Colorimetric analysis, sample preparation

Experiment 19 Colorimetric Analysis of Prepared and Real Water Samples for Iron... 

Assessing the resources available for method development should also be done before beginning a project. The resources available include not only HPLCs, detectors, and columns, but also tools for sample preparation, data capture and analysis software, trained analysts, and especially samples representative of the ultimate analyte matrix. Also, it should be considered whether a fast, secondary method of analysis can be used to optimize sample preparation steps. Often, a simple colorimetric or fluorimetric assay, without separation, can be used for this purpose. A preliminary estimate of the required assay throughput will help to guide selection of methods. 

Kimerle [27] reviewed the ecotoxicology of LAS focusing on the results rather than on the method of analysis, for which the author referred to the review undertaken by Painter and Zabel [30], alluding only to two papers on biota sample preparation. Litz et al. [31] determined the concentration of LAS in rye grass by Azure A active substances (AzAAS). AzAAS is a non-specific colorimetric method, which has not been used as frequently as MBAS (see Chapter 3.1). Briefly, it consists of the formation of an ion association complex with a dyed solution of Azure A (cationic). The complex formed is solvent-extractable and is separated from unreacted dye prior to colour measurement. 

A. Analysis of Wastewater and Natural Waters. The presence of certain anions in wastewater effluents can cause deterioration of natural water systems. Phosphorous and nitrogen can be present in several chemical forms in wastewaters. Phosphorous is usually present as phosphate, polyphosphate and organically-bound phosphorus. The nitrogen compounds of interest in wastewater characterization are ammonia, nitrite, nitrate and organic nitrogen. Analyses are often based on titrimetric, and colorimetric methods (3). These methods are time consuming and subject to a number of interferences. Ion Chromatography can be used to determine low ppm concentrations of these ions in less than thirty minutes with no sample preparation. 

Measurement of trace metals, including nickel in seawater can be completed using an in-line system with stripping voltammetry or chronopotentiometry (van den Berg and Achterberg 1994). These methods provide rapid analysis (1-15 minutes) with little sample preparation. The detection limit of these methods for nickel was not stated. Recommended EPA methods for soil sediment, sludge, and solid waste are Methods 7520 (AAS) and 6010 (ICP-AES). Before the widespread use of AAS, colorimetric methods were employed, and a mrmber of colorimetric reagents have been used (Stoeppler 1980). 

Glucose was analyzed by using the Folin-Wu procedure (32) modified as follows 25-mL glass-stoppered tubes were used, the final volume was 15 or 20 mL, and colorimetric analysis was performed at 650 nm. The aqueous and acetonitrile column effluents were analyzed prior to further concentration 200- iL aliquots were used in the Folin-Wu test. Samples were prepared for Folin-Wu analysis by extracting them with dichloromethane to remove other species and evaporating the remaining aqueous phase to about 5 mL. 

Currently, high-performance liquid chromatography (HPLC) methods have been widely used in the analysis of tocopherols and tocotrienols in food and nutrition areas. Each form of tocopherol and tocotrienol can be separated and quantified individually using HPLC with either a UV or fluorescence detector. The interferences are largely reduced after separation by HPLC. Therefore, the sensitivity and specificity of HPLC methods are much higher than those obtained with the colorimetric, polarimetric, and GC methods. Also, sample preparation in the HPLC methods is simpler and more efficiently duplicated than in the older methods. Many HPLC methods for the quantification of tocopherols and tocotrienols in various foods and biological samples have been reported. Method number 992.03 of the AOAC International Official Methods of Analysis provides an HPLC method to determine vitamin E in milk-based infant formula. It could probably be said that HPLC methods have become dominant in the analysis of tocopherols and tocotrienols. Therefore, the analytical protocols for tocopherols and tocotrienols in this unit are focused on HPLC methods. Normal and reversed-phase HPLC methods are discussed in the separation and quantification of tocopherols and tocotrienols (see Basic Protocol). Sample... 

