Pipetting & Calibration

Norway June 2008

2
Pipettors calibration & Service
History
Engaged in metrology and calibration
2th company in the world with ISO 17025-accreditation
Over 10 years of know-how
75.000 pipette-calibrations a year.
Customer focused
Provide choice
Onsite
ISO 8655 or customer driven
Specifications
Quality focused
ISO 17025 accreditated
SOPs based
Known uncertainty budget
Skilled technicians
Pipettors calibration & Service
ISO 17025 - accreditation
Multilateral agreement
Country Body Country Body
Austria BMWA Lithuania LA
Germany DKD Netherlands RvA
Czech Republic CAI Norway NA
Denmark DANAK Portugal IPQ
Estonia EAK Romania RENAR
Finland FINAS Slovakia SNAS
France COFRAC Slovenia SA
Belgium BKO Greece ESYD
Spain ENAC Latvia LATAK
Sweden SWEDAC Ireland INAB
Switzerland SAS Italy SINAL
United Kingdom UKAS
Standard for all countries is ISO 17025
Customer made calibration / ISO 8655;
Gravimetric method;
Complete programme;
As found calibration;
Maintenance;
Calibration;
Adjustment;
Repair
Multichannels, measurement of all channels;
Qualified technicians;
Validated software;
ISO 17025 calibration-certificates;
Warranties responsibility;
Due time reminder system.
Pipettors calibration & Service
Pipettes performed 2007:
59.812 pieces;
Coverage: Europe
Manufacturer: 113x
Problem solved: 98,2%
Rejection: 1.913x
Turnaroundtime: 3,2 days
Brand deviation
40%
18%
16%
7%
6%
1%
3%
2%
7%
Gilson
Finnpipette
Eppendorf
Biohit
Socorex
Brand
Rainin
VWR
Others
Pipette-Calibration at Competence Center
8
67 Headcounts
Technicians: 48
Quality: 4
Dispatch / administr: 7
Logistic: 2
Sales: 2
HR: 1
Financial: 3
Pipette Calibration-facilities VWR
= VWR technicians
= VWR partner
= ISO 17025 accredited calibration-lab
Pipetting??
Every lab-
technician can do
that!!!
- Principle pipettors
- Liquid Handling group
- Pipetting technics
- Terminology
- Calibration
- Wrong Pipetting
- Maintenance & Repair
- Measure uncertainty
Accuracy and Precision
Precision
Precision is an agreement between replicate
measurements. Precision is quantified by imprecision
High precision, i.e. small imprecision means very little
variation between repeated measurements
Accuracy
It is possible to be very consistent, but
consistently wrong
Inaccuracy is the numerical difference between the
mean of a set of replicate measurements and the true
value
Accuracy and Precision
Accurate Precise Accurate Not Precise
Precise Not Accurate Not Precise Not Accurate
Liquid Handling
Glassware
Pipettors, mechanical / electronical
Bottle top dispenser
Burette
Automatic Transfer Pipette station
Basics of a pipettor
Vertical movement
Diameter
Length of the piston-movement
Pipettor types
Monochannel
Multichannel
Stepper
Multistepper
Pipettor working principles
Air displacement
Air between liquid and piston
Length and diameter of piston define the amount
Watery liquids
Sensitive for contamination
Worse accuracy
Cheap system
Pipettor working principles
Positive displacement
Contact between liquid and piston
Length, diameter and tip of piston makes the amount
Viscosity liquids
Volatile liquids
Non-Sensitive for contamination
Good accuracy
Expensive system
Monochannels, fix and variable volume
Air- and positive displacement system
Pipettor types
Multichannels, fix and variable volume
Air- displacement system
Stepper, variable volume
Air- and positive displacement system
Multistepper, variable volume
Air- displacement system
Forward technique
Pipetting technique
Reverse technique
Whole blood technique
Repetition technique
Forward Technique
- Most common technique
- Only for watery liquids
- Manufacturer adjustable
- VWR / TSL common technique
Pipetting technique
The worst pipetting technique
Reverse Technique
- Best technique for viscosity liquids
- Best technique for volatile liquids
- Best technique for small volumes
- Only with air displacement pipettor
Pipetting technique
Best technique for lab-technicians who cant
pipetting
Reverse technique
97.5
98
98.5
99
99.5
100
100.5
101
101.5
1 2 3 4 5 6 7 8 9 10
Subsequent pipettings

