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Liquid Handling:Theory and practice

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Authored by: Doug Gurevitch, University of California, San Diego

 

At its most basic, liquid transfer devices obtain a volume of liquid from a source container by creating suction, described as aspirating, and dispensing this liquid when this volume has been repositioned over the destination container by creating positive pressure. The fluid being transferred is commonly referred to as the sample fluid. The sample fluid is usually held in a structure described as a “tip”. Tips can be permanent structures resembling thin tubes or large hypodermic needles made from metal or plastic, or disposable conical pieces made from injection molded plastic. They are designed for accurate placement into the source and destination containers and to provide the best liquid transfer possible. Many systems have more than one tip, allowing the system to speed up the transfer of liquids by aspirating from or dispensing to multiple containers at the same time. The normal nomenclature for automated liquid handling (ALH) systems terms each working tip on a system as a channel. Currently the majority of automated liquid handling systems operate in the microliter to milliliter range of volumes, which is what will be covered in this article. One of the main advantages of this technology is the ability of the laboratory to use the repeatability of automation to allow for miniaturizing assays from the milliliter range in to the microliter range. This allows laboratories to save money in reagents and samples, as well as to increase throughput.

Contents

Automated liquid handler fluid drive types and characteristics

Piston pump

The manipulation of pressures to achieve liquid transfer can be accomplished by a number of different methods, based on different mechanical pump designs. The simplest, as embodied in the manual syringe, is a piston pump. The piston fits snugly with an air-tight seal in a tube. Withdrawing the piston creates negative pressure, which in turn creates suction, aspirating liquid into the tip. Advancing the piston creates positive pressure, which forces fluid out of the tip[1]. Usually there is an inherent volume of air or other liquid between the piston and the sample fluid. This volume of fluid, air or liquid, between the sample fluid and the piston, or any other type of pump, is referred to as the working fluid as it is the medium through which the work of the piston or other pumping device is transferred to act on the sample fluid. In a small number of devices, capillary action creates initial suction, and pre-aspirates the sample fluid to be in direct contact with the piston. This volume of fluid becomes the working fluid. The piston then protrudes into the tip to control the aspiration and dispense of the sample. These devices are said to use positive displacement[1], in which the piston is in direct contact with the sample fluid. In most other devices, such as manual syringes or pipettes, there is an inherent volume of air that is made of the volume designed in the tips between the piston and the sample fluid. This volume of air is usually referred to as an air gap.

In automated liquid handling systems, piston pumps can take many forms. They can be actual syringe pumps directly connected to the working tip or located at a distance and connected via tubing. They can also be arrays of pistons attached to a plate that work within drilled holes in another structure. Most directly connected automated liquid handling systems are described as being air displacement systems as there is an inherent air gap that functions as the working fluid. In systems where the syringes are mounted at a distance and connected by tubing, a pre-loaded liquid is usually used to act as the working fluid. These systems are described as liquid or hydraulic displacement systems. Piston based pumps are used on the majority of automated liquid handling systems.

Vacuum/air pump

(See also Pressure-based_microvolume_dispensing_for_high-throughput_screening)

Other liquid handling systems utilize a pressure driven system where small air pumps or pressurized reservoirs create the vacuum for suction or pressure for dispense. Pumps are usually mounted just above the tips. To achieve accuracy these systems include active valve and pressure sensing technology with advanced feedback-control algorithms that utilize the data collected from the pressure sensors to control the valves and pumps[2],[3].

Gear motor/diaphragm/peristaltic flow through pump

The last type of liquid handling system utilizes flow through pumps. These can be found as stand-alone devices or on piston based systems for bulk dispensing of reagent or diluting fluid (usually described as the diluent). In this incarnation they are usually diaphragm, gear pumps or peristaltic pumps. There is one novel system that utilizes a unique flow through gear pump that can reversibly pump highly accurate small volumes of liquid[4]. For normal operation the fluid moved by the pump is a liquid working fluid, thus making this system a liquid or hydraulic displacement type.

Automated liquid handler pipetting channel configurations

Single or multi-channel

Automated Liquid handling systems can vary from a single channel to an array of 1,536 channels. These are broken down into three main variations: single channel, multi-channel with small numbers of tips and multi-channel with large numbers of tips. Single channel systems were the original automated liquid handling systems and can still be found. They have great flexibility, but also have throughput limitations due to having only one channel. Currently, multi-channel systems make up the majority of the automated liquid handlers produced.

Small or large numbers of channels

Systems with small numbers of channels usually have 4, 8, 12 or 16 channels. Systems with large numbers of channels usually have 96, 384 or 1,536 channels. The systems with smaller number of channel systems hold the edge for flexibility as the majority of systems have the ability to change spacing between the channels. The most common method employs a gearing system that spaces the channels equally, known as VariSpan™. These are produced in 4 or 8 channel options. Less common are systems that allow for variable spacing between the channels. These can be found with 4, 8, 12 or 16 channels. In both cases, these automated liquid handling systems allow for moving samples between tubes or other large containers to microplates, frequently described as reformatting. Microplates are trays produced to hold multiple samples in a single tray or plate in separate compartments known as wells. They are most commonly made from injection molded plastics but can also be purchased formed from glass. They can be purchased with as few wells as 4 and as many as 3,456. The most common microplate formats produced are 96, 384 and 1,536 wells.