Wet chemical methods involve sophisticated sample preparation and standardization with National Bureau of Standards reference materials but are not difficult for the analytical chemist nor necessarily time consuming (Figure 1). The time from sample preparation to final results for various analytical methods, such as GFAA (graphite furnace atomic absorption), ICP (inductively coupled plasma spectroscopy), ICP-MS (ICP-mass spectrometry), and colorimetry, ranges from 0.5 to 5.0 h, depending on the technique used. Colorimetry is the method of choice because of its extreme accuracy. Typical results of the colorimetric analysis of doped oxides are shown in Tables I and II, which show the accuracy and precision of the measurements. 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

For colorimetric analysis, plasma and urine samples are prepared by alkalinization and chloroform extraction. Methocarbamol couples with diazotized dinitroaniline to give an orange-red compound33. Alternatively, colorimetric determination with chromotropic acid is possible after alkaline hydrolysis (10 mins, in boiling water) of the carbamate and periodate oxidation to formaldehyde34. 

Sodium and potassium levels are difficult to analyze by titrimetric or colorimetric techniques but are among the elements most easily determined by atomic spectroscopy (2,38) (Table 2). Their analysis is important for the control of infusion and dialysis solutions, which must be carefully monitored to maintain proper electrolyte balance. Flame emission spectroscopy is the simplest and least expensive technique for this purpose, although the precision of the measurement may be improved by employing atomic absorption spectroscopy. Both methods are approved by the U.S. (39), British (40), and European (41) Pharmacopeias and are commonly utilized. Sensitivity is of no concern, due to the high concentrations in these solutions furthermore, dilution of the sample is often necessary in order to reduce the metal concentrations to the range where linear instmmental response can be achieved. Fortunately, the analysis may be carried but without additional sample preparation because other components, such as dextrose, do not interfere. 

The direct determination of zinc in diet, tissue and in body fluids can be accomplished by a variety of methods. A common limitation is the chance of sample contamination prior to analysis. Some early studies using less sensitive methods may not have recognised this problem and reported erroneously high results. Older colorimetric methods required that the biological sample be efficiently digested or otherwise deproteinised, prior to formation of a coloured zinc complex. These techniques have largely been superseded by atomic absorption spectrometry which is more sensitive yet less prone to interferences. For fluids such as plasma or urine, simple dilution is all that is required prior to analysis. Tissue or diet samples only require to be dissolved in mineral acid. These simpier sample preparation procedures limit the chances of contamination. 

The samples preparation and extraction of phenolics from source materials is the first step involved in their analysis. While colorimetric methods are used for determination of different classes of phenolics, chromatographic and... 

In plastics analysis, there are a variety of analytical methods for characterization of morphological differences related to degradation of resins from reprocessing. These techniques include FTIR, thermal analysis (TGA, DSC, TMA, DMA), Colorimetric analysis, ESCA or XPS, and GPC. These methods can be laborious and require technical expertise and equipment not always readily available. An imaging method correlated to one of these techniques, especially Melt Flow Index, is extremely useful. Such a method is rapid to obtain by imaging, reduces sample preparation time, and provides a scale of intensities for purposes of correlation. The method is surface sensitive. 

Analytical Techniques. Sorbic acid and potassium sorbate are assayed titrimetricaHy (51). The quantitative analysis of sorbic acid in food or beverages, which may require solvent extraction or steam distillation (52,53), employs various techniques. The two classical methods are both spectrophotometric (54—56). In the ultraviolet method, the prepared sample is acidified and the sorbic acid is measured at 250 260 nm. In the colorimetric method, the sorbic acid in the prepared sample is oxidized and then reacts with thiobarbituric acid the complex is measured at - 530 nm. Chromatographic techniques are also used for the analysis of sorbic acid. High pressure Hquid chromatography with ultraviolet detection is used to separate and quantify sorbic acid from other ultraviolet-absorbing species (57—59). Sorbic acid in food extracts is deterrnined by gas chromatography with flame ionization detection (60—62). 

