Back to Basics

Incognito goes back to basics with standard laboratory skills. How do you use your pipette properly?

Who taught you basic laboratory skills such as using a balance, a glass or automatic-pipette, or a pH meter? Were you shown the "proper" way? But, what is the "proper" way and who decides what this is? I originally intended this instalment to be about statistics and uncertainty of measurement with reference to the way we use "control" samples in chromatography; however, a simple piece of research turned into one of my most eye-opening experiences in the laboratory.

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While writing a worked example on error propagation, I wandered into the laboratory and asked a colleague to confirm his perception of the accuracy of a Grade A 10 mL pipette. He answered: "Depends which way you use it." I said: "Pardon?" He said: "Depends if you blow all of the liquid out of the bottom of the pipette." I know we have a basic skills training module, and I know that I regularly show new folks in the laboratory how to carry out basic operations, so where could this misunderstanding have come from?

I asked three other colleagues to pipette 10 mL of water into a volumetric flask. Every single one of them did something incorrectly, and it turned out that for two of these three I had signed their competency assessments. I guess people get lazy, forget things, learn the bad habits of their peers, and, dare I say it, don't care about the basics as much as they should. Furthermore, I have seen many de facto operating procedures for volumetric and gravimetric analysis, with senior laboratory personnel willing to argue hard that their version is absolutely the way to do it. Who specifies the way you should use glassware and balances? Do you know?

Most volumetric glass pipettes these days are Grade A, which means that they are highly accurate, with a 10-mL bulb pipette and a tolerance of around ±0.012 mL and a repeatability of around ±0.004 mL.¹ These figures can be used in calculations of uncertainty and, because the price of volumetric glassware is relative low, most laboratories standardize on Grade A equipment. Of course, many factors have to be considered when assuming the above accuracy and intermediate precision:

1. The liquid being measured is at ambient temperature using the correct equipment.

2. The glassware is calibrated and free from chips, defects, and adsorbed solids.

3. The pipette is used correctly.

Most pipettes are made of borosilicate glass and don't suffer from a great deal of thermal expansion or contraction. An error of ±0.01% at ±10 °C around the 20 °C standard temperature is fairly typical. The major problem with temperature is impatience and not allowing the measured liquid to cool (or warm) to the ambient temperature. As a result, major quantitative volumetric errors can occur when pipetting hot or cold liquids — the magnitude of the error depending upon the liquid temperature and its coefficient of expansion.

Bulb pipettes are the most accurate, followed by graduated pipettes, automatic pipettes, burettes, and measuring cylinders. The label on the glassware, or its corresponding certificate, will give details such as those shown in Figure 1:

Figure 1: Typical label markings on a Grade A pipette.

Ex indicates the volume "expired" rather than "in", which would indicate the volume included within (held by) the pipette. TD means "to deliver", which is the volume delivered by draining over 20 s at 20 °C (which depends upon the orifice size at the pipette tip). BS is the quality standard under which the pipette is produced and calibrated. Check the certificate for details of the calibration, which will usually state something like 10.01 mL with a calibration accuracy of ±0.012 mL

Onto my second point, do you know when the pipette is out of calibration? Do you have a calibration department or process within your organization? Do you know when— or if — a pipette should be recalibrated? If not then I suggest you find out!

Using a volumetric glass pipette is relatively straightforward; however, one needs to know the potential pitfalls and the best experimental technique. For a glass bulb pipette follow these steps:

1. Select a clean pipette with clear graduation marking. Don't dry glassware in an oven as this can affect their accuracy. Make sure the graduation mark is not faded. Ensure there are no chips and that the internal surface is dust, particle, and grease free. If you are unsure, dispense water from the pipette and look for discrete droplets of retained liquid — this indicates that the pipette is not clean enough and should be washed with a surfactant, thoroughly rinsed, and allowed to air dry.

