0000000000117617
AUTHOR
M.j.medina Hernández
Continuous-flow tristimulus colorimetry: a new approach for gradient scanning techniques
Abstract A flow-injection gradient scanning technique for colour evaluation by means of tristimulus colorimetry is described. Equipment and data acquisition requirements are discussed. The program CHROMA.FIA data the treatment and comparative chromatic analysis is presented. The chemical and flow conditions were optimized. Comparative studies using metallochromic indicators with both the flow-injection and the conventional batch procedures were made. The continuous-flow procedure provides good results and is more than fifteen times faster than the manual titrimetric procedure.
Some observations on the automation by flow injection analysis of the spectrophotometric determination of amino acids and proteins witho-phthalaldehyde
Automation by flow injection analysis with Spectrophotometric detection of the determination of total amino acids and proteins witho-phthalaldehyde is not straightforward. The use of spectrophotometry, instead of spectrofluorimetry, and of N-acetyl-L-cysteine, instead of the conventional mercaptoethanol is advantageous because of the lower variability of absorptivities with respect to fluorescence yields, and the larger stability of the derivatives. Under adequate working conditions and with leucine as reference, the procedure can be used for the evaluation of total amino acids. A similar procedure is proposed for the analysis of proteins in a sample. Limits of detection are ≈ 1 × 10−5M for…
Thermal lens spectrometric determination of cerium with oxine
Abstract The spectrophotometric and photothermal (TLS) procedures for cerium determination using 8-hydroxyquinoline (oxine), after extraction into chloroform, are compared. Photothermal measurements are made using a coaxial pump/probe thermal lens spectrometer. The use of high-purity reagents at low concentrations permits a decrease in the TLS blank signal and noise, leading to a limit of detection of 9 × 10−9 M (cerium extract concentration), 40-fold lower than the spectrophotometric value. The dynamic range extended up to 6 × 10−7 M and the relative standard deviation for 5 × 10−7 M cerium was 3.9%.
Spectrophotometric Determination of Cystine with O-Phthalaldehyde in the Absence of Thiol
Abstract A spectrophotometric method for the determination of cystine with o-phthalaldehyde (OPA) in the absence of thiol is described. When cystine is heated at 60[ddot]C for 30 min in a low excess of OPA (pH 9.5), a very stable derivative with 1:2 stoichiometry (cystine:OPA) and an absorption maximum at 335 nm (E = 4600) is formed. At pH < 1 the derivative is protonated (protonation constants: log K1 = 5.88 and log K2 = 3.70 at I = 0.1 and 20[ddot]C) and another absorption band at 440 nm (E = 3800) appears, which allows the determination of cystine in the presence of other amino acids.
Use of the o-Phthalaldehyde and N-Acetyl-L-Cysteine the Evaluation of Milk Proteins
Abstract o -Phthalaldehyde and N-acetyl-Lcysteine are used in the determination of milk proteins. Three procedures are proposed and compared. One of them is based on reaction of o -phthalaldehyde and N-acetyl-Lcysteine with the intact proteins and the two others on reaction of the reagents with the released amino acids after total acid hydrolysis of the protein samples. When the protein sample is hydrolyzed, calibration is performed either with a hydrolyzed protein standard or with isoleucine. A procedure for the measurement of the degree of enzymatic hydrolysis of milk proteins without separation of the unhydrolyzed protein, which makes use of the same reagents, is also described. In all c…
Spectrophotometric determination of cystine by formation of an o-phthalaldehyde/N-acetyl-l-cysteine derivative
Abstract Cystine reacts with o -phthalaldehyde (OPA) in the absence and presence of a thiol compound to yield different compounds. The use of N -acetyl- l -cysteine as thiol leads to the formation of two derivatives, likely simple and double isoindoles, where the disulfide bond remains unbroken. In contrast, mercaptoethanol gives rise to the reduction of the amino acid to form a cysteine derivative. Obtaining cystine isoindoles makes it possible to spectrophotometrically determine the amino acid after Chromatographic separation and is further evidence of the large stabilization effect produced by N -acetyl- l -cysteine in the formation of OPA-thiol derivatives.
Determination of total free amino acids with o-phthalaldehyde and N-acetyl-l-cysteine
Abstract A spectrophotometric procedure is proposed for the determination of total free amino acids after reaction with o-phthalaldehyde and N-acetyl- l -cysteine, using isoleucine as the reference. The procedure was applied to the analysis of five samples of widely different composition. Recoveries were 98–105%.
Available Lysine in Protein, Assay Using o-Phthalaldehyde/ N-Acetyl-L-cysteine Spectrophotometric Method
An assay was based on reaction of free e-amino groups in proteins with the o-phthalaldehyde/N-acetyl-L-cysteine reagent to form isoindoles, which absorb at 335 nm. The procedure was suitable for proteins or mixtures of proteins with available lysine contents of more than 5 moles lysine/mole protein and required absence of free amino acids and peptides. This method was simpler and more convenient than other methods, since it did not require hydrolysis, amino acid analysis, long heating periods or solvent extraction.
Evaluation of the proteolysis degree with the o-phthalaldehyde/N-acetyl-L-cysteine reagent
The o-phthalaldehyde/N-acetyl-L-cysteine (OPA-NAC) reagent is applied to the spectrophotometric evaluation of the proteolytic activity of enzymes. The high stability of the OPA-NAC isoindoles makes a strict control of the time of reaction unnecessary. A mathematical expression is proposed to calculate proteolysis degrees, where the absorbance decrease of the OPA-NAC derivative of the protein itself during the hydrolysis process is taken into account. The method is applied to bovine serum albumine, caseine, lysozyme, lactoglobuline and protamine sulphate as substrates, and pronase, papaine, trypsin and chymotrypsin as enzymes.
High-performance liquid chromatographic determination of diuretics in urine by micellar liquid chromatography.
The use of micellar liquid chromatography for the determination of diuretics in urine by direct injection of the sample into the chromatographic system is discussed. The retention of the urine matrix at the beginning of the chromatograms was observed for different sodium dodecyl sulphate (SDS) mobile phases. The eluent strengths of a hybrid SDS-methanol micellar mobile phase for several diuretics were compared and related to the stationary phase/water partition coefficient with a purely micellar mobile phase. The urine band was appreciably narrower with a mobile phase of 0.05 M SDS-5% methanol (v/v) at 50 degrees C (pH 6.9). With this mobile phase the determination of bendroflumethiazide an…