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Once the liquid handling is complete, it’s valuable to be able to double check the automated liquid handler and make sure that the liquid was transferred at the correct volume. As part of our Automated Liquid Handling Guide, this page describes methods to measure transferred liquid volumes to ensure success. Liquids can be measured through a wide range of tests, from simple visual checks to more complex measurements that involve dyes or special scales.

Keep in mind that measurement described on this page only provides you with a measure of the volume transferred.

The purpose of measuring liquid transfers is to check for a “true” and “precise” result. Each automated liquid handler has expected performance at various liquid volumes and tip sizes. These specifications can be used as a reference during verification, but keep in mind that differences in laboratory conditions, pipetting approaches, and liquid types can result in different performance. It is a good practice to consider the pipetting specifications to be the best case scenario and to consider your own method’s pipetting performance requirements when evaluating verification results.

Formula for Trueness in methods to measure transferred liquid volumest

Trueness is defined as Yield (% trueness / bias). It is a percent error between the average volume of solution measured and the expected or accepted value. A result of high trueness is equivalent to a small percent error.

Formula for precision in methods to measure transferred liquid volumes

Precision is defined as Reproducibility (% CV). It is the closeness of a set of values obtained from
identical measurements of the same volume. A high amount of precision is equivalent to a small percent coefficient of variation.

In addition to trueness and precision, the term “accuracy” is often used in discussions about automated liquid handlers. In 2015, liquid handler manufacturers defined and standardized the terms to be used across the industry. The manufacturers also standardized the volumetric performance determination for Automated Liquid Handling Systems (ALHS). For reference, the standardization decisions are logged in ISO International Workshop Agreement (IWA) 15 titled “Specification and method for the determination of performance of automated liquid handling systems.”

ISO IWA 15 describes accuracy as the relationship between trueness and precision. Definitions from the ISO document include the following:

Trueness — An average which is very close to the true value. As trueness improves, there is a decrease in systematic errors.

Precision — Highly consistent results. As precision improves, random errors decrease.

Accuracy — Knowing that each measurement correctly represents what is present in the sample. As accuracy is obtained, uncertainty is decreased.

ISA IWA 15 accuracy relationship model between trueness and precision

Download this information and much more with the Liquid Handling Reference Guide.

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Photometric Measurement

A photometric approach uses a dye and a plate reader to analyze results. Add dye to the liquid and verify using a plate reader. Testing can generally be done in the actual assay labware (plates). Keep in mind that it can be difficult to choose an appropriate dye for the specific test, and dyes can alter physical characteristics of the solutions being tested.

Advantages

  • Allows testing to be done in the actual labware used in the process, which makes for a truer comparison.
  • Is good at gathering large amounts of data. For example, photometric measurement is useful for testing with a 96 or 384 multi-probe head since all transfers can be performed and measured at the same time. Each well in a 96- or 384-well plate corresponds to a channel on the multi-probe head. Since an absorbance value is collected per well, the transfer volume can be determined per channel.
  • Is the only method that works for liquid filled systems because of a potential dilution effect that cannot be detected by weighing the sample. In contrast to the gravimetric approach, the plate reader absorbance read could detect unwanted dilution relative to reference standard or manually pipetted transfer of the sample. Since the photometric approach is required for liquid filled systems, it is therefore a popular and accepted standard of measurement for any type of automated liquid handling system.
  • Works with off-the-shelf products and readily available laboratory equipment. Options exist to purchase a ready-made solution or create your own at less cost.

Disadvantages

  • Can be challenging to develop a test from scratch, since it requires identifying the optimal wavelength, using and/or acquiring an appropriate reader, and setting up the necessary calculations. For reference, the photometric measurement process is described in detail below.
  • Can be difficult to choose an appropriate dye for the specific test. The addition of dyes to the liquid can alter the physical characteristics. This makes the liquid type not truly representative of the one you are trying to measure.
  • Can limit the amount volume to be tested. The transfer volume may be limited to the labware required for analysis in the plate reader. A microplate typically holds about 300 μL of liquid. If more volume is required, additional testing procedures must be used that are different from the liquid transfer settings in the method.
  • Requires the use of a plate reader and further calculations in order to obtain results. Data collection can therefore be slower using this approach.
  • Not ideal for small volumes under 10 μL. The path length for the light to travel through the liquid would be minimal and the approach may not be sensitive enough for reliable measurement. This depends especially upon well geometry. To avoid this issue, small volumes may require additional buffer, which creates additional variables. Such variables include the potential for uneven path lengths and variable path length and concentration which complicates the application or Beer’s Law.

