What a buffer does and where it stops working
A buffer is a mixture of a weak acid and its conjugate base. Added acid is absorbed by the base form and added base by the acid form, so the pH moves far less than it would in unbuffered water.
The capacity to do that depends on having appreciable amounts of both forms present, which is only true near the pKa. At the pKa the split is exactly even and capacity is at its maximum. One pH unit away the ratio is ten to one and capacity has fallen substantially; two units away it is a hundred to one and there is effectively nothing left to absorb a challenge from one direction.
The useful range is pKa plus or minus one
This is the standard working rule and it follows directly from the ratio. Within one unit of the pKa both forms are present in workable amounts; outside it, one of them has essentially run out.
A target of pH 5.0 is therefore well served by acetate at pKa 4.76 and poorly served by phosphate at pKa 7.2, even though a phosphate solution can be titrated to pH 5.0.
Choosing between the common systems
Phosphate at pKa 7.2 is the workhorse for physiological pH, and it has two well-known drawbacks: it precipitates with calcium and magnesium, and it shifts pH sharply on freezing as sodium phosphate crystallises out. HEPES at 7.48 avoids both and is the usual choice for cell work.
Tris at 8.06 is standard for biochemistry and has a strong temperature dependence, about minus 0.028 pH units per degree Celsius. A Tris buffer adjusted to pH 8.0 at room temperature is nearer pH 8.5 in a cold room, which is a real and frequently overlooked effect.
- •Acetate, pKa 4.76: useful pH 3.8 to 5.8
- •MES, pKa 6.15: useful pH 5.2 to 7.2
- •Phosphate, pKa 7.2: useful pH 6.2 to 8.2, precipitates with divalent cations
- •MOPS, pKa 7.20: useful pH 6.2 to 8.2
- •HEPES, pKa 7.48: useful pH 6.5 to 8.5, standard for cell culture
- •Tris, pKa 8.06: useful pH 7.1 to 9.1, strongly temperature dependent
Matching the buffer to the peptide
The pH has to keep the peptide away from its isoelectric point, which is a solubility constraint rather than a buffer one. A peptide with a pI of 7.2 should not be handled in a phosphate buffer at 7.4.
Concentration matters too. 10 to 25 millimolar is typical for cell work, where ionic strength has to stay physiological. 50 to 100 millimolar is common where the buffer needs to resist a substantial acid or base challenge and the ionic strength is not constrained.
Preparing a buffer to a target pH
The Henderson-Hasselbalch calculation gives the acid and base amounts that should produce the target pH. It is a starting point, not a substitute for measuring: activity coefficients, temperature and the presence of other ions all shift the actual result.
Prepare from the calculated amounts, then check and adjust with a calibrated meter at the temperature the buffer will be used at. For Tris in particular, adjusting at the wrong temperature is a real error rather than a refinement.
How the buffer composition is calculated
Henderson-Hasselbalch for the acid and base split, and the Van Slyke expression for buffer capacity, which peaks at the pKa and falls away either side.
ratio = [base]/[acid] = 10^(pH - pKa) [base] = total x ratio / (1 + ratio) [acid] = total - [base] capacity = 2.303 x total x ratio / (1 + ratio)^2 capacity is maximal at pH = pKa, where it equals 0.576 x total
- Compute the base to acid ratio. Ten raised to the difference between the target pH and the pKa. At the pKa the ratio is one and the split is even.
- Split the total concentration. The ratio determines what fraction of the total is the base form, and the acid form is the remainder.
- Convert to amounts for your volume. Millimolar times millilitres divided by a thousand gives millimoles of each component for the volume you are preparing.
- Compute the buffer capacity. The Van Slyke expression, in millimoles per litre per pH unit. It shows how much acid or base the buffer absorbs before the pH moves by one unit.
- Rank the systems by fit. Buffers whose useful range covers the target are recommended first, then ordered by how close their pKa is to it.
What this method cannot tell you
- •Henderson-Hasselbalch assumes ideal behaviour. Activity coefficients at real ionic strengths shift the actual pH by a tenth or two.