Analysis of antioxidant activity by performing a FRAP assay was proposed by Benzie and Strain [23]. It involves colorimetric determination of the reaction mixture in which the oxidants contained in the sample reduce Fe ions to Fe. At low pH, Fe(in)-TPTZ (ferric-tripyridltria-zine) complex is reduced to the ferrous (Fe ) form and intense blue colour at 593 nm can be observed. The FRAP reagent is prepared by mixing 2.5 ml of TPTZ (2,4,6-tris (l-pyridyl)-5-triazine) solution (10 mM in 40mM HCl), 25 ml acetate buffer, pH 3.6, and 2.5 ml FeCl3 H20 (20 mM). The colour of Fe(II)(TPTZ)2 which appears in the solution is measured colorimetri-cally after incubation at 37°C. The measurement results are compared to those of a blank sample, which contains deionised water instead of the analysed sample. The duration of the assay differs from one study to another 4 min [23, 24], 10 min [25] to 15 min [26]. The analysis results are converted and expressed with reference to a standard substance, which can be ascorbic acid [26], FeS04 [23, 25], Trolox [27,18]. 

Meat discoloration studies typically involve a maximum of 5 days, with discoloration analy-sis being performed every day or on alternate days. The actual experimental time involved in the objective assessment of discoloration is not extensive and depends on the number of samples being analyzed. Colorimetric measurements with hand-held colorimeters are very rapid (three measurements per meat surface in < 1 min). Spectral scans of meat surfaces require 1 to 2 min. Extraction and analysis of ground meat products has the added step of homogenization and filtration prior to spectrophotometry, but relative to many laboratory procedures, this is relatively quick. Isolation and purification of preparative amounts of myoglobin requires only 2 to 3 days once appropriate preparations are made. Finally, metmyoglobin can be reduced to oxymyoglobin in 15 to 20 min. 

The preparation of a standard calibration curve is required for many colorimetric and gas chromatography analyses. A fresh calibration check standard at any selected concentration should be prepared daily and analyzed prior to sample analysis. If the response for the check standard falls outside of 15% standard deviation for the same concentration in the standard calibration curve, then a new calibration curve should be prepared. 

Perform a blank analysis using the same volume of caustic soda dilution solution. Prepare a series of cyanide standards and plot a calibration curve from pg CN vs. absorbance. The efficiency of the sample distillation step may be checked by distilling two of the standards and then performing the above colorimetric test. Distilled standards must agree within 10% of undistilled standards. 

The most widely used chemical method is the antimony trichloride colorimetric method. The method is applicable to vitamins D2 and D3 for their analysis in pharmacopeial preparations. The reaction product of vitamin D3 with antimony trichloride is believed to be isovitamin D3 (46, See Scheme IV). Antimony trichloride reacts with vitamin A also. Vitamin A occurs along with D3 in many biological samples and is also an ingredient in many commercial products. Therefore, it is necessary to remove it and other interfering substances prior to reaction with... 

Takeuchi et al. published a mechanized assay of serum cholinesterase by specific colorimetric detection of the released acid [40]. The cholinesterase reaction was carried out on a thermostatted rack at 30° C with a reaction mixture of serum (10 pL), 50mM barbitone-HCl assay buffer (pH 8.2 140 pL), and 12.5 mM acetylcholine solution (50 pL). The solutions were prepared by programmed needle actions, and a sample blank was also prepared. The reaction was stopped after 9.7 min by injection of the mixture into a flow injection analysis system to determine the quantity of acetic acid formed. The carrier stream (water, at 0.5mL/min) was merged with a stream (0.5mL/min) of 20 mM 2-nitrophenylhydrazine hydrochloride in 0.2 M HC1 and a stream (0.5 mL/min) of 50 mM 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride in ethanol containing 4% of pyridine. The sample was injected into this mixture (pH 4.5), passed through a reaction coil (10 m x 0.5mm) at 60°C, 1.5M NaOH was added, and, after passing through a second reaction coil (lm x 0.5 mm) at 60°C, the absorbance was measured at 540 nm. 

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