1. Detailed Technical Information
‘Air-displacement pipettes are used to perform so many analytical methods that they are often taken for granted. Pipettes are complex precision instruments subject to error due to mechanical failure and improper operator technique. Pipettes may contribute more inaccuracy and imprecision to laboratory results than any other single source (Conners and Curtis 1999). In the forensic laboratory, where data integrity must be above reproach, it is vital for criminalists and managers to understand and address the likely sources of error related to pipette function and technique.’
Abstract from
Eliminating Sources of Pipetting Error in the Forensic Laboratory
Forensic Science Communications October 2003 – Volume 5 – Number  4
David M. Epstein, Ian R. Tebbett, Shannon E. Boyd.

The abstract above is an excellent description of how a pipette is perceived in the working laboratory. Its importance to the reliability of data is fundamental and both regular preventative maintenance and calibration have to be carried out in order to eliminate the error due to the use of a pipette.

Types of Pipettes

The main types of pipette are shown below. The manual single channel pipette air-displacement pipette, where there is a cushion of air between the liquid and the pipette contains one channel and is available as a fixed or variable volume model.
The manual multi channel pipette is available as an 8, 12 or 16 channel model and is used to pipette into well plates. This is usually available as a variable volume model.
The electronic pipette, usually available as a variable volume model, in both single and multichannel options has the added advantage that it has a higher precision than a manual pipette due to its highly repetitive plunging and deplunging action and also it combats RSI among its users. However, the main disadvantage is that they are more expensive than manual pipettes
The positive displacement pipette, which has the piston in contact with the sample, is useful for delivery of viscous and high vapour pressure liquid samples. These pipettes are available where the piston is reused after each liquid delivery or where the piston is discarded after liquid delivery. The choice of pipette type used depends on what medium your sample is contained in, and what are you using your pipette for.

Pipette Operating Principles

When the push-button is pressed on an air-displacement pipette (A), the piston inside the instrument moves down to let air out resulting in air being displaced by the piston.
On placing the tip of the pipette into the liquid and the push button being released, a vacuum is created resulting in the liquid being drawn into the pipette due to atmospheric pressure.
To expel the liquid the push button is pressed, resulting in an increase in air pressure within the tip and expulsion of the liquid from the pipette.
The main advantage of air displacement pipettes is that the sample and the piston never come into contact due to the cushion of air between them so cleaning of the pipette between samples is unnecessary, unless there is an operator error whereby which the liquid is accidentally aspirated into the body of the pipette.

Air Displacement and Positive Displacement working principle

The positive displacement pipette (B) is used when handling liquids such as oils, emulsions, high vapour pressure liquids (e.g. Acetone) or liquids prone to foaming. The piston is in direct contact with the sample.
Positive displacement pipettes work on a principle like that of a syringe. After the volume is set the push button is pressed and the tip placed into the liquid.
The push button is released which results in the liquid being drawn into the pipette again due to atmospheric pressure. Pressing of the push button expels the sample from the pipette.
The main advantage of positive displacement pipettes is that the results are reproducible, regardless of the pipetting speed, fluid viscosity or vapour pressure.

Pipetting Techniques

There are two main modes for the drawing and expulsion of liquids from pipettes. These are known as the forward (normal) and reverse mode of pipetting.

Forward Mode

1. Depress the plunger to the first stop (a) using the thumb and place the tip of the pipette 2-3mm into the liquid making sure that the pipette is held in a vertical position.
2. Slowly, steadily and consistently retract the plunger and remove the thumb when the plunger has retracted to the upper stop and hold the tip in the liquid for one second (b). Slowly withdraw the tip and wipe any drops on the tip on the walls of the vessel the liquid is drawn from. This completes the aspiration step
3. In order to dispense the liquid, hold the tip at an angle of around 30-45º against the wall of the receiving container. Depress the plunger to the first stop (c), hold for one second and then push the pipette to the second stop (d) while sliding the pipette against the walls of the container. This second depressing of the plunger is known as the “Blow out” function. Releasing the plunger at this point returns it to its uppermost position (e).

