THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROEDURE
PROCEDURE NO:
MOD:
PAGE:
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
1.
GEP 014
1st Issue
1 of 13
Risk Assessment:
TASK PERFORMED
Operation of Pipettes
HAZARDS
1. RSI (Repetitive Strain Injury)
2. Exposure to biohazards/hazardous substances
RISK ASSESSMENT
1. The risk of developing a muscle strain that leads to RSI is present whenever pipettes are
used, however, risk of injury increases as the period of use increases.
2. The risk of exposure to biohazards and hazardous substances while using a pipette is
present whenever these substances are being handled. Exposure may occur as a result of
splashing or contact with vapours.
RISK CONTROL
1. See Safety Precautions (point 4.1) in this GEP.
2. Ensure that biohazards and hazardous substances are only pipetted in appropriate
environments such as in biohazard hoods or fume cupboards, refer to the MSDS. Always
wear gloves (and safety glasses where necessary) when pipetting any substance. Draw and
dispense volumes slowly and with care to avoid splashing.
2.
Calibration:
2.1
Frequency
All pipettes should be calibrated at six-month intervals,
or after major cleaning.
2.2
Method - Calibration Set up
WRITTEN BY
NAME (signed)
DATE
Distributed To:
GEP Master File / GEP Lab File
CHECKED BY
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
2.2.1
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 2 of 13
Pipettes must be calibrated using the forward pipetting technique (see step 4
- General Operation.
2.2.2
GILSON does not guarantee that its' pipettes conform to specifications
when using other manufacturers tips.
2.2.3
Balances should be checked and if possible, calibrated before use in pipette
calibration.
2.2.4
P2 and P10 GILSON pipettes must be sent away for calibration as they
require a 6 decimal place balance.
2.2.5
A NATA calibrated thermometer must be used for all temperature
measurements (see GLP 001 NATA Thermometer Calibration).
2.2.6
Double de-ionised or distilled water must be used in the calibration.
2.2.7
The weighing vessel should be filled to a volume of 10 - 50 times the test
volume. The vessel should have a narrow neck (such as a conical flask) to
minimise the effects of evaporation. Heat from a light source or radiated
heat from the hands may increase the evaporation rate. Use a lid/cap for
volumes below 20μL. Only a high ambient humidity will negate the need for
a cover. The cover/lid should be made of a non-porous material and
tweezers must be used to lift it on and off the flask.
2.2.8
For all models other than the P5000 and P10mL, racked tips must be used
for the calibration. Do not touch the tip or white shaft of the pipette with the
hands while fitting the tip. For the P5000 and P10mL models, take a tip
(between the thumb and forefinger) and place it onto the pipette firmly with
a slight twisting motion to ensure a positive and airtight seal.
2.2.9
It is vital that anyone attempting to calibrate pipettes can pipette accurately
and repetitively. This can be tested by performing a series of 10 -15
weighings with a single pipette and noting the results. If repetitive results
cannot be achieved, then someone else must perform the calibration. Even a
pipette that is out of the correct calibration range should dispense repetitive
volumes unless it is leaking or sticking. See step 6. Leak Test.
2.3
Method - Calibration
2.3.1
Allow the temperature of the pipettes, tips, thermometer, a beaker of water and
an empty measuring vessel to eqilibrate for one hour. They should be placed as
close as possible to the balance being used.
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
2.3.2
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 3 of 13
The balance should be capable of weighing to at least one tenth of the maximum
allowable error deviation. Therefore a 6-digit balance is required for volumes less
than 20µL, a 5 digit balance for volumes less than 200µL and a 4-digit balance for
volumes greater than 200µL.
2.3.3
An Estimation of Evaporation Loss is only required when calibrating the
P20 model, as the evaporation loss for measurements above 20µL is
insignificant. Evaporation loss measurements should be taken before and
after the calibration measurements. Estimation of Evaporation Loss is
determined as follows:
a)
Fill the weighing vessel to half its level with temperature stabilised
distilled or double de-ionised water
b)
Cover the weighing vessel with the lid
c)
Tare the balance
d)
Take off the lid with tweezers
e)
Aspirate a sample
f)
Dispense the sample in a dummy vessel outside the balance
g)
Cover the weighing vessel
h)
Read and record the result (a negative value)
i)
Repeat the steps d - h three times
j)
Calculate the mean of these readings, e, usually between 20nL and
30nL, under normal conditions.
Record all measurements and the mean (e) on the Pipette (Gilson)
Calibration Record Sheet (Illustration 9.1).
2.3.4
Set the volume of the pipettes as per the following table.
MODEL
SET VOLUME TO
PERMITTED RANGE
P2 (0.1 - 2µL)
0.5µL
0.483 - 0.517µL
P10 (0.5 - 10µL)
1.0µL
0.983 - 1.017µL
P20 (2.0 - 20µL)
2.0µL
1.93 - 2.07µL
P100 (20 - 100µL)
20.0µL
19.8 - 20.2µL
P200 (50 - 200µL)
50.0µL
49.7 - 50.3µL
P1000 (200 - 1000µL)*
200µL
198 - 202µL
P5000 (1000 - 5000µL)
1000µL
993 - 1007µL
P10mL (1000 - 10000µL)
1000µL
979 -1021µL
* The P1000 pipette must only be calibrated using C200 tips.
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 4 of 13
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
2.3.5
Use the Calibration Record Sheet (Illustration 9.1) to record all measurements.
2.3.6
Place a tip firmly onto the cone tip.
2.3.7
Measure the water temperature to the nearest 0.1oC and record it on the
record sheet.
2.3.8
Fill the weighing vessel to 10-50 times the test volume. Place the weighing vessel
onto the balance and press Tare. Pre-rinse the tip by drawing up the set volume of
liquid and discarding it outside the weighing vessel. Pipette distilled water into
the vessel at least ten times, recording the mass to the nearest 0.0001g each time.
This should be done as quickly as practicable but without compromising the
integrity of the liquid delivery, the precision of the operator technique or the time
intervals.
2.3.9. If required, perform another 4 measurements for the Estimation of Evaporation
Loss (step 2.3.3) Record measurements on the Calibration Record Sheet.
2.3.10 Measure and record the water temperature again and use the average of the
two measurements as the calibration temperature.
2.3.11 Use the table below to determine the conversion factor (z) and record it on
the Calibration Record Sheet.
Temp oC Conversion
Temp oC
Factor(z)
Conversion
Temp oC
Factor(z)
Conversion
Factor(z)
15
1.0020
20.5
1.0030
26
1.0043
15.5
1.0020
21
1.0031
26.5
1.0044
16
1.0021
21.5
1.0032
27
1.0045
16.5
1.0022
22
1.0033
27.5
1.0047
17
1.0023
22.5
1.0034
28
1.0048
17.5
1.0024
23
1.0035
28.5
1.0050
18
1.0025
23.5
1.0036
29
1.0051
18.5
1.0026
24
1.0038
29.5
1.0052
19
1.0027
24.5
1.0039
30
1.0054
19.5
1.0028
25
1.0040
20
1.0029
25.5
1.0041
2.3.12 Calculate the average mass of the balance readings (m). Record the result
on the record sheet.
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 5 of 13
2.3.13 Calculate the average volume using the following formula:
V = ( m + e ) X z, where;
V = average volume
m = average mass
e
= average evaporation loss*
z = temperature conversion factor
* used only in P20 model calibration.
2.3.14 Compare the average volume result with the values given in the Permitted Range
column of the table in step 2.3.4. If the volume is within this range, then
calibration is complete. Proceed to step 2.4. If the volume is outside this range, the
pipette needs to be adjusted, as follows.
2.3.15 To adjust the pipette, dismantle as follows:
♦ Pull the tip ejector down and unscrew the connecting nut.
♦ Separate the handle from the bottom part of the pipette.
♦ Remove the piston from the tip holder then inspect the piston surface, oring and seal. (Never disassemble the upper part of the pipette).
♦ Clean the piston, o-ring and seal with detergent and water. Rinse with
absolute alcohol and leave to dry.
♦ If required, replace the o-ring and seal. Always replace the o-ring and
seal at the same time using genuine parts only. (These have a life span of
approximately 12 months.)
2.3.16 Reassemble the pipette and repeat the calibration (steps 2.3.1 - 2.3.14). If, after the
second calibration test the volume is still outside the Permitted Range, the pipette
may need to be reseated. Do not attempt this step without seeking assistance from
an experienced staff member.
2.3.17 Wind the volume adjuster right down to the bottom where it should read
V2. If not, wind the volume adjuster up until it comes right out. Push the
volume and piston back in until it rests against the lug but is not winding.
Move the adjuster to read V1, then push down to engage the thread. Wind
the volume adjuster to the bottom again, where it should read V2.
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 6 of 13
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
2.3.18 Repeat the calibration procedure (step 2.3.1 - 2.3.14). If the pipette is still out
of range, then the calibration may be adjusted using the special calibration
tool. Adjust as described in the following table:
PIPETTE MODEL
VOLUME INCREASE/DECREASE PER FULL
TURN (μL)
P20
1
P100
4
P200
10
P1000
40
P5000
200
P10000
400
2.3.19 Repeat the calibration procedure (step 2.3.1 - 2.3.14) and 2.3.18 if required. If
the pipette is still out of range - send it to John Morris Scientific for
professional diagnosis and calibration.
2.4
Calibration Control
2.4.1
Once successful calibration is complete a calibration control test must
be performed. Set the pipette as indicated in the table below:
MODEL
SET VOLUME TO
CONTROL LIMITS
P20
20.0µL
19.9 - 20.1µL
P100
100µL
99.5 - 100.5µL
P200
200µL
199 - 201µL
P1000
1000µL
995 - 1005µL
P5000
5000µL
4979 - 5021µL
P10mL
10000µL
9985 - 10042µL
2.4.2
Perform one pre-rinsing (aspirate a sample, and dispense it).
2.4.3
Using the same procedure described in 2.3, perform three
consecutive measurements.
2.4.4
Calculate the mean volume dispensed for the three measurements,
and compare these with the CONTROL LIMITS in the table above. If
the mean value is out of the control limits, the pipette must be recalibrated. If the mean value is within these limits, the pipette has
been calibrated successfully.
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
2.5
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 7 of 13
Records
Record all calibration measurements and adjustments on the Calibration Record Sheet
(multiple sheets may be required - staple them together) and file in the appropriate
section of the Equipment Records folder when complete.
3.
Monitoring:
4.
General Operation:
4.1
N/A
SAFETY PRECAUTIONS - excessive pipette use can lead to RSI (Repetitive Strain
Injury). Therefore the following guidelines should be noted:
4.1.1
Wrists should be held in a straight, neutral position - it may be necessary to
incline the sample holders or solution flasks, rather than the wrists.
4.1.2
The workload should be shared between right and left hands.
4.1.3
Elbows should be held as close as possible to the users' side.
4.1.4
Reaching may be reduced by using shorter pipettes, shallower waste
containers for used tips and shallower solution containers.
4.1.5
Items should be positioned on the bench and as close as possible to the
worker to minimise twisting of the neck and torso.
4.1.6
The chair should provide adequate lower back and thigh support.
4.1.7
Regular breaks should be taken - even very short breaks help.
4.1.8
Work processes should be evaluated to spread pipetting throughout the
day.
4.1.9
Pipetting should be rotated among several staff.
4.1.10 Pipetting tasks should be automated wherever possible.
4.2
The delivery volume is set using the device seated in the pipette handle. Ensure that the
desired volume is in line with the indicator and that the digits are completely visible on
the handle display. Do not set volumes outside the pipette's specified volume range.
4.3
The pipette is fitted with a tip ejector system on the top of the tip holder. Direct the tip
towards a suitable waste receptacle and press on the ejector mechanism, disposing of the
tip safely.
4.4
Forward Pipetting Technique
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 8 of 13
4.4.1
Depress the operating button to the first stop.
4.4.2
Lower the tip under the surface of the liquid and slowly release the operating
button. Withdraw the tip from the liquid, touching it against the side of the
container to remove excess liquid.
4.4.3
Deliver the liquid by gently depressing the operating button through the first stop
all the way to the second stop. This action will empty the tip.
4.4.4
Release the operating button to the ready position. Replace tip if necessary and
continue with pipetting.
4.5
Reverse Pipetting Technique
4.5.1
The reverse technique is suitable for pipetting liquids having high viscosity, a
tendency to foam easily or for very small volumes.
NB Performing a serial dilution is more accurate than a straight dilution,
especially when very small volumes are involved (less than 10μL). For example, it
would be more accurate to perform a serial dilution involving two dilutions of
1/10 than a straight dilution of 1/100.
4.5.2
Depress the operating button all the way to the second stop.
4.5.3
Lower the tips under the surface of the liquid and slowly release the operating
button. This action will fill the tip. Withdraw the tip from the liquid touching it
against the side of the container to remove excess liquid.
4.5.4
Deliver the preset volume by gently depressing the operating button to the first
stop. Some liquid will remain in the tip and should not be included in the
delivery. It should either be discarded or pipetted back into the container.
4.6
Filter tips should be used when pipetting corrosive substances to prevent damage
to the internal parts of the pipette. After contact with corrosive liquids, the piston
should be cleaned with alcohol and a soft tissue.
5.
Cleaning:
5.1
Frequency
5.1.1
Before use - A routine external cleanliness check should be performed and the
pipette cleaned if required.
5.1.2
Six Monthly - Disassemble and calibrate.
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
5.2
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 9 of 13
Method
5.2.1
Before Use
Check for dust and dirt on the outside surfaces of the pipette. Particularly the tip
cone. Use absolute alcohol or detergent and water to clean exterior surfaces as
required. If the inside of the cone tip is dirty, disassemble and clean, according to
step 2.3.15, then perform a calibration control test as described in step 2.4.
5.2.2
Six Monthly
Disassemble and clean, as described in step 2.3.15. Then Calibrate the
pipette, as described in step 2.
5.3
Records
Record all cleaning/calibration details on the Maintenance Record Sheet
(Illustration 9.2).
6.
Maintenance:
6.1
6.2
Frequency
6.1.1
Ongoing maintenance - Leak Test
6.1.2
Ongoing maintenance - Diagnostic Test
6.1.3
Periodic Maintenance - Six monthly Clean and Calibration.
Method
6.2.1
Ongoing Maintenance - Leak Test
To test the pipette for leaks (for pipettes P1000 to p10mL);
♦ Set the pipette to the maximum setting and draw some water into the
tip, hold the pipette vertically and let it stabilise for 20 seconds.
♦ If a drop appears at the end of the tip, there is a leak.
To test for leaks (for pipettes P2 to P200);
♦ Set the pipette to the maximum setting and draw some water into the
tip, hold the pipette vertically and let it stabilise for 20 seconds.
♦ If a drop appears at the end of the tip, there is a leak. If no drop appears,
re-immerse the tip in the test liquid. While immersed, the level of the
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 10 of 13
liquid in the tip should remain constant. If the level in the tip goes
down, there is a leak.
Leaks may be caused by the following:
♦ a scratched or damaged tip holder
♦ use of non-Gilson tips
♦ use of non-Gilson seals
♦ a damaged seal
♦ vapour pressure from organic solvents. When an organic solvent is used
with an air-displacement pipette, leaks may occur. This is due to the
difference in the solvent vapour pressure and the vapour pressure of the
air cushion between the piston and the sample. To solve this problem is
to use a positive-displacement pipette, as it has no air-cushion.
Alternatively, saturating the air cushion of the pipette with solvent
vapour by aspirating and distributing solvent repeatedly. The leak will
stop when the pressure equilibrium is reached.
6.2.2
Ongoing Maintenance - Diagnostic Test
To check if the pipette is performing normally:
♦ Go through the entire volume range using the push -button adjusting knob
(or thumb wheel on the older pipettes). The knob should move smoothly.
♦ The min and max settings should correspond to the pipettes normal
volume range.
♦ Check the alignment and the movement of the volumeter display.
♦ Set the volume at the maximum volume for that pipette and depress the
push-button slowly. The movement should be smooth. 'Hitches' in the
motion or variations in friction may be due to a scratch or corrosion on
the piston, or to a bent operating rod. Listen for a spring noise, which
would indicate incorrect positioning of the spring inside the pipette.
♦ Any problems may require an internal inspection or be sent away for a
service. For dismantling and cleaning instructions, see step 6.1.3, Periodic
Maintenance - Six Monthly Clean and Calibration
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
6.1.3
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 11 of 13
Periodic Maintenance - Six Monthly Clean and Calibration
♦ All pipettes must be cleaned and calibrated every six months.
Disassemble and clean pipettes, as described in step 2.3.15. Then
calibrate the pipette, as described in step 2.
6.3
Records
Record dates and details of all maintenance performed on the Maintenance
Record Sheet (Illustration 9.2) and file completed sheets in the appropriate section of
the Equipment Records folder.
7.
Instruction Manual:
7.1
8.
9.
10.
See specific ERN for availability.
Repairs:
8.1
Check ERN for Service Company and warranty details.
8.2
Contact the University Workshop for advice and repairs.
Illustrations:
9.1
Pipette (Gilson) Calibration Record Sheet
9.2
Pipette (Gilson) Maintenance Record Sheet
Change History:
10.1
Issue Number:
1st Issue
Date Issued:
10.2
Issue Number:
Reason for Change:
Date Issued:
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 12 of 13
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
ILLUSTRATION 9.1
CALIBRATION RECORD SHEET
PIPETTE (GILSON)
ERN/MODEL:
/
INITIALS:
DATE:
Acceptable Weight Range:
oC
Average Water Temp
Conversion Factor (z)
Water temp 1
Water temp 2
EVAPORATION ESTIMATION MASSES (for P20 model only)
BEFORE CALIBRATION MASS
M1
M2
M3
M4
AFTER CALIBRATION MASS
M5
M6
M7
M8
AVERAGE (e):
READING
1
2
MASS
AVERAGE
MASS (m)
AVERAGE
VOLUME
V=(m+e)X z
3
4
5
6
7
8
9
10
THE UNIVERSITY OF NEWCASTLE - DISCIPLINE OF MEDICAL BIOCHEMISTRY
STANDARD OPERATING PROCEDURE
PROCEDURE NO: GEP 014
MOD: 1st Issue
PAGE: 13 of 13
Procedure Type:
General Equipment Procedure
Title:
Pipette (Gilson)
ILLUSTRATION 9.2
MAINTENANCE RECORD SHEET
PIPETTE (GILSON)
ERN:
Date
Maintenance Performed
Signed