Diode Array Spectrophotometer Research Articles

Study of high-molecular weight compounds in sugar using gel-permeation chromatography with an evaporative light scattering detector

Raw sugars and other sugar process materials are studied by GPC (Gel Permeation Chromatography) using a Superose 12 column. A solution of 30% acetonitrile with 0.005 M ammonium acetate was used as eluent. A spectrophotometric Diode Array Detector (DAD) and Evaporative Light Scattering Detector, in series, were used as detector. By this arrangement, both chromophoric and non-chromophoric compounds are detected simultaneously. High sensitivity of both detectors allows a rapid detection of high molecular weight compounds without pre-concentration of samples.

Gas chromatography with UV-vis molecular absorption spectrometry detection: Data acquisition and treatment when using a diode-array spectrophotometer

This article describes a theoretical/practical study of the parameters which affect the chromatograms obtained when using a molecular diode-array spectrometer as the detector in gas chromatography. The objective is to offer some rules which permit the identification of the optimum signal/noise relation. To achieve this, we study the effect of the different parameters which affect the noise and how to reduce their impact, as well as alternatives for increasing the signal. All the options tested can be applied by correctly programming the spectrometer with BASIC programs, within reach of any use who has even a small understanding of programming. Finally, we consider the effect that the selection of the acquisition conditions may have on the chromatographic resolution. All the studies are carried out using a mixture of alcohols and phenols.

Simultaneous determination of arsenic, antimony, selenium and tin by gas phase molecular absorption spectrometry after two step hydride generation and preconcentration in a cold trap system

A cold trap system for the simultaneous determination of arsenic, antimony, selenium and tin by continuous hydride generation and gas phase molecular absorption spectrometry is described. The hydride generation is carried out in two steps; first, tin hydride is generated at low acidity and second, arsenic, antimony and selenium hydrides are formed at higher acidity. All the hydrides are collected in a liquid nitrogen cryogenic trap and transported to the flow cell of a diode array spectrophotometer, where molecular absorption spectra are obtained in the 190–250 nm range. Five calibration solutions containing arsenic, antimony, selenium and tin are solved using multiple linear regression analysis. Tests are performed in order to extend the same manifold to other hydrides but no signals are obtained for bismuth, cadmium, lead, tellurium and germanium. Under the optimum conditions found and using the wavelengths of maximum sensitivity (190, 198, 220 and 194 nm), the analytical characteristics of each element are calculated. The detection limits are 0.050, 0.020, 0.12 and 1.1 μg ml −1 and the RSD values are 3.7, 3.1, 3.5 and 3.0% for As, Sb, Se and Sn, respectively. The method is applied to As, Sb, Se and Sn determination in natural spiked water samples.

High-pressure solubility of 1,4-bis-( n-alkylamino)-9,10-anthraquinones in near- and supercritical carbon dioxide

Solubilities of 1,4-bis-( n-alkylamino)-9,10-anthraquinones (with n-alkyl=methyl (AC01), ethyl (AC02), butyl (AC04), pentyl (AC05), octyl (AC08)) in CO 2 were measured using a flow method. The concentrations were determined in the temperature range from 299 to 345 K and at pressures from 7 to 20 MPa. The apparatus mainly consists of a supercritical fluid chromatograph using a diode array spectrophotometer for the detection of the dissolved dyestuff. The solubilities are discussed with respect to the pressure and density of the fluid phase. Intersection points of the concentration versus pressure solubility isotherms are found here for the methyl-derivative and discussed as a density effect. Additionally, the results of the different derivatives are compared and discussed with respect to the chain length of the alkyl-substituent. A maximum of the dyestuff solubility is obtained for the pentyl-derivative.

Elastotic degenerative change and yellowish discolouration in photoaged skin.

