The Study of UV and VIS Absorption Spectra of the Complexes of Amino Acids with Ninhydrin

Abstract

The analysis of amino acids with post column derivatization using ninhydrin is usually carried out with automatic analyzers at wavelengths of 570 nm (for the quantitation of α-amino acids) and 440 nm (for the imino acids), or at only one wavelength, 520 nm, for the total acid content. The α-amino acids are reported to give an absorption maximum at 570 nm, after complex formation with ninhydrin; and α-imino acids have a γ at 440 nm, 520 nm is a compromise wavelength.

This paper presents a study of the absorption of the complexes of amino acids with ninhydrin between 275 and 700 nm. Two new wavelengths are suggested for use, 290 and 404 nm.

In the near UV (290 nm) all the tested acids show a common absorption maximum and good sensitivities. In the visible region at 404 nm, the sensitivities of the α-amino acids are very similar to those observed at 570 nm. At 520 nm the αamino acids have the lowest sensitivities.

The α-imino acids (proline and oxyproline) show absorption maxima only at 290 nm; after that their sensitivities decrease with increasing wavelength.

The paper offers some practical hints on changing the detector wavelength, and to adapt the HPLC's detector to work at one of these two, newly suggested, bands (290 or 404 nm).     

Analysis of fluorine and iodine derivatives of tyrosine

Separation of tyrosine, fluorotyrosine, monoiodotyrosine and diiodotyrosine was achieved by reversed-phase high-performance liquid chromatography (HPLC) using a gradient of acetonitrile with water and using trifluoroacetic acid for ion pairing. No derivatization of the amino acids, prior to separation, was needed. The spectral properties of Tyr and its fluorine and iodine derivatives and the dependence of their absorbance maxima on pH, made it possible to analyze and differentiate between these derivatives in the free amino acid form or in peptides. This analysis was accomplished by adjusting the post column HPLC eluate from two identical runs to different pH values and then comparing the spectra of the peaks from these two runs with a diode array detector. Hydrolysis in 6 M hydrochloric acid was totally destructive to mono- and diiodotyrosine. However, base hydrolysis in 13.5 M sodium hydroxide for 30 min at 121 degrees C in an autoclave caused no destruction and allowed excellent recovery of all of the Tyr derivatives. This is the first report of simple methods for the detection and analysis of these amino acids and of a hydrolytic method which protects against their loss. A method of storage was also proposed.     

Analysis of aliphatic car☐ylic acids and amino acids in effluents of landfills, composting plants and fermentation plants by ion-exclusion and ion-exchange chromatography

Short chain aliphatic acids, including volatile fatty acids (VFAs), di-/tricar☐ylic acids, hydroxy- and keto-acids were analyzed in landfill leachates and related water samples by two independently operated ion-exclusion chromatographic systems, differing mainly in the retention characteristics of the separation columns (Merck Polyspher OA-HY, Dionex HPICE AS6), and in the detection mode (UV absorbance at 210 nm, conductivity). The amino acid content of the samples was determined by ion chromatography. Because methods for amino acids analysis are widely standardized, the main efforts were undertaken to optimize the determination of car☐ylic acids. The VFAs (7 compounds) contributed between 33% and 89% to the sample's dissolved organic carbon (DOC) content. The DOC proportions of the multifunctional acids (9 compounds) ranged from 1.1–49%. Between 0.9% and 13% of the DOC content was apportioned to amino acids. Main components were alanine, valine and leucine. The analytical efficiencies of the ion-exclusion chromatography systems were compared and the specific application properties are discussed.     

Effects of three characteristic amino acid residues of pharaonis phoborhodopsin on the absorption maximum

Phoborhodopsin (pR or sensory rhodopsin II, sRII) or pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) has a unique absorption maximum (lambda max) compared with three other archaeal rhodopsins: lambda max of pR or ppR at ca 500 nm and others at 560-590 nm. Alignment of amino acid sequences revealed three sites characteristic of the shorter wavelength-absorbing pigments. The amino acids of these three sites are conserved completely among archaeal rhodopsins having longer lambda max, and are different from those of pR or ppR. We replaced these amino acids of ppR with amino acids corresponding to those of bacteriorhodopsin, Val-108 to Met, Gly-130 to Ser and Thr-204 to Ala. The lambda max of V108M mutant was 502 nm with a slight redshift. G130S and T204A mutants had lambda max of 503 and 508 nm, respectively. Thus, each site contributes only a small effect to the color tuning. We then constructed three double mutants and one triple mutant. The opsin-shifts of these mutants suggest that Val-108 and Thr-204 or Gly-130 are synergistic, and that Gly-130 and Thr-204 work additively. Even in the triple mutant, the lambda max was 515 nm, an opsin-shift only ca 30% of the shift value from 500 to 560 nm. This means that there is another yet unidentified factor responsible for the color tuning.     

