Gilson guide to pipetting Gilson guide to pipetting
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LT303409-01 Copyright © 2005 Gilson, Inc. Allrights reserved.Printed in the USA.
Second Edition Second Second Edition Louis Pasteur the father of modern biology 1822-1895
The next significant improvement on pipettes came in the late 50’s with the introduction of a hand-held, piston-operated pipette as a safe alternative to potentially dangerous mouth pipetting. The first hand-held pipettes had a pre-established volume setting (fixed volume pipettes). Further improvement came with the introduction of a more flexible, stepper volume setting (variable volume pipettes). Pipetting since Louis Pasteur In 1972, Dr. Warren Gilson introduced a high precision pipette which could be adjusted for any volume within its range… even fractional (continuously adjustable). Its originality… to avoid error, the selected volume was clearly displayed on the Gilson pipette (direct digital readout). Twenty-five years later, Gilson precision pipettes are still the world standard for accuracy, precision and reliability.
Photo courtesy of the Institut Pasteur.
Pipette guiderev11012006.indd 1 18/07/2007 16:45:08 Chapter 1 Selecting the best pipette for your application
Consider the physical properties of your sample ------17 n Aqueous samples n Viscous samples n Radioactive or corrosive samples n DNA, RNA, biological samples
The working principle of air-displacement and of positive-displacement pipettes - - 18 Learning more… All air-displacement pipettes are not the same Learning more… Mistake-free pipetting
Consider the volume of liquid you want to transfer ------14 n Volume range of Pipetman and Pipetman Ultra n Volume range of Microman n Volume range of Distriman Learning more… How do I read the volume?
Chapter 2 Selecting the right tip for your pipette
Evaluating tips ------17 Learning more… More than meets the eye
Choosing the best tip for your application ------18 n Racked tips n Sterilized tips n Filter tips Learning more… How to prevent aerosol contamination?
Chapter 3 Correct operation
Organize your work station ------21
Fit a disposable pipette tip ------22 Learning more… How to mount tips on a Pipetman Ultra multichannel pipette?
Adjust the volume setting ------24
Follow the instructions for forward or reverse mode pipetting ------25 Learning more… Pre-rinsing
Eject the used tip and store the pipette in an upright position ------32 Learning more… Why do I need a pipette holder?
Pipette guiderev11012006.indd 2 18/07/2007 16:45:08 Chapter 4 Preventing contamination
Types of contamination and how to prevent them ------35 n Personal exposure n Pipette-to-sample n Sample-to-pipette n Sample-to-sample
Decontaminating your Pipetman ------36
Chapter 5 Caring for your pipette
Quick Diagnosis • The Pipetman Two-minute Inspection ------39 Learning more… How old is my pipette? Learning more… How can I prevent piston corrosion? Learning more… Tips do not fit well. What should I do? Learning more… What are the main causes of leaks? Learning more… How do organic solvents cause leaks?
Repair in the lab or return for service? ------43
Chapter 6 Control and calibration
What are published specifications? ------45 Learning more… What is accuracy? What is precision?
How to calculate volumetric accuracy and precision ------46
Using the gravimetric method ------47
Performance check procedure ------49
When to perform the test ------49
Appendix APPENDIX I • Example of a performance check ------52
APPENDIX II • Z factor ------53
APPENDIX III • Evaporation loss ------54
Pipette guiderev11012006.indd 3 18/07/2007 16:45:08 Pipette guiderev11012006.indd 4
Air-displacement pipette PIPETMAN number Serial AA50001 Identification mark Filter (also known as tip holder) tip as known (also volume adjustment ring) adjustment volume • buttonPush • (also known as fine as known (also Volumeterdisplay Tip ejector buttonejector Tip adjustment knob adjustment Largevolume Disposable tip Disposable Thumbwheel Tip ejector Tip Shaft
Sealing Level mark ring 18/07/2007 16:45:10 Pipette guiderev11012006.indd 5 capillary • • Volumeknob adjustment buttonPush • and piston and Ejector of capillary of Ejector capillary and piston and capillary Volumeterdisplay capillary holder) capillary (also known as known (also Disposable Shaft
piston number Serial
AA50002 18/07/2007 16:45:11 MICROMAN positive-displacement pipette Selecting the best pipette for your application
Today, there is a pipette for virtually every requirement... aqueous solutions, dense, viscous, or radioactive
compounds, DNA sequencing, Aqueous samples distributing aliquots, filling microtiter plates, etc.
The type of analysis you are performing, the physical Viscous properties of the liquid and samples the volume range of the pipette will determine which pipette Radioactive and corrosive samples and tip you should use. Microman pipettes are specially designed to handle aggressive liquids without danger for the user or damage to the instrument. Microman
Radioactive corrosive… samples
DNA - RNA biological samples Chapter Chapter 1 6
Pipette guiderev11012006.indd 6 18/07/2007 16:45:13 1.1 Consider the physical properties of your sample Chapter Chapter 1
Aqueous samples Ultra
Correctly operated and maintained, Pipetman® PUM pipettes will give precise results with most Pipetman Distriman
liquids commonly found in the laboratory. If you Pipetman have a large number of fractions to dispense, use a Distriman™ repetitive pipette.
Viscous samples You may find it easier to use a Gilson Microman® pipette. Microman gives reproducible results with
highly viscous samples like glycerol, detergent Microman and honey. Microman is also recommended for highly volatile (chloroform) or dense liquids (mercury) which are often difficult to aspirate.
Radioactive and corrosive samples Microman pipettes are specially designed to handle aggressive liquids without danger for the user or damage to the instrument. Microman
Radioactive corrosive… samples
DNA, RNA, biological samples PUM The choice is yours. You can use either a ® Pipetman
Pipetman with Diamond autoclavable filter Pipetman tips (0.1 µl - 1 ml) or an autoclavable Microman (Model M10 and M100) with sterilized capillaries and pistons. Properly used, these instruments will assure precise, contamination-free results. For multiple dispensing, opt for a ™ Distriman Distriman repetitive pipette with a DistriTip Microman gamma-sterilized micro syringe.
Pipette guiderev11012006.indd 7 18/07/2007 16:45:19 1 .2 The working principle of air-displacement and of positive-displacement pipettes
The most important difference between Pipetman, Microman and Distriman is their working principle.
Ultra Pipetman (single and multi-channel)
PUM are air-displacement pipettes. Pipetman Pipetman Pipetman
Microman and Distriman are positive-displacement pipettes. Distriman Microman
Important Piston perfect When you set the volume on an air-displacement pipette, the piston regulates the volume of the air cushion, which, in turn, determines the volume of liquid which is to be aspirated. The piston has to be perfect if the sample volume is to correspond exactly to the selected volume. That’s why Pipetman pistons are examined individually to make sure there are no flaws. They are even cleaned individually to make sure there are no dust particles.
Since the piston is a permanent part of Pipetman pipettes, a perfect piston means perfect measurement every time.
Pipette guiderev11012006.indd 8 18/07/2007 16:45:20 Chapter Chapter 1
What are What are air-displacement pipettes? positive-displacement pipettes?
Three things to remember Three things to remember 1 Recommended for aqueous samples and 1 Recommended for problem samples for general laboratory work (viscous, dense, volatile, radioactive, corrosive) 2 Always have a cushion of air (dead volume) between the pipette piston and 2 Direct contact of the piston with the sample the liquid sample (no air cushion) 3 The piston is a permanent part of the 3 Disposable piston (not a permanent part of pipette the pipette)
disposable piston disposable capillary
air cushion disposable tip
Pipette guiderev11012006.indd 9 18/07/2007 16:45:21 Set 1 the volume The required volume is set. The piston moves to the appropriate How do air-displacement pipettes work? position.
When the push-button is pressed on an air-displacement pipette, the piston inside the instrument moves down to let air out. Air is displaced by the piston. The volume of air displaced is equivalent to the volume of 1 liquid aspirated. 0 0 The schematic drawings (on the right) show how the piston determines the volume of air displaced and subsequently the volume of sample aspirated.
Set 1 the volume The required volume is set. The piston moves down to the appro- How do positive-displacement pipettes work? priate start position.
Positive-displacement pipettes work like a syringe. There is no air cushion between the disposable piston and the sample. With no elastic air cushion to expand or contract, the aspiration force remains constant, 1 unaffected by the physical properties of the sample. 0 0 This allows the Microman operator to pipette very viscous or high density samples, such as mercury or toothpaste.
