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Brinkmann/Eppendorf SOP,,, Standard Operating Procedure for Pipettes

Brinkmann/Eppendorf SOP Standard Operating Procedure for Pipettes

Contents

1 Measuring station and accessories 2 Testing conditions and pipetting conditions 3 Calibrating pipettes and bottletop dispensers / mode of operation 3.1 Full certification program 3.2 Standard calibration procedure 3.3 Brinkmann PACE calibration procedure 4 Individual evaluation 5 Sterilizing and cleaning the pipette 6 Checking for leaks 7 Troubleshooting 8 Adjustment 8.1 Adjusting pipettes (adjustable-volume) 8.2 Adjusting pipettes (fixed-volume) 8.3 Adjusting for liquids with a density different to that of bidistilled, degassed water 9 Factor Z for distilled water 10 Technical specifications of Eppendorf pipettes 10.1 Fixed-volume pipettes 10.2 Adjustable pipettes 10.3 Multichannel pipettes 10.4 Repeater 10.5 Bottletop dispensers 10.6 Error limits in accordance with DIN 12 650

1 Measuring station and accessories

Test conditions in accordance with ASTM E-1154, DIN 12 650, ISO 8655, and NCCLS (referred to as The Standards).
These test conditions are valid for all Eppendorf pipettes and dispensers in the appropriate form. No special reference is made to the different tip ejection mechanisms or the differences between manual and electronic pipettes.

Our expert staff offers cGMP/GLP service, in accordance with your SOP.

The following components are required for a measuring station for calibrating/adjusting pipettes:
Balance tested by Board of Weights and Measures (NIST) (e.g. Sartorius, Mettler, Ohaus or AnD)
Certified Barometer, Thermometer, and Hygrometer

Resolution/Accuracy:
The relationship between the error limits of the pipettes to be tested and the graduation scale of the balance ensures that scattering within a measurement series is recorded with sufficient accuracy to evaluate the accuracy and precision in accordance with the DIN standard. Manufacturer specifications sometimes deviate from the DIN standard and demand a high degree of weighing accuracy (see Technical specifications).

Nominal volume of the pipette
to be tested (l)
Error limits of the device
to be tested (l)
Required accuracy
of balance (g)
1 50* 0.1 0.8 0.00001 > 50 1,000 1.0 10 0.0001 > 1,000 > 10 0.001 * A balance with six decimal places or, where appropriate, a photometric test should be used for volumes <= 1 l. Evaporation protection:
Moisture trap or other equipment which prevents evaporation.

Electrostatic charge:
For test volumes <= 50 l, electrostatic discharge to all parts which come in direct or indirect contact with the pipettes must be prevented. The tubes provided by Eppendorf and the moisture trap with lid are made of glass, which minimizes electrostatic charge.

Measuring station:
Calibration software and accessories for your balance (PICASO, catalog no.: 22 44 501-4) are recommended for electronic processing of measuring data.

Measuring station and accessories for test volumes Up to 50 l 50 l and more Balance setting int 1 (integration time)
asd 2 (standstill time) int 1
asd 2 Weighing vessel, vessel adapter Vessel, diameter 15 mm (cat. no. 22 47 493-6, 3 pcs.) with vessel adapter dia. 15 mm Vessel diameter 24 mm (cat. no. 22 47 492-8, 3 pcs.) with vessel adapter dia. 24 mm Evaporation protection Glass moisture trap Approx. 10 ml distilled water in a dish inside the balance For pipettes with a maximum volume of 5,000 l and above, the weighing plate with a vessel of an appropriate size, such as a 50 ml volumetric flask, is used instead of the special Eppendorf balance adapter. The moisture trap is not used in these cases.

2 Testing conditions and pipetting conditions

Room temperature:
20 25C, 0.5C during measurement.

Temperature differences:
Factors which affect the temperature of the pipettes and measuring station (e.g. direct sunlight) should be avoided.

Please ensure that the ambient temperature and the temperatures of the test liquid and the pipettes used match the temperature of your tips. It is advisable to temperature-stabilize all components for approximately 3 4 hours prior to calibration.

Location of the balance:
The balance should be free of drafts and vibrations and should be placed in a temperature-controlled environment.

Test liquid:
Degassed, bidistilled water which is at room temperature (20 25C).

