Spectrophotometrically calibrated measurements

The worksheet functions SLOPE known ys, known xs) and INTERCEPT(/f/ioiv/i ys, known xs) return the slope, m, and intercept, b, respectively of the least-squares straight line through a set of data points. For example. Figure 11-1 illustrates some spectrophotometric calibration curve data (concentration of potassium permanganate standards in column B, absorbance of the standards in column C). The formula =SLOPE(C4 C8,B4 B8) in cell C10 was used to obtain the slope of the straight-line calibration curve. The SLOPE and INTERCEPT functions should be used with some caution, since they do not provide a measure of how well the data conforms to a straight line relationship. 

The photometric calibration also contributes to the uncertainty of the measured spectrum. Flux standard stars are typically measured at widely spaced wavelengths (50 A is common), and the sensitivity function of the instrument is determined by fitting a low-order polynomial or spline to the flux points. Such fits inevitably introduce low-order wiggles in the sensitivity function, which will vary from star to star. Based on experience, the best spectrophotometric calibration yield uncertainties in the relative fluxes of order 2-3% for widely-spaced emission lines the errors may be better for ratios of lines closer than 20 A apart. Absolute fluxes have much higher uncertainties, of course, especially for narrow-aperture observations of extended objects. 

Comparative Measurements of Oxygen Consumption with Spectrophotometrically Calibrated Substrates... 

Glass pH electrodes are convenient in the lab but are not well suited to longterm environmental measurements because electrode potentials drift We compensate for drift in the lab by frequent calibration in standard buffers. To monitor pH in natural waters, a spectrophotometer measures the ratio of absorbance of light at wavelengths corresponding to the two colored forms of an indicator. This ratio provides stable measurements without calibration. The precision (repeatability) of shipboard spectrophotometric pH measurements is 0.000 4 pH units. 

An illustrative example generates a 2 x 2 calibration matrix from which we can determine the concentrations xi and X2 of dichromate and permanganate ions simultaneously by making spectrophotometric measurements yi and j2 at different wavelengths on an aqueous mixture of the unknowns. The advantage of this simple two-component analytical problem in 3-space is that one can envision the plane representing absorbance A as a linear function of two concentration variables A =f xuX2). 

A fifth spectrophotometric method for the quantitative determination of the concentration of Pb + in blood uses a multiple-point standard addition based on equation 5.6. The original blood sample has a volume of 1.00 mb, and the standard used for spiking the sample has a concentration of 1560 ppb Pb +. All samples were diluted to 5.00 mb before measuring the signal. A calibration curve of Sjpike versus Vj is described by... 

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. 

The three normal means of presenting the spectrophotometric data are described below by far the most common procedure is to plot absorbance against wavelength (measured in nanometres). The wavelength corresponding to the absorbance maximum (or minimum transmission) is read from the plot and is used for the preparation of the calibration curve. This point is chosen... 

Stintzing, unpublished observations). Analogous aspects were recently addressed in an extended study of anthocyanin quantification. Spectrophotometric determination is still the most preferred method that has been shown to compare well with HPLC measurements." Hence, only in rare cases have purified standards from a particular food commodity been used for calibration by HPLC-DAD. " ... 

It is always wise to calibrate physical methods of analysis using mixtures of known composition under conditions that approximate as closely as practicable those prevailing in the reaction system. This procedure is recommended because side reactions can introduce large errors and because some unforeseen complication may invalidate the results obtained with the technique. For example, in spectrophotometric studies of reaction kinetics, the absorbance that one measures can be grossly distorted by the presence of small amounts of highly colored absorbing impurities or by-products. For this reason, when one uses indirect physical methods in kinetic studies, it is essential to verify the stoichiometry of the reaction to ensure that the products of the reaction and their relative mole numbers are known with certainty. For the same reason it is recommended that more than one physical method of analysis be used in detailed kinetic studies. 