Pipette guiderev11012006.indd 10 18/07/2007 16:45:22 Prepare Aspirate the Dispense the 2 for aspiration 3 sample 4 sample The push-button is pressed prior As the push-button is released, a The push-button is pressed again. to sample aspiration. The piston partial vacuum is created inside Air pressure increases inside the How do air-displacement pipettes work? descends and expels a volume of the tip. The ambient atmospheric shaft and the tip. The compressed air equal to the selected volume pressure forces the desired vol- air pushes the liquid out of the Chapter Chapter 1 of liquid. ume of liquid through the orifice tip. into the tip.
Prepare Aspirate the Dispense the 2 for aspiration 3 sample 4 sample The push-button is pressed prior The orifice is then immersed The push-button is pressed to sample aspiration. The piston below the liquid surface. As the again. The piston moves down descends down to the end of the push-button is released, the pis- and expels the liquid out of the How do positive-displacement pipettes work? capillary. ton moves up and the ambient capillary. pressure forces the desired vol- ume of liquid through the orifice into the capillary.
Pipette guiderev11012006.indd 11 18/07/2007 16:45:24 12 Pipette guiderev11012006.indd 12
Learning more performance less sensitive to to sensitive makes variations in temperature and liquid less sample and performance piston the Min P10.Model exceptional 2 µl for Model P2 and 0.5 to 10 µl for provide to 0.1 from precision, and accuracy tiny, models Pipetman require two samples, valuable, that procedures sequencing DNA as such applications For same the not are pipettes air-displacement All of leaks and contamination. and leaks of G cross-contamination. or contact piston, Sele density.and pressure vapor as such properties rease-free Pipetman also introduce contaminating particles into the body of the pipette. the of body the into particles contaminating introduce also can Grease leaks. of cause another pipetted… liquids from vapors by altered be Greased pistons must be regreased regularly or leaks may occur. The grease may most pipettesUnlike with greased pistons, Pipetman uses “dry seal” technology. mm i space air imum ct a pipette with a protected To grease or not to grease? to Tonot or grease so there is no risk of sample and biochemical biochemical and You should never grease a Gilson requires less maintenance, reduces the risk between between Important
with a protected piston protected a with tip holder piston seal disposable tip enclosed piston Pipette Pipetman.
18/07/2007 16:45:24 ex disposable p osed pist piston se tip hold o t e Pipette guiderev11012006.indd 13
Learning more pipetting Mistake-free They are ejected automatically so… automatically ejected are They changedeachafterbe sample. may piston and Tocapillary the contamination, prevent glue. to glycerol from sample, viscous any almost of dispensing accurate assuring wall, capillary internal the wipes piston the dispensing, During instrument. the entering from aerosols corrosive or radioactive biohazardous, prevents seal piston The completely. aspirated is volume desired The samples. of temperature the or density the to response in contract or expand to cushion air no is There sample. the with (positive-displacement) contact direct into comes piston the pipettes, Microman With … you do not have to touch them.
13 Chapter 1 1.3 Consider the volume of liquid you want to transfer
The eight models of the Gilson Pipetman and Important Pipetman Ultra are designed to transfer precise volumes from 0.1 µl to 10 ml. Pipetman is also The model and the useful volume Pipetman available in fixed volumes from 2 µl to 1 ml. range on Pipetman, Pipetman Ultra and Microman model volume range pipettes are shown P2 / U2 0.2 - 2 µl* on the top of the push-button Ultra P10 / U10 1 - 10 µl* P20 / U20 2 - 20 µl P100 / U100 20 - 100 µl
Pipetman Pipetman P200 / U200 50 - 200 µl* / 20 - 200 µl P1000 / U1000 200 - 1000 µl P5000 / U5000 1 - 5 ml P10ml / U10ml 1 - 10 ml
* With a precise pipetting technique P2 and U2 may be used to aspirate volumes down to 0.1 µl, P10 and U10 at 0.5 µl, and P200 at 30 µl.
The six models of the Gilson Microman speciality pipettes are designed for the precise transfer of problem liquids in volumes from 1 µl to 1000 µl. Microman model volume range M10 1 - 10 µl M25 3 - 25 µl M50 20 - 50 µl M100 10 - 100 µl Important M250 50 - 250 µl M1000 100 - 1000 µl The total volume that can be aspirated is shown on the side of each Distriman syringe The Distriman repetitive pipette is available with three different syringes (micro, mini and maxi), so you can distribute aliquots in any volume (even Distriman fractional) from 1 µl to 1.25 ml.
syringe volume range (total syringe volume) of aliquots Micro (125 µl) 1 to 12.5 µl Mini (1250 µl) 10 to 125 µl Maxi (12.5 ml) 100 µl to 1.25 ml 14
Pipette guiderev11012006.indd 14 18/07/2007 16:45:29 Pipette guiderev11012006.indd 15
Learning more Q A Pipetman P200 Pipetman P20 Pipetman volumeterdigital the on shown is volumeThe volume? the read I do How Pipetman P1000 Pipetman 200 µl 50 µl 2 µl MIN. MIN. MIN. 0 5 0 2 0 0 0 2 0 0 5 12.5 µl 750 µl 125 µl INT INT INT 2 1 5 7 0 5 2 1 . . .
1000 200 µl 20 µl MA MA MA 2 0 0 1 0 0 0 2 X X X . . . µ l
models P20, P200 and P1000. and P200 P20, models pipettes, Pipetman used commonly most the from taken Examples manual. instruction your consult models, other the on correspondancecorrect the For P1000. the on (ml) milliliters and P20 the on (µl) microliters of tenths represent red in numbers The ofintermediate volume settings (INT.). (MAX.)volume settings. leftshows theminimum (MIN.) andmaximum areread from toptobottom. Thefigure onthe Thevolume isdisplayed asthree digits which Pipetman the for settingsvolume of Examples
Italso shows examples
15 Chapter 1 Selecting the right tip for your pipette
Micropipettes such as Guaranteeing manufacturing quality Pipetman and Microman must Every Diamond® precision tip always be used with a suitable is individually marked with the Gilson logo and with an tip attached. Tips for Microman identification number. are also known as “capillaries”. With this number, it is possible to identify the mold and even Tips for Distriman are also to locate the exact cavity which known as “syringes”. produced the tip.
Used correctly, all of these pipettes provide guaranteed accuracy and precision provided that proper Gilson tips are used. Safety first! Gilson capillaries and pistons for Microman Guaranteeing traceability are unbreakable. The risk of The batch number on every personal injury associated box and bag makes it possible with glass capillaries is totally to trace the itinerary of the tips from packaging to delivery to eliminated. the laboratory.
QUAL ED IT I Y IF T Sterilized T I R filter tips P E S C DF10ST
S I BATCH No. A P I M T O N ND IO PRECIS Chapter Chapter 2 16
Pipette guiderev11012006.indd 16 18/07/2007 16:45:31 2.1 Evaluating tips Chapter Chapter 2
Although they may look alike, all tips are not More than meets the same. The choice of a poor quality tip will jeopardize your results. Prefer a tip recommended the eye by the pipette manufacturer and always check the following points: These photos taken under the microscope show Tip Evaluation Sheet some of the differences Physical Aspects yes no between good and poor Clean and free of dust particles? . . . . . quality tips. Perfectly formed collar? ...... (for air-tight fit) Smooth and regular surface? ...... Learning more (minimal retention of liquid) Fine point finish? ...... (prevents formation of droplets) Translucent ...... (no contaminating additives or dyes) Chemical resistance ......
Tip manufacturer’s Guarantee yes no perfect point Brand marked on the tip? ......
Identification number on the tip? ......
Batch number on the box? ......
Quality certificate? ......
imperfect rough excess Important point surface plastic aperture Gilson Diamond tips are supplied with typical values for trace metal release. These concentrations are lowered to the analytical noise level after 5 to 10 rinsings with concentrated acid.
