Amino-groups, estimation

Estimation of the Number of Amino Groups in Aniline. First Method. 

If the molecular weight of the aniline is known, the number of amino groups can be calculated alternatively, if the aniline is known to be a monacidic base, its molecular weight can be calculated. If the molecular weight and the acidity of the aniline are both known, then dearly the method can be used to estimate the amount of aniline in a given sample. The method is general for many primary and secondary amines, aniline being used solely as a typical member of the former class. 

Another important method of following protein hydrolysis is that due to Van Slyke, and consists in estimating the free amino groups liberated by treatment with nitrous acid, whereby gaseous nitrogen is evolved and measured in a special apparatus. 

Conjugation of the amino group of an aiylfflnine with a second aiomatic ring, then a thud, reduces its basicity even further. Diphenylamine is 6300 times less basic than aniline, whereas triphenylamine is scarcely a base at all, being estimated as 10 ° times less basic than aniline and 10 " times less basic than ammonia. 

A strictly dehned region of chemical shifts of C2, C4, and C5 atoms in A-oxides of 4A-imidazoles allows to dehne clearly the position of the A-oxide oxygen atom (102). Chemical shifts of the a-C nitrone group in a-N-, O-, and S-substituted nitrones are located in the region of 137 to 150 ppm (388, 413). On the basis of 13C NMR analysis of 3-imidazoline-3-oxide derivatives, the position of tautomeric equilibria in amino-, hydroxy-, and mercapto- nitrones has been estimated. It is shown that tautomeric equilibria in OH- and SH-derivatives are shifted toward the oxo and thioxo forms (approximately 95%), while amino derivatives remain as amino nitrones (413). In the compounds with an intracyclic amino group, an aminonitrone (A) - A-hydroxyaminoimino (B) tautomeric equilibrium was observed (Scheme 2.76), depending on both, the nature of the solvent and the character of the substituent in position 2 of the heterocycle (414). 

The determination of amino nitrogen before and after acid hydrolysis of urine has frequently been used for the quantitative estimation of the amount of urinary peptides (H5, M4). The number of liberated a-amino groups represents, in fact, the whole of formerly combined amino groups, not necessarily attached to a second amino acid partner. Besides, considerable losses connected with decomposition of some amino acids occur in the course of hydrolysis thus limiting the true quantitative value of this procedure. 

The imidazole side-chain of histidine has a value of 6.0, making it a weaker base than the unsubstituted imidazole. This reflects the electron-withdrawing inductive effect of the amino group, or, more correctly the ammonium ion, since amino acids at pH values around neutrality exist as doubly charged zwitterionic forms (see Box 4.7). Using the Henderson-Hasselbalch equation, this translates to approximately 9% ionization of the heterocyclic side-chain of histidine at pH 7 (see Box 4.7). In proteins, plCa values for histidine side-chains are estimated to be in range 6-7, so that the level of ionization will, therefore, be somewhere between 9 and 50%, depending upon the protein. 

The H-NMR spectrum of lra/3s-4-aminocyclohexane carboxylic acid ethyl ester is enriched in a mixture containing ca. 10 % of the cis isomer is seen in Figure 3. The signals belonging to the protons next to the ester and amino groups in the trans isomer appeared at chemical shifts of 5=2.20 ppm and 5=2.65 ppm, respectively. The intensity of signals in the NMR spectra allows estimating the ratio of stereoisomers. 

It had been found previously [106] that cell adhesion on the surface of HA copolymers decreased with increasing ambient pH. With polyHEMA, on the other hand, no pH-dependency was observed. We considered the adhesion to be caused primarily by ionic interaction between the lymphocytes and the HA surfaces. The degree of protonation (a) of amino groups in HA copolymer was estimated by acid-base titration of an HCI solution of polyamine macromonomer with NaOH solution. In physiological conditions (pH 7.2-7.4) about 50% of the amino groups of the macromonomer are protonated. In this pH range, the polyamine macromonomer was found to be insoluble. 

The tautomeric ratio of B to A for histidine in water (Eq. 2-6) has been estimated, using 15N- and 13C-NMR, as 5.0 when the a-amino group is proto-nated and as 2.5 when at high pH it is unprotonated.17 This tautomerism of the imidazole group is probably important to the function of many enzymes and other proteins for example, if Ne of structure A (Eq. 2-6) is embedded in a protein, a proton approaching from the outside can induce the tautomerism shown with the release of a proton in the interior of the protein, perhaps at the active site of an enzyme. The form protonated on Ns (B of Eq. 2-6), which is the minor form in solution, predominates in some positions within proteins.18... 

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