How to pipette small volumes

pipette small volumes in beaker

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Pipetting is a foundational scientific laboratory technique critical for accurate and precise liquid handling. It’s used in diverse fields such as biochemistry, molecular biology, and clinical testing. However, even minor inaccuracies can significantly impact experimental results when dealing with small volumes. 

Thus, acquiring expertise in precisely manipulating small volumes using a pipette is vital to upholding the validity and consistency of scientific research outcomes. Pipetting small volumes will be discussed in this blog post, providing valuable insights and techniques to improve accuracy and precision, ultimately enhancing the reliability and reproducibility of scientific experiments.

pipette small volumes

Pipette Small Volumes

Understanding Small Volumes in Pipetting

In scientific laboratories, precise measurement and transfer of liquids are critical tasks performed using a technique known as pipetting. When we talk about ‘small volumes’ in the context of pipetting, we generally refer to measurements ranging from 0.1 µL (microliters) to 1000 µL or 1 mL (milliliter). These volumes are typically handled with micro-pipettes, specialized devices designed for accuracy and precision when working with such tiny quantities.

Techniques for Measuring Small Volumes

Various methods are available for calculating tiny volumes, each offering different degrees of precision. The two main types are air displacement pipetting and positive displacement pipetting.

The technique of air displacement pipetting is most frequently used. It involves a piston in the pipette, creating an air cushion between the liquid sample and the piston itself, making it ideal for aqueous solutions.

Alternatively, positive displacement pipetting involves a piston driving the liquid right out from the pipette’s end, similar to the mechanism of a syringe. This method is best suited for handling viscous, volatile, or hazardous liquids where air displacement might lead to inaccuracies.

pipette

Role of Small Volume Pipetting in Molecular Biology, Biochemistry, and Analytical Chemistry

Pipetting small volumes is an everyday task in molecular biology, biochemistry, and analytical chemistry. For instance, in molecular biology, the extraction and amplification of DNA or RNA often require precise pipetting of small volumes of reagents and samples. 

Similarly, in biochemistry, enzyme assays and protein studies demand accurate pipetting of small volumes to ensure correct reaction conditions and reliable results.

In analytical chemistry, techniques such as High-Performance Liquid Chromatography (HPLC) and Gas Chromatography-Mass Spectrometry (GC-MS) rely on precise small-volume pipetting for sample preparation and injection. For example, an HPLC analysis might require accurately pipetting small volumes of a sample solution into a vial to inject the chromatograph.

The Significance of Small-Volume Pipetting

The importance of small-volume pipetting in these fields cannot be overstated. Reliability and reproducibility of experimental results are directly impacted by the accuracy of these measurements. Inaccurate pipetting can introduce significant errors, leading to incorrect conclusions and potentially invalidating hours of painstaking work.

Moreover, precise small-volume pipetting allows for increased sensitivity in experimental procedures, enabling the detection and quantification of substances at lower concentrations. It also contributes to cost-effectiveness, as working with smaller volumes reduces the quantity of expensive reagents required.

The Pipetting Equipment

Selecting the Right Pipette Size

The pipette’s size, which refers to its maximum capacity, dramatically influences the accuracy and exactness of your measurements. You’d use smaller pipettes for precise transfer of minuscule volumes, while larger ones are better for handling more enormous volumes.

As an example, suppose your task requires handling volumes under 1 milliliter. In this case, a micropipette, available in sizes like 10µl, 20µl, 200µl, and 1000µl, would be your go-to instrument. 

On the other hand, if you’re dealing with volumes between 1 and 50 milliliters, a macro-pipette would be more suitable. Don’t forget that choosing the correct pipette size is integral to maintaining your experimental findings’ accuracy, precision, and consistency.

how to pipette small volumes

Importance of Accuracy, Precision, and Repeatability

In scientific experiments, accuracy (how close a measurement is to the actual value) and precision (how consistent repeated measurements are) are essential. Selecting the correct pipette size aids in achieving both.

Repeatability is another crucial factor, referring to the degree of agreement between measurements under unchanged conditions. Utilizing the appropriate pipette size enhances repeatability, providing reliable and consistent results.

Tips for Identifying the Perfect Pipette Size

Understand Your Volume Requirements: Determine the volume of liquid you need to transfer. This will directly influence your choice of pipette size.

Consider the Liquid’s Properties: The nature of the liquid (viscosity, volatility, etc.) can affect the accuracy of pipetting. For viscous or volatile liquids, positive displacement pipettes are recommended.

