Ultrasonic liquid level sensing
Authored by: Caliper Life Sciences
(reprinted with permission)
Ultrasonic-based liquid level sensors (also known as tranceivers when they both send and receive) have been used in industrial bulk environments for some time, but have only recently appeared in the laboratory workstation environment (i.e. the Caliper Life Sciences “PING!” ) as advances in piezoelectric crystal technology have enabled miniaturization. This approach uses a series of brief high-voltage pulses delivered to a piezoelectric crystal to cause the crystal to oscillate and create ultrasonic vibrations that travel through the surrounding air. As these vibrations travel outward from the crystal and are reflected back off the nearest surface, the voltage to the crystal is turned off. The reflected vibrations impact the crystal, causing it to oscillate and generate a voltage that is monitored. The distance to the encountered surface is calculated using the signal transmission and return time together with the known velocity of the signals in the surrounding media (air). In a laboratory workstation environment, the liquid level in a vessel can be determined by measuring absolute distance from the piezoelectric sensor, or by measuring the location of a liquid meniscus relative to the top of the vessel or the bottom of an empty vessel.
- Zephyr® GW equipped with PING! ultrasonic sensor (Caliper Life Sciences)
- PING! sensor operated at 800kHZ, with a pulse interval of 2ms
- Waters / Pall Sirocco Protein Precipitation Filter Plate (96 well)
- Deionized water (NERL CAT# 9801)
Ultrasonic monitoring has the advantage of being a non-contact technique (Figure 1), thus enabling a large number of measurements to be executed in a very short time. The technique is based upon acoustic reflections at points of density change from air, and so is not affected by the polarity, ionic strength, density, color or opacity of the liquid media nor by the media of the surrounding vessel. Changing ambient light levels have no effect. The measurement has a resolution of 0.1 to 0.3mm, and is not subject to the somewhat “fuzzy” signal state change that characterizes contact sensing techniques near and at the air/liquid interface. Because ultrasonic scanning measures distance, not just presence, it can also be used to determine when a vessel contains no liquid or has been drawn down to contain a specified level of liquid or dryness – a measurement that would risk a tip collision using a contact technique. Measurements (the speed of the ultrasonic signal) can be slightly affected by the ambient temperature and to a lesser degree by humidity, but these factors may be accounted for by measuring one surface, such as the surface of a liquid, relative to another surface, such as the top of the microplate. The velocity of sound in dry air at 20C is about 343,000 millimeters per second. Irregular liquid surfaces (bubbles, froth) can affect ultrasonic distance measurements (contact techniques are also hampered by these conditions), but multiple ultrasonic measurements can be quickly made across a liquid surface (at a rate of 2 milliseconds / measurement) and the median value calculated to compensate for surface irregularities.
|Figure 1: How an ultrasonic sensor works|
In some applications, the ultrasonic approach allows the monitoring of conditions which are not at all possible with contact approaches. One such example is the monitoring of the evacuation of filtration vessels (wells) to dryness. Determination of the proper endpoint of automated membrane filtration has long been problematic. This is especially so for multi-well filter plates, where each filtration well may perform differently based on varying membrane and liquid characteristics. As a whole, automated microplate filtration is best described as “unreliable”.
Using Caliper Life Sciences “PING!” ultrasonic sensing approach together with Zephyr® or SciCloneTM liquid handling workstations, automated microplate filtration can be made much more reliable. (Figures 2 and 3) The “PING!” sensor can scan each well repeatedly to monitor filtration progress. An entire 96 well plate (Figure 4) can be scanned and mapped in about 25 seconds. The scan of one row of 12 empty wells is shown below in Figure 5. The scan is executed from a height of about 20mm above the top of the filter plate. A vertical distance of “0” on the graph equates to the workstation deck, the bottom of empty filtration wells are indicated at about 5mm and the top of the filter plate is at about 25mm vertical distance from the deck.
|Figure 2: PING! Detection hardware||Figure 3. ScicloneTMG3 equipped with PING!|
|Figure 4: Waters / Pall Sirocco Protein Precipitation Filter Plate (96 well)|
|Figure 5: Scan of one row (12) of an empty filter plate|
The scan is performed by moving the sensor (embedded in the pipetting head) along the length of the filter plate at a linear velocity of 60mm/sec. Each data point seen in Figure 5 represents the Median of 5 ultrasonic scans taken at 2msec intervals. Thus each data point represents a linear distance of 0.6mm.
|Figure 6: Clogged (well 4) filter plate scan|
The Method written for the Automated Liquid Handling Workstation specifies a Threshold (Figure 6) above which any well is assumed to be "not empty" after the specified evacuation time and the specified number of retries. For example, the Method may be written to allow up to 10 retries with evacuation times up to 3600 seconds. Evacuation is complete and the process terminates when the liquid level in all wells is determined to be below the threshold level.
A sample protocol is as follows:
- Wells will be scanned beginning at Row A. Scanning will continue for Row A until the liquid level in all wells is below the threshold level or the specified vacuum time has elapsed.
- The scanning process will continue to the next row as soon as the liquid level in all wells of the currently scanned row are below the threshold level.
- Once the specified vacuum time has elapsed, any rows that have not been previously scanned will be scanned.
- The above process will be repeated for the specified number of retries.
- The process will complete with the liquid level in all wells of the plate is below the specified threshold level or the specified number of retries has been completed.
- Any clogged wells are then reported to the operator.
Sample throughput is increased vs. conventional timed methods because the evacuation time is controlled by actual liquid levels in the wells rather than simply a pre-set time. In the example shown in Figure 7, the system operator is notified that wells A2, B4, F6 and G2 are clogged. The operator may attempt to clear the clogs and press "Retry" or press "Abort" to terminate the process. A "Retry" will attempt another filtration for the time specified in the method, and again at the end of the process any clogged wells will be reported.
|Figure 7: PING! GUI display of clogged wells|
- ↑ Patent Application 700-02600 Provisional
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