Calibration Graph Method

The normal calibration curve is asymptotic to the value of (B), which represents the transmission of unabsorbed light. Unabsorbed light may be due to straylight or non-absorbing lines from the radiation source (cathode material or filler gas) which pass within the spectral bandwidth of the monochromator. The slope of the calibration graph is also dependent on the ratio of the half-widths of the emission line (zt e) and absorption line w (i) The curve is linear when wjw l/5 (ii) The curve is slightly curved when /b wjw / (hi) The initial slope starts to decrease when 1/1  

Modern spectrometers determine the calibration function automatically and preset the analytical results in desired units. Most instruments calculate the slope of the calibration graph according to the least squares fit procedure. 

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Quantitative Analysis Using Calibration Graph Method... 

PVSA-SG film was used for determination of Fe(Phen) + and Zn + as ternary complex Zn +-Phen-bengal rose by spectrophotometric method. The calibration graph was linear in the concentration 5T0 -5T0 mol/lfor Fe(II) and FlO - 5T0 mol/1 for Zn(II). The film can be regenerated and reused. LG-PDMDA-SG film was shown to be perspective modificator of the PG electrode surface and used for voltammetric detection of Mo(VI) at ppb level. 

It was shown that Zn + adsorbed onto SG-PVSA composite film as Zn(Phen) complex. It can be detected spectrophotometrically after treatment with anionic dye Bengal Rose (BR). Ternary complex Zn + - Phen-BR formed on the surface under optimal conditions. SG-PVSA film was used for determination of Zn + by spectrophotometric method. The calibration graph was linear in the concentration range 2,5T0 - STO mol/l. 

Following this procedure urea can be determined with a linear calibration graph from 0.143 p.g-ml To 1.43 p.g-ml and a detection limit of 0.04 p.g-ml based on 3o criterion. Results show precision, as well as a satisfactory analytical recovery. The selectivity of the kinetic method itself is improved due to the great specificity that urease has for urea. There were no significant interferences in urea determination among the various substances tested. Method was applied for the determination of urea in semm. 

Once a linear relationship has been shown to have a high probability by the value of the correlation coefficient (r), then the best straight line through the data points has to be estimated. This can often be done by visual inspection of the calibration graph but in many cases it is far better practice to evaluate the best straight line by linear regression (the method of least squares). 

The following procedure has been recommended by the Analytical Methods Committee of the Society for Analytical Chemistry for the determination of small amounts of arsenic in organic matter.20 Organic matter is destroyed by wet oxidation, and the arsenic, after extraction with diethylammonium diethyldithiocarbamate in chloroform, is converted into the arsenomolybdate complex the latter is reduced by means of hydrazinium sulphate to a molybdenum blue complex and determined spectrophotometrically at 840 nm and referred to a calibration graph in the usual manner. 

In many cases when methods involve internal or external standards, the solutions used to construct the calibration graph are made up in pure solvents and the signal intensities obtained will not reflect any interaction of the analyte and internal standard with the matrix found in unknown samples or the effect that the matrix may have on the performance of the mass spectrometer. One way of overcoming this is to make up the calibration standards in solutions thought to reflect the matrix in which the samples are found. The major limitation of this is that the composition of the matrix may well vary widely and there can be no guarantee that the matrix effects found in the sample to be determined are identical to those in the calibration standards. 

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. 

Szathmary and Luhmann [50] described a sensitive and automated gas chromatographic method for the determination of miconazole in plasma samples. Plasma was mixed with internal standard l-[2,4-dichloro-2-(2,3,4-trichlorobenzyloxy) phenethyl]imidazole and 0.1 M sodium hydroxide and extracted with heptane-isoamyl alcohol (197 3) and the drug was back-extracted with 0.05 M sulfuric acid. The aqueous phase was adjusted to pH 10 and extracted with an identical organic phase, which was evaporated to dryness. The residue was dissolved in isopropanol and subjected to gas chromatography on a column (12 m x 0.2 mm) of OV-1 (0.1 pm) at 265 °C, with nitrogen phosphorous detection. Recovery of miconazole was 85% and the calibration graph was rectilinear for 0.25 250 ng/mL. 

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. 

Byeon et al. [23] described a fluorimetric method for (z>)-penicillamine using 9-fluorenylmethyl pentafluorophenyl carbonate and acetonitrile. Capsules containing penicillamine were extracted with water and then filtered. The solution was incubated at 70 °C for 40 min with borate buffer solution. After cooling, the mixture was extracted with diethyl ether and the fluorescence of the aqueous phase measured at (excitation = 260 nm, emission = 313 nm). The calibration graph was linear for 0.4-5.0 pM of penicillamine with a coefficient of variation of 0.4%. 

Russell and Rabenstein [43] described a speciation and quantitation method for underivatized and derivatized penicillamine, and its disulfide, by capillary electrophoresis. Penicillamine and penicillamine disulfide were determined by capillary electrophoresis on a capillary (24 cm x 25 pm i.d. or 50 cm x 50 pm i.d. for underivatized thiols) with detection at 357 nm (200 nm for underivatized thiols). The run buffer solution was 0.1 M phosphate (pH 2.3). Detection limits were 20-90 pM without derivatization, and 5-50 pM after derivatization. Calibration graphs were linear from 1 pM to 5 mM thiols. 

