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Learning the basics – how to work with volumetric instruments. Volumetric Measurement in the

FIRST CLASS·BRAND

Introduction

Volumetric measurement plays a central role in the laboratory. The user has to determine the degree of accuracy required for each measurement. Based on this, he can choose the appropriate volu- metric instrument. Reliable measurements require the use of precision instruments and their proper handling. To provide a better understanding of volumet- ric instruments and their operation, this booklet explains the most important terms for their classification and handling, and illustrates them by using BRAND laboratory equipment as examples. The brochure 'Information on Volumetric Measurement' is designed to give the reader a quick overview of volumetric instruments. It is not intended to replace the operating manuals of the liquid handling instruments described. By all means, read the operating manuals supplied with these instruments before use – for your own safety and success.

Please do not hesitate to contact us if you have any further questions on the subject of volumetric measurement.

BRAND GMBH + CO KG P.O. Box 11 55 97861 Wertheim/Main · Germany

Telephone: +49 9342 808-0 Fax: +49 9342 808-98000 E-Mail: [email protected] Internet: www.brand.de

© BRAND GMBH + CO KG · All rights reserved

Contents

n Volumetric Instruments – an Overview 5 n Manufacture of Volumetric Instruments 6 From raw material to finished precision instrument 6 Identification of volumetric instruments 8 Accuracy classification of volumetric instruments 9 n Working with Volumetric Instruments 10 Meniscus of a liquid 10 Delivery and waiting times 11 , general 12 Handling of pipettes 13 Handling of volumetric flasks 15 Handling of graduated and mixing cylinders 15 Handling of 16 Handling of density 17 Working with pipetting aids 18 n Working with Liquid Handling Instruments 21 Dispensing with bottletop dispensers 22 Titrating with bottletop burettes 24 Pipetting with air-interface pipettes 25 Pipetting with positive-displacement pipettes 29 Dispensing with repetitive pipettes 30 n Accuracy Defined 32 n Monitoring of Measuring Instruments 33 Procedure for volumetric testing 34 Calibration software 36 Calibration service 36 n Conformity and Calibration Certificates 37

® n BLAUBRAND USP volumetric instruments 38 n IVD Directive 39 n Quality Management 40 n Cleaning of Laboratory Equipment 41 n Safety Information 43

Volumetric Instruments – an Overview Volumetric Instruments – an Overview

The volumetric measurement of liquids is a routine operation in the laboratory. Therefore, Volumetric instruments volumetric instruments, such as volumetric flasks, bulb pipettes, graduated pipettes, graduated glass/plastics cylinders and burettes are standard equipment. They can be made from glass or plastic. Sup- pliers offer volumetric instruments in varying qualities. Graduated beakers, beakers, Erlenmeyer flasks, dropping and the like are not volumetric instruments! They are not precisely calibrated, and the scale serves only as an approximate guide. A selection of volumetric instruments by BRAND is illustrated below:

Volumetric flasks Bulb

Liquid handling instruments

To meet the ever growing demands on volumetric measuring in the lab, such as testing in series, new devices are constantly being developed, e.g., for dispensing, pipetting and titrating. Usually, the instruments designed by different manufacturers for a particular purpose are more or less similar in functional principle. However, major differences are apparent when the focus is on constructional details and design. This selection of liquid handling instruments by BRAND illustrates the range of instruments avail- able:

Bottletop dispenser Bottletop dispenser Bottletop burette

Single-channel Multichannel Positive displace- Repetitive pipette, Repetitive pipette, air interface pipettes air interface pipettes ment pipette manual electronic

BRAND

5 Glass Volumetric Instruments · Manufacture Manufacture of Glass Volumetric Instruments From raw material to finished precision instrument

Blanks

The glass types used are soda- This results in optimum me- sterilizer without any resulting Therefore: lime glass (e.g., AR-GLAS®) chanical stability, a requirement volume changes. Always place glass instruments for bulb and graduated pi- for keeping the volume con- As with all glass instruments, into a cold drying cabinet or pettes, and stant despite any subsequent however it should be noted, sterilizer; then heat slowly. At (e.g., Borosilicate glass 3.3) temperature fluctuations. that uneven heating or sudden the end of the drying or steril- for volumetric flasks, graduated Therefore, BLAUBRAND® and temperature changes produce izing period, allow instruments cylinders and burettes. These SILBERBRAND volumetric thermal stresses, which may to cool off slowly inside the glass types meet instruments can be heated up result in breakage. switched-off drying cabinet or the stringent laboratory re- to 250 °C in a drying cabinet or sterilizer. Never heat volumetric quirements for chemical and instruments on a hotplate! physical resilience.

High-quality blanks and strict statistical testing of the re- quired quality characteristics are the basis for producing high-quality volumetric instru- ments. For example, thermal stress in the glass blanks must be eliminated by a controlled heating and cooling process.

Blanks for graduated cylinders

Calibration

Every glass volumetric instru- Volumetric instruments are either calibrated 'to contain' ('In') ment is individually calibrated at or 'to deliver' ('Ex'). BRAND, i.e. the instrument is accurately filled with a defined Calibration 'to contain' Calibration 'to deliver' Reference temperature quantity of water, and a calibra- (TC, In): (TD, Ex): The standard reference tem- tion mark is applied at the low- The contained quantity of liquid The delivered quantity of liquid perature, i.e. the temperature est point of the meniscus. In corresponds to the capacity corresponds to the capacity at which volumetric instruments the case of graduated instru- printed on the instrument. printed on the instrument. will contain or dispense their ments, two calibration marks These instruments include e.g., The wetting residue remaining volumes, is 20 °C. are applied. graduated cylinders, volumetric in the instrument has already If the adjustment or calibra- Computer-controlled systems flasks, and capillary pipettes up been taken into account in the tion is performed at a temper- ensure maximum precision in a to 200 µl. calibration. These instruments ature that deviates from this fully automated production line. include e.g., graduated and standard, the corresponding 'Statistical Process Control' bulb pipettes, and burettes. measurement values must be (SPC) guarantees production of corrected. volumetric instruments with the smallest deviation from nominal Note: capacity (accuracy) and narrow Due to the small coefficient of scatter of individual values expansion of the glass material, (coefficient of variation). the reference temperature is of minor significance in practical use since the measurement de- viations resulting from volume expansion of the measuring in- strument are generally smaller than the error limit.

Calibration of graduated pipettes BRAND

6 Glass Volumetric Instruments · Manufacture

Silk-screening Screening inks

Calibration is followed by BRAND uses quality inks manufactured especially for glass volumetric instruments: silk-screen printing of marks and inscriptions. BRAND uses stretchable screen stencils for Blue enamel: White enamel: Amber diffusion stain: all graduated pipettes, burettes, High contrast, optimum White enamel is used for Diffuses into the glass sur- graduated cylinders, and mix- combination of resistance and SILBERBRAND volumetric face and can only be removed ing cylinders. These stencils legibility. Blue enamel is used instruments (class B). by abrasion. It is used for can be stretched to match the for BLAUBRAND® volumetric volumetric instruments that are calibration marks accurately, so instruments (class A/AS). exposed to especially aggres- that the measuring precision is sive cleaning conditions. An maintained for all intermediate amber diffusion dye is used volumes. for BLAUBRAND® ETERNA volumetric instruments (class Pipettes are additionally marked A/AS) as well as for SILBER- with 'color-code' rings at their BRAND ETERNA volumetric upper end, which make it easier instruments (class B). to identify similar pipette sizes clearly. The ISO 1769 industry standard defines color-coding for different nominal volumes.

Automatic silk-screening of volumetric flasks

Firing Quality assurance

Firing or annealing the Quality assurance is imple- screened ink is the last step on mented at BRAND through the way from the blank to the ongoing testing during produc- finished volumetric instrument. tion and statistical testing in the A carefully controlled anneal- final inspection. (For detailed ing process, along with the information, see page 39.) specially produced quality inks, is a prerequisite for durable graduations. This involves a gradual heating and cooling of the volumetric instruments. De- pending on the type of glass, temperatures of 400 to 550 °C are reached in this process. Silk-screened graduated cylinders before firing

BRAND

7 Glass Volumetric Instruments · Manufacture

Identification of volumetric instruments

Batch number

Example: BLAUBRAND® bulb pipette

is the symbol for the con- formity certification of BRAND, according to 'Eichordnung', the German Federal Weights and Measures Regulations and to DIN 12 600 Manufacturer Designation of the standard BRAND trademark for volumetric instruments Country of origin class A/AS

Reference temperature (20 °C), waiting time (5 sec.), Nominal volume calibration (TD, Ex = to deliver)

Error limit Class 'A', the highest quality grade, Volume unit 'S' for swift delivery

The labeling below must be printed The following information may on every volumetric instrument: also be added:

n Nominal capacity l Country of origin

n Unit symbol: ml or cm³ l Error limit

n Calibration temperature: 20 °C l Manufacurer's trademark (here: BLAUBRAND®) n Calibration: Ex or In l Standard, e.g., ISO 648 n Class: A, AS or B l Batch number n Waiting time (if necessary): in the format 'Ex + 5 s'

n Manufacturer’s name or logo

BRAND

8 Glass Volumetric Instruments · Manufacture

Accuracy classification

Volumetric instruments are generally available in two accuracy classes:

Class A/AS

Volumetric instruments of class A and AS have identical error lim- Class A/AS its as established by DIN EN ISO. These are generally implement- ■ Always stands for the ed only in glass volumetric instruments. Exceptions are BRAND highest-grade accuracy plastic volumetric flasks made from PFA and PMP and plastic ■ 'S' stands for swift delivery graduated cylinders made from PMP, which are designed to meet (pipettes and burettes) the highest requirements and likewise correspond to class A. For class AS volumetric instruments, calibrated to deliver (TD, Ex), ■ Only class A/AS is DE-M the additional 'S' means swift delivery. marking Class AS volumetric instruments have become quite well estab- ■ Graduation: lished. The risk of clogging is low in pipettes and burettes with The long graduation marks a larger tip opening. The delivery behavior of various liquids is extend over at least 90% compensated by observing the defined waiting time (see 'Delivery of the tube perimeter or are and waiting times' on page 11). present as ring markings.

