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Best Laboratory Practices - Pipette #2 - Understanding the impacts of the air gap spring effect
Laboratory Best Practices - Pipette #2
Understand the Impacts of the Pipette Air Gap Spring Effect
Standard pipettes are designed with an air gap in the system. This air gap is contained within the pipette tip and the nozzle of the pipette in the air chamber. The air gap is there to serve as a barrier between the sample and the pipette to eliminate, or reduce below detectable levels, sample cross contamination while pipetting. The air gap is very important but it does present some challenges when trying to accurately pipette. Here are a couple tips for pipetting followed with the why they are needed and important and when you should use them.
1) Use appropriate, consistent and smooth aspiration speed
1) Use appropriate, consistent and smooth aspiration speed
2) Pause appropriate time after aspiration with tip still in liquid
3) Use the smallest pipette volume and tip that you can
| Why The Spring Effect When the plunger is released in the pipette, it draws up into the shaft of the pipette creating a vacuum in the air gap within the pipette shaft and pipette tip. It is this vacuum that draws up the sample into the pipette. I like to think of this air gap as acting like a spring connecting the pipette plunger to my sample. It is important to visualize that the connection between the plunger and the sample is not a solid connection but rather a soft spring like connection. The reason that we need to release the plunger with an appropriate and consistent speed and pressure and pause after aspiration, is that we need to reproduce consistently the spring action of the air gap and allow the “spring” or air pressure time to stabilize. Again, visualize a soft spring pulling up your sample into the pipette tip. Allow that spring to softly stretch out and slowly pull your sample into the pipette and allow it to stabilize before withdrawing your pipette tip from the liquid. When describing the aspiration speed and pause time I deliberately use the word “appropriate”. Typically, when pipetting samples, you want to aspirate with a consistent smooth speed and pause for 1-2 seconds after the plunger is fully released. This smooth speed and pause allows the pressure to equalize and for the sample volume to stabilize or, in our spring analogy, for the spring to stop bouncing. If you use too fast of an aspiration speed this will increase the vacuum pressure and more importantly the linear velocity of the sample up the pipette tip. This increased velocity will allow the sample to over aspirate as the fluid actually compresses the air gap as the high linear velocity of the liquid translates into a higher than normal momentum for the fluid. If the pipette is drawn out of the liquid during this stabilization time, it will aspirate air into the tip decreasing the volume of liquid in the pipette tip. |  |
When
Your aspiration speed and pause time have to be adjusted for some various reasons. Sample properties, specifically viscosity and air gap size, will change the appropriate aspiration speed and pause time. High viscosity samples will aspirate more slowly into the tip requiring a slower aspiration speed and a longer stabilization pause before withdrawing from the liquid.
The size of the air gap will impact the speed and pause time. Larger air gaps act as softer springs when compared to smaller air gaps and take longer to stabilize. As previously discussed in the Quasar Instruments Best Practices #1, selecting the proper pipette size is important. The proper pipette size is not only important for better accuracy due to limiting mechanical absolute errors but for minimizing the air gap size. Using a 1000 uL pipette and tip to aspirate 100 uL instead of a 100 uL pipette will create a much larger air gap. If forced to use a pipette tip combination with a large air gap, you must pause longer after releasing the plunger as the weaker spring forces take longer to stabilize.
Also, with some pipettes, you have the choice of larger or smaller volume tips. These larger tip options might help you reduce lab consumable costs like when a 20 uL pipette uses the larger 200 uL tips instead of the smaller 10 uL tips (yes, a 10 uL tip will hold 20 uL). Sometimes the manufactures make “long” tips with a greater volume to reach into longer neck tubes. For the best accuracy select the smallest pipette tips that is appropriate for your experiments.
To improve your pipetting accuracy, you need to understand the concept of the “air spring” and why you need to use a slow and constant aspiration speed combined with a short 1-2 second pause after aspiration. You also need to understand that if you increase the air gap volume or have a sample with a different viscosity you have to modify this technique to allow the liquid time to stabilize within the tip.
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