GUIDE TO PIPETTING Third Edition HOW THE PIPETTE STORY BEGAN... PIPETTE HOW THE
HOW THE PIPETTE STORY BEGAN…
Over a century ago, Louis Pasteur invented the glass Pasteur pipette to reduce contamination when transferring samples. The Pasteur pipette is still in use today. The next significant improvement on pipettes occurred in the late 1950s with the introduction of a handheld, piston-operated pipette as a safe alternative to potentially dangerous mouth pipetting. The first handheld pipettes had pre- established volume setting (fixed volume pipettes). Further improvement was provided with the more flexible stepper volume setting (variable volume pipettes). In 1972, Dr. Warren Gilson invented the first continuously adjustable pipette. As an original error-preventing feature, the selected volume was now clearly displayed on the Gilson pipette (direct digital readout). Today, Gilson precision pipettes are still the world standard for accuracy, precision, and reliability. Today, the company still maintains its continuous effort of creativity and offers innovative, robust, and reliable pipettes to help scientists in their daily work. The pipetting system is our core expertise, and we truly enjoy sharing this knowledge and experience with pipette users so that they may achieve their goals. This guide will provide you with keys to understand how pipettes should be used and to get the most out of them. Examples given are based on our own pipette range, but the techniques described are equally applicable to other brands of pipettes and tips.
Gilson is one of the winners of the Red Dot Award: Product Design for MICROMAN E.
GILSON GUIDE TO PIPETTING TABLE OF CONTENTS TABLE OF CONTENTS TABLE CONTENS OF
CHAPTER 1 - SELECTING THE RIGHT PIPETTE | 8 Appendix | 45 Working Principle of Pipettes | 8 Appendix A: Pipetting Terms | 45 The Right Choice for your Application | 11 Appendix B: Example of a Performance Check | 47 SpecificP ipettes for SpecificV essels | 13 Appendix C: Z Factor | 48 High Throughput and Repetitive Pipetting | 14 Appendix D: Evaporation Loss | 49 Appendix E: Chemical Resistance of Plastics | 50 TABLE OF CONTENTS TABLE CHAPTER 2 - PIPETTING TECHNIQUES | 16 Adjust the Volume Display | 16 FAQ | 51 Forward or Reverse Mode Pipetting | 17
Tips for Mistake-Free Pipetting | 23 Pipetting Ergonomics | 23
CHAPTER 3 - SELECTING THE RIGHT TIP | 26 Fitting a Disposable Pipette Tip | 26 Choosing the Best Tip for Your Application | 28 Evaluating Tip Quality | 30
CHAPTER 4 - PREVENTING CONTAMINATION | 32 Types of Contamination and How to Prevent Them | 32 Decontaminating Your Pipette | 34
CHAPTER 5 - PIPETTE SERVICE & MAINTENANCE | 36 Pipette SpecificationsA ccording to ISO 8655 | 36 Repair in the Lab or Return for Service? | 36 Quick Pipette Diagnosis | 37 How to Calculate Volumetric Accuracy and Precision | 38 Pipette Calibration | 39 Calibration with the Gravimetric Method | 41 Performance Check Procedure | 43
3 TABLE OF CONTENT | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 4 Air-Displacement Pipette Positive-Displacement Pipette PSTV-IPAEET PIPETTE POSITIVE-DISPLACEMENT AIR-DISPLACEMENT PIPETTE
5 GLOSSARY | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 6 Chapter 1 SELECTING THE RIGHT PIPETTE SELECTING THE RIGHT PIPETTE SELECTING THE RIGHT PIPETTE RIGHT THE SELECTING
Working Principle of Pipettes
Air-Displacement Pipettes Positive-Displacement Pipettes • Recommended for aqueous samples • Recommended for problem samples and for general laboratory work. (viscous, dense, volatile, radioactive, • Always have a cushion of air (dead corrosive, contaminating, hot and cold). volume) between the pipette piston • Direct contact of the piston and the liquid sample. with the sample (no air cushion). • The piston is integrated into the • The disposable piston is part of the tip lower part of the pipette. (not integrated into the pipette).
Disposable piston Disposable capillary
Air cushion Disposable tip
7 SELECTING THE RIGHT PIPETTE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 8 Working Principle of Air-Displacement Pipettes Working Principle of Positive-Displacement Pipettes When the push button is pressed on an air-displacement pipette, the piston inside the Positive displacement pipettes, such as MICROMAN, work like a syringe. There is no air cushion instrument moves down to let air out. Air is displaced by the piston. The volume of air between the disposable piston and the sample. With no elastic air cushion to expand or contract, displaced is equivalent to the volume of liquid aspirated. the aspiration force remains constant, unaffected by the physical properties of the sample. SELECTING THE RIGHT PIPETTE RIGHT THE SELECTING The schematic drawings show how the piston determines the volume of air displaced and This allows the positive-displacement operator to pipette very viscous or high density samples, subsequently the volume of sample aspirated. such as glycerol and blood.
1 2 1 2 1 1 0 0 0 0 Set Volume Prepare for Set volume Prepare for Aspiration aspiration The required The required volume is set. The push button volume is set. The push button SELECTINGTHE RIGHT PIPETTE The piston is pressed prior to The piston is pressed prior moves to the sample aspiration. moves down to to sample appropriate The piston the appropriate aspiration. position. descends and start position. The piston expels a volume descends down of air equal to the to the end of the selected volume of capillary. liquid.
3 4 3 4
Aspirate sample Dispense Aspirate sample Dispense sample sample As the push button The orifice is then is released, a The push button is immersed below The push button partial vacuum is pressed again. Air the liquid surface. is pressed again. created inside the pressure increases As the push button The piston tip. The ambient inside the shaft is released, the moves down atmospheric and the tip. The piston moves up and expels the pressure forces the compressed air and the ambient liquid out of the desired volume of pushes the liquid pressure forces the capillary. liquid through the out of the tip. desired volume orifice into the tip. of liquid through the orifice into the capillary.
