Amino acids have two ionizable groups always (the alpha amino and alpha carboxyl) plus one more if the R group is ionizable. At any given pH, some of those groups are protonated and some are not. Combining those charge states across three groups gives every amino acid its personality on a titration curve.
Titration and the isoelectric point
Acid-base
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Scroll sideways to see the whole map.
What a plateau isA flat stretch means the solution is buffering: half the group is protonated and half is not, so adding base barely moves the pH. The pH at the centre of a plateau is the pKa of whatever group is being titrated there.
How to find pIThe isoelectric point is where net charge is zero, which is always the average of the two pKa values that flank the neutral species. For glycine that is (2.3 + 9.6) / 2 ≈ 6.0. Add an acidic side chain and you average the two lowest pKa values; add a basic one and you average the two highest.
Why it matters downstreamAt a pH below its pI a molecule carries net positive charge; above it, net negative. That single rule is what makes isoelectric focusing and ion-exchange chromatography work, and it is why proteins clump and precipitate when the pH reaches their pI.
The curve is a charge readout, not just a chemistry plot. Read left to right and the molecule loses protons one group at a time: fully protonated and positive, then neutral and zwitterionic, then fully deprotonated and negative. Everything the exam asks about pI is somewhere on that sentence.
You will see this concept tested two ways: (1) “what is the charge at pH X” and (2) “what is the pI.” Master both and you can handle every acid-base amino acid question.
The Zwitterion
A zwitterion is a molecule with both a positive and a negative charge that cancel out to give a net charge of zero. At physiological pH (7.4), every amino acid with a nonionizable R group exists almost entirely as a zwitterion: the alpha amino group is protonated (-NH3+) and the alpha carboxyl is deprotonated (-COO-).
Titration Curve of a Simple Amino Acid (Glycine)
Glycine has two ionizable groups. Titrating from low pH (excess H+) with added base gives a curve with two plateaus and three charged species.
At very low pH: fully protonated. Both -NH3+ and -COOH. Net charge = +1.
As pH rises past pKa1 (~2.3): the -COOH loses its proton to become -COO-. Now -NH3+ and -COO-. Net charge = 0. This is the zwitterion. The flat region near pKa1 is a buffering zone.
As pH rises past pKa2 (~9.6): the -NH3+ loses its proton to become -NH2. Now -NH2 and -COO-. Net charge = -1.
Calculating the Isoelectric Point (pI)
The pI is the pH at which the amino acid carries no net charge. For an amino acid with only the two backbone ionizable groups, the pI is simply:
With an Ionizable Side Chain
If the R group is also ionizable, the amino acid has three pKa values. The pI is the average of the two pKas that flank the zwitterionic (net-zero) form.
Acidic amino acid (Asp, Glu): pI = average of the two lowest pKas (both the side-chain carboxyl and the alpha carboxyl lose protons to produce the neutral form). The pI is low (around 3 to 4).
Basic amino acid (Lys, Arg, His): pI = average of the two highest pKas (both amino/basic groups must still be protonated for the zwitterion). The pI is high (around 7.6 to 11).
Neutral amino acid with an ionizable side chain (Cys, Tyr): treat it like a neutral amino acid; pI is the average of the alpha-carboxyl pKa and the alpha-amino pKa.
Worked Examples: Three Types, Three Calculations
The MCAT loves these. Work through all three types once and the pattern will feel obvious on test day.
The shortcut: acidic side chain → pI = average of the two lowest pKas; basic side chain → pI = average of the two highest pKas. This works because you always sandwich the zwitterion between the two pKas closest to it on the protonation ladder.
Charge at Any pH
Two simple rules beat every “what is the charge” question:
If pH is below pKa, the group is protonated.
If pH is above pKa, the group is deprotonated.
For an amine (-NH3+ / -NH2): protonated is +1, deprotonated is 0.
For a carboxyl (-COOH / -COO-): protonated is 0, deprotonated is -1.
Apply the rule to every ionizable group on the molecule and sum the charges.
Henderson-Hasselbalch for Amino Acids
The Henderson-Hasselbalch equation applies to each ionizable group:
What is a zwitterion?
Click to reveal answer
A molecule that has both a positive and a negative charge which cancel to give a net charge of zero. For a typical amino acid at physiological pH, the alpha amino is protonated (-NH3+) and the alpha carboxyl is deprotonated (-COO-) simultaneously.
How do you calculate pI for a neutral amino acid (no ionizable side chain)?
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pI = (pKa1 + pKa2) / 2, the average of the alpha-carboxyl pKa (~2) and the alpha-amino pKa (~9). For glycine: (2.34 + 9.60) / 2 = 5.97.
How do you find the pI for an acidic amino acid like glutamate (pKa values ~2.2, 4.1, 9.7)?
Click to reveal answer
Average the two pKas that flank the zwitterionic (net-zero) form. For glutamate, both carboxyls (alpha-COOH pKa ~2.2 and side-chain pKa ~4.1) must be deprotonated for net-zero, so pI = (2.2 + 4.1) / 2 ≈ 3.15. Acidic amino acids have low pI values; basic amino acids have high pI values.
