Pipettes are laboratory equipment used to measure and transfer very small volumes of liquids. Accuracy in pipette measurements is very important because differences in the volume dropped by the pipette can affect the experimental results. To ensure accuracy, it is important to check the pipette calibration every few months. The calibration process is useful for checking whether this equipment is dripping liquid in the right volume, so that repairs can be made if needed.
Step
Part 1 of 2: Checking Calibration
Step 1. Gather the necessary materials
To check the calibration of the pipette, what is needed is a pipette, pipette tips, distilled water, beaker, thermometer, balance, and weighing cup. The balance used must be in microgram scale to calibrate the micropipette with a maximum value of 1 L.
- You will not need more than 5 mL of water. Fill the beaker with water.
- Make sure the pipette tip is correct and fits snugly into the pipette.
Step 2. Measure the temperature of the distilled water
Put the thermometer in the water and let it sit for at least a minute. If the red line of the thermometer is still moving, wait another minute. After one minute, record the temperature. Take the thermometer and dry it when you're done.
The temperature of the water is important to know, because it will be used for calculations performed to check calibration
Step 3. Place the weighing cup on the balance and zero the balance
Ideally, the balance used has a door and an insulated space inside. Place the weighing cup into the balance chamber and close the door. If your balance doesn't have a cubicle and a door, just put a weighing cup on the balance. Press the “Zero” or “Tare” button and wait for the scale to show zero.
Zeroing the scale reduces the weight of the plastic cup and allows you to weigh only the weight of the substance placed in the cup
Step 4. Prepare the pipette for calibration
Wipe the pipette with ethanol before starting and make sure nothing is clogging the tip of the pipette. Attach the correct pipette tip to the pipette tip and determine the volume to be tested.
For calibration, test the smallest and largest volumes that can be removed from the pipette
Step 5. Wash the pipette tip before calibrating it
Press the button to the first limit and dip the tip of the pipette into the distilled water to a depth of approximately 2 mm. Release the button to absorb some of the liquid and then release the liquid again by pressing the button. Repeat this step three times to wash the pipette tips before use.
Press the button to the second limit to remove the remaining liquid on the pipette tip, then take the pipette out of the water
Step 6. Suck up the calibration volume
With the pipette tip positioned outside of the distilled water, press the button to the first limit. Dip the pipette tip 2 mm deep into the distilled water and release the button to suck the liquid into the pipette tip. Wait about 1 second before removing the dropper tip from the water.
Make sure the pipette tip is fully submerged during the aspiration process. There should be no bubbles in the dropper tip or the result may be wrong
Step 7. Pour the liquid into the weighing dish on the balance
Place the pipette tip on the bottom of the weighing cup and then press the pipette button to the first limit. Move to another point, a little bit away from the water, then press the button to the second limit. Keeping the button pressed, lift the pipette tip from the weighing cup.
Leave the tip attached to the pipette as you will be using it again for some other calibration tests
Step 8. Record the weight shown on the balance
Close the balance booth door if you are using a door balance. Wait until the numbers don't change anymore. Write in your notebook.
It's important to wait until the numbers on the scale don't change again before writing them down. Notes will go wrong if you don't wait
Step 9. Repeat this process to do the reading at least 10 times
Zero the scale again, wash the pipette tips before use, suck the same volume of liquid, remove the volume, then record the weight. Record the weight of distilled water for the same volume, then average all your notes.
You can repeat this process for different volumes if each volume is tested multiple times
Part 2 of 2: Calculating the Calibration Result
Step 1. Define the formula for the calculated volume
The formula for calculating the volume of liquid ejected by the pipette is V = w * Z, w is the weight of the water, Z is the conversion factor based on the density of the water, while V is the volume of the amount of water removed.
- Variable Z can be obtained by calculating the density of water using the temperature recorded at the beginning of the experiment.
- For example: If the water temperature is 23°C, the Z value is 1.0035 g/mg.
Step 2. Calculate the overall average of the pipette calibrations
The volume of water released by the pipette has been weighed at least ten times. To average all these values, add them up and divide by 10. If you did more or less of the process, add up the overall results and then divide by the number of trials performed.
- For example: The weight of water from the 10 experiments you performed with the 10µL pipette is as follows: 9, 89, 10, 01, 10, 02, 9, 99, 9, 95, 10, 04, 9, 96, 10, 01, 9, 99, and 9, 98.
- The mean is: (9, 89 + 10, 01 + 10, 02 + 9, 99 + 9, 95+ 10, 04 + 9, 96 + 10, 01 + 9, 99 + 9, 98)/10 = 99, 84 /10 = 9,984
Step 3. Plug the variables into the equation and solve
Once you determine the correct number for each variable, plug it into the formula and complete the calculated volume. To solve this equation, simply multiply the average weight of all trials by the Z value.
For example: V = w * Z = 9,984 * 1.0035 = 10, 019
Step 4. Calculate the accuracy of the pipette
Use the equation A = 100 x Vavg/V0 to calculate pipette accuracy. A indicates pipette accuracy, Vavg is the calculated mean volume, and V0 is the value set on the pipette. The accuracy value should be between 99-101%.
- If the pipette is properly calibrated, the calculated value should be very close to the actual value set on the pipette.
- For example: A = 100 x Vavg/V0 = 100 x 10, 019/10 = 100 x 1.0019 = 100.19%
- This pipette is properly calibrated.
Step 5. If necessary, send the pipette for calibration
If your pipette doesn't pass the calibration test, don't use it for experimentation right now. Pipettes are very fragile and expensive laboratory equipment. You can't fix the calibration yourself, the pipette has to be sent in for repair. Alternatively, several companies will come to your laboratory and calibrate the pipette on site.
