An analytical scientist is required to accurately dispense volumes of liquids from μl to ml levels with a high degree of accuracy. This is true in applications ranging from classical to advanced instrumental analysis .
Most advanced instruments these days come equipped with auto-samplers where pre- programmed volumes of samples and wash solvents are transferred unattended thereby eliminating human errors. However, even for such techniques there is need for sample preparation which may involve serial dilutions or accurate transfer of sample standard reference solutions.
In earlier days glass pipettes (graduated or single mark) were popular. Such pipettes even to this day have widespread presence in school/college laboratories, industrial laboratories and even sophisticated laboratories equipped with latest analytical facilities. However, such pipettes have one severe limitation, i.e, their inability to deliver small volumes (μl levels).This severely limited scope of applications with limited availability of sample amounts. Another main benefit of micro and sub-micro analysis is saving on costs of expensive reagents and standard materials.
Introduction of micro-pipettes extended the limits of analytical chemistry to micro-level determinations. It became possible to measure and deliver volumes from μl to ml levels with high accuracy. Another major advantage offered by micro-pipettes is adjustment to permit variable volumes from the same device ranging from μl to ml levels. However, the benefits offered by micro-pipettes can be exploited to the fullest only if required skills are acquired in their use and suggested precautions are taken during usage.
Handling of Micro-Pipettes
The skills required for handling of micro-pipettes can be acquired through experience but it is important to be aware about the precautions that will help ensure highly precise delivery of volumes every time.
Choice of Appropriate Micropipette
It is important to select the correct micropipette for the requirement at hand. The micro-pipettes are available over a range of volumes that can be adjusted, e.g., 1-20μl, 20 – 200μl 200 – 1000 μl. It can be seen that there can be overlap in volume when you decide on the micropipette. Always opt for smaller volume range micropipette for greatest accuracy, for example if your requirement is for 50 μl volume transfer select the 20 – 200 μl range over the 100 – 1000 μl range micropipette.
Adjustment of Transfer Volume
Volume adjustment knob helps you to set the required volume. Carefully and slowly set the digits of the indicator dial taking care not to turn beyond the upper and lower limits. This can introduce free play and damage to the piston.
The volume of a liquid is dependent on the temperature of surroundings. Allow the sample solution and pipette tips to equilibrate to room temperature for adequate time for transfer of accurate volumes or avoiding evaporation loss due to expansion of captive air inside the barrel on account of temperature variation from extended period of holding in hand. This can result in smaller delivery volumes than set values.
Immersion Depth of Tip
The tip immersion depth into solution can influence the volume uptake. Too shallow immersion can increase uptake of air along with solution and deeper immersion can increase risk of adherence of fine droplets to the tip and larger intake due to increased hydrostatic pressure outside the tip. Optimally the tape should be immersed between 2-6 mm into the solution
Accuracy and precise solution transfer essentially requires development of micropipette handling skills. These involve:
- Application of sufficient force to the tip in the box to produce an airtight seal between the tip and the shaft of the micropipette
- Smoothly depress the plunger to first stop. Pause for a moment
- Keeping the barrel vertical immerse into the solution and depress to second stop. Release plunger gradually to ensure smooth filling without turbulence or splash as a splash can contaminate the barrel
- Wipe only the side of the tip without contacting the nozzle
- Do not place the filled micropipette on the bench as this can contaminate the barrel
- Deliver the contents to the receiving tube or vial by touching the tip against the side wall of the container
- Press the ejector to dispense the used tip
- Reset the volume to zero after using the micropipette
Micropipettes are versatile devices which are capable of delivery of consistent volumes every time provided the recommended precautions are adopted in their handling and use.