Methods to Measure Transferred Liquid Volumes

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.
   
   
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.
   

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.