HomeIntroductionLabsAppendices

Instructions for Pipetting/Dilution for Lab Practical

You will be asked to make a dilution using a volumetric pipette and a volumetric flask. You will dilute 5.00 mL of colored solution to make a total of 10.00 mL solution. You will be asked to describe what you are doing as you do it.

Below are instructions for proper pipetting/dilution procedure. This list will be referred to when grading the lab practical. Please ask ahead of time if you have any questions about what is meant by the description of the different steps.

1. Wear safety glasses.
2. Never dip a pipette directly into a reagent bottle. Place your reagent in a beaker and pipette from the beaker. You will want 10-15mL more liquid than you need, but don't be wasteful.
3. Rinse your volumetric pipette with your solute-containing solution:
• Before inserting pipette into solution, squeeze bulb and press against top of pipette.
• Draw solute-containing solution into the pipette until it starts to fill the bulb in the center of the pipette.
• Wipe off the outside of the pipette barrel using a Kimwipe.
• Rinse your pipette by rotating it on its side, being sure to rinse above the fill line.
• Touch pipette tip to inside of waste beaker. Let rinse solution drain into beaker.
• Though we will not be doing it for this course, it is good practice to rinse your pipette three times.
4. Filling the volumetric pipette:
• Before inserting pipette into solution, squeeze bulb and press against top of pipette.
• Draw solute-containing solution into pipette, until the meniscus is a few cm above line.
• Remove bulb and quickly make a seal with your finger on the top of the pipette so the liquid does not drain. It is recommended that you use your dominant hand to seal the pipette at this point. If you cannot maintain a seal, try moistening your finger.
• Wipe off the outside of the pipette barrel using a Kimwipe to avoid transferring extraneous liquid.
• Hold the pipette vertically, with the fill line at eye level. Touch the tip of the pipette to the inside of a beaker held at an angle. Use your non-dominant hand to hold both the beaker and the lower part of the pipette.
• Drain the pipette until the bottom of the meniscus is at the fill line, while maintaining contact between the tip of the pipette and the side of the beaker. This will take some practice. One trick that may work for you is to rotate the pipette while maintaining a light seal with your finger - this releases liquid very slowly.
5. Draining the pipette:
• Holding the pipette vertically, touch tip of pipette to inside of the volumetric flask, which should be held at an angle.
• Let liquid drain by gravity.
• Some solution will remain in the tip of the pipette. This is intentional (it is accounted for when the pipettes are calibrated). Allow the remaining solution to remain in the pipette.
• This amount has been accounted for in the volume of the pipette. at end of dispensing. Volumetric flask:
6. Filling the volumetric flask:
• Obtain a beaker of deionized water.
• If you like, you may pour deionized water into the flask until the liquid level is a few cm below the fill line. Be careful not to over-fill!
• With the volumetric flask on the benchtop, move your eye to be level with the fill line.
• Use a disposable pipette to add deionized water until the bottom of the meniscus is at the fill line. Be careful not to over-fill!
7. Put stopper on flask and mix by inverting 4-5 times. Make sure that the air leaves the neck of the flask when inverting.
8. After mixing, place the flask on the counter, move your eye to be level with the fill line, and make sure that liquid level is still at line.
9. If the liquid level is below the line, add more deionized water, mix, and check again. Repeat as necessary.
10. Absorbance of your solution should be within 0.01 of the correct value.