Reliable TLDA-microvolume UV spectroscopy with applications in chemistry and biosciences for microlitre analysis and rapid pipette calibration

A TLDA-microvolume (transmitted light drop analyser) accessory for use with a standard UV-visible fibre spectrophotometer is described. The physics of the elegantly simple optical design is described along with the experimental testing of this accessory. The modelling of the arrangement is fully explored to investigate the performance of the drop spectrophotometer. The design optimizes the focusing to deliver the highest quality spectra, rapid and simple sample handling and, importantly, no detectable carryover on the single quartz drophead. Results of spectral measurements in a laboratory providing NIST standards show the closest correlation between modelled pathlength and experimental measurement for different drop volumes in the range 0.7-3 mu l. This instrument accessory delivers remarkably accurate and reproducible results that are good enough to allow the accessory to be used for rapid pipette calibration to avoid the laborious weighing methods currently employed. Measurements on DNA standards and proteins are given to illustrate the main application area of biochemistry for this accessory. The accessory has a measurement range of at least 0-60 A units without sample dilution and, since there exists an accurate volume-pathlength relationship, the drop volume used in any specific measurement or assay should be optimized to minimize the photometric error. Studies demonstrate that the cleaning of the drophead with lab wipes results in no measurable carryover. This important practical result is confirmed from direct reading of the accessory and an analytical balance which was used to perform carryover studies.