Types of Pipettes

Pipetting techniques

Errors

Terminology used

Factors affecting accuracy of pipetting

Proper techniques of pipetting

Types of Pipettes                    

• Air Displacement Pipettes
  • The plunger pushes out air and liquid is aspirated in.
  • Less accurate, especially the continuously adjustable pipette.
  • Should expect 1.5 to 2 % error
• Fixed Volume Pipette
  • Error expected is about 1%
• Positive Displacement
  • Very accurate, especially for viscous solutions.

Forward Pipetting: For Aqueous liquids

• Press the push button to the first stop.
• Place the tip a few millimeters below the surface of liquid
• Release the button slowly to aspirate the liquid in to the tip
• To dispense the liquid, press the button all the way down to second stop.

Withdraw the tip and release the button back to the starting position

Reverse Pipetting
For Viscous and easily foaming liquids

• Push the button all the way down to the second stop.
• Place the tip few millimeters bellow the surface of liquid.
• Release the button slowly to aspirate the liquid slowly in to the tip.
• To dispense the liquid, press the button to the first stop.
• To perform continued reverse Pipetting keep the button in the first stop

Errors

• Systematic Error.
• Random Error.
• Systematic error: This error is consistent, due to instrument calibration, or operating conditions. The pipette is generally inaccurate.
• Random error: This error is inconsistent and un predictable and is generally seen as imprecision or scatter, causes? Defective seal or other parts.

Inaccuracy and Imprecision

• Accuracy: The closeness of a dispensed volume to the volume specified by setting of the instrument.
• The accuracy is expressed in terms of error, the difference between set and true volume. Smaller the error more accurate the instrument.

Precision:  Agreement between replicate measurements. Precision has a numerical value and it’s quantified by imprecision

Sources of error in Pipetting

• Tips
• Pipetting angle.
• Density of the liquid
• Temperature
• Altitude

The Right Tip

• Use the tip from the manufacture of the pipette.
• Aerosol tips prevent cross contamination.
• Pre rinse the tip before Pipetting.
• Whenever possible keep the tips and the liquid being dispensed at same temperature.

Temperature

• Variation in the temperature of the solution being pipetted is the second largest cause of Pipetting error.
• Solutions that were warmed to 30 c consistently delivered lower volumes than room temperature samples.
• Similarly, solutions that were cooled to 8.5 c delivered higher volumes than ambient samples.

Pipetting Angle

• The effect of Pipetting position
• Pipette vertical with the tip dipped about 1 cm into the liquid.
• Pipette vertical with tip dipped about 3 cm into the liquid.
• Pipette tilted at 30º-40º angle with the tip dipped about 3-4cm into the liquid.

Dialing the volume

• Turn the volume adjustment knob 1/3 revolution above the desired setting.
• Then slowly turn down until required volume shows on the indicator.
• If you pass the desired setting, turn the knob 1/3 revolution above and re set.
• Always dial down to the desired volume. This prevents mechanical backlash from affecting the accuracy