Spectrophotometer absorbance

The program for monitoring the absorbance at a specific wavelength of maximum absorbance pre-determined by taking the whole spectrum of each adsorbate was then ran on the software of the spectrophotometer. Absorbance data were recorded in programmed time intervals of 2 min imtil the eqtrilibrium is reached. Then these data were converted into concentration data using calibration relatiorrs predetermined at the wavelength of interest for each adsorbate. 

Table 20-1 gives the absorbance of a solution that you can prepare to test the accuracy of absorbance measurements on your spectrophotometer. Absorbance accuracy is affected by all components of the spectrophotometer, as well as stray light. Standards are also available to measure wavelength accuracy.9... 

Solution The other solutes may also absorb light at 615 nm. The best negative control would be an identical mixture with the chemical of interest entirely absent. Known concentrations of the chemical could then be added to the negative control to create positive controls (external standards) and develop a calibration curve of the spectrophotometer absorbance reading at 615 nm as a function of concentration. 

Color was determined by the method of Somers and Evans (10). All absolbance measurements were conducted using a Beckman (Irvine, CA) DU-6 spectrophotometer. Absorbance of the wine was measured at 420 and 520 nm with a 1 mm length path cuvette. Thirty yl of 20% sodium metabisulfite solution were added to another 2 ml of wine. Samples were mixed by inversion and allowed to stand at 20°C for 45 min and absorbance measured at 520 nm. One hundred yl of wine were added to 10 ml of 1 M HC1, mixed and held at 10°C for 3 hr and absorbance measured at 520 nm. All absorbance values were adjusted to a 10 mm length path and corrected for dilution. Color expressions were calculated as follows ... 

In addition, a calibration graph of sulfonate concentration versus ultraviolet absorbance was obtained for each microemulsion prior to adsorption. A small known volume of microemulsion was diluted with methanol ("spectro" grade) to obtain a clear sulfonate stock solution which was used to prepare four still more dilute methanol solutions of varying sulfonate concentration. The UV absorbance spectra of these solutions were measured on a Cary 118 spectrophotometer absorbances were determined at 271 nm for PDM-334 microemulsions and at 259 nm for TRS 10-410 microemulsions. All calibration plots were linear and passed through the origin. 

The two other detectors are based on absorbance. The fixed-wavelength UV detector essentially is a marriage of a flow cell and a UV/VIS spectrophotometer. Absorbance at one wavelength at a time can be detected, but most such detectors can operate at more than one wavelength. A photodiode... 

To test a spectrophotometer for its accuracy, a solution of 60.06 ppm K2Cr207 in 5.0 mM H2SO4 is prepared and analyzed. This solution has a known absorbance of 0.640 at 350.0 nm in a 1.0-cm cell when using 5.0 mM H2SO4 as a reagent blank. Several aliquots of the solution are analyzed with the following results... 

The accuracy of a spectrophotometer can be checked by measuring absorbances for a series of standard dichromate solutions that can be obtained in sealed cuvettes from the National institute of Standards and Technology. Absorbances are measured at 257 nm and compared with the accepted values. The results obtained when testing a newly purchased spectrophotometer are shown here. Determine if the tested spectrophotometer is accurate at a = 0.05. 

Finally, values of sx are directly proportional to transmittance for indeterminate errors due to fluctuations in source intensity and for uncertainty in positioning the sample cell within the spectrometer. The latter is of particular importance since the optical properties of any sample cell are not uniform. As a result, repositioning the sample cell may lead to a change in the intensity of transmitted radiation. As shown by curve C in Figure 10.35, the effect of this source of indeterminate error is only important at low absorbances. This source of indeterminate errors is usually the limiting factor for high-quality UV/Vis spectrophotometers when the absorbance is relatively small. 

When using a spectrophotometer for which the precision of absorbance measurements is limited by the uncertainty of reading %T, the analysis of highly absorbing solutions can lead to an unacceptable level of indeterminate errors. Consider the analysis of a sample for which the molar absorptivity is... 

The methods dependent upon measurement of an electrical property, and those based upon determination of the extent to which radiation is absorbed or upon assessment of the intensity of emitted radiation, all require the use of a suitable instrument, e.g. polarograph, spectrophotometer, etc., and in consequence such methods are referred to as instrumental methods . Instrumental methods are usually much faster than purely chemical procedures, they are normally applicable at concentrations far too small to be amenable to determination by classical methods, and they find wide application in industry. In most cases a microcomputer can be interfaced to the instrument so that absorption curves, polarograms, titration curves, etc., can be plotted automatically, and in fact, by the incorporation of appropriate servo-mechanisms, the whole analytical process may, in suitable cases, be completely automated. 

Procedure. Weigh out 0.0226 g of hydrated ammonium iron(III) sulphate and dissolve it in 1 L of water in a graduated flask 50 mL of this solution contain 100 g of iron. Place 50.0 mL of the solution in a 100 mL separatory funnel, add 10 mL of a 1 per cent oxine (analytical grade) solution in chloroform and shake for 1 minute. Separate the chloroform layer. Transfer a portion of the latter to a 1.0 cm absorption cell. Determine the absorbance at 470 nm in a spectrophotometer, using the solvent as a blank or reference. Repeat the extraction with a further 10 mL of 1 per cent oxine solution in chloroform, and measure the absorbance to confirm that all the iron was extracted. 

The above procedure may be adapted to the determination of molybdenum in steel. Dissolve a 1.00 g sample of the steel (accurately weighed) in 5 mL of 1 1 hydrochloric acid and 15 mL of 70 per cent perchloric acid. Heat the solution until dense fumes are evolved and then for 6-7 minutes longer. Cool, add 20 mL of water, and warm to dissolve all salts. Dilute the resulting cooled solution to volume in a 1 L flask. Pipette 10.0 mL of the diluted solution into a 50 mL separatory funnel, add 3 mL of the tin(II) chloride solution, and continue as detailed above. Measure the absorbance of the extract at 465 rnn with a spectrophotometer, and compare this value with that obtained with known amounts of molybdenum. Use the calibration curve prepared with equal amounts of iron and varying quantities of molybdenum. If preferred, a mixture of 3-methylbutanol and carbon tetrachloride, which is heavier than water, can be used as extractant. 

The scales of spectrophotometers are often calibrated to read directly in absorbances, and frequently also in percentage transmittance. It may be mentioned that for colorimetric measurements I0 is usually understood as the intensity of the light transmitted by the pure solvent, or the intensity of the light entering the solution /, is the intensity of the light emerging from the solution, or transmitted by the solution. It will be noted that ... 

The basic principle of most colorimetric measurements consists in comparing under well-defined conditions the colour produced by the substance in unknown amount with the same colour produced by a known amount of the material being determined. The quantitative comparison of these two solutions may, in general, be carried out by one or more of six methods. It is not essential to prepare a series of standards with the spectrophotometer the molar absorption coefficient can be calculated from one measurement of the absorbance or... 

The colour filters used with absorptiometers should be examined from time to time by measuring the absorbance in a spectrophotometer if the results depart markedly from those expected for the filter, it should be replaced. Normally a filter as supplied with the instrument will be used, but if necessary, filters from the Wratten range supplied by Kodak Ltd or from the Spectrum Filter or Bright Spectrum Filter series supplied by Ilford Ltd may be used. 

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