10 steps to improve pipetting accuracy

Step 1

Aspirate and fully expel an amount of the liquid at least three times before aspirating for delivery. Failure to pre-wet the tip increases evaporation within the tip air space, which can cause significantly lower delivery volumes. Pre-wetting increases the humidity within the tip, thus reducing evaporation.

Step 2

Allow liquids and equipment to equilibrate to ambient temperature prior to pipetting. The volume of liquid delivered by air displacement pipettes varies with relative humidity and vapor pressure of the liquid – both of which are temperature-dependent. Working at a constant temperature minimizes variation of pipetted volume.

Temperature differences cause thermal expansion and shrinking in the air space. After temperature equilibrium, the influencing factor is liquid density. Cold liquid is more dense and hot liquid is less dense compared to room temperature liquids.

Step 3

Before dispensing, carefully remove droplets from the outside of the tip by touching off the side of the reservoir, being sure to stay clear of the tip opening to avoid wicking liquid out of the tip. After dispensing, and before releasing the plunger, deliver any residual liquid remaining in the tip by touching the tip to the side of the container. Surface tension will help draw the remaining liquid out of the tip.

Step 4

Depress the plunger to the first stop, immerse the tip into the liquid, and aspirate by releasing the plunger. Remove the pipette from the liquid and depress the plunger to the second stop to dispense the entire contents. Standard (or forward) mode pipetting yields better accuracy and precision than reverse mode for all but viscous or volatile liquids. Reverse mode often results in over-delivery. Hence, it’s recommended to evaluate the effect of possible over-delivery in the experiment and make adjustments if needed.

In this experiment 200µL of  viscous liquid (glycerol) was pipetted 10 times by using both forward and reverse pipetting techniques. The pipette used was adjusted for glycerol using forward pipetting. The chart describes the accuracy and precision obtained with both techniques.

Using the reverse method a smaller deviation between doses was observed and therefore reduced imprecision.

The reverse method gave bigger doses as the liquid column in the tip is taller and therefore the liquid amount above the dose presses a larger dose out.

Step 5

After aspirating, and before removing the tip from the liquid, pause for one second. Make this pause as consistent as possible. Liquid continues to flow into the tip for a short time after the plunger stops. At the same time, evaporation within the tip is occurring. Pausing consistently balances these two effects and ensures correct aspiration.

Step 6

When aspirating liquid, hold the pipette vertically and pull the pipette straight out from the center of the reservoir. This technique is especially important when pipetting small volumes (less than 50µL). Holding the pipette at an angle as it is removed from the liquid alters the aspirated volume.

Step 7

Hold the pipette loosely and utilize the finger rest. Remember to return the pipette to the pipette stand between deliveries. Avoid handling pipette tips or reservoirs with bare hands. Body heat transferred during handling disturbs temperature equilibrium, which leads to variations in delivered volume.

Step 8

Step 10

Depress the plunger smoothly until coming to rest with a light and consistent force at the first stop. Immerse the tip, and  then release the plunger at a constant rate. Repeatable actions produce repeatable results.

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