Intuition The big pictureAn amino acid carries two opposite personalities at once : an acidic group (–COOH, wants to give a proton) and a basic group (–NH₂, wants to take a proton). So instead of staying neutral, the molecule donates a proton to itself , becoming an internal salt with a + and a – on the same molecule. That internal-salt form is the zwitterion . Everything else (high melting points, water solubility, behaviour in electric fields, the pI) flows from this single idea.
A molecule with both a carboxyl group (–COOH) and an amino group (–NH₂) attached to the same carbon (the α-carbon), plus an H and a side chain R .
H 2 N − C ∣ R H − COOH \text{H}_2\text{N}-\underset{\underset{\text{R}}{|}}{\text{C}}\text{H}-\text{COOH} H 2 N − R ∣ C H − COOH R .
The α-carbon (except in glycine, where R = H) has 4 different groups → it is a chiral centre, so amino acids are optically active and occur naturally as the L-form .
Intuition Why the proton jumps–COOH is a moderately strong acid (pKa ≈ 2). –NH₂ is a good base (its conjugate acid –NH₃⁺ has pKa ≈ 9–10). When a stronger acid meets a stronger base inside the same molecule , the acidic proton simply hops onto the amine. The result is lower in energy → it is the dominant form in the solid state and in neutral water.
The dipolar ion of an amino acid: the carboxyl is deprotonated (–COO⁻) and the amino is protonated (–NH₃⁺), giving net charge zero but two formal charges.
H 3 N + − CHR − COO − \text{H}_3\overset{+}{\text{N}}-\text{CHR}-\text{COO}^{-} H 3 N + − CHR − COO −
Consequences of being an internal salt:
High melting points (ionic lattice) — they char rather than melt cleanly.
Soluble in water, poorly soluble in non-polar solvents.
They are amphoteric (react with both acids and bases).
Worked example Reacting with acid vs base
Add H⁺ (low pH): –COO⁻ grabs the proton → molecule becomes the cation H 3 N + − CHR − COOH \text{H}_3\overset{+}{\text{N}}-\text{CHR}-\text{COOH} H 3 N + − CHR − COOH Why this step? In excess acid there are plenty of protons; the weakest base present (carboxylate) protonates.Add OH⁻ (high pH): –NH₃⁺ loses its proton → molecule becomes the anion H 2 N − CHR − COO − \text{H}_2\text{N}-\text{CHR}-\text{COO}^- H 2 N − CHR − COO − Why this step? In base, the most acidic proton (on –NH₃⁺) is pulled off.
So as pH rises, charge goes cation → zwitterion → anion : from +1 to 0 to –1.
Definition Isoelectric point (pI)The pH at which the amino acid exists predominantly as the zwitterion , so its average net charge = 0 and it does not migrate in an electric field.
A simple amino acid has two acidic ionisations. Number them by the proton lost:
H 3 N + − CHR − COOH ⏟ net + 1 → p K a 1 ( ≈ 2 ) H 3 N + − CHR − COO − ⏟ zwitterion, net 0 → p K a 2 ( ≈ 9 ) H 2 N − CHR − COO − ⏟ net − 1 \underbrace{\text{H}_3\overset{+}{\text{N}}-\text{CHR}-\text{COOH}}_{\text{net }+1}\ \xrightarrow{pK_{a1}\ (\approx2)}\ \underbrace{\text{H}_3\overset{+}{\text{N}}-\text{CHR}-\text{COO}^-}_{\text{zwitterion, net }0}\ \xrightarrow{pK_{a2}\ (\approx9)}\ \underbrace{\text{H}_2\text{N}-\text{CHR}-\text{COO}^-}_{\text{net }-1} net + 1 H 3 N + − CHR − COOH p K a 1 ( ≈ 2 ) zwitterion, net 0 H 3 N + − CHR − COO − p K a 2 ( ≈ 9 ) net − 1 H 2 N − CHR − COO −
We want the pH where net charge = 0 , i.e. where the zwitterion is exactly flanked by equal amounts of cation and anion.
