Pipetting Accuracy and Volumetric Error in Peptide Work

Concentration is mass divided by volume, and most attention goes to the mass. The volume is quietly assumed to be whatever the pipette was set to, and that assumption is where a good deal of unexplained variability in peptide work actually comes from.

Two errors, and only one of them is visible

A pipette has accuracy — how close the delivered volume is to the setting — and precision, how consistent successive deliveries are. They fail differently and matter differently.

An accuracy error is systematic. Every solution prepared with that instrument is off in the same direction by roughly the same proportion, and because everything is consistent, nothing looks wrong. A precision error is random, and it shows up as scatter between replicates, which at least announces itself.

The systematic one is the more dangerous of the two for exactly that reason. A pipette delivering three percent low will produce a coherent, reproducible, internally consistent set of results that are all three percent wrong.

Where the specification stops applying

Manufacturers quote accuracy as a percentage of the nominal volume, and the figure applies at the top of the range. Toward the bottom of the range the same absolute error becomes a much larger proportion.

A 200 µL pipette set to 20 µL is operating at the edge of its useful range, and the tolerance there may be several times the headline figure. The rule that follows is simple and widely ignored: use the smallest pipette that covers the volume, and avoid settings below about ten percent of a pipette’s maximum.

Air displacement and what it assumes

An ordinary pipette does not touch the liquid. It moves a column of air, and the liquid follows. Everything about its accuracy depends on that air column behaving predictably, which it does for water at equilibrium and does not for several things routinely used in peptide work:

• Volatile solvents. Acetonitrile and methanol evaporate into the air column, raising its pressure and pushing liquid out of the tip. The delivered volume is high and it drips.
• Viscous solutions. Concentrated glycerol or a dense buffer flows slowly, and a tip drawn up at the normal rate is not full when it leaves the liquid.
• Dense solutions. An air-displacement pipette is calibrated for the density of water; a solution appreciably denser delivers a different mass for the same indicated volume.
• Temperature difference. A cold solution pipetted in a warm room, or the reverse, changes the air column’s volume during the operation. Solutions taken from a freezer should reach room temperature before volumes are taken from them, for this reason as well as the condensation one.

Positive-displacement pipettes, where a piston contacts the liquid directly, remove all four problems and are the correct instrument for volatile or viscous work.

Technique variables that are larger than they look

• Pre-wetting. The first aspiration into a dry tip partly saturates its air space with vapour and coats its walls. Discarding the first draw and using the second is worth a percent or more, and it doubles as the adsorption mitigation described in adsorptive loss to surfaces.
• Immersion depth. Too shallow draws air; too deep leaves liquid clinging to the outside of the tip which then contributes to the delivered volume. Two to three millimetres is the usual target for small volumes.
• Speed. Releasing the plunger quickly aerosolises the sample into the tip and the shaft. Slow and even is not fussiness, it is the calibration condition.
• Angle. The instrument is calibrated vertical. Holding it at an angle changes the hydrostatic head on the air column.
• Warmth. Holding a pipette in a closed hand for a long session warms the air column measurably. This is one of the arguments for working in short runs.

Forward and reverse, and when to switch

Forward pipetting — draw to the first stop, dispense to the second — is the default and is correct for aqueous solutions. Reverse pipetting draws past the first stop, dispenses to the first stop only, and discards the remainder with the tip.

Reverse mode delivers more consistently for viscous and foaming solutions, at the cost of wasting a small volume of sample every time. For a peptide solution that took real material to prepare, that trade is worth making deliberately rather than by habit.

Checking an instrument without a service contract

Gravimetric verification needs a balance and ten minutes. Pipette distilled water onto a tared balance and weigh it: one microlitre of water weighs one milligram closely enough for this purpose at room temperature. Ten replicates at the nominal volume give both figures at once — the mean against the setting is accuracy, the spread is precision.

Doing this at the top and bottom of the range, and recording the result, converts an assumption into a measurement. It is also the single cheapest way to find out that the instrument used for every standard on the bench has been reading low since someone dropped it.

Why this belongs in the same conversation as a certificate

A supplier’s certificate reports what a laboratory measured in a solution that laboratory prepared. Everything downstream — a dilution, a working standard, a comparison between two lots — rests on volumes measured locally, and a systematic volumetric error is indistinguishable from a real difference in the material.

Before a lot is judged weaker than the last one, the arithmetic that produced both numbers is worth checking, along with the other sources of apparent disagreement set out in why certificates disagree on purity.