Automated measurement of dye coverage in photographic negatives by flow-injection analysis.

A system was designed to automate the determination of three image dyes in an instant photographic material. The method involves extracting negative samples with dimethylsulfoxide and filtering the extract, followed by quantitating the dye coverage (dye per unit area of negative) through a spectrophotometric flow-injection procedure. Significant spectral overlap exists among the dyes, and as a result calculation of coverage requires solving three simultaneous equations. A microcomputer and data acquisition system were employed for controlling the detector and flow-injection system, acquiring and integrating the detector response, calculating coverage, producing and displaying control charts, and automatically transferring results to a VAX based corporate database. In addition to automating sample preparation and measurement steps as much as possible, the goal of this project was to automate the data manipulation and transfer steps.

Accuracy of peak deconvolution algorithms within chromatographic integrators.

The soundness of present-day algorithms to deconvolve overlapping skewed peaks was investigated. From simulated studies based on the exponentially modified Gaussian model (EMG), chromatographic peak area inaccuracies for unresolved peaks are presented for the two deconvolution methods, the tangent skim and the perpendicular drop method. These inherent inaccuracies, in many cases exceeding 50%, are much greater than those calculated from ideal Gaussian profiles. Multiple linear regression (MLR) was used to build models that predict the relative error for either peak deconvolution method. MLR also provided a means for determining influential independent variables, defining the required chromatographic relationships needed for prediction. Once forecasted errors for both methods are calculated, selection of either peak deconvolution method can be made by minimum errors. These selection boundaries are contrasted to method selection criteria of present data systems' algorithms.

LC-EC determination of nucleotides and nucleic acids: application to enzyme assays and the analysis of DNA fragments.

High-performance liquid chromatography (HPLC) coupled to an electrochemical detector in an oxidative mode was used to analyze purine bases, nucleosides, and nucleotides as well as restriction fragments of nucleic acids. Ligands were separated by liquid chromatography with electrochemical detection (LC-EC) using size exclusion, ion-exchange, or reverse phase techniques. Using an amperometric electrochemical detector the determination was characterized with respect to sensitivity, selectivity, and capacity factor. It was observed from hydrodynamic and cyclic voltammetry that the optimum oxidation potential differed for the three major classes of purines, permitting an enhancement in selectivity when compared to detection. Guanylyl moieties demonstrated a half-wave potential at 0.800 V vs Ag/AgCl, while those for the adenylyl and inosylyl groups are above 1,000 V vs Ag/AgCl. The facility of the method to analyze components of a complex biological milieu was demonstrated by examining the purine pools of crude and partially purified eye lens homogenates as well as by comparing the traditional hexokinase assay to the newly developed LC-EC technique. Additionally, LC-EC was compared to detection for determination of the purine-metabolizing enzyme activities, adenylate deaminase and adenylosuccinate synthetase from crude cellular lysates of the cellular slime mold, Dictyostelium discoideum. Finally, the technique was used to assay the fragments from lambda-DNA cut with the restriction endonuclease Pst-1.