The water in the liquid supply or in the weighing vessel must be changed every hour and must not be reused.

The humidity over the liquid surface of the weighing vessel should be maintained at a value between 60% and 90% of the relative humidity.

Instruction manual:
In view of the many different types of volume measuring devices, it is particularly important to refer to the instruction manual during testing. DIN 12 650, Part 6 states that, in accordance with DIN 8418, the instruction manual forms part of the delivery package of the device and the contents of the instruction manual must be observed.

3 Calibration of pipettes and bottletop dispensers/mode of operation

3.1 Full Certification Procedure
(As performed at Eppendorf manufacturing facility and by special request at Brinkmann Instrument, Inc.)
Documentation supplied with certification includes:
- actual volume measurements for each measured volume
- standard calibration report
- pipette calibration sticker

Adjustable pipettesthree different volumes are tested:
- the nominal volume (10 measured values)
- approximately 50% of the nominal volume (10 measured values)
- the smallest adjustable volume, which should not be less than 10% of the nominal volume (10 measured values)

Fixed-volume pipettesthe nominal volume is tested (10 measured values).

Multichannel pipettesthe following volumes are tested for each channel:

- the nominal volume (3 measured values)
- approximately 50% of the nominal volume (3 measured values)
- the smallest adjustable volume, which should not be less than 10% of the nominal volume (3 measured values)

Bottletop dispensersthe nominal volume is tested (10 measured values).

Repeater Pipettetesting is carried out with the Eppendorf Combitip in Position 1 of the Repeater Pipette (48 measured values) with a 12.5 ml Combitip.

Repeater Plus Pipettetesting is carried out with the Eppendorf Combitip Plus Tip in Position 1 of the Repeater Plus Pipette (50 measured values) with a 5 ml Combitip.

3.2 Standard Calibration Procedure
(As performed as standard procedure at Brinkmann Instruments, Inc., Westbury, NY facility)

Documentation supplied with calibration:
- standard calibration report
- pipette calibration sticker

Adjustable pipettesthree different volumes are tested:
- the nominal volume (5 measured values)
- approximately 50% of the nominal volume (5 measured values)
- the smallest adjustable volume, which should not be less than 10% of the nominal volume (5 measured values)

Fixed-volume pipettesthe nominal volume is tested (5 measured values).

Multichannel pipettesthe following volumes are tested for each channel:
- the nominal volume (2 measured values)
- the smallest adjustable volume, which should not be less than 10% of the nominal volume (2 measured values)

Bottletop dispensersthe nominal volume is tested (10 measured values).

Repeater Pipettetesting is carried out with the Eppendorf Combitip in Position 1 of the Repeater Pipette (48 measured values) with a 12.5 ml Combitip.

Repeater Plus Pipettetesting is carried out with the Eppendorf Combitip Plus Tip in Position 1 of the Repeater Plus Pipette (50 measured values) with a 5 ml Combitip.

3.3 Brinkmann PACETM Calibration Procedure:
(As performed at customer account or through the PACE mail-in program)

Documentation supplied with Brinkmann PACE calibration includes:

- PACE calibration report
- pipette calibration sticker

Adjustable pipettestwo different volumes are tested:
- the nominal volume (4 measured values)
- the smallest adjustable volume, which should not be less than 10% of the nominal volume(4 measured values)

Fixed-volume pipettesthe nominal volume is tested (4 measured values).

Multichannel pipettesthe following volumes are tested for each channel:
- the nominal volume (1 measured values)
- the smallest adjustable volume, which should not be less than 10% of the nominal volume(1 measured values)

Repeater Pipettefill Combitip, dispense 48 times (without measuring), the steps must not skip, proceed to measure 4 weighings with a full Combitip (Step# 48, 47, 46, & 45), 4 volumes with a half-full Combitip (Step# 25, 24, 23, & 22), 4 volumes at the low-volume Combitip (Step# 4, 3, 2, & 1).

Repeater Plus Pipettefill Combitip, dispense 100 times (without measuring), the steps must not skip, proceed to measure 4 weighings with a full Combitip (Step# 100, 99, 98, & 97), 4 volumes with a half-full Combitip (Step# 51, 50, 49, & 48), 4 volumes at the low-volume Combitip (Step# 4, 3, 2, & 1).

