The Purity Test

It is true that, in order to see if it was strong and fit to stand a cut, he drew his sword and gave it a couple of slashes, the first of which undid in an instant what had taken him a week to do. 

Partial proteolytic digestion provides quick information. However, because the sensitivity of a protein as opposed to proteases strongly depends on its conformation, this information is only dependable if both samples are completely denatured during the digestion and contain sufficient SDS ( 0.05%) (Walker and Anderson 1985). 

Cleveland, D., et al. (1977). Peptide Mapping by Limited Proteolysis in Sodium Dodecyl Sulfate and Analysis 

Walker, A., and Anderson, C. (1985). Partial Proteolytic Protein Maps Cleveland Revisited, Anal. Biochem. 

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This system was found optimal for the purity test. 

As required by regulatory authorities [1-3, 6] and International Conference on Harmonisation (ICH) guidelines [7], analytical method validation is especially important in establishing the assay methods and procedures of quantitative or semi-quantitative measurement of target substances or compounds. Among the specification items for drug products, assay, content uniformity, and dissolution are typical of those which require almost full analytical validation. The purity test, related... 

Figure 67 shows, as an example, the purity test of cimetidine on 10x10 cm or 5 x 5 cm HPTLC plates. The amount applied to the 10 x 10 cm plate was reduced to half the amount prescribed in the DAB, and the amount applied to the 5x5 cm plate was V4 of this. Whereas the limiting concentrations on the larger plate are just detectable after derivatization with iodine, a zone can be seen only on lane 2 of the smaller plate. 

The use of reaction sequences is also common in the analysis of pharmaceuticals. For example, for the purity test for hydrochlorothiazide (HCT) the DAB specifies a nitration reaction followed by reaction with naphthylethylenediamine dihydrochloride, in which the reaction sequence is performed as in the example given below. HCT is a diuretic which is often used in combination with beta-blockers for the treatment of hypertension (high blood pressure). Diuretics of this type are also abused in sport (doping) and appear on the list of banned substances prepared by the IOC (International Olympic Committee). In the quantitative determination of HCT in urine, the red azo dyes formed in the derivatization (parent substance and metabohtes) is regarded as a doping-positive result [111]. The complete specification for this analytical method, known as Application A-43.2, can be obtained from CAM AG. 

Example of a reaction sequence for derivatization detection limit test for known and unknown compounds in the purity testing of ambroxol hydrochloride... 

Figure 46. Effect of the presence of 1 % ammonia solution in the solvent system on the purity testing of dimenhydrinate according to the DAB (shots in 254-nm UV light)...

ANOVA can also be used in situations where there is more than one source of random variation. Consider, for example, the purity testing of a barrelful of sodium chloride. Samples are taken from different parts of the barrel chosen at random and replicate analyses performed on these samples, in addition to the random error in the measurement of the purity, there may also be variation in the purity of the samples from different parts of the barrel. Since the samples were chosen at random, this variation will be random and is thus sometimes known as a random-effect factor. Again, ANOVA can be used to separate and estimate the sources of variation. Both types of statistical analysis described above, i.e. where there is one factor, either controlled or random, in addition to the random error in measurement, are known as one-way ANOVA. The arithmetical procedures are similar in the fixed- and random-effect factor cases examples of the former are given in this chapter and of the latter in the next chapter, where sampling is considered in more detail. More complex situations in which there are two or more factors, possibly interacting with each other, are considered in Chapter 7. 

The semiquantitative estimation of impurities by visual comparison is generally used in different pharmacopoeias for the purity testing of active raw materials and formulated products, as well. Four purity criteria can be defined ... 

For isolation of the core peptides of PS I (CPI) the 66 kDa region was cut out after SDS-PAGE and was eluted from the gel by electroelution at 200 V for 8 h. The purity test of the obtained CPI was carried out by SDS-PAGE and by Western blot procedure. For analysis of lipids the PS I particles and the isolated CPI (PS I core peptides) were dialysed against water, precepitated with acetone, centrifuged at 5.000 x g for 20 min and washed two times with water. The sediments were extracted with boiling methanol and subsequently extracted with methanol/chloroform (1 2 v/v). For the HPLC analysis a lipid solution in methanol was used. The analysis was carried out with a RP-18 column with acetonitrile/water/phosphoric acid (85%) 60.39, 9 0, 1 (v/v/v). The column pressure was 5,5 bar and the flow-rate 0.5 ml/min. Detection of eluted lipids was obtained with a spectral-detector at 205 nm. 