Pipette guiderev11012006.indd 17 18/07/2007 16:45:31 2.2 Choosing the best tip for your application
Gilson tips are available in a variety of formats:
n Loose in bulk packaging An economical solution for routine. May be hand loaded in empty tip racks for convenience or for autoclaving in the laboratory.
n Racked for easy mounting with no hand contact Hinged lids protect against dust. Convenient 96-well format for filling microtiter plates with a multichannel Pipetman. Color-coded for easy identification. Ready for autoclaving in the laboratory. Tip-racks may be reused.
n Racked and pre-sterilized for working in sterile conditions Factory irradiated and delivered in a sealed tip rack.
n Racked and with autoclavable filter Tips with a filter prevent contaminating aerosols from entering the pipette. Gilson Diamond filter tips can be successfully autoclaved in the laboratory.
n Racked and pre-sterilized with filter Factory irradiated filter tips delivered in a sealed tip rack.
n Individually wrapped and pre-sterilized Opened just before use so the benefit of sterilization is assured right up to the last minute. A good solution when you only need a few tips.
individually wrapped 18
Pipette guiderev11012006.indd 18 18/07/2007 16:45:32 Pipette guiderev11012006.indd 19
Learning more solutions: two offers Gilson etc.samples, materials,radioactive infectious solutions, DNA/RNA pipetting areyou when or methods, amplification other and PCR using youare when preventcontaminationtoaerosol indispensable is It contamination?aerosol preventto How Microman Pipetman For more information, see page 35. page see information, more For • • • 2 • • • 1 avoiding cross-contamination. avoiding samples aqueous pipetting conditions sterile in working avoiding cross-contamination. avoiding samples viscous pipetting conditions sterile in working
Use a Microman pipette with pre-sterilized pipettewith Microman a Use time: same the at followingproblems the of two least at face haveto you when tips filter sterilized pre- Diamond pipettewith Pipetman a Use at the same time: same the at followingproblems the of two least at face haveto you when pistons and capillaries
19 Chapter 2 Correct operation
To optimize your results, the Take a few minutes to get organized pipette is important but don’t • Adjust your chair or stool so forget to use it properly. that the work surface is at the right height when you are sitting 1 First organize your work straight station for maximum • If possible, always try to work with your hands below shoulder height efficiency and minimum • Try to evaluate if you can reduce fatigue. the height of applications such as gel loading 2 Make sure the tip is properly • Adjustable tables/workbenches are mounted and fits well a good solution before you set the volume.
Important 3 Adjust the volume. A good test is to see 4 Choose the mode of if you can rest your elbow pipetting (reverse or forward comfortably on the work surface mode) adapted to your Recipient at right sample. height
5 Eject the used tip and store the pipette in an upright
position to avoid damage Recipient too high and cross-contamination.
Recipient too low Chapter Chapter 3 20
Pipette guiderev11012006.indd 20 18/07/2007 16:45:33 3.1 Organize your work station Chapter Chapter 3
Take time to relax
• If possible, try to switch periodically between different types of work.
• Keep an appropriate, unrushed working speed. Let go of the pipette from time to time and give the fingers/hand a (micro) break.
• Take frequent short breaks. Change your sitting position. Lean back and relax your shoulders and arms.
Special attention should be paid to smooth pipetting
• To favor uniform timing and motion, have all necessary objects within easy arm’s reach
• Place the most frequently used objects in front of you. The more rarely used items can be placed a little further away from you
• The opening of the recipient for used tips should be at the same height as the end of your pipette 21
Pipette guiderev11012006.indd 21 18/07/2007 16:45:34 3.2 Fit a disposable pipette tip
To fit a disposable tip on a Pipetman, hold Fitting a disposable tip the micropipette in one hand and use a slight on Pipetman twisting movement to seat the tip firmly on
Pipetman the shaft of the micropipette and to ensure an air-tight seal. Diamond tips for Pipetman are available in TIPACK racks for easy mounting with no hand contact.
To mount DistriTips on a Distriman, consult the instruction manual. Distriman
Press down with Avoid stabbing the rotation motion tip as though the pipette were a knife
To fit a capillary and piston on a Microman, press the plunger button to the second stop. The jaws on the pipette will open
Microman automatically and seize the piston, mounting the capillary and piston in the correct position. For maximum protection against contamination, capillaries and pistons for Microman are available pre-assembled, racked and pre- sterilized.
To fit tips on a Pipetman Ultra 8X300
PUM multichannel pipette. When you are using a multichannel pipette with a standard flat-bed tip rack, it is not always easy to mount all eight tips at the same time. You have to push down Important hard, or even hammer and pound. The patented Always choose a tip recommended by G-F.I.T. system™ provides a firmer fit of the Gilson Diamond® Tips and most standard tips the pipette manufacturer (see page16). while decreasing the force to fit and eject, pick- up is fast, and tips won't fall off. Important Never attempt to use a micropipette without a suitable tip attached.
Pipette guiderev11012006.indd 22 18/07/2007 16:45:36 Pipette guiderev11012006.indd 23
Learning more steady level around 17N. aroundlevelsteady a forceremainsat ejection the regulartip-holders of that of half as much as by G-F.I.T.System the using tips Diamond eject forceand requiredThe fit to tips. standard most with G-F.I.T.System pipetttes.Ultra Pipetman forforces ejection low and seals reliable providesinstant G-F.patented The I.T.System™ multichannel pipette? multichannel UltraPipetman a on tips mount to How reliable seal reliable instant provides 1 stop a and guide a as works 2 st nd ridge: instant ridge: Small contact areas areas contact Small low
+ = can be reducedbe can is compatible is Tip
. N,Above 40 forces seals
areas contact Small
rocky movement to secure the rockysecure movementto followed by a slight lateral slight followeda by Exert a light vertical force vertical light a Exert tip fitting tip 18/07/2007 16:45:41
23 Chapter 3 3.3 Adjust the volume setting
To adjust the volume Hold the body of the micropipette in one hand and use the other hand to rotate the thumbwheel - or the push-button. With the push-button, volume can be easily adjusted with one hand. Push-button volume adjustment is available on all Microman pipettes and on Pipetman pipettes manufactured after April 1995.
A helpful hint for improving reproducibility Always finish setting clockwise. In this way, if any mechanical backlash should occur, it will always be in the same position. Here’s how to obtain a clockwise volume setting: • when decreasing the volume setting, turn the thumbwheel slowly until the desired setting is displayed. Do not overshoot the required position, Thumbwheel • when increasing the volume setting, rotate the thumbwheel approximately 1/3 of a turn above Push-button the desired setting, and then slowly turn back to decrease the volume until you reach the desired setting.
A helpful hint for improving accuracy
1 1 1 1 0 0 0 0 0 0 0 The electronic0 patented multifunctional LCD of the Pipetman Ultra offers greater readability and zero risk of parallax errors.
Important To avoid internal damage Incorrect alignment : Correct alignment : to your pipette, never attempt error accurate reading To avoid parallax errors, make sure that the volume to force the volume setting indicator and the volume marking selected are in beyond the limits shown in the your direct line of vision. At close range, you may tables on page 14. find it helpful to close one eye. 24
Pipette guiderev11012006.indd 24 18/07/2007 16:45:42 3.4 Follow the instructions for forward or reverse mode pipetting Chapter Chapter 3
The choice of operating mode can Pre-rinsing significantly affect the results of an analysis. Greater uniformity and Gilson pipettes are calibrated for forward mode precision of dispensing are pipetting. Manufacturers should list all pertinent usually obtained by providing modes of operation and specify for which mode identical contact surfaces for the instrument is calibrated. all aliquots. This is achieved The forward mode is the usual way of by pre-rinsing with the same pipetting with an air-displacement pipette liquid that is being dispensed. like Pipetman. To pre-rinse, aspirate with the See page 26. tip and then dispense back
Learning more into the original reservoir or The forward mode for positive-displacement to waste. pipettes like Microman eliminates the purge stroke. See page 30.
The reverse mode is only possible with air-displacement pipettes. It is used for solvents or slightly viscous liquids. Q When should I pre-rinse See page 28. the tip?
Wiping If necessary (viscous liquids such as cream), wipe the APre-rinsing should be outside of the tip or the capillary with a clean medical wipe. Do not touch the orifice. Choose a tissue which performed is resistant, lint-free, and inert to acids and solvents. • every time you change a tip Dispose of the tissue in a safe, hygienic manner. • every time you increase the volume setting Important When working with high risk Don’t forget to pre-rinse specimens, do not wipe • capillaries and pistons for the disposable part. Make sure fluid Microman depth penetration does not exceed • DistriTips for Distriman 3 mm. Then touch-off from the sidewall of the vessel.