Erk [20] described a spectrophotometric method for the simultaneous determination of metronidazole and miconazole nitrate in ovules. Five capsules were melted together in a steam bath, the product was cooled and weighed, and the equivalent of one capsule was dissolved to 100 mL in methanol this solution was then diluted 500-fold with methanol. In the first method, the two drugs were determined from their measure d%/dk values at 328.6 and 230.8 nm, respectively, in the first derivative spectrum. The calibration graphs were linear for 6.2—17.5 pg/mL of metronidazole and 0.7—13.5 pg/mL of miconazole nitrate. In the second (absorbance ratio) method, the absorbance was measured at 310.4 nm for metronidazole, at 272 nm for miconazole nitrate and at 280.6 nm (isoabsorptive point). The calibration graphs were linear over the same ranges as in the first method. 

Besada [12] described a spectrophotometric method for determination of penicillamine by reaction with nitrite and Co(II). Penicillamine is first treated with 1 M NaN02 (to convert the amino-group into a hydroxy-group), then with 0.1 M CoCl2, and finally the absorbance of the brownish-yellow complex obtained is measured at 250 nm. The process is carried out in 50% aqueous ethanol, and the pH is adjusted to 5.4— 6.5 for maximum absorbance. The calibration graph is linear over the concentration range of 0.25-2.5 mg per 50 mL, and the mean recovery (n = 3) of added drug is 99.7%. Cystine, cysteine, methionine, and other amino adds do not interfere. 

Another assay that is very similar to the ABTS assay is the AGV-dimethyl-p-phenylenediamine (DMPD assay). In the presence of a suitable oxidant solution at an acidic pH, DMPD is converted to a stable and colored DMPD radical cation (DMPD +). Antioxidants capable of transferring a hydrogen atom to the radical cause the decol-orization of the solution, which is spectrophotometrically measured at 505 nm. The reaction is stable, and the endpoint is taken to be the measure of antioxidant efficiency. Antioxidant ability is expressed as Trolox equivalents using a calibration curve plotted with different amounts of Trolox (Fogliano and others 1999). This method is used to measure hydrophilic compounds. The presence of organic acids, especially citric acid, in some extracts may interfere with the DMPD assay, and so this assay should be used with caution in those extracts rich in organic acids (Gil and others 2000). 

The error in (a) is stated to compare favourably with calibration from benzene, since the absolute value of R90 is hardly known to this accuracy. In (b) the concentration of DNA was measured spectrophotometrically via the molar phosphorous extinction coefficient of 6415 (with a standard deviation of 2%). The low error in (c) arises from low levels of dust achieved as well as the integration over a period of 10 secs of the readings on a digital output. The specific refractive index increment used in (d) was an experimental one from the literature. In point of fact the assess-... 

A standard Lowry-based protein assay has been adjusted to the special conditions encountered with skin [126], Basically, proteins reduce an alkaline solution of Cu(II)-tartrate to Cu(I) in a concentration-dependent manner. Then, the formation of a blue complex between Folin-Ciocalteau reagent (a solution of complex polymeric ions formed from phosphomolybdic and phosphotungstic heteropoly acids) and Cu(I) can be measured spectrophotometrically at 750 nm. A calibration curve can be obtained by dissolving known amounts of stratum corneum in 1 M sodium hydroxide. A piece of tape that has not been in contact with skin is subjected to an identical procedure and serves as negative control. The method was recently adapted to a 96-well plate format, notably reducing analysis times [129],... 