9 SELECTING THE RIGHT PIPETTE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 10 The Right Choice for Your Application Accuracy and Precision While Pipetting Problem Liquids The type of analysis to perform, the physical properties of the liquid, and the volume to be Positive-displacement pipettes like MICROMAN are the right solution for complete and rapid handled will determine which pipette to use. It is recommended to select a pipette with a pipetting of viscous and dense liquids such as oil, syrup, cosmetic cream, liquefied food, paint, nominal (maximum) volume as close as possible to the desired volume to transfer. glycerol, or buffers. SELECTING THE RIGHT PIPETTE RIGHT THE SELECTING Positive-displacement pipettes are the unique solution to avoid leakage when pipetting high vapor pressure liquids such as acetone, chloroform, alcohol, or other solvents. Recommendations for Pipetting Different Volumes
0 µL 10 µL 50 µL 100 µL 200 µL 1000 µL 5000 µL 10 mL Volume of sample % Systematic error 5 Regular air-displacement pipette Molecular biology 4.5 4 Microbiology 3.5 Clinical assays Cell culture 3
Immunoassays Analytical chemistry 2.5 2 1.5 Air-displacement SELECTINGTHE RIGHT PIPETTE P2, P10, P100, P200, P1000 P5000, P10mL P20 pipettes 1
Positive-displacement 0.5 M10, M25, M50 M100, M250, M1000 pipettes 0 MICROMAN® E positive-displacement pipette Consider the Physical Properties of Your Sample 0 40 60 80 85 90 95 100 % Glycerol For volumes higher than 10 mL, it is suggested to work with a pipette filler like the Viscosity/Density MACROMAN with plastic or serological pipettes. Regardless of the volume you require, the nature of the sample directly impacts precision and accuracy. Air-displacement pipettes will be better for aqueous liquids whereas positive- Figure 3 displacement pipettes should be used for problem liquids. MICROMAN® E, Positive-displacement pipette, Systematic error
SAMPLE TYPES EXAMPLES RECOMMENDED PIPETTES % Random error 3.5 Aqueous Water, sucrose, Tris, buffers with a pH of 7 Air-displacement. Regular air-displacement pipette 3 Biological DNA, RNA, proteins Air-displacement with filter tips Viscous Glycerol, surfactants, oil 2.5 Volatile Ethanol, hexane, formaldehyde 2 Radioactive isotopes, blood, infectious 1.5 Hazardous bacteria or viruses 1 Acids such as hydrochloric acid or sulfuric acid, bases such as ammonium hydroxide, 0.5 Corrosive salts such as sodium chloride Positive-displacement 0 MICROMAN® E positive-displacement pipette
0 40 70 80 85 90 95 100 % Glycerol Viscosity/Density
Figure 4 MICROMAN® E, Positive-displacement pipette, Random error
11 SELECTING THE RIGHT PIPETTE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 12 SpecificP ipettes for SpecificV essels High Throughput and Repetitive Pipetting When pipetting in a high throughput setting it is important to have reliable results and to be as efficient as possible. Reliable results means not only having reproducible results with one technician's samples, but also among all technicians SELECTING THE RIGHT PIPETTE RIGHT THE SELECTING in the lab. There are a variety of ways to improve reliability and efficiency, some of Test tubes and centrifuge tubes are which include using motorized pipettes and/or repetitive pipettes. used with all single channel pipettes for sample preparations, such as qPCR templates. User-to-User Variability Motorized pipettes can help reduce variability between users. There are many factors that can affect your pipetting results, which include setting the volume, Long test tubes, also called assays tubes, pipetting technique, and the rate of aspirating and dispensing. With a motorized are used with positive-displacement pipette you can set the exact volume on the digital display — the motor uses pipettes and pipette fillers with plastic or the same pipetting force every time and maintains the same rate of speed when glass pipettes: these devices are specially aspirating and dispensing a sample. designed to reach the bottom of these tubes. Aliquoting SELECTINGTHE RIGHT PIPETTE To deliver several aliquots without refilling, Reagent reservoirs are ideal for you may either choose the repetitive mode dispensing reagents, especially with of a motorized air-displacement pipette,
multichannel pipettes. or use a positive-displacement repeater. Repeaters enable up to 125 aliquots, whereas the number of aliquots with 96-well and 384-well microplates, as air-displacement motorized pipettes will well as 8-well strips, are commonly used depend on the pipette volume. with air-displacement multichannel pipettes for applications like ELISA, For operations fewer than ten aliquots, but also with single channel pipettes. using a motorized air-displacement pipette might be the better option. Multichannel pipettes allow transferring 8 to 12 different samples in one shot and filling a microplate 8 to 12 times faster than a single channel pipette.
13 SELECTING THE RIGHT PIPETTE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 14 PIPETTING TECHNIquES 16
Volume Accuracy Improving Improving djusting the the djusting A GILSON GUIDE TO PIPETTING TO GUIDE GILSON With the push button, the volume the volume the push button, With To avoid internalTo damage to your pipette, never attempt to force the volume setting limits. the beyond int for int for NOTICE eproducibility and eproducibility When decreasing the volume setting, slowly setting, slowly the volume decreasing When not to making sure setting, the required reach the setting. pass and then slowly of a turn 1/3 by value required not to making sure the volume, reach to decrease the setting. pass can be easily adjusted with one hand. Push button one hand. Push with button can be easily adjusted on all MICROMAN is available adjustment volume (except pipettes and on PIPETMAN pipettes April 1995. after L) manufactured PIPETMAN Reading and Reading in one hand and the micropipette Hold the body of or the thumbwheel rotate to use the other hand the push button. R To avoid parallax, hold the pipette in a horizontal in a horizontal hold the pipette parallax, avoid To until the indicator the volume position. Adjust volume. is lined up with the desired A Helpful H best for clockwise finish setting Always obtain a clockwise to is how This reproducibility. setting: volume • the setting, pass the volume increasing When • Chapter 2
isplay 1 D 0 Incorrect Incorrect PIPETTING TECHNIquES 0 alignment: error olume olume V
shown on shown 0 accurate reading accurate The volume is volume The Correct alignment: alignment: Correct the volumeter Volume Volume indicator Adjust theAdjust Thumbwheel Push button UIDE TO PIPETTING TO UIDE ILSON G G
PIPETTING TECHNIquES PIPETTING TECHNIQUES PIPETTING 15 Forward or Reverse Mode Pipetting
Air-Displacement / Forward Mode TECHNIquES PIPETTING The forward mode is the standard way of pipetting with an air-displacement pipette like PIPETMAN. In general, precision in forward mode depends on precise draining by air pressure (air-displacement pipettes) or internal wiping of the pipette barrel (positive-displacement pipettes). 1 2 3 4 5
Preparation Aspiration Dispense Purge Home VOLUME IMMERSION DEPTH (MM) Hold the Immerse the Place the pipette Wait one second, then Allow the plunger to 1 instrument in a pipette tip in the tip at an angle depress the plunger to move up to the rest 0.1 —1 µL nearly vertical liquid*. Allow the (10° to 45°) against the second stop position. 1—100 µL 2–3 PIPETTINGTECHNIquES position. Depress plunger to move the inside wall position. This purge 101—1000 µL 2–4 the plunger up smoothly to the of the receiving stroke removes any 1001 µL— 10 mL 3–6 smoothly to the rest position. Wait vessel. Depress the remaining sample first stop position. one second so that plunger smoothly from the tip. Remove all the liquid has to the first stop pipette tip end from Pre-Rinsing time to move up position. sidewall by sliding into the tip. it up the wall. To obtain greater uniformity and precision of dispensing, it is better to provide identical contact surfaces for all aliquots. Rest position This is done by pre-rinsing with the same liquid as the one dispensed. First stop For pre-rinsing, aspirate with the tip, and then dispense back into the original reservoir or to waste. Second stop Pre-rinse again when adjusting the volume. (purge)
* The immersion depth of your tip can have a significant effect on your results (for depth per model, see table above). 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.
17 PIPETTING TECHNIQUES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 18 Forward or Reverse Mode Pipetting
Air-Displacement / Reverse Mode In reverse mode pipetting, the purge stroke is used during preparation. During aspiration, an The reverse mode is only possible with air-displacement pipettes. It is sometimes used to amount of liquid equal to the amount of purged air is added. This amount compensates for the TECHNIquES PIPETTING pipette slightly viscous liquids. liquid that remains inside the tip during dispensing.
1 2 3 4 5 Preparation Aspiration Dispense Re-Aspiration Complete Purge Hold the instrument Immerse the pipette Place the pipette tip at If the pipette tip is to be Wait one second and purge. in a nearly vertical tip in the liquid. Allow an angle (10° to 45°) reused for the same sample, or If the pipette tip is not to be position. Depress the the plunger to move against the inside wall maintain the plunger in the re-used, depress the plunger plunger smoothly up smoothly to the of the receiving vessel. intermediate position for to purge position over an PIPETTINGTECHNIquES to the second stop rest position. Wait one Depress the plunger subsequent immersion for appropriate waste container position. second so that all the smoothly to the first the next pipetting cycle and and then eject the tip. liquid has time to move stop position. Wait one restart operation 2. up into the tip. second.