Pipette guiderev11012006.indd 25 18/07/2007 16:45:42 Air-displacement / Forward mode
In general, the precision of the forward mode relies on precise 1 Preparation 2 Aspiration draining by air pressure Hold the instrument in a nearly Immerse the pipette tip in the (air-displacement pipetters) or vertical position. Depress the liquid*. Allow the plunger to internal wiping of the pipette plunger smoothly to the first move up smoothly to the rest barrel (positive-displacement stop position. position. Wait one second so that pipetters). all the liquid has time to move up into the tip.
second stop or purge
* The immersion depth of your tip can have a significant effect on your results. If the tip is immersed too deeply, droplets will form on the outside of the tip and they will be deposited along with your sample. If the tip is not immersed deeply enough, vortexing will occur and your pipette will not aspirate the selected volume.
volume immersion depth µl mm 0.1 - 1 1 1 - 100 2-3 101 - 1000 2-4 1001 µl -10 ml 3-6 26
Pipette guiderev11012006.indd 26 18/07/2007 16:45:43 Air-displacement / Forward mode Chapter Chapter 3
3 Distribution 4 Purge 5 Home Place the pipette tip at an Wait one second, then depress Allow the plunger to move up angle (10 to 45°) against the the plunger to the second stop to the rest position. inside wall of the receiving position. This “blow-out” stroke vessel. Depress the plunger removes any remaining sample smoothly to the first stop from the tip. Remove pipette position. tip end from sidewall by sliding it up the wall.
Pipette guiderev11012006.indd 27 18/07/2007 16:45:44 Air-displacement / Reverse mode
In reverse mode pipetting, the purge stroke is used during 1 Preparation 2 Aspiration preparation. During aspiration, Hold the instrument in a nearly Immerse the pipette tip in the an amount of liquid equal to the vertical position. Depress the liquid*. Allow the plunger to amount of purged air is added. plunger smoothly to the second move up smoothly to the rest This amount compensates for stop position. position. Wait one second so that the liquid that remains as film all the liquid has time to move up inside the tip during dispensing. into the tip.
second stop or purge
* See page 26. 28
Pipette guiderev11012006.indd 28 18/07/2007 16:45:44 Air-displacement / Reverse mode Chapter Chapter 3
3 Distribution 4 Re-aspiration 5 complete purge Place the pipette tip at an If the pipette tip is to be reused Wait one second and purge. If angle (10 to 45°) against the for the same sample, maintain the pipette tip is not to be inside wall of the receiving the plunger in the intermediate re-used, depress the plunger to vessel. Depress the plung- position for subsequent immer- purge position over an appro- er smoothly to the first stop sion for the next pipetting cycle and priate waste container and then position. Wait one second. restart operation 2. eject the tip.
Pipette guiderev11012006.indd 29 18/07/2007 16:45:45 Positive-displacement
In positive displacement pipettes, the piston enters into 1 Preparation 2 Aspiration direct contact with the liquid; Press the plunger button to the Immerse the capillary/piston in there is no air interface. first stop. The piston moves to the liquid*. Release the plunger the appropriate position. letting it move up to the home Direct contact enhances position. The piston moves up and accuracy and precision for the ambient pressure forces the liquids which are too heavy desired volume of liquid through or too viscous to be displaced the orifice into the capillary. by air. Direct contact allows aspiration of volatile liquids without evaporation. In addition, the absence of air permits rapid pipetting without cavitation.
* See page 26. 30
Pipette guiderev11012006.indd 30 18/07/2007 16:45:46 Positive-displacement Chapter Chapter 3
3 Distribution 4 Ejection Press the plunger button to the Press the plunger all the way first stop. The piston moves down down to the second and last and expels the liquid out of the stop. Capillary and piston are capillary. ejected without hand contact.
Important The piston and capillary are the volumetric components of positive displacement pipettes. As both parts are in contact with liquid, they must both be replaced frequently to avoid cross- contamination. 31
Pipette guiderev11012006.indd 31 18/07/2007 16:45:46 3.5 Eject the used tip and store the pipette in an upright position
To avoid touching contaminated tips, hold the pipette over the trash can and Important press the tip ejector push-button. Discarded tips contain liquid residues, particularly when a pipette is used in the reverse mode. Take suitable precautions when discarding disposables.
Autoclavable bags are available for collecting, autoclaving and/or eliminating biohazardous waste. Special bags and anti-beta containers are also available for solid and liquid radioactive waste.
PIPETMAN MICROMAN no-touch no-touch disposal of disposal of tip capillary and piston Always eject used tips. The residual liquid can When to change a tip damage your pipette. Repetitive dispensing of samples. For repetitive dispensing of the same liquid (diluent, buffer or reagent), use the same pipette tip. This method is economical and efficient. It is advisable to pre-rinse the tip at the beginning of the test series.
Transferring single samples of different liquids. Select a new pipette tip for each new liquid. It is recommended to pre-rinse every new pipette tip. 32
Pipette guiderev11012006.indd 32 18/07/2007 16:45:46 Chapter Chapter 3 Q Why do I need a pipette holder?
ATo prevent corrosion, contamination and breakage Learning more
Do not leave your pipette lying on the workbench where it can come into contact with chemicals or fall off and break.
Always store your pipette vertically to prevent liquids from running inside the shaft of the pipette.
Pipette guiderev11012006.indd 33 18/07/2007 16:45:46 Preventing contamination
Avoidance of contamination in Personal exposure the laboratory requires the use n of strict precautions. Among Prevention
these are decontamination • Wear a lab coat.
of pipettors, wearing gloves • Wear gloves.
and choosing an appropriate • Wear protective glasses.
pipette tip. • Wear a mask.
• Wipe work bench The importance of these before and after with an appropriate cleaner for precautions is evident when your application we consider the extreme (cell culture, radio-active components, pathogenic sensitivity of modern samples…).
techniques such as PCR, • Work under hood.
which allows detection of a • Work behind a radioactivity single molecule. We must also shield. bear in mind the dangers of • Avoid touching used tips. radioactivity and the risk of • Use unbreakable capillaries and pistons. personal contamination from a pathogenic micro-organism. Chapter Chapter 4 34
Pipette guiderev11012006.indd 34 18/07/2007 16:45:47 4.1 Types of contamination and how to prevent them Chapter Chapter 4
Pipette-to-sample Sample-to-pipette Sample-to-sample (also known as Contaminated tips or a Contamination can occur if sample carry-over) contaminated pipette will, in the sample or aerosols from turn, contaminate samples. the sample are allowed to A portion of sample A can enter the body of the pipette. adhere to the inside wall of n the tip after sample delivery. Prevention n The left-over portion of Prevention • Use sterilized tips and sample A can mix with the clean or autoclave the • To prevent liquids from next sample (B) and may parts of your pipette which running into the pipette cause a false test result. are in contact with the body, avoid inclining sample (see pages 36-37). your pipette excessively n Prevention and always store the • Change the tip after each instrument vertically. Change the tip after each sample. sample. •Release the push-button slowly.
• To prevent aerosol Microman contamination, use filter Pipetman Pipetman with with with capillary tips with Pipetman or standard tip filter tip Sample carry-over and piston choose a Microman positive-displacement pipette with built-in aerosol barrier.
protected air space
Piston Filter seal aerosol barrier
non-protected air space (aerosol) left-over Contamination sample A of sample B inside the tip
Risk of aerosol No risk of aerosol contamination contamination 35
Pipette guiderev11012006.indd 35 18/07/2007 16:45:47 4.2 Decontaminating your Pipetman
If your pipette is potentially contaminated, one of the following procedures should be carried out before further use or maintenance.
Autoclaving n This is the most usual means of sterilization. Gilson Diamond tips and certain parts of Pipetman pipettes may be sterilized in the laboratory under the following conditions : moist heat/121°C/20 minutes/1 bar.
Note : Autoclaving has a limited spectrum of action and will not destroy RNase for example.
Autoclavable parts of Pipetman and Pipetman Ultra n The Pipetman and Pipetman Ultra lower parts (light blue) can be autoclaved.
IMPORTANT: The piston assembly (dark blue) of Pipetman cannot be autoclaved.
