pH, buffers and the charge of a peptide

A peptide's charge is not a fixed property. It is set by the pH of the solution around it, through the ionizable groups along the chain, and it decides whether the peptide dissolves, whether it sticks to a surface, how it runs on a column and how it behaves in an assay. Buffers exist to hold that pH where you put it. This article covers the groups that carry charge, the equation that connects pH to charge, the isoelectric point, and how to choose and make a buffer that does its job without interfering.

The groups that carry charge

Ionizable groups in a peptide and their approximate pK_a values

Group	pKa (typical)	Below the pKa	Above the pKa
C-terminal carboxyl	3.1–3.6	Neutral (COOH)	Negative (COO−)
Aspartate side chain	3.9	Neutral	Negative
Glutamate side chain	4.3	Neutral	Negative
Histidine side chain	6.0–6.5	Positive	Neutral
N-terminal amine	8.0–9.0	Positive (NH3+)	Neutral
Cysteine thiol	8.3–8.5	Neutral	Negative
Tyrosine phenol	10.1	Neutral	Negative
Lysine side chain	10.5	Positive	Neutral
Arginine side chain	12.5	Positive	Neutral

The values shift by a few tenths with neighbors and with ionic strength, which is why different tables disagree slightly and why a calculated isoelectric point is an estimate. An acetylated N-terminus or an amidated C-terminus has no charge at any pH, which is one reason those modifications change a peptide's behavior so much.

The Henderson–Hasselbalch equation

For each group, the fraction in the charged form is set by the difference between the pH and the pK_a: pH = pK_a + log([base]/[acid]). One unit of pH above the pK_a, a group is 91% in its basic form; two units, 99%. Summing the fractional charges of every group gives the peptide's net charge at that pH, which is exactly what the peptide calculator plots across pH 0–14. The equation assumes dilute solution and independent groups; it is accurate enough to choose a buffer and roughly right for the rest.

The isoelectric point

Where the net charge crosses zero is the isoelectric point, pI. A peptide with only acidic groups has a low pI and is negative at neutral pH; one with only basic groups has a high pI and is positive. Near the pI, molecules neither repel nor are strongly hydrated, and solubility is at its minimum; most aggregation and precipitation problems in why a peptide will not dissolve trace to a working pH within a unit of the pI. The practical rule: keep the pH at least two units away, on the side the sequence makes natural, acidic for basic peptides and basic for acidic ones.

Choosing a buffer

A buffer holds pH best within one unit of its own pK_a. Choose one whose pK_a is near the pH you want, at a concentration high enough to absorb what the peptide stock brings with it, and with a chemistry that does not interfere with the experiment.

Common buffers for peptide work

Buffer	pKa (25 °C)	Useful range	Notes
Acetate	4.76	3.8–5.8	Volatile; good for basic peptides and for HPLC sample preparation
MES	6.15	5.5–6.7	A Good's buffer; low metal binding
Phosphate	7.20	6.2–8.2	Physiological; precipitates calcium and some metals; pH shifts on freezing
HEPES	7.55	6.8–8.2	Standard in cell culture; forms radicals under light with some dyes
Tris	8.06	7.5–9.0	pKa falls 0.03 per °C; reacts with aldehydes; a primary amine
Ammonium bicarbonate	≈ 6.3 and 9.2	7.5–8.5	Volatile; ideal before freeze-drying or mass spectrometry
Glycine	9.6	8.8–10.6	Amino acid; interferes with amino acid analysis

Buffer capacity and what a stock brings in

A peptide dissolved in 10% acetic acid and diluted 1:100 into a 10 mM phosphate buffer adds 17 mM of acetic acid to a buffer with 10 mM of total phosphate: the pH drops by well over a unit. The same stock diluted 1:1,000 adds 1.7 mM, which 10 mM phosphate absorbs with a shift of a few hundredths. The dilution factor and the buffer concentration decide, and when the stock's acid or base is significant the buffer should be 50–100 mM or the pH checked after dilution. The dilutions article gives the scheme that keeps the acid out of the working solution.

Making a buffer

Weigh the buffer salt, dissolve in most of the water, adjust the pH with acid or base at the temperature of use with a calibrated meter, then make up to volume. Adjusting before making up to volume, and at the right temperature, are the two steps commonly skipped; Tris in particular reads 0.5 pH units different between 4 °C and 25 °C. Filter through 0.22 µm if the buffer will be stored, and record the date, since phosphate and Tris buffers grow microbial contamination within weeks at room temperature. Degassing helps peptides with cysteine or methionine.

Ionic strength

Salt screens the charges that keep a peptide dissolved, so solubility of a charged peptide often falls as ionic strength rises, while a hydrophobic peptide may salt in at low salt and salt out at high. Physiological saline is 150 mM; a buffer made for solubility may do better at 10–50 mM and a buffer made for an assay must match the assay. The reagent solvents article explains what 0.9% sodium chloride contributes.

Frequently asked questions

Why is the calculated pI different from the one on another site?

Different pK_a tables. The values vary by a few tenths between sources, and a pI calculated with any of them is an estimate good to about half a unit.

Does plain water have a pH?

Pure water is pH 7, but water open to air dissolves carbon dioxide and drifts to pH 5.5–6.5. That is enough to matter for a peptide near its isoelectric point, and it is why a buffer gives reproducible results where water does not.

Which buffer for mass spectrometry samples?

A volatile one: ammonium bicarbonate, ammonium acetate or formate. Phosphate and Tris are not volatile and suppress the signal.

Can I use the same buffer for dissolving and for the assay?

Often, if the assay's buffer is at least two units from the peptide's pI. If not, dissolve in a small volume of acid or base and dilute into the assay buffer at a factor that leaves its pH alone.

Sources & Further Reading

1. Po HN, Senozan NM. The Henderson–Hasselbalch equation: its history and limitations. Journal of Chemical Education 2001;78(11):1499–1503. doi:10.1021/ed078p1499
2. Good NE, Winget GD, Winter W, Connolly TN, Izawa S, Singh RMM. Hydrogen ion buffers for biological research. Biochemistry 1966;5(2):467–477. doi:10.1021/bi00866a011
3. Bjellqvist B, Hughes GJ, Pasquali C, Paquet N, Ravier F, Sanchez JC, Frutiger S, Hochstrasser D. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 1993;14(10):1023–1031. doi:10.1002/elps.11501401163
4. Grimsley GR, Scholtz JM, Pace CN. A summary of the measured pK values of the ionizable groups in folded proteins. Protein Science 2009;18(1):247–251. doi:10.1002/pro.19

Cosmic Peptides. (2026, October 30). pH, buffers and the charge of a peptide. Cosmic Peptides Research Library. Retrieved October 30, 2026, from https://cosmicpeptides.com/research/methods/ph-buffers-and-peptide-charge

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  author       = {{Cosmic Peptides}},
  title        = {pH, buffers and the charge of a peptide},
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  year         = {2026},
  month        = oct,
  url          = {https://cosmicpeptides.com/research/methods/ph-buffers-and-peptide-charge},
  urldate      = {2026-10-30},
  note         = {Last reviewed 2026-10-30}
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