Garcia et al. [45] determined penicillamine in pharmaceutical preparations by FIA. Powdered tablets were dissolved in water, and the solution was filtered. Portions (70 pL) of the filtrate were injected into a carrier stream of water that merged with a stream of 1 mM PdCl2 in 1 M HC1 for determination of penicillamine. The mixture was passed though a reaction coil (180 cm long) and the absorbance was measured at 400 nm. Flow rates were 1.2 and 2.2 mL/min for the determination of penicillamine, the calibration graphs were linear for 0.01-0.7 mM, and the relative standard deviation (n = 10) for 0.17 mM analyte was 0.8%. The method was sufficiently selective, and there were no significant differences between the labeled contents and the obtained results. 

Vinas et al. [47] determined penicillamine routinely by using batch procedures and FIA. A capsule was dissolved in water, diluted to 250 mL, and a suitable portion of the solution treated with 1 mM Co(II) solution (2.5 mL) and 2 M ammonium acetate (2.5 mL). The mixture was diluted to 25 mL and the absorbance of the yellow complex was determined at 360 nm. Calibration graphs were linear for 0.02-0.3 mM of penicillamine. The method was modified for flow injection analysis using peak-height or peak-width methods, but in both cases the flow rates were maintained at 3.3 mL/min. For the peak-height technique, calibration graphs were linear for 0.1-2 mM, and the sampling frequency was 150 samples per hour. For the peak-width method, the response was linear for 50 pM to 0.1 M, and this method was particularly useful for routine determinations. 

Mann and Mitchell [58] described a simple colorimetric method for estimation of (-D)-penicillamine in plasma. Blood containing 2-50 pg of penicillamine was mixed with 0.1 M EDTA solution in tromethamine buffer solution. 0.1 mL of this solution was adjusted to pH 7.4 and centrifuged. To a portion of the plasma was added 3 M HCL, the mixture was freeze-dried, and a suspension of the residue in ethanol was centrifuged. The supernatant liquid was mixed with tromethamine buffer solution (pH 8.2) and 10 mM 5,5 -dithiobis-(2-nitrobenzoic acid) in phosphate buffer solution (pH 7.0), the mixture was shaken with ethyl ether, and the absorbance of the separated aqueous layer was measured at 412 nm. The mean recovery was 60% (four determinations), and the calibration graph was linear for the cited range. 

Shaw et al. [64] described a (D)-penicillamine detection method in blood samples that had been treated with EDTA, deproteinized with trichloroacetic acid, and analyzed within 1 h. Penicillamine was detected at a vitreous-carbon electrode operated at +800 mV after HPLC separation. A linear calibration graph was obtained, and the method had a limit of detection equal to 5-20 ng. The method was useful in clinical and in pharmacokinetic studies. 

Vishwavidyalaya et al. [22] used a difference-spectrophotometric method for the estimation of primaquine phosphate in tablets. One portion of powdered tablets, equivalent to 7.5 mg of primaquine phosphate, was extracted with hydrochloric acid-potassium chloride buffer (pH 2) and a second portion was extracted with phosphate buffer (pH 10). Primaquine phosphate was determined from the difference in absorbance of the acid and alkaline extracts at 254.2 nm. The calibration graph was rectilinear from 2 to 14 pg/mL of primaquine phosphate. Recovery was 98.6% and no interference was observed from excipients. Results compared with those by the British Pharmacopoeial method. 

Ibrahim et al. [30] described a fluorimetric method for the determination primaquine and two other aminoquinoline antimalarial drugs using eosin. Powdered tablets or ampule contents containing the equivalent of 50 mg of the drug was extracted with or dissolved in water (100 mL). A 10 mL aliquot was mixed with 10 mL of aqueous ammonia, 1 mL of 0.001% eosin (C.I. acid red 87) in dichloro-ethane, and dichloroethane was added to volume. Primaquine was determined fluorimetrically at 450 nm (excitation at 368 nm). Calibration graphs were rectilinear for 0.1-5 pg/mL of primaquine. Recoveries were quantitative. The method could be readily adapted for determination of the drug in biological fluids. 

Cheng et al. reported the use of a synchronous fluorimetric method for the determination of primaquine in two-component antimalarial tablets [31]. Ground tablets were dissolved in water and the mixture was filtered. The fluorescence intensities of chloroquine phosphate and primaquine phosphate, in the filtrate, were measured at 380 nm (excitation at 355 nm) and 505 nm (excitation at 480 nm), respectively. The calibration graphs were linear from 1 to 8 pg/mL of chloroquine phosphate and 10 to 110 pg/mL of primaquine phosphate. The mean recoveries were 98.2-101.49% and the relative standard deviations were 2.23%. 

Three anticonvulsant drugs including valproic acid were determined using different dyes as ion-pair reagents. Gentian violet was used for the spectrophotometric detection at 588 nm and acridine orange for the fluorimetric detection at 470 nm after excitation at 297 nm. Calibration graphs were linear for 5-50 pg/mL 2.5 0.50 pg/mL for the spectrophotometric and fluorimetric methods, respectively [15]. 

Salgado Ordonez et al. [28] used di-2-pyridylketone 2-furoyl-hydrazone as a reagent for the fluorometric determination of down to 0.2 pg aluminium in seawater. A buffer solution at pH 6.3, and 1 ml of the reagent solution were added to the samples containing between 0.25 to 2.50 pg aluminium. Fluorescence was measured at 465 nm, and the aluminium in the sample determined either from a calibration graph prepared under the same conditions or a standard addition procedure. Aluminium could be determined in the 10-100 pg/1 range. The method was satisfactorily applied to spiked and natural seawater samples. 

In this method, inorganic lead in seawater samples are converted to tetra-ethylead using sodium tetraethylboron (NaB(C2H5)4) which is then trapped in a graphite furnace at 400 °C. Quantitation is achieved by using a simple calibration graph prepared from aqueous standards. An absolute detection limit of (3relative standard deviation. 

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