Use of a wedge cuvette in thin layer photometry and its application to oximetry.

A wedge cuvette was constructed by fixing 2 glass plates at a known angle with a spacer at one end. This resulted in a thin layer with thickness varying from 0 to 250 micrometer. By measuring the intensity of a beam of light through the thin layer as a function of distance along the wedge (and thus layer thickness), the absorption coefficient at the light wavelength used could be obtained without a separate measurement of I0, the reference light intensity. In addition, the difficult problem of determining accurate layer thickness as encoutered in conventional thin layer photometry has been avoided. Tests of the wedge cuvette method with Evans Blue and Malachite Green serial dilutions as well as with haemoglobin solutions at several oxygen saturations demonstrate that accuracy of the order of 1% can be obtained. Application of the wedge cuvette in experiments on oxygen uptake by layers of haemoglobin solution are discussed.