The depressing and releasing action can be summarised in the following diagram.

Forward Mode

Reverse Mode
The reverse mode is used when working with, viscous and volatile liquids.

• Depress the plunger to the second stop and place the tip 2-3mm into the liquid, remembering to hold the pipette vertically.
•  With the pipette in the vertical position slowly, steadily and again consistently, retract the plunger and remove the thumb when the plunger has retracted to the upper stop. This may take a little time when using viscous liquids. Again wipe any drops on the outside of the tip on the vessel wall.
• In order to dispense the liquid, depress the plunger to the first stop only, while whipping the tip against the wall of the vessel. To ensure that the correct volume is delivered the liquid remaining in the tip should be discarded with the tip. Releasing the plunger at this point returns it to its uppermost position (d).

Reverse Mode

Why calibrate pipettes?

Calibration of a new pipette takes place before delivery. When the pipette leaves the factory it satisfies the manufacturer specifications in regards to the volume, as well as the accuracy and precision the manufacturer defines for its pipettes. The calibration of pipettes is governed by the standard EN ISO 8655 1-6:2002; EN ISO 8655 7:2005. This standard details the terminology, general requirements, user recommendations and protocols for the calibration of Piston Operated Volumetric Apparatus (POVA).
Every pipette is subject to a normal wear and tear. Faults may appear without notice at any time.  Leaks and wear of moving parts cannot easily be determined/observed and may influence the result and lead to significant errors. Changes in environmental conditions (the main environmental conditions that affect pipette performance are temperature, humidity and atmospheric pressure) or the varied techniques of operation by different users may lead to deviations.
Regular calibration regimens should be implemented to ensure a minimisation of these faults. The calibration sequence calculates both the accuracy and precision. If the pipette is not within specifications it must be adjusted, eventually damages will be recognised immediately and can be fixed at the same time.
Only by calibrating a pipette regularly, can its actual condition be identified and documented in a traceable way.

2. Guidance for Better Pipetting
(1) Pre-wet the pipette tip.
Aspirate and expel an amount of the sample liquid at least once times before aspirating a sample for delivery. There is a difference of up to 10% in the volume taken up by a “dry” tip than a “wet” tip. Source: Royal Free Campus, London.    

(2) Work at room temperature.
Allow liquids and equipment to equilibrate to ambient temperature.
           
(3) Aspirate your sample with the pipette held in the upright position and dispense the sample at and angle of 30-45º into the receiving container.
Aspiration at an angle up to 30º can result in a 5% volume difference between measured and delivered values.

(4) Use Forward Mode pipetting.
Use ‘reverse mode’ pipetting only for viscous samples.
           
(5) Pause consistently after aspiration.
Pause for a moment with the tip still in the liquid after aspirating.
                       
(6) Place the pipette back on its rack between samples.
Placement of the pipette on the rack avoids transfer of body heat and ensures the pipette will not be damaged.

(7) Ensure immersion of the tip to the correct depth.
ISO 8655: 6 (2002) Section 7.2.5 (b) suggests immersing the tip 2-3mm into the liquid to be aspirated.

 (8) Employ the correct manufacturer’s tips of those recommended by the manufacturer.
Securely attach a tip designed for use with the pipette. Press the tip on firmly.

(9) Carry out a consistent rhythm while pipetting.
Depress and release the plunger smoothly and consistently for each sample.

(10) Calibrate the pipette regularly.
This reduced error and improves downtime.