One clinical sign of photodamage is a sallow discolouration of the skin, and solar elastotic degenerative change in the upper dermis may cause this change. We have attempted to quantify these phenomena and relate them to each other and to age. Twenty-seven healthy volunteers (age 18 to 61 years) were recruited. We examined the back of the hand of photodamaged individuals and the inner upper arm to demonstrate changes due to ageing. Each site was assessed for cutaneous blood flux using a laser Doppler fluxmeter (PF4000, Perimed UK), and for melanin pigmentation using a Melanin Index meter (Dermotronics Ltd, Cardiff, UK). Skin colour was measured using a diode array spectrophotometer (MCPD-1000, Photal, Japan), which computes CIE coordinates L*, a*, b*. Biopsies from each site were obtained from 13 of the subjects and were processed and stained with Gomori's aldehyde fuchsin. The percentage area of elastic staining material in the dermis was measured using image analysis. Sections were measured in four bands of 200 micron depth, and four fields were measured per band. Neither melanin content nor skin blood flux correlated with age for either skin site. Percentage area of elastosis correlated with age on the exposed site from the uppermost 600 microns of the dermis, (<200 micron depth, r=0.74, P<0.01, 200<depth<400, r=0.57, P<0.05; 400<depth<600, r=0.57, P<0.05). No correlation was found on the unexposed inner upper arm site. On the back of hand site, a linear relationship was found between a parameter derived from the L*, a*, b* coordinates of (b*-5a*) and both age (r=0.904, P<0.001) and percentage area of elastosis of the uppermost 200 microns of dermis (r=0.74, P<0.01). This was not seen for the inner upper arm site. On exposed sites, the coefficient (b*-5a*) correlates with solar elastosis and these both correlate with age, which is related to exposure. This data confirms that solar elastosis accounts for the yellowish discolouration of sun damaged skin, and suggests that the coefficient b*-5a* could be used for the non-invasive quantification of solar elastosis.

Hemoglobin–Oxygen Equilibrium Curves Measured during Enzymatic Oxygen Consumption

A rapid, new method to measure hemoglobin–oxygen equilibrium curves is described using the protocatechuic acid/protocatechuic acid 3,4-dioxygenase system [C. Bull and D. P. Ballou (1981)J. Biol. Chem.256, 12673–12680] to deoxygenate hemoglobin solutions enzymatically. The reaction is followed by simultaneous measurements of hemoglobin spectra using a diode array spectrophotometer and oxygen tensions using a polarographic O2microelectrode. Multicomponent analysis allows the determination of fractions of oxyhemoglobin, deoxyhemoglobin, and high-spin and low-spin methemoglobins in each spectrum collected as the reaction proceeds. Fractional saturation as a function of oxygen partial pressure is calculated as the ratio of oxyhemoglobin to oxy- plus deoxyhemoglobin. Several advantages are offered by this method: (i) Hemoglobin–O2binding curves are obtained rapidly and reproducibly; (ii) the speed of the reaction limits methemoglobin formation by autooxidation; (iii) there is no gas–liquid interface, eliminating protein denaturation at the surface; and (iv) direct calculations of fractional saturation are made using spectral analysis, thus avoiding the assumption of a linear transition between deoxy- and oxyhemoglobin.

Partition Analysis of an Enzyme Acting Concurrently upon Two Substrates in a Continuous Multiwavelength Assay

We describe a multiwavelength method for measuring an enzyme's discrimination of one substrate over another by continuously monitoring the reactions of the two substrates simultaneously. This method is generally applicable to ultraviolet–visible diode array or rapid-scanning spectrophotometers and the measurement requires a single incubation of enzyme with two substrates. Rates at each of the wavelengths may be fit globally by using a nonlinear least-squares fitting procedure which provides adequate statistical analysis. The specificity of trypsin forN-α-benzoyl-l-argininep-nitroanilide (BRpNA) overN-t-butyloxycarbonyl-l-alanine-p-nitrophenylester (BocApNP) was 2.1 as measured by the multiwavelength partition method and 2.3 by comparing the individualkcat/Km's for the two substrates. Multiwavelength analysis was applied to two enzymes in the biosynthetic pathway for fungal melanin: scytalone dehydratase and trihydroxynaphthalene reductase fromMagnaporthea grisea.The specificity of trihydroxynaphthalene reductase for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one compared toscytalone, a natural substrate for the enzyme, was 95. Scytalone dehydratase was eight-fold more specific for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one than it was for scytalone. Multiwavelength analysis was also used to measure an equilibrium constant of 0.040 for the reaction {dihydroorotate + oxonic acid ↔ orotate + dihydrooxonic acid} catalyzed by dihydroorotate dehydrogenase. Advantages, limitations, and further applications of this steady-state method, which directly measures relative substrate specificities, are delineated. All studies described in this paper were at pH 7.0 and 25°C.

