If you work at the bench, accurate pipetting is crucial — without accurate it your experiments would be non-reproducible, stock solutions inaccurate and assays would have such large errors that comparing them would be meaningless. But luckily, there’s no need to worry – your trusty, precision micro-pipettes take care of all that for you.
Or do they?
Precision instruments they may be, but the accuracy of micro-pipettes depends on you. You need to maintain your pipettes well, practice good technique and have an understanding of how they work before you can claim to have anything like precision instruments at your disposal.
Know how your pipette works
1. Most likely, you use an air displacement pipette. It works a bit like a syringe, except that there is an air-filled cushion between the piston and the sample. The air cushion prevents the piston from coming into contact with the solutions, which is good, but it also puts some limitations on the pipette.
The volume of the air cushion is affected by temperature and pressure, and volatile solvents can evaporate into it. Each of these affects pipetting accuracy. The barrel of air displacement pipettes is also vulnerable to contamination by the pipetted solution. If you are working with corrosives or bio-hazardous material, this can be a problem.
2. Don’t use an air displacement pipette (depending on your application!) Most of the information in this article relates to air displacement pipettes, but in certain situations a positive displacement pipette may be a better option. Positive displacement pipettes also work like a syringe, but unlike air displacement pipettes, they don’t have an air cushion. This makes them more accurate for pipetting volatile solvents, and more suitable for pipetting corrosives and bio-hazardous material. They are expensive because the barrel is replaced as part of the tip, but can be a good option in some cases. A cheaper alternative is to use and air displacement pipette with barrier tips, but these only address some of the problems.
Take care of your pipette
3. Have your pipette serviced every 6-12 months, or more frequently, depending on how accurate you need to be. The service should include re-calibration, greasing of the moving parts and replacement of any work out seals or other parts. It’s best to have this done by an experienced pipette doctor – if you work in university or large company there may be pipette clinics you can use, otherwise you can send your pipettes off to Eppendorf or other companies who will do it for you.
4. Check your pipettes daily for damage to the nose of the barrel (where the tip is fitted) or any other obvious damage. If there is a problem, have it serviced because it is unlikely to be fit for the job you need it for.
5. Clean your pipettes every day before use – a wipe with some 70% ethanol should do it.
6. Store your pipette vertically, using a pipette holder. This prevents any liquids that have sneaked into the barrel of the pipette from getting any further inside and corroding it.
7. Never put your pipette on it’s side with liquid in the tip. The liquid might get into the pipette barrel and cause some serious corrosion damage.
8. Use well-fitting tips. Poorly fitting tips allow air to escape when drawing up and dispensing, leading to inaccurate results. This report from Rainin provides more information.
Practice Good Pipetting Technique
9. Make sure you know how to pipette properly. For the basics, take a look at Eppendorf’s pipetting technique manual. The most important rules to follow are:
- Pipette with a slow, smooth action
- Hold the pipette vertically when drawing liquid in
- Only immerse the tip slightly when drawing liquid in – otherwise you will coat the outside of the tip with liquid, which will be transferred along with the volume inside the pipette
- When dispensing the liquid, hold the pipette vertically but keep the sidewall of the receiving vessel at 45 degrees. Pipette against the sidewall or into the liquid that’s already there.
10. Test how good you are. Check the accuracy of your pipetting by dispensing 100 microlitres onto a fine balance. The mass of the droplet you make should be around 0.1 g. Now do the same thing 10 times and record the masses you obtain. If the variation is more than +/- 0.5% you need to either have your pipette looked at, or you need to practice!
Know the tricks that increase your accuracy
11. Pre-wetting. When you dispense liquid from your pipette a coating of the is left on the tip, making the expelled volume slightly less than it should be. Pre-wetting the tip before you pipette will help this. Just draw up the liquid into your pipette then dispense back into the original vessel. The coating is now on the tip so when you now draw up the liquid again and dispense it into the receiving vessel, none of it will be lost to wetting. This is only recommended for volumes greater than 10 microlitres.
12. Reverse pipetting is a good technique for pipetting viscous liquids or volatile solvents. Push the piston down to the purge position (the second stop), then draw the liquid up. There is too much liquid in the tip at this point but when the liquid is dispensed by pushing the piston to the aspirate position (the first stop), the extra liquid is left inside the tip. Using this method the tip is “automatically” pre-wetted but the extra liquid also helps when pipetting volatile solvents because some of the solvent will tend to evaporate into the air cushion.
13. Take the ambient temperature into account. Your pipette will have been calibrated at room temperature. If you are working at a different temperature (e.g. in a cold room) your pipette will not be dispensing the displayed volumes.
14. Take the sample temperature into account. In a recent Nature Methods publication, Millet and Barthlen observed a strange phenomenon where, when repeatedly pipetting cold samples, the first dispensed volume is always larger than expected, but subsequent pipetting with the same tip gave the correct volume. The same was true for hot samples, except that the first dispensed volume was smaller than expected. Their solution was simple – dispense the first volume back into the original vessel, then start pipetting.
15. Use a sensible pipette for the volume you want to dispense. The accuracy of your pipette decreases as the dispensed volume approaches the minimum the pipette can handle. So for dispensing 15 microlitres, for example, a 1mL pipette would be terrible, a 200 microlitre pipette not so good and 20 microlitre pipette ideal.
16. Use the largest volume possible. Large volumes are easier to pipette accurately than small. Say you are performing an assay where you have to accurately pipette 5 microlitres. Pipetting 5 microlitres accurately is not easy and will likely contribute greatly to the statistical error in your results. On the other hand, if you diluted the stock solution 10 times and pipetted 50 microlitres, this would be much more accurate, giving you much tighter error bars.
17. And finally, here’s how to get maximum accuracy. An analytical balance is more accurate than any pipette. For maximum accuracy, use your pipette to dispense the volume you need but do it into a tared container on a balance. Then calculate the actual pipetted volume from mass (volume=mass/density). Of course, this only works for solutions of known density – but for aqueous solutions, which have a density of 1, this is not a problem.
Do you have any questions, did I miss something or do you have some tips of your own? Let me know.