l
P rP d (4x25ul)
Pipetting Viscous Liquids
(Glycerol)
Whole blood Technique
- Accuracy for pipetting whole blood
- Best technique for measurement of or in whole
blood (erythrocytes)
Pipetting technique
Terminology
- Calibration
- Specifications
- Adjustment
- Validation
- Traceability
- Nominal volume
- Dead volume
Calibration methods
- Gravimetrical
- Titrimetrical
- Photometrical
- Radio-active
Traceability chain
Primary standard
National standard
Reference standard
Working standard
Pipettor
Gravimetric method
Given a certain mass of water with a known specific gravity, its volume
can then be predicted.
- Only official, described method
- Most common calibration method
- Method is several times accreditated
- Uncertainty is known
- Expensive method
- Time consumable
ISO 8655 gravimetric method
Given a certain mass of water with a known specific gravity, its volume
can then be predicted.
Test room: Stable environment. Constant 0,5C and humidity > 50%
Evaporation: < 50l precautions
Test volumes: Nominal volume / 50% of nominal volume / the lower limit of the
volume range or 10% of the nominal volume (whichever is the greater)
Number of measurements: 10 per test volume
Test liquid: Deionized water grade 3 as in ISO 3696, degassed
During calibration: measurement of aquadest with 0,2 C accuracy
Multichannel: all channels!!!!!
ISO 8655 gravimetric method
Given a certain mass of water with a known specific gravity, its volume
can then be predicted.
Weighing equipment:
1 l < V < 10 l: Microscale / resolution 0,001 mg.
10 l < V < 100 l: Semi-microscale / resolution 0,01 mg.
100 l < V < 1.000 l: Analytical scale / resolution 0,1 mg.
1 ml < V < 10 ml: Analytical scale / resolution 0,1 mg.
10 ml < V < 200 ml: Scale / resolution 1 mg.
ISO 8655 gravimetric method
Given a certain mass of water with a known specific gravity, its volume
can then be predicted.
Weighing vessel: Never plastics!!!!
Thermometer: Uncertainty of < 0,2 C
Hygrometer: Uncertainty of < 10%
Barometer: Uncertainty of < 0,5 kPa
Certified weights!!!!
Most important: Technicians who are trained in pipetting
ISO 8655 procedure
Preparation
As found-calibration + certificate
Preventive maintenance
Calibration
Meet specifications?
Adjustment / Repair
As left-calibration + certificate
Pipette service
Preventative Maintenance:
Disassemble pipette;
Inspect and clean exterior and interior parts;
Clean Piston & tipholder & seals;
Check Spring tension;
Grease/oil;
Reassemble Pipette.
Functional test
Leakage test
Pipette service
Calibration
Check temperature and humidity;
Pre-wet tips by pipetting 4 times;
Calibrate pipette by 10 measures on lowest and highest volume;
Pipette-technique is Forward Technique;
Meeting specifications for accuracy and precision;
Check temperature;
Adjustment or repair if necessary;
Recalibration;
ISO 17025 - Calibration certificate
Calibration sticker with Cal. Date and recal.-date
Specifications
- Manufacturer
- Customer; validation!
- ISO 8655 (DIN 12650)
You are responsible, not the manufacturer or auditor!
Depends on the asked accuracy in the
analytical test
Calibration certificate
Calibration
Stable testroom
Scales
Instruments
Traceability
Acclimatisation
Trained technicians
Small volume !!!
Temperature
Biohit Proline 200 - 1000 l
900
920
940
960
980
1000
1020
1040
5.2 22.2 28.1
Water temperature (C)
T
e
s
t

v
o
l
u
m
e

(

l
)
Pipet t or at 28C
Pipet t or at 22C
Pipet t or at 5C
Common failures at pipetting
No pre-rinse of pipettortip
Use of other pipetting-technique as at calibration
No wipping of pipettortip
Multi-use of pipettortip
Uneven movement of push button
Angle at aspirate and deliver
Temperature-difference instrument and materials
Poorly seated pipettips
Keep the pipettor vertical
Wrong pipettor for the amount of liquid
Wrong pipettor for the sort of liquid
Pipettors are precision-instruments!!!
Use them in that way!!!!
Questions, please.