Pipetting Made Easy - Part 2: Challenging Liquids

Taming Tricky Liquids: A Deep Dive into Advanced Pipetting

If you've ever struggled with pipetting thick gels, foamy detergents, or fast-evaporating solvents, you already know that not all liquids behave the same. Pipetting may seem straightforward, but achieving accurate and reproducible results becomes much more challenging when your sample isn't water. Viscous, volatile, or otherwise demanding liquids can test even the skills of experienced laboratory professionals.

The good news? With the right techniques and the right tools, you can overcome these challenges and achieve precise, reliable results in every pipetting task.

Air and Adhesion: The Hidden Challenges of Accurate Pipetting

Most laboratory pipettes use an air-displacement system. They are designed to deliver highly accurate volumes of aqueous solutions, making them ideal for routine laboratory work. Inside the pipette, a small air cushion separates the piston from the liquid. As the plunger is pressed and released, this air cushion expands and contracts, drawing liquid into the tip and dispensing a defined volume.

This principle works extremely well for water-based samples. However, liquids with different viscosity, volatility, density, or temperature behave differently. Since the air cushion is compressible, changes in air pressure can affect the transferred volume. In addition, some liquids tend to adhere to the inner surface of the pipette tip, further reducing dispensing accuracy.

KNAUER: Figure 1 - Air-displacement system. An air cushion separates the liquid from the piston

The Ideal Solution: Positive Displacement Pipettes

For non-aqueous and difficult-to-handle liquids, positive displacement pipettes are often the best choice. They are particularly suitable for highly viscous, volatile, infectious, or otherwise challenging samples. Instead of an air cushion, these pipettes use a disposable piston inside the capillary tip that comes into direct contact with the liquid.

Without an air cushion to compress or expand, sample properties such as density and vapor pressure no longer influence pipetting accuracy. The piston-capillary design also minimizes errors caused by liquid remaining on the inner walls of the tip.

As a result, positive displacement pipettes provide the highest accuracy and precision when transferring liquids that differ significantly from water.

KNAUER: Figure 2 - Positive-displacement system. The liquid is in direct contact with the piston. No air cushion is involved.

When a Positive Displacement Pipette Isn't Available

Of course, not every laboratory has access to a positive displacement pipette. If you're working with challenging liquids using a standard air-displacement pipette, several simple adjustments can significantly improve accuracy.

Switching to reverse pipetting, reducing aspiration and dispensing speed, and recalibrating the pipette using the actual sample instead of water can all make a noticeable difference. Remember that air-displacement pipettes are factory calibrated with water under controlled conditions, so their performance may differ with other liquids.

Let's take a closer look at how different liquid properties influence pipetting performance—and how to compensate for them when using air-displacement pipettes.

Reverse Pipetting – A Simple Technique with Big Benefits

When handling viscous, volatile, or foaming liquids with an air-displacement pipette, reverse pipetting is often the most effective way to improve accuracy and reproducibility.

The technique involves aspirating slightly more liquid than required while dispensing only the desired volume, leaving a small residual amount inside the tip. This extra volume compensates for liquid retained on the tip walls and helps prevent dripping or air bubble formation.

Although reverse pipetting takes a little practice, it can greatly improve consistency when working with difficult samples.

KNAUER: Reverse Pipetting – The Best Kept Secret for Challenging Liquids

High- and Low-Density Liquids

• Examples: Chloroform, ethanol, sulfuric acid, phosphoric acid
• Challenge: Liquid density influences the size of the air cushion inside the pipette, which affects the aspirated volume. High-density liquids exert greater force on the air cushion, resulting in less liquid entering the tip than expected.
• Solution: Recalibrate the pipette using the liquid being transferred.

KNAUER: High- and Low-Density Liquids

Viscous Liquids

• Examples: Glycerol, DMSO, Tween® 20, oils
• Challenge: Viscous liquids flow slowly. Fast aspiration can introduce air bubbles, while the liquid itself tends to adhere to the tip walls, reducing the dispensed volume.
• Solution: Use reverse pipetting, aspirate and dispense slowly, and allow additional waiting time after each step. Recalibrate the pipette using the viscous liquid.
• Tip: Low-retention and wide-bore tips reduce adhesion and allow viscous samples to enter the tip more easily.

KNAUER: Viscous Liquids

Volatile Liquids

• Examples: Acetone, acetonitrile, ethanol
• Challenge: Volatile liquids evaporate into the air cushion, increasing internal pressure. This can force liquid out of the tip, leading to droplet formation and sample loss.
• Solution: Pre-wet the tip at least five times to equilibrate vapor pressure. Use reverse pipetting at a relatively fast speed and avoid unnecessary delays between aspiration and dispensing.

KNAUER: Volatile Liquids

Foaming Liquids

• Examples: Tween® 20, BSA solutions
• Challenge: Foam traps air bubbles, reducing the amount of liquid actually transferred.
• Solution: Pipette slowly using the reverse pipetting technique to minimize bubble formation.
• Tip: Wide-bore tips reduce foaming, while filter tips protect the pipette from aerosol contamination.

KNAUER: Foaming Liquids

Low Surface Tension Liquids

• Example: Detergents such as Tween® 20
• Challenge: Liquids with low surface tension readily spread across plastic surfaces and tend to remain inside standard pipette tips.
• Solution: Use slow reverse pipetting together with high-quality low-retention tips to minimize liquid adhesion.

KNAUER: Low Surface Tension Liquids

Infectious, Corrosive, Toxic, or Radioactive Liquids

• Examples: Blood samples, DNA/RNA PCR reagents
• Challenge: Aerosol formation can contaminate the pipette cone, increasing the risk of cross-contamination.
• Solution: Use standard forward pipetting together with filter tips to prevent contamination. Regularly disinfect the pipette with 70% ethanol or autoclave it when appropriate.

KNAUER: Infectious, Corrosive, Toxic, or Radioactive Liquids

Liquids with Significant Temperature Differences

• Examples: Buffers at 37°C, nucleic acid reagents stored at 4°C or below
• Challenge: Temperature differences change the volume of the air cushion. Warm liquids expand the air cushion during aspiration, causing under-delivery, while cold liquids contract it, often resulting in over-delivery.
• Solution: Whenever possible, allow samples and pipettes to reach the same temperature. If this isn't practical, use a fresh tip for every transfer and avoid pre-rinsing the tip.

KNAUER: Liquids with Significant Temperature Differences

The Takeaway

Working with challenging liquids doesn't have to compromise your results. Whether you're transferring viscous gels, volatile solvents, or sensitive biological samples, successful pipetting depends on understanding how each liquid interacts with your pipette.

KNAUER: Table 1 - Recommended pipetting techniques and best practices for various liquids.

By selecting the appropriate technique—such as reverse pipetting, adjusting aspiration speed, pre-wetting tips, choosing low-retention consumables, or using a positive displacement pipette—you can significantly improve accuracy, precision, and reproducibility.

Mastering pipetting isn't just about technique—it's about understanding the science behind every transfer.