Question

Experimental Biochemistry pKa of Amino Acids.. ** PLEASE ANSWER IN CLER HAND WRITING PLEASE HELP AND ANSWER AS SOON AS POSSIBLE**

B M Volume PH Unknwn 3 600 vu WNA 1.82 1.9 2.01 2.14 2.31 2.6 3.44 6.01 7.42 8.31 8.79 9.19 9.59 10.09 10.63 11.26 Titration curve of Amino Acid 10 11 12 11.26 11.6 10.63 13 10.09 9.59 14 15 9.19 8.79 8.31 16 11.6 pH 3.44 2.6 1.82 1.9 2.01 2.14 2.31 o 2 4 6 8 10 Volume of NaOH (mL) 12 14 16 18

1- where the pKas are located (for pKa1, pKa2 and if applicable, pKa3) for your unknown. 2-write their values on the correct parts of the graph. c) mark where the buffering regions are.

3-Record the pKa values below, that you obtained from the graphpKa1=_______ pKa2=__________ ; pKa3 (if present)=_________

4-For pI value: Show the correct formula to use for your match-1: Calculate the pI as per the formula and show your work. On the graph, Mark where this pI value is located and label it.

Answer 1

Background knowledge

Amino acids have both basic (-NH₂) and acidic groups (-COOH) along with variable side chains which may be neutral, charged, acidic, or basic. Whether they show acidic (-COOH donates H⁺ and amino acid acquires negative charge) or basic behaviour (-NH₂ group gains H⁺ and amino acid acquires positive charge) is dependent on the pH of the solution in which they are present. The pH of the solution when the net charge on amino acid is zero (i.e. -COOH has donated H⁺ and -NH₂ has gained H⁺ at the same time) is called its isoelectric point or pI. At this point it exists as zwitter-ion or dipolar ion.

Amino acid titration curves are graphs of pH of the solution containing aminoacid plotted against the volume of titrant added. In the present case, the titrant in NaOH. These titration curves provide important information regarding pKa values of amino acid (more than one because amino acids contain two or more ionizable groups), pI, and buffering range.

At very low pH, the amino acid is fully protonated, i.e. it exists in the following form:

R-CH-C—Он NH3 (I)

When pH is raised, the amino acid starts to lose H⁺ and become like the following structure:

R-CH C-O: NH3 II

But, initially, as the pH is being raised, the proportion of structure II is less than that of structure I (i.e. II < I).

A stage comes (as more NaOH is being added), when II = I and at this stage, the solution starts to act as a buffer because protons being lost by structure II are being accepted by structure I. This is pKa₁. This point is marked in the modified picture of the graph attached at the end of this answer. In the present case, pKa₁ = 2.14

Further increase in pH of the solution causes the amino acid to become zwitterion as shown in structure II. The point in the middle of vertical ascent of the graph is the position where it predominantly exists as structure II. It is marked in the graph and in this case PI=5.8

As the pH increases, -NH₃⁺, it loses H+ to become -NH₂ and attain the following structure:

R-CH CO NH2 III

When proportions of structure II and III become equal, the pKa₂ value is reached and in this case, it is the middle point of the graph where it begins to flatten (at volume 8) and where it begins to rise again (at volume 16). It is marked in the modified graph attached at the end. pKa2 = 9.5

PI is the average of pka₁ and pka₂ in this case.

pI = (pKa₁ +pKa₂)/2 = (2.14 + 9.5)/2 = 5.82

There are no additional pKa's as the curve does not show the elongated initial region before rise (this is shown in case an addition acidic group is present in side chain) and also does not show two vertical rises (this is shown in case of additional basic group present in side chain)

The buffering region starts at pKa₁ and ends at pKa₂(see modified graph)

Titration curve of Amino Acid 14 11.6 12 11.26 10.63 10.09 9.59 10 9.19 pka2=9.5 8.31 7.42 8 Buffering region 6.01 6 pI=5.8 3.44 4 pkal 2.6 2.31 2.01 2.1 1.82 1.9 2 0 2 4 6 8 10 12 14 16 18 Volume of NaOH (mL) O Hd