Key takeaways

• Deprotonation removes H⁺ to give a conjugate base. How readily it occurs depends on how well the resulting negative charge is stabilised.
• Choose a base by comparing pKa values. Deprotonation is favourable when the base’s conjugate acid has a higher pKa than the substrate — a difference of two units or more gives essentially complete transfer.
• In acetate, the negative charge is delocalised over the two oxygen atoms, not onto the adjacent carbon. That symmetrical two-oxygen resonance is the whole reason carboxylic acids are acidic.
• NaBH₄ and LiAlH₄ are reducing agents, not bases for carbonyls. They deliver hydride to a carbonyl carbon, giving an alcohol. The hydride reagent used as a base is sodium hydride, NaH.
• Grignard reagents are already organometallic. They act as powerful bases and nucleophiles — they are not produced by deprotonation.
• Proton sponges are a specific class of exceptionally basic diamines. 2,6-lutidine is not one; it is a mildly basic hindered pyridine.
• In biology, protonation state sets enzyme catalysis, protein charge and buffer choice — which is why buffer pKa, not just pH, determines whether a buffer works.

Contents

• What deprotonation is
• pKa: the governing number
• What makes a proton acidic
• Choosing a base
• Reagents used for deprotonation
• Reagents commonly mistaken for bases
• Deprotonation in biology
• Where it shows up in the lab
• References
• Choosing buffers
• Frequently asked questions

Buffers and pH-dependent reagents

Every reagent below works because of protonation state — two of them change colour precisely because a proton moves.

Protein assay

Bradford Protein Assay

ColorimetricProtein lysates

Coomassie G-250 shifts from its brown-red protonated form to the blue anionic form on binding protein — the assay is a protonation change.

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TMB substrate

ELISA Accessory Pack: TMB Substrate and Stop Solution

TMB + stopELISA

Acidic stop solution protonates the oxidised TMB product, shifting absorbance from 650 nm to 450 nm and halting the reaction.

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TBS buffer

SDS-PAGE Tris-Glycine SDS Running Buffer (10X)

Running bufferSDS-PAGE

Tris has a pKa near 8.1, which is why it buffers well at the slightly alkaline pH these separations need.

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Transfer buffer

Western Blotting Membrane Transfer Buffer (10X)

Transfer bufferWestern blot

Transfer depends on protein net charge, which is set by pH relative to each protein’s isoelectric point.

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Lysis buffer

GenieLyse RIPA Lysis Buffer

Lysis bufferCells & tissue

Buffered to hold pH constant while cellular contents — including acids — are released.

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Lysis buffer

Cell Lysis Buffer (ELISA & Western Blot)

Lysis bufferELISA / WB

An alternative buffered lysis system for downstream immunoassay and blotting.

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What deprotonation is

Deprotonation is the transfer of a proton (H⁺) from an acid to a base, leaving the conjugate base of the acid. It is one half of every acid-base reaction — the base is protonated in the same step.

A free proton does not exist in solution in any meaningful sense; it is always transferred to something. That framing matters practically, because a deprotonation only proceeds if there is a base willing to accept the proton — and how willing it is can be read directly from pKa values.

pKa: the governing number

The acidity of a proton is expressed as pKa, the negative logarithm of the acid dissociation constant. A lower pKa means a stronger acid and a more readily removed proton.

The span is enormous — more than fifty orders of magnitude — which is why “strong base” is not a useful description on its own. Hydroxide and LDA are both called strong bases, yet they differ by roughly twenty pKa units in what they can deprotonate.

What makes a proton acidic

Acidity is determined by the stability of the conjugate base left behind. Four factors dominate:

• Electronegativity. A more electronegative atom bears negative charge more comfortably, so O–H is far more acidic than N–H, which is more acidic than C–H.
• Atomic size. Larger atoms spread charge over greater volume, so acidity increases down a group — H–I is a stronger acid than H–F despite fluorine’s electronegativity.
• Resonance. Delocalising the charge stabilises it. This is the dominant effect in carboxylic acids and phenols.
• Induction. Nearby electron-withdrawing groups pull charge density away and stabilise the anion — trichloroacetic acid has a pKa near 0.7 against acetic acid’s 4.8.

The acetate example, stated correctly

Deprotonating acetic acid gives acetate, and the resulting negative charge is delocalised equally over the two oxygen atoms. The two resonance forms are identical, and the real structure is a symmetrical hybrid with two equivalent C–O bonds of intermediate length.

The charge is not delocalised onto the adjacent carbon. Carbon is neither electronegative enough to stabilise it nor part of the pi system carrying the charge. This distinction is the reason a carboxylic acid (pKa ~4.8) is around fifteen orders of magnitude more acidic than an alcohol (pKa ~16): both give an oxygen anion, but only the carboxylate spreads it across a second oxygen.

Choosing a base

The practical rule is a direct comparison of two pKa values:

Deprotonation is favourable when the conjugate acid of the base has a higher pKa than the substrate.

Put another way, the proton ends up on whichever species holds it more tightly. A difference of about two pKa units gives roughly 99% conversion; a larger gap makes the reaction effectively irreversible.

Sodium hydride illustrates a useful additional point: the by-product is hydrogen gas, which leaves the solution. Removing a product drives the equilibrium to completion regardless of how finely balanced the pKa comparison is.