Pipette guiderev11012006.indd 36 18/07/2007 16:45:48 Pipette guiderev11012006.indd 37 Ethylene oxide and its reaction product ethylene chlorohydrin are highly toxicchlorohydrin ethylenehighly product are reaction its and oxideEthylene n Ethyleneoxide : Note disposables. of quantities large of producers for reserved is irradiation installations, special requiring and Costly disposables. pipette manufacture to used plastics “sterilized“.inert relatively the for effective highly are rays penetrating The label the under sold products for manufacturers by used is method This n ( Beta pipette. the inside penetrate cannot rays UV vapors, or liquids contaminating unlike because, pipettesminutes. for 15 recommended not is method This for light UV 300nm to exposure by decontaminated be may Worksurfaces n radiation UV : Note thoroughly.dry to and water with pipette the rinse to recommended strongly is It solution. fungicidal and bactericidal virucidal, a in immersed are parts the Next, instruments. laboratory for recommended detergent a with bath ultrasonic an in immersed service. fully and dismantled is Pipetman The for returned pipettes decontaminating for Gilson by used techniqueis This n cleaning Chemical PCR is a trademark of Hoffman LaRocheHoffman of trademark a is PCR * irradiation. by altered be whichmay materials plastic of sterilization mutagenic. the and for reserved is it Therefore A. Prince and L. Andrus, PCR : How to kill unwanted DNA, BioTechniquesunwantedDNA, kill to How : PCR L. Andrus, and Prince A. manufacture the pipette or the disposable. the or pipette the manufacture The choice of gamma or beta rays is determined by the type of plastic used to used plastic of type the by determined is rays beta or gammachoice of The liquid can counter every risk. every counter can liquid single no risk, given a counter to properties right the with liquid a find to possible always is it However,while laboratory*. PCR the in templates DNA of elimination 10%Johnson).Clorox and A 2% formaldehyde solution is a common choice(CIDEX common a is solution formaldehyde 2% A b ) or gamma ( gamma or ) ®
g has been recommended as a decontaminant for decontaminant a as recommended been has ) radiation ) ® from Johnson from 1992 , 12 , 359-360. , 18/07/2007 16:45:48
37 Chapter 4 Caring for your pipette
For long life and optimum Step 1 Check the records performance, pipettes should be returned for complete Step 2 service once a year. Assess pipetting functions If you do encounter a minor technical difficulty, chances are Step 3 Leak test very good that the malfunction can be repaired right in your Step 4 own laboratory. Disassembly
Warning: use only genuine Step 5 Reassembly Gilson replacement parts available exclusively from your authorized Gilson representative.
The Pipetman Two-minute Inspection will help you diagnose faults and decide whether the pipette should be
repaired in your lab or returned Important to your representative for service.
Never handle an unknown pipette without wearing gloves. It could be contaminated. Chapter Chapter 5 38
Pipette guiderev11012006.indd 38 18/07/2007 16:45:48 Pipette guiderev11012006.indd 39 • • • • must keep detailed records of every step of step every of records detailed keep must GLP (Good Laboratory Practice) laboratories KeepingRecord servicing. last of daterecords the for n n recordsthe Check 1 Step to computerized Quality management management Quality systems. computerized to turning are laboratories more and more but This information may be classified manually, • to able be must suchas: information produce laboratory the used, pipette is a When procedure. analytical the The Pipetman The Two-minuteInspection 5 Environmental conditions Environmental method Control Specifications etc.) operator’sname, checks,calibration lab, the in repairs servicing, of (dates pipette the of History number) (identification Identity of the pipette the of Identity Check laboratoryCheck age. determineto its and pipettethe identify to serialnumber the Use . 1 ‑ Quick Diagnosis:Quick
Q A carry a serial number number serial a carry pipettes Gilson All pipette? my is old How of manufacture of date the and pipette the identifies which Jan. 1984 - Dec. 2005 Dec.Jan. - 1984 Letter Month Number ProductionMonth Year G 80 12345 80 G After2006 January Year Month Production Number Production Month Year Year Production Number Month YearNumber Production A A 50001 A A A 10369 H 10369 A M N G H D A K C B E L J Before1984 September November December February January October August Month March June April May July Letter W M U Q N G X H D A Y S R K C B Z T P E L J
2000 2022 2000 2005 2027 2005 2026 2004 2025 2003 2024 2002 2001 2023 2001 1987 2009 1987 2008 1986 2007 1985 198 1999 2021 1999 2020 1998 1996 20181996 20171995 20161994 20151993 20131991 20121990 1992 20141992 1988 20101988 1997 20191997 1989 20111989 4 2006 4 Year 18/07/2007 16:45:49 Learning more 39 Chapter 5 40 Pipette guiderev11012006.indd 40 - - n n - 24). page More, LearningSee 1995.May before pipettesdated on thumbwheel the (Use n functions pipetting Assess 2‑ Step The minimum and minimum The smoothly.move page 14).page see (or manual instruction the in indicated pipette’sthe as to range volume normal correspond should settings maximum The thumbwheel should thumbwheel The of the volumeter display.volumeter the of Check the alignment and the movement the and alignment Checkthe the ejector.the ejector tip- depressthe and tip Gilson a Fit Pipetman. the inside spring the of positioning incorrect indicate whichwould noise spring a for Listen rod. operating bent a to or piston, the on corrosionscratch or a to due be may friction the in variations or motion the in smooth. “Hitches”be should movement push-buttonslowly.depressthe and valuemaximum volumeterat the Set knob. push-buttonadjusting the rangevolumeusing entirethe through Go to verify the proper operation of operation proper the verify to
The Q Q A A liquids, a piston should be be should piston a liquids, corrosivewith contact After corrosion?piston preventI can How • do? I should What well.fit not Tipsdo 8. page see pistons, about information more For scratches. Takeor avoid shocks to care tissue. soft a and alcohol with cleaned • • Always use Gilson tips for tips AlwaysGilson use Gilson representative. Gilson yourfrom part new a order attacked, chemically been has or worn is it alcohol.If with holder tip the Clean properly. positioned is it suremake to ejector tip- the upwardon Push pipettes. Gilson 18/07/2007 16:45:49 Learning more Pipette guiderev11012006.indd 41 - at appears drop a If - - - there is a leak. a is there down, goes tip the in level the If - liquid. test the in tip the re-immerse appears, drop no If leak. a is there tip, the of end the at appears drop a If n leak. a is there tip, the of end the n test Leak 3‑ Step constant. remain should tip the in liquid While immersed, the level of the of level the immersed, While seconds. 20 for Stabilize water.with tip the fill and setting volume maximum Stabilize for 20 seconds. 20 for Stabilize tip. the fill and setting Select the maximum volume maximum the Select the Select P2 to P200 to P2 pipettesPipetman For P10mltoP1000 pipettes Pipetman For
Q Q A A 2 2 1 1 Solution • • leaks? of causes main the areWhat (see page 9). page (see sample the and piston the between cushion air the of pressure and solventthe of pressurevapor the differencebetween the by caused occur.may leaks are leaks These pipette, air-displacement an with used solventis organic an When leaks? cause solventsorganic do How • seals non-Gilson of Use • it has no air cushion. (See page 6.) page (See cushion. air no has it Use a positive-displacement pipette because pipette positive-displacement a Use Tip holder scratched or damaged scratchedor Tipholder Vapororganic frompressure solvents. Use of non-Gilson tips non-Gilson of Use If you are using an air-displacementan using pipette,are you If pressure equilibrium is reached.is equilibrium pressure repeatedly. solvent when stop will leak The distributing and aspirating by vapor solvent with pipette your of cushion air the saturate 18/07/2007 16:45:50 Learning more 41 Chapter 5 Step 4‑ Disassembly
Important 4 Pipetman ultra micro pipettes, Models P2 and P10, include miniaturized parts. It is best to avoid dismounting these pipettes in the laboratory.
n Disassemble the bottom part of the pipette to confirm your diagnosis. 2
n Disassembly protocol: - Eject the tip. - Pull the tip-ejector 1 down. 7 - Unscrew the connecting nut 2. - Separate the handle 4 from the bottom 3 8 part of the pipette. - Remove the piston 5 from the tip holder 6. 5 - Check the piston 5 surface, the seal 7 and the O’ring 8.
Never disassemble the upper part 3 of your pipette 1 Step 5‑ Reassembly 6 n To avoid losing or damaging fragile parts, reassemble the pipette immediately.
Make sure to respect the correct order of parts: the piston seal 7 should always be positioned before the O’ring 8.
