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An improved and semi-automated method has recently been reported by Vanderslice and Maire for determining very low concentrations of the B6 vitamers by HPLC ]. The method is sensitive enough for the analysis of whole blood and has a detection limit of 0.01 ng/ml for the metabolite pyridoxic acid and 0.10 ng/ml for the B6 vitamers. Separation of the vitamers is significantly improved over previous methods, and no interference is observed from the other water-soluble vitamins 2-5 ]. A Perkin-Elmer 650-40 fluorescence spectrophotometer and a Shimadzu CR-1A integrator are primary components of the analytical system. These instruments have so called "smart" features which are implemented by single keystroke commands and which provide capabilities that would be difficult or impossible to duplicate manually. The PerkinElmer spectrophotometer functions used for vitamin B6 analysis execute changes in zero-suppression and both excitation and emission wave-lengths. The integrator was programmed to integrate and plot the analog detector signal from the spectrophotometer, print results and control elements of the HPLC system. These "intelligent" capabilities generally reduce the time and manipulative skill required of the operator. Figure illustrates the commands entered at the instrument keyboards as they were required during the manual analysis of vitamin B6. The integrator requires a START command to begin integrating data and controlling the HPLC system for each sample. The operator must also change the spectrophotometer excitation and emission wave-lengths and zero-suppression four times during the 75 minute analysis to optimise detection of the individual B6 vitamers. These requirements interrupt the operator’s other activities and obviously prohibit unsupervised overnight operation. The need for frequent human attention during the analysis is an obstacle to full automation of the analytical system. Automating the spectrophotometer/integrator system to analyze multiple samples without human attention was accomplished using a simple, inexpensive and non-traditional technique which can be implemented on many analytical systems without special skills. The method involves interfacing a microcomputer to the instrument keyboards and associated external servo-controls and using software that allows the microcomputer to "learn" and then imitate operator actions during an analysis. The microcomputer "learns" how to carry out the analysis simply by observing what the operator does; explicit programming is unnecessary.

Control programming without language: automation of vitamin B6analysis

Control programming without language: automation of vitamin B₆analysis