High Performance Liquid Chromatographic Methods for the Quantitation of Vitamin A Palmitate in Liquid Multivitamin Formulations

Abstract

Several normal-phase high performance liquid chromato-graphic methods for the quantitation of Vitamin A palmitate in liquid multivitamin formulations using both radially-compressed and conventional silica columns are described. The methods are reproducible, require minimal sample preparation and resolve the predominant cis-trans isomers of vitamin A (13-cis, all-trans, 9-cis and 9,13-dicis). Good agreement is obtained between HPLC and USP assay for samples containing a small amount (6% or less) of the vitamin A as the 13-cis isomer.     

~1H-NMR studies on cis-trans isomers of spheroidenes

All-trans spheroidene was extracted from the cells of Rhodobacer sphaeroides 2.4.1 and a set of cis-trans isomers were isolated from an isomeric mixture obtained by iodine-sensitized photo-isomerization of the all-trans isomer by high-pressure liquid chromatography (HPLC). On the basis of assignment of the 1H-NMR spectra (COSY, long-range COSY, rekyed COSY, ROESY and NOESY) of the all-trans isomer, the configurations of cis isomers were determined by the isomerization shifts of the olefinic 1H signals. The peaks of HPLC could be assigned as follows: A: 9,13'-cis, B1: 5,13-cis, B2: 13,9'-cis, C: 13-cis, E: 9-cis, F: 13'-cis, G: 5,9'-cis, H: 9'-cis, I: all-trans.     

SIMPLE EXPLANATION OF THE MISSES IN THE COOPERATION OF CHARGES IN PHOTOSYNTHETIC O2 EVOLUTION

Abstract —In the model of Forbush et at. (1971) the observed damping of the flash yield sequence of photosynthetic O₂ evolution was related to a certain percentage of ‘misses’ (α; i.e. centers not converted). The possibility of a miss was supposed to be equal for all states S_0.1,2,3. We propose a new model and a new recurrence law that gives better quantitative agreement with the O₂ yield oscillations observed in Chlorella during a sequence of flashes. We find a better fit with all experimental results by assuming very unequal misses; the misses occur nearly exclusively on S₂ (and also sometimes on S₃). In the simpler case of only one miss on one state, half of S₂ exists as an inactive form S₂₊^- because it is in apparent equilibrium with pool A. The active form of S₂ is converted to S₃ in a flash and the unchanged inactive form S₂₊^- explains the miss: S ₁ hv→S₂₊^-=S₂hv→S₃ (S₂₊^- is a transition state between S₁ to S₂ associated with Q^-). In the dark, the apparent equilibrium constant K_A between pool A and Q (i.e. S₀, S₁ in the dark) is very large; this explains why there is no miss on these states. In light, the experimental value of K_A between pool A and Q (i.e. S₂, S₃ in the light) is 1, and this explains why the misses are large for states S₂, S₃; i.e., S₂₊^-/S2- 1 and sometimes S₃₊^-/S₃?0–1. This new model predicts that the total number of active states ΣS_i=S₀+S₁+S₂+S₃ is an oscillating function of the flash number. This sum 2S, is also the number of trapping centers for excitons. As fluorescence is proportional to excitons that are not trapped, our model explains why the fluorescence oscillates as a function of the flash number. We find also that the initial rates of O₂ evolution after (n - 1) flashes vs the 02 yield of the n^th flash are not exactly on a straight line, which also favors our model.     

On the “anomalous” flourescence of 3,4-benzypyrene in the vapour phase

An analysis of the fluorescence of 3,4-benzpyrene in the vapour phase shows two contributions to the “anomalous” fluorescence: (i) the emission from the second excited state to the ground state and (ii) a vibronically induced S₁ → S₀ emission originating from the +520 cm^?1 vibrational level. A comparison between the intensities of the emissions indicates that in the vapour phase the vibrational redistribution from the 520 cm^? vibrational level of S₁ to modes of lower frequencies is relatively slow.     