Pipette guiderev11012006.indd 42 18/07/2007 16:45:51 Pipette guiderev11012006.indd 43 P2 and P10and P2 Models for and damage, other all For Problem ejector tip or O’ring,holder seal, tip piston nut, connecting push-button, the to damage identified have you U10,P10and U2, P2, than other models For Problem months 12 past the within serviced been not has it that show records and old year one than more is pipette The Problem returnor service? lab for the in Repair 5.2 ••••••••••••••••••••••••••••••• •••••••••••••••••••••••••••••••
helps prevent costly repairs. costly prevent helps Good routine maintenance routine Good
Spare parts may be orderedyourfrombe may Spareparts Solution representativeservice. for pipetteyourself, returnGilson your to it the service cannot you If 38).page Performa Two-minute(see Inspection Return the pipetteReturnservice. forthe Solution pipette. performanceyoureffect the of no on laboratorywith the changedin be representative.Gilson mayparts These Solution
43 Chapter 5 Control and calibration
A pipette is to liquids what a laboratory balance is to solids. Like balances and all other precision instruments, pipettes should be periodically inspected, cleaned, maintained and calibrated so that they will perform within the specifications established by the manufacturer.
In chapter five, we learned about inspecting, cleaning, and maintaining pipettes. In this When Big Ben chimes... chapter, we will learn about Every day at five ‘o’clock specifications and find out I synchronize my watch how to determine whether or with Big Ben's chimes.
not your pipette needs to be If my watch sometimes adjusted. shows 4:59, sometimes 5:01, it is said to be fairly accurate, but not very precise.
If my watch systematically shows 5:02, it is precise, but not very accurate.
If it always shows 5:00, my watch is both accurate and precise. Chapter Chapter 6 44
Pipette guiderev11012006.indd 44 18/07/2007 16:45:51 Pipette guiderev11012006.indd 45
anand n i ue udr h same the indicated. those as conditions operating under used is and maintained 1000µl, or 500 properly been has pipette 200, the that provided at set P1000 Model performance Pipetman any from obtain could you which the shows below table The setting.volumecertain a at model given a and brand given a of pipettes performance all of the precision, the and accuracy by of terms established in guarantee, manufacturer.They are Specifications 1000 1000 specifications? arepublished What
6 P1000 Model Model Volume Pipetman P 1000 P Pipetman permissible errors established by the ISO 8655 committee. 8655 ISO the by established errors permissible the specifications of the pipette must be within the maximimum standard, 8655 ISO IMPORTANT:the with acordance in Tobe tip. the changing and of the handwarming normal both to due error of components conditions all include other given the °C. values 30 all The and 15 between and with stabilized water the used distilled in the is of method used temperature gravimetric pipette The for mode. defined forward are specifications These (µL) accurate but not precise not but accurate . 500 ± 4 ≤ 1 ≤ 4 ± 500 0.6 ≤ 3 ± 200 1 error (µL) error (µL) error (µL) error (µL) error (µL) error (µL) error (µL) error Systematic Random Systematic Random Systematic Random Systematic Permissible Errors PermissibleErrors PermissibleErrors Gilson Maximum ISO 8655 Maximum 8655 ISO Maximum Gilson
± 8 ≤ 1.5≤ 8 ± ± 8 ≤ 3 ≤ 8 ± 3 ≤ 8 ± ± 8 ≤ 3 ≤ 8 ± precise but not not but precise
accurate Learning more ** For complete definitions, see glossary P.50.glossary see definitions, complete For ** 07-001,X NF 1994P.: * (F), AFNOR 41. Q Q A A of a measuring instrument measuring a of ability the Accuracyis accuracy? is What reproducibility.** and/or repeatability as to referredalso is Precision (measurements). responses similar closely provideto instrument an of ability the is Precision precision? is What value.*true a to close responsesgive to accurate and precise and accurate
45 Chapter 6 6.2 How to calculate volumetric accuracy and precision
“Accuracy” and “precision” are qualitative terms. The corresponding quantitative terms are “systematic error” and “random error”.
• Evaluation of accuracy • Evaluation of precision The specified accuracy is the limit to the The specified precision is the limit to the random systematic error, which is the difference between error, which is the distribution of the measured the mean volume of actual measurements values around a mean value. For pipettes, and the true value of the volume set on the precision refers to a within-series group of data, instrument. and therefore to repeatability. The systematic error (E) can be estimated as The random error is then quantified by the follows : standard deviation of measurements performed _ at a given volume setting under the same E = V - V 0 measuring conditions. The standard deviation (SD or “s”) can be E systematic error estimated as follows : _ V0 nominal volume n 2 _ (V - Vi) V mean volume SD = i=1 n - 1 _ n _ 1 V = Vi V mean volume n i=1 _ Vi individually measured volume 1 n V = Vi n number of measurements n i=1
The accuracy of a pipette can be expressed Vi individually measured volume as a percentage of the nominal volume: _ n number of measurements (minimum 10) E% = V - V 0 x 100 V0 The precision of a pipette can also be expressed as a percentage of the mean Important volume. This is known as relative standard The mean value and number of deviation (RSD) or coefficient of variation (CV), and is estimated as follows : replicates must be stated, and the experimental procedure used must SD be described in such a way that other RSD = _ x 100 workers can repeat it. See example V of specifications section 6.1.
Pipette guiderev11012006.indd 46 18/07/2007 16:45:54 Pipette guiderev11012006.indd 47 Using the gravimetricthe method Using over-warmedhandling. extensive by be not must evaluated being instrument the However, series. test the the during handwarming for normal of effects compensate to designed are pipettes Gilson considerationsGeneral the and conditions strict environmental equipment. the controlled and adequate of use systematic requires of method monitoring this of the of Implementation determination the on pipette. the by delivered samples based water of weight is It 8655). (ISO pipette by manufacturers and international recommended standard organizations is method gravimetric The A calibrated barometer to check the atmospheric atmospheric the check to pressure. barometer calibrated A of Bar • constant the check to test. the during air the in humidity hygrometer calibrated A Hyg • series. test the of end the at and beginning the at temperatures water and ambient both A calibrated thermometer readable to 0.1°C to measure of uncertainty standard a with thermometer Calibrated • Requiredequipment theconversion table (µl/mg). See appendix II. waterof aspirated willhavecorrected beto according to outside these conditions, the weight of the setting :volume Note 1013hPa 6.3 50-75% 21.5°C o ±1.5 ±25 0.2 ometer with a standard uncertainty of uncertainty standard a with ometer rometer with a standard uncertainty of uncertainty standard a with rometer f h ppte ae sd n teeoe checked therefore and used are pipettes the If °C the build-up of electrostatic charges.electrostatic of build-up the evaporation rate and to control to and rate evaporation Averagebarom Relative humid Relative The in the test lab should be should lab test the in maintained at 50 - 75% - 50 at maintained in order to reduce the reduce to order in 101325hPa(mbar) ± temperature 21.51.5°C± (293-296 K) (293-296 etricpressure ity should be should should be should
0.5 KPa0.5 o 14.5 psi 14.5 50-75% 70.7 °F 70.7 10% ±0.36 ±2.7 et qimn sol crepn t te following the indications: to correspond should equipment Test • Vessels or exceed the following performances: following the exceed or meet should test the for required balances Laboratory Balances • temperature; room at be shall water air-equilibrated.or degassed 3696, ISO in The specified as 3 grade conforming waterdeionized or distilled Use water Distilled • 1 µl 1 10 µl < 10µl 100 µl < µl 100 V 1 ml < ml 1 V U1000- U5000 P1000- P5000 of apparatusof Instruments F250- F5000 U100- U200 M50- M250 M10to M25 P100- P200 volume (V) volume under test under PUMx300 F25- F200 PUMx20 U Selected P2- P20 F2- F10 U10ml o M1000 P10ml M100 2 - U V V o o o o 20 10ml 1000 µl 1000 100 µl 100 10µl > > 0. Volumes > 2 50 5 t 5 1to 0 to 200 µl 200 to 200 Resolution 00 µl 00 o 10ml o Balance 2 0µl 0.001 0.01 m 0.1 0.1 g Reservoir Sample Ø50 mm Ø3 Ø H70 mm H50 mm H 250ml beaker 50 mm 35mm 5mm Repeatability and linearityand Weighing Ø H100 mm Ø40 mm Ø3 Ø2 H5 H50 mm H1 Vessel 10.5 mm 0.002 0.02 mg 0.2 0.2 3mm 0mm 5mm 1mm Resolution Balance 0. 0.01mg 0.1 0.1 001mg mg mg uncertainty of uncertainty measurement Standard 0.002 18/07/2007 16:45:55 0.02 mg 0.2 0.2 Tw equip. Other F ilters eezers Lid Lid Li L id d
47 Chapter 6 Some remarks about balances Estimation of the Z factor (conversion factor) With modern analytical balances, a laboratory needs only two balances to check an entire stock of pipettes ranging The Z factor is not just equal to the density of water from 0.1µl to 10ml. A good combination would be one adjusted to the local temperature and pressure six-digit balance and another one that works on two parameters. It must also take into account the air scales, for example 50g with sensitivity 0.01mg and density and the density of weights used to calibrate 200g with 0.1mg sensitivity. the balance. The test balances should be calibrated, maintained and For very low volumes, application of the Z factor may recognized by the national department of weighing and not affect the final result. measurements. The detailed formula as well as the table indicating the To minimize vibration, the balances should be on a factor Z to take into account are given in Appendix II. marble table or the equivalent. Keep the balance area free of draughts and the ambient area free of dust. Estimation of the e factor (evaporation loss) From weight to volume Evaporation that occurs during the gravimetric test Conversion to volume must take into account the depends mainly on temperature, humidity and cycle density of the liquid as well as evaporation during the time of work. It may have a noticeable effect on small cycle time. For each measurement, the corresponding volume measurements (particularly for Pipetman P volume (Vi) can be calculated as follows : models P2, P10 and P20, Pipetman F models F2, F5, _ F10 and F20 and Microman models M10 and M25). Vi = ( Wi + e ) Z Evaporation loss is estimated by running a series of four simulated weighing cycles and calculating the Wi is the weight as read on the balance mean weight loss per weighing cycle in mg. Perform _ each weighing cycle without adding the aspirated liquid e is the mean evaporation loss during to the vessel. Dispense the liquid into a dummy vessel. the cycle time The mean evaporation e, is calculated as follows : _ Z expressed in l/mg, is a conversion 1 factor incorporating density of water e = (e1 + e2 + e3 + e4) buoyed in air, at test temperature 4 and barometric pressure A procedure for the determination of e is given in Appendix III. NB : For measurements higher than 20 µl, the evaporation factor can be disregarded.
