Mastering Accurate Volume Measurement with Microlit Pipettes

Adjustable pipettes, which you will use in practically every experiment, are arguably the most significant scientific instrument that you will utilize in this semester. Pipettes are precision tools used to transfer volumes in the microliter range accurately and precisely. In your lab notebooks and lab reports, you may use microliters or milliliters as volume units, but be sure to always state the volume unit that you are using. Remember the following volume unit relationships:

Accuracy and precision

The accuracy of the pipette is dependent on it delivering the correct volume. Exact results can be reproduced. Let's use a target as an example to show the difference between accurate and precise results. Consider four students attempting to hit the bulls-eye five times. Students A and B are exact, whereas students A and C are correct.

Micropipettes' accuracy and precision are tested by transferring predetermined volumes of distilled water to a drop, which is then weighed on an analytical balance. At 25 degrees Celsius, the density of water is 1.0 gram per mL. During the calibration process, the operation is performed numerous times, and the data is utilized to calculate the accuracy and precision of a micropipette.

The micropipette's performance in respect to a standard (the desired) value is referred to as accuracy. The difference between the actual volume dispensed by the pipette and the anticipated volume is used to calculate accuracy. Be aware that this number may be either positive or negative. The accuracy of micropipettes is often given as a percentage of the chosen value while calibrating them. The accuracy of micropipettes is supposed to be a few percent (about 3%) of the target value. When micropipettes are programmed to give volumes that are somewhat near to the lowest values in their range, their accuracy suffers a little.

Without mentioning a standard, precision provides data on reproducibility. Precision is the result of random errors that are never completely removed from a process. A normal or binomial distribution (opposite) should be produced as a result of a series of repeated measurements. The standard deviation (s) of the collection of measurements is used to express precision. In a normal distribution, approximately two-thirds of measurements will be within one standard deviation of the average or mean (x), and approximately nine out of ten measurements will be within two standard deviations of the mean. Using the formula below, the standard deviation for a collection of n measurements may be determined.

Choosing the micropipette

The standard deviation describes the measurement distribution in relation to the mean value.

In the laboratory, we employ three distinct sizes of micropipettes: P20, P200, and P1000. Our micropipettes came from a variety of vendors, but the operating principles remain the same. The numbers following the "P" represent the maximum number of microliters that the micropipette is capable of transporting. It should be noted that the ranges of the various micropipettes overlap somewhat. Both the P200 and P20, for example, can transfer 15 l, but the P20 is more accurate within that range. As a general guideline, choose the smallest volume pipette that will transmit the volume.

Choosing the Transfer Volume

The volume indicator has three numbers on it. By adjusting the volume adjustment knob on each pipette, you can set the volume to three digits. The vernier markers on the lower dial allow you to extrapolate between the lowest numbers as well. With the micropipettes, the majority of your measurements will be accurate to four significant figures!

Transferring volumes properly

Air displacement is the method by which micropipettes function. An internal piston is moved to one of two positions by the operator depressing a plunger. The micropipette tip is filled at the first stop, and its contents are released at the second stop. An internal piston moves a volume of air equal to the volume indicated on the volume indicator dial as the operator depresses the plunger to the first stop. Only the tip's contents are released through the second stop.

Aspirating the reagent with Micropipette

• The box with the appropriate size micropipette tips should have its cover removed. P-20 and P-200 tips are either yellow or clear, whereas P-1000 tips can be either blue or clear.
• By placing the micropipette's shaft into the tip and firmly pressing down, you can attach the tip (see figure on the right). This should result in an airtight seal between the micropipette's tip and shaft.
• To maintain the sterility of the remaining tips, replace the tip box lid. Because the tips are sterile, avoid touching them, especially the thinner end.
• Push the micropipette's plunger all the way down to the FIRST stop.
• Immerse the tip in the solution being pulled up into the pipette a few millimeters below the surface. When pipetting, the pipette is most accurate when held vertically. For optimal results, keep the angle from vertical to less than 20 degrees.
• Slowly release the plunger, enabling the tip to fill smoothly. Allow a brief pause to ensure that the entire volume of the sample has entered the tip. Do not allow the plunger to snap. When transferring larger volumes, this is especially important because a splash could contaminate the shaft of the micropipette. If you accidentally contaminate the shaft, clean it right away.

Dispensing the micropipette's contents

• Place the micropipette tip against the receiving test tube's side. Surface tension will aid in the dispensing of the micropipette contents. Do not try to eject the micropipette contents into “Thin Air.”
• Depress the plunger smoothly to the first stop. Pause for a moment, then depress the plunger to the second stop. The pipette's contents should have been mostly released at the first stop. The second checkpoint verifies that you have discharged the "last drop."
• To remove the tip, use the tip ejector.

To know more about the pipette, visit our product guide "Micropipette Product Guide".

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