140121 pg-hdb automatic pipette verification eng pg-hdb automatic... · 2016-06-03 · 140121 pg-hdb automatic pipette verification eng.doc 5 / 15 3. Good working practice: Tips:

Instituut voor Tropische Geneeskunde Institut de Médecine Tropicale Institute of Tropical Medicine Instituto de Medicina Tropical

Nationalestraat, 155

B – 2000 Antwerpen

Practical notes

__________________________

AUTOMATIC PIPETTES

"Fast is fine, but accuracy is everything."

Wyatt Earp

Philippe Gillet, Hilde De Boeck, Jan Jacobs, [email protected] [email protected], [email protected]

JANUARY 2014

140121 pg-hdb automatic pipette verification eng.doc 2 / 15

AUTOMATIC PIPETTES

What this unit is about

This unit gives an introduction about the use and different types of automatic pipettes. It explains how to properly use automatic pipettes work and how to verify their accuracy and precision.

What you will learn:

On completion of this unit, you should:

• Mastering good practices for volume measurement

• Understand working principles of pipettes

• Be aware of common errors in pipetting techniques

• Know principles of calibration/verification of pipette.

• Know factors to consider for the calibration/verification of automatic pipette.

• Be able to perform pipette check and how to interpret results

• Factors to consider in the use of automatic pipette.

What this unit includes:

• Course notes: “Automatic pipettes”

• Pwt-presentation: “140121_PG_Calibration+VERIF PIPETTE”

• Exercise:

Pre-requirements: Be able to operate a pipette using the forward pipetting technique If you are not familiar with pipette operation, first read chapter 3 of this unit for general operating procedures and/or consult the “instructions for use” of your pipette.

Work in groups of 2 persons:

Task 1: - Verify the performance of an automatic pipette (variable volume 100 µl – 1000 µl).

o Read chapter 5 of this module o Make sure you have a necessary materials to perform pipette verification o Use the verification report (annex 2) to record obtained data o Calculate accuracy and precision, you can use the excel file on the desktop of

the lab computer, make a copy of the template for your group in a new worksheet. Excel file: “120103-PG-HDB-Automatic Pipette Check”

- Present your evaluation and comments on the pipette.

Task 2: - Answer to the post lab questions at the end of this unit (p15/15) Results will be discussed in class


Materials needed for this exercise:

• Micropipette + disposable tips

• Analytical balance and calibrated weight set (at least 0,001 g and 100g).

Volumes to check Balance sensitivity

0.5 - 10 µl ≥ 0.001 mg

10-100 µl ≥ 0.01 mg

> 100 µl ≥ 0.1 mg

• Thermometer (readable to 0.1 °c)

• Barometer (readable to ± 25 mbar)

• Distilled water (at room temperature) • Weighing vessel (10-50 times the test volume). • Calculator

• Verification form (Annex 2)

• References and further readings (1-4):

1. Blues, J.; Bayliss, D.; Buckley,M.; (2004) Measurement Good Practice Guide N° 69 –

The Calibration and Use of Piston Pipettes, NPL, UK. 2. Guide to Pipetting, Accutek laboratories.

http://www.pipette.com/public/staticpages/guidetopipetting.aspx 3. WHO (2008), Maintenance Manual for Laboratory Equipment, 2nd Edition, chapter 16 4. ARTEL (2009) – 10 Tips to improve your pipetting techniques.

http://www.artel-usa.com

All references listed can be easily downloaded on our website: www.labquality.be

Go to section: Laboratory Management > Equipment > Maintenance


AUTOMATIC PIPETTES

1. Introduction

Micropipettes with disposable tips are frequently used to measure small volume. They are available in a variety of volumes, ranging from ..5 µl to 5 ml. There are many types of pipettes, but they work on essentially the same principle: by putting pressure, a piston (or plunger) is displaced over a fixed length thereby forcing a pre-determined volume of liquid out of the orifice of the pipette (1). Two main types of pipettes are available: fixed volume or variable volume. Fixed volume pipettes are less complicated, and cost less.

Both types can be further subdivided in:

• Air-displacement pipettes: a layer of air separates the piston from the liquid. Used for standard pipetting applications

• Positive displacement pipettes: direct contact between liquid and piston. Used for PCR and DNA amplification techniques, most accurate, most expensive

• Multi-channel pipettes: several pistons and cylinders combined into one unit. Used for dosing liquids in microtitre plates In this course, we will limit us to air-displacement pipettes as they are most common type used in district laboratories.

