Common Errors in Micropipetting and How to Avoid Them
Common Errors in Micropipetting and How to Avoid Them
Blog Article
Introduction
Micropipettes are essential instruments in contemporary chemical and biological labs that enable accurate measurement and transfer of tiny liquid quantities. However, using the right micropipetting techniques is crucial to the precision and dependability of investigations. Particularly in sensitive applications like PCR, ELISA, or spectrophotometric analysis, even little mistakes might cause notable variations in the results. Common micropipetting errors nevertheless happen despite their frequent use, frequently as a result of poor technique, handling, or insufficient training.
In order to ensure higher accuracy and consistency in lab work, this blog examines the most common micropipetting errors and provides helpful advice on how to avoid them.
The wrong pipette was chosen.
Selecting the incorrect micropipette for a given volume range is one of the most fundamental yet frequently disregarded problems. Every micropipette is made to produce a specific volume range; for instance, a P20 can deliver 2–20 µL, while a P200 can deliver 20–200 µL. Because of the limited resolution and mechanical restrictions at the lower end of its range, using a P1000 to dispense 10 µL is extremely imprecise.
Always choose a micropipette that lets your required volume fall between the middle and upper range of its capacity to guarantee precise pipetting. This lowers variation and guarantees improved control.
Incorrect Attachment of the Tip
A lot of lab professionals neglect to firmly fasten the micropipette's disposable tip. Air leakage from a loosely fitted tip might lead to insufficient aspiration and imprecise volume transfer. Furthermore, the issue may get worse if universal tips are used, which do not fit all pipettes well.
Do not twist the micropipette too much; instead, press it firmly into the tip. Maintaining a tight and airtight seal requires using premium, suitable tips made for your pipette model.
Using the Incorrect Angle for Pipetting
Accuracy can be significantly impacted by the angle at which you hold the micropipette during inhalation and dispensing. The actual volume drawn may be decreased if it is held at an angle greater than 20 degrees from vertical during aspiration, allowing air bubbles to enter.
Always aspirate while holding the micropipette upright to prevent this. Consistency is crucial for dispensing, however a small angle of up to 45 degrees is allowed.
Not Wetting the Tip First
Surface tension from a dry tip can result in liquid retention, particularly in volatile or viscous liquids. Delivery volumes become irregular and lower than anticipated as a result.
Before taking your real measurement, aspirate and dispense the liquid one or two times to get rid of this problem. By reducing evaporation and residual liquid sticking to the tip, pre-wetting the tip conditions the inner surface and increases precision.
Quick Plunger Action
Micropipetting calls for a steady, slow hand. When aspirating or dispensing, pressing the plunger too fast might result in splashing and air bubbles, which can lead to erroneous volume transfer and possible sample loss.
The plunger should always be pressed and released smoothly and slowly. For micro-volume transfers in particular, the most accurate outcomes will come from a motion that is steady and quiet.
Making Use of the Second Stop While Aspiring
There are two stops on each micropipette plunger. While the second stop is utilized to remove any liquid that may still be present from the tip, the first stop measures the predetermined volume. Pressing the plunger to the second stop while aspirating is a typical mistake that causes excessive volume intake, especially for novice users.
Always only press the plunger to the first stop when aspirating. To guarantee that all of the inhaled liquid is given, just use the second stop for dispensing.
Variability in Immersion Depth
Accuracy of inhalation and volume can be impacted by the depth to which the tip is submerged in the liquid. Air may be pulled in if the tip is submerged too shallowly. Changes in pressure and depth can compromise accuracy.
For small volumes, the tip should ideally be submerged only 2–3 mm below the liquid surface; for greater volumes, it should be submerged up to 6 mm. Maintaining a constant depth guarantees uniform pressure distribution and inhibits the production of bubbles.
Effects of Temperature
Volume accuracy may be impacted when pipetting hot or cold liquids without taking temperature variations into consideration. The temperature of the liquid, pipette, and surrounding environment can change the results because air displacement pipettes are sensitive to thermal expansion.
To minimize this issue, ensure that your liquids, pipettes, and tips are all at room temperature. Use positive displacement pipettes, which are less impacted by temperature fluctuations, for extremely delicate processes.
Ignoring Typical Calibration
Micropipettes may become out of calibration over time as a result of mechanical wear and tear or frequent use. When uncalibrated pipettes are used repeatedly, systemic mistakes and inaccurate data are produced.
Depending on usage, follow a regular calibration schedule, which is typically every three to six months. Many labs send pipettes to expert calibration services or use in-house calibration. Additionally, if available, conduct routine checks using gravimetric techniques.
Concluding remarks
Micropipetting is both a science and an art. Even the most advanced equipment might provide bad results if not used correctly. Researchers and lab staff can greatly increase their accuracy, repeatability, and confidence in experimental findings by comprehending and resolving the main mistakes related to micropipetting.
Mastering micropipetting requires careful skill, regular maintenance, and appropriate training. The micropipette becomes an effective instrument in any laboratory setting when certain procedures are regularly followed, allowing for accuracy in anything from standard tests to ground-breaking investigations.
Investing time on micropipetting best practices is an easy yet powerful technique to guarantee high-quality outcomes when you are starting a new lab or teaching a new group of researchers.