Reagents used for deprotonation

• Hydroxides and carbonates — NaOH, KOH, K₂CO₃. Convenient and cheap for acidic substrates such as carboxylic acids and phenols.
• Alkoxides — sodium ethoxide, potassium tert-butoxide. Bulky alkoxides such as KOtBu favour deprotonation over competing substitution.
• Metal amides — LDA and LiHMDS. Strong and sterically hindered, so they remove a proton rather than adding to a carbonyl. This is why LDA, not a hydroxide, is used to make enolates cleanly.
• Sodium hydride — the hydride reagent that genuinely acts as a base, giving H₂ as the by-product.
• Organolithiums and Grignards — exceptionally strong bases, though usually valued as nucleophiles. Their basicity is why they must be kept scrupulously dry.
• Proton sponges — 1,8-bis(dimethylamino)naphthalene and relatives, whose strained geometry makes them unusually basic while remaining poorly nucleophilic.
• Enzymes — in biology, active-site residues perform proton abstraction with precise positioning rather than brute basicity.

Reagents commonly mistaken for bases

Three points here are worth stating plainly, because getting them wrong leads to the opposite reaction.

Borohydride and aluminium hydride are reducing agents

NaBH₄ and LiAlH₄ do not deprotonate carbonyl compounds. They deliver hydride to the carbonyl carbon in a nucleophilic addition, converting the ketone or aldehyde into an alcohol. That is reduction — adding H⁻ — which is chemically the opposite of removing H⁺.

The confusion arises because hydride is formally basic, and sodium hydride is indeed used as a base. But NaH is ionic and non-nucleophilic, while NaBH₄ and LiAlH₄ are hydride delivery reagents. Using borohydride on a ketone expecting an enolate gives an alcohol instead.

Grignard reagents are organometallics, not products of deprotonation

A Grignard reagent is formed by inserting magnesium into a carbon-halogen bond, not by removing a proton. Once formed it is a very strong base as well as a nucleophile — which is precisely why traces of water, alcohol or terminal alkyne destroy it, quenching the reagent to the alkane.

2,6-Lutidine is not a proton sponge

Proton sponges are a defined class — 1,8-bis(dimethylamino)naphthalene and analogues — in which two amine nitrogens are forced into proximity, making protonation unusually favourable and giving a conjugate acid pKa above 12. 2,6-lutidine is a hindered pyridine with a conjugate acid pKa near 6.7, roughly a million-fold weaker as a base. It is useful as a mild, non-nucleophilic acid scavenger, which is a different job entirely.

Deprotonation in biology

• Enzyme catalysis. The serine protease triad works by acid-base chemistry: histidine deprotonates the serine hydroxyl, generating an alkoxide nucleophile, while aspartate orients and stabilises the histidine. The enzyme achieves at neutral pH what would otherwise need a strong base.
• Amino acid side chains. Ionisation state at physiological pH follows side-chain pKa — aspartate and glutamate deprotonated, lysine and arginine protonated, and histidine near its pKa of about 6, which is exactly why histidine is so often the catalytic residue.
• Protein charge and pI. Net charge is the sum of these ionisations. At its isoelectric point a protein carries no net charge, is least soluble, and does not migrate in an electric field — the basis of isoelectric focusing.
• Nucleic acids. The phosphodiester backbone is fully deprotonated and anionic at physiological pH, which is why DNA migrates to the anode and why cationic reagents are used to complex it for transfection.

Where it shows up in the lab

• Buffer selection. A buffer works within roughly one pH unit of its pKa. Tris has a pKa near 8.1, suiting slightly alkaline work; phosphate has a second pKa near 7.2, suiting physiological pH. Choosing on pH alone without checking pKa is the commonest buffer mistake — and Tris in particular shifts markedly with temperature.
• Colorimetric assays. The Bradford reagent works because Coomassie G-250 changes protonation state on binding protein, shifting from brown-red to blue with an absorbance maximum at 595 nm.
• ELISA stop solutions. Adding acid to oxidised TMB protonates the product, shifting the absorbance maximum from 650 nm to 450 nm while simultaneously halting the peroxidase reaction.
• Electrophoresis and transfer. Migration depends on net charge, which depends on pH relative to the protein’s pI — so buffer pH determines direction and rate of transfer.
• Sample handling. Lysis releases acidic cell contents; an adequately buffered lysis solution prevents a pH shift that would alter protein charge and antibody binding.

References

• Fischer, J., & Wentrup, C. (2002). Deprotonation Reactions in Organic Synthesis. Chemical Reviews, 102(7), 2477–2510. doi:10.1021/cr010193e
• Yamamoto, H. (2004). Recent advances in the deprotonation of organic molecules. Pure and Applied Chemistry, 76(6), 1097–1109. doi:10.1351/pac200476061097
• Carey, F. A., & Sundberg, R. J. (2007). Advanced Organic Chemistry: Part A: Structure and Mechanisms. Springer.
• March, J. (2007). Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. Wiley.
• Silverstein, R. M., Webster, F. X., & Kiemle, D. J. (2014). Spectrometric Identification of Organic Compounds. Wiley.
• Smith, M. B., & March, J. (2006). March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure. Wiley.

Frequently asked questions