Choosing the right pipette for laboratory tasks is critical, highlighting the importance of pipetting accuracy and efficiency throughout scientific and medical research. This white paper provides an overview of the two main categories of pipettes—air-displacement and positive-displacement—and provides tips for optimal use of pipettes based on specific workflow requirements.
1 Introduction The ability to handle and transfer small amounts of liquid using pipettes is fundamental to many scientific and medical procedures. Therefore, it is crucial to choose the right pipette and use the right technique. Choosing the right pipette for a specific application can significantly impact throughput and workflow, ultimately improving results and increasing productivity. Different types of pipetting tools help achieve optimal results and higher productivity, while offering additional benefits such as improved ergonomics and better functionality for specific applications. There are two main types of pipettes: air displacement and positive displacement. Both displacement types use the diameter and stroke length of the piston to determine the amount of liquid dispensed. This white paper provides an overview of common types of pipettes and provides valuable tips on how to get the most out of them based on your workflow.
2 Air-displacement pipettes Air-displacement pipettes are the most common pipetting instruments in laboratories. These pipettes operate by placing the tip end into the liquid sample and then releasing the plunger button. As the pipette piston moves upward within the body, a partial vacuum is created and liquid sample is drawn into the tip to fill the void. Air displacement pipette:
Pipetting Cycle and Technical Pipetting Cycle When using any air displacement pipette, the pipetting cycle consists of four main steps:
Pipetting Technique Pipetting technique is arguably one of the most critical factors in delivering accurate volumes, yet it is often overlooked. Insufficient training, incorrect assumptions, and lack of understanding of sample properties can greatly affect experimental results and reproducibility.
Proper pipetting technique is essential to achieve high precision. It is generally accepted that the results obtained with air displacement pipettes depend on technique. There are two different but powerful techniques when using air displacement pipettes: forward and reverse pipetting. Each technique uses the same pipetting cycle, but some steps are slightly different. For the application or for each technique, the biggest difference between the two techniques depends on the nature of the sample and the temperature at which the protocol needs to be performed. The forward technique delivers accurate volumes when pipetting aqueous solutions, while reverse pipetting is highly recommended when working with challenging liquids (e.g. viscous, dense). The main difference between forward and reverse pipetting lies in the first two steps of the pipetting cycle (e.g. liquid aspiration). When performing the forward technique, the plunger is depressed to the first stop (Figure 3A). Using the reverse technique, the plunger is depressed to the second stop (Figure 3B).
3 Positive Displacement Pipettes Although less common than air displacement pipettes, positive displacement pipettes are often seen in laboratory settings. These pipettes use a disposable piston and capillary system to physically void the selected volume. The piston is in direct contact with the sample, and as the piston moves upward, the sample is drawn into the capillary (Figure 4).
Positive displacement pipettes provide high precision when pipetting aqueous solutions, but are generally recommended for viscous, dense, volatile and corrosive solutions. Positive displacement pipettes use disposable capillaries and pistons that are more expensive than disposable air displacement pipette tips, so when they produce the same results, air displacement pipettes are recommended.
4 Sample properties and pipette selection Sample type and viscosity
Certain types of pipettes are better suited than others for different sample types. For example, viscous samples may require different techniques or pipettes to achieve good accuracy in an experiment - smaller random errors (precision) and/or lower systematic errors (fidelity). Sample viscosity often adversely affects the ability of air-displacement pipettes and tips to completely aspirate and expel samples during pipetting. This problem is further exacerbated when pipetting liquids that interact hydrophobically with the polypropylene tip surface.
Aqueous Liquids The composition and properties of the liquid can affect pipetting accuracy. The three physical mechanisms that determine the physical properties of aqueous liquids are:
Surface tension refers to the tendency of a liquid to coalesce into a shape with minimal surface area. Cohesion is a property of the molecules that form a liquid that determines its physical structure and allows it to resist fragmentation when subjected to various physical forces. Adhesion refers to the tendency of a liquid to interact with its surface. The presence of surface tension and cohesion in water samples is directly attributed to hydrogen bonding. Adhesion may be due to hydrophobic, electrostatic, or other types of interactions. Other liquid types, whether organic or inorganic, may also exhibit substantial amounts of cohesion and surface tension. Cohesion and surface tension often aid in the movement of liquids during aspiration and dispensing, allowing for optimal pipetting accuracy. Adhesion forces often impede liquid movement and adversely affect pipetting accuracy. Liquids with reduced cohesion and surface tension, as well as liquids that exhibit adhesive forces, generally exhibit greater pipetting inaccuracies. Many additives can significantly reduce hydrogen bonding in aqueous liquids and may adversely affect pipetting accuracy. These additives include alcohols and other water-soluble organics, surfactants, fatty acids and glycols.
Volatile Organic Liquids Organic liquids generally have lower surface tension than aqueous liquids and may also be volatile. Volatile organic liquids have high vapor pressure at ordinary room temperature. This high vapor pressure is due to a lower boiling point, causing a large number of molecules to evaporate from the liquid and into the surrounding air, creating its characteristic "organic" smell. The expulsion of organic molecules into a closed system (such as the air space between the top of the tip column and the piston within the pipette) often results in an increase in pressure. This increase in pressure, combined with reduced surface tension and reduced viscosity, often results in volatile organic liquids dripping from the end of the pipette tip. Liquid loss results in a significant loss of pipetting accuracy since the volume of liquid transferred is now significantly different from the preset volume on the pipette. It should be noted that these two air displacement pipetting techniques are not recommended when working with highly volatile or high vapor pressure liquids. In this case, the reproducibility of air displacement pipettes may be poor.
4 Pipette Options Manual Single-Channel Pipettes Compact and ubiquitous, single-channel pipettes are arguably the most commonly used instruments in any life sciences facility. While new technologies and techniques facilitate groundbreaking discoveries, single-channel pipettes will always be necessary. Furthermore, the design and sizing principles of modern pipettes have remained largely unchanged (the modern pipette was invented in 1957) and have withstood the latest trends in assay development. Few instruments in the life sciences are so versatile. Single-channel pipettes are used in nearly every branch of the life sciences - from basic research to the latest trends in biotechnology, molecular biology, genomics, gene therapy and immunotherapy - and are compatible with any assay format and container, from single ratio Cuvettes, vials and centrifuge tubes to Petri dishes, flasks and multi-well plate formats (up to 384-well microplates). Single-channel pipettes are ideal for experimental work that uses a single tube and typically smaller volumes, such as siRNA transfections, or for techniques such as cell culture or Western blotting.
Electronic single-channel pipettes Electronic pipettes have been available since the mid-1980s. In electronic air displacement pipettes, aspiration and dispensing are controlled by a microprocessor and initiated by pressing the trigger rather than pressing or releasing the plunger button with your thumb. Most users will find that using electronic pipettes allows for more consistent sample pickup and dispensing, improves accuracy and reproducibility, and virtually eliminates user-to-user variability. Modern electronic pipettes should be simple to operate, have a good user interface and a color screen. These pipettes are versatile and can accurately perform complex tasks such as:
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