The appearance of fine structure in the phosphorescence spectra of organic molecules in solid solutions under T1 ← S0 laser excitation

Phosphorescence spectra of coronene. 1,2-benzypyrene and chrysene in bromobutyl at 4.2 K under laser excitation directly in the region of the T₁ ← S₀ transition have been studied. Under these conditions, the phosphorescence spectra acquire a line structure. Vibrational analysis of the spectra has been performed and the frequencies of the normal vibrations of the investigated molecules in the S₀ state have been determined. A “multiplet” structure of the phosphorescence spectra appearing for monochromatic excitation to vibrational sublevels of the T₁ state has been studied. It is shown that the frequencies of the molecular vibrations in a triplet state can be found from an analysis of this structure.     

IRRADIATION-ENHANCED PHYTOCHROME PELLETABILITY IN AVENA: IN VIVO DEVELOPMENT OF A POTENTIAL TO PELLET AND THE ROLE OF Mg2+ IN ITS EXPRESSION IN VITRO

The enhanced phytochrome pelletability that results from in vivo irradiation of Avena shoots may be divided into two operationally defined sequential stages: the in vivo development of a “potential to pellet” and the “expression” of this potential in vitro. Kinetic studies confirm previous findings that the generation of this “potential to pellet” is a very rapid (complete in < 10 s, 25°C), genuinely intracellular process, itself photoreversibly induced by P_fr. In addition, it is shown that the sustained development of the “potential to pellet”, that proceeds in the dark at 0°C following a red pulse, requires P_fr continually in the cell over the entire development period. Far red light immediately terminates further development of the red-induced “potential” at any point during the development phase. No immediate reduction is observed, however, in that level of “potential pelletability” already attained at the time of the far red pulse. This indicates that the level of “potential pelletability” established in vivo is insensitive to the form of the pigment at extraction regardless of the level reached. “Expression” of the “potential to pellet” refers to the actual detection in homogenates of an enhanced physical association of phytochrome with pelletable material. Maximum “expression” requires the presence of a divalent cation in the medium during homogenization. Rapid posthomogenization addition of Mg²⁺ to Mg²⁺-free extracts sustains enhanced pelletability but with rapidly declining effectiveness over the fmt 1–2 min after extraction. The rate of decline is faster if the phytochrome is present as P_fr than as P_r in the homogenate. Neither these nor previous data permit a distinction to be made between (a) preservation by the cation of a pre-existing intracellular interaction, and (b) a Mg²⁺-mediated induction of an artifactual, in vitro association predetermined in the cell by a genuine phyto-chrome-controlled process. Various formalistic models are discussed in the context of these and other data.     

Vibrational analysis of 2-fluoropropane and 2-fluorobutane

Vibrational spectra were obtained for 2-fluorobutane in the vapor, liquid, and solid states. The S_HH and Spy conformers are present in the solid, and the S_HH conformer is present in the liquid and in lower concentration in the vapor. Vibrational assignments were made with the aid of normal coordinate calculations that fit thirty-six frequencies of 2-fluoropropane and S_HH 2-fluorobutane with an average error of 3.7 cm^?1. Only a few vibrations of 2-fluorobutane seem to be dependent on conformation. The “C—F stretching” frequency cannot be used to distinguish the conformers because of extensive interaction between this coordinate and others.     

STEREOELECTRONIC PROPERTIES OF PHOTOSYNTHETIC AND RELATED SYSTEMS—VI. AB INITIO CONFIGURATION INTERACTION CALCULATIONS ON THE GROUND AND LOWER EXCITED SINGLET AND TRIPLET STATES OF ETHYL BACTERIOCHLOROPHYLLIDE-a AND ETHYL BACTERIOPHEOPHORBIDE-a‡

