Syringe Pump Accuracy
Syringe pumps are inherently very accurate, but that does not gurarantee you will dispense the liquid quantity you expect or, more importantly, that you correctly measure what you dispensed. The following is a checklist of things that may cause you problems.
Syringe pumps are typically accurate to a fraction of a percent. They depend on precise linear movement of a plunger being converted to liquid volume displacement. In general, the small errors that are present in a syringe pump are due to mechanical backlash and to the behavior of liquids as they come out a dispense needle.
FTA200 and FTA1000 Direct Drive Pump Setup
These pumps can take a variety of syringes, so the software must be told what syringe is present in the pump. The software must know the capacity, internal diameter, and scale length of the syringe. These three values are entered on the Pump | Syringe tab. Since it is difficult to measure the internal diameter of the syringe, the diameter will be computed if you enter the total capacity and the length of the marked scale on the syringe. For Hamilton Gas Tight syringes, the length is 60mm. Thus you might enter 500 microliters for the capacity and 60mm for the length.
FTA200 systems have a choice of pump motors. The correct "gear ratio" must be present.
FTA1000 Kloehn Pump Setup
The Kloehn pump can use different capacity syringes, although they are all the same length so setup is easier. You must enter the capacity of the syringe on the Pump | Syringe tab.
The consequence of an incorrect capacity entry is that all volumes will be scaled by the ratio of the correct value to the entered value.
Air bubbles in a pumping system can interfere with the conversion of plunger movement to liquid volume displacement. Liquids move because of applied pressures. Air bubbles can absorb the pressure generated by the plunger and result is a non-responsive system. Air bubbles can be generated by cavitation when the pressure is lowered too much during an aspiration or sucking cycle. When you are "pumping in" you should limit your rates to a few microliters per second. 10 ul/s should be considered an upper limit.
If you are relying on video measurements of volume, it is imperative that magnification be calibrated correctly. Any error in magnification will be multiplied to the third power in the volume calculation. An easy way to check the magnification is to check the measured width of the needle in the image (assuming you are using a straight needle and you know its diameter). If you have a zoom microscope, you must recalibrate magnification each time you change the zoom.
The Laplace relationship says there is significant back pressure generated across a small spherical surface. The pressure is inversely proportional to the radius and proportional to the the surface tension. If you think about a drop emerging from a needle, the time of maximum pressure occurs just as the new droplet makes a hemisphere shape at the tip (because its radius of curvature is minimized). This means the liquid may seem to "hesitate" as pressure is built up by the plunger. Once the drop becomes larger, the Laplace pressure drops rapidly and no longer plays a role. This effect is most noticeable in needles smaller than #22GA (0.711mm OD).
One way to avoid problems with Laplace pressure effects is to use the Dispense Volume macro available on the Pump | Setup and Tools tab. The software forms a real time control loop to dispense the volume you request.
If you believe the Laplace pressure is causing problems, and there are no bubbles in the system, you may need to use a "stiffer" tubing to connect the pump and needle.
Sessile Drop Volume
The actual volume delivered to a surface will vary from the pendant drop volume because some liquid will be left attached to the needle. Also, it is more difficult to measure a sessile drop's volume (as the shape is not as well defined) than it is to measure a pendant drop's.
A Simple Test
The following is a simple test to help you understand what type of problem you have. The idea is to continuously dispense liquid from a known tip and measure how long it takes, on average, for a drop to form and then fall off. A relationship known as Tate's Law predicts the volume of liquid that can be supported by a tip of known diameter. (We assume the liquid wets to the outside diameter.) For water, Tate's Law predicts the following volumes:
- 17 GA, 1.499mm OD, 35.0ul
- 18 GA, 1.270mm OD, 29.7ul
- 19 GA, 1.067mm OD, 24.9ul
- 20 GA, 0.914mm OD, 21.3ul
- 21 GA, 0.813mm OD, 19.0ul
- 22 GA, 0.711mm OD, 16.6ul
- 23 GA, 0.635mm OD, 14.8ul
- 24 GA, 0.584mm OD, 13.6ul
- 25 GA, 0.508mm OD, 11.9ul
- 26 GA, 0.457mm OD, 10.7ul
- 27 GA, 0.406mm OD, 9.48ul
Run the pump constantly at, say, 1 microliter per second. Time the period between drops falling off the tip. Vibration may cause the actual period to be a per cent or two less than the theoretical period. As an example, using a #20 needle, one would expect a drop to fall each 21.3 seconds according to Tate's law. In practice it might be every 20 to 21 seconds.
You can now compare the "drop rate" with what you asked for and with what volumes are measured by the video system. Certain problems will not affect the drop rate experiment:
- Laplace pressure
- magnification calibration
However the following will affect the drop rate:
- syringe capacity or ID entry
- FTA200 motor gear
- needle OD
- liquid's IFT and density
The logic behind this is that if the drop rate is OK, but you are having dispense problems, your difficulty is in the first table with bubbles, Laplace pressure, or magnification calibration.