2. Transfer the liquid to be pipetted to a clean, dry beaker (don't pipette directly from stock or reagent bottles).

3. Immerse the pipette tip into the liquid to a sufficient depth to prevent air ingress during aspiration and use a pipette filler to fill the pipette 5 to 20 mm above the graduation mark.

4. Discard the solution to waste. Repeat with a second washing of the pipette.

5. Avoid handling the pipette bulb as heat transfer from hands may affect the accuracy of the measurement.

6. Aspirate the solution to 5–20 mm above the gradation mark and wipe any excess liquid from the outside of the pipette tip with laboratory paper.

7. Remove the pipette filler and use a dry finger or the release valve on a three-way filler bulb to dispense liquid to waste until the bottom of the solution meniscus within the pipette is tangential to the pipette graduation mark (measure at eye level with the pipette vertical to avoid parallax error).

8. Remove any drop of liquid adhering to the pipette tip by gently touching it against the side of the beaker or container.

9. Discharge the liquid into the receiving vessel, with the tip of the pipette touching the wall of the vessel at an angle between 10° and 45°.

10. Allow the liquid to run out under gravity and check that the time taken to empty the pipette approximately corresponds to the emptying time stated on the label — if not then there may be a problem with the pipette.

11. Once the flow has stopped, leave the pipette tip in contact with the vessel wall for 3 s.

12. DO NOT agitate or blow out the remaining liquid; this is not part of the graduated volume.

Easy and straightforward? How does this compare with your own technique? The accuracy of an automatic/micropipette will be less than a glass pipette but these instruments are routinely used for the quantitative measurement of solutions less than 1 μL. A 100 μL pipette may be used to dispense volumes between 10 μL and 100 μL and a typical accuracy would be ±0.8 μL. These pipettes are calibrated to standard ISO 8655 and their calibration status and accuracy should be regularly checked. Operating guidelines do vary between manufacturers; however, shown below is a typical protocol that serves to highlight the important aspects of volumetric measurement with these devices:

1. Select the correct pipette and tip volume. Fit the tip with a slight twisting motion to ensure a good seal.

2. Holding the pipette vertically, press the plunger to the first-stop position and immerse into the liquid to the required depth to avoid droplet formation on the outside of the tip or vortexing during sample aspiration (immersion depth is typically between 1 and 7 mm — see instructions from the manufacturer).

3. Release the plunger to aspirate into the tip, withdraw, and discard to waste. Repeat several times with a new tip to wet the inner tip surface.

4. Aspirate the required volume by depressing the piston to the first stop, immersing the tip into the liquid to the required depth, and slowly releasing the piston, waiting for a few seconds at the top position to allow more viscous liquids to fill into the tip.

5. Withdraw from the sample and remove any excess liquid from the tip by gently touching against the vessel wall.

6. Dispense by holding the pipette vertically and tilting the receiving vessel to an angle of 10° to 40° and depress the piston to the first-stop position. Wait one second and then depress the piston to the second-stop position to "blow" the remaining liquid from the pipette tip.

7. Remove from the receiving vessel by sliding the tip up the vessel wall.

So, there we are, a short primer on using pipettes. I know the vast majority of you will have SOPs to cover this subject. When was the last time you read them?

Some of you will be saying "That's not right!", but on what basis and whose regulatory standards are you basing your challenge? Did you ever measure your own accuracy or precision by dispensing and weighing water? Give it a try, you'll be surprised at how much better you can become.

I haven't had a chance to tell you how bad my conversations got when discussing weighing and pH measurement (!) — more on that next time.

Reference

1. Richard Lawn and Elizabeth Pritchard, Practical Laboratory Skills Guide, Measurement of Volume (Royal Society of Chemistry Publishing, 2003).

Contact author: Incognito

E-mail: admin@chromatographyonline.com

This article is from The Column. The full issue can be found here:http://images2.advanstar.com/PixelMags/lctc/digitaledition/November20-2014-uk.html