Top 10 Crimes Committed Against Pipettes

How to NOT use a pipette:

#1: Not Proactively Replacing Common Parts at Routinely Scheduled Intervals:

Pipettes must have common parts replaced routinely. The following pipette parts should be inspected, tested, and replaced proactively in order to maintain good accuracy and precision.

• Seal and o-ring: The internal polyethylene seal begins to wear and needs replacing at least once every year (12 months. The rubber o-ring is typically more durable and typically should be replaced every 1-2 years (12-24 months). If you’re replacing the seal, it is recommended to replace the o-ring at the same time to allow both parts to wear/break-in together. Pipette Supplies, Inc stocks and sells many different pipette models seals and o-rings.
  
• Pipette Shaft: The pipette shaft is highly durable, but should be replaced every 2-3 years (24-36 months). Over time the constant friction of loading and ejecting pipette tips, chemical splash from liquids, etc. begins to wear down the shaft enamel, causing the pipette tip to potentially slip and not seal as strongly as designed. Internally, the shaft can become pitted or scratched and prevent the seal and o-ring from sealing properly, leading to liquid leaking from the tip.
  • Think of pipette shafts like your teeth enamel. Liquids, friction, and acids from food and drink attack and try to wear down teeth enamel. Without routine brushing and flossing your teeth enamel can become compromised. The same is true with the pipette shaft and can lead the pipette to not fully aspirate and dispense the desired volume, thus impacting accuracy and precision.

• Piston Assembly: The internal piston assembly should be replaced every 3-4 years (36-48 months). Piston materials vary by pipette manufacturer. Pistons are typically made of 316-gauge stainless steel, porcelain/ceramic, plexus-glass, or other high-grade plastics. The process of aspirating and dispensing liquids takes its toll on the piston assembly, especially when vapors, aerosols, chemical splash, etc. come in contact with the piston assembly and piston lubricant (on those models that require it).
  
  When liquids or chemical splash come in contact with the piston, it can cause the stainless-steel piston to rust, corrode, become scratched, and change the consistency of the piston lubricant (if applicable). Also, repeated autoclaving of the pipette and piston assembly may expand and contract the plastics of the piston. Long term, the piston may not be as linear/straight as it was when new.
  
  
• Pipette Lubricant: Some pipette models use a high-grade lubricant on the internal piston assembly, seal, and/or o-ring. Not all lubricants are alike and is recommended that only the recommended manufacturer lubricant be used. On most models that require lubricant, it is recommended that the lubricant be removed, parts cleaned, and re-lubricated with the manufacturer’s recommended lubricant at least once every 6 months (1/2 year). Lubricant is like a magnet and attracts dust, vapors, aerosols, and chemicals.
  • Over the course of use and operation, the lubricant can migrate and become dirty due to chemical splash, vapors, aerosols, etc. This is highly visible if this has occurred, as the pipette would visibly leak when set to the nominal volume. Keep the lubricant clean to ensure good accuracy and precision.

• Batteries (Electronic Pipettes): Over the years, pipette manufacturers commonly selected Nickel Cadmium (Ni-Cd), Nickel Metal Hydride (Ni-mH), Lithium-Ion (Li-Ion), etc., as their preferred batteries of choice. The style of battery is imprinted on the battery itself.
  • Recharging of Ni-Cd batteries have a high memory effect and should only be recharged once the battery is depleted. Consistent recharging of the battery when not depleted or left sitting on the charging stand will degrade the battery life cycle. Most Ni-Cd batteries last 1-2 years for the simple fact that the end user does not let the battery fully deplete itself before recharging. Once the battery is fully recharged, unplug the stand.
  • Recharging of Ni-mH batteries have a moderate memory effect and like the Ni-Cd batteries should only be recharged once the battery is depleted. Consistent recharging of the battery when not depleted or left sitting on the charging stand will degrade the battery life cycle moderately. Most Ni-mH batteries last 2-3 years. Once the battery is fully recharged, unplug the stand.
  • Recharging Lithium-Ion (Li-ion) batteries have a low memory effect and can be left on the charging stand or recharged when not fully depleted. Most Li-Ion batteries have a small PCB chip embedded into the battery that will allow to top off the battery when recharging and not accept any further recharging when fully recharged. This means the battery will not shut itself off when fully recharged, thus protecting itself and life cycle. Most Li-Ion batteries last 3-5 years.

#2 Not Protecting Pipette Plunger Rod When Stored on The Pipette Stand, Carousel, or Pipette Hang-Ups:

Storing your pipette at the nominal volume (i.e., at the 100% volume) can increase the probability of a damaged plunger rod and other internal damage, if dropped. Think about this analogy. When is a turtle the most vulnerable?  When the turtle’s neck, head, or legs are exposed from its shell. A turtle is most protected when its neck, head, and legs are safely retracted inside its shell. Pipette plunger rods are a lot like a turtle’s neck, head, and legs. The plunger rod is most protected against damage when it is retracted to the 10% volume. The 10% volume provides the most protection to the plunger rod should it be knocked off the pipette stand or dropped on the floor. This helps to ensure accuracy and precision and costly repairs due to bent plunger rods.

#3 Not Storing Pipettes Vertical When Not in Use:

Pipettes like to be stored vertically! Be good to your pipettes and store them on a pipette stand, in the middle of the lab bench away from edges and opportunities to be knocked off.

Placing a pipette horizontally on the lab bench, in a drawer, on the counter, etc. can increase the probability of damage to the pipette shaft, body, and plunger rod. Pipettes stored horizontally are more costly to keep accurate and precise than pipettes stored vertically.

#4 The Selection of Non-Manufacturer Tips -Versus – Manufacturer Tips:

Tight budgets and spending restrictions are common in most labs. Selection and procurement of pipettes tips is one of the most common overlooked crimes against pipettes. Many of us know very little about the intricacies of pipettes, pipette tips, and filtered tips. If the tip fits and allows for the aspiration and dispensing of liquids, then it must be okay, right? Not exactly.

Most pipette manufacturers engineer, injection mold, and publish their accuracy and precision specifications for their pipettes when used with their tips. However, published accuracy and precision specifications may or may not be obtained when using manufacturer pipette tips with a competitor pipette. Most pipette and tip manufacturer’s leave it to the lab technician, supervisor, manager, or purchasing agent to decide if the desired volume delivered will meet their internal requirements, SOP’s, and expectations.

Think about this before placing your next pipette tip order:

• Are you purchasing the same manufacturer tips as your pipette?
  • Using manufacturer tips on the same manufacturer’s pipette (for example: Gilson Diamond tips with a Gilson Pipetman, or Eppendorf Research-Plus with Eppendorf EP tips) will provide the best accuracy and precision results, guaranteed! Why? The tips are engineered and designed to fit, form, seal, and deliver the best results over and over again. The problem lies when manufacturer tips are used with a pipette from a different manufacturer. Why? Once again, manufacturers design their tips to deliver the best results when used with their pipettes. When different pipettes and tips are interchanged, end users can typically expect up to a 10% margin of error in the volume aspirated and dispensed due to different variables. This could be due to the differences in:
    • Micron size of the filter to allow air flow to permeate and pass (if filter tips are used).
      The smaller the pore size of the micron <20um, the greater restriction of air-flow through the filter
    • Tip orifice size (wide orifice, normal orifice).
      Wide orifice tips have larger swings in both accuracy and precision due to larger orifice. The larger the orifice opening, the more liquid that wants to enter or leave
    • Thin-walled tips, thick-walled tips, yip orifice beveled/orientation.
      When dispensing, liquids like to cling to beveled and thick wall tips. This can have an impact on accuracy and precision. Thin-walled tips allow for better dispensing and droplet touch-off.
    • Embedded tip sealing ring(s) location when sealing to a pipette shaft. The internal tip sealing ring is in different locations on different manufacturer’s tips. This may prevent the tip from properly sealing to the shaft fully and allowing desired aspiration and dispensing with the pipette.
    • Type of liquid aspirated & dispensed (aqueous, dense, volatile, viscous, high-surface tension). Air displacement pipettes excel with aqueous-like liquids and can be impacted by other liquids. The internal air cavity inside the shaft will not allow the pipette to achieve proper aspiration or leak with non-aqueous-like liquids. If the air cavity is weaker than the liquid aspirated, you selected the incorrect pipette. When the liquids are dense, volatile, viscous, or have high surface tension, use a positive displacement pipette.
• Does your application assay require the use of filtered pipette tips?
  • Not all filtered tips are the same. Do your research to ensure that the filtered tips will meet your specific application needs. This includes but is not limited to:
    • What material is the tip filter made from? Most filters are made from molded polyethylene or use a cellulose gum additive (i.e., self-sealing when it gets wet). Some filters may flake or leach out and potentially compromise your samples.
    • What is the average pore size of the tip filter being used? Air-displacement pipettes require the displacement of air in order to aspirate and dispense your sample. Most tip manufacturer’s filters are between 25microns -45+ microns that will successfully allow air to permeate through the filter in order to aspirate and dispense your sample. Once the pore size of the filter decreases lower than 20 microns or smaller, then the air flow needed in order to aspirate and dispense liquids becomes more restricted. This may lead to lower-than-expected volumes of liquids aspirated and dispensed.
    • Self-Sealing Filters: Are a great concept, but only work when they get wet. If a portion of the filter gets wet, it will self-seal and limits air flow. Of course, this is mostly obvious if the entire filter becomes wet because no liquid will be aspirated.
• Does your application assay require the use of low retention tips?
  • Low retention tips are great in helping to fully expel costly liquids from the tip or those liquids that are troublesome at fully dispensing. Many tip manufacturers either injection mold or dip their tips into a fluoropolymer like fluoropel to give them low retention properties. Keep in mind that if using low retention tips at volumes of 5uL or lower, the probability of aspirating and dispensing the desired volume may be lower than expected. Most fluoropel proprieties are gone after 1-3 aspiration and dispensing cycles.
• Does your application require the use of wide orifice (wide bore) tips?
  • Wide orifice tips have a slightly larger tip orifice opening. Wide orifice tips are great when aspirating and dispensing viscous, dense, high surface tension liquids that get caught up at the tip orifice with a normal sized tip orifice. However, wide orifice tips have a tendency to aspirate and dispense slightly more liquid during the pipette cycle due to the larger tip orifice. This may impact the accuracy and precision of the pipette used under test. The pipette gets the blame when the volume delivered is not what was expected. Be sure that it is not the wide orifice tip. Also, wide orifice tips should not be used for the calibration of pipettes for the same reason. The accuracy and repeatability may not be as good as expected when compared to a normal tip orifice pipette tip.
• Do you autoclave your tips?
  • The common concern with autoclaving tips is that they are autoclaved multiple times. Pipette tips should be autoclaved ONLY once at 121°C for 10 minutes with a 5-minute drying cycle at 110°C. Filtered or presterilized tips NEVER should be autoclaved as the process of becoming sterilized through gamma radiation or electron-beam radiation could make the polypropylene tips brittle and crack if autoclaved. Not to mention, the tip filter will become brittle and flake during the autoclaving process.
• Do you store your tips under UV light?
  • Pipette tips can be stored in a hood under UV light in a pipette tip rack with the lid closed. Leaving the rack uncovered for an extended period of time with the UC light on will make the tips brittle and crack, especially when loading a tip onto a pipette shaft. Also, the internal sealing ring in the pipette tip may become compromised if expose to UV light for an extended period of time in an uncovered rack. UV light can impact tips, thus providing compromised accuracy and precision results that are often a crime blamed on the pipette.

#5 Not Adhering to Good Pipetting Techniques:

Learning, using, and maintaining good pipetting techniques is vital to the life and longevity of any pipette. The following pipetting techniques below will help put you on the road to providing your pipettes with a long life.

• Reduce mechanical back-lash: Mechanical back-lash is the art of setting the pipette to its desired volume. Most mechanical pipettes have a three- or four-digit volume display and the spacing (commonly referred to slop) in-between the digits can lead to minor volume mis-settings. Winding the pipette 1/3 of a turn above the desired volume (i.e., 203uL ad then back to 200uL) will help minimize mechanical slop in-between the dials. Thus, the desired volume delivered should be slightly more accurate. Every little bit helps, right?

• Pre-Rinse at least twice for aqueous-like liquids: Pipette tips are equilibrated to air temperature and are hydrophilic. By wetting the tip through two prerinses at the desired volume makes the tip hydrophobic and wettable. Also, the tip is now equilibrated as close to the aqueous-like sample temperature. Pre-rinse of at least two times, can improve accuracy up to 0.2% of the desired volume under test. Water versus air temperature may vary from 0.5-1.0°C. By prerinsing at least twice helps to minimize evaporation impacts and deliver better accuracy and precision.

• Tip immersion depth:One of the common mistakes end user make that lead to larger swings in accuracy and precision is not following industry-wide tip immersion depths. Adopting this will aid in delivering better accuracy and precision. Recommended tip immersion depths by nominal volume are below. This means submerge the pipette tip “x” mm beneath the surface of liquid before aspiration:
  • 2uL -10uL: 1-2mm
  • 20uL -50uL: 2-3mm
  • 100-300uL: 3-4mm
  • 1000uL – 2000uL: 4-6mm
  • 5000uL and up: 6-12mm

• Pipetting verticalness: Not holding the pipette vertical during aspiration and dispensing may lead to inaccurate volumes. This means you may aspirate too little, too much or not fully get all of the liquid out of the tip during dispensing. Holding the pipette greater than 20% of vertical exacerbates the volume aspirated and dispensed, and may lead to up to 0.2% less volume.

• Reduce hand-warming: Holding and using a pipette (especially in gloved hand) for at least 15-30 minutes non-stop can impact the amount of volume aspirated and delivered. As we know already, air-displacement pipettes use a partial air cavity in order to aspirate and dispense liquids. When the air cavity gets heated through our hands, it will expand, causing the liquid aspirated and dispensed to be less.  By holding the pipette loosely, using the finger hook (if applicable) or placing the pipette in the stand in-between pipetting cycles, will minimize hand warming impacts. Pipetting 30 minutes consecutively may impact accuracy of the desired volume under test by 0.2% or more. Don’t let your pipette air cavity get warm.

• Speed, time, smoothness, and care: Establishing a good pipette rhythm is helpful to ensure good accuracy and precision. Do it the same way every time. Care is given to not aspirate and dispense too fast. When aspirating, gently control the aspiration upward with mechanical pipettes and pause 1-2 seconds after full aspiration to ensure that a full aspiration is received before removing your tip from the sample. Not pausing could lead to air bubble, or not achieving a full aspiration.
  • Dispensing should be in a smooth, controlled rhythm to ensure the sample flows smoothly out of the tip. When the sample is fully dispensed, pause 1-2 seconds, then proceed to blow-out and touch off against the side the reservoir. Not pausing before blowout and then touching off may impact the amount of volume delivered, especially if the tip is a thick-walled tip or has a beveled orifice that may have liquid clinging to it after dispensing.

• 60-Second pipette liquid sealing system test: On a routine basis, based on frequency of use, end users should conduct a 60 second leak test to confirm there are no visible leaks with the pipetting sealing system. This can be accomplished as such:
  • Set the pipette volume to nominal (i.e., 100% volume)
  • Fully load a pipette tip onto the shaft
  • Immerse the tip into deionized water or distilled water at the recommended tip immersion depth
  • Gently aspirate liquid into the tip in a smooth, controlled rhythm. Pause 1-2 seconds after full aspiration
  • Remove the tip from the sample and hold the pipette loosely by the finger hook or body and count to ten. We are looking for any visible leaks from the pipette tip orifice.
  • If no leaks are detected, the internal sealing system is maintaining a proper air vacuum.
  • Lastly, visibly inspect the exterior of the shaft for wear, scratches, marks, etc. where the pipette tip seals to the shaft.

If a leak is detected, please contact Pipette Supplies, Inc for additional troubleshooting support and possible part(s) replacement.

#6 Not Performing Routine Preventive Maintenance and Calibration:

Pipettes require routine preventive maintenance and verification to ensure accuracy and precision are maintained. Common replacement parts that customers can replace include but are not limited to: seals and o-rings, shafts, piston assemblies, and lubricant as needed.

The accuracy and precision of a pipette should be verified at least once every year (12 months), based on frequency of use, or every 6 months for pipettes that use internal grease lubricant on the piston assembly (i.e., 5000uL and larger sizes).  This includes calibration adjustments as needed. There are many qualified calibration service providers out there than can verify the accuracy and precision. This ensures that your pipettes remain in tip-top shape and continue to provide many hours of reliable use.

#7 Selection of Calibration Service Providers – They’re Not All the Same:

The selection of a calibration service provider is almost as important as purchasing a new pipette itself. A good calibration service provider will extend the life of your pipette and keep it accurate and precise. Adversely, selecting the incorrect calibration provider can be a crime against your pipette and shorten it’s lifespan. When selecting a pipette calibration provider, the following should be considered:

• Are they accredited to a calibration standard such as ISO 9001, ISO 17025, etc. If yes, ask for a copy of their calibration procedure (SOP’s) and accreditation certificate(s)
• Do they calibrate pipettes to manufacturer’s specifications, ISO 8655 tolerances, or customer tolerances?
• What are their calibration service offerings and pricing?
• Do they calibrate pipettes with manufacturer tips?
• Do they use manufacturer parts or manufacturer equivalent?
• What are their calibration lab temperature and humidity requirements?
• Do they verify pipettes gravimetrically or photometrically?
• What is the balance resolution used for the calibration of pipettes of different volume sizes?
• What is their current turn-around-time (TAT)?
• Do they provide onsite calibration service at the customer’s facility?
• Are they able to provide calibration service for most pipette models in the market?
• Can they provide training records of qualifications for different pipette models?
• Do they provide true preventive maintenance, replacement of parts, and calibration?

#8 Selecting the Incorrect Pipette for The Application Assay:

Another common crime committed against pipettes is asking the pipette to do an application assay that it is not designed to handle. There are three common pipette systems and selecting the correct one(s) is vital. These three pipette systems are:

• Air-Displacement: Air displacement pipettes are the most commonly used pipettes and can be found in almost every lab throughout the world. This style of pipette uses an internal piston assembly, seal, and o-ring to create a partial air vacuum inside of the white colored shaft. The partial air vacuum allows for the displacement of air that is needed for the aspiration and displacement of liquids.
  • Excels at: Aspirating and dispensing aqueous (water-like) liquids.
  • Does not excel with: More dense, viscous, volatile, high surface tension liquids.
  • Comments/Observations: If you find that the air-displacement pipette does not work as well with your liquids or application assay, consider the positive displacement pipette.
  • Examples: Eppendorf Research Plus, Gilson Pipetman, Rainin Pipet-Lite XLS, Finnpipette, Sartorius Tacta, etc.
• Positive Displacement (Pos-D): Positive Displacement pipettes are almost as common as air displacement pipettes. The Pos-D uses an external capillary and piston (CP) I order to aspirate and dispense viscous, volatile, aqueous, and high surface tension liquids. This style of pipette does not have an “internal air vacuum” as the external piston comes in direct contact with the liquid in order to aspirate and dispense t from the capillary.
  • Excels at: Aspirating and dispensing viscous, dense, volatile, high surface tension, and yes; aqueous (water-like) liquids.
  • Does not excel with: N/A, the accuracy and precision specifications of Pos-D pipettes are wider/larger than air displacement pipettes due to direct contact of liquid with the piston, friction, coefficient of expansion of the plastics in the capillary, etc.
  • Comments/Observations: While the Pos-D can be used with aqueous-like liquids, the cost of purchasing capillary and pistons (CP’s) is higher than the cost of polypropylene (PP) tips used for air displacement pipettes.  PP tips cost is ~10 -15 cents each, CP’s ~50-75 cents each.
  • Examples: Eppendorf Repeater X-Stream, Brand Handy-Step, Gilson Microman, RAININ Pos-D, etc.
• Serological: Serological pipettes are used for the transfer of large liquids from one container to another. These pipettes use an external, graduated plastic or glass tube that allows the user to aspirate and dispense liquids.
  • Excels at: Transferring aqueous-like and other liquids through an external glass/plastic graduated tip that attaches to the unit.
  • Does not excel with: N/A, serological pipettes are not calibrated instruments. The graduated plastic/glass tube is sold calibrated.
  • Comments/Observations:  Internal filters, tubing, and motor need to be checked time-to-time for liquid, debris and or replaced.
  • Examples: Pipet-Aid Filler, Eppendorf Easy-Pet, etc.

#9 Not Maximizing the Pipette Potential:

You can severely minimize damage to your pipettes by giving the following recommendations serious consideration:

• Perform proactive parts replacement with manufacturer parts or manufacturer equivalent
• Implement and demonstrate good pipetting practices
• Use manufacturer’s tips with that manufacturer’s brand of pipette (best accuracy and precision)
• Conduct routine pipette liquid inspection checks at least monthly
• Conduct routine calibration verification (every 6 – 12 months based on pipette model & volume size)

#10 Doing Nothing:

The biggest crime against pipettes is doing nothing at all. Pipettes are sometimes viewed as consumables that can be tossed and replaced. Having trusted resources that you can turn to when needed for advice, recommendations, or support is only a call or e-mail away. We are here to assist when you need us.

Phone: 817-483-9883 or Contact Us

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