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AOAC Official Method 994.12 Amino Acids in Feeds
AOAC Official Method 994.12 Amino Acids in Feeds
Titration Curve of Amino Acids Amino Acids Have Characteristic Titration Curves
Acid-base titration involves the gradual addition or removal of protons. Figure 1 shows the titration curve of the diprotic form of glycine. The plot has two distinct stages, corresponding to deprotonation of two different groups on glycine. Each of the two stages resembles in shape the titration curve of a monoprotic acid, such as acetic acid, and can be analyzed in the same way. At very low pH, the predominant ionic species of glycine is the fully protonated form, +H3 NCH2-COOH. At the midpoint in the first stage of the titration, in which the OCOOH group of glycine loses its proton, equimolar concentrations of the protondonor (+H3 N-CH2-COOH) and proton-acceptor (+H3 NCH2-COO-) species are present. At the midpoint of any titration, a point of inflection is reached where the pH is equal to the p K a of the protonated group being titrated. For glycine, the pH at the midpoint is 2.34, thus its OCOOH group has a p K a of 2.34. (Recall that pH and p K a are simply convenient notations for proton concentration and the equilibrium constant for ionization, respectively. The p K a is a measure of the tendency of a group to give up a proton, with that tendency decreasing tenfold as the p K a increases by one unit.) As the titration proceeds, another important point is reached at pH 5.97. Here there is another point of inflection, at which removal of the first proton is essentially complete and removal of the second has just begun. At this pH glycine is present largely as the dipolar ion +H3 N-CH2-COO-.We shall return to the significance of this inflection point in the titration curve shortly. The second stage of the titration corresponds to the removal of a proton from t he -NH3+ group of glycine. The pH at the midpoint of this stage is 9.60, equal to the p K a for the -NH3+ group. The titration is essentially complete at a pH of about 12, at which point the predominant form of glycine is +H2 NCH2-COO-. Titration of an amino acid. Shown here is the titration curve of 0.1 M glycine at 25 °C. The ionic species predominating at key points in the titration are shown above the graph. The shaded boxes, centered at about p K 1 = 2.34 and p K 2 = 9.60, indicate the regions of greatest buffering power.
Effect of the chemical environment on p K a.
The p K a values for the ionizable groups in glycine are lower than those for simple, methyl-substituted amino and carboxyl groups. These downward perturbations of p K a are due to intramolecular interactions. Similar effects can be caused by chemical groups that happen to be positioned nearby — for for example, in the active site of an enzyme. Titration Curves Predict the Electric Charge of Amino Acids
Another important piece of information derived from the titration curve of an amino acid is the relationship between its net electric charge and the pH of the solution. At pH 5.97, the point of inflection between the two stages in its titration curve, glycine is present predominantly as its dipolar form, fully ionized but with no net electric charge. The characteristic pH at which the net electric charge is zero is called the isoelectric point or isoelectric pH, designated pI. For glycine, which has no ionizable group in its side chain, the isoelectric point is simply the arithmetic mean of the two p K a values:
A titration curve of an amino acid is a plot of the pH of a weak acid against the degree of neutralization of the acid by standard (strong) base. This curve empirically defines several characteristics; the precise number of each characteristic depends on the nature of the acid being titrated: 1) the number of ionizing groups, 2) the pKa of the ionizing group(s), 3) the buffer region(s).
ionizing groups, 2) the pKa of the ionizing group(s), 3) the buffer region(s).
Based on the number of plateaus on a titration curve, one can determine the number of dissociable protons in a molecule. The one plateau observed when acetic acid is titrated indicates that it is a monoprotic acid (i.e., has only one dissociable H+). Many organic acids are polyprotic (have greater one dissociable H+). The protein building blocks, amino acids, are polyprotic and have the general structure:
The majority of the standard amino acids are diprotic molecules since they have two dissociable protons: one on the alpha amino group and other on the alpha carboxyl group. There is no dissociable proton in the R group. This type of amino acid is called a “simple amino acid”. A simple amino acid is electrically neutral under physiological conditions. NOTE: Under this definition it is possible to have a simple amino acid which is triprotic. Ionization of a diprotic amino acid will proceed as follows: As more of the strong base (titrant) is added to the aqueous solution, more of the weak acid is converted to its conjugate base. During this process, a buffer system forms and the pH of the system will follow the Henderson- Hasselbalch relationship. The titration curve of the neutralization of acetic acid by NaOH will look like this:
When a weak monoprotic acid is titrated by a base, a buffer system is formed. The pH of this system follows the Henderson-Hasselbalch equation. This curve empirically defines several characteristics (the precise number of each characteristic depends on the nature of the acid being titrated): 1) the number of
The order of proton dissociation depends on the acidity of the proton: that which is most acidic (lower pKa) will dissociate first. Consequently, the H+ on the αCOOH group (pKa1) will dissociate before that on the α-NH3 group (pKa2). The titration curve for this process looks similar to the following:
This curve reveals, in addition to the same information observed with a monoprotic acid, an additional characteristic of polyprotic acids and that is the pH at which the net charge on the molecule is zero. This pH defines the isoelectric point (pI) of the molecule, a useful constant in characterizing and purifying molecules. Using a titration curve, the pI can be empirically determined as the inflection point between the pKa of the anionic and cationic forms. Mathematically, the pI can be determined by taking the average of the pKa for the anionic and cationic forms. The ionic form of the molecule having a net charge of zero is called the zwitterion. A few amino acids are classified as triprotic. This is because, in addition to the ionizable protons of the αCOOH and α-NH3 groups, they also have a dissociable proton in their R group. Although triprotic amino acids can exist as zwitterions, under physiological conditions these amino acids will be charged. If the net charge under physiological conditions is negative, the amino acid is classified as an acidic amino acid because the R group has a proton that dissociates at a pH significantly below pH 7. The remaining triprotic amino acids are classified as basic amino acids due to a) their having a net positive charge under physiological conditions and b) an R group dissociable proton with a pKa near or greater than pH 7. Titration curves for triprotic amino acids generate the same information as those for the diprotic amino acids. The pI for a triprotic amino acid can be determined graphically, although this is somewhat more challenging. Graphical determination, as was the case with the diprotic acids, requires one to know the ionic forms of the amino acid and finding the inflection point between the cationic and anionic forms. Mathematically, the pI for an acidic amino acid is the average of pKa1 and pKaR (the pKa of the dissociable proton in the R group); for a basic amino acid, it is the average of pKa2 and pKaR .