Abstract— Ab initio quantum mechanical calculations on ethyl bacteriochlorophyllide-a (Et-BChl-a) and ethyl bacteriopheophorbide-a (Et-BPheo-a) are presented, including self-consistent-field (SCF) molecular orbital studies on the ground states using the molecular fragment procedure, and configuration interaction (CI) calculations on the low-lying singlet and triplet states and absorption spectra. A characterization and comparison of many of the higher-lying molecular orbitals obtained from the SCF studies is presented. The estimated first ionization potentials are 5.66 and 5.97 eV for Et-BChl-a and Et-BPheo-a, respectively. Excited state calculations show that the visible spectrum of both molecules consists of an intense, y-polarized S₁← S₀ transition and a weakly-allowed, x-polarized S₂← S₀ transition. Both S₁ and S₂ states are ¹(π, π*) in character, and are described by a four-orbital model. Transitions to the remaining calculated states, S₃-S₁₂, appear in the Soret region of the spectrum of both molecules. However, only transitions to S₉(‘x’), S₁₀(‘x’) and S₁₁(‘y’) of Et-BChl-a, and S₇(‘x’) and S₁₀(‘y’) of Et-BPheo-a are of high intensity. The composition of the high intensity Soret states is ¹(π, π*) and strongly “four-orbital” in nature. The lowest triplet state, T₁, is predicted to lie 9752 cm^-1 and 7880 cm^-1 above S₀ for Et-BPheo-a and Et-BChl-a, respectively. In each molecule T₂ and S₁ are nearly degenerate, suggesting a favorable pathway for intersystem crossing. Calculated T_n← T₁ transitions indicate that the y-polarized T₁₂← T₁ transition in Et-BChl-a corresponds to the observed intense 24,400 cm^-1 absorption in the triplet-triplet spectrum of BChl-a. A similar type spectrum is also predicted for BPheo-a.     

THERMOLUMINESCENCE and OXYGEN EVOLUTION FROM A THERMOPHILIC BLUE-GREEN ALGA OBTAINED AFTER SINGLE-TURNOVER LIGHT FLASHES*

Abstract— Oxygen evolution and thermoluminescence (TL) studies on a thermophilic blue-green alga Synechococcus vulcanus Copeland revealed the following: (a) The deactivations of the S₃ and S₂ states of the Oxygen Evolving Complex, at room temperature, have half-times of ～200 and 75 s, respectively, instead of 30 and 20 s found in mesophilic plants, (b) The TL band(s) “B”, due to the recombination of the state S₂ or S₃ and Q_B, the reduced secondary quinone acceptor, is(are) at50–55°C instead of25–30°C; the intensity of this band oscillates with a period of 4 with maxima on the 2nd and the 6th flashes, (c) The TL band “D” in the presence of diuron, due to the recombination of S₂ and the reduced primary quinone acceptor Q_A,^? occurs at ～35°C instead of0–10°C. (d) Furthermore, the ratio of Q_B^? to Q_B in dark-adapted S. vulcanus cells is close to 1 as in intact spinach leaves, but not 0.43 as in isolated thylakoids from spinach.     

LOCATION OF THE CAROTENOID 2Ag-STATE AND ITS ROLE IN PHOTOSYNTHESIS

The location of the carotenoid 2A_g-state (S₁) was studied by fluorescence spectroscopy in two series of carotenoids. One consisted of natural polyenes like phytoene, phytofluene, β-carotene, and neurosporene, and the other of minicarotenes, i.e., compounds similar to β-carotene, but with a smaller number of double bonds (n=3–9). A decrease of the S₁-S₀ energy gap with n was observed in both series, and extrapolation to n= 11 gave the energies 14 500 and 13 200 cm^?1 for β-carotene and lycopene, respectively. The neurosporene S₁ state was located at ca 16 000 cm^?1. A good relationship between the nonradiative relaxation rate and the number of conjugation was observed. The rate increased by a factor of 2.5 per double bond. The possible role of the S₁ state in reversible carotenoid ← chlorophyll electron-exchange energy transfer is discussed.     