Second stop (purge)
19 PIPETTING TECHNIQUES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 20 Forward or Reverse Mode Pipetting
Positive-Displacement / Forward Mode PIPETTING TECHNIquES PIPETTING
1 2 3 4 Wiping Preparation Aspiration Dispense Ejection Press the plunger Immerse the capillary/piston Press the plunger Press the plunger all the To avoid distorted results linked to the volume pipetted, button to the first in the liquid*. Release the button to the first stop. way down to the second ensure that no liquid is on the outside part of the tip. stop. The piston plunger, letting it move up The piston moves down and last stop. Capillary If necessary (viscous liquids, such as cream), wipe the moves to the to the home position. The and expels the liquid and piston are ejected outside of the tip or the capillary with a clean medical appropriate position. piston moves up and the out of the capillary. without hand contact. wipe. Do not touch the orifice. Choose a tissue that ambient pressure forces the is resistant, lint-free, and inert to acids and solvents. PIPETTINGTECHNIquES desired volume of liquid Dispose of the tissue in a safe, hygienic manner. through the orifice into the capillary. When working with high risk specimens, do not NOTICE wipe the disposable part. Make sure fluid depth penetration does not exceed the recommended Rest position immersion depth*.
Ejection Pre-Rinsing To pre-rinse, aspirate with the tip and then dispense back into the original reservoir or to waste.
When working with high risk specimens, do not NOTICE wipe the disposable part. Make sure fluid depth penetration does not exceed the recommended immersion depth.
* The immersion depth of your tip can have a significant effect on your results (for depth per model, see table page 20). 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.
21 PIPETTING TECHNIQUES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 22 Tips for Mistake-Free Pipetting Take Time to Relax 1. If possible, try to switch periodically between different types of work. How to Avoid Typical Pipetting Mistakes 2. Keep an appropriate, unrushed working speed. Let go of the pipette from time to time and give your fingers/hand a (micro) break. MANY FACTORS MAY IMPACT PIPETTING ACCuRACY TECHNIquES PIPETTING 3. Take frequent short breaks. Change your sitting position. Lean back and relax your shoulders and arms. INFLUENCING PARAMETERS AND EFFECTS CORRECTIVE MEASURES Leaky/poorly seated pipette tips may affect accuracy using original or recommended by 0.5% to 50% pipette tips Special Attention Should be Paid to Smooth Pipetting
Reuse of pipette tips may affect accuracy by up to 4% using pipette tips only once 1. To facilitate uniform timing and motion, keep all necessary items within The straightness of pipette tips may affect accuracy arm's reach. by up to 10% using appropriate tips only 2. Place the most frequently used objects in front of you. The more rarely used The difference in vapor pressure of the liquid to be items can be placed a little further away from you. pipetted versus that of the water used for adjustment
PIPETTINGTECHNIquES may affect accuracy by up to 2% Sufficient pre-wetting of pipette tips 3. The opening of the receptacle for used tips should be at the same height as Failure to wipe pipette tip on the vessel wall can affect Wiping of the pipette tip on the vessel the end of your pipette. accuracy by up to 3% wall (wiping distance 8 to 10 mm)* Pipette tip immersion depth and handling angle during Holding pipette in a vertical position pipetting may affect accuracy by up to 1% while pipetting Use a Pipette Holder Irregular rhythm and timing during pipetting can Applying a consistent pipetting Protect your pipette and always store it vertically on a affect accuracy by up to 1.5% technique pipette holder. Pipettes left on a workbench or stored in Regularly checking the pipette a drawer can easily come into contact with samples and A leaky piston system can affect accuracy by 1% to 50% and the volume aspirated become contaminated. Uneven piston movement can affect accuracy by up to 0.5% Smooth operation of piston
Information extracted from ISO 8655-2 - Annex B * Gilson recommends touching the tip to the vessel wall at an angle of 10° to 45°.
Take a Few Minutes to Get Organized and Ensure You Have: 1. An appropriate posture 2. The right material Gilson offers various pipettes with forces adapted to user preferences. The forces of PIPETMAN L are some of the lowest. 3. The appropriate gestures 4. A good work organization
A good test is to see if you can rest your elbow Download the Gilson comfortably on the work surface. If not, your Ergonomics Poster receptacle may be too low or too high, find the right for increased working height. comfort in your lab Figure 5 Figure 6 SINGLE® Pipette Holder POWER CAROUSSEL® Stand
23 PIPETTING TECHNIQUES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 24 Chapter 3 SELECTING THE RIGHT TIP SELECTING THE RIGHT TIP SELECTING THE RIGHT TIP RIGHT THE SELECTING
Fitting a Disposable Pipette Tip
To Fit a Disposable Tip on a PIPETMAN Single Channel Hold the micropipette in one hand, lower the pipette into the tip, and use a slight twisting movement to seat the tip firmly on the tip holder of the micropipette to ensure an air-tight seal. To protect your pipette, avoid tapping the tip onto the pipette like a hammer. Tips are available in TIPACK racks for easy mounting with no hand contact.
Press down with Avoid hammering a rotating motion the tip into the pipette
Exert a light vertical force followed by a To Fit a Disposable Tip on a slight lateral rocking PIPETMAN Multichannel movement to secure the tip fitting To avoid damage to your pipette, Gilson does not recommend hammering or pounding on the tips. The patented ROCKY RACKTM system available on TIPACK, TOWERPACK, BLISTER REFILL and RELOAD PACK makes it extremely easy to fit tips on a multichannel pipette. Tips will not fall off nor will they have to be positioned manually.
Figure 7 Fit a Disposable Pipette Tip on Single and Multichannel Pipettes
25 SELECTING THE RIGHT TIP | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 26 To Fit a Capillary Piston (CP) Choosing the Best Tip for Your Application on a MICROMAN PIPETMAN Tips are available in a variety of packaging options to suit virtually all needs and 1. Place the plunger button to the second 33 applications: stop. SELECTING THE RIGHT TIP RIGHT THE SELECTING 2. Place the pipette over the tip. The jaws on Autoclavable Tips the pipette will open automatically and seize the piston. Loose in bulk packaging An economical solution for routine applications. May be hand loaded in empty tip racks for 3. Press the pipette down to attach the convenience or for autoclaving in the laboratory. capillary to the pipette. Racked for easy mounting with no hand contact 4. Return the plunger to the rest position. TIPACK have a hinged lid to protect against dust. 5. Press the plunger to the first stop to Convenient 96-well format for filling microplates with a PIPETMAN multichannel and color-coded complete tip attachment. A1 for easy identification. Ready for autoclaving in the laboratory. Tip racks may be reused. For an easy CP fitting, choose the new 22
SELECTING THE RIGHT TIP MICROMAN E. The QuickSnap feature makes it Racked and sterilized for working in sterile conditions as easy to use as a regular pipette. Factory sterilized and delivered in a sealed tip rack. TOWERPACK refill system 1. Press the MICROMAN E onto the capillary Figure 8 CP Fitting on MICROMAN E High quality tips in an economic, easy-to-use and eco-friendly rack refill system. piston until it is firmly seated. The reload box is reusable and can be repeatedly autoclaved. 2. Pick up the CP from the rack. Also available in sterilized packaging. 3. Slowly press the plunger button until you feel and hear a slight click and continue to Sterilized Filter Tips press to the first stop. Then pipette the liquid. PIPETMAN MICROMAN TIPACKS are racked and sterilized with filter no-touch no-touch Tips with a filter prevent contaminating aerosols from entering the pipette. disposal of tip disposal For maximum protection against PIPETMAN filter tips are factory irradiated delivered in a sealed tip rack. of capillary contamination, capillary pistons for and piston MICROMAN pipettes are available pre- STERILPACKS are individually wrapped and sterilized assembled, racked and presterilized. 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.