As microplates gained favor, automated liquid handlers were produced with matching numbers of channels to the most common plate formats, 96, 384 and 1,536. These channels are built into a structure normally called a head with permanent spacing matching the spacing of the wells in the microplates. The majority of these systems employ piston based pumps for their operation. High number of channel automated liquid handler systems are routinely used for operations between microplates and for dispensing sample liquids from a source/mother microplate to a large number of destination/daughter microplates. This last operation is referred to as plate replication.

Many of these systems have some capability to determine if the tip is within the source liquid. This is accomplished by a number of means, usually electronic in nature. The most common is capacitive sensing, where electronics determine the electrical capacitance of the tip suspended in free air and look for a change as the tip is submerged into the source or destination liquid.

Uses of automated liquid handlers

Since their introduction into biology laboratories in the 1980s, automated liquid handling robots have matured and become commonly used for a number of biological laboratory processes. These include, but are not limited to, the following processes or experimental assays:

  • PCR Preparation
  • IC50 Plate Creation
  • Compound Purification
  • Metabolic Stability
  • P450 Inhibition
  • Solubility
  • ELISA
  • Cherry-picking
  • Variable Input/Output
  • Worklists
  • Pipetting with tubes/vials
  • Reformatting Plates
  • Time-Course Studies
  • Titration Studies
  • Combinatorial Chemical Synthesis
  • Plate Replication
  • Screening Assays
  • Cell-Based Assays

Processes such as cherry-picking, in which specific samples from multiple sources are collected into a single destination plate, and reformatting, are handled by systems with a single channel or smaller numbers of channels with flexible tip spacing. The smaller number of channel systems also can have milliliter range pumping, and are the only choice for pipetting needs where greater than 200 microliters needs to be transferred in a single operation. Plate replication is specifically the domain of the higher number of channel systems. However, the maximum volume for these systems, even at 96 channels (allowing for larger tips and pistons) is 200 microliters.

With this wide range of uses, automated liquid handlers have found their place in laboratories involved in drug discovery, pharmaceutical development, pharmaceutical quality assurance, clinical reference and testing, forensics, biosecurity, food and agriculture development and safety, and materials science[5].

Practical liquid handling issues

Critical liquid handling parameters

Reliable and repeatable liquid transfer in the microliter to milliliter range depends heavily on the junction of fluid and electromechanical dynamics. The fluid dynamic characteristics of both the sample and working fluids and the electromechanical dynamics of the system components are critical for proper and consistent performance. For example, the selection of materials for the tips can be critical to the fluid dynamics factors that affect the accuracy and precision of the liquid transfer process. Listed below are the most common characteristics that affect liquid transfer in automated systems[6]:

  • Accuracy – how close an electromechanical system can approach a given goal, be it position in three dimensions or volume dispensed. Both are important for automated liquid handlers, providing limits for how small is the smallest volume that can be sampled or the smallest container that can be accessed.
  • Aqueous – a solution in which the solvent is water.
  • Backlash – the amount of play or clearance between mating components in a mechanical system. In automated liquid handlers, it is the play back and forth in the gearing of the pumping mechanism that is most important.
  • Cavitation - when a liquid is pumped or moved too quickly and drops below its vapor pressure and vaporizes, creating bubbles. For automated liquid handling systems, this is possible when pipetting highly viscous fluids.
  • CV – Coefficient of Variation – essentially the standard deviation normalized to a percentage by dividing by the mean. This is usually how the precision of automated liquid handling system pipetting capabilities are given, as in 1 microliter ± 5%
  • Diffusion mixing – the amount of mixing that can be expected of a sample in a diluent based solely on concentration differences. It is usually slow and very uneven throughout a given volume.
  • Flow rate or speed – how fast the pumping system can actually pump fluids, usually measured in microliters per second.
  • Foaming – the propensity of many biological reagents, buffers and enzymatic solutions to create foamy froths when dispensed or mixed too aggressively.
  • Laminar flow – where a fluid (liquid or gas) flows in parallel streamlines, or “smooth” flow. This is determined by the flow rate, tubing and tip diameters, and wettability of all the surfaces. Laminar flow is preferred due to its less chaotic state, making for more repeatable performance. 
  • Liquid level tracking – the capability of most automated liquid handlers to either track the level of the liquid being aspirated or dispensed in order to prevent aspirating air.
  • Liquid type or liquid performance file – most automated liquid handler manufacturers have optimized pipetting speeds, air gaps and pre-aspiration volumes already determined for their systems. These sets of performance parameters are stored in liquid type databases or performance files. Water, DMSO, alcohol and glycerol based solutions are common. These can also be modified or used as a basis for custom parameter optimization. 
  • Precision/repeatability – usually defined as the measure of how likely it is for an electromechanical system to achieve its goal.
  • Standard Temperature and Pressure – STP also known as calibration conditions. For automated liquid handling systems this is essentially standard room temperature and pressure at 20°C and 1 atmosphere pressure (101.325 kPa = 14.696 psi).
  • Surface wetting/wettability – is a measure of a drop on a surface and how much of an angle the contact edge makes with the surface. It is a measurement of how easily a liquid spreads on a surface. Low wettability means that bubbles can be easily entrained in a sample fluid causing turbulent flow and reducing consistency.
  • Surface adhesion – is a measure of how well a liquid sticks on a surface. Liquids with very low surface adhesion, such as some alcohols and DMSO may drip out of a pipetting tip during transfers.
  • Surface tension – the characteristic of the surface portion of a liquid to be attracted to another surface. This characteristic is primary in determining the wettability and depends on the liquid and the material of the other surface.
  • Tubing or tip diameter – the size of the internal dimension of the tubing between the pump and the tip and the tip itself.
  • Turbulent flow – where a fluid flows in a chaotic fashion, with swirls and eddies, frequently described as “rough” flow. This is not desirable for liquid transfer as it makes the process less consistent. 
  • Viscosity – the resistance of a fluid to shear motion – its internal friction. This can be thought of as its “thickness” or “gooiness”. Water is a low viscosity fluid. Viscosities much higher than water can be a real challenge for automated liquid handling systems as they have higher surface adhesion and lower surface wettability. High viscosity fluids will cause turbulent flow at lower flow rates than low viscosity fluids.