Use Henderson–Hasselbalch for each equilibrium:
pH = p K a + log [ base form ] [ acid form ] \text{pH}=pK_{a}+\log\frac{[\text{base form}]}{[\text{acid form}]} pH = p K a + log [ acid form ] [ base form ]
At the pI the cation (+1) and anion (–1) are present in equal concentration (they cancel), and zwitterion is maximal. Mathematically, set up:
pH = p K a 1 + log [ zwit ] [ cation ] and pH = p K a 2 + log [ anion ] [ zwit ] \text{pH}=pK_{a1}+\log\frac{[\text{zwit}]}{[\text{cation}]}\quad\text{and}\quad\text{pH}=pK_{a2}+\log\frac{[\text{anion}]}{[\text{zwit}]} pH = p K a 1 + log [ cation ] [ zwit ] and pH = p K a 2 + log [ zwit ] [ anion ]
Add the two equations:
2 pH = p K a 1 + p K a 2 + log [ zwit ] [ cation ] ⋅ [ anion ] [ zwit ] 2\,\text{pH}=pK_{a1}+pK_{a2}+\log\frac{[\text{zwit}]}{[\text{cation}]}\cdot\frac{[\text{anion}]}{[\text{zwit}]} 2 pH = p K a 1 + p K a 2 + log [ cation ] [ zwit ] ⋅ [ zwit ] [ anion ] 2 pH = p K a 1 + p K a 2 + log [ anion ] [ cation ] 2\,\text{pH}=pK_{a1}+pK_{a2}+\log\frac{[\text{anion}]}{[\text{cation}]} 2 pH = p K a 1 + p K a 2 + log [ cation ] [ anion ]
At the pI, [ anion ] = [ cation ] [\text{anion}]=[\text{cation}] [ anion ] = [ cation ] = log 1 = 0 =\log 1 = 0 = log 1 = 0
Intuition Why "average the two flanking pKa"The zwitterion sits between two ionisations. To make exactly as much cation as anion, you must sit at the midpoint pH of the two reactions that create those charges. Hence the arithmetic mean.
If R itself ionises (e.g. Asp/Glu have an extra –COOH; Lys/Arg have an extra basic N), there are three pKa values. Rule:
Worked example Glycine, pKa₁ = 2.34, pKa₂ = 9.60pI = 2.34 + 9.60 2 = 5.97 \text{pI}=\frac{2.34+9.60}{2}=5.97 pI = 2 2.34 + 9.60 = 5.97 Why this step? Glycine has no ionisable R, so just average its two pKa's. At pH 5.97 it sits still in electrophoresis.
Worked example Aspartic acid, pKa(α-COOH)=2.0, pKa(side-COOH)=3.9, pKa(NH₃⁺)=9.9The neutral species is created by losing the first two protons (both COOH). So:
pI = 2.0 + 3.9 2 = 2.95 \text{pI}=\frac{2.0+3.9}{2}=2.95 pI = 2 2.0 + 3.9 = 2.95 Why this step? Two acidic groups must both be deprotonated to reach net-zero charge; we average the two lowest pKa's. The acidic side chain pulls pI down .
Worked example Lysine, pKa(α-COOH)=2.2, pKa(α-NH₃⁺)=9.0, pKa(side-NH₃⁺)=10.5Two basic groups → average the two highest :
pI = 9.0 + 10.5 2 = 9.75 \text{pI}=\frac{9.0+10.5}{2}=9.75 pI = 2 9.0 + 10.5 = 9.75 Why this step? You must remove protons up to where the molecule is neutral; the basic side chain raises pI.
Electrophoresis link (Forecast-then-verify): Forecast — at pH 7, will glycine, aspartate, lysine move toward + or – electrode? Verify: pH 7 > pI(Gly 5.97) → net –, moves to anode (+). pH 7 > pI(Asp 2.95) → strongly –, anode. pH 7 < pI(Lys 9.75) → net +, moves to cathode (–). ✔
Essential amino acids : the body cannot synthesise them; must come from diet. (e.g. Valine, Leucine, Isoleucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan — Histidine, Arginine essential for children.)Non-essential amino acids : the body can make them itself (e.g. Glycine, Alanine, Glutamic acid, Aspartic acid, Serine, Proline).
Definition By side-chain nature
Acidic (extra –COOH): Asp, Glu → low pI.Basic (extra –NH₂/N): Lys, Arg, His → high pI.Neutral : the rest → pI near 6.
Mnemonic Remember the essentials & the pI ruleEssentials: "PVT TIM HaLL" → P henylalanine, V aline, T hreonine, T ryptophan, I soleucine, M ethionine, H istidine, L eucine, L ysine.
pI rule: "Average the two pKa's hugging the zwitterion." Acidic = average the low ones (pI low); Basic = average the high ones (pI high).
Common mistake Steel-manning the wrong ideasWrong 1: "pI = average of all the pKa's."
Why it feels right: averaging seems symmetric. Fix: only the two pKa's flanking the neutral form matter; the rest are spectators at that pH.
Wrong 2: "Zwitterion has a net charge because it shows + and –."