Biochemical research often requires the quantitative measurement of protein concentrations in solutions. Several techniques have been developed however, most have limitations because either they are not sensitive enough or they are based on reactions with specific amino acids in the protein. Since the amino acid content varies from protein to protein, no single assay will be suitable for all proteins. In this section we discuss five assays three older, classical methods that are occasionally used today and two newer methods that are widely used. In four of the methods, chemical reagents are added to protein solutions to develop a color whose intensity is measured in a spectrophotometer. A standard protein of known concentration is also treated with the same reagents and a calibration curve is constructed. The other assay relies on a direct spectrophotometric measurement. None of the methods is perfect because each is dependent on the amino acid content of the protein. However, each will provide a satisfactory result if the proper experimental conditions are used and/or a suitable standard protein is chosen. Other important factors in method selection include the sensitivity and accuracy desired, the presence of interfering substances, and the time available for the assay. The various methods are compared in Table 2.3. 

Spectrophotometric measurements of CD and CT values in oils or lipid extracts requires 45 min depending on the number of samples to be analyzed. The classical PV determination requires a bit of ti me for preparation of reagents and standardization of the sodium thiosulfate solution. The analysis itself is not very time consuming ( 30 min), but in all, about 3 to 3.5 hr should be anticipated. The FOX method has an assay time of < 10 min however, with sample preparation and time to construct the calibration curve, 2 to 3 hr should be expected. 

Results and Discussion. The 2-ethyl polyaniline concentration in the silica gel film was determined by constructing a Beer s law calibration curve from solutions of known concentration. Assuming an average molecular weight of 5000, the 2-Et PANi concentration in the silica gel was found to be 9.6 x 10 4 M. The refractive indices of CS2 and 2-Et PANi SiC>2 were estimated to be 1.6 and 1.4 at 1.06 im, respectively. The emeraldine base doped silica gel was found to have low losses due to scatter, and exhibited good transparency at 1.06 im. Spectrophotometric measurements at 1.06 fim yielded absorption coefficients of 0.1 cm-1 (> 99% T over 1 mm pathlength) for the CS2 reference and 4 cm 1 (96% T over 1 mm pathlength) for the 2-Et PANi doped silica film. 

An early spectrophotometric method [ 1 ] for aluminium in soil involves the use of a Technicon sample changer, proportioning pump and automatic colorimeter. The method is based on the measurement of the rate of colour development in the reaction between aluminium and xylenol orange in ethanolic media. The calibration graph is rectilinear up to 2.7 mg/1 aluminium and the coefficient of variation is 4.5%. 

The correlation coefficient (y) is a measure of linear relationship between two sets of data. It can attain a value which may vary between 0 and 1. A value of+1 (or-1, when the slope is negative) indicates the maximum possible linearity on the other hand, a zero y indicates there is absolutely no link between the data. In environmental analysis, especially in spectrophotometric methods, y is calculated to determine the linearity of the standard calibration curve, y may be calculated from one of the following equations. 

This paper has examined the role of calibration and evaluation of measurement uncertainty in clinical laboratories arising from the request for traceability assurance. To produce results which are accurate and reliable within the stated uncertainty, all uncertainties of the quality measurement process and the traceability chain should be demonstrated. Also, the quality of a spectrophotometric result depends critically on RMs and photometric systems whose traceability have been properly demonstrated. 

In spectrophotometric analyzers, interference filters are selected for desired wavelengths, as determined from the spectral relationship curves. Photodetectors are least sensitive in the blue end of the spectrum. This can be dealt with by using prefilters or narrow spectral ranges, which are calibrated for more sensitivity. Improvements in spectrophotometers include a flashed xenon light source with dual-beam measurement. Dual-beam machines measure the spectrum of both the light source and the reflected light for each measurement. 

Hu et al. described an ultraviolet spectrophotometric method for the analysis of dipyridamole [25]. A sample (50 mg) of the powdered tablet was dissolved and diluted to 100 mL with 0.01 M hydrochloric acid. A 2 mL portion of the solution was diluted to 100 mL with 0.01 M hydrochloric acid, and the absorbance of the final solution was measured at 283 nm (or at 403 nm for pure dipyridamole). The calibration graph was linear for upto 12 or 60 pg/mL, and suitable for high-content determinations. Results were in good agreement with those obtained by titration with bromate. 

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