1.1 To lay down a procedure for handling, operation, maintenance and calibration of Micropipettes.
2.0 SCOPE
2.1 This procedure applies to the Pharmaceutical Manufacturing Facility of __________________________________________________________
3.0 RESPONSIBILITY
3.1 Microbiologists.
4.0 ACCOUNTABILITY
4.1 Head-Quality control
5.0 PROCEDURE
5.1 PRELIMINARY CHECKS :
5.1.1 Push and release the push button slowly at all times particularly when working with high viscosity liquids.
5.1.2 Do not allow the push button to snap back.
5.1.3 Always make sure that the tip is firmly attached to the tip cone.
5.1.4 Always hold the pipette in an upright position while aspirating liquid.
5.1.5 At the beginning of each working day, the pipette should be checked for dust and dirt on the outside surfaces and specifically the tip cone and it should be cleaned with 70% ethanol
5.2 BASIC OPERATION :
5.2.1 Use only clean and dry tip. Fix the tip to tip holder with slight twist motion to ensure positive airtight seal.
5.2.2 Hold the pipette in an upright position and depress the push button to the first stop.
5.2.3 Dip the tip under the surface of the liquid in the liquid sample.
5.2.4 Withdraw the tip from the liquid sample touching it against the edge of the glassware to remove excess liquid. To deliver push button to first stop then, after one second, continue to press the push button all the way to second stop to empty the tip.
5.2.5 Release the push button to the ready position.
5.2.6 In case, of liquids that have a high viscosity or foaming property press the push button all the way to the second stop and follow step 5.2.3-5.2.4.
5.2.7 Transfer the preset volume by gently pressing the push button to the first stop only. The remaining liquid in the tip should be discarded.
5.3 Removing the Tip:
5.3.1 Eject the tip with the help of tip ejector button.
5.3.2 If different liquid are to be used change pipette tip.
5.4 MAINTENANCE:
5.4.1 Externally pipette can be cleaned with moist lint free duster. If required, use 70% isopropanol to moisten the cloth.
5.4.2 When the Micropipette is not in use, then it should be stored in an upright position.
5.4.3 If the push button movement is not free, the pipette needs to be disassembled and cleaned internally. For this press the tip ejector and insert the plier under the ejector bar to release the tip ejector.
5.4.4 Remove the tip cone using the maintenance pliers. Pull out the piston.
5.2.7 Transfer the preset volume by gently pressing the push button to the first stop only. The remaining liquid in the tip should be discarded.
5.3 Removing the Tip:
5.3.1 Eject the tip with the help of tip ejector button.
5.3.2 If different liquid are to be used change pipette tip.
5.4 MAINTENANCE:
5.4.1 Externally pipette can be cleaned with moist lint free duster. If required, use 70% isopropanol to moisten the cloth.
5.4.2 When the Micropipette is not in use, then it should be stored in an upright position.
5.4.3 If the push button movement is not free, the pipette needs to be disassembled and cleaned internally. For this press the tip ejector and insert the plier under the ejector bar to release the tip ejector.
5.4.4 Remove the tip cone using the maintenance pliers. Pull out the piston.
5.4.5 Remove the O-ring, O-ring support and spring from the tip cone.
5.4.6 Rinse the tip holder, piston and O-ring with purified water and allow to dry Grease the cleaned parts with the lubricant provided with the pipette before reassembling the pipette.
5.4.7 While reassembling the pipette, never try to over tighten any connection of the pipette. Slide parts over the piston pressing down the large spring. Attach the tip cone to the handle so that the adapter opening is on the tip ejector side and press in the snap joints.
5.4.8 Recalibrate the pipette after every internal cleanup.
5.4.9 Pre rinse each new tip if to be used with one of the following liquids:
a) Liquids with density different than water (e.g. 70 % Isopropyl Alcohol)
5.4.6 Rinse the tip holder, piston and O-ring with purified water and allow to dry Grease the cleaned parts with the lubricant provided with the pipette before reassembling the pipette.
5.4.7 While reassembling the pipette, never try to over tighten any connection of the pipette. Slide parts over the piston pressing down the large spring. Attach the tip cone to the handle so that the adapter opening is on the tip ejector side and press in the snap joints.
5.4.8 Recalibrate the pipette after every internal cleanup.
5.4.9 Pre rinse each new tip if to be used with one of the following liquids:
a) Liquids with density different than water (e.g. 70 % Isopropyl Alcohol)
b) Liquids with high viscosity (e.g. glycerol).
5.5 TROUBLE SHOOTING:
Defects
Possible Reason
Solution
5.5 TROUBLE SHOOTING:
Defects
Possible Reason
Solution
Leakage
Tip incorrectly attached
Foreign particles between tip and tip cone
Foreign particle between the piston, O-ring and cylinder
Insufficient amount of grease on cylinder and O-ring
O-ring damaged
Attach firmly.
Clean tip cones attach new tips.
Clean grease O-ring & cylinder Grease accordingly
Change the O-ring
Inaccurate dispensing
Incorrect operation
Tip incorrectly attached
Calibration altered
Follow instructions carefully
Attach firmly
Recalibrate according to instructions
Inaccurate dispensing with certain liquids
Unsuitable calibration.
High viscosity liquids may require calibration.
Recalibrate with the liquid In question .
5.6 CALIBRATION
5.6.1 Calibration Frequency: Half Yearly
5.6.2 Preparation of Sample
5.6.1 Collect purified water into a clean dried glass stoppered bottle and maintains the temperature at 22°-25°C.
5.6.3 Gravimetric Measurement of Volume:
5.6.3.1 Take the Micropipette and adjust the set volume for calibration with the help of the knob provided with the top of pipette
5.6.3.2 A new tip is first pre-wetted 3-5 times and series of ten pipetting are done using purified water in the pipette tip.
