We report a detailed analysis of the effects of photometric errors in absorption spectroscopy. We show that photometric errors can be divided into five categories, each having a different effect on the relative concentration error for equilibrium and kinetic analyses. Error coefficients defined for each category are incorporated into mathematical equations that permit one to calculate both systematic and random components of the error. We show that the transmittance for minimum error depends largely on the types of errors that predominate, and equations are presented that can be used to predict the optimum point for any combination of systematic or random error coefficients. These equations are applied successfully to one commercial spectrometer for which data were published recently and to two custom-designed spectrometers with very different performance characteristics. We think that this error analysis can be applied successfully to virtually any spectrometer currently used in clinical laboratories and that the results of the analysis will identify the nature and origin of the major sources of error. We believe this to be an essential step in any program aimed at improving reliability of analytical results.

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We studied the kinetics of the reaction between cholesterol and sulfuric acid in acetic acid-acetic anhydride medium. Results have been used to establish near-optimal conditions for the fast kinetic determination of cholesterol in serum. The reaction rate measured during the first 20 s of the reaction is proportional to cholesterol concentration. There is good agreement (<2% deviation) between pseudo-first-order rate constants for cholesterol standards and sera. Recoveries of standard cholesterol added to sera range from 99% to 104% (average, 100.7%). Values for serum cholesterol by this kinetic determination tend to be somewhat lower than equilibrium values reported by local hospital laboratories. Whether bilirubin interferes depends on reaction conditions; under optimal conditions, each milligram of bilirubin is kinetically equivalent to about 1 mg of apparent cholesterol. Kinetic data are included to show how results are degraded by operating under nonoptimal conditions, and considerations involved in changing experimental conditions are discussed.

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