Spectrophotometers calibration

Fortunately, protein concentration methods are relatively simple (low-tech) and inexpensive. The simplest assays require only a spectrophotometer calibrated for wavelength and absorbance accuracy, basic laboratory supplies, and good pipetting techniques. Protein concentration assays are quite sensitive, especially given the typical detection limits required for most biopharmaceuticals. 

The qualification of a Raman spectrometer is described in USP chapter < 1120>. In particular, the tests for the operational and performance qualification of a Raman spectrometer are described x-axis precision, photometric precision, laser power precision and accuracy. The x-axis of the Raman spectrometer is the Raman shift measured in wavenumbers. Before the Raman shift can be determined, both the laser wavelength and spectrophotometer calibration must be determined. The precision of the Raman shift can then be measured using an American Society for Testing and Materials (ASTM) Raman standard material [20]. A commonly used Raman standard material is acetaminophen. The peak position of the known reference peaks can be determined visually, but is better done with a peak location algorithm. The USP chapter on Raman specifies that the peak location should not vary more than... 

Holmium is used in glass for spectrophotometer calibration (Allen 2007). Also, holmium is used as a colorant for cubic zirconia (imitation diamond) and glass, which yields yellow or red colors (Fig. 5.7). 

This exercise affords students the opportunity to use several instrumental techniques to which they have previously been introduced in the laboratory in order to conduct a comparison of soil types with respect to determining the ratio of Cr(III) to Cr(VI). The exercise includes pH measurement, calibration of an UV vis spectrophotometer, calibration of an atomic absorption spectrophotometer in the flame mode (FIAA), and sample preparation techniques. 

The following is not strictly a calibration table. More accurately, this table provides guidance in checking the wavelength reliability of typical ultraviolet-visible (UV-Vis) spectrophotometers. Calibration is usually not straightforward and is typically done in the facilities of spectrophotometer manufacturers. 

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. 

Procedure. Allow the whole of the sample solution (1 L) to flow through the resin column at a rate not exceeding 5 mL min . Wash the column with 250 mL of de-ionised water and reject the washings. Elute the copper(II) ions with 30 mL of 2M nitric acid, place the eluate in a small conical flask (lOOmL, preferably silica) and evaporate carefully to dryness on a hotplate (use a low temperature setting). Dissolve the residue in 1 mL of 0.1 M nitric acid introduced by pipette and then add 9 mL of acetone. Determine copper in the resulting solution using an atomic absorption spectrophotometer which has been calibrated using the standard copper(II) solutions. 

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 ... 

Double-beam spectrophotometers. Most modern general-purpose ultraviolet/ visible spectrophotometers are double-beam instruments which cover the range between about 200 and 800 nm by a continuous automatic scanning process producing the spectrum as a pen trace on calibrated chart paper. 

A calibration curve for the range 0.2-10 mg fluoride ion per 100 mL is constructed as follows. Add the appropriate amount of standard sodium fluoride solution, 25 mL of 2-methoxyethanol, and 10 mg of a buffer [0.1 Af in both sodium acetate and acetic (ethanoic) acid] to a 100 mL graduated flask. Dilute to volume with distilled water and add about 0.05 g of thorium chloranilate. Shake the flask intermittently for 30 minutes (the reaction in the presence of 2-methoxyethanol is about 90 per cent complete after 30 minutes and almost complete after 1 hour) and filter about 10 mL of the solution through a dry Whatman No. 42 filter paper. Measure the absorbance of the filtrate in a 1 cm cell at 540 nm (yellow-green filter) against a blank, prepared in the same manner, using a suitable spectrophotometer. Prepare a calibration curve for the concentration range 0.0-0.2 mg fluoride ion per 100 mL in the same way, but add only 10.0 mL of 2-methoxyethanol measure the absorbance of the filtrate in a 1 cm silica cell at 330 nm. 

Procedure. To 100 mL of the neutral sample solution (containing not more than 0.4 mg nitrite) add 2.0 mL of solution A and, after 5 minutes, 2.0 mL of solution B. The pH at this point should be about 1.5. Measure the absorbance after 10 minutes in the wavelength region of 550 nm (yellow-green filter), in a spectrophotometer against a blank solution prepared in the same manner. Calculate the concentration of the nitrite from a calibration plot prepared from a series of standard nitrite solutions. 

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. 

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. 

