Standard additions, Color Plate

The white plate is used by most investigators for standardization, but there may be instances where the colored standard is preferred. Whichever is chosen, that same plate must be used throughout the discoloration study. Additionally, the exact specifications of the standardization plate should be indicated in any scientific report. 

If the pH of the solution is above 10, add about 0.2 g Na2C03 and mix. Add a drop of phenophthalein indicator which will turn the solution red (or pink) in alkaline medium. Add 1 N HC1 dropwise till the color disappears. Place 3 drops of the above pretreated sample, 3 drops of distilled water, and 3 drops each of thiocyanate standards (0.05, 0.1, and 0.2 mg SCNYL) in the cavities of porcelain spot plate. Add 1 drop of chloramine-T solution to each cavity and mix with a clean glass rod. This is followed by the addition of 1 drop of pyridine-barbituric acid to each cavity. Again, mix the contents and allow it to stand for a minute. If thiocyanate is present, the sample spot will turn pink to red, depending on the concentration of SCN- in the sample. If deep red coloration is produced, dilute the sample and repeat the test. 

Testing of contrast sensitivity is an additional screening procedure that may detect early macular dysfunction, particularly in patients younger than 40 years of age. Color vision should be evaluated with a color vision test designed to detect both mild blue-yellow and protan red-green deficiencies. Tests that meet these criteria are the Standard Pseudoisochromatic Plates Part 2 and the American Optical Hardy-Rand-Ritter. 

Direct quantification is carried out in situ rather than after spot elution. The simplest direct method involves visual comparison of sample zone size and/or intensity (color) variation according to concentration against reference standards developed on the same plate.f This qualitative/semiquantitative approach is specified in various pharmacopeias for the purity analysis of active raw materials and formulated products. These pharmacopeial methods are designed for analyses at several levels 1) simple detection of impurities as additional spots 2) detection and identification of impurities by comparison to the R( values distances of standards or 3) detection, identification, and estimation of amounts of impurities by comparing intensities between samples and standard dilutions of the same compounds. ... 

The identification of unknown pesticide zones is initially based on the comparison of the migration of sample zones relative to standards developed on the same layer and colors obtained with selective chromogenic and fluorogenic detection reagents. Many densitometers can record in situ UV and visible absorption and fluorescence excitation spectra to confirm compound identification by the comparison of unknown spectra with stored standard spectra obtained under identical conditions or spectra of standards measured on the same plate. Additional confirmation methods include off-line and on-line combination of TLC with infrared, Raman,... 

Substrate (1 mg/mL ABTS, 1 mg/mL urea hydrogen peroxide in citrate buffer 0.024 M citrate, 0.047 M phosphate, pH 5.0) was added and color was allowed to develop for 30 min. The color reaction was stopped by the addition of 100 uL 0.5 M citric acid. Absorbance of each well was measured at 405 nm with a microtitre plate reader. Absorbance values of the standards and the samples (A) were divided... 

In a typical ELISA test on a microtiter plate (Fig. 5). the carrier is first coated with analytebinding antibody. The sample or standard is added after washing off excess antibody with a surfactant solution. The volume added is typically 100 - 200 pL. After (optional) preincubation, a constant quantity of tracer, e.g., enzyme-labeled hapten, is added to the sample or standard. This initiates a competitive reaction, because only a limited number of antibodies are available for binding. After the tracer incubation period, sample (or standard) and excess reagents are washed away. The bound tracer concentration is inversely proportional to that of the analyte. The amount of bound tracer can be determined via an enzyme substrate reaction with a chromogenic substrate. After a reaction time long enough to produce sufficient dye, the enzyme is denatured (enzyme reaction stopped) by addition of acid. Subsequently, the depth of color formed in the individual cavities of the microtiter plate can be automatically determined with a plate reader (photometer). 

Nuttall and Bush (102) described a TLC chromatographic method for the analysis of multivitamin preparations. After extraction of fat-soluble vitamins, water-soluble vitamins and water-soluble materials were separated in three TLC systems. Biotin was resolved with acetone-acetic acid-benzene-methanol (1 1 14 4) as solvent and visualized by spraying o-toluidine-potassium iodide. Standards can be included if quantitative results are required. However, the reproducibility of the technique has not been tested. Groningsson and Jansson (105) worked out a TLC method for the determination of biotin in the presence of other water-soluble vitamins. After dissolution of the lyophilized preparation and addition of the internal standard (2-imidazolidone), the sample was applied on a silicagel plate and eluted with chloroform-methanol-formic acid (70 40 2). Biotin was visualized by spraying with p-DACA and determined in situ by reflectance measurements. The sensitivity of the method could be increased by spraying with paraffin after the coloring procedure. LFnder these conditions the detection limit was 10 ng. 

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