Quantitation of sodium hydrogensulfite in parenteral samples by a flow-injection method

Sodium hydrogen sulfite reacts with 5,5′-dithiobis(2-nitrobenzoic acid) at pH 6.0 to produce a colored 2-nitrothiobenzoate ion with maximum absorbance at 410 nm. The absorbance varies linearly with concentration over the range of 1 ng-1 μg of sodium hydrogensulfite injected and the absolute detection limit is 0.3 ng. The relative standard deviation at 10 ng or greater is typically 1.5%. Matrix components in pharmaceutical parenteral solutions (e.g., amino acids, drugs, dextrose, buffers) do not interfere.     

Complex formation of uranium(VI) with the amino acids l-glycine and l-cysteine: A fluorescence emission and UV–Vis absorption study

The complexation of uranium(VI) with the amino acids l-glycine and l-cysteine has been investigated by time-resolved laser-induced fluorescence spectroscopy (TRLFS) and UV–Vis spectroscopy at a low pH range. The identified 1:1 and 1:2 uranyl-l-glycine complexes fluoresce and have similar absorbance properties. In contrast to the glycine system, uranyl forms two different non-fluorescent 1:1 complexes with l-cysteine, showing individual absorbance properties under the given experimental conditions. The corresponding complex formation constants were calculated using the spectroscopic data.     

Development of effective cross-linking method for bioactive substance--enzyme immobilization using glutaraldehyde oligomers

When a 10% aqueous solution of glutaraldehyde (GA) was alkalized to pH 8.5 in borate buffer solution and heated at 60 degrees C, the ultraviolet spectrum of GA solution showed two distinct absorption maxima. The one at 280 nm with a weak absorbance ascribable to the C = O bond in the aldehyde group shifted to near 300 nm after 50 min with a slight increase in its intensity. Another maximum at 235 nm with a strong absorbance was ascribable to the C = C bond of the alpha,beta-unsaturated aldehyde group which was formed by aldol condensation reaction of GA monomer, and its absorbance increased markedly with increasing reaction time. The high performance liquid chromatography (HPLC) analysis with detection at 235 nm indicated that several GA oligomers were formed by the alkali treatment and their concentrations increased. The cross-linking ability of these oligomers was examined by immobilizing enzymes (alcohol dehydrogenase (ADH), glutamate dehydrogenase (GLDH] to an aminated polymer gel matrix by reaction with the treated GA solution. The enzyme activities increased with increasing concentration of GA oligomers. Then, the GA oligomers were isolated and used as the cross-linking agent. The activities of ADH and GLDH were 4-fold and 13-fold higher, respectively, than those obtained by using untreated GA solution, while the total amounts of immobilized enzymes were almost unchanged. These results suggest that GA oligomers may act as cross-linkers in a manner different from the generally accepted Schiff base formation reaction; a possible mechanism may involve addition reaction of an amino group to the double bond in the aldol condensate of GA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Simultaneous Spectrophotometric Estimation of Rabeprazole Sodium and Itopride HCl

Abstract

Two simple, rapid, accurate, and economical analytical methods have been developed for the simultaneous estimation of Rabeprazole Sodium and Itopride Hydrochloride in combined capsule dosage form. First method is based on the determination of Q‐value and second method is based on simultaneous equation method. Rabeprazole Sodium has absorbance maxima at 284 nm and Itopride Hydrochloride has absorbance maxima at 258 nm in methanol AR. The absorption ratio (Q‐value) was determined at 266.6 nm (Iso‐bestic point) and 284 nm (λmax of Rabeprazole Sodium). Both the drugs obey Beer's law in the concentration ranges employed for these methods. Both the methods were found to be simple, rapid, accurate, and can be adopted in routine analysis of drugs in formulations. The accuracy and reproducibility of the proposed method was statistically validated by recovery studies     

Effects of organic and aqueous solvents on the electronic absorption and fluorescence of chloroaluminum (III) tetrasulphonated naphthalocyanine

