Color differentiation method

For many situations, a simple total anthocyanin determination is inappropriate because of interference from polymeric anthocyanins, anthocyanin degradation products, or melanoidins from browning reactions. In those cases, the approach has been to measure the absorbance at two different pH values. The differential method measures the absorbance at two pH valnes and rehes on structural transformations of the anthocyanin chromophore as a function of pH. Anthocyanins switch from a saturated bright red-bluish color at pH 1 to colorless at pH 4.5. Conversely, polymeric anthocyanins and others retain their color at pH 4.5. Thus, measurement of anthocyanin samples at pH 1 and 4.5 can remove the interference of other materials that may show absorbance at the A is-max-... 

The pH differential method was described as a fast and convenient assay for the quantitation of monomeric anthocyanins by Giusti (2001). It was approved by the Association of OfQcial Analytical Chemists (AOAC) in 2005 as a standard method to evaluate total monomeric anthocyanin pigment content in fruit juices, beverages, natural colorants, and wines. The degradation index is the ratio between total and monomeric anthocyanins (Table 6.3.1). The content of total anthocyanins can be obtained by the single pH method and the monomeric anthocyanin by the pH differential method. ... 

Lee, J., Durst, R., and Wrolstad, R., AOAC official method 2005.02 total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method, in Official Methods of Analysis of AOAC International, Horowitz, H., Ed., AOAC, Washington, D.C, 2005. 

Basic Protocol 1 Total Monomeric Anthocyanin by the pH-Differential Method FI. 2.1 Basic Protocol 2 Indices for Pigment Degradation, Polymeric Color, and... 

Anthocyanin pigments undergo reversible structural transformations with a change in pH manifested by strikingly different absorbance spectra (Fig. FI.2.1). The colored oxonium form predominates at pH 1.0 and the colorless hemiketal form at pH 4.5 (Fig. FI.2.2). The pH-differential method is based on this reaction, and permits accurate and rapid measurement of the total anthocyanins, even in the presence of polymerized degraded pigments and other interfering compounds. 

The pH-differential method is of special importance for investigations of anthocyanins suspectedly adulterated with synthetic red food colorants. Adulteration with synthetic food colorant is overlooked by the simple UVA IS measimement described before as those food colors are stable imder extraction/hydrolysis conditions and may add substantial absorption at 510-540 mn masking perfectly the lack of anthocyanins. 

However, the pH-differential method serves as quick and simple check for adulteration as synthetic food colorants are not pH-sensitive in UV-absorption. As seen in Table 10, spiking with synthetic food colors to mask a 50 % lack of anthocyanin content passes successfully conventional UV/VlS-spectroscopy but fails in the pH-differential method. 

For confirmatory purposes, aqueous bilberry extract solutions resulting fi om the test protocol of the pH-differential method were injected onto a HPLC system used in om- laboratory for the determination of synthetic food colorants (adopted from [138]). 

Thermal Properties. The thermal stabiUty of cellulose esters is deterrnined by heating a known amount of ester in a test tube at a specific temperature a specified length of time, after which the sample is dissolved in a given amount of solvent and its intrinsic viscosity and solution color are deterrnined. Solution color is deterrnined spectroscopically and is compared to platinum—cobalt standards. Differential thermal analysis (dta) has also been reported as a method for determining the relative heat stabiUty of cellulose esters (127). 

In a more modified approach, differential display proteomics can also be done with no separation of proteins. This is called the protein chip approach. In this method, a variety of bait proteins such as antibodies, peptides, or protein fragments may be immobilized in an array format on specially treated surfaces. The surface is then probed with the samples of interest. Proteins that bind to the relevant target can then be analyzed by direct MALDI readout of the bound material (Nelson, 1997 Davies et ah, 1999). Lor example, well-characterized antibodies can be used as bait. Protein samples from two different cell states are then labeled by different fluorophores, mixed together, and used as probe. In such a case, the fluorescent color acts as an indicator for any change in the abundance of the protein that remains bound to the chip (Lueking et ah, 1999). A number of technical problems would still need to be overcome before applying this technique for large-scale analysis of proteins. 

JJ)M,A. Laccetti, S. Semel M. Roth, AnalChem 31, 1049—50(1959). The fermus sulfate-sulfuric acid method is used to differentiate HMX and RDX from NGu, Tetryl, TEGN, PETN, NG K nitrate. Following the procedure given (which requires the colorimetric measurement of the color produced on dissolving the sample in the reagent) reveals that HMX and RDX have Beet s Law... 

Table B1.1.5 demonstrates the relative degree of protein-to-protein variation that can be expected with the different protein assay methods. This differential may be a consideration in selecting a protein assay method, especially if the relative color response ratio of the protein in the samples is unknown. As expected, the protein assay methods that share the same basic chemistry show similar protein-to-protein variation.

Total nitrogen of TNT in wastewater was determined by preliminary digestion with K2S203 in alkaline soln and the N03 released was reduced by an automated Cd reduction method (Ref 133), Trinitrobenzoic acid was dissolved in water and titrated with NaOH without an indicator until a weak pink color appeared and persisted even in the presence of TNT and TNB (Ref 34). A gas comparison pycnometer used in conjunction with a Velidyne D15 differential pressure transducer with He yields a density accuracy of 0.002g/ml (Ref 134). 

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