Extinction calibration curves

Measure the intensity of molar extinction at 302 nm against that of a blank soluhon prepared by diluting 15 mL of methanolic KOH to 25 mL with methanol. Determine the carbon disulhde content from a calibration curve obtained by plothng the carbon disulhde concentrations of different standard solutions on the abscissa versus the absorbance on the ordinate. 

One cm3 of the reactant/product/catalyst mixture was sampled periodically during the reaction for the transmission infrared analysis (Nicolet Magna 550 Series II infrared spectrometer with a MCT detector). The concentrations of reactants and products were obtained by multiplying integrated absorbance of each species by its molar extinction coefficient. The molar extinction coefficient was determined from the slope of a calibration curve, a plot of the peak area versus the number of moles of the reagent in the IR cell. The reaction on each catalyst was repeated and the relative error for the carbamate yield measured by IR is within 5%. 

Theory Folic acid (I) undergoes cleavage by reduction with Zn-Hg in acidic medium to yield p-aminobenzoylglutamic acid (II). The primary aromatic amino group present in the latter is subsequently diazotized in the usual manner and coupled in acidic solution with N-(l-naphthyl)-ethylenediamine hydrochloride in the absence of light (caution). The colour thus produced has a maximum absorption at 550 nm and the extinction (E) is consequently compared with a calibration curve obtained from / -aminobcnzoic acid (PABA) that has been duly diazotized and coupled exactly in the same fashion as the/ -aminobcnzoylglutamic acid. 

Halohydrin dehalogenase activity was determined by monitoring halide liberation at 30 °C in tris-S04 buffer (50 mM, pH 8.0) containing 5 mM 1,3-dichloropropanol or 1,3-dibromopropanol as the substrate. All buffers used for activity assay were prepared with bidest water. From the incubation mixture, 0.5 ml samples were taken and mixed with 1.6 ml of H2O, 0.2 ml or halide reagent 1 and 0.2 ml of halide reagent II. Absorbances were read at 460 nm. A calibration curve of 0-1 mM of chloride or bromide was used to calculate the concentration of halide. The extinctions at 460 nm should be below 0.4 (for chloride) or 0.8 (for bromide). 

The Parr turbidimeter is an extinction type instrument, which consists of a cylinder to contain the turbid suspension, a lamp filament of fixed intensity at the base, and an adjustable plunger through which visual observation is made. Measurement is made of the depth of turbid medium necessary to extinguish the image of the lamp filament Standard suspensions are used to prepare a calibration curve, which is a plot of depdi vj. concentration. 

The concentration of arsenic can be determined at 600 nm because the Sb-Ag DDTC complex does not absorb light of this wavelength. The molar absorptivity of the antimony complex with Ag DDTC reaches its maximum value at 504 nm, but there is also appreciable light absorbance from the As-Ag DDTC complex at this wavelength. The antimony concentration can be calculated from the total extinction value measured at 504 nm by subtracting the extinction value (at 504 nm) that corresponds to the previously determined arsenic concentration. It is clear that calibration curves of arsenic at 504 and 600 nm and of antimony at 504 nm are needed to perform the calculation. 

Baseline resolution of the compounds must be achieved in the chromatographic analysis so that a peak height or area can be assigned to one compound alone. In addition, it is important that the calibration curves in HPLC are produced from the same batch of solvent in which the sample is to be analysed. This is particularly important because small differences in pH can lead to different extinction coefficients when measuring UV absorptions, thus leading to inaccuracies in the quantification process. 

A measured quantity of alkaloidal solution (containing about 0.05 to 0.15 mg alkaloid) is evaporated to dryness on a water-bath, the residue is nitrated with fuming nitric acid (0.2 to 0.3 ml) and evaporated again to dryness. The residue is transferred to a 10-ml graduated flask with the aid of small quantities of dimethylformamide, 25% w/w aqueous solution of tetramethylammonium hydroxide (0.3 ml) is added to the flask which diluted to volume with dimethylformamide. The resulting mixture is allowed to stand for 5 minutes and the extinction of the developed color is measured at 540 nm in 1-cm cells against dimethylformamide. The alkaloidal content is ascertained from a calibration curve which is linear (59,78). 

Following Beer s law given in Equation 6.1, for a given wavelength, equivalent concentrations of species with a different molar extinction coefficient will not have an equivalent absorbance. Under these circumstances, the use of calibration curves determined from compounds dissolved in organic solvents and therefore present only in the neutral form will not be appropriate for the determination of the concentration of a species that is ionized. 

This problem can be overcome by determining a scan of the samples across a range of wavelengths, typically 200-400 nm and selecting the wavelength at which the extinction coefficient is equivalent for all species, the isosbestic point (DMSO absorbance will cause interference if scanning is done at wavelengths much lower than 230 nm), to plot the calibration curves and thus calculate the concentration of the analyte present. 

On the other hand, the presence of microabsorption and extinction does not invalidate the internal standard method, provided these effects are constant from sample to sample, including the calibration samples. Microabsorption and extinction affect only the values of the constants and in Eq. (14-13), and therefore the constant in Eq. (14-16), and the latter constant determines only the slope of the calibration curve. Therefore, microabsorption and extinction, if present, will have no effect on the accuracy of the internal standard method as long as the crystals of the phase being determined, and those of the standard substance, do not vary in degree of perfection or particle size from one sample to another. 

Chemicals used were of standard analytical grade. Fe(lll) was analysed by complexomentric (EDTA) titration [5] with NH4SCN indicator at pH 3. Fe(Il) was estimated by deducting Fe(III) from total Fe found after oxidation of a mix of Fe(IlI) and Fe(II) solution. Chromium was estimated spectrophotometrically at mO 373 as chromate with molar extinction co-efficient of 4815 in IM NaOH solution. Sulphate was analysed by a Nephelometer against a calibrated curve of standard BaS04 suspension. 