2. Air Displacement Pipettes:

Fig. 1 Internal principle of an air displacement pipette (adapted from AccuTek Laboratories™)

Working principle:

• The piston moves to appropriate position when volume is set

• When button is pressed to first stop, the piston expels the same volume of air as indicated on the volume setting

• After immersing the tip into the liquid, the button is released which creates a vacuum that aspirates the specified volume in the tip

• When pressed to the first stop again, the air dispenses the liquid. To empty the button is pressed to the second stop (blow out)

Air displacement pipettes are highly accurate, but conditions as temperature, atmospheric pressure, gravity and viscosity of the solution may have an effect on performance.


3. Good working practice:

Tips: The best pipetting results are achieved by using tips recommended by the pipette manufacturer to ensure complete compatibility. Most manufacturers of pipettes will not recommend the reuse of plastic tips. In some situation, however, tips may need to be decontaminated, washed and reused. To reuse, the tips must be well cleaned (immediately with soap) to remove all traces of protein, then decontaminated. All traces of protein or disinfectant must be removed (rinse well), and the tips must be completely dry before to be reused.

Operation:

Forward pipetting technique (Figure 2)

1. Press the plunger gently until it reaches the first limit. (B) Until this point, the tip of the pipette must not touch the liquid.

2. Put the extremity of the tip in the liquid. (2-3mm) Hold in vertical position.

3. Release the plunger gently for the pipette to aspirate the liquid (position 2A). Wait at least two seconds before removing the pipette’s tip from the liquid.

4. Place the pipette’s tip against the wall of the receiving tube. (position 3A). Use an angle between the pipette’s tip and its wall is between 30 and 45°. If the receiving tube already contains liquid, avoid the pipette’s tip from being submerged

5. Dispense the contents of the pipette by pressing the plunger gently but firmly, until reaching the first limit (position 4B). Maintain contact between the pipette’s tip and the wall of the receiving container.

6. Press the plunger gently until it reaches the second limit on the piston’s path (position 5C). This expels any fraction of liquid still in the pipette’s tip.

7. Keep the plunger pressed at the second limit while the pipette is removed from the receiving tube.

8. Discard the pipette’s tip. To do this, press the expulsion mechanism’s button (position 6)

Figure 2 - Different steps in forward pipetting (Adapted from WHO 2008)


Operator technique has a major impact on pipetting performance. Following these ten tips (Artel, Incorporated 1998) will enable operators to obtain more accurate and precise results in laboratory conditions. The tips are listed in order of importance.

• Pre-wet the pipette tip—Aspirate and expel an amount of the sample liquid at least three times before taking a sample for delivery. This reduces the chance of air bubbles and evaporation in the tip which can cause a significant loss of sample before delivery. Pre-wetting increases humidity in the tip, thus reducing the amount of and the variation in sample evaporation.

• Work at temperature equilibrium—Allow liquids and equipment to equilibrate to ambient temperature. The volume delivered varies with air pressure, relative humidity, and vapor pressure of the sample; all of which are temperature dependent.

• Examine the pipette tip before dispensing—Wipe the tip carefully against the side of the vessel if any drops adhere. Dispense the liquid by touching the tip against the wall of a receiving vessel above the liquid vessel and at an angle of 30-45°

• Use standard-mode pipetting—For all but viscous samples, standard-mode (also called forward-mode) pipetting yields better accuracy and precision than reverse-mode pipetting. In reverse-mode, the plunger is depressed completely (e.g., past the first stop) to aspirate the sample.

• Pause after aspiration—Pause with the tip in the liquid for one to two seconds after aspirating the sample. This is important because the liquid in the tip bounces slightly when the plunger stops.

• Hold pipette in vertical position when aspiration and lift the pipette straight out—Do not touch the tip to the sides of the container. Surface tension causes the sample to vary if the exit angle varies, particularly for small volumes.

• Minimize handling of the pipette and tip—Set the pipette down between deliveries. Body heat transferred to equipment during handling disrupts temperature equilibrium.

• Immerse the tip properly—Immerse the tip 2-5mm below the meniscus and well clear of the container walls and bottom during sample aspiration, otherwise volume is affected.

• Use the correct pipette tip—Securely attach a high-quality tip designed for use with the pipette and appropriate for the size of the container.

• Use consistent plunger pressure and speed—Depress and release the plunger smoothly. Pipettes are precision instruments and give more consistent results when operated with care.

Note: Always check manufacturer’s instructions to check for any recommended deviations from the suggested practice. Only set the volume within the range specified for the pipette!

Maintenance and care

Preventing contamination A contaminated pipette or contaminated tips can cause contamination of samples. Samples or aerosols from samples can be prevented of entering the body of the pipette by:

• Check pipette at beginning and end of working, if needed wipe with 70% ethanol

• Keeping the pipette vertical when pipetting to prevent liquid running into the body

• Release the push-button slowly

• Never put the pipet on its side when there is liquid in the tip.