3. Measurement Uncertainty
An exact measurement does not exist. There is therefore a need to give some measure of the reliability and the uncertainty of measurement of the results calculated. The main concern of a metrologist is to estimate the size of the systematic errors compared to the random errors.
It can be defined as a parameter, associated with the result of a measurement, which characterises the dispersion of the values that could reasonably be attributed to the measurand.
There is no point in quoting how accurately a measurement was taken if the equipment was not set up properly in the first place.
Errors arise naturally in the process of taking measurements and can occur from a number of factors. Typical causes of errors include those arising from:

• Mistakes – e.g. recording an incorrect reading.
• Human factors – errors arising from the skill of the operator.
• Instrument limitations.
• Changes occurring during observation – e.g. trying to read a fluctuating meniscus on a manometer.
• External factors – e.g. drafts while weighing.

Types of Uncertainty
1.     Random errors are errors that arise due to accidents, misuse or unplanned events. In the laboratory these failures can be prevented by frequent scheduled maintenance due to the fact that these failures are not dependent on predictable factors such as the frequency of usage. Random failures can occur at any point in a maintenance cycle. Research has shown that approximately 90% of errors are random.
2.     Systematic errors are those that arise from simple wear, as a function of the pipette usage and frequency of calibration. These errors can be prevented and predicted by adjusting the service cycle and calibration interval based on a review of the as-found performance history of the pipette. [American Biotechnology Laboratory, Bertermann R, 2004].
3    Personal Errors (Blunders) occur when the analytical test is not carried out correctly: the wrong chemical reagent or equipment might have been used; some of the sample may have been lost; a volume or mass may have been recorded incorrectly resulting in a transcription error; etc. It is partly for this reason that analytical measurements should be repeated a number of times using freshly prepared laboratory samples.  Blunders are usually easy to identify and can be eliminated by carrying out the analytical method again more carefully.
4. Accuracy and Precision
Accuracy: This is a measure of reliability and is the difference between the True Value of a measured quantity and the most probable value, which has been derived from a series of measures. The True Value is, of course, never known.
Precision: This is a measure of repeatability, i.e. the degree of agreement between individual measurements of a set of measurements, all of the same quantity.
Repeatability and Reproducibility are also related to accuracy and precision and are defined as follows,

Repeatability is how close a set of measurements is to each other when made at the same time on the same equipment.
Reproducibility is how close different sets of measurements are to each other when made at different times and/or on different sets of equipment of the same type.
Accuracy & Precision Visual
Pitching onto a Golf Green indicates the difference between accuracy and precision.

At CTL we hold a large library of articles under the following headings. If you require any of these documents please contact us at 
service@calibrationtech.ie

5. Measurement Uncertainty

What is Uncertainty?
UKAS Website.

GUM: The Principles.
National Weights and Measures Laboratory, UK.

European Directorate for the Quality of Medicines (EDQM): Uncertainty of Measurement Part 1.
European OMCL Network.

Estimating Uncertainties in Testing
Keith Birch and Alex Williams, British Testing and Measurement Association.

Uncertainty of Measurement of the analysis of Glutaraldehyde in air by DNPH Filter.
Greg O’Donnell, Laboratory Services Unit, Workcover NSW, Australia.

CITAC EURACHEM Workshop on Measurement Traceability and Uncertainty in Analytical Chemistry.
Alan Squirrell ILAC Secretary.

Why do we have Measurement Uncertainty?
Matthias Rösslein EMPA – ST. Gallen / Switzerland.

Introduction to EURACHEM/CITAC Uncertainty Guide 2 Uncertainty Guide 2nd edition.
Matthias Rösslein EMPA – ST. Gallen / Switzerland.

Implementing Measurement Uncertainty in Analytical Chemistry.
S Ellison LGC.

Interpretation of ISO/IEC 17025 Requirements for Measurement Uncertainty and Traceability.
Bernard King, NARL, Australia.

Meeting the ISO/IEC 17025 Requirements for Traceability and Measurement Uncertainty APLAC Approaches.
Dr Bernard King NARL, Australia.

The Estimation of Uncertainty by the Utilization of Validation and Quality Control Data.
Greg O’Donnell and Robert Geyer Laboratory Services Unit, Workcover NSW, Australia.