Additional Services available:
- As Found Data available for an addition charge
- Proper Pipetting Technique Seminars: available to meet your cGMP/GLP training requirements

Mode of operation: - Attach the appropriately-sized Eppendorf Pipette Tip onto the nose cone of the pipette, or insert the appropriately-sized Combitip into the Repeater pipette - Adjustable pipettesset to the smallest volume which is to be tested
Bottletop dispensersset the nominal volume
Repeater pipettesset the volume selection dial to Position 1
The first value of the Repeater Pipette is discarded (when the Combitip is fully pulled). In the case of the Repeater Plus Pipette, the empty stroke is executed (when the Combitip Plus Tip is fully pulled) - Tare the balance - Pre-wet the tip (not applicable for Combitips) - Aspirate and then dispense the set volume three times. Execute blow-out Aspirate the volume which is to be tested from the liquid supply as follows: - Hold the pipette vertically in the liquid supply - Immerse the tip approximately 2 3 mm into the test liquid (see instruction manual) - Aspirate the test volume slowly and uniformly. Observe the waiting period of 1 3 sec. (for the Series 2100 (Research), 500 5,000 l: 5 sec.)
(The waiting time depends on the size of the pipette tip; see instruction manual) - Remove the pipette tip from the test liquid slowly and uniformly. Remove any remaining liquid by wiping the pipette tip against the inside of the vessel Dispense the test volume into the weighing vessel as follows: - Place the filled tip at an angle of 30 against the inside of the weighing vessel - Dispense the test volume slowly and uniformly up to the first stop (measuring stroke) and wait for 1 3 sec. (This applies to manual pipettes only) - Press the control button to the second stop (blow-out) and dispense any liquid remaining in the tip (not applicable when bottletop dispensers and the Repeater pipettes are used) - Hold down the control button and pull the tip up along the inside of the weighing vessel - Document the value which appears in the display of the balance immediately after the display has come to a standstill - Document all measured values from a measurement series as described above. Evaluate the inaccuracy and the imprecision - With adjustable pipettes, proceed in the same way, using the nominal volume and 50% of the nominal volume - With multichannel pipettes, the measured values of all channels per test volume are summarized for evaluation of the inaccuracy and imprecision Please note: When user testing, follow section 3.1 Full Certification Procedure.

4 Individual evaluation

The following calculation is carried out from all measured values within the evaluation limits: Mean value = x Number of measured values = n Standard deviation = s Coefficient of variation = CV (imprecision) Inaccuracy = d Range = Difference between maximum and minimum value For exact calculation, Z factor is used to correct changes in the test liquid regarding temperature and air pressure.

The manufacturer specifications for the pipette can be found in the instruction manual or in Technical specifications

5 Sterilizing and cleaning the pipette

Sterilization
The instruction manual of the pipette contains information on cleaning with solvents or autoclaving(121 C, 20 min.).

Eppendorf pipettes should be autoclaved as follows:

The Model 4800 and the Model 4810, the Series 2000 (Reference), fixed-volume and adjustable-volume, and the Positive Displacement Pipette (Model 4830) are fully autoclavable.

Before autoclaving, unscrew the upper housing from the lower housing of the pipette by rotating the central junction once. This allows vapor to enter the device easily. Do not reassemble until the pipette cools down to room temperature.

Only the lower parts of the Series 2100 (Research) and Multichannel Pipettes are autoclavable.

Series 2100 (Research):
The tip ejector is pulled off the pipette and the lower part of the pipette is unscrewed. The lower part and the ejector can now be autoclaved. Do not reassemble the parts until they have reached room temperature.

Titermate: Turn the control button to the uppermost stop. Press your thumb against the grip of the pipette and push the lower part forward through the slit. Do not reassemble the parts until they have reached room temperature.

Brinkmann Bottletop Dispenser: must be completely disassembled before autoclaving begins.

Brinkmann ChemSaver Dispenser: Move the discharge valve toggle into the dispensing position (->). Disengage the volume setting button, push it into the middle position and leave it disengaged. Place the dispenser onto a cloth and autoclave the device. Ensure that the device does not come into contact with hot metal surfaces. Do not use the dispenser until it has cooled down to room temperature.