For your reference, a complete example is given in Appendix I.
Pipette guiderev11012006.indd 48 18/07/2007 16:45:56 Pipette guiderev11012006.indd 49
16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1 checkprocedure Performance
Place distilled or deionized water from the from waterdeionized or distilled Place calculate accuracy and precision, see page 46.) page see precision, and accuracy calculate (Tomanufacturer’sspecifications. with compare and precision the and accuracy the Calculate II. appendix See (Z). needed correction the determine to pressure barometric and temperature of values second the and first the of average the Use limits. recommended within still are values conditions.Checkthat test Record times. (m) several repeat and (ei) value absolute Record container.weighing the to sample any adding actually without but weighing sample normal a as 10exactly to 8 steps repeating by loss evaporation estimate µl, 50 to equal or below sample For weights of to series W1 a as W10.recorded been have measurements ten until cycle test the Repeat weight. the record stabilize, to display allowing After container,weighing door, retrieve balance Open balance). (reset mass tare Determine volume. test the Pipette once. tip the Pre-wet tip. Change calculations. for account into take not do but pipette, the of volume dead the in equilibrium reachhumidity to times five tip pipettePre-wet valid are specifications manufacturer's (the pipette the to assembly capillary/piston or tip the Fit pipette. piston variable-volume your of volume test the Select and (ambient conditions test the Record mm. 3 least at of depth a to vessel weighing the in container the door.the close and balance the on replace sample, deliver tips). manufacturer's the with performed is test when only humidity,barometric relative temperature, water pressure).
the pipetting tasks and the and tasks pipetting the * a pipette. a of precision) and (accuracy performance the testing for method used widely practical, a is analysis Gravimetric compared manufacturer’sspecifications. with regularly be pipettes individual of performance the that imperative is it results, analytical of quality the influence on direct a have precision and accuracy Since to performtestto the When 6.5 Frequency Frequency should be adapted to the type of sample, the number of number the sample, of type the to adapted be should Frequency Annually months Weekly faults (see faults Two-minute38) page Inspection, Every three Daily Always checkAlwaysmechanical for pipette your before performing a gravimetric test. gravimetric a performing before * maintenance maintenance maintenance maintenance Preventive Preventive Preventive Complete Control Important environmental conditions of the laboratory.the of conditions environmental Adjustment -Calibration Adjustment -Calibration Cleaning &Diagnosis: parts ofsecondlevel Replacement spare Replacement spare (Piston, volumeter, - Visual inspection Leak test(See the parts level offirst - Check functions Accuracy check (seals/ O-rings, operating rod) “Two-minute tip holder…) inspection”) Action
Authorized End-users End-users End-users End-users 18/07/2007 16:45:56 Service Service Center Center Gilson Who &
49 Chapter 6 50 Pipette guiderev11012006.indd 50
GLOSSARY Accuracy* a percentage of the true value (relative error). (relative value true the of percentage a as error),or (absolute measured is whichquantity the in units the in either expressed be may negative) or (positive deviation or difference this : Commentary measurand. the of The result of a measurement minus a true value Errormeasurement)*(of Dispenser Aliquot cushion Air air cushion oe “cuay i a concept. qualitative is “accuracy” Note: value. true a to give close responses to measuring instrument a of ity abil the is Accuracy etc. mixture, solution, any of ples sam multiple to ring refer term general A entity. homogeneous portionMeasured a of the sample. of level surface the and piston pipette the between located air ion “dead cushvolume”, air the called Also ntuet r con externally.nected or the instrument into integrated be may reservoir The reservoir. a from uid mined volumes of liq predeter for de-livering instrument An s h vlm of volume the is lower part of the of part lower ------
minus a true value of the measurand. the of value true a minus conditions repeatability under out carried and infinite measur an same the of measurements from of number result would that mean The Error(systematic)* error.random of estimate an only determine to possible is it made, be can measurements of number finite a only Because 2 1 Random error is equal to error minus systematic error,: Notes conditions. repeatability under out carried of measurand same number the of measurements infinite an from result would that mean the minus measurement a of result The Error(random)* appearing on the volumeter of a pipette. a of volumeter the on appearing single-markvolumetricflask, volume* Nominal pressure. and ture tempera time, as such quantities about statements require may measurand a of specification the : Note at20°C. : Example measurement. to subject Particularquantity Measurand* planned, reported. and recorded are monitored, performed, studies laboratory which under conditions the and process organizational the Good Laboratory Pratice (GLP) is concerned with LaboratoryPracticeGood pipette. a of accuracy of error the quantifies error systematic : Commentary 2 1 Systematic error is equal to error minus random error,Notes : be completely known. Liketrue value, systematic error andits causes cannot 0 0 1 vapor pressure of a given sample of water of sample given a ofpressure vapor 100 µ
h vle akd n a on marked value the : Examples use. its to guide a instru provides that ment measuring a of characteristic of a value proximate ap- or rounded The b) 100 µl as the setting the as µl 100 a) 18/07/2007 16:45:58 1 L as as L 1
- - - Pipette guiderev11012006.indd 51 • ditions the of same measurand measurements carried out of results the between Reproducibility is the closeness of the agreement measurements)resultsof (of Reproducibility* minimum. a to time) cycle (example: operator the to due variations mize mini to necessary is it pipetting, For : Commentary time. of period short a over repetition • • • • out include: conditions Repeatability carried conditions same measurand the same under the measure of successive ments of results agreement the of between closeness the is Repeatability measurements)resultsof (of Repeatability* Precision Pipette/Pipetter the same location, same the conditions, same the h sm mauig ntuet ue under used instrument, measuring same the operator/observer,same the procedure, measurement same the ofmeasurement (ex: different operator).