Class B Volumetric instruments of class B are available in glass or plastic. Class B Class B instruments generally have twice the error limits of class ■ Generally twice the error A/AS. For class B measuring instruments calibrated to deliver limits of class A/AS (TD, Ex), no waiting time is specified. ■ Graduation: The long graduation marks extend about 20 - 40% of the tube perimeter.

The choice of volumetric instruments – glass or plastic?

There is no all-round material The accuracy of bulb pipettes, Graduated cylinder made of to meet every single require- graduated pipettes, volumetric PMP, class A ment in the laboratory. The flasks, and graduated cylinders decision to use glass or plas- made from PP corresponds to tic is guided by the application, that of class B error limits. product design and after con- PMP and PFA are also used for sidering the specific properties measuring instruments that cor- of the materials and econom- respond to class A error limits, ic aspects. Volumetric instru- e.g., volumetric flasks (PMP/ ments of plastic excel by their PFA) and graduated cylinders high resistance to breakage (PMP). Due to its higher purity, and low weight. PP, PMP and PFA is preferably used in trace PFA are proven materials. analysis applications.

BRAND

9 Volumetric Instruments of Glass · Measuring the Volume Working with Volumetric Instruments

The liquid meniscus

The term meniscus describes a If the liquid molecules are If the diameter of a pipette is If the cohesion force of a fluid curvature in the surface of the attracted more strongly by narrow enough – as with capil- is stronger than the adhesion liquid. the glass wall (adhesion) than lary pipettes – the adhesion force of the glass wall, an The meniscus may be curving by their own kind (cohesion), force is strong enough to pull upwardly curved (convex) me- upwards or downwards. The the meniscus is curved down- up not only the edge but also niscus is formed. This happens curvature develops as a func- wards, or concave; the edge the entire liquid level (capillary with mercury, for example. tion of the interplay between of the liquid surface is slightly effect). the adhesion and cohesion raised. This is the case, e.g., forces. with aqueous solutions.

Meniscus setting

Correct meniscus setting is a prerequisite for accurate volumetric measurement.

Important note: The temperature of the liquid and the environment during use are important. While the expansion of a glass volumetric instrument is negligible, the expansion of the liquid at dif- ferent temperatures must be Concave meniscus in a Convex meniscus in a Appearance of the meniscus taken into account. To minimize graduated pipette. graduated pipette. at the Schellbach stripe in a volume errors as much as burette. possible, the volumes of all the liquids in contact with one In the case of a concave me- In the case of a convex me- The Schellbach stripe is a another should be measured niscus, the volume has to be niscus, the volume has to be narrow blue band at the center at a common (prevailing daily) read at the lowest point of the read at the highest point of the of a white stripe. Schellbach temperature. Especially in the liquid level. The lowest point of liquid level. The highest point stripes are imprinted on the preparation of standard solu- the meniscus has to touch the of the meniscus has to touch back of volumetric instruments tions, for example, the pipetting upper edge of the graduation the lower edge of the gradua- to improve readability. of the samples and the mark. tion mark. The refraction of light causes should be done at the same two arrow points to appear temperature to the extent pos- at the meniscus. The reading sible. Significant temperature point is where the two arrows differences between the mea- meet. suring instrument and the liquid should likewise be avoided. Reading the meniscus

For parallax-free adjustment of the meniscus, the volumetric instrument is held upright and the observer’s eye must be at the same height as the menis- Ring mark / cus. In this position the ring graduation mark will appear as a line. mark Meniscus The meniscus will appear dark- er and more easily readable in front of a light background if a piece of dark paper is held behind the instrument immedi- ately beneath the ring mark or graduation mark.

BRAND

10 Volumetric Instruments of Glass · Measuring the Volume

Delivery and waiting times

In volumetric instruments for liquid delivery (calibrated to deliver, TD, Ex), the volume delivered is always smaller than the volume contained in the . This is caused be the fact, that a certain amount of liquid remains as a film on the inner surface of the instrument. The volume of this liquid film depends on the delivery time and should be taken into account when calibrating the measuring instrument.

Possible volume errors: The volume delivered from a pipette or burette becomes smaller if the tip is broken off (shorter deliv- ery time), or increases if the tip is not clean and the liquid outflow is impeded (longer delivery time). Likewise, the volume increases if the residual liquid in the tip after pipetting is blown out by mistake. (for the proper handling of pipettes, see page 13.)

Delivery time Waiting time

The delivery time is defined as The waiting time begins when Waiting time for class AS: the period of time required for the meniscus comes to rest at The established waiting time of the free fall of the meniscus the lower volume mark or in the 5 s for class AS bulb and (discharge of water due to tip. During the waiting time, graduated pipettes is the time gravity) from the upper volume residual liquid continues to flow after which the meniscus visibly mark to reach the lower volume down from the glass wall. comes to rest in the tip, and mark or the tip. This is con- before the tip can be removed nected with the defined waiting from the inner surface of the time for class AS volumetric receiving vessel. instruments. The waiting time of 5 s must be indicated on the pipette by the manufacturer (see page 8).

Examples of delivery and waiting times for different classes (25 ml bulb pipette)

Class A (DE-M marking)

Delivery time 25 - 50 sec. (no waiting time)

Class AS (DE-M marking)

Delivery time 15 - 20 sec. + Waiting time 5 sec.

Class B

Delivery time 10 - 50 sec. (no waiting time)

BRAND

11 Glass Volumetric Instruments · Handling Working with Volumetric Instruments Pipettes, general Pipettes are volumetric instruments for measuring volumes of liquid and are generally calibrated 'to deliver'. During the manufacturing process, they are individually volumetrically calibrated and provided with one or more calibration marks. We distinguish between bulb and graduated pipettes (calibrated to deliver, TD, Ex) and disposable micropipettes up to 200 µl (calibrated to contain, TC, In).

Bulb pipettes Graduated pipettes

■ Calibration: ■ Calibration: Class AS: 'Ex + 5 s' Class AS: 'Ex + 5 s' Class B: 'Ex' Class B: 'Ex' ■ Generally higher measure- ■ Scaling permits the reading ment accuracy than gradu- of partial volumes ated pipettes ■ Types of graduated pipettes: ■ Bulb pipette models: Type 2 – nominal volume at The most important model is top, total delivery the bulb pipette with 1 mark also for partial (total delivery). volumes Less common is the model Type 1 – nominal volume at with 2 marks (partial deliv- bottom, partial de- ery). livery for all volumes Type 3 – nominal volume Bulb pipettes are also called at bottom, total single volume pipettes. delivery only for the nominal volume

Bulb pipette with 1 mark Graduated pipette, type 2, nominal volume at the top

Capillary pipettes Capillary pipettes e.g., BLAUBRAND® intraMark e.g., BLAUBRAND® intraEnd

■ Calibrated to contain (TC, In) ■ Calibrated to contain (TC, In) ■ One ring mark ■ No ring mark ■ Volume limited by one end ■ Volume limited by both ends and the ring mark (end-to-end capillaries)

BRAND

12 Glass Volumetric Instruments · Handling

Handling of pipettes

Pipettes calibrated 'to deliver' ('TD, Ex') Proper pipetting with bulb pipettes with 1 mark (here: nominal volume 25 ml) and graduated pipettes type 2, class AS (here: partial volume 3 ml) Utility: pipetting aid (see page 18)

Filling Bulb pipette Type 2 Type 1 Type 3 1 mark total partial total 1. Fill the pipette with a pipet- delivery, delivery, delivery, ting aid to approx. 5 mm nominal zero point zero point above the selected gradua- volume at the top at the top tion mark. at the top 2. Remove any liquid remaining Set meniscus on the outside of the tip of 2 times Set meniscus 2 times the pipette with a tissue. 3. Set the meniscus. 4. Wipe off any drop of liquid Set meniscus 1 time adhering to the tip.

Delivering 5. Hold the pipette vertically. Deliver the liquid with the tip of the pipette in contact with the inner surface of the inclined receiving vessel. 6. When the meniscus comes to a rest in the tip, the wait- ing time of 5 s begins (class AS only). Type 1 and type 3 7. After the waiting time, draw the pipette tip upwards When using type 1 or type 3 along the inner wall through graduated pipettes (zero point a distance of about 10 mm Set meniscus at top), the meniscus must to remove residual liquid. 1 time 1.) first be set at the zero point and then the liquid be run out to slightly above the Note: desired partial volume. The residual liquid still 2.) After a waiting time of 5 s, left in the tip has already the meniscus should be set been taken into account a second time. during calibration and must not be discharged into the vessel, such as by blowing out.

Working with type 2 graduated pipettes is significantly quicker and simpler. With type 1 and type 3 pipettes there is a risk that with the always neces- sary second meniscus setting too much liquid is released, and that the sample needs to be prepared again (as also with bulb pipettes that have 2 marks).

Fill pipette Wipe off Set meniscus Deliver

BRAND

13 Glass Volumetric Instruments · Handling

Handling of pipettes

Pipettes calibrated 'to contain' (TC, In) Correct pipetting with capillary pipettes Utility: pipetting aid (see page 18)

Capillary pipettes are pipettes with a very narrow internal diameter. They are filled either with a pipetting aid, or automatically by capillary action. After emptying, the capillary pipette must be rinsed repeatedly with the diluting medium.

Filling

■ Aspirate liquid accurately to the desired mark. ■ Hold the pipette horizontally and carefully wipe off with a tissue.

Delivering

■ To empty capillaries blow out the liquid with a pipet- ting aid, and rinse two to three times with the diluting medium (required due to calibration 'to contain'). Fill capillary Wipe off Deliver ■ End-to-end capillaries are often placed directly in the dilution solution and washed out by shaking.

