1. Buffers resist change. pH buffers resist change in pH when acid (H+) or base (OH-) is added.
2. pH = -log [H+] - where bracket indicates molar concentration. If [H+] = 10^-7 M, then pH = 7. This is neutral pH since [H+] = [OH-] and it is middle of pH scale. Draw a pH scale by calculating the pH of 1 M HCl and 1 M NaOH. Use the equation Kw = 10^-14 = [H+] x [OH-], to find the pH of 1 M NaOH.
3. pH of a buffered solution can be found by using the Henderson-Hasselbach equation:
pH = pK + log([A-]/[HA])
All acids and bases have a conjugate acid (HA) and a conjugate base (A-).
At [HA] = [A-], pH = pK (prove this with the above equation).
At the pK, a buffer maximally resists change in pH and...
the buffering zone is considered to be 1 pH unit above and below the pK.
Useful Concepts:
Buffering zone = pK +/- 1 pH unit.
At pH = pK, [HA] = [A-].
At pH below pK, [HA] > [A-].
At pH above pK, [HA] <[A-].
4. Amino acids (AAs) are more complex than simple buffers and...
have at least two pK values because AAs have at least two ionizing groups:
Glycine structure
For example, Glycine has an alpha-amino group and alpha-carboxylic acid group.
Each ionizable group has a pK (called pK-1 and pK-2).
Glycine pK-1 = 2.3 (alpha-carboxylic acid); pK-2 = 9.6 (alpha amine).
Draw a titration curve for Glycine for practice.
In this class, we treat all ionizing groups with in a molecule,
as acting independently of other ionizing groups.
The pH on the titration curve where an AA has no net charge is called the pI or isoelectric point.
The pI is calculated by averaging the two pK values on either side of the neutral form (ie form with no net charge).
All AAs must have at least 3 ionic forms: "AA+1", "AA0", & "AA-1".
Where "AA+1" = AA form with net charge of +1,
"AA0" = neutral form with no net charge, &
"AA-1" = AA form with net charge of -1.
While an AA may have other ionic forms, only these 3 forms count when finding pI:
AA+1 goes to AA0 via pK-below
AA0 goes to AA-1 via pK-above.
USEFUL CONCEPTS:
pI = average of (pK-below) + (pK-above)
where pK-below is the pK between AA+1 and AA0
and pK-above is the pK between AA0 and AA-1.
5. For more complex AAs, like Asp, Glu, Lys, Arg & His - with 3 ionizing groups,
other forms exist with charges other than +1, zero, & -1.
For these AAs, the best approach is to start with the fully protonated form
(ie. One with all the protons it can take on its ionizable groups).
Calculate the net charge on this group (it must be positively charged),
then titrate the AA to remove the first proton and find net charge again.
Continue doing this until all the protons have been removed.
Keep track of this by making a simple model (see below for His).
Then, find the form with no net charge (ie. AA0) and use the pK values,
which govern the transition from AA+1 to AA0 and AA0 to AA-1,
as the ones to calculate the pI.
His has 3 ionizing groups, alpha-carboxylic acid (pK 1.8),
side-chain amino (pK 6.0) and alpha-amino (pK 9.2):
His+2 goes to His+1 via pK 1.8;
His+1 goes to His0 via pK 6.0;
His0 goes to His-1 via pK 9.2.
Therefore, pI = (6.0 + 9.2)/2 = 7.6.
6. Some of you have trouble figuring out which group ionizes first.
Here are some rules to apply to help you:
A. Carboxylic acids ionize at acidic pH; ie. Carboxylic acids give up their protons at acid pHs.
B. Amino groups ionize at basic pH; ie. Amines give up their protons at basic or alkaline pHs.
C. When groups with a similar chemical nature are present:
i. Carboxylic acids near an amino group have a more acidic pK than isolated carboxylic acids.
ii. Amino groups near a carboxylic acid have a more acidic pK than isolated amines.
iii. Aromatic amines (like in His side chain) have a pK near neutral.
Apply the above rules and concepts to calculate the pI for all the other complex AAs:
Asp, Glu, Lys, and Arg.
Their pK values and structures are shown below.
Go To Lecture 3 for more details!
©Wilbur H. Campbell, 1995; wcampbel@mtu.edu