I have noticed that more solution is used for reverse pipetting than for normal pipetting. What is the reason for this?

Reverse pipetting means uptake of liquid including the blow-out volume and dispensing of liquid without the blow-out volume. Reverse pipetting is recommended to reduce the error with viscous or foaming liquids or when dispensing very small sample volumes. The excess solution aspirated remains in the tip and is discarded.

Can you recommend any literature on reverse pipetting?

Userguides 20 and 21 describe the technique and give application examples. They can be downloaded from the Knowledge Base on our website.

Should the pipette tip be wetted with liquid before pipetting volumes smaller than 10 µL?

Regardless of the volume, the air cushion should be wetted by multiple filling and emptying of the tip before dispensing.

What is the best way to pipette viscous liquids?

When pipetting viscous liquids, we recommend positive displacement instruments like Biomaster, Multipette/Repeater, and Varipette or an air cushion pipette (Research plus, Reference 2) in combination with the reverse pipetting technique (see Userguide 20 and 21 for details).

Is there an Eppendorf pipette which can be used to pipette 40 % polyethylene glycol 8000 (PEG)?

Yes, for pipetting PEG instruments operating according to the positive displacement principle like the Biomaster or a Multipette/Repeater can be used.

What is the best way to pipette chloroform?

When pipetting chloroform, we recommend positive displacement instruments like Biomaster, Multipette/Repeater, and Varipette or an air cushion pipette (Research plus, Reference 2) in combination with the reverse pipetting technique (see Userguide 20 and 21 for details).

What is the best way to pipette diethyl ether?

Diethyl ether has such a high vapor pressure that it would leak out of the tip immediately when an air cushion pipette is used. For this reason, it can only be pipetted using a positive displacement pipette, such as Biomaster, Multipette/Repeater, and Varipette.

Are Eppendorf pipettes resistant to acetonitrile?

Yes, Eppendorf pipettes are resistant to acetonitrile but care must be taken to avoid damage to printing and display window.

What should be considered when pipetting methanol with air cushion pipettes?

Due to the increased vapor pressure, we recommend that the tip is prewetted at least 3 times and that the reverse pipetting technique is used.

What is the best pipette for pipetting a 99% solution of n-amyl alcohol?

A 99 % solution of n-amyl alcohol is a highly viscous solution. We recommend working with an instrument that operates according to the positive displacement principle like Multipette/Repeater, Varipette, or Biomaster.

What is the best way to dispense organic solvent?

It is best to use a positive displacement device like the Multipette/Repeater. Positive displacement devices are most suitable for precise and drip-free dispensing of liquids with high vapor pressure. As the liquid is enclosed in a tip with integrated piston, the solvent vapor does not come into direct contact with the instrument.

I would like to pipette dichloromethane and n-hexanol very precisely. Can I use air cushion pipettes for this application?

Both dichloromethane and n-hexanol have a very high vapor pressure. For that reason, positive displacement instruments like the Biomaster and the Multipette/Repeater should be used.

Are your pipettes free of nucleic acids?

No, we cannot guarantee this. Of course we work carefully and cleanly in production, but no special measures are taken to prevent the infiltration of nucleic acids. To protect your pipette from contamination, we recommend using positive displacement pipettes, for example, our Biomaster or filtertips (ep Dualfilter T.I.P.S. or ep Dualfilter T.I.P.S. SealMax).

How can radioactive contamination be removed from Eppendorf pipettes?

Following contamination of pipettes with radioactive liquids, we recommend placing the contaminated parts in a complex-forming liquid or special cleaning solution. Then rinse very thoroughly with distilled water and dry. Grease piston lightly afterwards.

Do Eppendorf pipettes always need to be greased?

No, the piston only has to be greased if grease-dissolving solutions like organic solvents have been used.

Can the pipette withstand evacuation (applying a vacuum)?

No, air cushion pipettes should not be used under vacuum, as the pressure ratios are not right.

How should warm or cold liquids be pipetted?

Exactly in the same way as liquids at room temperature, however, please note that solutions may behave differently. In addition, the pipetting accuracy / precision may be outside the permissible error limits. In order to achieve maximum accuracy / precision, all components should have the same temperature: pipette, tips, liquid, and the surrounding air.

Are the handles of Eppendorf pipettes made of a special material to minimize temperature effects during pipetting?

Temperature effects are minimized in our pipettes by their design. We use a special polypropylene which satisfies the special requirements on a pipette handle.

Do Eppendorf pipettes always have to be kept in a stand or can they also be put down on the laboratory bench?

They can be put down on the laboratory bench - but never with a full tip.

I often work with different chemicals. Can you tell me when I need to adjust my pipettes?