Pipette holder with end-to-end-capillaries

BRAND

14 Glass Volumetric Instruments · Handling

Handling of volumetric flasks

Volumetric flasks, class A and How to prepare a standard solution with a : B, are calibrated to contain (TC, In), and are mainly used 1. Insert the precisely weighed amount of substance, or rinse in a for the preparation of highly standard solution concentrate. accurate dilutions and standard solutions. 2. Fill the flask with distilled water to about half. Swirl the flask to dissolve and mix the contents thoroughly. Modern analytical methods require small-capacity volumet- 3. Top up the flask with distilled water to just below the ring mark. ric flasks. Small-sized conven- tional volumetric flasks (up to 4. Top up the remaining volume, using a wash or pipette, approx. 50 ml) tend to tip over until the meniscus is exactly at the ring mark. Important: me- easily due to their high center niscus must be read at eye level. The wall of the flask must not of gravity and their small base be wetted above the mark. area. Trapezoidal volumetric flasks are much more stable. 5. Close the flask and shake upside down to mix contents. Their center of gravity is lower, and their base area is more than twice as large compared to the equivalent standard flasks.

Handling of graduated and mixing cylinders

Graduated cylinders: Handling Mixing cylinders Graduated cylinders, class A ■ Fill with liquid. The mixing cylinder is cali- Mixing cylinders can be used in and B, are measuring instru- brated to contain (TC, In), just the preparation of standard so- ■ Set the meniscus to the ments that are calibrated to as the graduated cylinder. They lutions and dilutions, the same required mark (read at eye contain (TC, In), i.e. they indi- are additionally supplied with a as volumetric flasks. level!) cate the exact volume taken up. and . ■ After portions of various ■ The wall of the cylinder liquids have been measured must not be wetted above out, these can then be the mark. mixed by shaking directly in ■ The indicated volume is the the mixing cylinder. amount of liquid contained. Note: Note: When two liquids are mixed, a In the laboratory, graduated volume change may occur. cylinders are frequently used just like measuring instruments that are calibrated to deliver (TD, Ex). Measurements with water showed that the dispensed volume is reduced by approxi- mately the amount of the error limit of the graduated cylinder due to the residue amount from wetting. Prerequisite: The liquid is slowly dispensed in one por- tion, and to finish delivery of the liquid the cylinder is held at an incline for a further 30 s.

BRAND

15 Glass Volumetric Instruments · Handling

Handling of burettes

Burettes are glass volumetric instruments calibrated to deliver Calibration (TD, Ex) which are used for titration in volumetric analysis. Class AS: 'Ex + 30 s' Class B: 'Ex'

Note on waiting time: Compared to pipettes, the handling of burettes is different during In practice the time required for this dropwise addition is the same practical use and calibration. Typically, the volume used in a titra- or even longer than the established waiting time. As a result, it is tion is less than the nominal volume, and the standard solution is not necessary to observe the established waiting time of 30 s with added dropwise when close to the color change to avoid overtitra- class AS burettes during practical use. tion.

Types of burettes:

Handling 1. Rinse the burette with the standard solution to be used, and orient it so that the burette tube stands upright. Make sure that the solution is completely homogenous, i.e., there must be no cloudiness, flocculation or precipitation present. 2. Fill the burette slightly above the zero mark. To prime the stop- cock, drain the burette no further than the nominal capacity. Should a small air bubble persist inside, hold the burette at an angle and lightly tap a finger against the location of the bubble. 3. Refill titrant to approx. 5 mm above the zero mark. The wall of the burette should not be wetted further upwards. 4. Drain liquid to set the zero point accurately. Important: Menis- cus must be read at eye level (parallax-free level). Automatic burettes: Fill to approx. 5 mm above the zero mark. This is ad- justed automatically after air release. 5. Wipe off any drops adhering to the discharge tip. 6. Open the stopcock and slowly add titrant to the sample (con- taining the indicator). The stopcock must not touch the wall of the vessel. Keep swirling the sample vessel lightly while adding titrant, or place it on a . For easiest recognition of the color change, place the sample vessel on a white surface. When the color changes, close the stopcock. The titration is finished. 7. Read the discharged volume at eye level. The waiting time re- quirement (class AS: 30 s) is already fulfilled in practice dur- ing the titration process. It only needs to be taken into account during the calibration of the measuring instrument. 8. Any drops remaining on the tip of the stopcock should be wiped against the vessel wall and rinsed down. It is part of the titrated volume.

■ Before each new titration, reset the zero point and start the Burette with lateral Automatic burette, Automatic burette, titration from there. stopcock Pellet pattern Dr. Schilling pattern In addition to burettes, the following equipment is required for titrating: Volumetric flasks, bulb pipettes, Erlenmeyer flasks.

BRAND

16 Glass Volumetric Instruments · Handling

Handling of density bottles

Density bottles are mainly used to determine the density of liquids of moderate viscosity. They are not volumetric instruments, however, they are calibrated 'to contain' as in the case of volumetric flasks.

Types of density bottles:

Handling 1. Determine the weight of the dry and empty density bottle. 2. Fill the density bottle with liquid, avoiding bubbles. The ground neck should be covered to about 1/3. 3. In a thermostatic bath, adjust the temperature of the bottle and contents to 20 °C. 4. Align the stopper respectively, the of the density bottle according to the marking, and insert carefully. The capil- lary tube fills up and the displaced liquid comes out. 5. Carefully dry the outer surfaces of the stopper (respectively, the side capillary) and the density bottle with tissue. ATTENTION: Be careful not to remove any liquid from the capillary. The sample liquid must be exactly level with the upper end of the capillary. 6. Determine the weight of the filled density bottle.

Density bottle Density bottle with Calculate the density from the mass (weight) and the volume of with stopper thermometer and the liquid at the reference temperature of 20 °C. The volume is side capillary engraved on the bottle. The equation is: (recommended for liquids with an in- Density (ρ) = Mass (m)/Volume (V) creased vapor pres- sure) Take the buoyancy of the air into account for weighing.

Note: Calibrated density bottles always carry a unique identification number on all component parts. Use only parts with the same number together.

BRAND

17 Glass Volumetric Instruments · Pipetting Aids

Working with pipetting aids

Pipetting aids are indispensable for working with pipettes. Pipetting using the mouth or with a tube and mouthpiece Types of pipetting aids: is not allowed. A pipetting aid should always be used for this. This significantly decreases the risk of infection or ■ motorized pipetting aids injury. ■ manual pipetting aids

Motorized pipetting aids

Motorized pipetting aids are ideal for pipetting longer series (e.g., for cell cultures). e.g., accu-jet® pro by BRAND

The variable motor speed control and a special valve system An integrated check valve in connection with a membrane filter enable sensitive operation with pipettes from 0.1 to 200 ml. effectively protects against penetrating liquids. To protect against corrosion, an active pressure compensation diverts vapors outside.

Handling Pipetting is controlled through two large function buttons:

Filling Delivery To fill the pipette, press the Discharge rate is con- upper button. Intake rate tinuously variable by trigger is continuously variable by pressure. trigger pressure. Choose: Free delivery or Power delivery

Dispensing liquid: free delivery or power delivery?

The choice of delivery modes is the delivery and waiting times. determined by the application. In microbiology, however, In the analytical laboratory, the volumetric accuracy is less 'free delivery' mode is often significant. Here, the uniform preferred in order to obtain and rapid delivery of nutrient the required volumetric accu- solutions, etc. is of primary racy. To achieve the accuracy importance. Therefore, the indicated on the pipettes, it is 'power delivery' mode is necessary to allow the liquid to usually preferred in this field. run off freely, and to observe

BRAND

18 Glass Volumetric Instruments · Pipetting Aids

Manual pipetting aids Manual pipetting aids are used when pipetting a short series, primarily in chemical . e.g., macro pipette controller by BRAND The macro is compatible with the full range of bulb and graduated meniscus adjustment. A hydrophobic membrane filter protects the pipettes from 0.1 to 200 ml. The special valve system allows easy system against penetrating liquid.

Handling

Create negative pressure Filling Adjust meniscus / Blow out dispensing by ‘free delivery’

Squeeze bellow. Move pipetting lever upward. Press pipetting lever slightly When pipetting viscous media The farther up the lever is downward. The meniscus falls with 'free delivery', the pipette pressed, the faster the pipette – release the lever, the menis- tip frequently does not empty will fill. cus stops. completely. In these cases, To drain the pipette, press the blow out any remaining resi- lever all the way down. dues by pressing on the rubber To comply with class A accura- bulb of the macro pipette cy, do not blow out the residual controller. liquid!

Pipette filler The classic standard pipetting aid for bulb and graduated pipettes.

Handling Blow out Attention!

A 1. Insert top of pipette To blow out viscous media, the The pipette filler should not be side opening must be closed stored in the primed state, so 2. Press 'A' and squeeze the and the small ball compressed. that no liquid will be taken up! ball (create negative pres- sure)

3. Press 'S' to aspirate liquid just above the desired mark

S 4. Press 'E' to discharge liquid to the desired mark, or to drain the pipette

E

BRAND

19 Glass Volumetric Instruments · Pipetting Aids

Manual pipetting aids for small-volume pipettes up to 1 ml Pipetting using the mouth or with a tube and mouthpiece is not allowed. A pipetting aid should always be used for Special pipetting aids have been developed for these pipettes. this. This significantly decreases the risk of infection or They are used in the medical field with capillary pipettes, blood injury. diluting pipettes and blood sugar pipettes up to a max. of 1 ml.

e.g., micro pipette controller by BRAND

Handling

Filling / Delivery Dispensing by ‘free delivery’ Eject Turn the thumb wheel to take in To deliver liquid from pipettes The large ejector key allows and deliver liquid. calibrated to deliver (TD, Ex), ejection of used pipettes with- Pipettes calibrated to contain press the air release button out hand contact. (TC, In) should be washed (observing the waiting time if multiple times by filling and necessary). emptying with dilution solution.

e.g., micro-classic pipette controller by BRAND Due to its angled design, it is especially suitable for work under a in IVF laboratories and in medical laboratories.

Handling

Attaching the pipette Filling Delivery Always attach the short end Turn the thumb wheel back- Pipettes calibrated of the pipette, i.e. hold at the ward until liquid reaches the 'to contain': color code of the pipette and desired mark. Turn the thumb wheel forward carefully slide it into the adap- until liquid is delivered. Rinse tor. the pipette at least three times with the dilution solution. Pipettes calibrated 'to deliver': For 'free delivery' press the air release button until liquid has run out (observe waiting time if required).