General rules

Detailed article: Small Volume Pipetting

The majority of automated liquid handling systems pipette quite accurately aqueous samples at STP and at volumes that are above the minimum 5% of their mechanical pumping range. These conditions are used as the design goal for most systems. As an example, a system that has a 200 microliter tip and pipettes a minimum accuracy of 1 microliter can pipette 20 microliters of an aqueous sample at STP with a precision/repeatability of ± 1% CV. However when working outside these conditions, precision decreases and CV increases. The same ALH working at its lowest limit of 1 microliter, the precision, due to the increased relative influence of the minimum motion and backlash of the pump, is now ± 5%. Likewise, pipetting at faster than recommended flow rates and pipetting more viscous fluids will also decrease the precision. In both cases, the sample fluid is more easily forced into turbulent flow.  In the worst case scenario, viscous fluids can also experience cavitation if moved at too high a flow rate. This essentially replaces critical sample fluid volume with bubbles, and reduces precision.

Other confounding factors include changes in temperature or air pressure. For air displacement or active air pressure based systems, changes in temperature affect the behavior of the working fluid, air. Air is a compressible fluid at STP and its behavior changes as you change the ambient environment. This can decrease precision and accuracy greatly, especially at extremes. As such, most air pressure based systems sample temperature and ambient air pressure to correct for these influences. Fluid/hydraulic driven systems are less susceptible to these changes, but not immune. Even though the working fluid is now a liquid, and usually water based, water is not truly incompressible. Given the length of the tubing between the pump and the tip, the effect ambient conditions have on the liquid transfer process can be quite substantial, also decreasing the precision and the accuracy, if potentially less than for air displacement systems.
Another confounding factor is foaming. Many biological assay buffers, reagents and enzymes include components that can foam or froth when aggressively mixed. Problematic buffers include BSA (Bovine Serum Albumin) and glycerol. Most enzymes, including taq polymerase, include both glycerol to protect the enzyme when frozen for storage and shipping, and surfactants (a chemical that lowers the surface tension of a solution, also known as a wetting agent). Both froth if mixed too aggressively, or even if dispensing at too fast a flow rate. These foamy “heads” can then cause a great deal of difficulty in two ways. First, they essentially turn needed sample into unavailable bubbles, causing a shortage of sample liquid. Second, they can be sensed as the start of the liquid in the container. The liquid detection technology can be fooled by such “heads” and cause them to be aspirated instead of the actual remaining sample liquid. This also causes a shortage of sample liquid.

Automated liquid transfer systems offer the ability to adjust pipetting parameters such as flow rates, air gaps, movement speeds and even offer automated touch off (in which the tip is touched to the side of the destination container to remove external drops). These parameters can be adjusted to optimize liquid transfer for almost any sample fluid. Testing with the appropriate solvents, buffers and inexpensive model reagents allow the user to determine the optimal settings. Lastly, regular testing with dyes or by weight, known as gravimetric testing, can guarantee the user knows when components have worn enough to affect accuracy or precision.

References

  1. 1.0 1.1 “Air-Displacement and Positive-Displacement Pipetting Techniques”, www.gilson.com/PDFs/pdfid240.pdf
  2. Product information - Hamilton robots, http://www.hamiltonrobotics.com/liquid-handling-products/star-line-liquid-handling-workstations.html
  3. Product information - Xiril AG, http://www.xiril.com/xiril/products/index.html#0330e598fb0a12b08
  4. Unique fluid system with low maintenance pumps, http://www.sias.biz/OEM.htm#pumps
  5. Liquid Handling Boot Camp, short course, LabAutomation2005-2008, D. Gurevitch, P. Stojadinovic, J. Provchy.
  6. Definitions excerpted, en.wikipedia.org

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