This belongs to the permissible errors of your specific application. In general an adjustment of air cushion pipettes, in other words altering the stroke of the pipette, is required for solutions of a density higher 1.2 mg/µL. Please be aware that variable manual pipettes turn into "fixed-volume pipettes" when being adjusted: They can only be adjusted to a specific liquid to one volume and not across the entire dispensing range. Eppendorf manual pipettes have a user adjustment for temporary adjustment purposes. This user adjustment can easily be set back to manufacturer settings without the need for recalibration. For the correct procedure and examples for settings of user adjustments for different liquids please see the operating instructions and CD enclosed to your pipette.

How do I determine the volume values when calibrating gravimetrically?

The measuring values of the analysis scales are multiplied by the correction factor Z (see SOP pipettes). This takes into account the density of the water at the relevant temperature and the relevant air.

What does EN ISO 8655 cover?

ISO 8655 addresses the needs of suppliers, test houses, and users and specifies the general requirements for piston-operated liquid handling devices. It is structured in seven chapters: ISO 8655-1: Terminology, general requirements, and user recommendations ISO 8655-2: Piston pipettes ISO 8655-3: Piston burettes ISO 8655-4: Dilutors ISO 8655-5: Dispensers ISO 8655-6: Gravimetric method for the determination of measurement error ISO 8655-7: Non-gravimetric methods for assessment of equipment performance (titration, photometry)

What do the terms random error and systematic error mean in a pipette calibration?

The systematic error describes the deviation of the dispensed volume from the nominal volume or from the selected volume of the piston stroke device, and is determined by establishing the mean value of (usually) 10 measurements. The random error describes the mean variation of the dispensed volumes from the mean value of the dispensed volumes, and is determined by calculating the repeat standard deviation of (usually) 10 measurements.

Why are the Z factors listed in our SOP different from factors which can be derived from density reference values (of water)?

The Z factor is included in EN ISO 8655-6 and was incorporated into our SOP. As we understand it, it was incorporated in the standard by the Physikalisch Technische Bundesanstalt in Braunschweig. The Z factor describes the density of water at a particular temperature using a 5th degree polynomial. Furthermore, the density of water is corrected by the density of air (derived from air pressure) and a fixed correction value for balances. These corrections are the key reasons for the deviation from density values in various books of tables.

At which volumes should a variable pipette be calibrated?

According to the EN ISO 8655 standard, a variable pipette should be calibrated to 3 volumes during conformity testing: 1. Nominal volume, 2. 50 % of nominal volume, 3. Smallest value that can be set or 10 % of nominal volume.

What is the nominal volume of a variable pipette?

The nominal volume of a variable pipette is always the maximum volume. For a 100 - 1,000 µL pipette, it is therefore 1,000 µL.

I very frequently work with different chemicals. Can I still perform pipette calibration using water?

Calibration, i.e. checking a pipette on a balance, is always performed using distilled or deionized water (see EN ISO 8655).

During weighing (pipette calibration) I obtained steadily decreasing measuring results. Why is this?

The weight fluctuations you measured would appear to be a temperature problem. Please ensure that the pipette, the distilled water used for testing, the tips etc. are all at the same temperature. It is recommended that the pipettes be placed in the weighing room at least 2 hours before the test is performed. While measuring, ensure that transfer of heat from your hands is kept to a minimum (otherwise weight fluctuations may occur!) Tip: do not constantly grasp the pipette in the palm of your hand while waiting for each measurement result! We recommend our standard operating procedure (SOP) for pipettes as a source of basic information on pipette calibration.

How are multi-channel pipettes tested?

According to ISO 8655, all channels of multi-channel pipettes are to be tested. All channels have to be filled but only the channel to be tested is measured. Because of the high number of measurements (8-channel pipette: 240 measurements, 12-channel pipette: 360 measurements), it makes sense to send the multi-channel pipette to a professional calibration lab. These labs have special constructions for balances to ensure that all channels are filled but only one channel is measured.

Is the pipette grease free of resins?

Yes, the pipette grease supplied by Eppendorf is free of resins.

How is the wall mount for pipettes attached to the wall?

The pipette holder is affixed to the wall with adhesive.

How many times can Reference 2 be autoclaved?

Our pipettes are checked for a minimum lifetime of five years. This means a normal use with 200,000 strokes, 26,000 volume changes, 100,000 tip ejections, and 175 autoclavings without the pipetting results and pipette material being affected.

Do I have to regrease the Reference 2 after autoclaving?

If the pipette grease is not removed actively (e.g., by cleaning with alcohols before autoclaving), there is no need to regrease the piston-cylinder system. During endurance testing (200,000 strokes, 26,000 volume changes, 100,000 tip ejections, and 175 autoclavings), the Reference 2 was not regreased and gravimetrically checked after each autoclaving. No decline in systematic and random measurement error was observed. If regreasing is needed, Eppendorf grease for Research plus and Reference 2 must be used. Only this grease withstands the heat in the required manner.