• Store the pipette vertically

• Avoid contamination to or from fingers by using tip ejector and tip-boxes.


4. Calibration of pipettes

Calibration means determining the difference between the dispensed volume and the selected volume. A series of pipettings is performed and accuracy and precision are calculated (see formulas sections below). Inaccuracy is the difference between the dispensed volume and the selected volume of a pipette. Precision refers to the repeatability of the pipettings. It is expressed as standard deviation (s) or coefficient of variation (cv). If the calculated results are within the limits, the pipette calibration is correct. If not, the pipette has to be adjusted and checked again. An adjustment means altering the pipette so that the dispensed volume is within certain specifications. Instuctions for adjustements can be founf in the manufacturer’s “Instructions for use”.

When verifying the performance of an instrument, pipetting must be repeated at least 10 times to estimate accuracy and at least 30 times to estimate within-run precision. For subsequent control evaluations, the estimate for within-run precision should be made after pipetting at least 10 times, and the estimate of accuracy after pipetting at least 4 times.

Precision = reproducibility

Check by performing several runs

Accuracy = correctness

Check by using a different method

Fig. 2 Distribution data of pipette calibration (adapted from AccuTek Laboratories™)

Determining the frequency of calibration

There are two main factors to consider when setting up a calibration program. First, you must find the Mean Time Before Failure (MTBF). This is the average rate at which a failure will occur. Take a group of pipettes and determine how long it takes for each one to fail. After you have the MTBF, you can estimate how long a pipette can be expected to stay within a certain accuracy and precision.

Secondly, you must establish a level of target reliability of liquid delivery for the pipette. This reliability level is expressed as a percent, for example, 95% reliability means that 95% of the times, 95% of the pipettes in your lab are operating correctly.

The above two values along with a consideration of frequency of use, storage and mandates of your specific lab, will serve as a basis for determining your calibration frequency. The FDA (cGLP, cGMP, and QSR) requires that test equipment in all development and manufacturing processes involved in measurement be checked for performance.

But, the precision of the pipetting should be evaluated periodically (e.g. each 3 or 6 months), to ensure the accuracy of results.


5. Calibration/Verification Procedure

The general procedure is based on gravimetric analysis of water samples delivered by the instrument. If need, the values are corrected for evaporation. True mass and volume are then calculated simultaneously, based on the density of water at specific temperatures, and corrections for air buoyancy

Objective: This procedure provides a method for testing the (in)accuracy and the (im)precision of automatic micropipettes (fixed or variable volume pipettes).

Requirements:

• Distilled water (at room temperature)

• Analytical balance and calibrated weight set (at least 0,001 g and 100g).

Volumes to check

Balance sensitivity

0.5 - 10 µl ≥ 0.001 mg

10-100 µl ≥ 0.01 mg

> 100 µl ≥ 0.1 mg

• Thermometer (readable to 0.1 °c)

• Barometer (readable to ± 25 mbar)

• Weighing vessel (10-50 times the test volume). The location should be out of direct sunlight, free of drafts and vibrations.

Procedure Prior to beginning your verification, be sure that all components, pipettes, tips, balance and test liquid are temperature stabilized. Volume errors may occur because of changes in air displacement.

Verification Data need to be recorded on a verification Report. (Annex 2)

1. Determine the volume at which you will calibrate Typically it’s at least two points, preferably 3 points. For variable volume pipettes, verification and calibration should be done at the highest, the smallest, and the mid-value.

2. Select the desired mode, direct (or reverse), pipetting.

3. Measure and record the temperature of the water to 0.1 °C, before and after the weighing procedure. The temperature (T) is the average of the two measurements of water temperature, rounded to the nearest 0.5 °c.

4. Measure and record the atmospheric pressure.

5. Deliver a total of n samples into a weighing vessel and weigh each sample after delivery. Replicate as precisely as possible all motions and time intervals in each sampling cycle. Use a randomly selected pipette tip either only once for each sample weighing or repeatedly for the n weighings (in this case pre-wet your pipette tip 3 times).

6. Estimate evaporation if: - Balance without built-in taring (which allows rapid measurement), and/or

- Volume < than 50 µl, and/or - Relative humidity < 55 %

Then: Dispense volume (1.) into the weighing vessel, note the reading. Then duplicate all motions and time intervals as in normal pipetting, with the exception that no more liquid is added to the weighing vessel. Check how much the reading decrease. Use the resultant mean loss of weight as the correction value for evaporation.


Calculations

Calculate the mean volume (Vm) delivered at the test temperature (T) from the mean weighing result (xm) by adding the mean evaporation (e) and correcting the sum by an appropriate factor that allows for density and buoyancy corrections when water is weighed in air, at the test temperature and pressure, and standard humidity.