The Series 2100 (Research), fixed-volume, the MaxipettorTM, the Repeater Pipette, and the Repeater Plus Pipette are not autoclavable.

Cleaning:
If not otherwise stated in the instruction manual, all parts of the pipette can be cleaned with a soap solution or isopropanol. The parts must then be rinsed with distilled water. Do not reassemble the parts until they have dried completely.

After cleaning, manual pipettes should be lubricated lightly using the special silicone grease included with the pipette. (To reorder: catalog no.: 22 34 851-5)

6 Checking for leaks

For pipettes with volumes 100 l and up:
To check for leaks, hold the pipette vertically for approximately 15 seconds with a filled tip. Do not touch the pipette tip. Observe the meniscus of the liquid in the tip. If there is a leak, a droplet is visible on the pipette tip.

For all volume pipettes:
Special equipment can be used to test for leaks in the system, such as the Brinkmann Instruments Universal Pipette Tester (catalog no:. 22 47 490-3).

7 Troubleshooting

(See instruction manual for further details)
Error Cause Solution
Droplets visible on the inside of the pipette tip - Uneven wetting of the plastic wall - Attach a new pipette tip
Pipette drips; incorrect pipetting volume - Tip is loose - Incorrect pipette tip Pipette leaks because: - the piston is contaminated - the piston is damaged - the seals are damaged - the nose cone is loose - Push tip on tightly - Use Eppendorf Pipette Tip - Clean the piston and lubricate lightly - Replace the piston and piston seals and lubricate lightly - Replace all seals and lubricate the piston lightly - Tighten the nose cone slightly using the key provided; replace the nose cone if necessary
Air bubble present in Combitip Plus; liquid cannot be aspirated correctly;
dripping occurs - Combitip Plus Tip is leaking - Replace the Combitip Plus Tip - Temperature of Combitip Plus Tip has risen - Ensure that the temperature is uniform as liquid expands when heated
Control button jams or does not move smoothly - Piston is contaminated - Seal is contaminated - Piston is damaged - Solvent vapors have entered device - Clean the piston and lubricate lightly - Disassemble the pipette; clean all seals and replace if necessary; lubricate the piston lightly - Replace the piston and piston seals and lubricate lightly - Unscrew the pipette at the central junction and air the pipette. Clean the piston and lubricate lightly

8 Adjustment

The pipettes have been calibrated by the manufacturer with bidistilled, degassed water in accordance with the Standards under the measuring conditions described in Section 2.

If the precision of the pipetted volume is questionable, consider the following points before readjusting the pipette:
- Has the pipette been checked for leaks? (see Sec. 7)
Exception: Positive Displacement Pipette (Model 4830). - Is the temperature of the liquid the same as:
- the temperature of the device? - the ambient temperature? - Is there a draft in the room? - Has the correct volume been set? - Have the density of the liquid and the air pressure been taken into consideration? - Does the density of the test liquid deviate from that of bidistilled, degassed water? - Is the mode of operation correct? (see Sections 3 and 4) - Have Eppendorf Pipette Tips been attached (pipette + pipette tip -> pipetting system)?
Volume errors may occur during the pipetting of liquids which have a high vapor pressure or a density which is significantly different to that of water.
Do not adjust the device until all of these points have been examined. 8.1 Adjusting pipettes (adjustable-volume)

The adjustment of adjustable-volume pipettes Series 2100 (Research), Model 4810, Series 2000 (Reference)
adjustable-volume, Positive Displacement Pipette (Model 4830),
and Titermate is a zero-point shift.
  1. In accordance with the Standards, Part 6; the temperature of the device, the Eppendorf Pipette Tip, the test liquid, and the ambient conditions must be the same (20 25 C constant to 0.5 C) during testing.
  2. Set the device to the smallest possible volume, which should not be less than 10% of the nominal volume.
  3. When using single-channel pipettes, attach an appropriate Eppendorf Pipette Tip to the pipette. With multichannel pipettes, the tip can be attached to any one channel.
  4. Pipette the volume ten times. The actual volume is calculated from the mean value obtained from these weighings (use the Z factor to convert mg into l).
  5. To adjust the device, insert the tool provided into the appropriate opening and set the actual volume (see Fig. 1 8 or consult the instruction manual).
  6. Check the volume which has been set by carrying out a measurement once again. If the nominal volume does not correspond with the result of the measurement, repeat points 2 5.
  7. After adjustment, check the accuracy of the measured values of the nominal volume and of 50% of the nominal volume.
8.2 Adjusting pipettes (fixed-volume)

Fixed-volume pipettes are adjusted in the same way as adjustable-volume pipettes (with fixed-volume pipettes,
10 measured values of the nominal volume are checked).