With the best observing conditions, it is possible for the trained observer to compete with photoelectric colorimeters for detection of small color differences in samples which can be observed simultaneously. However, the human observer cannot ordinarily make accurate color comparisons over a period of time if memory of sample color is involved. This factor and others, such as variability among observers and color blindness, make it important to control or eliminate the subjective factor in color grading. In this respect, objective methods, which make use of instruments such as spectrophotometers or carefully calibrated colorimeters with conditions of observation carefully standardized, provide the most reliable means of obtaining precise color measurements. 

The homopolymers of styrene and acrylonitrile were not soluble In the acetonitrile mobile phase. Calibration factors thus had to be derived from a combination of literature data and experimental measurements. To calibrate the UV detector for polystyrene, 254 nm absorbance of both monomer and polymer was measured with a conventional spectrophotometer, using chloroform... 

An additional requirement not noted in Table 1 is compliance with GLP7 These practices establish a paper trail for all procedures involved in the determination of residues. With regard to immunoassays, GLPs require calibration of measurement devices such as adjustable pipettors and dedicated spectrophotometers. Computer software output, as noted above, must be verified prior to use. This process can be simplified by limiting the application of specialized software to the operation of an instrument and carrying out the residue calculations in a broadly available spreadsheet such as Excel. On a positive note, in recent years, the software accompanying most microtiter plate readers has become generally easier to use and usually incorporates internal spreadsheets that are compatible with external systems. 

Ninhydrin Assays. Ninhydrin tests were performed using a modified procedme of Taylor et al. " APS Silica (10-75 mg) of various loadings (0.857, 0.571, and 0.343 mmol NH2/g Silica) was added to phosphate buffer (5 mL, 100 mM, pH 6.5), and 1 mL of a 5% w/v solution of ninhydrin in ethanol was added to the sluny. After stirring for an hour in a boiling water bath, the mixture was allowed to cool slowly to room temperature. The silica was then filtered and washed three times with 70°C distilled water. The filtrate was collected, added to a volumetric flask, diluted to 100 mL, and the absorbance of this solution at 565 mu was measured using a UV-visible spectrophotometer. The reference solution was prepared as above with unmodified amine-free silica. Calibration standards were prepared with aliquots of a 1 mg/mL solution of APS in ethanol. 

The final optically clear solution is evaluated as to intensity of color at 555 m/x, and with slit width at 0.02 to 0.04 mm., on a Beckman spectrophotometer adjusted to 100% transmittance with an untreated sample freshly processed in parallel with the unknown sample. From a standard calibration curve (see Figure 3), the concentration of parathion may readily be ascertained, and an appropriate factor converts this value to micrograms of parathion present in the original field sample. 

Three methods for quantitative analysis of niclosamide at concentrations of 0.5-2.0 ppm were given. For in situ analysis, safranine dye solution was added to the sample and the extraction solution added which formed the upper phase. The niclosamide content was determined by the color intensity of the upper phase. The colors were compared with blanks of known concentration. When an accurate determination was required, niclosamide was extracted from the water sample with amylacetate, a methanol solution of sodium hydroxide was added to the extraction, and the resulting yellow color was measured at 385 mft in a spectrophotometer. Third method made use of a calibration curve [60],... 

The term calibration is often used when in fact what is meant is verification . Calibration of an instrument or a piece of equipment (e.g. glassware) involves making a comparison of a measured quantity against a reference value. For example, to calibrate a spectrophotometer response, the appropriate reference material is selected and the spectrophotometer response to it, under the specified conditions, is measured. Then, the measured value is compared to the value quoted in the literature. Either a correction is made to the results from subsequent measurements or an adjustment is made to the instrument. 

Extraction of mustard agent from the samples was realized with ethyl alcohol. The remains of the toxic substance in samples were determined with the spectrophotometer SPECOL according to the thymolphtalein reaction [1, 2] with preliminarily made calibration curve. 

Scenario A student mixed 5.0 mL of 0.015 M Pb(N03)2 with 3.0 mL of 0.030 M KI in 2.0 mL of 0.200 M KN03. The test tube was shaken for 15 minutes and the solid precipitate of lead (II) iodide was allowed to settle. The tube was centrifuged to remove any excess lead (II) iodide from the supernatant. To the supernatant, she added KN02 to oxidize the 1 to I2. The supernatant was then analyzed for 1 by using a spectrophotometer. She then made known dilutions of a 0.10 M potassium iodide solution in acidified KN02 to create a calibration curve to be able to determine the F in the supernatant. 

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