component spectrophotometry

The mathematical technique of over-determined simultaneous equations has been applied to the analysis of chlorophyll extracts from plant materials. The choice of spectral range is important — it is shown that 50 data points in the range 500–700 nm give better results than a similar number in the range 400–700 nm, as they avoid interferences from carotenoids. Results agree with those calculated using Arnon's method (1949), but have a better precision of 2% rsd compared with 4%. The computing procedure also allows testing of the correctness of the results.Dedicated with best wishes to Professor Dr. K. Doerffel, on the occasion of his 70th birthday, remembering in particular how he helped to open cooperation with the west before reunification     

Homogeneous liquid-liquid extraction method for spectrofluorimetric determination of chlorophyll a

A new homogeneous liquid-liquid extraction using a fluorocarbon ionic surfactant, Zonyl FSA (FSA), having a diethylthioether group as a spacer between the perfluoroalkyl group and carboxyl group has been developed. In this FSA method, the phase separation phenomena were observed at mild pH (below pH 6). Moreover, by using this extraction method as a preconcentration, a highly sensitive spectrofluorometric determination of chlorophyll a was established. The results for the standard chlorophyll a were as follows. The concentration factor (V(w)/V(o)) was 200 (water phase, V(w), 20 cm(3), water-immiscible phase, V(o), 100 mul), the distribution ratio, log D, was 4.85, the extraction percentage, E, was 99.7%, and the procedure time was approximately 30 min. The calibration curve was linear in the concentration range 2 x 10(-11)-3 x 10(-7) mol dm(-3) and the detection limit (S/N=3) was 1 x 10(-11) mol dm(-3). The relative standard deviation was 0.72% for 10(-8) mol dm(-3) (five determinations).