Alternatives to the photometric measurement include the fluorometric approach, which can help address some of these disadvantages.

How to Perform Photometric Measurement

  1. Obtain a dye and known optimal wavelength and concentration for use. If unknown, determine it using spectral analysis of the color. Measure with scanning spectrometer (i.e. Molecular Devices Spectramax®) when possible to determine optimal wavelength and concentration. If not possible, guess based on dye color: (Yellow dye transmits yellow light, so it absorbs blue
    (450 nm)).
  2. Use a flat bottom plate for testing. For example, a round well plate can cause issues with the test due to light reflection and refraction on the curved part of the well. If needed, other plate types can be accommodated, but it takes modification in the reader software and equation.
  3. Test several concentrations to determine reader saturation point and aim for a test concentration 50% to 75% of saturation.
    Photometric Measurement diagram
  4. Apply Beer’s Law to determine volume: Absorbance (Abs) = eLc. Refer to the instructions below for details on the application of Beer’s Law. Use it to create a standard curve around the value of interest.
    Beer's Law
    Abs = eLc
    e = extinction coefficient
    L = pathlength (depth of liquid in well)
    c = concentration of dye
  5. Create a standard curve with same dye solution that is being used for unknown volumes. This allows constants to cancel out. The curve is obtained by collecting data below and above the volume of interest and fitting a linear curve to determine the slope and offset.
  6. Additional detail on Beer’s Law:
    1. Because e and c are constants that do not change from well to well, they can be viewed as a single proportionality constant, k, so Beer’s Law is simplified to: Abs = k* Volume
    2. Using the curve fit parameters, the slope is the value for k.
    3. There also exists an offset due to absorbance within the plate material itself that is the y-intercept.
  7. Apply the curve to calculate unknown data.
    1. Read a plate containing wells of liquid transfers of the volume to be tested. These are considered the unknowns in this test.
    2. Apply curve values to calculate unknown data.

Example Where the Transfer Volume of Interest is 50 μL:

  1. First, absorbance data is collected for several volumes of dye transfers below and above 50 μL. Specifically, 30, 40, 50, 60, and 70 μL. The table below shows the raw data from the reader and the average of replicates.
  2. A linear curve is fitted to determine the slope and offset. y = 0.0064x + 0.184 where y is the Absorbance and x is the Volume.
  3. A plate of unknown 50μL liquid dye transfers is read collecting absorbance values for each transfer.
  4. The curve values are applied to the unknown absorbance values to determine the volume for each transfer.

Fluorometric Measurement

Fluorometric measurement is similar to photometric approaches, because both types of measurement require light to be directed onto the sample well and then measured using a photomultiplier tube. However, there are some significant differences in the approach that can lead to one being a better option than the other depending on the circumstances.

In photometric measurement, the amount of light transmitted is subtracted from the amount of light generated at the source. This allows you to determine the total amount of light absorbed.

In contrast, when using fluorometric measurement, the dye is illuminated at a specific wavelength and absorbs light energy. The light energy then emits at a different wavelength. Since the light emits directly from the dye molecule, it emits in all directions equally. This means that a variety of plates can be used, for example black, opaque plates without an optically clear bottom. In fact, using black plates during testing reduces background interference, because they absorb scattered light.

To perform fluorometric measurement, follow the same general steps and data handling as used for photometric measurement

Fluorometric Measurement

Advantages

  • Allows testing to be done in the actual labware used in the process, which makes for a truer comparison.
  • Is good at gathering large amounts of data and is the only method besides photometric that works for liquid filled systems.
  • Is not effected by well geometry because the measurement is only dependent on the total amount of fluorescent molecule residing in the area illuminated by the light source.
  • Is effective for small volumes, but may still need a diluent. Fluorescent dyes tend to be sensitive and often require substantial dilutions from a stock solution to remain in the dynamic read range for a plate reader. This ensures accurate measurements of low volumes. Fluorescent measurement examines the total amount of fluorophore in the area that is excited, eliminating the effect of varying diluent volumes.