Although phenol gives these marked colour changes, the test is unsatisfactory with many other phenols, the precise tint obtained varying with the purity of the phenol, amount of reagents used, and temperature and time of heating. 

Influence of added substances upon the critical solution temperature. For a given pressure the C.S.T. is a perfectly defined point. It is, however, affected to a very marked extent by the addition of quite a small quantity of a foreign substance (impurity), which dissolves either in one or both of the partially miscible liquids. The determination of the consolute temperature may therefore be used for testing the purity of liquids. The upper consolute temperature is generally employed for this purpose. 

Testing the purity of a compound. If the spectrum of a sample of known purity is available, the presence of impurities in another sample can be detected from the additional bands in its infrared spectrum. 

The other two methods used by industry to examine the purity of maleic anhydride are the crystallization point (168) and color deterrnination of the sample (169). These tests determine the temperature at the point of solidification of the molten sample and the initial color properties of the melt. Furthermore, the color test also determines the color of the sample after a two-hour heat treatment at 140°C. The purpose of these tests is to determine the deviation in properties of the sample from those of pure maleic anhydride. This deviation is taken as an indication of the amount of contaminants in the maleic anhydride sample. 

Gra.de A. Types I and II both represent the requirements of the USP XX (26). The USP tests arose from original formal oxygen specifications made necessary by the low purity and certain contaminants, particularly CO and CO2, contributed by early chemical and cryogenic manufacturing methods. Containers marked Oxygen-USP must also indicate whether or not the gas has been produced by the air Hquefaction process (see also Fine chemicals). 

Both the Toth and Alcoa processes provide aluminum chloride for subsequent reduction to aluminum. Pilot-plant tests of these processes have shown difficulties exist in producing aluminum chloride of the purity needed. In the Toth process for the production of aluminum chloride, kaolin [1332-58-7] clay is used as the source of alumina (5). The clay is mixed with sulfur and carbon, and the mixture is ground together, pelletized, and calcined at 700°C. The calcined mixture is chlorinated at 800°C and gaseous aluminum chloride is evolved. The clay used contains considerable amounts of silica, titania, and iron oxides, which chlorinate and must be separated. Silicon tetrachloride and titanium tetrachloride are separated by distillation. Resublimation of aluminum chloride is requited to reduce contamination from iron chloride. 

Reference 38 is a good guide to the selection of plate thickness test methods. Test methods may vary with the purity and electrochemical activity of the deposit. Metals deposited from commercial plating solutions are seldom pure. For example, zinc deposits from the three commonly used baths, ie, cyanide, chloride, and zincate, vary significantly in purity and activity (39). Standard ASTM test methods for determining plate thickness are... 

One of the best tests of purity of dioxane is the formation of the purple disodium benzophenone complex during reflux and its persistence on cooling. (Benzophenone is better than fluorenone for this purpose, and for the storing of the solvent.) [Carter, McClelland and Warhurst Trans Faraday Soc 56 343 I 960], TOXIC. 

Evaluation of loop-test results Although the thermal loop test approximates to the conditions which obtain in a dynamic heat-transfer system, in evaluating the results it is necessary to be aware of those aspects in which the test differs from the full-scale unit, as otherwise unwarranted confidence may be placed in the data. Assuming that adequate attention has been paid to the purity and condition of components, etc., the following factors will, according to ASTM G68 1980, influence the observed corrosion behaviour ... 

If there is any doubt as to the purity of the reagents used, they should be tested by standard methods for the impurities that might cause errors in the determinations. It may be mentioned that not all chemicals employed in quantitative analysis are available in the form of analytical reagents the purest commercially available products should, if necessary, be purified by known methods see below. The exact mode of drying, if required, will vary with the reagent details are given for specific reagents in the text. 

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