Calibrate Regularly: Regular calibration ensures that your pipette delivers the correct volume consistently. This is particularly important when working with small volumes.

Making Necessary Adjustments

Even with the right pipette size, adjustments may be needed to maintain accuracy and precision. For instance, if you notice a consistent measurement discrepancy, it might indicate the need for recalibration. Moreover, the pipetting method, including speed and angle, can influence accuracy. Enhancing this skill through practice and training can be beneficial.

Choosing the right pipette size enhances the credibility of your scientific work by ensuring accurate, precise, and repeatable results. It’s crucial to achieving reliable outcomes in your experiments, lab equipment handling, or product testing. Recognizing the significance of pipette sizes and making appropriate selections can uphold the credibility and dependability of your scientific experiments.

Key Considerations Before Pipetting

Pipettes require calibration to ensure accurate volume measurements. It involves adjusting the pipette to dispense the correct amount of liquid confidently.

  • Regularly Check Calibration: Regular calibration checks are essential. Aim for at least once every six months or after every 10 hours of continuous use.
  • Use Professional Services: For accurate results, consider using professional calibration services. They use advanced technologies that can provide more precise measurements.
  • In-house Calibration: If you decide to calibrate in-house, follow the manufacturer’s guidelines strictly.

Prewetting Pipette Tips: An Overlooked Step for Improved Accuracy

Prewetting pipette tips can often be overlooked, but it’s a critical step that can improve the accuracy of your pipettes.

  • Why Prewet? : Prewetting increases the humidity inside the pipette tip, which reduces evaporation and ensures a more accurate volume.
  • How to Prewet? : To prewet the pipette tip, aspirate and expel the liquid 2-3 times before aspirating the sample for dispensing.

Maintaining Proper Pipetting Technique: A Game-Changer for Your Results

How you handle your pipette can significantly impact the results of your study. Here’s how to ensure a proper technique:

  • Pipetting Angle: Hold the pipette vertically while aspirating and dispensing. This ensures that the liquid’s total volume is aspirated or dispensed.
  • Speed: Pipette slowly to prevent the formation of bubbles, which can affect volume accuracy.
  • Consistency: Maintain a consistent technique, especially during repetitive pipetting, to ensure repeatability in your results.
pipette small volumes

Step-by-Step Guide: How to Pipette Small Volumes

Selecting the Appropriate Pipette Tip

  • Tip Selection: Choose a tip that fits your pipette snugly to prevent leaks. The tip should also be suitable for the volume you’re dispensing.
  • Avoid Reusing Tips: Use a new tip for each sample to prevent cross-contamination.
  • Essential Safety Tip: Never touch the tip with your hands. Use the ejector button to release used tips into a waste container.

Aspirating the Sample Like a Pro

  • Pre-wet the Tip: Aspirate and dispense the liquid 2-3 times before aspirating your sample. This step ensures the inside of the tip is coated with your sample, reducing evaporation.
  • Correct Angle: Hold the pipette vertically while aspirating.
  • Avoid Air Bubbles: Aspirate slowly to avoid creating air bubbles. If you see any, gently tap the pipette to move the bubbles to the top, then expel and re-aspirate.

Dispensing the Liquid Accurately

  • Dispense Slowly: Dispense the liquid against the side of the receiving vessel at a 45-degree angle. This technique helps reduce splashing and allows for more controlled dispensing.
  • Ensure Complete Dispensing: After dispensing, touch the pipette tip to the vessel’s side to allow any remaining liquid to flow out.
  • Don’t Blow Out: Only blow out the last liquid if blow-out pipette. Blowing out can lead to over-dispensing.

Techniques for Pipetting Small Volumes

Explanation and Benefits

The reverse pipetting technique is used primarily for handling viscous or volatile liquids or when dispensing small volumes requiring the highest accuracy.

Unlike forward (or regular) pipetting, where the liquid is drawn up to the set volume and then fully expelled, the reverse technique involves drawing up more liquid than needed and dispensing only the desired volume. The excess liquid is left in the pipette tip.

Benefits of Reverse Pipetting:

  • Reduced error: This method reduces error when dealing with difficult-to-pipette liquids, like those that are viscous or volatile.
  • Improved accuracy: Reverse pipetting enhances accuracy and precision when dispensing small volumes.
  • Prevents bubbles: This technique is less likely to create bubbles, which can affect volume accuracy.