BRAND

20 Liquid Handling Instruments Working with Liquid Handling Instruments

The ever increasing demands on the quality of analytical results, and the growing number of samples to be processed result in a need for volumetric instruments which help to handle the routine work in sample preparation as efficiently as possible. Manufacturers of laboratory equipment have responded to this need by developing specialized liquid handling instruments. These appliances represent an improvement over the traditional volumetric in- struments made of glass or plastic, and allow efficient work with a superior degree of precision and ease of operation. The liquid handling instruments from most manufacturers share a similar operating principle, however the design details and materials used differ somewhat from one manufacturer to the next. On the following pages, we will explain the functional principles and application of some of the most common liquid handling instruments, using appliances made by BRAND as examples.

Single-channel Multichannel Bottletop dispenser Bottletop dispenser Bottletop burette air interface pipette air interface pipette

Dispensette® seripettor® Titrette® Transferpette® S Transferpette® S -8/-12 (manual) (manual)

Single-channel Multichannel Positive displace- Repetitive pipette Repetitive pipette air interface pipette air interface pipette ment pipette

Transferpette® electronic Transferpette®-8/-12 Transferpettor HandyStep® HandyStep® electronic electronic (manual) BRAND

21 Liquid Handling Instruments · Dispensing

Dispensing with bottletop dispensers

Definition of 'dispensing': The term 'dispensing' is understood to mean the discharge of defined quantities.

For easy, rapid and precise dispensing of reagents, bottletop dispensers are widely employed to replace poured transfers into gratuated cylinders. They can be mounted directly on commercial labo- ratory bottles, either directly or by means of adapters. It is no longer necessary to transfer or decant chemicals. Serial dispensing in particular is greatly facilitated.

Functional principle of bottletop dispensers

By an upward movement of the piston, the liquid is released piston, the preset amount of through a valve system and the liquid is aspirated from the re- discharge tube. agent bottle into the dispenser There is no need to set a me- cylinder. By the subsequent niscus or to observe a waiting downward movement of the time.

We distinguish between bottletop dispensers with floating piston and with wiping-seal piston.

Bottletop dispensers with floating pistons This system requires no piston seal and is therefore very durable and maintenance-friendly. The piston fits into the dispensing cylinder without contact. Piston and cylinder are separated by a gap only a few thousandths of a millimeter wide and filled with liquid. This film of liquid acts as a lubricant which makes the piston glide very smoothly. e.g., Dispensette® by BRAND

Range of application Materials

For dispensing aggressive Depending on the require- reagents, e.g., concentrated ments, parts that come into

acids such as H3PO4, H2SO4, contact with liquid are made bases like NaOH, KOH, saline from various specially resis- solutions, as well as many tant materials, e.g., ceramic, organic solvents: platinum-iridium, tantalum, Dispensette® III. ETFE, PFA. For dispensing organic sol- vents, such as chlorinated and fluorinated hydrocarbons (e.g., trichlorotrifluoroethane and dichloromethane), concen- trated acids such as HCl and

HNO3, as well as for trifluoro- acetic acid (TFA), tetrahydro- furan (THF) and peroxides: Dispensette® Organic. Not suitable for for dispensing hydrofluoric acid (HF).

BRAND

22 Liquid Handling Instruments · Dispensing

Safety always comes first! Monitoring of measuring instruments / calibration

When choosing a bottletop Likewise, the suitability of the In regard to the monitoring of instruments should be checked dispenser, the safety features dispenser for the medium to be measuring instruments accord- regularly and recalibrated if of the instrument should be dispensed should be checked ing to ISO and GLP guidelines, necessary (see page 33). kept in mind. For example, by the user. Information about the accuracy of volumetric does it reduce the risk of this can generally be found in injury due to glass breakage? the operating manual in the How does it avoid accidental chapter 'Function and limita- splashing when the instrument tions of use'. When in doubt, is primed? How is contact with contact the manufacturer the medium minimized when directly. Information on main- the dispensing tube is closed? tenance and monitoring of measuring instruments are also found in the operating manual.

Bottletop dispensers with wiping-seal piston In addition to the 'floating piston' operating principle, instruments with 'wiping-seal' piston are also used. It is frequently reported that these systems require higher operating forces and that the frictional wear can cause defective seals. e.g., seripettor® by BRAND The design of the system allows a replacement of the complete The present example reflects a competitively-priced dispenser for dispensing cartridge. The somewhat higher operating forces dur- simple dispensing tasks in the volume range of 0.2 to 25 ml. ing filling are minimized by a spring with automatic lifting action.

Range of application and materials

The range of application in- The seripettor® pro dispenser cludes the daily routine dispens- is suitable for dispensing acids ing of bases, acids in low con- such as concentrated HCl, centration, biological buffers, polar solvents such as acetone, cell culture media, biological essential oils and UV-sensitive detergents and polar solvents. media. In contrast to the seripettor® dispenser, valves made from chemically resis- tant materials are used in this instrument.

BRAND

23 Liquid Handling Instruments · Titrating

Titrating with bottletop burettes

Definition of 'titration': Titration is a volumetric method used for the quantitative analysis of a dissolved substance.

How to titrate?

Using a bulb pipette, a defined portion of a sample (liquid with an unknown fraction of dissolved material, e.g., acetic acid) is placed in an . After dilution with water, 3 drops of an indicator solution are added. Then, with continuous swirling, a suitable titrant of known concentration (e.g., 0.1 M NaOH) is added from a burette until a color change in the indicator signals the endpoint of the titration. Using the chemical equation and the volume of titrants used, the amount of substance dissolved in the sample can be calculated.

Functional principle of bottletop burettes Reading the volume

Bottletop burettes are mounted By the subsequent downward The discharged volume can be directly upon the reservoir bot- movement of the piston, the read directly from the display of tle. By the upward movement liquid is released slowly, and the bottletop burette. There are of the piston, liquid is aspirated added to the sample through no meniscus reading errors. from the into the the discharge tube until the burette cylinder. titration is finished, e.g., by change of color. e.g., Titrette® by BRAND in 10 ml, 25 ml, and 50 ml sizes The piston moves when the hand wheels are turned, and this takes up or discharges the liquid. The electronics of the in- strument automatically recog- nize the direction of rotation, whether filling or titration is taking place. The liquid can be taken in quickly, and can then be deliv- ered exactly, very slowly, drop by drop. A recirculation valve makes it possible to run the liquid back into the bottle during priming. Thus, air bub- bles can be removed without loss of medium. The instrument can readily be disassembled in the laboratory for cleaning and maintenance.

Range of application Materials

It can be used in many ap- Parts that come into contact plications for aqueous and with liquid are made from non-aqueous solutions (e.g., various specially resistant ma- alcoholic KOH) up to 1 M. terials, e.g., borosilicate glass,

PTFE, platinum-iridium, Al2O3 ceramic.

BRAND

24 e.g., Transferpette Manual single-channelpipettes Pipetting withairinterfacepipettes range. Itoperatesbytheairinterfaceprinciple. An airinterfacepipetteisusedforpipettingaqueousliquidsinthemicrolitertomilliliter Pipetting istheaccurateone-timeuptakeanddeliveryofliquids. Definition of'pipetting': Disposable tip Pipette shaft Air column ® Reagent S byBRAND Operation 2. 1. Aspirate reagent interface piston-operatedpipettestoday. In theroutinelabandinresearch,precisionfunctionalityarestandardstoexpectfromair any carry-over.Thisisparticu- only once,whicheliminates enters thetip.Tipsareused instrument; theliquidonly There isnowettingofthe Benefits column. Asaresult,liquidis positive pressureoftheair shaft createsanegativeor of thepistoninsidepipette The upanddownmovement Functional principle range Volume > 1000µl > 100µl-1000 > 1µl-100 0.1 µl-1 liquid willbeaspirated. slowly slideback sothatthe Allow thepipettingbuttonto the tipintoliquid. vertically andimmerse first stop.Holdthepipette Press pipettingbuttontothe

depth inmm Immersion 3 -6 2 -4 2 -3 1 -2

➠ ➠ Waiting time in s 3 1 1 1 ➀ ➁

allowed. required, ornocarry-overis where sterileconditionsare larly importantforapplications separated fromthepiston. (air interface)keepstheliquid expelled fromit.Theaircolumn either aspiratedintothetipor 2. 1. Discharge reagent 10 mm. over adistanceofapprox. pipette tiponthevesselwall second stopandwipethe pipetting buttondowntothe the tipcompletely:press The blow-outstrokeempties hold itdown. slowly tothefirststopand press thepipettingbutton the wallofvesseland Place thepipettetipagainst

Liquid HandlingInstruments·Pipetting

➧ ➠ ➀ ➁ page 33). and adjustedifnecessary(see brated regularly(i.e.checked) instruments shouldbecali- the accuracyofvolumetric ing toISOandGLPguidelines, measuring instrumentsaccord- In regardtothemonitoringof Calibration Press thetipejector. Eject tip BRAND 25 Eject tips Press the tip ejector. ➁ ➀

➠ ➧ Place the pipette tips Place the pipette tips against the wall of the vessel and press the pipet- ting button slowly to the first stop and hold it down. The blow-out stroke emp- ties the tips completely: press the pipetting button down to the second stop and wipe the pipette tips on the vessel wall over a distance of approx. 10 mm. Discharge reagent 1. 2.

➁ ➀

➠ ➠ Press pipetting button to the Press pipetting button to the first stop. Immerse the tips 2 to 3 mm into the liquid. Allow the pipetting button to slowly slide back so that the liquid will be aspirated. Immunofluorescence (IF) Immunofluorescence (RIA) Radio immunoassay Enzyme immunoassay (EIA, ELISA) Cell culture dilution Aspirate reagent 1. 2. Operation Analytical techniques ■ ■ ■ ■ -8/-12 by BRAND S ® Clinical diagnostics Food analysis Cell culture 26 Immunology Biochemistry Liquid Handling Instruments · Pipetting Instruments Handling Liquid BRAND e.g., Transferpette ■ ■ ■ ■ ■ Multichannel pipettes are ideal for the efficient transfer of samples, for serial dilutions and for wash- are ideal for the efficient transfer of samples, Multichannel pipettes ing of microtiter plates. Areas of application Microtiter technology requires pipetting into microtiter plates of 8 x 12 cavities (96-well plates) with with cavities (96-well plates) plates of 8 x 12 pipetting into microtiter technology requires Microtiter of minute quantities of proteins. This technology allows e.g., the detection standardized spacing. pipettes. be employed efficiently with multichannel This method can only Manual multichannel pipettes multichannel Manual to or 12 pipetting operations They allow 8 the air interface principle. also function by These pipettes out simultaneously. be carried Liquid Handling Instruments · Pipetting

Ergonomics and Strain Intensive, repeated operations done on mechanical instruments without proper ergonomic design result in prolonged stress that can lead to a number of muscular problems, known collectively as RSI (repetitive strain injuries). At particular risk are the muscles in the neck area, shoulders, arms and thumbs. Thus, laboratory work is often accompanied by the appearance of, inter alia, tendonitis and carpal tunnel syndrome. Especially with microliter pipettes, the need for fatigue-free operation is paramount.