环丙酮光解离和热异构机理的从头算研究

运用精确的量子化学计算方法CASSCF, B3LYP和MP2, 结合cc-pVDZ基组, 优化了环丙酮的基态和激发态势能面上的驻点结构, 计算了它们的相对能量. 在此基础上, 深入探讨了环丙酮光解离反应的机理. 在292～365 nm波长的光的激发下, 环丙酮被激发至S₁态, 最可能的初始过程是α C—C键断裂. 我们的理论研究发现, 在α C—C键断裂途径上, 存在基态和第一激发势能面的交叉点, 它在随后的反应过程中起着重要作用. 一方面可形成单态双自由基中间体, 然后发生另一个C—C键的断裂, 生成基态产物一氧化碳和乙烯. 另一方面, 经过S₁/S₀交叉点可以回到热的基态. 在这种情况下, 体系具有足够的能量, 克服基态途径上的势垒, 生成同样的基态产物乙烯和一氧化碳. 此外, 还对环丙酮基态异构化反应进行了理论研究.     

Fluorescence spectra and intramolecular vibrational redistribution in jet-cooled perylene

The jet-cooled fluorescence spectra of perylene excited to the S₁ state with E_vib = 0–1600 cm^?1 are recorded and analyzed. For E_vib <800 cm^?1 only the resonant fluorescence was detected. Ground- and excited-state frequencies of 14 low-frequency normal modes are determined. A drastic change in frequency of the “butterfly” modes upon electronic excitation shows that perylene slightly deviates from planarity in its ground state and is more rigid in the excited singlet state. For a number of levels in the E_vib = 800–1600 cm^?1 range, the fluorescence is composed of the resonant emission and of non-resonant (“‘relaxed’”) bands. It is shown that apparently single bands in the fluorescence-excitation spectrum correspond to ovelapping bands pumping different molecular eigenstates resulting from the intrastate coupling. The relative role of the anharmonicity and of the Coriolis interaction are discussed. The data are treated in terms of a selective coupling between doorway and hallway states with the coupling constant rapidly decreasing with the difference in the overall vibrational quantum number between initial and final state.     

Beiträge zur Chemie des Phosphors. 72. Über die Alkyl-cyclomonocarba-phosphane (PR)4CH2, R = CH3, C2H5, t-C4H9

Contributions to the Chemistry of Phosphorus. 72. About the Alkyl Cyclomonocarba Phosphanes (PR)₄CH₂, R = CH₃, C₂H₅, t-C₄H₉ The alkyl-substituted cyclomonocarbaphosphanes (PR)₄CH₂ (R = CH₃ 1 , C₂H₅ 2 , t-C₄H₉ 3 ) are obtained in very good yield by the reaction of the corresponding dipotassium alkylphosphides K₂(PR)_n (n = 2, 3, 4) with methylene chloride. Besides, small amounts of the homocyclic rings characteristic for the given substituent and of the five-membered cyclodicarbaphosphanes (PR)₃(CH₂)₂ with isolated CH₂ groups are formed. For the alkylcyclomonocarbaphosphanes 1 and 2 , configuration isomers could be identified for the first time. The “all-trans” forms are always predominant; the relative amounts of the other isomers decrease strongly with increasing number of cis relationships between the substituents at adjacent phosphorus atoms. The ³¹P n.m.r. parameters for three of the all together six isomers of 1 distinguishable by n.m.r. spectrometry and for the “all-trans” isomers of 2 and 3 are reported and discussed. A definite separation is possible between substituent and configuration influence on the chemical shifts as well as on the coupling constants.     

Excited States of Weak Interacting Complexes of Formaldehyde and Alkali Metal Ions

The electronically excited states of formaldehyde and its complexes with alkali metal ions are investigated with the time-dependent density functional theory (TD DFT) method. Vertical transition energies for several singlet and triplet excited states, adiabatic transition energies for the first singlet and triplet excited states S₁ and T₁, the adiabatic geometries and vibrational frequencies of the ground state S₀ and the first singlet and triplet excited states S₁ and T₁ for formaldehyde and its complexes are calculated. Better agreement with the experiment than that of the CIS method is obtained for CH₂O at the TD DFT level. The nonlinear C=O?M⁺ interaction in the excited states S₁ and T₁ is weaker than the linear interaction in the ground state. In the S₀ and S₁ states, the C=O bond is elongated by cation complexation and its stretching frequency is red-shifted, but in the T₁ state the C=O bond is shortened and its frequency is blue-shifted.     

THE PREPARATION AND CHARACTERIZATION OF Cd(XCN)4M TYPE COMPOUNDS

Abstract

A series of compounds of the type Cd(XCN)₄M (X=S and Se; M=Mn, Fe, Co and Zn) have been prepared and characterized. These compounds crystallize in the tetragonal system: space group I4[sbnd]S₄ ². The bidentate thiocynate and selenocyanate ligands coordinate to the “hard” M(II) ions through the nitrogen atom and the “soft” Cd(II) ion through the sulfur or selenium atom. The Cd[sbnd]Se bond in Cd(XCN)₄ M is slightly stronger than the Cd[sbnd]S bond while the M[sbnd]NCSe bond is slightly weaker than the M[sbnd]NCS bond. The M(II) ions tetrahedrally coordinated by the nitrogen end of XCN^? are in the high spin state. The [sbnd]NCS and [sbnd]NCSe ligands are located at about the same place in the spectrochemical series.     

SOLVENT EFFECT ON SPHEROIDENE IN NONPOLAR AND POLAR SOLUTIONS AND THE ENVIRONMENT OF SPHEROIDENE IN THE LIGHT-HARVESTING COMPLEXES OF Rhodobacter sphaeroides 2.4.1 AS REVEALED BY THE ENERGY OF THE 1Ag−→1Bu+ ABSORPTION AND THE FREQUENCIES OF THE VIBRONICALLY COUPLED C=C STRETCHING RAMAN LINES IN THE 1Ag− AND 1Bu− STATES

Abstract –The ¹A_g^?→¹B_u⁺ electronic absorption band and the vibronically coupled, C=C stretching Raman lines in the ¹A_g^? and 2¹A_g^? states were recorded for spheroidene free in nonpolar and polar solvents as well as for spheroidene bound to the LH1 and LH2 complexes of Rhodobacter sphaeroides 2.4.1. The ¹B_u⁺ energy exhibited a linear dependence on R(n) = (n² - 1)/(n²+ 2) in both nonpolar and polar solvents; the line for polar solvents had a gentler slope and crossed the line for nonpolar solvents at R(n) = 0.3. The above characteristic of polar solvents was ascribed to the electric field generated by fluctuation of the solvent permanent dipoles; it stabilizes the ¹B_u⁺ energy and reduces the polarizability of the solvent. The vibronically coupled, C=C stretching frequencies in the ¹A_g^? and 2¹A_g^? states [ν(A_g) and [ν(2A_g)] also showed similar dependence on R(n), which is explained in terms of vibronic coupling among the ¹A_g^?, 2¹A_g^? and 3¹A_g^? states. The environment of spheroidene in the LH2 and LH1 complexes was assessed on the basis of the ¹B_u⁺ energy and the ν(A_g) and [ν(2A_g) frequencies: Spheroidene in the LH2 complex is located in an environment with high polarizability, while spheroidene in the LH1 complex is located in an environment with lower polarizability.     

Structural and Mechanistic Insights into CO2 Activation by Nitrogenase Iron Protein

The Fe protein of nitrogenase catalyzes the ambient reduction of CO₂ when its cluster is present in the all-ferrous, [Fe₄S₄]⁰ oxidation state. Here, we report a combined structural and theoretical study that probes the unique reactivity of the all-ferrous Fe protein toward CO₂. Structural comparisons of the Azotobacter vinelandii Fe protein in the [Fe₄S₄]⁰ and [Fe₄S₄]⁺ states point to a possible asymmetric functionality of a highly conserved Arg pair in CO₂ binding and reduction. Density functional theory (DFT) calculations provide further support for the asymmetric coordination of O by the “proximal” Arg and binding of C to a unique Fe atom of the all-ferrous cluster, followed by donation of protons by the proximate guanidinium group of Arg that eventually results in the scission of a C−O bond. These results provide important mechanistic and structural insights into CO₂ activation by a surface-exposed, scaffold-held [Fe₄S₄] cluster.     