6. Ergonomics

Ergonomics Risk Analysis.
Melanie Alexandre and Candace Quick, Lawrence Livermore National Laboratory, University of California.

Laboratory Ergonomics: Pipetting, microscope use and hood work.
Tamara Mitchell.

Guide to Laboratory Ergonomics.
Environment Health and Safety Office, California Institute of Technology.

Selection and Use of Pipettes.
Cindy Burt, UCLA Ergonomics.

Reducing the risk of musculoskeletal injury in healthcare laboratory technologists performing pipetting tasks.           
Project update; Neil Squire Foundation, British Colombia Institute of Technology.

Safe Operating Procedure: Laboratory Ergonomics Pipetting.
University of Nebraska, Lincoln.

7. Clinical

FDA Information

Pipettes and GMP Compliance

21 CFR 11: Electronic Records; Electronic Signatures.

21 CFR 58: Good Laboratory practice for NonClinical Laboratory Studies.

21 CFR 211: Current Good Manufacturing Practices for Finished Pharmaceuticals.

21 CFR 862: Clinical Chemistry and Clinical Toxicology Devices.

21 CFR 880: General Hospital and Personal Use Therapeutic Devices.

862.2050 Product Classification Database.

Current CLIA Regulations

CLIA- Clinical Laboratory Improvement Act.

493.1250  Condition: Analytic Systems.

493.1251  Standard:  Procedure Manual.    

493.1252  Standard:  Test Systems, equipment, instruments, reagents, materials, and supplies. 

493.1253  Standard: Establishment and verification of performance specifications.
 
493.1254   Standard: Maintenance and function checks.

1. Standard:  Calibration and calibration verification procedures. 

8 Pipette User Manuals
We have available over 100 up to date User Manuals for the most popular brands and models of pipette on the market today, such as Gilson, Eppendorf, Brand, and Cappelen etc.

9. Technical Articles

How to Choose the Appropriate Pipettes and Pipette Tips for Your Applications.
Leora Schiff, Cal Lab September/October 1997.

Gravimetric & Spectrophotometric Errors Impact on Pipette Calibration Certainty.
John P. Clark & A. Harper Shull, Cal Lab Jan/Feb/Mar 2003.

Eliminating Sources of Pipetting Error in the Forensic Laboratory.
David M. Epstein, Ian R. Tebbett, Shannon E. Boyd, Forensic Science Communications October 2003 – Volume 5 – Number 4.

Guide for the determination of the mass within the scope of reference measurement procedures in medical reference measurement laboratories.
Translation of Leitfaden für die Massebestimmung bei Referenzmessprozeduren in medizinischen Referenzlaboratorien, PTB-Mitteilungen 109 (1999) No. 5, pp. 379-383.

Performance verification of manual action pipettes, Part I and II.
Richard H. Curtis, American Clinical Laboratory 1994.

Performance verification of small volume mechanical action pipettes.
Richard H. Curtis, Cal Lab 1996.

Effects of common techniques on accuracy and precision of pipetting results.
Gail Pentheny, American Clinical Laboratory 1997.

Pipetting error: A real problem with a simple solution.
Michael Connors et al, American Laboratory News 1999.

Controlling pipette performance in the real world.
Richard H. Curtis, Cal Lab 2000.

Pipet Calibration Software: Understanding regulatory and Laboratory requirements.
David L. Bohnsack, American Biotechnology Laboratory 2002.

Pipet performance verification: An important part of method validation.
Richard H. Curtis et al, American Laboratory News 2004.

Pipet Quality Control: A microliter of prevention.
Ralph Berterman, American Biotechnology Laboratory 2004.

Evaluation of Methods for Estimating the Uncertainty of Electronic Balance. .
John P. Clark & A. Harper Shull, Westinghouse Savannah River Company.

Software Validation in Accredited Laboratories: A Practical Guide.        
Gregory D. Gogates, Fasor Inc.

International Labmate

The art of good pipetting.                                                                             
Jenni Quinn, December 2000.

