Pipettes and volumetric glassware: which to use and how accurate each is

Laboratories measure liquids with a range of glassware and pipettes, and they are not equally accurate. A beaker's markings are a rough guide. A volumetric flask or pipette is made to hold or deliver one volume very precisely. This guide explains what each piece is for, what the markings on glassware mean, and how to use and check them.

Terms are defined in the glossary.

The main types, from least to most accurate

A rule of thumb: use the most accurate tool the job needs. A buffer topped up to roughly 1 L in a beaker is fine for rinsing. A standard solution for an assay needs a volumetric flask.

Class A and Class B

Volumetric glassware comes in two accuracy classes:

• Class A has the tighter tolerance. Each piece, or each batch, comes with a statement or certificate of its accuracy. Use it for standards, calibrations and anything you'll report.
• Class B tolerances are usually about twice as wide. It is cheaper and fine for routine work.

The tolerance is often printed on the glass, for example "100 mL ± 0.10 mL". Tolerances depend on the size and the standard the glass is made to. US Pharmacopeia tolerances can be tighter than ISO ones for the same size (DWK Life Sciences).

Reading the markings

Glassware carries a short code that tells you how it was calibrated:

• "In" or "TC" (to contain). The glass holds the stated volume when filled to the mark. Volumetric flasks are marked this way. Pouring the liquid out leaves a film behind, so you get slightly less than the stated volume.
• "Ex" or "TD" (to deliver). The glass delivers the stated volume when emptied. The film left on the inside has already been allowed for. Volumetric pipettes and burettes are marked this way.
• "Ex + 5 s" or a similar waiting time. After the liquid stops flowing, wait that long before removing the pipette, to let the last of it drain.
• "Blow out". Some pipettes are made to be emptied completely by blowing out the last drop. Don't blow out a pipette that isn't marked this way.
• "20 °C". The temperature the glass was calibrated at. Liquids and glass both expand when warm, so the volume is only exact near that temperature.

Reading a meniscus

Water and most water-based solutions curve up the glass, forming a dip called a meniscus. To read a volume:

1. Hold or set the glassware upright.
2. Bring your eye level with the mark. Looking from above or below makes the reading wrong, an effect called parallax.
3. Read from the bottom of the curve. For dark liquids where the bottom can't be seen, read the top edge instead and do so consistently.

A white card with a dark stripe held behind the glass makes the meniscus easier to see.

Using a volumetric pipette

1. Always use a pipette filler or bulb. Never draw liquid up by mouth.
2. Rinse the pipette with a small amount of the liquid you'll measure, then discard it.
3. Draw the liquid up past the mark.
4. Wipe the outside of the tip with a lint-free tissue.
5. Let the liquid run down until the bottom of the meniscus sits on the mark.
6. Run the liquid into the receiving vessel, with the tip touching the inside wall.
7. When flow stops, wait for the time marked on the pipette, then touch the tip to the wall and remove it. A small drop stays in the tip. That is expected unless the pipette is marked "blow out".

Using a volumetric flask

1. Dissolve the solid in less than the final volume, ideally in a beaker, then transfer it to the flask. Rinse the beaker into the flask too.
2. Fill the flask to just below the mark.
3. Add the last few drops with a Pasteur pipette until the bottom of the meniscus sits on the mark.
4. Stopper the flask and invert it several times to mix.

Let warm solutions cool to room temperature before making up to the mark, or the volume will be wrong once they cool. For the maths of making solutions, see molarity, mg/mL and dilutions.

Micropipettes

A micropipette (also called a piston pipette or air-displacement pipette) measures small volumes using a piston and a disposable plastic tip. Most are adjustable within a range, for example 2–20 µL, 20–200 µL or 100–1,000 µL.

Good technique matters as much as the pipette:

• Choose the right size. Accuracy is best near the top of a pipette's range and gets worse near the bottom. To measure 15 µL, a 2–20 µL pipette beats a 20–200 µL one.
• Pre-rinse the tip. Draw up and expel the liquid once or twice before the real measurement. This wets the inside of the tip.
• Hold it upright and dip the tip only a few millimetres below the surface.
• Use the stops correctly. In forward pipetting, press to the first stop, draw up, then deliver by pressing to the second stop to push out the last drop.
• Try reverse pipetting for thick, foamy or volatile liquids. Press to the second stop, draw up, then deliver only to the first stop, leaving the excess in the tip.
• Draw up and release smoothly. Letting the plunger snap back causes bubbles and errors.
• Match the temperature. Liquid, pipette and tip should all be at room temperature.

ISO 8655 sets the maximum errors a piston pipette may have at each volume (Eppendorf). Manufacturers usually publish tighter specifications for their own models. Errors are given as two numbers: systematic error (how far the average is off, a measure of accuracy) and random error (how much repeated deliveries vary, a measure of precision). See accuracy, precision and significant figures for the difference.

Positive-displacement pipettes have a piston inside the tip itself, with no air gap. They suit very thick or very volatile liquids, where an air-displacement pipette is inaccurate.

Pasteur pipettes

A Pasteur pipette is a simple glass or plastic tube with a narrow tip and a rubber or squeezable bulb. It moves liquid from one place to another, for example to top up a volumetric flask to the mark. Some plastic ones have rough markings, but they aren't measuring tools.

Checking accuracy by weighing

Labs check pipettes and glassware gravimetrically, by weighing the water they deliver. At 20 °C, 1 mL of water weighs about 0.998 g. Weighing several deliveries on an analytical balance, and converting to volume with the water's density at the measured temperature, shows both the average error and the spread. ISO 8655 describes this method for piston pipettes, and ISO 4787 for glassware (ISO 4787).

Micropipettes are usually checked or calibrated at least once a year, and more often if they're used heavily or dropped.

Care of volumetric glassware

• Clean promptly with a suitable detergent, rinse well with tap water, then purified water. Water should drain in an even film. Droplets that cling show the glass is greasy and will deliver less than its stated volume.
• Avoid high heat. Drying volumetric glassware in a hot oven, or heating it, can change its volume. Air-dry it, or rinse with the liquid you're about to measure.
• Don't store strong alkaline solutions in volumetric glass. They slowly etch it.
• Retire chipped glass. A chipped tip on a pipette changes its delivery.

Related guides

For why glass vials are made of borosilicate, see borosilicate glass vials, stoppers and crimp seals. For making up a buffer, see buffers and pH. Every lot has its own COA, published on the product page and on that lot's own page. See Quality.