- •pKa values are temperature dependent, dramatically so for Tris at about minus 0.028 units per degree Celsius.
- •Polyprotic systems such as phosphate and citrate have several pKa values, and only the relevant one is modelled here.
- •Carbon dioxide absorption acidifies alkaline buffers over time, so a stored basic buffer drifts.
pH buffer calculator: frequently asked questions
Pick one whose pKa is within about one pH unit of the target. That is where both the acid and base forms are present in workable amounts.
For pH 7.4, HEPES at 7.48 or phosphate at 7.2 are both good fits. Acetate at 4.76 is not, whatever pH it can be titrated to.
pH equals pKa plus the logarithm of the ratio of base to acid. Rearranged, it gives the ratio needed to reach a target pH.
At the pKa the logarithm is zero, so the pH equals the pKa and the two forms are present in equal amounts.
How much strong acid or base the buffer absorbs before the pH moves by one unit, in millimoles per litre per pH unit.
It peaks at the pKa, where it equals 0.576 times the total buffer concentration, and falls away in both directions.
10 to 25 millimolar for cell work, where ionic strength has to stay physiological. 50 to 100 millimolar where the buffer needs to resist a substantial challenge.
Capacity scales linearly with concentration, so doubling the concentration doubles the capacity.
Its ionisation has a large enthalpy change, so its pKa shifts by about minus 0.028 units per degree Celsius.
A Tris buffer adjusted to pH 8.0 at 25 degrees is nearer pH 8.5 at 4 degrees. Adjust it at the temperature it will be used at.
Three situations recur:
- •With calcium or magnesium, which precipitate as insoluble phosphates
- •For anything that will be frozen, since sodium phosphate crystallises out and the pH of the remaining liquid drops sharply
- •Where phosphate itself participates in the reaction being studied
One of a family designed by Norman Good for biological work: buffering in the physiological range, water-soluble, membrane-impermeant, minimally metal-binding and not absorbing in the visible or near-ultraviolet.
HEPES, MES and MOPS are the ones most often used with peptides.
HEPES at 10 to 25 millimolar is the usual choice: it buffers near physiological pH, it does not precipitate with divalent cations and it is stable in a carbon dioxide incubator.
Bicarbonate is the physiological buffer and only works in a carbon dioxide atmosphere, which is why it is paired with HEPES rather than replaced by it.
Weigh the calculated amounts of the acid and base forms, dissolve in most of the final volume, check the pH with a calibrated meter, adjust, then make up to volume.
Adjusting after making up to volume changes the volume again, which is why the order matters.
Because Henderson-Hasselbalch assumes ideal behaviour and real solutions are not ideal. Activity coefficients at real ionic strengths shift the result by a tenth or two.
Temperature, carbon dioxide absorption and meter calibration account for most of the rest. The calculation is a starting point; the meter is the answer.
Away from the peptide's isoelectric point, and generally slightly acidic for storage, around pH 4 to 6, where deamidation and disulfide exchange are slower.
The working pH is usually set by the assay rather than by the peptide, in which case the constraint is to avoid the pI rather than to choose freely.
Yes, and it covers a wide range because citric acid has three pKa values at 3.13, 4.76 and 6.40.
It chelates divalent metals strongly, which is useful for suppressing metal-catalysed oxidation and unhelpful when a metal is required.
Through pH, primarily: deamidation accelerates above neutral and disulfide exchange is faster under mildly alkaline conditions.
Some buffer components have their own effects. Phosphate catalyses certain degradation reactions, and Tris has a primary amine that can react with aldehydes.
Weeks refrigerated if sterile-filtered. The failure mode is usually microbial growth rather than chemical change.
Alkaline buffers drift acidic over time as they absorb carbon dioxide from the air.
For anything going into cell culture, yes, through a 0.22 micrometre filter. For chemistry, it is optional and extends the useful life.
PBS is phosphate buffer with sodium chloride added to bring it to physiological ionic strength. Plain phosphate buffer has no added salt.
The buffering behaviour is the same; the ionic strength is not, and ionic strength affects peptide solubility and adsorption.
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