Ejecting the Used Tip Does not apply to 5 & 10 mL Capillaries and Pistons for Positive-Displacement Pipettes To avoid touching contaminated tips, hold pipettes the pipette over the waste container and Safety first! press the tip ejector push button. Gilson capillary pistons for MICROMAN are made of plastic, eliminating the risk of injury associated To eject the tip from MICROMAN, depress with broken glass. the push button completely to the second stop. Discarded tips contain liquid residues, particularly when a pipette is used in reverse mode. Take suitable precautions Figure 9 when discarding disposables. Ejecting the Tips
When to Change a Tip? When transferring single samples of different liquids, select a new pipette tip for each new liquid. It is strongly recommended to pre-rinse every new pipette tip. 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.
27 SELECTING THE RIGHT TIP | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 28 Tip Sterilization Methods Evaluating Tip Quality 1. Beta or gamma radiation of consumables Although they may look alike, all tips are not equal. The choice of a poor quality tip may This method is used by manufacturers for products sold under the label “sterile“. The jeopardize your results. Choose the pipette tip recommended by the pipette manufacturer for penetrating rays are highly effective for the relatively inert plastics used to manufacture the best accuracy, precision and tip fit, and always check the following points: pipette disposables. The choice of gamma or beta rays is determined by the type of TIP RIGHT THE SELECTING plastic used to manufacture the tips. PIPETMAN DIAMOND Tips are sterilized using gamma rays and a sterility assurance level SAL 10-6 is guaranteed. Quality of the Tip's Raw Material 2. Ethylene oxyde gas There are many different brands of tips made of varying quality plastics. If the type of plastic to be sterilized cannot withstand beta or gamma radiation, ethylene Gilson selects a specific polypropylene because it is naturally hydrophobic and a low oxide is used instead. EtO is notably used to sterilize CPs. retention material.
Absence of Potential Contaminants Gilson Tip Packaging Cleanness of tips is very important as production residues, such as dust or biological Gilson offers a wide range of PIPETMAN Tips packaging to suit all your needs. contaminants coming from the production site, may contaminate your samples. Additionally, tips should be chemically resistant and free of additives, such as silicone, dyes, biocides, antistatic SELECTING THE RIGHT TIP agents, as well as traces of metal, such as aluminum, nickel, or zinc. TOWERPACK A trace metal certificate can be obtained from the manufacturer upon request. Tower of racks
RELOAD PACK 2 stacks of racks ECO & EASYPACK Bags of tips Tip Manufacturer’s Guarantees GuARANTEEING TRACEABILITY With the batch number on each box and bag, the history of the tips can be traced from packaging TIPACK to delivery to the laboratory.
GuARANTEEING PRODuCTION quALITY Figure 11 PIPETMAN DIAMONDS Tip, Every PIPETMAN DIAMOND precision tip is individually Identification Label marked with an identification number. With this number, the mold can be identified, and even the exact cavity which produced the tip can be located.
Figure 12 PIPETMAN DIAMONDS Tip Guaranteed Figure 10 Traceability PIPETMAN Tips Packaging Options
Check Gilson Manual Check the tip compatibility table in Gilson Product Liquid Handling Catalog Guide to match your pipette model with standard or filter tips. www.gilson.com/ecatalog/index.htm
29 SELECTING THE RIGHT TIP | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 30 Chapter 4 PREVENTING CONTAMINATION PREVENTING CONTAMINATION PREVENTING CONTAMINATIONPREVENTING
Types of Contamination and How to Prevent Them
Personal Protective Equipment Sample to Pipette PREVENTION Contamination can occur if the sample or The specific personal protective aerosols from the sample are allowed to enter equipment recommended depends on the body of the pipette. your laboratory and can include: PREVENTION • Wear a lab coat. • To prevent your sample from contaminating • Wear gloves. the body of your pipette, do not turn the pipette upside down when there is sample in • Wear protective glasses. the tip. Always store your pipettes vertically. • Wear a mask. • Release the push button slowly. • Wear protective footwear. • Use filter tips to reduce contamination risk. LAB BENCH • Use the corrosion protection kit available • Wipe workbench before and after for PIPETMAN P1000 (Neo, G, and L). with an appropriate cleaner for your • For complete protection of the pipette, application (cell culture, radioactive choose MICROMAN E for problem liquids. components, pathogenic samples…). • Work under a hood. PIPETMAN PIPETMAN MICROMAN with with with capillary • Work behind a radioactivity shield. standard tip ﬁlter tip piston • Avoid touching used tips.
Pipette to Sample Protected Contaminated tips or a contaminated air space pipette will contaminate your samples. PREVENTION • Store pipettes vertically on a holder. Piston Filter seal • Eject tips into a designated container. Aerosol barrier • Follow laboratory protocol to clean Non-protected your pipette. air space (aerosol) • Use sterile tips when appropriate. • Change the tip after each sample to avoid cross contamination.
Risk of aerosol Reduced risk of No risk of aerosol contamination aerosol contamination contamination
Figure 13 Solutions Against Contamination
31 PREVENTING CONTAMINATION | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 32 Sample to Sample (Carryover) Decontaminating Your Pipette CHANGE THE TIP AFTER EACH SAMPLE. The solutions mentioned below are options and other solutions may be used. Make sure A portion of sample "A" can adhere to the your decontamination technique is compatible with your pipette material and refer to your inside wall of the tip after sample delivery. laboratory decontamination procedure. PREVENTING CONTAMINATIONPREVENTING The leftover portion of sample "A" can mix with the next sample "B" and may cause a CONTAMINATION DECONTAMINATION CLEANING GUIDELINES false test result. CAUSES TECHNIQUES
Disassemble the lower part of your pipette. Fully immerse the How to prevent aerosol contaminated parts* into an ultrasonic bath with a detergent or contamination? cleaning solution recommended for laboratory instruments. It is strongly recommended to rinse the pipette several times with water and dry it thoroughly. It is essential to prevent aerosol contamination when using PCR and other Radioactive Detergent—cleaning Always make sure that radioactivity has decreased to amplification methods, or when pipetting compounds solution an acceptable level. DNA/RNA solutions, infectious materials, Work surfaces may be decontaminated by exposure to 300 nm UV radioactive samples, etc. light for 15 minutes. UV will not damage Gilson PIPETMAN materials.
Gilson offers two solutions: Note that the UV rays cannot penetrate inside the pipette and
PREVENTINGCONTAMINATION Left-over Contamination Viruses, bacteria, cannot be considered as a decontamination protocol for the 1. use a PIPETMAN pipette with sample A of sample B mycoplasma, fungi uV radiation internal components of the pipette. PIPETMAN DIAMOND sterilized filter inside tips when faced with the following Disassemble the lower part of your pipette. Fully immerse the the tip contaminated parts* into at least 3% sodium hypochlorite for
situation: at least 15 minutes. Rinse thoroughly with distilled water and dry.
• Working under sterile conditions. DNA, RNA, 10 % bleach solution Exposure to UV light for 30–60 minutes will further reduce DNA biological samples or uV radiation contamination, but not fully eliminate it from the pipette surface. • Pipetting aqueous samples. Aqueous solutions • Avoiding cross-contamination. and buffers 2. use a MICROMAN pipette with Disassemble the lower part of your pipette. Rinse the sterilized capillary pistons when faced Acids/alkalis Water cleaning contaminated parts thoroughly with distilled water and dry. with the following situation: Disassemble the lower part of your pipette. Fully immerse the Figure 14 Organic solvents contaminated parts* into an ultrasonic bath with a detergent • Working under sterile conditions. Sample Carryover Detergent—cleaning or cleaning solution recommended for laboratory instruments. Proteins solution Rinse the pipette several times with water and dry it thoroughly. • Pipetting viscous samples. • Avoiding cross-contamination. If pipette brands other than Gilson are used, please make sure the material is compatible with the appropriate cleaning solutions. * Check the User's Guide for specific parts to clean by immersion.