Adjusting the Series 2000 (Reference),
fixed-volume:
To facilitate relocation of the initial setting, attach the label provided (calibration aid) to the control button, as described in the instruction manual.
Using side B of the universal tool provided, loosen the inner screw until the control button can be rotated (see Fig. 1). Adjust the control button to the volume which has been obtained under point 4 (see instruction manual). One revolution of the control button is equivalent to the following (figures based on water):

Reference fix Volume/rotation 1, 2, 5, 10 l approx. 0.5 l 10, 20 l approx. 1 l 25, 50 l approx. 2.4 l 100 l approx. 5 l 200, 250 l approx. 12 l 500, 1,000 l approx. 46 l 1,500, 2,000, 2,500 l approx. 118 l
Adjusting the Series 2100 (Research), fixed-volume: To align to the volume displayed, insert side D of the wrench horizontally into the alignment opening (Fig. 2) in the pipette grip. (When doing so, hold the pipette vertically.) Rotate the wrench into a vertical position.
To reduce the volume, turn the setting ring clockwise. To increase the volume, turn the setting ring counterclockwise. One rotation is equal to the following:

Reference fix Volume/rotation 5 l approx. 0.8 l 10l approx. 0.8 l 20 approx. 0.8 l 25l approx. 4 l 50 l approx. 4 l 100 l approx. 4 l 200 l approx. 38 l 250 l approx. 38 l 450 l approx. 38 l 500 l approx. 38 l 1,000 l approx. 38 l
Remove the wrench and move the setting ring backwards and forwards until the counter and the stroke system lock together. Adjusting the Series 2000 (Reference), adjustable-volume: Turn the universal tool provided to set the digital indicator of the pipette to its actual volume (the measurement in point 4). The piston stroke should remain unchanged. See Fig. 3.
Remove the tool. Set the Series 2000 (Reference) to its nominal volume in the usual way (see instruction manual).

Adjusting the Series 2100 (Research single and multichannel) adjustable-volume: Insert side D of the key provided horizontally into the upper half of the calibration opening at the side of the pipette grip (see Fig. 4). Move the key into a vertical position.
When the setting ring is rotated (either in the + or - direction), the piston stroke of the pipette is altered (although the volume-setting dial remains unchanged).
One rotation is equal to the following:

Research, adjustable-volume 0.1-2.5 l approx. 0.1 l 0.5-10 l approx. 0.1 l 2-20 l approx. 0.8 l 10-100 l approx. 4 l 20-200 l approx. 4 l 100-1,000 l approx. 4 l 500-5,000 l approx. 38 l Research, multichannel 0.5-10 l approx. 0.5 l 10-100 l approx. 5 l 30-300 l approx. 10 l
After removing the key, move the adjusting ring to and fro until the digital indicator system and the stroke system click together (see instruction manual). Adjusting the Model 4810: Using a Phillips screwdriver (size 0), set the digital indicator of the pipette to the actual volume (the measurement in point 4). Make sure that the control button is fully pushed in. See Fig. 5.
Remove the screwdriver. Set the Model 4810 to its nominal volume in the usual way (see instruction manual).

Adjusting the Positive Displacement Pipette (Model 4830): Turn the universal tool provided to set the digital indicator of the pipette to its actual volume (the measurement in point 4). The piston stroke should remain unchanged. See Fig. 6.
Remove the tool. Set the Positive Displacement Pipette to its nominal volume in the usual way (see instruction manual).
Adjusting the Titermate: Using a Phillips screwdriver (size 0), set the digital indicator of the pipette to the actual volume (the measurement in point 4). Make sure that the control button is fully pushed in. See Fig. 7.
Remove the screwdriver. Set the Titermate to its nominal volume in the usual way (see instruction manual). Adjusting the ChemSaver Bottletop Dispenser: Adjust the ChemSaver Bottletop Dispenser by turning the fine adjustment (see Fig. 8). One rotation is equivalent to the smallest dispensing step.
Rotation of the button in the + direction: increase in volume.
Rotation of the button in the direction: decrease in volume.
(Factory-set adjustment at 20 C with degassed, bidistilled water; see instruction manual.)
8.3 Adjusting for liquids with a density different to that of bidistilled, degassed water It is possible to adjust the pipettes described on the previous pages for one volume of a liquid with a density different to that of water so that the volume displayed corresponds with the pipetted volume.

