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reagent and the analyt in question, as well as factors (besides analyt concentration) contributing to the color intensity of the sample and standard respectively. The chemical selectively relies on the actual color reagent used and the analyt being examined and will be discussed in the individual chapters, but a few general remarks can be given on the subject of color intensity variations. Compounds containing an acid or base group will most likely have a pH dependent UV-visible absorption spectra. If there are large changes in the visible region it will be acknowledgeable as a change in color, a property forming the basis of pH indicators. Only compounds where the acid or base group contributes with their n or pi electrons to the conjugated electron systems of the molecule will show marked changes in absorption spectrum and in appearance. An obvious example is the pH indicator methyl red, which is red in its acid form (Figure 5.1) and change to its yellow basic form (Figure 5.2) at a pH near 5.1. This marked change in absorption behavior is equally evident in the absorption spectras measured at pH on both sides of the pKa of the indicator (Figure 5.3). As many of the color reagents used by the pharmacopoeia do contain an acid or base group even after reaction with the analyt, this behavior is often encountered in test and standard solutions. It means that a difference in pH and ionic strength between the standard and sample solution can give a difference in color intensity, if not even a difference in color, even with equal concentrations of the colored product. In this discussion, it is important to remember the less obvious acid and bases, for example enolisable ketones and aldehydes. These compounds will be found partly in their enol resonans form (Figure 5.4). In alkaline conditions, the equilibrium is likewise but with the enolate ion (Figure 5.5).
COO H N N
Figure 5.1 Methyl red, red acid form.
N(CH3)2
COO N N
Figure 5.2 Methyl red, yellow basic form.
N(CH3)2
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pH 2
pH 7
0.00A
350.0nm
650.0nm
O R C
OH C CH2
O R C CH2 R
O C CH2
Even though the colored compound in question does not contain a carboxylic acid group or a nitrogen base, but does contain, for example, an oxo group, it might have a proteolytic ability and show a pH dependent absorbance or appearance. If the acid or base group of a compound participates in a salt formation, the absorption spectra and in some cases the color intensity or appearance of the parent compound will be affected. Same behavior is seen when a compound participates in a complex formation with a metal, and this is widely used in determining metal ions with the aid of organic color reagents. The same effect can, however, also be the cause of unintended interference in cases were a test solution or reagent solution in a color reaction is contaminated with metal ions. Complex forming metals are usually found in the d block of the periodic table as listed in Table 5.1 (or the rare f block) shown in Table 5.1. They form
100 Pharmaceutical Chemical Analysis: Methods for Identication & Limit Tests
Table 5.1 D Block Elements
H Li Na K Rb Cs Fr Be Mg Ca Sr Ba Ra B Al Ni Cu Zn Ga Pd Ag Cd In Pt Au Hg Tl C Si Ge Sn Pb N P As Sb Bi O S Se Te Po F Cl Br I At He Ne Ar Kr Xe Rn
Sc Y La Ac
Ti Zr Hf Unq
V Nb Ta Unp
Cr Mo W Unh
Mn Tc Re Uns
Fe Ru Os Uno
Co Rh Ir Unn
complexes due to their ability to act as Lewis bases, and the tendency of the complexes to be colored is because their d and f electrons are easily excitable. A Lewis acid is an electron pair acceptor and a Lewis base is an electron pair donator. A complex is formed when a Lewis acid and a Lewis base share a pair of electrons and form a bond. When an organic compound is used as a ligand in a complex formation with a metal it acts as a Lewis acid and the combination in many cases produces real acids. This means that the metal Lewis bases compete with the acidic loose protons for the sharing electron and will be replaced by the proton at pH values that are low compared to the pKa of the ligand in question. The metals of the d block, on the other hand, are known for forming insoluble hydroxides or oxides at neutrality or high pH. So a complex is often stable only inside the pH interval between ligand deprotonation and metal hydroxide precipitation. The intensity of a condensated color product is similarly pH dependent, but through a somewhat different mechanism. Here two conditions have to be fullled. The analyt and the reagent should be mixed in a test solution with a pH facilitating formation of the colored condensate, and when this product is formed it is desirable to shift the test solution pH toward a value giving the optimum color intensity the best stability achievable. It is very likely that the pH optimum of the reactions and of the product are alike, and a difference between the standard and test solution pH can therefore give rise to color intensity differences through two different mechanisms. Another general remark one could make regarding colored condensation products is that the chemical reactions affording them are in many cases reversible and that an equilibrium therefore is established. This equilibrium might not be shifted entirely to the right. A quantitative reaction is by no means a requisite for the analysis to be valuable, as long as the equilibrium constant in the test solution and standard are alike, giving a similar yield of colored product in both solutions. This again brings focus on ensuring that the chemical environment in the standard and test are alike since many factors inuence a chemical equilibrium, including pH, ionic strength reagent concentration, and temperature. The last parameter affects both the position of the equilibrium and the speed with which the equilibrium is reached. Many chemical reactions have reaction kinetics in which a 10C change in temperature affords a two- to threefold increase in reaction speed. The absorption spectra or even appearance of some colored compounds varies with the nature and concentration of the solvent in which they are dissolved. Polar solvents are capable of giving weak interactions with the
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electrons of a molecules conjugated system, whereas nonpolar solvents are not. Much effort has been given into elaborating systems capable of predicting the shift in absorbance maximum and intensity caused by a given change in solvent, and with some success too. An example of solvents affecting visual appearance is found in 3.19. Iodides. Much of the literature dealing with the individual color reactions found in the pharmacopoeia describe methods for quantitative spectrophotometric determinations, whereas in the pharmacopoeia they most often are simple colorimetric tests. In these cases, it is valuable to correlate the visual color of a complex with its absorbance maximum. This is tabulated below in Table 5.2 correlation, where the different maximum absorbance wavelength intervals are correlated with the expected color of the compound, hue (transmitted), and with the color of the absorbed wavelengths, complementary hue.
Table 5.2 Transmitted and Complementary Hue Wavelength (nm) 400435 435480 480490 490500 500560 560580 580595 595610 610750 Hue (transmitted) Violet Blue Greenish-blue Bluish-green Green Yellowish-green Yellow Orange Red Complementary hue Yellowish-green Yellow Orange Red Purple Violet Blue Greenish-blue Bluish-green