Electronic single- and multichannel pipettes

Operating principle Advantages of electronic pipettes

Pressing the pipetting button discharge are controlled by a The combination of motor-con- An additional advantage is the starts the aspiration or discharge microprocessor. trolled pipettes and ergonomic execution of pipetting programs mechanisms (incl. blow-out). Various pipetting programs can design enables stress-free, such as the gel electrophore- The pipette’s piston is moved be selected with the control fatigue-free operation. sis mode (with precise display by a motor, aspiration and keys. It also reduces the demands of delivered volume) and a on the thumb while carrying dispensing mode, which are not out lengthy series which would possible with manual pipettes. otherwise increase the risk of RSI syndrome! e.g., Transferpette® electronic single- and multichannel pipette by BRAND

Program keys

Pipetting button

Tip ejection key

With electronic single- and multichannel pipettes, handy design, balanced weight distribution, intuitive software and clear, readily understood technical documentation should be standard.

Operation

Aspirate reagent Discharge reagent Eject tips Immerse the tip in the reagent Press the pipetting button Press the tip ejector. and press the pipetting button again, and the liquid will be once – the set volume will be discharged. The blow-out takes aspirated. place automatically! During this process, wipe the tip on the vessel wall over a distance of approx. 10 mm. BRAND

27 Liquid Handling Instruments · Pipetting

Pipetting programs of the microliter pipette Transferpette® electronic

The display

Program

Battery capacity

Arrow symbol for 'intake'

Pipetting volume

The pipetting programs Additional functions

Pipetting Depending on the quality and The standard program. The set volume is aspirated by the pipette, design, electronic pipettes can and then discharged. offer further instrument-specific functions in addition to the pi- petting programs. The Transferpette® electronic, for example, offers a program to simplify and speed up the calibration of the instrument as Mixing of Samples well as a battery refresh func- Program for mixing of liquids. The sample is repeatedly aspirated tion. and discharged, and the number of mixing cycles is displayed.

Reverse Pipetting What does 'reverse' mean? Program specially designed for the pipetting of liquids with a A reversal of the stops used to high viscosity, high vapor pressure or foamy media. measure a volume. For a set volume, the blow-out volume is aspirated additionally. Works with mechanical pipettes This volume remains in the tip after delivery to prevent undefined as follows: running out, splashing, or the formation of foam or bubbles. To aspirate the reagent, press the pipetting button down to the second stop and let it slide all the way back. Then Pipetting with Electrophoresis press down just to the first Program for loading of electrophoresis gels. The required stop to discharge the set sample volume is aspirated. During discharge, the volume be- volume. ing dispensed is tracked continuously, allowing the user to stop discharge to avoid over-filling sample wells. The pipette records the exact volume dispensed to ensure accuracy of sample mass calculations. GEL mode may also be used for microtitrations.

Dispensing A program for the dispensing of liquids in a series of equal ali- quots. A volume that has been aspirated is dispensed in steps.

BRAND

28 Liquid Handling Instruments · Pipetting

Pipetting with positive-displacement pipettes

Positive-displacement pipettes are the ideal alternative for difficult applications where air interface pipettes reach their physical limits. They are well suited even for media of very high or low viscosity, high vapor pressure, or a tendency to foam.

Functional principle Benefits

Glass cylinder In contrast with air interface physical properties of the liquid. Highest accuracy and speedy pipettes, the positive-displace- There is no need to discard operation. The tips or capil- ment piston is in direct contact tips or capillaries after each laries are reusable. No need for Positive-displace- with the pipetted liquid. The pipetting operation, since the reading a meniscus for pipet- ment piston piston wipes the walls of the minimal residual wetting is ting. tips/capillaries completely negligible for most applications. clean – literally to the last drop If carry-over is a concern, such which can be observed leaving as with infectious or radioactive Reagent the tip orifice. This principle media, an air interface pipette always provides reproduc- with disposable tips should be ible results, regardless of the preferred.

e.g., Transferpettor by BRAND

Range of application

Highly viscous media, such Media with high vapor pressure Media which tend to foam, as highly concentrated protein such as alcohols, ether and such as tenside solutions. solutions, oils, resins and fats. hydrocarbons.

Operation (similar to air interface pipettes)

Volume setting Aspirate reagent Discharge reagent Select desired volume by turn- Press piston to the stop. Im- Place the capillary/tip against ing the volume setting knob. merse tip into reagent and the vessel wall and press the slowly let the piston return to pipetting button down a second aspirate reagent. time to the stop. Positive- displacement pipettes have no blow-out!

BRAND

29 Liquid Handling Instruments · Dispensing

Dispensing with repetitive pipettes

The distribution of liquids is one of the most important and common activities in medical, pharmaceu- tical and biological laboratories. The most common techniques are pipetting and dispensing. Dis- pensing refers to the repeated discharge of identical quantities of liquid. The dispensers described in this chapter eliminate the need for repeated intake after each step – a major time-saving compared to pipetting. Since dispensing is such a common technique, the ergonomic design of the devices plays an essential role. Dispensing tasks in the laboratory are rarely handled by fully automatic systems requiring no manual intervention. Generally, repetitive pipettes are used for these routine jobs.

Types of repetitive pipettes: ■ Manual repetitive pipettes ■ Motorized electronic repetitive pipettes

Functional principle

With manual repetitive pipettes, No intermediate volumes can pressure, high viscosity or the volume delivered in each be selected. A main advantage a tendency to foam pose step results from the length of these devices is their robust- no problem to the repetitive of the stroke, defined by the ness; their drawback is their pipettes. number of steps on a toothed often fatiguing operation. According to volume range, the rack, and the size of the tip. Repetitive pipettes work on the repetitive pipette can be fitted Therefore, only a limited num- proven positive-displacement with PD-Tips of different sizes. ber of defined dispensing steps principle. Therefore, even is available. difficult media with high vapor

Manual repetitive pipette e.g., HandyStep® by BRAND

The repetitive pipette simplifies serial pipetting by taking up a me- dium once and then delivering it step by step. With one filling, up to 49 steps between 2 µl and 5 ml can be dispensed, depending on the size of the PD-Tip. The volumes and number of steps are a combined result of the setting of the volume selector key (1-5) and the size of the tip used. Positive-displacement tips (PD-Tips) from BRAND are available in 10 different sizes, sterile or non-sterile. Compatible tips of other manufacturers may also be used.

Available combinations with positive-displacement tips (PD-Tips) of different sizes from BRAND Steps and volume ranges

Tip size (ml)

Setting 0.1 0.5 1 1.25 2.5 5 10 12.5 25 50 Steps

1 2 10 20 25 50 100 200 250 500 1000 49 1.5 3 15 30 37.5 75 150 300 375 750 1500 32 2 4 20 40 50 100 200 400 500 1000 2000 24 2.5 5 25 50 62.5 125 250 500 625 1250 2500 19 3 6 30 60 75 150 300 600 750 1500 3000 15 3.5 7 35 70 87.5 175 350 700 875 1750 3500 13 4 8 40 80 100 200 400 800 1000 2000 4000 11 4.5 9 45 90 112.5 225 450 900 1125 2250 4500 10 5 10 50 100 125 250 500 1000 1250 2500 5000 9

Volume (µl)

BRAND

30 Liquid Handling Instruments · Dispensing

Ergonomics and design Repetitive working with hand-operated appliances can lead to a variety of muscular ailments which may particularly affect the neck, shoulders, arms and thumb. Fatigue-free operation is therefore a crucial requirement for repetitive pipettes, since they are used almost exclusively for serial dispens- ing. Ergonomic design is indispensable for the stress-free performance of extended pipetting opera- tions in the same working position.

Motorized electronic repetitive pipettes

Functional principle

Intake and discharge are The ergonomic design results The positive-displacement tips controlled by a single key. The in fatigue-free operation. The allow the dispensing of media piston inside the tips is driven piston wipes the walls of the of high density, high vapor by a motor, with a microproces- tips completely clean, providing pressure or volatility, or with a sor controlling the volume and precisely reproducible results tendency to foam. number of steps. without the influences of an air interface.

e.g., HandyStep® electronic by BRAND The size of PD-Tips from dispensed and the desired BRAND is encoded in their pis- operating program can then be ton. After inserting the tip, the easily selected. When a new size is automatically recognized PD-Tip of the same size is and displayed. This prevents inserted, all instrument settings errors, and the volume to be are maintained.

In contrast to manual repetitive pipettes which only permit a lim- ited number of volume settings, the electronic repetitive pipettes allow the continuous selection of intermediate volumes, such as 1.01 ml.

Available working modes:

Dispensing standard mode Automatic Dispensing Pipetting

A predefined volume is dis- The instrument calculates the Works like a positive-displace- pensed repeatedly. average time interval between ment pipette. A predefined your three first dispensing volume is taken up and dis- steps, and automatically contin- charged. ues to work at this rhythm.

BRAND

31 Accuracy Precision Defined What do 'Error Limit, Accuracy, Coefficient of Variation and Precision' mean in volumetric measuring?

An illustration of Precision and Accuracy The dart board simulates the volume range around the centered specified value, the black dots simulate the different measured values of a specified volume.