The effects of various solvents on the ground and excited states of chloroaluminum (III) tetrasulphonated naphthalocyanine (AlNcS(4)) were studied. Both the absorbance and fluorescence spectra were found to be influenced by the hydrogen bond donating ability of various solvents. As the hydrogen bond donating ability of the solvent increased, hypsochromic and bathochromic shifts in the absorbance and fluorescence spectra were observed in protic and aprotic solvents respectively. Plots of the absorbance and fluorescence maxima versus the E(T)(30) solvent parameter showed linear relationships in binary mixtures of protic-protic (methanol-H(2)O) and aprotic-protic (DMSO-H(2)O) solvents. Aggregation was indicated by a broad band in the ground state absorption spectra and a low quantum efficiency 0.04 relative to the efficiency observed in organic solvents. A face-to-face conformation of the monomeric subunits of the dimer is suggested due to the red-shifted absorbance band. The acid-base properties of the dye were studied and were indicative of a multi-step process. In acidic conditions (pH 1), protonation of the bridging nitrogen atoms was identified by a broad band appearing red-shifted to those obtained at higher pH values. Under slightly acidic conditions a pKa value of 6.7 was determined for one of the meso-nitrogen. In alkaline conditions a pKa of 11.5 was determined for another meso-nitrogen and a second fluorescence band emerged at 804 nm, red-shifted to the emission maxima.     

Simple and low-cost high-performance liquid chromatographic method for determination of D- and L-amino acids

In this article, a simple and low-cost method for the analysis of amino acid enantiomers by using high-performance liquid chromatography (HPLC) is described. In this method, the amino acids are modified to diastereomers in order to be separated into enantiomers on a usual C(18) reversed-phase column. Methanol instead of acetonitrile is used as an elution solvent; the results of HPLC with methanol elution are comparable with those of HPLC with acetonitrile elution. Sub-nanomolar sensitivity is attained by measuring the absorbance at 340 nm in analysis of 15 amino-acid enantiomers.     

Capillary electrochromatography with thermo-optical absorbance detection for the analysis of phenylthiohydantoin-amino acids.

Capillary columns were packed with 3 microns C18 stationary phase, interfaced with an ultraviolet-laser based thermo-optical absorbance detector, and evaluated for separation of a mixture of phenylthiohydantoin-amino acids. These columns demonstrated consistent performance with a relative standard deviation (RSD) for migration time of less than 1.5% and a separation efficiency of 216,000 plates/m for the electroosmotic flow marker, thiourea. The thermo-optical absorbance detector was based on a 248 nm krypton-fluoride excimer laser. Detection limits (3 sigma) ranged from 1.6 to 4.8 x 10(-7) M phenylthiohydantoin (PTH)-amino acid injected onto the column, which is a factor of three superior to those obtained in micellar electrokinetic chromatographic analysis of these compounds. A mixture of 17 PTH amino acids was injected onto the capillary; 13 components were nearly baseline resolved in 14 min.     

pH DEPENDENCE OF THE ABSORPTION SPECTRA AND PHOTOCHEMICAL TRANSFORMATIONS OF THE ARCHAERHODOPSINS

Abstract —Two strains of archaebacteria have been found to contain light-driven proton pumping pigments analogous to bacteriorhodopsin (bR) in Halobacterium salinarium . These proteins are called archaerhodopsin-1 (aR-1) and archaerhodopsin-2 (aR-2). Their high degree of sequence identity with bR within the putative proton channel enables us to draw some conclusions about the roles of regions where differences in amino acids exist, and in particular the surface residues, on the structure and function of retinal-based proton pumps. We have characterized the spectral and photochemical properties of these two proteins and compared them to the corresponding properties of bR. While there are some differences in absorbance maxima and kinetics of the photocycle, most of the properties of aR-1 and aR-2 are similar to those of bR. The most striking differences of these proteins with bR are the lack of an alkaline-induced red-shifted absorption species and a dramatic (apparent) decrease in the light-induced transient proton release. In membrane sheet suspensions of aR-1 at 0.15 M KCI, the order of proton release and uptake appears opposite that of bR, in which proton release precedes uptake. The nature of this behavior appears to be due to differences in the amino acid sequence at the surfaces of the proteins. In particular, the residue corresponding to the lysine at position 129 of the extracellular loop region of bR is a histidine in aR-1 and could regulate the efficient release of protons into solution in bR.     

荧光素在明胶片中的光吸收及稳定性

由于对非线性光学材料的需求,近年来已有很多工作对荧光素的光学及光化学性质进行研究,研究对象既有溶液也有聚合物薄膜。本工作使用明胶片研究荧光素在明胶片中的光吸收、荧光及光稳定性。     

Spectrophotometric determination of tartaric acid with beta-naphthol

Tartaric acid (50-800 mug) is reacted with beta-naphthol (5 mg) in 90 % sulphuric acid (2 ml) for 30 min on a steam-bath. The solution is cooled and diluted to 25 ml with 90% sulphuric acid and the absorbance is measured at 603 nm. Several absorption maxima are observed and the ratio and intensity of these depend on the concentration of acid during and after the reaction, and on the beta-naphthol concentration. All tartaric acid isomers give essentially the same colour and absorbance. Interference studies are reported.     

Studies on fluorescein-V The absorbance of fluorescein in the ultraviolet, as a function of pH

The ultraviolet absorption spectum of an aqueous solution of highly-purified yellow fluorescein at ionic strength 0.10 has been measured at various pH values in the range from 0.15 to 8.70. The maxima at 227, 249 and 295 nm change little with pH, but the maximum found at 437 nm in acid medium changes greatly in absorbance and position on addition of alkali, resolving first into two maxima, at 455 and 475 nm, and finally becoming a single large maximum (at pH 8) at 490 nm. A unique feature of the absorption at 437 nm is that all four prototropic forms of fluorescein, H(3)Fl(+), H(2)Fl, HFl(-) and Fl(2-), absorb at this wavelength. The total absorbance at this wavelength first falls rapidly as the pH rises from 0.15, reaching a minimum at pH 3.63, then increases to a maximum at pH 5.3, and finally falls to steady value at pH > 8.0. The absorbance as a function of pH is defined by seven constants: three dissociation constants (K(H(3)Fl) = 6.61 x 10(-3), K(H(2)Fl) = 3.98 x 10(-5), K(HFl) = 4.36 x 10(-10)) and four molar absorptivities ((H(3)Fl) = 4.94 x 10(-4), (H(2)Fl) = 1.20(5) x 10(4), (HFl) = 2.16 x 10(4) and (Fl) = 7.61 x 10(3) 1.mole(-1).cm(-1)). Solutions of yellow fluorescein in water undergo rapid deterioration on exposure to daylight or fluorescent lighting but are stable in the dark.     

Enantioselective micellar electrokinetic chromatography of dl‐amino acids using (+)‐1‐(9‐fluorenyl)‐ethyl chloroformate derivatization and UV‐induced fluorescence detection

Chiral analysis of dl ‐amino acids was achieved by micellar electrokinetic chromatography coupled with UV‐excited fluorescence detection. The fluorescent reagent (+)‐1‐(9‐fluorenyl)ethyl chloroformate was employed as chiral amino acid derivatizing agent and sodium dodecyl sulfate served as pseudo‐stationary phase for separating the formed amino acid diastereomers. Sensitive analysis of (+)‐1‐(9‐fluorenyl)ethyl chloroformate‐amino acids was achieved applying a xenon‐mercury lamp for ultraviolet excitation, and a spectrograph and charge‐coupled device for wavelength‐resolved emission detection. Applying signal integration over a 30 nm emission wavelength interval, signal‐to‐noise ratios for derivatized amino acids were up to 23 times higher as obtained using a standard photomultiplier for detection. The background electrolyte composition (electrolyte, pH, sodium dodecyl sulfate concentration, and organic solvent) was studied in order to attain optimal chemo‐ and enantioseparation. Enantioseparation of 12 proteinogenic dl ‐amino acids was achieved with chiral resolutions between 1.2 and 7.9, and detection limits for most derivatized amino acids in the 13–60 nM range (injected concentration). Linearity (coefficients of determination > 0.985) and peak‐area and migration‐time repeatabilities (relative standard deviations lower than 2.6 and 1.9%, respectively) were satisfactory. The employed fluorescence detection system provided up to 100‐times better signal‐to‐noise ratios for (+)‐1‐(9‐fluorenyl)ethyl chloroformate‐amino acids than ultraviolet absorbance detection, showing good potential for d ‐amino acid analysis.     

Some characteristic properties of 3-alkyl-2-thiothiazon-4-ones and of intermediates in their synthesis

Acrylonitrile reacts with dithiocarbaminates to give nitriles of N-alkyl-S-thiocarbaminylthiohydracrylic acids, which are then hydrolyzed to the corresponding amides and acids. Dehydration of the latter gives 3-alkyl-2-thiothiazon-4-ones. The UV spectra of N-alkyl-S-thiocarbaminylthiohydracrylic acids, and amides and nitriles of them are characterized by two maxima in the 245–250 nm (thioamide band) and 266–280 nm (dithiocarbonate band) regions. Thiazan ring closure results in bathochromic shift of absorption maxima due to lengthening of the conjugated chain. The average values of the atomic refraction for sulfur in the molecules of the compounds investigated correspond to a thioketone structure.     

Rapid, selective, direct and derivative spectrophotometric determination of titanium with 2,4-dihydroxybenzaldehyde isonicotinoyl hydrazone

A simple and sensitive spectrophotometric method is developed for the determination of titanium in aqueous medium. The metal ion forms a reddish brown coloured complex with 2,4-dihydroxybenzaldehyde isonicotinoyl hydrazone (2,4-DHBINH) in the pH range 1-7. The complex shows two absorption maxima, one at 430 nm and the other at 500 nm. The reagent shows appreciable absorbance of 430 nm and negligible absorbance at 500 nm at pH 1.5. Beer's law is obeyed in the range 0.09 to 2.15 mug ml(-1) of titanium(IV). The molar absorptivity and the Sandell's sensitivity of the method are 1.35 x 10(4) 1 mol(-1) cm(-1) and 0.0049 mug cm(-2), respectively. A method for the determination of titanium by first-order derivative spectrophotometry is also proposed. The methods have been employed successfully for the determination of titanium in several alloy and steel samples.     

High-performance liquid chromatographic determination of cortolic and cortolonic acids as pyrenyl ester derivatives

A new procedure is described for the detection of the acidic metabolites of cortisol (cortoic acids) as the pyrenylmethyl-21-oic esters. The derivatizing reagent, diazomethylpyrene, was prepared by an improved procedure. The reagent was used at room temperature, required no catalyst, and was not restricted by stoichiometric requirements. The steroid esters were separated by reversed-phase high-performance liquid chromatography and analyzed simultaneously by their ultraviolet absorbance and fluorescence characteristics. Identities of the products were confirmed using the photodiode array detector to determine spectral profiles, absorbance maxima, and absorbance ratios. Further confirmation of identity of the cortoic acid esters used mass spectrometry under normal and collision-activated dissociation conditions. With the method described, a linear spectral response was obtained between 8 and 1680 fmol. Application of the technique to the analysis of steroid acids in human urine indicated the presence of cortoic acids.     

Electrochemistry and optical absorbance and luminescence of molecule-like Au38 nanoparticles

This paper describes electrochemical and spectroscopic properties of a well-characterized, synthetically accessible, 1.1 nm diam Au nanoparticle, Au(38)(PhC(2)S)(24), where PhC(2)S is phenylethylthiolate. Properties of other Au(38) nanoparticles made by exchanging the monolayer ligands with different thiolate ligands are also described. Voltammetry of the Au(38) nanoparticles in CH(2)Cl(2) reveals a 1.62 V energy gap between the first one-electron oxidation and the first reduction. Based on a charging energy correction of ca. 0.29 V, the indicated HOMO-LUMO gap energy is ca. 1.33 eV. At low energies, the optical absorbance spectrum includes peaks at 675 nm (1.84 eV) and 770 nm (1.61 eV) and an absorbance edge at ca. 1.33 eV that gives an optical HOMO-LUMO gap energy that is consistent with the electrochemical estimate. The absorbance at lowest energy is bleached upon electrochemical depletion of the HOMO level. The complete voltammetry contains two separated doublets of oxidation waves, indicating two distinct molecular orbitals, and two reduction steps. The ligand-exchanged nanoparticle Au(38)(PEG(135)S)(13)(PhC(2)S)(11), where PEG(135)S is -SCH(2)CH(2)OCH(2)CH(2)OCH(3), exhibits a broad (1.77-0.89 eV) near-IR photoluminescence band resolvable into maxima at 902 nm (1.38 eV) and 1025 nm (1.2 eV). Much of the photoluminescence occurs at energies less than the HOMO-LUMO gap energy. A working model of the energy level structure of the Au(38) nanoparticle is presented.