Cuvette absorbance

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. 

In the process of performing a spectrophotometric determination of Ee, an analyst prepares a calibration curve using a single-beam spectrometer, such as a Spec-20. After preparing the calibration curve, the analyst drops the cuvette used for the method blank and the standards. The analyst acquires a new cuvette, measures the absorbance of the sample, and determines the %w/w Ee in the sample. Will the change in cuvette lead to a determinate error in the analysis Explain. 

Absorbance. Analyte measurements in clinical analyzers using Hquid reagents are most commonly performed by transmission of light, ie, by absorbance photometry or colorimetry (Fig. 3a). The Hquid to be analyzed is either held in a cuvette or passed through a flowceU having transparent walls. 

Experimental A photometric method was found in the literature which seemed to suit the particular circumstances. Two cyanide stock solutions were prepared, and an electromechanical dispenser was used to precisely prepare solutions of 20, 40,. .., 240 respectively 10, 30, 50,. .., 250 fig CN /100 ml. 10 ml of each calibration solution were added to 90 ml of the color-forming reagent solution and the absorbance was measured using 1-cm cuvettes. (See Table 4.17 (left and middle panels) and data file CYANIDE.dat.)... 

Legend No number of measurement. Cone concentration in fig, CN"/100 ml Absorb absorbance [AU] slope slope of regression line t CV intercept see slope res. std. dev. residual standard deviation Srts -n number of points in regression LOD limit of detection LOQ limit of quantitation measurements using a 2-fold higher sample amount and 5-cm cuvettes—i.e., measured absorption 0. .. 0.501 was divided by 10. 

Porphyridium species are the sources of fluorescent pink color. The main Porphyridium phycobiliproteins are B-phycoerythrin and b-phycoerythrin. Maximum absorbance of a 1% solution of B-phycoerythrin in a 1-cm cuvette is at 545 inn, and the fluorescence emission peak is at 575 inn molecular weight is 240 kda. Batch culture of Porphyridium species outdoors yields approximately 2(X) mg of colorant per liter of culture after 3 days the phycoerythrin level in the colorant is about 15%. A higher concentration of phycoerythrin, up to 30%, can be achieved under optimal algal culture conditions. 

Pectolytic activity was also studied in batch reactors, following the reaction progress in thermostated quartz cuvettes. The reaction medium (3 cm ) was prepared with 1.5 g/L pectin in the standard buffer and 0.063 mg of enzyme. The absorbance of the reaction mixture against the substrate blank was continuously recorded at the spectrophotometer (Perkin Elmer Lambda 2, USA). Typical reaction time was 15 minutes, but initial reaction rates were estimated considering only the absorbances recorded during the first 200 seconds, range of totally linear response. 

To construct a standard curve of various biotin concentrations, first zero a spectrophotometer at an absorbance setting of 500 nm with sample and reference cuvettes filled with 0.05M sodium phosphate, 0.15M NaCl, pH 6.0. Remove the buffer solution from the sample cuvette and add 3 ml of the (strept)avidin solution plus 75 pi of the HABA-dye solution. Mix well and measure the absorbance of the solution at 500nm. Next add 2 pi aliquots of the biotin solution to this (strept)avidin-HABA solution, mix well after each addition, and measure and record the resultant absorbance change at 500 nm. With each addition of biotin, the absorbance of the (strept)avidin-HABA complex at 500 nm decreases. The absorbance readings are plotted against the amount of biotin added to construct the standard curve. 

To measure the response of the biotinylated protein sample, add 3 ml of the (strept)avidin solution plus 75 pi of the HABA dye to a cuvette. Mix well and measure the absorbance of the solution at 500 nm. Next, add a small amount of sample to this solution and mix. Record the absorbance at 500 nm. If the change in absorbance due to sample addition was not sufficient to obtain a significant difference from the initial (strept)avidin-HABA solution, add another portion of sample and measure again. Determine the amount of biotin present in the protein sample by using the standard curve. The number of moles of biotin divided by the moles of protein present gives the number of biotin modifications on each protein molecule. 

Beer s law allows us to determine concentration since absorbance is directly proportional to it. To negate influences due to cuvette differences, or differences between sample tubes, a reference solution is made containing all of the components except the species being analyzed. 

Cuvettes used with the spectrometer are not simply test tubes. They are specially made tubes or cuvettes and are often matched such that a set of tubes or cuvettes will all have the same absorbance characteristics. Cuvettes should never be used as test tubes they must be kept clean at all times, and care must be taken not to scratch them. When using cuvettes that have not been used before, they should be tested to make sure they are all the same. This is accomplished by inserting them into the spectrophotometer and noting their absorbance. All should be the same. Keep in mind that empty cuvettes will have a higher absorbance than when filled with water. This is because light is refracted at each surface, and when filled with water or solvent, there is less refraction at the surfaces. 

When using a spectrophotometer for a colorimetric analysis, both the 0% and 100% transmittance (oo and 0 absorbance) readings must be set. Once the instrument has warmed up, with the light beam blocked and with nothing in the sample compartment, the readout is set to 0% transmittance (oo abs.). Again, this measurement is done to set / in the absorbance equation shown earlier. A blank, a solution containing all the components used in the analysis except the analyte being measured, is placed in a cuvette, placed in the sample... 

In practical situations the absorbance of a sample is determined by making two measurements, the first to determine 70 and the second to determine I. The determination of I0 is used to cancel a large number of experimental factors that could affect the result. When measuring I0 the sample container must closely match the unknown container in all ways except for the analyte content. The cuvettes should be a matched pair if a double beam instrument is used and the same cuvette can be used for both the blank and sample with a single beam instrument. The blank solution filling the cuvette should be identical to the solvent that the sample is dissolved in, except for the sample itself. If done correctly, the least-squares line for the calibration graph will come very close to the 0,0 point on the graph. 

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