Photoisomerization Mechanism of Ruthenium Sulfoxide Complexes: Role of the Metal‐Centered Excited State in the Bond Rupture and Bond Construction Processes

Phototriggered intramolecular isomerization in a series of ruthenium sulfoxide complexes, [Ru(L)(tpy)(DMSO)]ⁿ⁺ (where tpy=2,2’:6’,2’’‐terpyridine; DMSO=dimethyl sulfoxide; L=2,2’‐bipyridine (bpy), n=2; N,N,N’,N’‐tetramethylethylenediamine (tmen) n=2; picolinate (pic), n=1; acetylacetonate (acac), n=1; oxalate (ox), n=0; malonate (mal), n=0), was investigated theoretically. It is observed that the metal‐centered ligand field (³MC) state plays an important role in the excited state S→O isomerization of the coordinated DMSO ligand. If the population of ³MC_S state is thermally accessible and no ³MC_O can be populated from this state, photoisomerization will be turned off because the ³MC_S excited state is expected to lead to fast radiationless decay back to the original ¹GS_S ground state or photodecomposition along the Ru²⁺?S stretching coordinate. On the contrary, if the population of ³MC_S (or ³MC_O) state is inaccessible, photoinduced S→O isomerization can proceed adiabatically on the potential energy surface of the metal‐to‐ligand charge transfer excited states (³MLCT_S→³MLCT_O). It is hoped that these results can provide valuable information for the excited state isomerization in photochromic d⁶ transition‐metal complexes, which is both experimentally and intellectually challenging as a field of study.     

Micellar effects on photoprocesses in retinyl polyenes

The effects of micellar solubilization on excited-state properties of several retinyl polyenes have been examined primarily by nanosecond laser flash photolysis. The relative intensity of band system III (254–256 nm) in the ground state absorption spectrum of 11-cis retinal decreases significantly on going from methanol to micellar solutions, suggesting that the 12-s-trans form of 11-cis retinal is relatively favored in the organized media. In addition to microsecond transient phenomena due to triplets, the laser flash photolysis of all-trans and 11-cis retinal and all-trans retinyl Schiff base incorporated into micelles leads to ‘permanent’ absorption changes attributable to photoisomerization (in the case of retinals) and protonation and/or complexation with water (in the case of Schiff base). All-trans retinol and retinyl acetate in micellar solutions undergo ionic photodissociation leading to long-lived retinyl carbocation (λ_max = 585–600 nm), the process being monophotonic in the case of retinyl acetate and predominantly bipho-tonic in the case of retinol. The trends in the location of ground-state absorption maxima (^IB_u^+*←^IA_g) and triplet yield of retinals, and photodissociation yield of retinyl acetate suggest that the polarity of the environment probed by the polyene systems increases in the order: Triton X-100 < CTAB < NaLS.     

EFFECT OF THE BROMINE ATOM UPON THE DISSOCIATION CONSTANTS OF BROMOPYRIDINES,BROMOQUINOLINES AND 4-BROMOISOQUINOLINE IN THEIR LOWEST EXCITED SINGLET AND TRIPLET STATES*

Abstract— The lowest excited singlet-state dissociation constants (pK^S_a) of bromosubstituted pyridines, quinolines, and isoquinolines were determined from the pH-dependent shifts in their electronic absorption spectra. The lowest excited triplet-state dissociation constants (pK^T_a) of bromosubstituted quinolines and 4-bromoisoquinoline were obtained from the shifts of the 0–0 phosphorescence bands measured in rigid aqueous solution at 77 K. The pK^S_a values indicate that the basicity of these brominated nitrogen heterocycles is increased in the lowest excited singlet state by 2 to 10 orders of magnitude as compared with the ground state. The pK^T_a values are found to be significantly different from the corresponding ground-state pK_a values, indicating that the basicity of bromoquinolines is increased in the lowest excited triplet state by 1.7 to 3.0 pK units. The enhancement of the excited singlet-and triplet-state basicity of brominated nitrogen heterocycle derivatives as compared with the unsuhstituted parent compounds is attributed to the increased electron-donor conjugative interactions of the bromine atom pπ orbitals with π orbitals in the lowest excited singlet and triplet state.