reproducibility. and/or repeatability as to referred often is Precision surements). (mea responses lar simi closely provide to instrument the of tive qualita a is Precision reservoir. a to connected not is pipetter A another. to liquid from one vessel of volume termined prede for a transferring instrument An under changed con f measurement. of term. It is the ability ------
07-001, NF X AFNOR: from abstracted *Definitions Sample carry-overSample Sample 1994 (F), pp. 20, 24, 26-28, 38. 26-28, 24, 20, 1994pp. (F), specified. is performance instrument which for conditions, ambient as well as range, temperature Totaland volume Workingrange measurement. perfect a by obtained be would that value a is Truevalue Truevalue pipette is less than from air-displacement pipette. V min Sa V m max pl e in the instrument after the sample of that is retained portion The ms usd recom limits. mended outside umes vol select not Do Note: a positive-displacement Carry-over :Note from samples. quent that may affect subse and delivery sample population”. term “random sample from statistical the with confusion to avoid necessary when used is sample“ “test term The be analyzed. to is wich liquid a of part resentative rep appropriate The - - - - 18/07/2007 16:45:59 GLOSSARY 51 APPENDIX I - Example of a performance check
Below is an example of how to evaluate 4.__ Calculate the mean volume the performance of Pipetman P10 at 1 µl. _ _ V = ( W+e ) x Z 1. Determine the mean value e of the For a temperature of 21.5°C and an air evaporation loss e that occurs dur- i pressure of 1013 hPa, the Z factor is equal ing your pipetting cycles. Proceed as to 1.0032 µl/mg (see table in Appendix II). described in appendix III to determine ei _ V = (0.964+0.018) x 1.0032 _ m _ 1 V = 0.985 µl i e = e m i=1 m : number of weighings 5. Evaluate accuracy Systematic error (E) : e1 = 0.016 mg e3 = 0.021 mg __ e2 = 0.018 mg e4 = 0.017 mg _ EE == VV -- VV00
APPENDIX e = (e1+e2+e3+e4) / 4 _ V0 true value set on the instrument e = (0.016+0.018+0.021+0.017) / 4 V0 true value set on the instrument _ EE == 0.9850.985 -- 11 == -- 0.0150.015 µlµl e = 0.018 mg/per cycle Relative error (E%) : 2. Change the pipette tip and perform __ the first weighing. Then, keep a regular E%E% == (V(V -- VV00)) xx 100100 // VV00 cycle and perform the 10 following E%E% == (-0.015(-0.015 xx 100)100) // 11 == -- 1.501.50 %% measurements.
wr = 0.957 mg w1 = 0.968 mg w6 = 0.966 mg 6. Evaluate precision (repeatability) w2 = 0.960 mg w7 = 0.955 mg Standard Deviation (SDW) w3 = 0.984 mg w8 = 0.972 mg _ w4 = 0.942 mg w9 = 0.958 mg n 2 (Wi - W) w5 = 0.969 mg w10 = 0.967 mg SDw = i=1 n - 1 wr rinsing measurement which is disregarded for the calculation n _ 2 1 2 SD = ( Wi - W ) w n - 1 i=1 3. Calculate the mean weight (0.968-0.964)2+ (0.960-0.964)2+ (0.984-0.964)2+ _ n 2 2 2 1 2 1 (0.942-0.964) + (0.969-0.964) + (0.966-0.964) + i SDw = 2 2 2 W = W 9 (0.955-0.964) + (0.972-0.964) + (0.958-0.964) + 2 n i=1 (0.967-0.964) n number of weighings SDw = 0.011 mg W weighing results _ i W = (0.968+0.960+0.984+0.942 Random error (SDv) : + 0.969+0.966+0.955+0.972 SDv = SDw x Z _ + 0.958+0.967) / 10 W = 0.964 mg SDv = 0.011 x 1.0032 = 0.011 µl 52
Pipette guiderev11012006.indd 52 18/07/2007 16:46:02 SD SD SD SD SD SD v v w w w = = = 2 w 2
= = = 0.011 x1.0032 SD 0.011 mg 9 1 n - w 1 xZ (0.967-0.964 (0.955-0.964 (0.942-0.964 (0.968-0.964 1 i=1 i=1 n n ( ) ) ) ) ( 2 2 2 2 W + (0.972-0.964 + (0.969-0.964 + (0.960-0.964 = 0.011µl W n - i i -W - _ 1 W _ ) 2 ) ) ) ) 2 2 2 + (0.958-0.964 + (0.966-0.964 + (0.984-0.964 2 ) ) ) 2 2 2 + + + Pipette guiderev11012006.indd 53
Where : Where waterpressuredistilled and for temperatureof function a as Valuesconversionfactor(µl/mg) the Z of g/ml. 8.0 were weights the of density the if as air in weighing when results give to adjusted been have OIML of 33 : No. RecommendationNote International to Weightsconforming : is equation calculation reference The APPENDIXII - Z Factor
Temperature 30 28.5 27 25.5 24 22.5 21 16.5 29.5 29 28 26.5 26 25 24.5 23.5 23 22 20.5 20 16 15.5 15 19.5 18 19 18.5 17 21.5 27.5 17.5 °C P P P A B W
= density of air at t°C. at air of density = = density of the balance weights. Use 8 g/cc for P for g/cc 8 Use weights. balance the of density =
= density of the test liquid at t°C. at liquid test the of density =
1.0039 1.0035 1.0032 1.0028 1.0025 1.0022 1.0052 1.0047 1.0043 1.0020 1.0050 1.0049 1.0046 1.0044 1.0042 1.0040 1.0038 1.0037 1.0034 1.0033 1.0031 1.0030 1.0027 1.0026 1.0024 1.0023 1.0022 1.0021 1.0018 1.0018 1.0019 800
1.0052 1.0048 1.0044 1.0040 1.0036 1.0032 1.0029 1.0026 1.0023 1.0020 1.0051 1.0049 1.0046 1.0045 1.0042 1.0041 1.0038 1.0037 1.0035 1.0033 1.0031 1.0030 1.0028 1.0027 1.0025 1.0024 1.0022 1.0021 1.0020 1.0018 1.0019 853 Z = [1/(P = Z
1.0053 1.0048 1.0044 1.0040 1.0036 1.0033 1.0030 1.0026 1.0024 1.0021 1.0051 1.0050 1.0047 1.0046 1.0043 1.0042 1.0039 1.0038 1.0035 1.0034 1.0032 1.0031 1.0028 1.0027 1.0025 1.0025 1.0023 1.0022 1.0020 1.0019 1.0019 907 Air pressureAir W hPa -P A )] [1-(P)] 1.0053 1.0049 1.0045 1.0041 1.0037 1.0033 1.0030 1.0027 1.0024 1.0022 1.0052 1.0050 1.0048 1.0046 1.0043 1.0042 1.0039 1.0038 1.0036 1.0035 1.0032 1.0031 1.0029 1.0028 1.0026 1.0025 1.0023 1.0022 1.0021 1.0020 1.0019 960
B A /P B )] 1.0054 1.0050 1.0045 1.0041 1.0038 1.0034 1.0031 1.0028 1.0025 1.0022 1.0052 1.0051 1.0048 1.0047 1.0044 1.0043 1.0040 1.0039 1.0036 1.0035 1.0033 1.0032 1.0030 1.0029 1.0027 1.0026 1.0024 1.0023 1.0021 1.0020 1.0020 1013
1.0055 1.0050 1.0046 1.0042 1.0038 1.0035 1.0031 1.0028 1.0025 1.0023 1.0053 1.0052 1.0049 1.0047 1.0045 1.0043 1.0041 1.0039 1.0037 1.0036 1.0033 1.0032 1.0030 1.0029 1.0027 1.0026 1.0024 1.0023 1.0022 1.0021 1.0020 1067
18/07/2007 16:46:02 APPENDIX 53 54 Pipette guiderev11012006.indd 54
APPENDIX 10. 1. check.gravimetric the for using be will you as balance and vessel weighing water, distilled same the Use of determination loss evaporation the for Procedure APPENDIXIII - Evaporation loss 9. 8. 7. 6. 5. 4. 3. 2.
e =(e _ Calculate the evaporation loss e using the using e loss evaporation the Calculate Half fill the weighing vessel with distilled with vessel weighing the fill Half between 0.01 mg and 0.03 mg. 0.03 and 0.01mg between usually is value this conditions, normal In : formula e obtain to times three 8, to 3 steps Repeat value). absolute e result negative the Read balance. the vessel and, using tweezers, replace the vessel on weighing the on lid the Replace Dispensethe sample into a dummy vessel. tweezers. Takewith lid the off balance. the of out vessel weighing the take and balance the Tare sample. a Aspirate tweezers. of pair a using balance the lid on it its place and with vessel weighing the Cover water. 2 , e , 3 , and e and , 4 1 4 . 1 + e 2 + 1 rcr the (record e _ 3 + e 4 )
18/07/2007 16:46:02 Pipette guiderev11012006.indd 55 18/07/2007 16:46:02 NOTES 55 56 Pipette guiderev11012006.indd 56
NOTES 18/07/2007 16:46:02 Gilson guide to pipetting Gilson guide to pipetting
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