Autoclaving This is a common method of sterilization. PIPETMAN DIAMOND Tips and 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.
* PIPETMAN parts can be autoclaved according to the User's Guide for the pipettes. Refer to your model user's Guide for the defined parts and the recommended conditions.
33 PREVENTING CONTAMINATION | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 34 Chapter 5 PIPETTE SERVICE MAINTENANCE PIPETTE SERVICE MAINTENANCE PIPETTE SERVICE MAINTENANCE SERVICE PIPETTE Pipette Specifications According to ISO 8655 The ISO 8655 standard gives the accuracy and precision limits as both absolute and relative values. Specifications will depend on the technique used (air-displacement, positive-displacement, repetitive pipettes).
What are Published Manufacturer Specifications? Specifications are established by the manufacturer. They guarantee, in terms of accuracy and precision, the performance of all pipettes of a given brand and a given model at a certain volume setting.
EXAMPLE OF MAXIMUM PERMISSIBLE MAXIMUM PERMISSIBLE ERRORS MAXIMUM PERMISSIBLE ERRORS ERROR FOR A 1000 μL PIPETTE: GILSON ISO 8655
VOLUME SYSTEMATIC RANDOM SYSTEMATIC RANDOM MODEL (μL) ERROR (μL) ERROR (μL) ERROR (μL) ERROR (μL)
100 ± 3 ≤ 0.6 ± 8 ≤ 3 500 ± 4 ≤ 1 ± 8 ≤ 3 P1000 1000 ± 8 ≤ 1.5 ± 8 ≤ 3
These specifications are defined for pipettes used in forward mode. The gravimetric method is used with the temperature of the distilled water and all other conditions stabilized between 15°C and 30°C. The values given include all components of error due to both normal handwarming and the changing of the tip.
NOTICE in order to comply with the ISO 8655 standard, the specifications of the pipette must be within the maximum permissible errors.
Repair in the Lab or Return for Service?
The conformity to the acceptable maximum permissible errors should be tested at least once a year (ISO 8655-1). If you do not have the required equipment or if the pipette fails a performance check, return the pipette to your Local Your pipette is more than one year old, and records Gilson representative for service. Between service periods, show that it has not been serviced within the past 12 Gilson recommends performing a two-minute inspection months. (Refer to quICK PIPETTE DIAGNOSIS on page 37).
For models other than microvolume pipettes (from 2 to 10 μL) you have identified damage to the Spare parts may be ordered from your Local Gilson push button, connecting nut, piston seal, O-ring, tip representative. These parts can be replaced on site holder, or tip ejector. without any impact on the performance of your pipette.
For all other damage, and for microvolume pipettes (from 2 to 10 μL). Return the pipette for service.
35 PIPETTING SERVICE MAINTENANCE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 36 Quick Pipette Diagnosis How to Calculate Volumetric Accuracy and Precision “Accuracy” and “precision” are qualitative terms. The corresponding quantitative terms are Pipette Maintenance “systematic error” and “random error”. Conversion from weight to volume must be calculated first.
Regular maintenance and service of your pipette willl ensure reliable results. Refer to your MAINTENANCE SERVICE PIPETTE product User's Guide for manufacturer's recommendations. Evaluation of Accuracy Evaluation of Precision TWO-MINuTE INSPECTION The specified accuracy is the limit to the The specified precision is the limit to the Use the PIPETMAN Quick Inspection video and the Two-Minute Inspection poster to diagnose systematic error, which is the difference random error, which is the distribution of the defects and decide whether the pipette should be repaired on site or returned to your between the mean volume of actual measured values around a mean value. For representative for service. Direct any maintenance or service questions to your local Gilson measurements and the true pipettes, precision refers to a within-series distributor. value of the volume set on the instrument. group of data, and therefore to repeatability. Refer to the Two-Minute Inspection video and the poster for a walk through of basic pipette The systematic error (E) can be estimated as The random error is then quantified by performance evaluation. follows: the standard deviation of measurements performed at a given volume setting under Good routine maintenance helps prevent costly repairs. the same measuring conditions. The standard deviation (SD or “s”) can be estimated as follows: PIPETTESERVICE MAINTENANCE
The precision of a pipette can also be expressed as a percentage of the mean volume. This is known as relative standard deviation (RSD) or coefficient of variation (CV), and is estimated as follows: The accuracy of a pipette can be expressed as a percentage of the nominal volume:
The mean value and number of replicates must be stated, and the experimental procedure Factors affecting accuracy: used must be described in such a way that other workers can repeat it. Download Gilson Watch the quick Temperature Two-Minute (temperature difference between Inspection video Inspection poster delivery device and liquid) NOTE Sample density (affects the liquid volume www.gilson.com/Inspection_Videos www.gilson.com/resources/inspection_poster.pdf aspiratedinto the tip)
37 PIPETTE SERVICE MAINTENANCE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 38 Pipette Calibration Pipette calibration should be carried out by trained personnel on a regular basis to quantify your pipette performance. PIPETTE SERVICE MAINTENANCE SERVICE PIPETTE Calibration of Pipettes in a Quality System The main objective of pipette calibration in a quality system is to ensure that dispensing is carried out with the intended accuracy. Frequently, error limits are adopted from the manufacturer’s specifications, although far less accuracy is needed to perform the task at hand. It should be kept in mind that in an (uncontrolled) laboratory environment, the manufacturer's specifications may not be achieved. Consequently, users should define their own acceptance limits according to the application involved and ambient conditions. Another option is to use the acceptance limits stated in standards, such as ISO 8655. The actual standard specifications, and for highest accuracy the manufacturer’s specifications, should be used only when testing can be performed in a controlled environment using distilled or deionized water.
Device Requirements and Test Conditions PIPETTESERVICE MAINTENANCE An analytical balance must be used. The scale graduation value of the balance should be chosen according to the selected pipette volume. The ISO 8655 standard states the accuracy and precision limits as both absolute and relative values. The values are specified for fixed single channel air-displacement pipettes. With variable volume pipettes, the nominal volume is the maximum selectable volume. The μL limit of the nominal volume applies to every selectable volume throughout the volume range. For example, for a 10–100 μL pipette the maximum permissible accuracy limit (systematic error) is 0.8 μL and the maximum permissible precision limit (random error) is 0.3 μL. With multichannel pipettes these values are further doubled.
Procedure to Check Calibration The pipette is checked with the nominal volume (maximum volume), approximately 50% of the nominal volume and with the minimum volume specified by the manufacturer or 10% of the maximum volume, whichever is higher. If the calculated results are within the selected limits, the adjustment of the pipette is correct.