Step-by-Step Guide to Reverse Pipetting

  • Set the Volume: Set the volume on your pipette to the desired amount.
  • Attach the Pipette Tip: Attach a clean, appropriate pipette tip firmly onto the pipette.
  • Depress the Plunger: You should depress the plunger until the second stop rather than stopping at the first stop.
  • Immerse the Tip: Immerse the pipette tip into the liquid, ensuring it is not too deep to avoid splashing or taking up too much liquid.
  • Aspirate the Sample: Slowly release the plunger to aspirate the sample. You will notice you’re drawing up more liquid than the set volume.
  • Dispense the Sample: Move your pipette to the receiving vessel, touch the tip to the vessel’s side, and depress the plunger smoothly to the first stop, dispensing the set volume of liquid.
  • Release the Remaining Liquid: Remove the pipette from the vessel without releasing the plunger. The excess liquid will remain in the tip.
  • Eject the Tip: Eject the pipette tip into a waste container.
how to pipette small volumes

Troubleshooting Common Issues

Dealing with Pesky Air Bubbles

  • Identify the Source: Bubbles often form during mixing or filling processes. Check your equipment for any signs of air leakage.
  • Solution: Adjust the speed of your filling or mixing process. Sometimes, slowing down the process can reduce the formation of air bubbles.
  • Prevention: Regularly maintain and check your equipment for leaks. Also, consider using anti-foaming agents if appropriate for your product.

Addressing Contamination Concerns

  • Identify the Source: Contamination can occur at any production stage – from raw materials to production processes, handling, and packaging.
  • Solution: Isolate and remove the contaminated batch immediately. Clean and sterilize the equipment before restarting the production process.
  • Prevention: Implement strict hygiene protocols, regular cleaning schedules, and rigorous quality checks on raw materials—train staff on proper handling techniques.

Recognizing and Correcting Inaccuracies in Volume Measurement

  • Identify the Issue: Regularly calibrate your measuring instruments to ensure their accuracy. If product batches are consistent, it could indicate accurate volume measurement.
  • Solution: Recalibrate your measuring instruments. Consider professional calibration services for high-precision instruments.
  • Prevention: Schedule regular calibration checks and maintenance of your measuring instruments. Ensure staff understand the importance of accurate measurements.
pipette small volumes

Maintaining Pipetting Equipment

Importance of Regular Maintenance and Calibration

Regular maintenance ensures your pipettes function correctly, providing accurate and consistent results. Calibration verifies that your pipettes are measuring accurately. These practices are crucial for data integrity, productivity, and prolonging the lifespan of your equipment.

Cleaning and Maintenance Guidelines

  • Daily: Wipe the exterior with 70% ethanol.
  • Weekly: Check for damage, such as cracks in the shaft, and clean the pipette tip holder using a brush and soapy water.
  • Monthly: Lubricate the piston and seals to your pipette model, if applicable. Check the manufacturer’s instructions for acceptable lubricants.
  • Every 3-6 months: Perform a more thorough cleaning. Disassemble the pipette according to the manufacturer’s instructions, clean all removable parts, and reassemble.

Note: Always refer to the manufacturer’s instructions, as cleaning procedures vary between models and brands.

Regular Calibration Practices

  • Frequency: Most manufacturers recommend calibration every 6-12 months. However, it may be needed more frequently if the pipette is used often or for critical measurements.
  • Procedure: Use a high-precision balance to weigh the volume of water dispensed by the pipette. Compare this to the expected weight (remember, 1 mL of water weighs 1 g). Adjust the calibration if the measured weight is outside the manufacturer’s tolerance range.
  • Documentation: Record each calibration, including the date, results, and adjustments.

Prolonging the Lifespan of Pipettes

  • Proper Use: Use the pipette within its specified volume range and avoid harsh     chemicals that can damage the seals and piston.
  • Storage: Always store pipettes upright to prevent liquid from entering the shaft.
  • Handling: Do not expose pipettes to extreme temperatures or humidity, affecting their performance.

Identifying When Replacement is Necessary

  • Repeated Calibration Issues: If you frequently need to recalibrate, this could indicate wear and tear.
  • Physical Damage: Any visible damage, like cracks or leaks, should prompt immediate replacement.
  • Performance Issues: It may be time for a new pipette if you notice consistent inaccuracies or inconsistencies in your measurements.

 

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Last words

Regular maintenance and calibration of pipettes are critical for accurate, consistent results and equipment longevity. Precision in pipetting, tiny volumes, is crucial to data integrity and productivity. 

Lab technicians must continue improving their skills through ongoing practice, leading to increased comfort and efficiency with the equipment. In the end, striving for precision in pipetting bolsters the credibility of experimental outcomes and cultivates an environment of perpetual learning and superior scientific standards.

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