Reading spectrophotometer

Advantages This assay in conjunction with the use of Multiwell plates, multichannel pipettes, microplate washer and shaker, together with a plate reading spectrophotometer, enables a large number of samples to be processed quickly. Interfacing the plate reader with a computer would provide an obvious additional advantage. 

Other methods of background correction have been developed, including Zee-man effect background correction and Smith-Iiieffje background correction, both of which are included in some commercially available atomic absorption spectrophotometers. Further details about these methods can be found in several of the suggested readings listed at the end of the chapter. 

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 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 absorbance and the percentage transmission of an approximately 0.1M potassium nitrate solution is measured over the wavelength range 240-360 nm at 5 nm intervals and at smaller intervals in the vicinity of the maxima or minima. Manual spectrophotometers are calibrated to read both absorbance and percentage transmission on the dial settings, whilst the automatic recording double beam spectrophotometers usually use chart paper printed with both scales. The linear conversion chart, Fig. 17.18, is useful for visualising the relationship between these two quantities. 

The experimental technique is simple. The cell containing the solution to be titrated is placed in the light path of a spectrophotometer, a wavelength appropriate to the particular titration is selected, and the absorption is adjusted to some convenient value by means of the sensitivity and slit-width controls. A measured volume of the titrant is added to the stirred solution, and the absorbance is read again. This is repeated at several points before the end point and several more points after the end point. The latter is found graphically. 

Procedure. Place 80 mL of the arsenic/antimony solution in the titration cell of the spectrophotometer. Titrate with standard bromate/bromide solution at 326 nm taking an absorbance reading at least every 0.2 mL. From the curve obtained calculate the concentration of arsenic and antimony in the solution. 

Procedure. Charge the titration cell (Fig. 17.24) with 10.00 mL of the copper ion solution, 20 mL of the acetate buffer (pH = 2.2), and about 120mL of water. Position the cell in the spectrophotometer and set the wavelength scale at 745 nm. Adjust the slit width so that the reading on the absorbance scale is zero. Stir the solution and titrate with the standard EDTA record the absorbance every 0.50 mL until the value is about 0.20 and subsequently every 0.20 mL. Continue the titration until about 1.0 mL after the end point the latter occurs when the absorbance readings become fairly constant. Plot absorbance against mL of titrant added the intersection of the two straight lines (see Fig. 17.23 C) is the end point. 

A double-beam atomic absorption spectrophotometer should be used. Set up a vanadium hollow cathode lamp selecting the resonance line of wavelength 318.5 nm, and adjust the gas controls to give a fuel-rich acetylene-nitrous oxide flame in accordance with the instruction manual. Aspirate successively into the flame the solvent blank, the standard solutions, and finally the test solution, in each case recording the absorbance reading. Plot the calibration curve and ascertain the vanadium content of the oil. 

Photoelectric-Colorimetric Method. Although the recording spectrophotometer is, for food work at least, a research tool, another instrument, the Hunter multipurpose reflectometer (4), is available and may prove to be applicable to industrial quality control. (The newer Hunter color and color difference meter which eliminates considerable calculation will probably be even more directly applicable. Another make of reflection meter has recently been made available commercially that uses filters similar to those developed by Hunter and can be used to obtain a similar type of data.) This instrument is not a spectrophotometer, for it does not primarily measure the variation of any property of samples with respect to wave length, but certain colorimetric indexes are calculated from separate readings with amber, blue, and green filters, designated A, B, and G, respectively. The most useful indexes in food color work obtainable with this type of instrument have been G, which gives a... 

About 5 ml of sample is withdrawn for every 4-6 hours. The absorbance reading of the sample at 580 nm was measured using a Hitachi U-2000 spectrophotometer. The sample is filtered in a vacuum through Whatman filter paper with a pore size of 2.5 pin and diameter of 47 mm. The dry weight of cells is measured to monitoring microbial cell population and cell density. A plot of optical density reading from the spectrophotometer against cell dry... 

There are available also several kits for the assay of calcium, in 10 or 20 microliter samples by chelate formation colorimetrically or fluorimetrically. (Pierce Chem. Co., Rockford, 111.). These are read either with the spectrophotometer or by spectrofluorometry. In our experience, while these systems can be used for approximate results, the plot of concentration versus reading curves are rather flat and only an approximation of the values can be obtained. This may be very important late at night or at times when the atomic absorption machine is down, but if the atomic absorption instrument is available it should be used in preference to these procedures. 

All colorimetric readings were made on the Beckman quartz spectrophotometer. Results and Discussions... 

Read the per cent transmittance at 555 m/< in a colorimeter or spectrophotometer against 85% sulfuric acid. 

Protein content The amount of protein in each extract can be determined by the Bradford method (Bradford, 1976), using BSA as a standard. Briefly, make a standard curve with 0,2,4,6,8,10,15 and 20 pg / mL BSA and mixed with 1 mL of Bio-Rad protein assay (diluted 1 4). Read standard curve and samples at A595 in a spectrophotometer, using as blank 1 mL of diluted Bio-Rad protein assay. 

Procedure The leaf disc (< ) 1cm) was placed in a vial containing 1 ml dimethyl sulfoxide and chlorophyll was extracted at 60 °C by incubating for 30 min. After cooling the OD values at 645 and 663 nm were read in a Shimadzu UV 160 spectrophotometer. Chlorophyll a and b content was calculated following the equation used by Amon (1949). The content of chlorophyll a and b was expressed in mg/g dry weight. 

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. 

Most manufacturers of dissolution testing devices offer semi-automated systems that can perform sampling, filtration, and UV reading or data collection. These systems automate only a single test at a time. Fully automated systems typically automate entire processes including media preparation, media dispensing, tablet or capsule drop, sample removal, filtration, sample collection or analysis (via direct connection to spectrophotometers or HPLCs), and wash cycles. A fully automated system allows automatic performance of a series of tests to fully utilize unused night and weekend instrument availability. 

In a further modification, Nordin and Bretthauer (N4) assay the residual UDPG by incubation with TNDPN. The final optical density is read at 400 mp, making an ultraviolet spectrophotometer unnecessary. 

The spectrophotometer used has two scales-absorbance (log scale, 0-2) and percent transmittance (linear scale, 0-100). Most readings are taken from the transmittance scale and then converted to absorbance through the relationship... 

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... 

The model immunoassay is the enzyme-linked immunosorbent assay (ELISA) in which a non-specific capture antibody is bound to a surface, such as a multi-well plate or small tube [13]. In the basic form of ELISA, a second antibody tagged with an enzyme interacts specifically with the analyte. The enzyme assay produces a colored product that is read with a spectrophotometer. There are many variations on the basic immunoassay format that serve to increase sensitivity, specificity, linear range, and speed. Many commercial instruments have been developed to take advantage of various technologies for reporter molecules. The immunoassay may be coupled to an electronic sensor and transducer, such as a surface acoustical wave (SAW) sensor. Electrochemiluminescence (ECL) is a method in which the detector antibody is tagged with a ruthenium-containing chelate [13-15]. When the tag is... 

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