For more details regarding pipette service, download Gilson Verification Procedure
39 PIPETTE SERVICE MAINTENANCE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 40 Calibration with the Gravimetric Method Some Remarks about Balances Estimation of the Z Factor (Conversion Factor) With modern analytical balances, a The gravimetric method is recommended • Barometer with a standard uncertainty of laboratory needs only two balances to check The Z factor is not just equal to the density of by pipette manufacturers and international max 0.5 KPa an entire stock of pipettes ranging from water adjusted to the local temperature and
standard organizations (ISO 8655). It is A calibrated barometer to check the MAINTENANCE SERVICE PIPETTE 0.1 μL to 10 mL. A good combination would pressure parameters. It must also take into based on the determination of the weight atmospheric pressure. be one six-digit balance and another one account the air density and the density of the of water samples delivered by the pipette. that works on two scales, for example 50 g weights used to calibrate the balance. • Distilled water Implementation of this method requires with sensitivity 0.01 mg and 200 g with Use distilled or deionized water conforming For very low volumes, application of the the strict monitoring of environmental 0.1 mg sensitivity. Z factor may not affect the final result. conditions and the systematic use of to grade 3 as specified in ISO 3696, The test balances should be calibrated, adequate and controlled equipment. degassed or air-equilibrated. The water shall The detailed formula as well as the table indicating the be at room temperature. maintained, and approved by the national Z factor to be taken into account are provided in department of weighing and measurements. APPENDIX B on page 47. General Considerations • Balances To minimize vibration, the balances should Laboratory balances required for the be set up on a marble table. Keep the Gilson pipettes are designed to compensate test should meet or exceed the following Estimation of the E Factor for the effects of normal handwarming balance area free of drafts and the ambient (Evaporation Loss) performances: area free of dust. during the test series. However, the Evaporation that occurs during the gravimetric instrument being evaluated must not be test depends mainly on temperature, over-warmed by extensive handling. SELECTED STANDARD REPEATABILITY humidity and cycle time of work. It may VOLUME BALANCE UNCERTAINTY From Weight to Volume AND PIPETTESERVICE MAINTENANCE (V) OF RESOLUTION OF have a noticeable effect on small volume LINEARITY APPARATUS MG MEASUREMENT Conversion to volume must take into The temperature MG UNDER TEST MG measurements (50 µL or less). 21.5°C should be 21.5 ± 1.5°C 70.7°C account the density of the liquid as well as (293–296 K) evaporation during the cycle time. For each Evaporation loss is estimated by running a ± 1.5 ± 2.7 1 μL < V ≤ 10 μL 0.001 0.002 0.002 measurement, the corresponding volume series of four simulated weighing cycles and 10 μL < V ≤ 100 μL 0.01 0.02 0.02 calculating the mean weight loss per weighing (Vi) can be calculated as follows: Relative humidity should be cycle in mg. Perform each weighing cycle 100 μL < V ≤ 1000 μL 0.1 0.2 0.2 maintained at 50%–75% Note: for measurements higher than 50 μL, the without adding the aspirated liquid to the in order to reduce evaporation factor can be disregarded. 1 mL < V ≤ 10 mL 0.1 0.2 0.2 vessel. 50-75%the evaporation rate and 50-75% to control the build-up of For your reference, a complete example is given in APPENDIX A on page 45. Dispense the liquid into a dummy vessel. The electrostatic charges. mean evaporation e, is calculated as follows: • Vessels Test equipment should correspond to the If the pipettes are used and therefore following indications: checked outside these conditions, the NOTE weight of the setting volume of water EXAMPLES aspirated will have to be corrected SAMPLE WEIGHING BALANCE OTHER is the weight as read on the balance OF VOLUMES according to the conversion RESERVOIR VESSEL RESOLUTION EQUIP. INSTRUMENTS table (μL/mg). See appendix. A procedure for the determination of e is given in is the mean evaporating loss APPENDIX C on page 48. P2 - P20 PM x20 during the cycle time. F2 - F10 Lid M10 to M25 0.1 to Ø 35 mm Ø 10.5 mm Tweezers Recommended equipment M100 20 μL H 50 mm H 13 mm 0.001 mg Filters • Calibrated thermometer with a standard P100 - P200 uncertainty of max 0.2°C F25 - F200 PM x300 > 20 to Ø 35 mm Ø 21 mm A calibrated thermometer readable to 0.1°C M50 - M250 200 μL H 50 mm H 50 mm 0.01 mg Lid to measure both ambient and water
temperatures at the beginning and at the P1000 - P5000 end of the test series. F250 - F5000 > 200 to Ø 50 mm Ø 35 mm M1000 5000 μL H 70 mm H 50 mm 0.1 mg Lid • Hygrometer with a standard uncertainty
of max 10% > 5 to 250 mL Ø 40 mm A calibrated hygrometer to check the P10 mL 10 mL beaker H 100 mm 0.1 mg Lid constant of humidity in the air during the test.
41 PIPETTE SERVICE MAINTENANCE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 42 Performance Check Procedure Pour distilled or deionized water from For samples below or equal to 50 μL, 1 the container into the weighing vessel 13 estimate evaporation loss by repeating to a depth of at least 3 mm. steps 8 to 10 exactly as a normal When to Perform the Test? sample weighing but without actually adding any sample to the weighing Since accuracy and precision have a direct influence on the quality of analytical results, MAINTENANCE SERVICE PIPETTE
it is imperative that the performance of individual pipettes be compared regularly with Record the test conditions (ambient container. Record absolute value (ei) manufacturer’s specifications. 2 and water temperature, relative and repeat several (m) times. humidity, barometric pressure). Gravimetric analysis is a practical, widely used method for testing the performance (accuracy and precision) of a pipette. Record test conditions. Check that Select the test volume of your values are still within recommended FREQUENCY* CONTROL ACTION WHO 3 variable-volume piston pipette. 14 limits.
Leak test (see the Preventive Two-Minute Inspection Daily maintenance poster) End-users 4 Fit the tip or capillary/piston Use the average of the first and the assembly to the pipette (the second values of temperature and Accuracy check manufacturer's specifications are Cleaning & Diagnosis: 15 barometric pressure to determine the Weekly to up Preventive - Visual inspection valid only when the test is performed correction needed (Z). to every three months maintenance - Function check End-users with the manufacturer's tips). Refer to APPENDIX B on page 47.
PIPETTESERVICE MAINTENANCE Replacement spare parts of first level (seals/O-rings, Gilson Authorized Pre-wet pipette tip five times to tip holder…) Service Center Calculate the accuracy and the Adjustment - Calibration & End-users 5 reach humidity equilibrium in the dead volume of the pipette, but do 16 precision and compare with not take into account for calculations. manufacturer’s or ISO 8655-2 Replacement spare parts specifications. (To calculate accuracy of second level (Piston, and precision, refer to HOW TO Complete volumeter, operating rod) Gilson Authorized Annually maintenance Adjustment - Calibration Service Center 6 Change tip. CALCuLATE VOLuMETRIC ACCuRACY AND PRECISION on page 38.)
* Frequency should be adapted to the type of sample, the number of pipetting tasks and the environmental conditions 7 Pre-wet the tip once. of the laboratory.
NOTE Always check your pipette for mechanical faults (Refer to TWO-MINuTE INSPECTION 8 Pipette the test volume. on page 37) before performing a gravimetric test.
9 Determine tare mass (reset balance).
Open balance door, retrieve weighing 10 container, deliver sample, replace on the balance and close the door.
After allowing display to stabilize, 11 record the weight.
Repeat the test cycle until ten 12 measurements have been recorded as
a series of weights W1 to W10.