5.6.3.3 Dispense the aspirated water into a clean, dry and tarred close beaker, in such a way that dispensing end of the pipette tip is in contact with the inner side of the beaker. Ensure that no drop is remaining in the pipette tip.
5.6.3.4 Weigh the flask and record the weight in g (w).Set and take the pipette readings at Minimum volume, Optimum volume and maximum volumes and record the readings in Calibration Data Sheet.
5.6.3.5 Calculate the volume dispensed in mL/ ml by using the formula.
Volume of water (mL) = (w + e) X Z
where, V = volume (mL)
w = weight (mg)
e = evaporation loss(mg)
Z = conversion factor for mg/ml conversion
Factor Z = 1.0032mL/mg at 22°C
(Always use evaporation trap or lid on the vessel used for pipette calibration to nullify evaporation loss.)
5.6.3.6 Volume dispensed should be equal to volume set. If not, then turn the pipette
5.6.3.7 knob to get the exact volume clockwise to increase or anticlockwise to decrease the volume.
5.6.3.8 Ensure that the pipette is dispensing the set volume and record ten observations for volume set.
5.6.3.9 Calculate average of the ten readings.
5.6.3.10 Record all the observations in the Annexure-1-Micropipette Calibration Data Sheet.
5.7 Acceptance Criteria :
Note : If equipment does not comply with any of the calibration parameters, then follow the relevant procedure of equipment breakdown.
6.0 ABBREVIATIONS :
6.1 W : Weight
6.2 Ml : milliliters
6.3 mL : microliters
6.4 g : grams
6.5 % : Percentage
7.0 REFERENCES
7.1 Nil
8.0 ANNEXURES
8.1 Annexure-1 : Micropipette calibration data sheet
END OF THE DOCUMENT
Tip incorrectly attached
Foreign particles between tip and tip cone
Foreign particle between the piston, O-ring and cylinder
Insufficient amount of grease on cylinder and O-ring
O-ring damaged
Attach firmly.
Clean tip cones attach new tips.
Clean grease O-ring & cylinder Grease accordingly
Change the O-ring
Inaccurate dispensing
Incorrect operation
Tip incorrectly attached
Calibration altered
Follow instructions carefully
Attach firmly
Recalibrate according to instructions
Inaccurate dispensing with certain liquids
Unsuitable calibration.
High viscosity liquids may require calibration.
Recalibrate with the liquid In question .
5.6 CALIBRATION
5.6.1 Calibration Frequency: Half Yearly
5.6.2 Preparation of Sample
5.6.1 Collect purified water into a clean dried glass stoppered bottle and maintains the temperature at 22°-25°C.
5.6.3 Gravimetric Measurement of Volume:
5.6.3.1 Take the Micropipette and adjust the set volume for calibration with the help of the knob provided with the top of pipette
5.6.3.2 A new tip is first pre-wetted 3-5 times and series of ten pipetting are done using purified water in the pipette tip.
5.6.3.3 Dispense the aspirated water into a clean, dry and tarred close beaker, in such a way that dispensing end of the pipette tip is in contact with the inner side of the beaker. Ensure that no drop is remaining in the pipette tip.
5.6.3.4 Weigh the flask and record the weight in g (w).Set and take the pipette readings at Minimum volume, Optimum volume and maximum volumes and record the readings in Calibration Data Sheet.
5.6.3.5 Calculate the volume dispensed in mL/ ml by using the formula.
Volume of water (mL) = (w + e) X Z
where, V = volume (mL)
w = weight (mg)
e = evaporation loss(mg)
Z = conversion factor for mg/ml conversion
Factor Z = 1.0032mL/mg at 22°C
(Always use evaporation trap or lid on the vessel used for pipette calibration to nullify evaporation loss.)
5.6.3.6 Volume dispensed should be equal to volume set. If not, then turn the pipette
5.6.3.7 knob to get the exact volume clockwise to increase or anticlockwise to decrease the volume.
5.6.3.8 Ensure that the pipette is dispensing the set volume and record ten observations for volume set.
5.6.3.9 Calculate average of the ten readings.
5.6.3.10 Record all the observations in the Annexure-1-Micropipette Calibration Data Sheet.
5.7 Acceptance Criteria :
|
Nominal volume |
Maximum permissible systematic error(ACC) |
|
|
µl |
±% |
±µl |
|
5 |
±1.4 |
±0.07 |
|
10 |
±0.09 |
±0.09 |
|
20 |
±0.12 |
±0.12 |
|
25 |
±0.15 |
±0.15 |
|
50 |
±0.3 |
±0.3 |
|
100 |
±0.4 |
±0.4 |
|
200 |
±0.4 |
±0.8 |
|
250 |
±0.4 |
±1.0 |
|
500 |
±0.3 |
±1.5 |
|
1000 |
±0.3 |
±3.0 |
|
2000 |
±6.0 |
±6.0 |
|
5000 |
±15.0 |
±15.0 |
|
10000 |
±30.0 |
±30.0 |
Note : If equipment does not comply with any of the calibration parameters, then follow the relevant procedure of equipment breakdown.
6.0 ABBREVIATIONS :
6.1 W : Weight
6.2 Ml : milliliters
6.3 mL : microliters
6.4 g : grams
6.5 % : Percentage
7.0 REFERENCES
7.1 Nil
8.0 ANNEXURES
8.1 Annexure-1 : Micropipette calibration data sheet
END OF THE DOCUMENT
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