Common Errors in Micropipette Use and How to Avoid Them
According to statistics conducted in Europe,
pipettes used in laboratories, particularly micropipettes, are used incorrectly approximately 55 percent of the time, nearly half of all instances.
For this reason, I will address micropipette use this time—a tool frequently used in laboratories and one that often leads to errors in its application.
Since this small instrument is extremely easy to use, I have prepared the following steps to prevent its misuse and have listed the matters that must be observed for correct pipetting.
[caption id="attachment_128350" align="aligncenter"]
Figure 1: You can see forward pipetting with pre-rinsing and without pre-rinsing. In pipetting with pre-rinsing, the desired volume and accuracy are achieved from the first pipetting onward, whereas without pre-rinsing, the desired volume and accuracy are reached only after 3-5 pipetting cycles. Consequently, pre-rinsing in forward pipetting is necessary to obtain correct results.[/caption]
How Can You Improve Your Correct Micropipette Use?
1. You Should Pre-wet the Pipette Tip
Before aspirating the sample liquid for pipetting, you should aspirate the same sample liquid and repeat this operation at least three times. You can significantly prevent sample loss due to evaporation in the tip, as pre-wetting increases moisture in the tip and thus reduces variation in sample evaporation. Pre-wetting is especially important when working with volatile samples (organic solvents).
[caption id="attachment_128351" align="aligncenter"] Figure 2: You can see an example of reverse pipetting shown here. In this graph, if you look at the pipetting numbers shown in the lower right, you are pipetting the correct volume from the first pipetting onward. Consequently, pre-rinsing is not necessary in reverse pipetting.[/caption]
2. You Should Insert the Pipette Tip to an Appropriate Depth While Drawing the Liquid Sample
Before aspiration, you should insert the tip sufficiently below the meniscus. For large-volume pipettes (1-5 mL), insertion to 5-6 mm is sufficient, while for smaller-volume pipettes, insertion to 2-3 mm will be adequate. Particularly with large-volume pipettes, inserting too shallowly can cause air aspiration. Inserting too deeply can cause samples to adhere to the outside of the tip. Touching the tip to the bottom of the container can restrict aspiration.
3. You Should Pause Briefly After Drawing the Liquid Sample
After aspirating the sample, it is best to leave the tip in the liquid for approximately one second. After the piston stops, it takes some time for fluid movement in the tip to cease; therefore, failing to do this will result in a significantly lower volume.
4. Use Consistent Piston Pressure and Speed with Your Micropipette
You should press the piston downward and release it gently and consistently. It is best to apply the same pressure and speed when aspirating and dispensing each sample. Repeatable actions produce repeatable results.
5. Hold the Pipette Vertically and Withdraw It in That Position
You should always hold the pipette vertically during sample aspiration and avoid touching the container walls. After sample aspiration, you should withdraw the pipette straight up from the center of the liquid in the container.
This technique is especially important when pipetting small volumes (<50 µL). Holding the pipette at an angle when withdrawing from the sample changes the aspirated volume. Touching the container walls can result in volume loss due to surface tension.
[caption id="attachment_128352" align="aligncenter"] Figure 3: You can see how the aspiration angle should be during pipetting.[/caption]
The figure above shows that the correct aspiration angle should be 90 degrees. In the figure on the right, when there is a deviation of 20 and 30 degrees from this 90-degree angle, it will result in an error rate of 0.7 percent. Consequently, when liquid is drawn into the pipette tip during pipetting, you must insert your pipette tip into the liquid at a 90-degree angle and draw the liquid in that manner.