The benefits of electronic pipetting.                                    
Sari Mannonen et al, 2002.

Transferpette electronic. Ergonomics-centered pipetting. 
Antonio Romaguera, 2003.

Pipette tip cone filters minimise the risk of contamination.
Sari Mannonen, 2000.

Don’t compromise on accuracy and precision-quality pipette tips are critical to optimal.
Carole Staniford, 2005.
           

Analytical Methods Committee (Royal Society of Chemistry)

Understanding and acting on scores obtained in proficiency testing schemes.
AMC Technical Brief, No.11 2002.
           
Is My Uncertainty Estimate Realistic?
AMC Technical Brief, No.15 2003.

Proficiency Testing: Assessing z-scores in the longer term.
AMC Technical Brief, 2004.

The Amazing Horowitz Function
AMC Technical Brief, No.17 2004.

General and Specific fitness functions for proficiency tests and other purposes- clarifying an old idea.
AMC Recommendation, AMCR No.2 2005.

Terminology - The Key to understanding analytical science Part 2. Sampling and Sample Preparation
AMC Technical Brief, No. 19 2005.    
           
American Laboratory

Reproducibility, reproducibility and reproducibility revisited.       
IS Krull, Oct. 2002

American Laboratory News

Surprising Statistics on Pipet Performance.
R. Pavlis, Mar 2004.

Clinical Chemistry

Improved Method for Using Eppendorf Pipettes for Accurate Delivery of Blood.
F. Medina, V. Cheong, C. Peck, T.A. Bensinger, 1977.

Technology of Manually Operated Sampler Pipets.           
Robert E Wenk and Jack A. Lustgarden, 1974.

Necessity of Prerinsing Disposable Polypropylene Pipet Tips.     
G.H. Zeman and N. S. Mathewson.

10. ISO 17025

Laboratory Self-Assessment Checklist ISO/IEC 17025.
Laboratory Accreditation Service, 2004.

A Guide to Writing a Quality Manual.
International Accreditation Service, Inc.

Individual Paragraphs of the ISO/IEC Standard 17025.
Lab Compliance.com website.

Performance Based Evaluation of Laboratory Quality Systems. An objective tool to identify QA program elements that actually impact data quality.
Sevda K. Aleckson and Garabet H. Kassakhian.

Quality Management. The Permanent Assurance of Quality.
Mettler Toledo Website.

Most Common Cited Deficiencies.
A2LA, 2002.

Lessons Learned Implementing 17025.
Dave Abell, Agilent Technologies, 2004.

Do You Really Need a 17025 Accredited Calibration?
Dave Abell, Agilent Technologies, 2003.

Accreditation or Registration?
Agilent Technologies Website.

The Four Faces of the ISO 17025 Calibration Standard
Agilent Technologies Website.

On Your Marks… ISO 17025 Calibration - essential information that will help you win
Agilent Technologies Website.

The "ISO 17025" Standard for Calibration Labs Why Calibrate?
Agilent Technologies Website.

Instrument and Equipment Documentation and Records.
FDA, 2003.

11. Balance and Calibration Weights

Terminology in relation to Balances.
Laboratories Procedures Manual, ORISE/ESSAP, 2001.

Testing Your Laboratory Balance.
IES Corporation.

Technical Guide: Laboratory Balance Calibration Requirements.
International Accreditation New Zealand, 2002.

Analytical and Top Loading Balance Use.
University and Community College System of Nevada implementing Procedure, August 2005.

Standard Operating Procedure for Calibration and Maintenance of Weigh Balances.
EPA/OPP Microbiology Laboratory, June 2002.

Commonly Asked Questions about Mass Standards.
Georgia L. Harris, Weights and Measurement Division, National Institute of Standards and Technology.

Weight Cleaning Procedures.
Excerpt from NBS handbook 145, Handbook for the Quality Assurance of Metrological Requirements, Nov 1986.