Disadvantages

  • Can be difficult to choose an appropriate dye for the specific test. The addition of dyes to the liquid can alter the physical characteristics. This makes the liquid type not truly representative of the one you are trying to measure. Fluorescent dyes are more expensive than the dyes used for photometry and also tend to be sensitive to light and sometimes temperature. In addition, fluorescent dyes do generally come with known excitation and emission wavelengths.
  • Can limit the volume to be tested. The transfer volume may be limited to the labware required for analysis in the plate reader. A microplate typically holds about 300 μL of liquid. If more volume is required, additional testing procedures must be used that are different from the liquid transfer settings in the method.
  • Requires the use of a plate reader and further calculations in order to obtain results. Data collection can therefore be slower using this approach. Plate readers for fluorescence measurements tend to be more expensive than those that just read absorbance.

Gravimetric Measurement

The gravimetric approach uses an analytical balance integrated directly on the automated liquid handler.
Once the liquid is weighed, the known density of the liquid can be used to calculate the volume.

Advantages

  • Uses only the liquid of interest with no dyes or other additives.
  • Covers a large range of pipette channel volume. It can cover a range of volumes used by all Hamilton pipetting devices from 0.5 μL to 5 mL.
  • Provides immediate feedback from the balance which allows for quicker optimization of liquid classes.

Disadvantages

  • Doesn’t use the actual labware used in the assay. Instead, you may be pipetting to a large tube on a balance instead of a plate or specific tube type.
  • Requires you to know the density of the liquid in order to correlate the weight to a volume. While it is easy to look up the density of common liquid types, it may be unknown for less common types or for mixtures.
  • Affected by temperature. The temperature can impact the density of the liquid, so the temperature needs to be monitored to properly check for trueness. See Section 2.3.2 for more information.
  • Allows only one transfer at a time to be weighed. This prevents the testing of multiple channels at the same time to collect individual measurements. Specifically, this limitation prohibits testing of all channels in a multi-probe head (96- and 384-) at the same time.
  • With small volumes, the balance quality and readability sensitivity can play a factor. It may not be possible to obtain significant digits with low volume transfers given the sensitivity of the balance and laboratory conditions.

Hamilton makes a gravimetric measurement system, known as the Liquid Verification Kit (LVK). The LVK is made up of a graphical user interface that directs liquid handling tests on an analytical balance that is placed on the automated liquid handler’s deck. The same Mettler Toledo WXS 205S analytical balance is used by customers who purchase the LVK and Hamilton field service engineers to conduct liquid testing. During execution, the LVK program displays data in real-time and creates reports on the pipetting performance. It is also possible to program custom methods using the Hamilton software that control the balance to verify liquid transfers without the use of the LVK interface.

Industry standard processes for gravimetric testing are defined in ISO 8655-6.

Combined Photometric and Gravimetric Measurement

It is also possible to combine the photometric and gravimetric approaches. The first liquid transfer containing dye can be weighed, then later be analyzed on a plate reader to determine the volume per well. This approach is used by Hamilton service engineers to perform installation and operational qualification testing of the automated liquid handlers as part of the Field Verification Kit 2. This process allows both the independent pipetting channels and the multi-probe heads (96- and 384) to be verified in a similar fashion.

With the weight measurement and the obtained optical density (OD) values, the volume of each well can be determined.

  1. Pipette dye liquid in a microplate.
  2. Weigh the plate.
  3. Add clear liquid like Borate Buffer, to make sure that the liquid sufficiently covers the bottom of the well. Mix the clear liquid and dye together.
  4. Measure the optical density by reading the plate with a photometric reader.

Additional Resources for Automated Liquid Handling

Check out our Automated Liquid Handling Platforms
See Hamilton Automated Plate Sealers and Small Devices
See the line of Hamilton Assay-Ready Robotic Workstations
Learn about all of Hamilton Company's Automated Liquid Handling solutions
Check out the homepage of our Automated Liquid Handling Guide
Read our recommendations for Step by Step Automated Liquid Handler Setup
The team at Hamilton gives recommendations for Best Practices for Liquid Handling Activities
Want to "own" the guide? Click for a PDF Downloadable Liquid Handling Guide
Read our tips to accurately Pipette Volatile Liquids
Learn how Liquid Properties affect automated liquid handling
Check this out to The Importance of Z-axis Control for Accurate Pipetting
Learn about Automatic XYZ Calibration
Read our advice on Addressing Challenges of Automated Pipetting