Measurement of total alkalinity of surface waters using a continuous flowing spectrophotometric technique

Methodology and instrumentation are described which were used for continuous measurement of total alkalinity (TA) in surface seawater during a Joint Global Ocean Flux Study (JGOFS) cruise in the Indian Ocean. The procedure involves a single step acid addition of HCl to a known volume of seawater with rapid spectrophotometric measurement of pH (3 to 4) and calculation of the TA after stripping out the CO2. The flowing total alkalinity system (FLTA) consists of a calibrated volumetric pipette (∼100 cm3), a thermostated mixing cell (∼200 cm3), a computerized piston titrator, a platinum thermometer, and a Hewlett Packard diode array spectrophotometer with a flowing spectrophotometric cell as the detector. All the electronic components have interfaces which are easily connected to a personal computer and manipulated using a Quick Basic program. The FLTA system makes continuous measurements of TA from the shipboard flowing seawater line. Laboratory results (N=127) on Gulf Stream seawater (GSSW) have shown a precision within ±2–3 μmol kg−1 in TA. Certified reference materials (CRMs) measured at sea (N=14) gave precise total alkalinities to ±2.2 μmol kg−1. Intercomparison of the total alkalinity of underway surface samples (N=16) obtained in the Indian Ocean using potentiometric and spectrophotometric titration systems gives average differences of ±3 μmol kg−1 in TA. The agreement between the two methods is very good considering the temporal and spatial differences in the sampling. The continuous measurement of TA using the FLTA system may reflect small variations of TA in the surface waters of the oceans which would not be visible when measuring TA in samples taken from CTD casts at individual stations. These data can also be combined with flowing pH or pCO2 data to fully characterize the carbonate system in surface waters.

Improved selectivity in multicomponent determinations through interference modelling by applying partial least squares regression to kinetic profiles

Binary and ternary mixtures of cysteine, tyrosine and tryptophan were used to show how kinetic methods together with partial least squares regression (PLSR) can be used to overcome interferences in multicomponent determinations. Two methods were compared: an equilibrium method which uses the characteristic UV spectra of amino acids and a kinetic method which follows the reaction kinetics of the oxidation of the amino acids by hexacyanomanganate(IV). In both cases, multi-wavelength detection with a diode-array spectrophotometer was used. The results obtained from the two methods are critically compared. Lower concentrations of tyrosine and tryptophan can be determined by the equilibrium method because of their high absorptivities. On the other hand, the kinetic method, in conjunction with PLSR, can be considered an example of how selectivity can be improved. This allowed the resolution of mixtures of tyrosine and tryptophan in the presence of several complex spiked matrices (aqueous extracts of feed samples that interfere to different extents) without any previous separation.

Multivariate Curve Resolution and Trilinear Decomposition Methods in the Analysis of Stopped-Flow Kinetic Data for Binary Amino Acid Mixtures

A stopped-flow method is proposed to carry out the kinetic development of the reaction between amino acids and 1,2-naphthoquinone-4-sulfonate by mixing analytes and reagent in a three-channel continuous-flow system. The process is monitored using a diode array spectrophotometer. Thus, every sample produces a data matrix built up from the spectra registered at regular steps of time. As the reaction is faster for secondary amino acids than for primary ones, it is possible to distinguish between the kinetic formation of their corresponding derivatives. The method is applied to the simultaneous determination of phenylalanine and proline by using second-order multivariate curve resolution. The derivatives of these two amino acids present some differences in both orders of measure, i.e., their spectra and kinetic profiles, which can be exploited advantageously to quantify one of the analytes in the presence of the other as interference, without including any information about this interference in the modeling of the system.

Intrinsic Specificity of the Reactive Site Loop of α1-Antitrypsin, α1-Antichymotrypsin, Antithrombin III, and Protease Nexin I