43 PIPETTE SERVICE MAINTENANCE | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 44 Nominal Value* Reproducibility* Rounded or (of Results of Measurements) APPENDICES approximate value Measurement precision under reproducibility of characterizing conditions of measurement.
quantity of a measuring APPENDICES 1 100 µL instrument or measuring 0 Sample Appendix A: Pipetting Terms 0 system that provides guidance for its The appropriate Accuracy* appropriate use. representative part of APPENDIX Examples: a liquid to be analyzed. Closeness of agreement Measurement Error* a) 1 L as the value marked on a The term “test sample“ is between a measured Measured quantity value minus a reference single-mark volumetric flask, quantity value and quantity value. b) 100 μL as the setting used when necessary to a true quantity value Comment: this difference or deviation (positive or negative) appearing on the volumeter of avoid confusion with the a pipette. Sample of a measurand. may be expressed either in the units in which the quantity statistical term “random is measured (absolute error), or as a percentage of the true Note: “accuracy” is a sample from population”. value (relative error). qualitative concept. Pipette/Pipetter An instrument Random Error* for transferring a Sample Carryover Component of measurement error that predetermined Air Cushion in replicate measurements varies in an volume of liquid The portion of the Also called “dead unpredictable manner. from one vessel to sample that is retained volume”, the air cushion Notes: another. A pipetter is in the instrument after is the volume of air 1. Random error is equal to error minus systematic error, not connected to a sample delivery and that 2. Because only a finite number of measurements can Air cushion located between the reservoir. may affect subsequent be made, it is possible to determine only an estimate samples. lower part of the of random error. pipette piston and the NOTE: Carryover from a positive displacement pipette surface level of the is less than from an air- sample. Systematic Error* displacement pipette. Component of measurement error that in Precision* True value replicate measurements remains constant or Closeness of agreement True value is a value that would be obtained varies in a predictable manner. between indications by a perfect measurement. Notes: Aliquot or measured quantity 1. Systematic error is equal to error minus random error, values obtained by Measured portion of a 2. Like true value, systematic error and its causes cannot replicate measurements homogeneous entity. be completely known. on the same or similar Working range A general term referring Comment: systematic error quantifies the error of objects under specified to multiple samples of accuracy of a pipette. Total volume and temperature range, as well conditions. any solution, mixture, as ambient conditions, etc. Good Laboratory Practice for which instrument performance is Good Laboratory Pratice (GLP) is concerned Vmin Vmax with the organizational process and the Repeatability* (of Results of specified. Measurements) Note: do not select volumes conditions under which laboratory studies are Measurement precision under a set of outside recommended limits. planned, performed, monitored, recorded, and repeatability conditions of measurement. * Definitions abstracted from reported. VIM (International Vocabulary Repeatability conditions include: Dispenser of Metrology). • the same measurement procedure, An instrument for delivering Measurand* • the same operator/observer, predetermined volumes Particular quantity intended to be measured • the same measuring instrument, of liquid from a Example: vapor pressure of a given sample of water at 20°C. used under the same conditions, reservoir. The reservoir Note: the specification of a measurand may require may be integrated statements about quantities such as time, temperature, • the same location, and pressure. into the instrument or • repetition over a short period of time. connected externally. Comment: for pipetting, variations due to the operator (e.g., cycle time) are to be minimized.
45 APPENDICES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 46 Appendix B: Example of a Performance Check Appendix C: Z Factor
The reference calculation equation is: Below is an example of how to evaluate 4. Calculate the mean volume Z = [1/(PW-PA)] [1-(PA/PB)] the performance of PIPETMAN P10 at 1 μL. For a temperature of 21.5°C and an air pressure Where: P A = density of air at t°C. of 1013 hPa, the Z factor is equal to 1.0032 μL/mg APPENDICES 1. Determine the mean value ē of the PW = density of the test liquid at t°C. evaporation loss e that occurs during your (see table in Appendix II). P = density of the balance weights. Use 8 g/cc for PB i B pipetting cycles. Proceed as described in appendix III to NOTE Weights conforming to International Recommendation No. 33 of OIML have been adjusted to determine ei give results when weighing in air as if the density of the weights were 8.0 g/mL.
APPENDICES 5. Evaluate accuracy Systematic error (E): Values of the conversion factor Z (μL/mg) as a function of temperature and pressure for distilled water.
Temperature AIR PRESSURE °C HPA 800 853 907 960 1013 1067 Relative error (E%): 15 1.0018 1.0018 1.0019 1.0019 1.0020 1.0020 15.5 1.0018 1.0019 1.0019 1.0020 1.0020 1.0021 16 1.0019 1.0020 1.0020 1.0021 1.0021 1.0022 16.5 1.0020 1.0020 1.0021 1.0022 1.0022 1.0023 17 1.0021 1.0021 1.0022 1.0022 1.0023 1.0023 17.5 1.0022 1.0022 1.0023 1.0023 1.0024 1.0024 2. Change the pipette tip and perform the 6. Evaluate precision (repeatability) 18 1.0022 1.0023 1.0024 1.0024 1.0025 1.0025 first weighing. Then, keep a regular cycle Standard Deviation (SD ) 18.5 1.0023 1.0024 1.0025 1.0025 1.0026 1.0026 and perform the 10 following w 19 1.0024 1.0025 1.0025 1.0026 1.0027 1.0027 measurements. 19.5 1.0025 1.0026 1.0026 1.0027 1.0028 1.0028
Wr = 0.957 mg 20 1.0026 1.0027 1.0027 1.0028 1.0029 1.0029 20.5 1.0027 1.0028 1.0028 1.0029 1.0030 1.0030 W1 = 0.968 mg W6 = 0.966 mg 21 1.0028 1.0029 1.0030 1.0030 1.0031 1.0031
W2 = 0.960 mg W7 = 0.955 mg 21.5 1.0030 1.0030 1.0031 1.0031 1.0032 1.0032 22 1.0031 1.0031 1.0032 1.0032 1.0033 1.0033 W3 = 0.984 mg W8 = 0.972 mg 22.5 1.0032 1.0032 1.0033 1.0033 1.0034 1.0035 W4 = 0.942 mg W9 = 0.958 mg 23 1.0033 1.0033 1.0034 1.0035 1.0035 1.0036 23.5 1.0034 1.0035 1.0035 1.0036 1.0036 1.0037 W5 = 0.969 mg W10 = 0.967 mg 24 1.0035 1.0036 1.0036 1.0037 1.0038 1.0038 24.5 1.0037 1.0037 1.0038 1.0038 1.0039 1.0039