The housing of Reference 2 contains different materials. What effects does this have on the pipette during autoclaving?

The Reference 2 is mounted in a way that all materials have room for expansion. Furthermore, only materials are used which are resistant to severe conditions, e.g. autoclaving (pressure, rise in temperature).

Is it possible to inactivate the spring-loaded tip cone of a Reference 2 single-channel pipette?

Yes. The scope of delivery includes a locking ring with which the spring-loaded tip cone can easily be inactivated. Disassemble the upper from the lower part of the pipette and push the locking ring onto the lower part from the top. Please refer to the operating manual for an illustrated description.

Is it possible to inactivate the spring-loaded tip cone of a Reference 2 multi-channel pipette?

Yes. The Reference 2 multi-channel pipettes have a switch on the lower part which inactivates/activates the spring-loaded tip cone.

Where and how can the Reference 2 pipettes be adjusted?

Reference 2 pipettes offer factory adjustment and user adjustment. The factory adjustment should be changed if the pipette was found to be out of specifications during calibration. Before adjusting, exclude other causes such as pipette handling, environmental errors, or worn-out parts. The user adjustment is an easy way of adjusting the pipette temporarily to different conditions (e.g. pipetting a liquid with high density). Please note that the adjustment is only valid for the chosen volume and not for the full volume range.

How often should pipettes be calibrated?

The user decides upon the frequency of calibration, the number of test volumes and the number of measurements for each test volume within the framework of monitoring his test materials. This can vary depending on the frequency of use of the pipette, the number of users of the device, the aggressiveness of the liquids to be dispensed and the error limits deemed acceptable by the user. However, an inspection should be carried out at least once a year. See also EN ISO 8655-6.

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Eppendorf Reference^® 2

Eppendorf Reference® 2 single-channel pipette along with 8- and 12-channel choices

Changing volume settings on the precise Eppendorf Reference® 2 pipette is quick and easy

Eppendorf Reference® 2 mechanical pipette in use with a tube

On/Off switch to deactivate spring-loaded tip cones on Eppendorf Reference 2 multi-channel lower part

Eppendorf Reference® 2 single-channel pipette family

Eppendorf Reference® 2 multi-channel pipette family

View of parts for the Eppendorf Reference® 2 mechanical pipette

epT.I.P.S.® macro volume tips facilitate easy reach to the bottom of e.g. 15 mL centrifugation tubes or flasks

The Pipette Carousel 2 with space for up to six Eppendorf pipettes

Laboratory technician using an Eppendorf Reference® 2 mechanical single-button pipette in the lab

Laboratory technician with Eppendorf Reference® 2 mechanical pipette in the lab

Laboratory technician adjusting Eppendorf Reference® 2 one-button pipette in the lab

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Choisissez la pipette mécanique Eppendorf Reference 2 à fonctionnement unique à un seul bouton ergonomique et bénéficiez de sa précision et de son exactitude remarquables avec des efforts de manipulation réduits. Étant complètement autoclavable et présentant une surface lisse, celle-ci peut être décontaminée de manière efficace, ce qui en fait un outil idéal pour le travail dans des conditions stériles. Disponible comme pipette monocanal à volume fixe ou variable et comme pipette à 8 et 12 canaux - toujours avec un fonctionnement unique à un seul bouton qui permet de gagner du temps. Une fois que vous aurez essayé cette philosophie de fonctionnement unique, vous ne pourrez plus vous en passer !

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Une pipette premium qui permet d’obtenir une précision et une exactitude exceptionnelles
La pipette mécanique à coussin d’air Eppendorf Reference 2 de précision et exactitude remarquables est extrêmement fiable et robuste, permettant de rallonger sa durée de vie. Grâce à son fonctionnement unique à un seul bouton, la Reference 2 permet une manipulation rapide et ergonomique avec des efforts d’utilisation réduits et une baisse réelle de la contamination par les aérosols. Le retour haptique permet d’éviter l’éjection accidentelle des pointes. Ne faites aucun compromis sur la qualité et la reproductibilité – choisissez la Eppendorf Reference 2 pour pipeter avec précision et en toute confiance vos échantillons les plus précieux et améliorer vos résultats.
La poignée solide en acier inoxydable protège la pipette des impacts et, en association avec les butoirs à amortisseur de force, la pipette Reference 2 a toutes les chances de rester étalonnée même en cas de chute. Ces niveaux de sécurité élevés font de la Reference 2 un outil idéal pour les applications nécessitant une exactitude extrêmement élevée.

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