Members of the serpin (serine protease inhibitor) family share a similar backbone structure but expose a variable reactive-site loop, which binds to the catalytic groove of the target protease. Specificity originates in part from the sequence of this loop and also from secondary binding sites that contribute to the inhibitor function. To clarify the intrinsic contribution of the reactive-site loop, alpha1-antichymotrypsin has been utilized as a scaffold to construct chimeras carrying the loop of antithrombin III, protease nexin 1, or alpha1-antitrypsin. Reactive-site loops not only vary in sequence but also in length; therefore, the length of the reactive-site loop was also varied in the chimeras. The efficacy of the specificity transfer was evaluated by measuring the stoichiometry of the reaction, the ability to form an SDS-stable complex, and the association rate constant with a number of potential targets (chymotrypsin, neutrophil elastase, trypsin, thrombin, factor Xa, activated protein C, and urokinase). Overall, substitution of a reactive-site loop was not sufficient to transfer the specificity of a given serpin to alpha1-antichymotrypsin. Specificity of the chimera partly matched that of the loop donor and partly that of the acceptor, whereas the behavior as an inhibitor or a substrate depended upon the targeted protease. Results suggest that, aside from the contributions of the loop sequence and the framework-specific secondary binding sites, an intramolecular control may be essential for productive interaction.

Quantitative Spot Tests Of Fe(III), Cr(VI) And Ni(II) By Reflectance Measurements

ABSTRACT This work shows that diffuse reflectance measurements, using fiber optical devices, can give rapid quantitative results for application with spot tests determination where little sample manipulation is a desirable feature. The experiments were performed with a Hewlett Packard diode array spectrophotometer HP8452A and a Labsphere RSA-HP-84 reflectance accessory. The quantitative reflectance measurements of different complexes such as Fe (III) with thiocyanate, Cr (VI) with diphenylcarbazide and Ni (II) with dimethylglyoxime could be obtained by forming the complexes on filter paper. Calibration curves were obtained for each metal by plotting the optical density of the reflectance signal (AR) vs. the log of the mol/dm3 concentration, from 1.5 × 103 to 3.6 × 102mol/dm3 for Fe (III), from 8.00 × 10−5 to 2.00 × 103 mol/dm3 for Cr (VI) and from 1.00 × 103 to 8.00 × 102 mol dm3 for Ni (II), with correlation coefficients of 0.9986 for Fe (III), 0.9878 for Cr (VI) and 0.9892 for Ni (II). The results obtained show that it is possible to perform quantitative spot-test analysis using reflectance measurements in the visible region of the spectrum.

The spectrophotometric determination of the pka of pyrrolidinedithiocarbamic and piperidinedithiocarbamic acids, based on diode array measurements

A spectrophotometric method, based on the use of a diode array spectrophotometer, is proposed in order to determine the pKa of pyrrolidinedithiocarbamic (HPyr-I) and piperidinodithiocarbamic (HPip-II) acids. The high speed of data acquisition of the equipment made it possible to minimize the problem of acid decomposition. The values obtained were 3.10(I) and 3.51(II) in I = 0.50 mol L-1 (NaClO4) at 25.0 °C, and are consistent with the acid decomposition mechanism. A discussion is presented based on the shape of the compound spectra in acidic and basic media and their relation to the order of the pKa values for I and II.

Enhanced decompositions of some cyano complexes during diode array spectrophotometric measurements

Acidic solutions of the Fe(III), Mo(V), W(V) and Mn(IV) cyano complexes are known to decompose photochemically. These reactions do not take place to an appreciable extent when the cyano complexes are subjected to spectrophotometric measurements by means of conventional spectrophotometers, but they do take place when diode array spectrophotometers are used. The reactions are autoaccelerated, at least in the cases of Mo(V), W(V) and Mn(IV), and consist of photoinduced reductions of the metal ion. This effect should be taken into account in all cases where the measured signal has to be used for quantitative purposes, but its significance must be emphasized mainly in the field of kinetic analysis, where it can lead to large errors. The extent of these reactions, and therefore the size of the errors, can be lowered by using UV cut-off filters and by taking discontinuous measurements with suitable integration times. All these possibilities, which are of general application, are discussed.

Using dynamic data exchange to exchange information between Visual Basic and Matlab: Application to a diode array spectrophotometer

Photodiode array spectrometers are increasingly being coupled to flow analysis and separation techniques. Computer programs are needed to control the data acquisition from each photodiode and to handle the data. The Microsoft Visual Basic programming language is used along with a DLL library and dynamic data exchange (DDE) to allow communication with the MATLAB computational environment.

Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs

The combination of chromatographic separation using reverse-phase high-performance liquid chromatography and continuous monitoring of the column eluate using a diode-array spectrophotometer allowed qualitative and quantitative pigment profiling of extracts of photosynthetic material in a single run. Carotenoids and the spectrally distinct types of chlorophyll and bacteriochlorophyll can be unambiguously identified even when imperfectly separated on the column. The resulting spectrochromatogram is a fingerprint useful for the rapid characterization of pure cultures or mixed populations. We have developed software to allow recording, manipulation, and presentation of the resulting spectrochromatograms and present results from photosynthetic microbes in pure and mixed cultures. We describe a number of approaches to the presentation of the resulting data in a readily comprehensible form.

Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs

The combination of chromatographic separation using reverse-phase high-performance liquid chromatography and continuous monitoring of the column eluate using a diode-array spectrophotometer allowed qualitative and quantitative pigment profiling of extracts of photosynthetic material in a single run. Carotenoids and the spectrally distinct types of chlorophyll and bacteriochlorophyll can be unambiguously identified even when imperfectly separated on the column. The resulting spectrochromatogram is a fingerprint useful for the rapid characterization of pure cultures or mixed populations. We have developed software to allow recording, manipulation, and presentation of the resulting spectrochromatograms and present results from photosynthetic microbes in pure and mixed cultures. We describe a number of approaches to the presentation of the resulting data in a readily comprehensible form.

Quality control of pharmaceuticals containing clenbuterol by thermal lens spectrometry

An ultrasensitive absorptiometric procedure for the determination of clenbuterol in pharmaceutical preparations was developed. Clenbuterol was diazotized with nitrite and coupled with 1-(naphthyl)ethylenediamine, and the absorbance of the azo dye formed was measured by both spectrophotometry and ultrasensitive thermal lens spectrometry (TLS). The TLS limit of detection was 1.5 ppb, 14-fold lower than with a Hewlett-Packard diode array spectrophotometer. Thus, the TLS procedure can be advantageously applied to quality control of clenbuterol at the individual dose level and in small samples. Repeatability as relative standard deviation was 1.5% (50 ppb, n = 6).

Determination of active components in insecticide formulations by liquid chromatography and resolution of overlapped peaks by multivariate analysis and derivative spectrophotometry

In liquid chromatography, complex mixtures of pesticides may show peaks with a great overlap under the experimental conditions commonly used. The use of partial-least squares (PLS) regression and derivative spectrophotometry makes it possible to discriminate between the spectral bands of the overlapping chromatographic solutes, taken from peaks with co-eluting species. In this paper, a procedure for the determination of insecticides in commercial formulations by using a diode-array spectrophotometer as chromatographic detector has been devised. The procedure consists of two steps. In the (first) chromatographic step, the active component peak (corresponding to fenitrothion or permethrin) which is isolated in time is determined by conventional liquid chromatography; the appropriate conditions permit to remove also the interference from solvents in the commercial formulations. In the (second) multivariate step, partial-least squares regression is applied to the overlapped spectra of the co-eluting components (piperonyl butoxide-neopynamine or dicofol-tetradifon). This method makes it possible to analyze compounds showing appreciable overlap of their chromatographic peaks, without modification of the experimental variables used.

Simultaneous kinetic spectrophotometric determination of o-, m-and p-aminophenol using partial least squares calibration

The oxidative coupling reactions of p-aminophenol with m-and p-aminophenol in the presence of octacyanomolybdate(v) were used as the basis of a differential kinetic method for the simultaneous determination of o-, m-and p-aminophenol (o-, m-and p-AP) in binary and ternary mixtures. The influence of Mov concentration, acidity, ionic strength and temperature was studied in order to establish optimum conditions. Several oxidants, including Ce4+, CrVI, MnVII, H2O2 and metaperiodate and FeIII, MnIV, MoV and WV cyano complexes were tried. The MoV cyano complex was chosen because it has a suitable redox potential and gives rise to kinetically useful runs. The reaction must be implemented under sub-stoichiometric concentrations of oxidant and so the concentration of each aminophenol in the mixture influences the reaction of the others, originating in a system with a high degree of non-linearity. As a result, most methods of data treatment cannot be used and multivariate calibration (PLS) was applied to the data acquired by a diode-array spectrophotometer between 340 and 500 nm. PLS calibration with a number of factors ranging between six and nine allowed satisfactory results for all kinds of mixtures. Aminophenols can be determined over the concentration range 0.1–1.0 mmol l–1 for o-AP, 0.3–1.0 mmol l–1 for m-AP and 0.25–3.0 mmol l–1 for p-AP with errors of less than 10% in all instances. No systematic errors were detected.