Why it feels right: you literally see two charges. Fix: they cancel → net charge zero ; that's exactly why it doesn't move in a field at the pI.
Wrong 3: "At pH below pI the amino acid is negative."
Why it feels right: low pH "sounds acidic = lots of O⁻." Fix: low pH = excess H⁺ → groups get protonated → molecule is positive (cation) , moves to cathode.
Wrong 4: "Essential = the most important amino acids."
Fix: "essential" is purely about diet — your body can't make them, so they're essential to eat , not chemically superior.
Recall Feynman: explain to a 12-year-oldImagine a tiny magnet-person with a "grabby hand" (the –NH₂) and a "give-away hand" (the –COOH). The give-away hand passes a little ball (a proton) to the grabby hand on the same person. Now one hand is + and one is –, but the whole person is balanced — that's the zwitterion. If you throw lots of balls at it (acid), both hands hold balls → it goes +. If you snatch balls away (base) → it goes –. The special "balance pH" where it stays perfectly still even between two magnets is the isoelectric point , and you find it by averaging the two pH levels where it flips.
What is a zwitterion? The dipolar internal-salt form of an amino acid with –COO⁻ and –NH₃⁺ on the same molecule and net charge zero.
Why do amino acids have high melting points? Because as zwitterions they form an ionic lattice held by strong electrostatic forces.
Define isoelectric point pI. The pH at which the amino acid is mainly zwitterion, has zero average net charge, and does not move in an electric field.
Formula for pI of a simple amino acid? pI = (pKa1 + pKa2)/2.
How do you find pI of an acidic amino acid (e.g. Asp)? Average the two LOWEST pKa values (the two that must lose protons to reach neutral).
How do you find pI of a basic amino acid (e.g. Lys)? Average the two HIGHEST pKa values.
At pH below its pI, what is the net charge of an amino acid? Positive (cation); it migrates to the cathode.
At pH above its pI, what is the net charge? Negative (anion); it migrates to the anode.
Why is the α-carbon chiral (except glycine)? It bears four different groups: H, COOH, NH2, and R (R = H in glycine, so achiral).
Define essential amino acid. One the body cannot synthesise; it must be supplied by diet.
Give pI of glycine and the calculation. (2.34 + 9.60)/2 = 5.97.
Why does an acidic side chain lower the pI? The extra –COOH adds a low pKa, so the two flanking pKa's averaged are both small.
Are amino acids amphoteric? Why? Yes — the zwitterion can react with acid (via –COO⁻) and with base (via –NH₃⁺).
Alpha-amino acid COOH plus NH2
High mp water soluble amphoteric
Intuition Hinglish mein samjhoDekho, amino acid ke paas do groups hote hain ek hi carbon par: –COOH (acid, proton dena chahta hai) aur –NH₂ (base, proton lena chahta hai). Toh molecule apne aap ko hi proton de deta hai — –COOH se proton nikal kar –NH₃⁺ ban jaata hai aur –COO⁻ ban jaata hai. Isi ko zwitterion kehte hain. Dono charges hain par net charge zero hota hai. Yahi reason hai ki amino acids ke melting points high hote hain aur ye paani me ghul jaate hain — basically internal salt jaisa behave karte hain.
Ab pI (isoelectric point) matlab woh pH jahan molecule mostly zwitterion roop me ho aur net charge zero ho — electric field me hilega nahi. Simple amino acid ke liye formula seedha hai: p I = ( p K a 1 + p K a 2 ) / 2 pI = (pK_{a1}+pK_{a2})/2 p I = ( p K a 1 + p K a 2 ) /2 un do pKa ko average karo jo neutral (zwitterion) form ko ghере rehte hain. Agar side chain acidic ho (Asp, Glu) toh do sabse chhote pKa average karo (pI kam aata hai); agar basic ho (Lys, Arg) toh do sabse bade pKa average karo (pI zyada aata hai).
Electrophoresis ka logic bhi simple hai: agar solution ka pH pI se kam hai toh molecule positive (cation) banta hai aur cathode (– plate) ki taraf jaata hai; agar pH pI se zyada hai toh negative (anion) ban kar anode (+ plate) ki taraf jaata hai. pI par bilkul still khada rahega.
Classification me ek aur cheez: essential amino acids woh hain jo body khud nahi bana sakti, diet se lene padte hain (PVT TIM HaLL mnemonic se yaad karo), aur non-essential woh jo body khud bana leti hai. Yaad rakho "essential" ka matlab "diet me zaroori" hai, "sabse important" nahi.
