Pipetting Technique

Jessiel S. Mondares, RMT

Laboratory Vessels
● Glassware-e.g., pipettes, flasks, beakers, and burettes

- Kimax/Pyrex (borosilicate), Corex (aluminosilicate), high silica, Vycor (acid and

alkali resistant), low actinic (amber colored), or flint (soda lime) glass

● Plasticware - polystyrene, polyethylene, polypropylene, Tygon, Teflon,

polycarbonate, and polyvinyl chloride
American Society for Testing and Materials (ASTM)
- is an international standards organization focused on developing technical
standards for a variety of industrial applications

-Those that satisfy Class A ASTM precision criteria are stamped with the letter “A”
on the glassware and are preferred for laboratory applications.

● Class A – Highest level of accuracy

● Class B – General purpose work calibrated to a lower level of accuracy
● To Contain (TC) - do not deliver that same volume when the liquid is
transferred into a container
● To Deliver (TD) -deliver the exact amount of liquid
Volumetric Flask

● volumetric flask is calibrated to hold

one exact volume of liquid (TC)
● The flask has a round, lower portion
with a flat bottom and a long, thin
neck with an etched calibration line.
Erlenmeyer flasks and Griffin beakers

● The Erlenmeyer flask - has a wide

bottom that gradually evolves
into a smaller, short neck.
● The Griffin beaker - has a flat
bottom, straight sides, and an
opening as wide as the flat base,
with a small spout in the lip
Graduated cylinders
- are long, cylindrical tubes usually held
upright by an octagonal or circular base
- The cylinder has calibration marks along
its length and is used to measure
volumes of liquids.
- Graduated cylinders do not have the
accuracy of volumetric labware. The
sizes routinely used are 10, 25, 50, 100,
500, 1,000, and 2,000 mL.
Pipettes

- are glass or plastic equipment used to transfer liquids

- they may be reusable or disposable
- Although pipettes may transfer any volume, they are usually
used for volumes of 20 mL or less
Pipette Classification
TC and TD pipettes

● TC pipette- holds or contains a

particular volume but does not
dispense that exact volume
● TD pipette - will dispense the
volume indicated
 Blowout vs Self-draining pipette
● A blowout pipette - has a continuous etched ring or two
small, close, continuous rings located near the top of the
pipette
- This means that the last drop of liquid should be expelled into
the receiving vessel
● A self-draining pipette - has no etched ring/ markings at the
top of the pipette
- the user allows the contents of the pipette to drain by gravity.
- The tip of the pipette should not be in contact with the
accumulating fluid in the receiving vessel during drainage.
With the exception of the Mohr pipette, the tip should remain
in contact with the side of the vessel for several seconds after
the liquid has drained.
● Volumetric Pipette- is
calibrated accurately
to deliver a fixed
volume of diluted
aqueous solution
● Ostwald-Folin Pipette
- is used to accurately
measure viscous fluids
such as blood
Correct and incorrect pipette positions
Manual Pipetting technique
1. Get the appropriate amount of the solution you wish to pipet in a small,
clean, dry beaker. Never pipet directly out of the stock bottles of
solution. This creates a contamination risk.
2. Insert the tip of the pipet into the beaker of solution so that it is about
1/4” from the bottom. Be sure not to press the tip against the bottom of
the container.
3. If you are right handed, hold the pipet in your right hand, leaving your
index finger free to place over the top of the pipet. With your left hand,
squeeze the pipet bulb. Press it firmly over the top of the pipet, but DO
NOT INSERT THE PIPET INTO THE BULB!! Release the pressure on the
bulb and allow the solution to flow into the pipet until it is above the
volume mark. Do not allow the solution to reach the bulb.
4. Quickly remove the bulb and place your index finger firmly over the
top of the pipet. Slowly roll you finger to one side and allow the liquid to
drain until the bottom of the meniscus is aligned with the volume mark.
With practice you will be able to lower the liquid very, very slowly.

5. When the bottom of the meniscus is even with the volume mark,
press your index finger firmly on the top of the pipet so no liquid leaks
out. Pull the pipet out of the solution and touch the tip once to the side of
the container.

6. To transfer the solution, place the tip of the pipet against the wall of
the receiving container at an angle of 10-20 degrees. Slowly allow the
liquid to drain from the pipet. Keep the flow slow so that no droplets
cling to the inside of the pipet.

7. When the solution stops flowing, touch the pipet once to the side of
the receiving container to remove any hanging drops. DO NOT blow out
the remaining solution. The pipet has been calibrated to deliver the
appropriate amount of solution with some remaining in the tip.
Automatic Pipettes
- Are pipetting devices that
permit rapid repetitive
measurement and delivery of
precise measures.
- It utilizes piston to generate
suction, which draws the sample
into a disposable tip
Fixed - vs Variable - volume pipette
● Fixed volume pipette - A pipette associated with only one volume

● Variable volume pipette - able to select different volumes

● Micropipette - A pipette with a pipetting capability of less than 1 mL
● automatic macropipette -pipette that dispenses greater than 1 mL

Types of Automatic Pipettes

1. air-displacement pipette - relies on a piston for creating suction to draw the

sample into a disposable tip that must be changed after each use. The piston does
not come in contact with the liquid.
2. positive-displacement pipette - operates by moving the piston in the pipette tip or
barrel, much like a hypodermic syringe. It does not require a different tip for each
use. Because of carryover concerns, rinsing and blotting between samples may be
required.
3. Dispensers and dilutor/dispensers - are automatic pipettes that obtain the liquid
from a common reservoir and dispense it repeatedly. The dispensing pipettes may
be bottle-top, motorized, handheld, or attached to a dilutor.
Air-displacement vs Positive-displacement pipette
Multichannel
pipette
Two Points of Resistance (Plunger)