Poor accuracy: Good accuracy: Poor accuracy: Good accuracy: Hits far off center. On average, hits are evenly Although all hits are close to- All hits are near the center, i.e., Poor reproducibility: distributed around center. gether, the center (true volume) the specified value. Hits widely scattered. Poor reproducibility: is still missed. Good reproducibility: Result: No gross errors, but hits widely Good reproducibility: All hits are close together. These volumetric instruments scattered. All hits are close together. Result: are of inferior quality. Result: Result: The volumetric instruments All deviations are 'equally Improperly controlled produc- have minute systematic errors, probable'. tion, with systematic variation. narrow scatter; the permissible Instruments exceeding the Instruments exceeding the limit is not exhausted. These permissible limit should be permissible limit should be instruments should remain in removed from service. removed from service. service.

To describe accuracy, the term "Error limit" is used for glass volumetric devices, while for liquid handling devices the statisti- cal terms "Accuracy [%]" and "Coefficient of Variation [%]" have become established.

➊ Error limit The term "Error limit" (EL) in the corresponding standards defines the maximum permissible deviation from the specified value. ➍ Partial volumes

Generally A and CV are related to the nominal volume (Vnominal). These values are in % and have to be converted for partial vol-

➋ Accuracy (A) umes (Vpart.). In contrast, there is no conversion for partial vol- Accuracy (A) indicates the closeness of measured mean volume umes, if A and CV are stated in volume units (e.g., ml). to the specified value, i.e., systematic measurement variation. Accuracy is defined as the difference between the measured _ ➎ Error limit of A and CV mean volume (V) and the specified value (V ), related to the spec. A good estimate for the error limit (EL) of the instrument, e.g., for specified value in percent. the nominal volume (Vnominal), can be calculated using the values for accuracy and coefficient of variation. ➌ Coefficient of Variation (CV) Coefficient of variation (CV) indicates the closeness of values of repeated measurements, i.e., random measurement variation. ➏ Precision (reproducilibity) Coefficient of variation is defined as standard deviation in percent, It describes the closeness in volume units between the different related to the mean volume. values in a set of measurements.

1 2 3 4 5

V - V s · 100 Vnominal A% + 2CV% A [%] = spec. CV [%] = A [%] = · A % EL ≥ Vmeasured - Vspec. · 100 part. nominal EL ≥ · Vnominal Vspec. V Vpart. 100%

(analog CVpart. [%] ) BRAND

32 Monitoring of Measuring Instruments Monitoring of Measuring Instruments

Which devices have to be monitored?

All measuring instruments which are used to confirm warranted product qualities are subject to monitoring.

Analytical laboratories have to verify and document the accuracy of all measuring devices used in or- der to achieve reliable results. This especially applies to laboratories which operate according to GLP guidelines, are accredited to DIN EN ISO/IEC 17 025, or are certified to DIN EN ISO 9001. All these standards and guidelines require the availability of written instructions describing the moni- toring procedure in detail. The error limits or accuracy and coefficient of variation have also to be defined, and there must be instructions on how to proceed if the acceptable limits are exceeded.

Timing and frequency of Monitoring of DE-M marking monitoring Testing procedures Time required for testing instruments

The instrument's accuracy and Volumetric instruments are The monitoring of measuring Volumetric instruments which measuring uncertainty must be tested gravimetrically. Testing devices should not become the are DE-M marking to DIN known and documented before for liquid handling instruments main occupation in the labora- 12 600 are also subject to the its admission for use. In addi- is performed according to ISO tory; it should be limited to a monitoring procedure. tion, the instrument has to be 8655; for glass volumetric reasonable extent. The demand There is no clear directive tested again at defined intervals instruments, ISO 4787 is ap- is for simple procedures which whether such instruments have (see DIN EN ISO 10 012). plied. are quick and inexpensive to to undergo initial testing or Many influences must be con- follow. not. The user is responsible for Reason: sidered when carrying this out. The combination of testing answering this question. The performance of measuring Therefore, BRAND provides instructions (SOP), specially However, to be on the safe instruments may be affected Standard Operating Procedures developed EASYCAL™ calibra- side, the initial test of a rep- by e.g., the use of aggressive (SOP), including detailed test- tion software together with resentative random sample chemicals, and the manner and ing instructions, for every type volumetric instruments deliv- is recommended. In addition, frequency of cleaning. Since of volumetric instrument. The ered with individual or batch such a test will document the the required accuracy largely testing procedure is outlined certificate are best suited to initial state in relation to subse- depends on the circumstances step by step. To make it even minimize the time needed for quent tests. of each application, the user easier, BRAND offers software this procedure. Another option would be the has to determine the intervals which can perform all calcula- purchase of volumetric instru- for routine testing. Typical tions, store them in a database, ments with a certificate from intervals for liquid handling in- and print out a detailed test the manufacturer. struments and plastic volumet- report. ric instruments are every 3-12 months, for glass volumetric instruments, every 1-3 years.

Terms used in the monitoring of measuring devices

Calibration Adjustment

Calibration in the strictest Adjustment consists of correct- Depending on the manufac- sense consists of determining ing the deviation of the mea- turer, the adjustment of liquid the actual volume delivered. sured value from the nominal handling instruments is gener- The calibration procedure value. ally accomplished by turning an should be quick and simple, adjustment screw. After set- eliminating potential sources of ting, a new calibration check is error. Therefore, BRAND pro- required. This procedure must vides detailed testing instruc- be repeated until the volume is tions for every type of volumet- within the error limits. ric instrument, free of charge.

BRAND

33 Monitoring of Measuring Instruments

Procedure for volumetric testing e.g., Microliter pipette Transferpette® adjustable volume, 20-200 µl We recommend a calibration of the Transferpette®, as described below, once every 3-12 months. Depending on frequency of use and pipetted media, shorter testing intervals may be defined by the user.

A Preparation: C Gravimetric test: 1. Verify instrument type and nominal capacity. 1. Determine temperature of the liquid for testing. 2. Read serial number. 2. Mount a new pipette tip. 3. If instrument is soiled, disassemble and clean if necessary 3. Condition the instrument: aspirate and release testing liquid (see operating manual). five times. This will improve the accuracy of the test. 4. Check for damage (housing, shaft tip, ejector, piston, seal). 4. Mount a new pipette tip and pre-rinse it once. Obtain spare parts as required. 5. Place the weighing vessel on the balance and tare. 5. Place the Transferpette® pipette (unpacked) including acces- 6. Remove weighing vessel from the balance. sories into the testing room for at least 2 hours to adjust to 7. Discharge testing liquid into weighing vessel, pressing the room temperature. pipetting key to the second stop to empty tip completely. 8. Place weighing vessel on the balance. Read and note value. 9. Retare the balance. B Functional test: 10. Repeat steps 2. to 9. ten times. Enter values obtained at 1. Mount a new pipette tip. 100%, 50% and 10% of nominal volume into the test record. 2. Pre-rinse the tip once with testing liquid (distilled/deionized water). Gravimetric testing values at Values obtained by gravi- 3. Hold the filled pipette vertically and observe for approx. 21.5 °C (Z = 1.0032) 10 seconds whether a drop is forming at the tip. Make sure metric testing only indicate the tip is not being heated, e.g., by the sun. Discharge the Tested volume (µl): 200.0000 the mass (weight) of the liquid. In case of smaller volumes (approx. < 50 µl) no drop Specified value (mg): 199.3620 pipetted volume. To obtain x 200.2000 the actual pipetted volume, will form even if there is a leak, due to surface tension. 1 x 199.6000 the values have to be multi- A tip for finding leaks in small volume pipettes: 2 x 199.4900 plied by a correction factor, Discharge a small drop from the filled tip so that a small air 3 x 199.7000 taking into account the tem- cushion (air bubble) is present below the liquid. If, during 4 x 199.7000 perature (see below). For observation, the air cushion falls, there is a leak. 5 x 199.2900 all BRAND liquid handling 6 instruments detailed testing x 199.3500 7 instructions are available for x 199.4100 8 download at www.brand.de. x9 199.2000

x10 199.1900

1. Calculation of the mean volume: 2. Calculation of accuracy: _ A mean volume ( x ) of the weighing values is calculated by divid- Extract from the table 'Factor Z for – liquid handling Instruments' V - V ing the sum of the weighings by the number of weighings made. A [%] = spec. · 100 This mean mass is then multiplied by_ a correction factor (Z, units Temperature Factor z Vspec. of µl/mg) to give the mean volume ( V ) delivered. The factor Z °C ml/g combines density of water at the testing temperature and effects 18 1.00245 200.1514 - 200 of atmospheric pressure. For a typical temperature of 21.5 °C and A [%] = · 100 air pressure of 1013 mbar (hPa), Z = 1.0032 µl/mg. 18.5 1.00255 200 19 1.00264 – – 19.5 1.00274 A [%] = 0.076 V = x · Z 20 1.00284 – x + x + x +... + x 20.5 1.00294 V = 1 2 3 10 · Z n 21 1.00305 21.5 1.00316 22 1.00327 – 200.2 +199.6 +199.49 + ... + 199.19 V = · 1.0032 22.5 1.00338 10 23 1.00350 – V = 199.513 · 1.0032 23.5 1.00362 24 1.00374 – 24.5 1.00386 V = 200.1514 25 1.00399 25.5 1.00412 26 1.00425 BRAND

34 Monitoring of Measuring Instruments

3. Calculation of the standard deviation, necessary for the determination of coefficient of variation

(x - x–)2 + (x - x– )2 + (x - x– )2 + ... + (x - x– )2 s = Z · 1 2 3 10 n - 1

(200.2 - 199.51)2 + (199.6 - 199.51)2 + (199.49 - 199.51)2 + ... + (199.19 - 199.51)2 s = 1.0032 · 9

0.8393 s = 1.0032 · 9

s = 0.306

4. Calculation of the coefficient of variation:

s · 100 CV [%] = – V

0.306 · 100 CV [%] = 200.1514

CV [%] = 0.153

The result for the calculated example is: What to do if the instrument exceeds the error limits:

Results of the gravimetric 1. Review the operating manual If despite these steps the Note: testing: to ensure that the instru- instrument still does not meet For checking partial volumes, ment was operated properly. the specifications, remove from Tested volume (µl): 200.0000 the values A [%] and service and contact the manu- nominal 2. Follow the troubleshoot- Mean volume (µl): 200.1514 CV [%] which are related facturer for support. nominal ing guide in the operating A [%] 0.076 to the nominal volume V nominal manual for assistance. CV [%] 0.153 must be converted. A [%] specified* 0.600 For a partial volume of 20 µl 3. Recalibrate the instrument in CV [%] specified* 0.200 this means: accordance with the operat- * Error limits in operating manual. ing manual. Vnominal A20 µl[%] = ·A nominal [%] ⇒ This pipette meets V20 µl specifications. 200 µl A [%] = · 0.5% If the calculated values for Ac- 20 µl 20 µl curacy (A [%]) and Coefficient of Variation (CV [%]) are less A20 µl[%] = 5% than or equal to the error limits, the instrument is calibrated to The calculation of CVpart operate within specifications. is analog.