The UV absorption in the 260 nm region is frequently used to evaluate styrene content in styrene-based polymers (2, 2, 3, 4, 5, 6, 7). Calibration curves for polystyrene solutions are usually based on the assumptions that the UV absorption of the copolymer depends only on the total concentration of phenyl rings, and the same linear relationship between optical density and styrene concentration that is valid for polystyrene holds also for its copolymers. These assumptions are quite often incorrect and have caused sizable errors in the analysis of several statistical copolymers. For example, anomalous patterns of UV spectra are given by random copolymers of styrene and acrylonitrile (8), styrene and butadiene (8), styrene and maleic anhydride (8), and styrene and methyl methacrylate (9, 10, 11). Indeed, the co-monomer unit can exert a marked influence on the position of the band maxima and/or the extinction... 

Figure 20 shows a calibration curve for a sufficiently rapid and technically simple quantitative sol-particle immunoassay (SPIA [18]). The assay is based on biospecific aggregation of 15-nm gold conjugates to Protein A due to interaction with hIgG molecules. We have found a direct correlation between the amount of the second added protein initiating aggregation and the spectral position of the extinction maximum. 

Fig. 7. Calibration curve for the extinction coefficient of benzene sorbed into zeolite H-ZSM-5 integrated absorbance of the typical benzene band at 1478 cm" vs. the barometrically measured amoimt adsorbed [132]...

The NMR experiment makes the direct observation of atoms possible. The integral of an NMR signal is strictly linearly proportional to the amount of atoms in the probe volume. The signals are a measure of molar ratios of molecules, independently of the molecular weight. There are no response factors such as those in UV-detection caused by varying extinctions dependent on molecular structures non-linear calibration curves such as those found with light-scattering detectors are unknown to NMR spectroscopy. 

An additional complication is that many metabolities/cofactors have temperature-dependent extinction coefficients those for NADH and for potassium ferri-cyanide, for example, are about 10% lower at 80° than at 20°. Fourage et al. point out that the effect of temperature on the absorbance and Xmax of p-nitrophenol can lead to substantial errors in fccat values measured by continuous release of p-nitrophenol, if the calibration curve is not determined at the same temperature as the assay. 

This process can be used when no chlorite is present in the range from 0.1 to approximately 0.002 mg/1 CIO2, using a 5-cm cuvette. Chlorine must first be eliminated with potassium bromide and sodium formate. In order to remove chlorine, 1.5 ml double-distilled water and 0.5 ml KBr solution are added to 50 ml of the water to be analyzed after 1 min. 0.5 ml sodium formate solution is added and allowed to stand for 15 mins. If no chlorine is present, 2.5 ml double-distilled water is simply added to the water to be analyzed. Approximately 3 drops of sulphuric acid are added in order to adjust to pH 2.5 with the aid of a pH meter. Then, 0.5 ml potassium iodine solution is added and mixed. The extinction is subsequently measured at 350 nm against a reference solution (52.5 ml double-distilled water, adjustment with approximately 3 drops H2SOz to pH 2.5 + 0.5 ml potassium iodide solution), using a 5-cm cuvette. The concentration of CIO2 is read off from the calibration curve. 

Where simple conversions do not achieve this objective, the calibration curves produced with chloramine-T must be produced for each determination variant, taking into consideration the volume of water and the volume of the reagent solutions. The extinction values for the iodine solutions resulting from every measuring process then serve to indicate... 

In order to plot the calibration curve, several solutions of known concentration are prepared of the substance to be determined and their extinction measured. These are then plotted on a graph as a function of concentration (Fig. 24). The measured extinction Ex provides the required concentration Cx of the solution of unknown concentration. 

The calibration curve provides information as to whether the Lambert-Beer law applies to the required concentration range, i.e. whether extinction E and concentration c are directly proportional. If this is the case, the calibration curve is a straight line. Deviations from the Lambert-Beer law in certain concentration ranges are indicated by the curved course of the calibration curve. 

Over a wide range, AAS obeys Beer s law, which describes a linear relationship between extinction and the concentration of the element in question. The concentration required is determined by multiplying the measured extinction by a calibration factor or by reading off with the aid of the calibration curve. 

Read off the sulphide sulphur content of the sample from the calibration curve on the basis of the measured extinction, and taking account of the volume of sample solution used for determination, recalculate the result in terms of 1 litre of the water sample. 

NO2 concentrations between 0.005 and 0.05 mg into 100 ml volumetric flasks and treat according to the method specified under "Determination". Prepare the calibration curve from the extinction values. 

Using the measured extinction, read the appropriate nitrite content from the calibration curve and convert the results for the volume of the water sample used to 1 litre. 

Transfer aliquots of the nitrate reference solution with between 0.01 and 0.5 mg NO3 to a glass or porcelain dish and treat according to the method given under "Determination". Either prepare a calibration curve or calculate the calibration factor from the extinction values obtained. 

Using either the calibration curve or a calibration constant, work out the content of iron (II) ions in mg from the extinction values as measured, and convert to mg Fe2+/1 taking into account the quantity of water transferred to the measuring flask at the sampling point. 

Read off the manganese content from the calibration curve (C) on the basis of the measured extinction value (or if appropriate the net extinction E[ - E2 = E). Using the formula... 

Read off the appropriate antimony content from the calibration curve on the basis of the extinction measured, taking the blank reading into account. Insert the volume of water sample used and recalculate for 1 litre of water. 

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