1. First Stop - is the point when the piston reached the

calibrated volume on the dial
2. Second Stop - is used to fully expel the aspirated volume
into receiving vessel (equivalent ti blowing out a glass
pipette)
Automatic Pipetting Technique - Forward
Pipetting
 Automatic Pipetting Technique - Reverse
Pipetting
***The reverse mode is only possible with air-displacement pipettes. It is sometimes
used to pipette slightly viscous liquids.***

● In reverse mode pipetting, the purge stroke is used during preparation. During
aspiration, an amount of liquid equal to the amount of purged air is added. This
amount compensates for the liquid that remains inside the tip during dispensing.
 Pipettes and Their Calibration
● Volumetric pipettes - are designed to deliver a single, specific volume of liquid
-They are referred to as TD (to deliver) devices.
- Liquid is drawn through the tip of the pipette, filling a bulb in the body of pipette, until the
liquid level reaches the calibration mark.
- The measured liquid is then drained for use. Volumetric pipettes are never to be blown out.
- It has a certificate of calibration from the National Institute of Standards and Technology
(NIST), stating that it is a Class A pipette.
Oswald-Folin pipettes

- type of volumetric pipette with bulbs near the delivery tip

- They are TD pipettes, but are designed for use with more viscous fluids
such as blood or serum.
- Etched rings near the mouthpiece of Oswald-Folin pipettes signify that
they are blow out pipettes, meaning that after the liquid has drained, the
residual film on the wall of the pipette is blown out.
Graduated/Serological pipettes
- They are not considered accurate enough to measure samples or
standards, and are therefore normally used for reagents.
- An etched ring near the top of graduated pipettes signifies that they are
blow out pipettes.
- All semi-automated pipettes and diluters should be of certified accuracy,
or checked for accuracy yearly by weighing the amount of water delivered
by the pipette or diluter, checking the volume displacement, or by a
colorimetric method
Calibration of Pipette (Spectrophotometric)

*** Pipette calibration can be performed using the colored reagents with
known absorbance at specific wavelengths, such as potassium dichromate
or p-nitrophenol****
Procedure
1. Prepare the reagents required
a. In a 100-ml volumetric flask, dissolve 105 mg of high purity p-nitrophenol (PNP) in
deionized water.
b. Fill in according to the specified level and thoroughly mix
c. Prepare various dilutions to the p-nitrophenol stock (PNP)
d. Fill in three 250 ml volumetric flasks halfway with 250 ml 0.01 mol/L NaOH
e. Using a different volumetric pipette, add 1 ml of stock PNP to each flask. Combine
thoroughly

2.Pepare 5 cuvettes at a time

3.To each of five cuvetes, add 2.5 ml of 0.01 mol/L NaOH

4. To each cuvette, add ten liters of PNP solution
5. Use a plastic cover to cover the cuvette
6. 8-10 times invert
7. Begin with water blank in the spectrophotometer using DH20. Adjust the absorbance zero point to
401 nm
8. Take absorbance readings for each cuvette and record them
9. Volume delivered in uL = (Absorbance obtained)/0.550 *0.003984 *2510
** The dilution of PNP is 1/251 = 0.003984, and 2510 is the total volume of each cuvette
10. From the 5 volumes delivered by the pipette, determine the mean, SD, and CV (measure of
precision)
11. Calculate the accuracy error using this formula:
(expected volume- Obtained volume) *100% / expected volume
NOTE: Accuracy should meet the manufacturer’s guideline or be <1%, whichever is smaller
Gravimetric Pipette Calibration

- Class A pipettes, like all other Class A labware, do not need to be

recalibrated by the laboratory.
- Automatic pipetting devices, as well as non–Class A materials, do need
recalibration.
Materials

● Pipette
● 10 to 20 pipette tips, if needed
● Balance capable of accuracy and resolution to ±0.1% of dispensed volumetric
weight
● Weighing vessel large enough to hold volume of liquid
● Type I/CLRW (Clinical Laboratory Reagent Water)
● Thermometer and barometer
Procedure
1. Record the weight of the vessel. Record the temperature of the water. It is recommended
that all materials be at room temperature. Obtain the barometric pressure.

2. Place a small volume (0.5 mL) of the water into the container. To prevent effects from
evaporation, it is desirable to loosely cover each container with a substance such as Parafilm.
Avoid handling of the containers.

3. Weigh each container plus water to the nearest 0.1 mg or set the balance to zero.

4. Using the pipette to be tested, draw up the specified amount. Carefully wipe the outside of
the tip. Care should be taken not to touch the end of the tip; this will cause liquid to be wicked
out of the tip, introducing an inaccuracy as a result of technique.
5. Dispense the water into the weighed vessel. Touch the tip to the side.

6. Record the weight of the vessel.

7. Subtract the weight obtained in step 3 from that obtained in step 6. Record the result.

8. If plastic tips are used, change the tip between each dispensing. Repeat steps 1 to 6 for a minimum of nine
additional times.

9. Accuracy or the ability of the pipette to dispense the Obtain the average or mean of the weight of the
water. Multiply the mean weight by the corresponding density of water at the given temperature and
pressure. At 20°C, the density of water is 0.9982.

10. Determine the accuracy or the ability of the pipette to dispense the expected (selected or stated) volume
according to the following formula:
● The manufacturer usually gives acceptable limitations for a particular pipette,
but they should not be used if the value differs by more than 1.0% from the
expected value. Precision can be indicated as the percent coefficient of
variation (%CV) or standard deviation (SD) for a series of repetitive pipetting
steps.
● Required imprecision is usually ±1 SD. The %CV will vary with the expected
volume of the pipette, but the smaller the %CV value, the greater the
precision. When n is large, the data are more statistically valid.