7. Calculate the mean weight (xm) from the individual weighings (xi): xm = [Σ xi] / n where n = number of samples.

8. If need, calculate the evaporation (e) from the number of determinations as follows:

e = [Σ ei] / ne where ei = individual determination and ne = number of control blanks.

9. Look up Z value = conversion factor (pl/mg) in function of temperature (T) and pressure (P)

(Values of Z for distilled water, at various test temperatures, are listed in Annexe 1)

10. Calculate the mean volume of the liquid samples (V) from the mean weight (xm):

Vm = (xm + e) x Z 11. Calculate the percentage accuracy (A%) of the instrument at the test temperature (T) as the

difference between the nominal volume of the instrument (Vo) and the calculated mean volume Vm:

A% = 100 x [(Vm - Vo) / Vo]

12. Calculate the standard deviation from the distribution of the individual weighings (xi) about

their mean (xm):

s = Z √ {[ Σ (xi-xm)²] / (n-1)} where Z = conversion factor (µl/mg) at the test

temperature (T)

and n = number of samples.

13. Calculate the within-run imprecision (coefficient of variation, CV) from the distribution of the

individual volume (Vi) about their mean (Vm):

CV % = 100 x s / Vm


Target values (or tolerances) for Labsystems®, Finnpipette color® :

Pipette Type

volume (µl)

Accuracy in % (A %)

Coefficient of variation in %

(CV %)

Tip immersion in the liquid

(in mm)

5-40 µl 5 ± 3 % ± 2,5 % 1 mm

40 ± 0,6 % ± 0,5 % 2 – 3 mm

40-200 µl 40 ± 1,1 % ± 0,8 % 2 – 3 mm

200 ± 0,6 % ± 0,4 % 3 – 6 mm

200-1.000 µl 200 ± 0,9 % ± 0,6 % 3 – 6 mm

1.000 ± 0, 5 % ± 0,3 % 3 – 6 mm

1.000-5.000 µl 1.000 ± 1,5 % ± 0,5 % 3 – 6 mm

5.000 ± 0,6 % ± 0,2 % 6 – 10 mm

Once you determine the inaccuracy and imprecision of your pipette you may determine you need to make adjustments to the unit. Follow the manufacturer’s instructions for adjustment.

Error can be caused by poorly trained personnel, method error such as using an adjustable volume pipette at the very lowest end of its range, a malfunctioning instrument or a poor quality tip.


ANNEXE 1 : Z value for distilled water


ANNEXE 2 : Pipette verification form cf. excel file : 120103-PG-HDB-Automatic Pipette check

Variable pipette is controled at smallest, highest and midvalue

Normally 10 measurements on every volume

Pipette type : . . . . . . . . . .N°…………

Name : . . . . . . . . . . Balance :

Date : . . . . . . . . . . Manufacturer :

Temperature : Start :. . . . . .End :….. . . Serial Number :

Pressure : . . . . . . . . .

e : …………..

Z : . . . . . . . .

N Volume ml (Vo) Weight g (xi) xi-xm (xi-xm)² Volume* 1

2

3

4

5

6

7

8

9

10

n = 10 Xm= * Volume in ml = (xi+e) x Z

xm= mean weight; Vm=mean volume

e = evaporation in mg during the reading time e may be considered as 0 if V0 > 50 µl and if relative humidity is > 55 % (and if balance with built in tarring system)

Inaccuracy A% = [100 x (Vm-Vo)] / Vo = %

Standard deviation s = Z √ {[ Σ (xi-xm)²] / (n-1)} = %

Coefficient of variation CV % = ( 100 x s) / Vm = %


ANNEXE 3: Example of pipette Instructions of use



Post lab Questions / write up :

1) Why are you required to use ddH2O to perform these calibration or verification measurements? If you use tap water, would that affect the random or systematic error of your measurements? Explain.

2) You are asked to repeat your measurements at each of three volumes (at least) six times.

• What is the advantage in making a limited number of replicate analyses rather than a single analysis?

• How can you use your data at three different volumes to assess any systematic error? 3) Think of and describe several sources of error, both random and systematic, that would result

in your pipetting the incorrect volume. 4) How precise were your measurements? Explain/Prove it with your numbers.

5) How accurate were your measurements? Explain/Prove it with your numbers.

6) Was this variation due to random or systematic error? How do You know? 7) Define for your pipette adjustments need.

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140121 pg-hdb automatic pipette verification eng pg-hdb automatic... · 2016-06-03 · 140121 pg-hdb automatic pipette verification eng.doc 5 / 15 3. Good working practice: Tips: