Liquid Types and Challenges – Tips for Successful Transfer

Aug 27, 2015

liquid-typesOne of the most basic pieces of laboratory equipment is the air displacement pipette.  Used for tasks ranging from manual assay development to fully-automated high throughput screening campaigns to routine quality control, this type of device consists of a piston that is coupled to the sample via air.  Fluid aspiration and dispensing occur as the air pocket moves up and down, respectively.

Modern air displacement pipettes allow one to achieve a high degree of accuracy and precision, especially when pipetting aqueous fluids. However, most labs pipette fluids that are less than ideal including blood, serum, proteins, detergents, oil, organic solvents, or salts.  By treating these fluid types the same as water, significant error can be introduced to an assay process, depending on volume.  Environmental factors must also be considered when pipetting fluids: temperature, humidity and atmospheric pressure are all capable of influencing results.  Other factors that can affect variability include operator technique, and random failures.

When pipetting liquids which differ from pure water, properties such as density, vapor pressure, viscosity, and surface tension also become critical to successful pipetting.

Density

Higher density liquids have greater mass per unit volume, and impose an increased gravitational force on the air space between the liquid and piston. The increased air space results in a smaller volume of liquid being aspirated into the tip. The effect is opposite when pipetting lower density liquids.  These effects can be compensated for by adjusting the pipette volume setting appropriately.  The adjustment is often an iterative approach whereby the volume setting is changed, the dispensed sample volume is measured, and the volume setting is again adjusted until the correct volume is being attained.

Vapor Pressure

Vapor pressure is a property that describes how fast a liquid evaporates into the atmosphere until it reaches equilibrium.  All liquids exist in equilibrium balance between their liquid and gas states.  In finding this balance, a liquid will continue to evaporate until a certain concentration is present in its surrounding atmosphere.  Liquids such as acetone and acetonitrile possess a high vapor pressure and thus evaporate quickly.  As soon as the liquid is aspirated into a pipette, evaporation begins.  The resulting pressure inside the pipette begins to build and eventually forces some of the liquid back through the tip orifice.   This is easily observed as a droplet hanging from the tip.  Pre-wetting the tip will help the air space in the tip to reach a concentration closer to the equilibrium point.

Viscosity

The property of the resistance to flow is another important fluid attribute that can affect dispensing.  High viscosity fluids such as molasses or glycerol flow very slowly and can leave a film on the wall of the tip during dispense.  When pipetting with same speed as water, liquid can remain in the tip, resulting in inaccurate dispenses.  To compensate for this effect, researchers often reduce the pipetting speed or use a reverse pipetting method.  Wide orifice tips can also help offset this phenomenon.  Another common problem is aspirating too quickly, in which case operators will often obtain air bubbles in the sample.  Again, slowing down the rate of aspiration can be very effective in improving volume accuracy and precision.

Surface Tension

Surface tension describes the elastic tendency of liquids which makes them acquire the least surface area possible. Water is an example of a liquid with a high surface tension and detergents such as Triton X-100 are examples of low surface tension liquids.  Low surface tension fluids tend to leave a thin film on the wall of the tip.  Similar to viscous fluids, slower pipetting and reverse pipetting can offset the effects of low surface tension fluid dispensing.

A summary of common liquid types and characteristics

Liquid Type Attributes Tips for Successful Transfer
Aqueous solutions and buffers Water-like properties – Follow normal pipetting practices
– Utilize forward mode pipetting
Acids/Bases Higher density, sometimes higher viscosity, sometimes higher volatility – Occasionally rinse the lower part of the pipette with distilled water if high-concentrations are pipetted frequently
– Volatile acids require rapid pipetting
– Follow normal pipetting practices and forward mode pipetting for dilute solutions
Salt solutions Elevated viscosity and density – Follow normal pipetting practices for dilute solutions
– Slower pipetting may be required for high concentrations
– Utilize forward mode pipetting
Cell culture solution Sometimes contains serum and therefore will behave like protein solution. Bacteria tend to remain suspend longer, while mammalian cells tend to settle quickly. Can contain glucose and yeast extract, which add to the complexity – Multiple rinses between aspirate/dispense cycles may be necessary
– Change tips between dispenses
– Use sterile filtertips
– Avoid rough pipetting when dispensing cells
Organic
solvents
Density can be higher (.e.g. chloroform) or lower (e.g. acetonitrile) than that of water; high vapor pressure – It may be necessary to adjust the pipette to compensate for density
– Pipetting should be carried out rapidly, due to the high vapor pressure and changes in the wetting pressure
– For best results, a positive displacement pipette should be used
– Pre-wet the tip
Protein solutions Elevated viscosity and density compared to water; prone to bubble formation – Slow and careful aspiration and dispensing is needed
– Reverse-mode pipetting may improve results
– Low adhesion and non-binding tips
Nucleic acid solutions DNA is highly negatively charged and can stick to surfaces; subject to acid hydrolysis when stored in water; genomic DNA is subject to shearing – Filtertips help reduce aerosol formation and contamination
– Excessive and rough pipetting should be avoided
– Wide orifice tips should be used for gDNA to avoid shearing
– Low adhesion and non-binding tips
Serum/Plasma Plasma composition is very complex, e.g., 90% water, 8% protein (50-70 mg/mL), 0.9% inorganic salts and 1.1% organic substances (lipids, amino acids) – Use wide orifice tips
– Utilize reverse-mode pipetting
– These types of solutions can stick to the outside of the tip. Ensure tip is only immersed to 1-2 mm below the surface or carefully brush tip against container to remove excess droplets
– Slow the dispense speed
Detergents Low surface tension (increased wetting ability). A very thin film of liquid remains on the tip walls, flowing down more slowly than most of the liquid mass – Rinse the tip with liquids in the receiving well to complete transfer
– Pipette slowly
– Utilize reverse-mode pipetting


Additional Resources


About the Author

Nat-HentzDr. Nathaniel Hentz is the Associate Director of the BTEC Analytical Lab at North Carolina State University. Dr. Hentz has served as an independent consultant working with Artel and his tenure in the HTS industry includes nearly two years as Sr. Research Investigator at Bristol-Myers Squibb and seven years at Eli Lilly RTP Laboratories in North Carolina. Dr. Hentz received his Ph.D. in analytical chemistry from the University of Kentucky in 1996 and joined Lilly as a postdoctoral scientist the same year.