A Problem in Calibrating a Spectrophotometer James G. Johnson Depaltment of Biology, University of Calgary, Calgary. Alberta. Canada T2N IN4
Here is a little lesson in simple trouble-shooting of a spectrophotometer. Our instrument was a single-beam UV-vis Gilford Model 260, but the lesson is applicable to other models and, indeed, to other types of instruments. We were estimating protein concentrations by measuring absorptions a t 280 nm. Twofold dilutions of each sample were made, and readings throughout the full range of the instrument (0-3 A) were exnected. We beean to notice nonlinearity a t absorbances than 1.2. it took a while for this to become a worry, as we tended just to take the lower readings which did agree a t each dilution. We only slowly realized that no high absorbance readings - were being encountered a t all. We then carried out the available calibration tests.
1) The wavelengths were correct aa tested with the factory-supplied
holmium oxide filter. 2) The lamp energies were acceptable. This was shown by comparing the slit widths necessary to zero the machine (no cuvettes, air reference) to the factory specificationsthat had been supplied with the new instrument. Also, our daily record of slit widths required for our standard procedures had shown no unusual increases. 3) There was n ~ electronic , problem preventing the photometer unit from displaying high ahsorhance values. This was checked by closing the slit completely and noting that the display went to its maximum value above 3.0 A. 4) Gilford's Model 202 factory-calibrated absorbance standards gave the proper readings. Model 202 is a solid metal block of the same dimensionsas the normal Cplace cuvette holder. Set in the block are neutral filter discs (14-mmdiameter) of nominal absorhances of 0.1,1.0, and 2.0 (550 nm). 5) Finally, in desperation, we prepared a series of standard solutions. In our cuvettes in the cuvette holder, the readings were linear up to about 1.2 A, but then they became nonlinear with a maximum around 1.5 A. The same samples, cuvettes, and holder gave the expected linear readings up to 2.8 A or more in a colleague's Gilford.
What a puzzle: one set of absorbance standards reads correctly, and one set does not! What is your diagnosis of the problem? In retrospect, of course, i t was simple. The problem had to be in the sample handling area, between the slit and the photometer unit. We found that the spring clamp on the cuvette carriage into which the cuvette holder is inserted was nearly broken. The cuvette holder was not being clamped firmly in the correct position in the light beam. An edge of the beam was strikine the side of the cuvette. The cuvette sides were transpare;, so some of this edgelight was reaching the photometer without passing through the sample. At ~
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high absorbances (when the sample allows very little light to pass through), this edge light dominated, holding the absorbance reading to a maximum of about 1.5 A. The alignment error could easily be seen by darkening the room and observing a visible wavelength with the cuvettes and cuvette holder in place and the sample compartment uncovered. The cure was to replace the spring clamp and to align the cuvettes visually with the sample compartment open as described. The same svmntoms and problem are caused bv using an aperture too broad for the c&ette width. Again, the sides of the light - beam pass through . the cuvette sidewall rather than through the sample volume. Correct alignment and correct aperture choice solve this problem. Self-masking, blackwalled cuvettes are a useful extra precaution. The important point is that the factory standards read correctly because they are constructed of a wide (14-mm) filter disc. The small alignment error (1 mm or less) was not sufficient to displace the light beam to the edge of the disc. In other words, the Model 202 standards are insensitive to small alignment errors. However, the same small misalignment drasticallv affected the narrow (4-mm broad samole region) semimi&o cuvettes used for daily work. The nonfinearity in absorbance readings was onlv aooarent when standard~solutionswere read inihese workin~cuvettes. The Gilford manual describes only calibrations 1.2, and 4. above, and all these are performed-without the cuvettes in place. The obvious minor lesson from our experience is that the simole alienment check should be added to rhe calihra~ ~ ~ - tion checks i n k e manual. Themore important lesson is that absorbance calibrations should also be carried out usine solutions in the actual working cuvettes. The Model 20i absorbance standards do not exactlv dunlicate workine conditions. The same may he true f i r other manufacGrers' spectrophotometers and their peculiar absorbance standards. The general lesson, applicable to all instruments, is that standardization checks should he carried out under exactly the same conditions as used for daily work with the instrument. The specific calibration checks are then useful for isolating any problem to the separate components of the instrument. Our oainful exoerience emuhasizes that these ~ specific calibration checks might all be' perfect, while the instrument as a whole is still delivering misleading data. Making your own calibration solutions may be a bit more work,' but is i t not worthwhile to out-maneuver Murnhv . occasionally? Note: Quick, convenient aqueous standards giving absorb a n c e ~near 3.0 are 0.018% (v/v) Triton X-100 (225 nm), 0.13%Triton (275 nm), and 0.20g/mlof C O ( N O ~ ) Y ~ H(500 IO linear nm). The exact absorbances are not importa&:as series of decreasing concentrations is made by accurate dilution from each of these stock solutions. ~~~
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Volume 62 Number 10 October 1985
885
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