The accurate transfer of minute liquid volumes is a core process in modern science, supporting fields from medical diagnostics to environmental analysis. Reliable results depend on pipettes, which are precision instruments designed to measure and move liquids with minimal error. Consistent delivery of the exact target volume requires both a properly functioning tool and a refined technique. This guide provides instructions for mastering these instruments to ensure experimental integrity.
Understanding Pipette Types and Function
The most common devices for handling small, variable volumes are air-displacement micropipettes. These operate using a piston that moves within a cylinder to create a vacuum. This system displaces a fixed volume of air, allowing an equal volume of liquid to be drawn into a disposable tip when the plunger is released. These instruments are adjustable, letting the user select any volume within the specified range, typically from 0.1 microliters up to 10 milliliters.
Other liquid handling tools include fixed-volume pipettes or volumetric pipettes, which dispense a single, highly precise volume. Serological pipettes are long, graduated tubes that rely on gravity or an external pump to dispense larger volumes, usually 1 to 50 milliliters. While volumetric glass pipettes offer superior precision for a fixed volume, the air-displacement micropipette is the standard tool for most small-volume laboratory work due to its adaptability and ease of use with aqueous solutions.
Step-by-Step Guide to Accurate Pipetting
The process begins by setting the desired volume on the digital indicator, ensuring the setting remains within the instrument’s specified maximum and minimum range. A clean, correctly sized disposable tip must then be attached firmly to the pipette’s cone. This creates an airtight seal necessary for the air-displacement mechanism; a loose tip compromises the vacuum and leads to inaccurate aspiration.
Before aspirating the sample, “pre-wet” the tip, especially when working with volatile or aqueous liquids. This involves aspirating and dispensing the liquid three to five times back into the source container. Pre-wetting saturates the air cushion inside the tip with vapor, reducing evaporation effects and minimizing volume loss due to surface tension.
To aspirate, depress the plunger only to the first stop before immersing the tip in the liquid. The tip should be immersed minimally, typically two to three millimeters below the surface for volumes up to 1,000 microliters, to prevent excess liquid from clinging to the outside. Release the plunger slowly and smoothly to avoid drawing air bubbles or splashing liquid into the pipette shaft.
After aspiration, pause for one to two seconds to allow the liquid column to stabilize before removing the pipette from the source. The instrument must be held vertically during aspiration to maintain the hydrostatic pressure required for the correct volume. When dispensing, place the tip against the inner wall of the receiving vessel at a slight angle (10 to 45 degrees) to aid smooth delivery.
Expel the liquid by depressing the plunger smoothly to the first stop, and then immediately continue to the second stop, or “blow-out.” The second stop ensures all the measured volume is dispensed by pushing out the small residual volume remaining in the tip. Hold the plunger down completely until the tip is withdrawn from the vessel, and eject the tip immediately afterward to prevent cross-contamination.
Troubleshooting Common Technique Mistakes
Deviation from proper technique is the primary source of volume inaccuracy, even with a calibrated instrument. A frequent error is incorrect immersion depth. Plunging the tip too deep causes liquid to adhere to the outer surface, leading to over-delivery. Too shallow an immersion risks sucking up air. Insert the tip only a few millimeters beneath the surface, ensuring the opening remains submerged as the liquid level drops.
Aspiration speed significantly impacts accuracy, especially with viscous liquids. Releasing the plunger too quickly creates a sudden, strong vacuum that causes turbulence, resulting in air bubbles or liquid splashing into the pipette shaft. A smooth, controlled release allows the liquid to enter the tip evenly and prevents volume errors.
The angle at which the pipette is held during aspiration also affects the result. If the instrument is tilted too far from vertical, the hydrostatic pressure changes, causing the aspirated volume to be inconsistent. For accurate measurement, hold the pipette as close to a 90-degree angle as possible during suction.
Temperature differences between the pipette, the liquid, and the environment can create errors due to thermal expansion or contraction of the air cushion. Allowing the liquid and the pipette to equilibrate to the same room temperature helps mitigate these volumetric changes.
Forgetting the pre-wetting step is detrimental when handling volatile solutions. Without pre-wetting, the initial aspiration causes the volatile liquid to evaporate into the dry air cushion. This increases internal pressure and forces some liquid out of the tip, resulting in an under-delivery. Pre-wetting ensures the air cushion is saturated, maintaining a stable volume during measurement.