Wr rinsing measurement which is disregarded 25 1.0038 1.0038 1.0039 1.0039 1.0040 1.0041 for the calculation 25.5 1.0039 1.0040 1.0040 1.0041 1.0041 1.0042 Random error (SDv): 26 1.0040 1.0041 1.0042 1.0042 1.0043 1.0043 26.5 1.0042 1.0042 1.0043 1.0043 1.0044 1.0045 3. Calculate the mean weight 27 1.0043 1.0044 1.0044 1.0045 1.0045 1.0046 _ n 1 27.5 1.0044 1.0045 1.0046 1.0046 1.0047 1.0047 W = Wi 28 1.0046 1.0046 1.0047 1.0048 1.0048 1.0049 ∑ 28.5 1.0047 1.0048 1.0048 1.0049 1.0050 1.0050 n i=1 29 1.0049 1.0049 1.0050 1.0050 1.0051 1.0052 29.5 1.0050 1.0051 1.0051 1.0052 1.0052 1.0053 n: number of weighings 30 1.0052 1.0052 1.0053 1.0053 1.0054 1.0055
Wi weighing results W (0.968 + 0.960 + 0.984 + 0.942 + 0.969 = + 0.966 + 0.955 + 0.972 + 0.958 + 0.967) / 10 W = 0.964 mg
47 APPENDICES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 48 Appendix D: Evaporation Loss Appendix E: Chemical Resistance of Plastics
Procedure for the Determination of Evaporation Loss. Product Steel PET Nitril EPDM LCP PA PBT PC PE PVDF TPX POM PP Acetamide ++ N/A ++ ++ N/A ++ N/A N/A ++ N/A N/A ++ ++ Use the same distilled water, weighing vessel, and balance as you will be using for the gravimetric Ethyl acetate ++ + - ++ ++ ++ ++ ++ ++ ++ + N/A ++ APPENDICES check. Acetone ++ + - ++ ++ ++ ++ - ++ ++ + + ++ Acetonitrile ++ N/A + ++ + N/A N/A - ++ + N/A N/A ++
20 % ++ ++ + ++ ++ ++ N/A ++ ++ ++ ++ ++ ++ 1 Half fill the weighing vessel with distilled water. Acetic acid 50 % ++ ++ + ++ ++ - N/A + ++ ++ ++ ++ ++ 100 % ++ ++ - ++ + - N/A - ++ ++ + + ++ 10 % - ++ ++ ++ ++ - ++ ++ ++ ++ ++ ++ ++
APPENDICES Hydrochloric acid 20 % - + + ++ ++ - + ++ ++ ++ ++ + ++ Cover the weighing vessel with its lid and place it on the balance using a pair of tweezers. 2 37 % - - - ++ ++ - - + ++ ++ ++ - ++ 20 % + + - ++ + - + ++ ++ ++ ++ + ++ Hydrofluoric acid 40 % - + - ++ - - - + ++ ++ ++ + ++ Aspirate a sample. Formic acid 100 % ++ N/A - ++ ++ - + - ++ ++ N/A + ++ 3 10 % ++ ++ + ++ ++ - ++ ++ ++ ++ ++ + ++ Nitric acid 30 % ++ + - + ++ - + ++ ++ ++ ++ - + 65 % ++ - - - + - - + + + ++ - - Tare the balance and take the weighing vessel out of the balance. 20 % ++ N/A + ++ N/A - ++ ++ ++ ++ ++ + ++ 4 Phosphoric acid 85 % ++ N/A - ++ N/A - ++ ++ ++ ++ ++ - ++ 50 % ++ - + N/A N/A ++ ++ + ++ ++ N/A - ++ Propionic acid Take off the lid with tweezers. 100 % ++ - - N/A N/A + ++ - ++ ++ N/A - ++ 5 20 % ++ ++ + + ++ + ++ ++ ++ ++ ++ + ++ Sulfuric acid 50 % ++ ++ - + ++ - + ++ ++ ++ ++ - ++ 95 % ++ + - - - - - + + + ++ - + 6 Dispense the sample into a dummy vessel. 20 % ++ N/A - N/A N/A + N/A ++ ++ ++ N/A ++ ++ Trifluoroacetic acid 80 % ++ N/A - N/A N/A - N/A + ++ ++ N/A + ++ 100 % ++ N/A - N/A N/A - N/A - ++ ++ N/A - ++ Benzyl alcohol ++ ++ - N/A N/A + N/A - ++ ++ ++ - ++ 7 Replace the lid on the weighing vessel and, using tweezers, replace the vessel on the balance. Aniline ++ - + ++ N/A ++ N/A - + ++ N/A + ++ Butanol / Butyl alcohol ++ ++ ++ ++ N/A ++ ++ ++ ++ ++ N/A ++ ++ Chloroform ++ - - - N/A + - - + ++ + + - 8 Read the negative result e1 (record the absolute value). Cyclohexane ++ ++ ++ - N/A ++ N/A ++ ++ - + ++ + Diacetone alcohol ++ ++ + N/A N/A N/A N/A N/A N/A + N/A N/A ++ Methylene chloride ++ + - - N/A - - - + ++ ++ ++ + 9 Repeat steps 3 to 8, three times to obtain e2, e3, and e4. Diethylene glycol ++ N/A ++ ++ ++ N/A N/A N/A ++ ++ ++ ++ ++ Dimethylformamide (DMF) ++ ++ - + ++ ++ ++ - ++ - ++ ++ ++ Dimethylsulfoxide (DMSO) ++ N/A - N/A N/A + N/A - ++ N/A N/A N/A N/A Dioxane (1,4) ++ ++ - + N/A ++ ++ - ++ + N/A ++ + 10 Calculate the evaporation loss ee using the formula: Ethanol ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ Ether ++ ++ ++ + N/A ++ ++ ++ + ++ + ++ ++ Formaldehyde ++ ++ ++ ++ N/A ++ N/A ++ ++ ++ ++ ++ ++ Hexane ++ N/A ++ - + ++ ++ ++ + ++ + ++ ++ Hydrogen peroxide 50 % ++ N/A + ++ N/A ++ ++ ++ ++ ++ ++ ++ ++ Ammonium hydroxide 20 % ++ ++ ++ ++ N/A N/A + - ++ N/A ++ ++ ++
10 % ++ + ++ ++ ++ ++ + - ++ ++ ++ ++ ++ Sodium hydroxide Under normal conditions, this value is usually between 0.01 mg and 0.03 mg. 40 % ++ - + ++ ++ ++ + - ++ ++ ++ ++ ++ Sodium hypochlorite 15 % Cl + N/A + ++ ++ ++ ++ ++ ++ ++ ++ - + Methanol ++ ++ ++ ++ + ++ ++ + ++ ++ ++ ++ ++ Methyl ethyl ketone ++ ++ - + ++ ++ ++ - ++ - + + ++ Pentane ++ N/A ++ - N/A N/A N/A ++ ++ ++ + ++ N/A Tetrahydrofuran (TH-) ++ ++ + + + ++ + - - + + N/A + Urea ++ ++ N/A N/A - ++ ++ N/A ++ ++ N/A ++ ++
PET = Polyethylene Terephthalate TPX = Polymethylpentene Nitril = Nitrile POM = Polyoxymethylene EPDM = Ethylene Propylene PP = Polypropylene LCP = Liquid Cristal Polymer ++ No chemical degradation PA = Polyamide + Medium resistance to chemical agents PBT = Polybutylene Terephthalate - Low resistance to chemical agents PC = Polycarbonate N/A No data available PE = Polyethylene PVDF = Polyvinylidene fluoride
49 APPENDICES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 50 FAQ Question: I have to pipette 100 μL, Question: Tips do not fit well. What NOTES several pipettes can be used, which one should I do?
should I choose? NOTES Answer: Answer: • Always use Gilson brand tips for Gilson FAq • According to the properties of the liquid pipettes. to be handled, air-displacement or • Push upward on the tip ejector to make positive-displacement should be chosen. sure it is positioned properly. • P100 is recommended for best precision. • Clean the tip holder with alcohol. If it is P200 can be used, but make sure that worn or has been chemically attacked, specifications are adapted to your order a new part from your Local Gilson protocol. representative. Question: When should I pre-rinse the tip? Question: What is accuracy? Answer: Answer: Pre-rinsing should be performed: Closeness of agreement between a • every time you change a tip, measured quantity value and a true quantity value of a measurand. • every time you increase the volume Notes: “accuracy” is a qualitative concept. setting. Definition abstracted from VIM Question: How can I prevent piston (International Vocabulary of Metrology). corrosion? Question: What is precision? Answer: After contact with corrosive liquids, a piston should be cleaned with Answer: alcohol and a soft tissue. Take care to Closeness of agreement between avoid shocks or scratches. indications or measured quantity values Use: obtained by replicate measurements on the same or similar objects under specified • filter tips, conditions. • corrosion protection kit, Note: Definition abstracted from VIM (International Vocabulary of Metrology). • positive-displacement pipette.
Accurate but not precise Precise but not accurate Accurate and precise
51 APPENDICES | GILSON GUIDE TO PIPETTING GILSON GUIDE TO PIPETTING | USER’S GUIDE 52 NOTES
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