BRAND

35 Monitoring of Measuring Instruments

Calibration software

The monitoring of measuring devices to GLP and DIN EN ISO 9001 is not exactly straightforward. Complex equations easily lead to calculating errors, and the documentation of results can be tiresome. To facilitate this tedious task, some manufacturers have developed special calibration software.

e.g., EASYCAL™ calibration software by BRAND EASYCAL™ performs all A demo version of the soft- calculations and generates ware can be downloaded from the complete documentation, the Internet (www.brand.de) or automatically. All you need is can be requested on CD-ROM an , a PC, without charge. a printer (optional) and EASYCAL™ software. ■ Suitable for instruments from all manufacturers ■ Specifications of many instruments preloaded ■ Testing according to ISO 4787, ISO 8655, etc.

Monitoring made easy

EASYCAL™ calibration soft- manual entry, or direct import ware facilitates the monitoring from the balance via cable, fol- of measuring devices to GLP lowed by automatic evaluation. and DIN EN ISO 9001, both After defining the error limits, for liquid handling instruments EASYCAL™ performs all cal- and for volumetric instruments culations automatically. By one of glass and plastic. push of a button, a comprehen- The software is user-friendly sive test record can be printed and intuitive to use. After out. All results are stored in determining the type of instru- a database. The test history ment to be tested, all neces- keeps track of all tested instru- sary data are entered step by ments, facilitating the monitor- step on the 'Entry of measuring ing over time. The test intervals results' screen. determined in relation to the Two options are available for testing instructions (SOPs) can entering the weighing results: be defined individually.

Calibration service for liquid handling instruments

Calibration can be a time-consuming procedure. For this reason, BRAND offers a full calibration service including instrument ad- justment and, if required, repair. Test record printout

BRAND

36 Declaration of conformity and Calibration Certificates Declaration of conformity and Calibration Certificates

Types of certificates: ■ Declaration of conformity ■ Certificates of performance (works certificates) ■ Calibration certificates (Eichamt, DAkkS certification)

Declaration of conformity Calibration certificates

Measurement and Calibration Regulation and DE-M marking Official calibration certificate

For volumetric instruments to be ready for use in legally regulated This certificate is issued by the 'Eichamt', the German State Of- areas such as the medical and pharmaceutical fields (manufacture fice of Weights and Measures, and is accepted in Germany and and testing of medicinal products), the German Measurement and many other countries. Both the instrument and the certificate Calibration Regulation of December 2014 requires a DE-M mark- show the individual serial number and the year of issue. ing instead of official calibration. This also applies for volumetri- cally relevant accessories (e.g., pipette tips for piston-operated pipettes). DAkkS Calibration certificate

The German Calibration Service (DKD) was founded in 1977 as Who certifies conformity? a joint institution of government and industry. It verifies the con- formity of measuring equipment used in industrial and research DE-M marking means: compliance of an instrument with the laboratories and testing institutions with the national standards German Measurement and Calibration Regulation. The procedure administered by the PTB (German Federal Institute of Physics and of DE-M marking is described in the German Measurement and Metrology). Based on the legal requirements the DKD Accredita- Calibration Regulation of December 2014. tion was successively transformed to the DAkkS Accreditation With the DE-M mark, the manufacturer declares that the instru- (Deutsche Akkreditierungsstelle GmbH), starting from 2010. ment in question meets the requirements of the German Measure- The DAkkS calibration certificate officially documents, at a high ment and Calibration Regulation and applicable standards. The level, the traceability of measuring results to national and inter- DE-M mark is generally printed directly onto the instruments and national standards, including SI units, as required by the groups on the packaging of any associated consumable products. of standards DIN EN ISO 9001 and ISO/IEC 17 025 for the monitoring of measuring instruments. A major difference between works calibration services and DAkkS calibration laboratories is the accurate and reliable determination Conformity symbol of the respective uncertainty of measurement, guaranteed by the accredited laboratory and supervised by the DAkkS. DAkkS calibration certificates are appropriate where calibrations DE-M marking by an accredited laboratory are required, where calibrations of the highest level are demanded and for the calibration of reference All BLAUBRAND® volumetric instruments are marked DE-M. The standards and for instruments used for comparative measure- manufacturer BRAND uses this mark to certify the conformity of the ments. instruments with the German Measurement and Calibration Regula- The DAkkS is a member of the International Laboratory Accre- tion. This sign 'DE-M' is printed directly on the instruments. ditation Cooperation (ILAC). A multilateral agreement assures BRAND lists all the test equipment used in each lot and individual obligatory recognition of the DAkkS calibration certificate in many certificate. countries. The certificate is issued in a variety of languages.

Certificates of performance Batch certificate Individual certificate A quality assurance system organized according to DIN EN ISO 9001 earns the right for a manufacturer to issue certificates of All instruments and certificates Both the instrument and the performance, or factory certificates. from one production batch certificate show an individual Factory certificates are available as individual or batch certificates. show the same batch number. serial number in addition to the All quality control results are documented and archived for a The certificate records the batch number. The certificate minimum of 7 years. If the batch or serial number is known, the mean value, the standard de- records the measured volume, individual results at the time of production can be accessed at any viation and the date of issue. the uncertainty of measurement time. and the date of issue.

BRAND

37 BLAUBRAND® USP volumetric instruments

Companies that manufacture pharmaceutical products for the American market and are therefore audited by US agencies such as the FDA (Food and Drug Administration) are obligated to meet the requirements of USP (United States Pharmacopeia). The current USP describes in Chapter 31 that the Class A error limits specified in the ASTM stan- dards are required for glass volumetric instruments. These error limits are listed in tables for volumet- ric flasks, bulb pipettes, and burettes. For graduated pipettes up to and including the 10 mL size, the error limits are listed in the text.

Error limits BRAND glass volumetric in- struments are manufactured according to current DIN EN ISO standards. Since the con- struction requirements defined in the DIN EN ISO standards differ from those in the ASTM standards, different error limits result for the individual measu- ring instruments. BRAND confirms with the en- closed USP certificates for all USP measuring instruments delivered that the Class A er- ror limits corresponding to the ASTM standards are observed.

DE-M marking USP batch certificate All BLAUBRAND® volumetric The mean value plus standard instruments are DE-M mark- deviation for the batch, along ing. With the sign DE-M, with the date of issue are docu- BRAND, as manufacturer, con- mented on the certificate (batch firms that the instruments are number: year of manufacture/ manufactured according to batch). 'Mess- and Eichverordnung', the German Federal Weights and Measures Regulations. This USP individual certificate the DE-M sign is printed directly on the instruments, according to The measured volume, mea- DIN 12 600. surement uncertainty, and the date of issue are documented on BRAND lists all the test equip- the certificate (individual serial ment used in each lot and indi- number: year of manufacture/ vidual certificate. batch/consecutive instrument number).

BRAND

38 IVD Directive IVD Directive

Implications and consequences

How to define In-Vitro-Diagnostic IVD Directive of EU Medical Devices (IVD)? What is a Medical Device? CE Marking

On December 7th, 1998, the An 'In-Vitro-Diagnostic Medical The definition of a 'Medical The CE mark is the official EU directive for 'In-vitro-Diag- Device' is any medical device Device' includes any instru- marking required by the Eu- nostic Medical Devices' (IVD used in-vitro for the examina- ment, apparatus, appliance, ropean Community. It shows Directive) was published in the tion of specimens, including material or other article, includ- the user, that this product Official Journal of the European blood and tissue donations, ing the software necessary for fulfills all essential safety and Communities and became ef- derived from the human body. its proper application, intended environmental requirements fective since June 7th, 2000. IVD can be a reagent, calibra- by the manufacturer to be as defined in the so-called tor, control material, kit, instru- used for human beings for the European Directives. The ment, apparatus, equipment, purpose of: manufacturer marks the instru- system, or specimen recepta- ment and produces a declara- ■ diagnosis, prevention, cles, intended by the manufac- tion of conformity describing monitoring, treatment or al- turer to be specifically used for the instruments' fulfillment with leviation of disease, injury or in-vitro diagnostic examination. the guidelines and technical handicap IVD are mainly used to provide requirements. information ■ investigation, replacement or modification of the anat- BRAND medical products are ■ concerning a physiological omy or of a physiological all included in the class of in- or pathological state process vitro diagnostic (IVD) devices. ■ concerning a congenital ■ control of conception. This includes, for example: abnormality Excluded are pharmacological – blood counting chambers ■ to monitor therapeutic or immunological means, which – haemacytometer cover measures. are regulated by appropriate drug laws. – disposable capillary pipettes – micro haematocrit capillaries – haematocrit sealing com- pound – sample cups for analyzers – urine beaker – feces container – cryogenic tubes – pipette tips – PD-Tips – Transferpette® microliter pipettes – HandyStep® repetitive pipettes

BRAND

39 Quality Management Quality Management

Quality management is indispensable. Ideally, it should already begin at project stage, and should ac- company a product’s design, development and manufacturing process. This guarantees the greatest possible security in working with laboratory equipment, and the reliability of analytical results.