Caution: With adjustable pipettes, all other values are then adjusted as well!

The adjustment process is similar to the procedure described in Sec. 8.1 and 8.2. The difference is the mean value of the weighings is converted into microliters using the following formula:

Mean value of weighings Pipetting volume=
Density of liquid weighed
The resulting value is the actual value to which the volume display of adjustable-volume pipettes or the volume of fixed-volume pipettes is adjusted.

Caution: The value set for this particular liquid must be checked gravimetrically. A pipette set in this way only provides a value corresponding with the digital indicator display/volume ring for the liquid used and for the volume set. If necessary, the setting must be corrected and checked again.

After the pipette has been adjusted to another value, the device must be clearly identifiable (e.g. a label should be applied).

9 Factor Z for distilled water

Factor Z (l / mg) in accordance with ISO DIS 8655/3 as a function of the temperature and air pressure for distilled water

10 Technical specifications of Eppendorf pipettes

10.1 Fixed-volume pipettes

10.2 Adjustable pipettes





Liquid: Bidistilled water Reference temperature: 20 25 C, constant to 0.5 C Number of determinations: 10, in accordance with the Standards
with Eppendorf Pipette Tips



Liquid: Bidistilled water Reference temperature: 20 25 C, constant to 0.5 C Number of determinations: 10, in accordance with the Standards
with Eppendorf Pipette Tips 10.3 Multichannel pipettes Liquid: Bidistilled water Reference temperature: 20 25 C, constant to 0.5 C Number of determinations: 10, in accordance with the Standards
with Eppendorf Pipette Tips 10.4 Repeater Plus Pipette 10.5 Bottletop dispensers Liquid: Bidistilled water Reference temperature: 20 25 C, constant to 0.5 C Number of determinations: 10, in accordance with the Standards
with Eppendorf Pipette Tips 10.6 Error limits in accordance with DIN 12 650 In accordance with DIN 12 650, the error limits are always based on the overall system of pipette and tip together. The error limits of pipettes with a nominal volume between the nominal volumes given in the table must correspond to the relative error limits for the next-highest nominal volume. If the nominal volume of the pipette is exactly between two nominal volumes given in Table 1, the relative error limit for the lower volume is applicable.

The error limits in accordance with the latest version of the DIN 12 650 standard and based on volume are contained in the following tables: The error limit in accordance with the DIN standard is defined by F = IAI + 2 s, whereby:
F = Error limit |A| = Systematic measurement deviation
(Deviation of the mean value of 10 measurements of the nominal volume = Inaccuracy) s = Random measurement deviation
(Repeated standard deviation of the 10 measurements = Imprecision)

Reference sources for pipetting standards

Information concerning proper pipetting technique and gravimetric testing procedure can be obtained by requesting copies of ISO (International Standard Organization) or DIN (Deutsche Industrial Norm) Standards from the American National Standard Institute (ANSI).

Request designation number ISO 8655 or DIN 12 650
ANSI
11 West 42nd Street
New York, NY 10036
Telephone: (212) 642-4900; Fax: (212) 398-0023
E-mail: info@ansi.org

Also, the ASTM Standard can be requested by contacting:
ASTM (American Society for Testing and Materials)
100 Barr Harbor Drive
West Conshohocken, PA 19428-2959
Telephone: (610) 832-9585; Fax: (610) 832-9555
E-mail: service@astm.org

Request designation number E 1154-89 Standard Specification for Piston or Plunger Operated Volumetric Apparatus.


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... is the smallest electronic pressure controller available ... Parker configured this unit specifically for the ... Used in carrier gas flow control, microfluidic ... and hydro-dynamic focusing, the OEM-EPC offers external ...
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