Quality management at BRAND

Quality management is briefly described for liquid handling instruments and BLAUBRAND® volumetric instruments

Quality management at BRAND The quality management All measuring instruments used All test results are docu- begins at product conception system applied at BRAND and in quality control are regularly mented and filed for 7 years. and continues through the certified to DIN EN ISO 9001 checked and are referenced to If the batch or serial number is design process and production. is a combination of process the national standards of PTB known, each specific test result Routine checks throughout the monitoring and random checks. (The German Federal Institute on the date of production can entire manufacturing process The accepted quality level of Physics and Metrology). be traced. result in volumetric instruments (AQL) is at the very least 0.4., Quality management accord- As BRAND manufactures with the smallest possible i.e., the limiting values are met ing to DIN EN ISO 9001 is the DE-M marking volumetric in- deviation from the true volume with a statistical certainty of at basis for issuing of calibration struments, the quality of prod- (accuracy) and narrow scatter least 99.6 %. certificates (e.g., our certifi- ucts is automatically supervised of individual values (coefficient cates of performance). by the 'Eichamt', the German of variation). The final step of State Office of Weights and this Statistical Process Control Measures. The requirements is random finished product for monitoring of measuring sampling according to DIN instruments, traceability to ISO 3951. national standards, and staff qualification are fully met.

BRAND

40 Cleaning Cleaning of Laboratory Equipment

Manual and machine cleaning

Glass and plastic labware can be cleaned manually, in an immersion bath, or in a laboratory washing machine. Labware should be cleaned immediately after use – at low temperatures, with brief soaking times, and low alkaline detergents. Labware which has come into contact with infectious substances should first be cleaned and afterwards, if necessary, autoclaved. This is the only way to prevent baking of the substance, and subsequent damage to the labware by any adhering chemical residues.

Note: Carefully disinfect labware before cleaning when there is a risk of injury during cleaning procedure.

Wiping and scrubbring method Immersion method Ultrasonic bath

The generally accepted wiping For the immersion method, lab- Both glass and plastic labware and scrubbing method with ware is soaked in the cleaning may be cleaned in an ultrasonic a cloth or sponge soaked in solution for 20 to 30 minutes at bath. However, direct contact cleaning solution is the most room temperature, then rinsed with the sonic membranes must popular cleaning method. with tap water, and finally with be avoided. Labware must never be treated distilled water. Only for stub- with abrasive scouring agents born residues should the soak- or pads which might damage ing time be extended and the the surface. temperature increased.

Machine cleaning

Machine cleaning with a laboratory washing machine is Glass labware Cleaning in trace analysis more gentle to labware than cleaning in an immersion bath. With glass labware, prolonged To minimize metallic traces, The labware is only exposed immersion times in alkaline laboratory equipment is placed to the cleaning solution for the media above 70 °C should into 1N HCl or 1N HNO3 at relatively short flushing periods be avoided. Such treatment, room temperature for not more when sprayed by the jet or particularly with volumetric than 6 hours. (Glass laboratory ejector nozzles. instruments, might cause equipment is often boiled for volume changes through glass 1 hour in 1N HNO .) ■ Lightweight objects will not 3 corrosion, and destruction of It is then rinsed with distilled be tossed and damaged by graduations. water. To minimize organic the jet if they are secured in contamination, laboratory washing nets. equipment can first be cleaned ■ Labware is protected Plastic labware with alkalis, or a solvent such against scratching when the as alcohol. wire baskets in the washing Plastic items generally have machine are plastic coated. smooth, non-wetting surfaces and can usually be cleaned effortlessly under low alkalinity conditions. Polystyrene or polycarbonate labware, e.g., centrifuge tubes, must only be cleaned manually with neutral detergents. Pro- longed exposure even to low alkaline detergents will impair their strength. The chemical resistance of these plastics should be verified in each case.

BRAND

41 Cleaning

Gentle cleaning

50 For gentle treatment of labware, clean immediately after use – at . low temperatures, with brief soaking times, and at low alkalinity. Glass volumetric instruments should not be exposed to prolonged 40 immersion times in alkaline media above 70 °C, as such treatment . 30 causes volume changes through glass corrosion, and destruction of graduations. 20 . layer attack (µm) 10 weight loss (mg/100 cm²) Information 0 6810 12 14 temperature (°C) pH

At 70 °C, a 1N sodium hydrox- 1.4 µm, or tenfold more, will be Alkali attack on Boro 3.3 in relation to Alkali attack on Boro 3.3 in relation to ide solution will corrode a layer removed. Therefore, clean- temperature, calculated from weight pH value, at 100 °C. Exposure time: loss. c (NaOH) = 1 mol/l. Exposure 3 hours. of approx. 0.14 µm off the ing temperatures above 70 °C time: 1 hour. surface of Boro 3.3 (borosili- should be avoided and low cate glass 3.3) within 1 hour. alkaline cleaning agents are (Graphs are from the brochure 'Technische Gläser' by SCHOTT AG, Mainz.) However, at 100 °C, a layer of preferrable.

Disinfection and sterilization

Disinfection Steam sterilization The following points must be observed:

Laboratory instruments that Steam sterilization (autoclaving) ■ Efficient steam sterilization is assured only if the steam is satu- have come into contact with is defined as the destruction rated and has unrestricted access to all contaminated areas. infectious material or geneti- or irreversible inactivation of all ■ To prevent pressure build-up, containers or vessels must al- cally modified organisms must reproducible microorganisms ways be open. be disinfected prior to reuse/ under exposure to saturated ■ Contaminated reusable labware must be cleaned thoroughly disposal; i.e., they must be steam at 121 °C (2 bar) ac- before steam sterilization. Otherwise, residue will bake on brought to a condition in which cording to DIN EN 285. during sterilization and microorganisms may not be effectively they no longer pose a risk of For correct sterilization pro- destroyed if they are protected by the residue. Furthermore, infection. cedure, including biological any adhering chemical residues may damage the surfaces due Therefore laboratory instru- security, please contact your to the high temperatures. ments can be treated with dis- sterilization officer. infecting detergents for exam- ■ Not all plastics are resistant to steam sterilization. Polycarbon- ple. If necessary and suitable, ate, e.g., will lose its strength. Polycarbonate centrifuge tubes the items may subsequently be cannot be steam sterilized. sterilized (autoclaved). ■ During sterilization (autoclaving), plastic equipment in particular should not be mechanically stressed (e.g., do not stack). Thus, to avoid shape deformation, beakers, flasks, and graduated cylinders should be autoclaved in an upright position.

Thermal resistance

All reusable BLAUBRAND® and SILBERBRAND volumetric instruments can be heated up to 250 °C in a drying cabinet or a sterilizer, without any subsequent volume deviations. However, as with all glass instruments, irregular heating or sudden temperature changes produce thermal stresses which may result in breakage. Therefore: ■ Always place glass instruments into a cold drying cabinet or sterilizer; then heat slowly. ■ At the end of the drying or sterilizing period, allow instruments to cool off slowly inside the switched-off . ■ Do not heat up volumetric instruments on a . ■ Pay attention to the maximum operating temperatures of plastic instruments.

BRAND

42 Safety Information Safety Information

Handling of hazardous substances

The handling of hazardous chemicals, infectious, toxic or radioactive substances and genetically modified organisms, calls for a high degree of responsibility on the part of everyone involved, to en- sure personal and environmental protection. The relevant regulations must be scrupulously observed including laboratory, professional association, environmental, radiation, waste disposal and generally accepted technical standards and guidelines (e.g., DIN or ISO).

Important information on safety

■ Before use, laboratory in- ■ In the course of repeated ■ Instruments to be repaired ■ It is not permissible to sim- struments must be examined use, laboratory instruments must be cleansed of all res- ply truncate defective grad- by the user for suitability should be examined for idues and be sterilized, as uated cylinders. This short- and functionality. eventual damage, especially necessary. Radioactive- ens the distance between instruments subjected to ly contaminated items must the upper graduation mark ■ Always hold pipettes near pressure or vacuum (e.g., be decontaminated as pre- and the spout, as defined the suction end, and care- desiccators, filter flasks, scribed by the radiation pro- by DIN, resulting in an in- fully insert the pipette into etc.). tection authorities. Volumet- creased danger of chemicals the adapter of the pipette ric glass instruments (e.g., being spilled and the oper- controller until it is secure- ■ The hazards of working with volumetric flasks, graduated ational safety is no longer ly and firmly seated. Do not defective labware should cylinders, etc.) should not guaranteed. use force. Broken glass can never be under-estimated be repaired when damaged. cause injury! (e.g., cuts, burns, risk of ■ Waste must be disposed of Exposure to heat may result infection). If a professional according to local laws and in residual stress within the repair is not practical, prop- regulations. This applies glass (greatly increasing the erly dispose of such items. also to disposable articles. probability of breakage), or It must not pose a hazard to an uncontrolled cooling pro- human beings or the envi- cess may lead to permanent ronment. volume alterations. ■ Laboratory equipment must be disposed of according to the materials they are made of, and in a clean state, in accordance with the regula- tions in force. Working with glass Please note that is not recyclable. When working with glass, it is essential to consider its limitations regarding resistance to thermal shock and to mechanical stress. Strict safety measures must be observed:

■ Do not heat volumetric ■ Glass instruments must instruments, measuring never be exposed to sudden cylinders and flasks on hot temperature changes. When plates. taking them out of a drying cabinet while hot, never ■ Exothermic reactions such place on a cold or wet lab as diluting sulfuric acid or bench. dissolving solid alkaline hy- droxides must always be ■ For compressive loads, only carried out while stirring and glass instruments intended cooling the reagents, and in for this purpose may be suitable vessels such as Er- used. For example, filter- lenmeyer flasks – never in ing flasks and desiccators graduated cylinders or volu- may be evacuated only after metric flasks! confirming that they are in perfect condition.

BRAND

43 For you notes. Trademark Index

accu-jet®, BLAUBRAND®, BRAND®, Dispensette®, EASYCAL™, HandyStep® S, seripettor®, Titrette®, Transferpette®, ® as well as the word and design marks shown here are brands of BRAND GMBH + CO KG, Germany.

Insofar as other owners’ brands or protect- ed terms, symbols or illustrations are used, ® this occurs merely for reference purposes and without any claim of right. Reproduced brands are the property of the respective owner. BRAND GMBH + CO KG · P.O. Box 1155 · 97861 Wertheim · Germany Tel.: +49 9342 808-0 · Fax: +49 9342 808-98000 · E-Mail: [email protected] · Internet: www.brand.de 9946 43 · Printed in Germany 21/0215