Before explaining how an HPLC digital flow meter works here's an explanation of what digital flow meters do:
Digital flowmeters are used to measure the accuracy of high performance liquid chromatography (HPLC) pumping systems in order to meet FDA regulatory requirements. These instruments are widely used during installation qualification (IQ), operational qualification (OQ) and performance qualification (PQ) of HPLC equipment used in the pharmaceutical and biopharmaceutical industries as well as other analytical labs.
HPLC demands accurate, reproducible column retention time as an analyte (what is being analyzed) is pumped through the system.
These precision digital flow meters are not to be likened to flow meters used on industrial process lines measuring flow rates in, for example, thousands of gallons per minute but instead in fractions of milliliters per minute.
Why HPLC Pump Accuracy Matters
As suggested above, meeting regulations (and protecting a company's reputation) is the reason. Consistent accurate performance is an absolute necessity. Complicating things, these pump systems are complex devices that include parts prone to leaks and wear when particulates settle in their components.
Further complicating the topic, we are dealing with an accuracy within 1% of low flow rates, down to as low as 0.05 mL/min. This demands care to avoid or at least minimize wear on HPLC pump system components.
But equipment wear does occur and accuracy impacted meaning HPLC pump system testing is required. Here's where digital flow meters come into play and why they should be considered versus traditional methods, which we explain here.
Traditional Methods for Measuring HPLC Pump Flow
A manual method for checking flow rates is either of the following:
- Volumetric measurement, using a graduated cylinder
- Gravimetric measurement, using an analytical balance
But the weak point, as we see it at Tovatech, is that both methods require a stopwatch, excellent eyesight and a high degree of patience on the part of the technician doing the testing.
In a typical volumetric test, the technician collects solvent in a graduated cylinder while simultaneously operating a stopwatch. After a measured volume has been collected, the elapsed time is used to calculate the flow rate in mL/min. The position of the meniscus is somewhat subjective and prone to operator interpretation. This tedious but critical process repeats over and over until a standard deviation can be calculated. Two sources of measurement error are interpretation of the meniscus position and solvent evaporation.
Imagine, if you will, a lab with several HPLC pump systems that have to be tested.
For gravimetric testing similar exercises are performed but using a precision scale or analytical balance to weigh the sample. Here as well, solvent evaporation can introduce errors.
One user at a pharmaceutical company reported that they have 100 HPLC systems in their facility. A pharmaceutical industry service supplier tests dozens of HPLC pump systems at several locations. The service representative noted that it takes about 20 minutes to do a gravimetric test.
A digital liquid flow meter such as the FlowCal 5000 offered by Tovatech replaces these time-consuming error-prone manual methods.
How a Digital HPLC Flowmeter Works
Finally we get to the topic of this post. Thank you for your patience.
Our example is the 12-oz, compact portable FlowCal 5000 digital flow meter connected to the outlet of HPLC system being tested. This enables the solvent effluent to be directed into the FlowCal 5000 for measurement before the solvent is discharged into a collection container for disposal.
Here is the sequence of operation that typically takes less than a minute for each reading:
- When solvent enters the measuring tube, the meniscus passes an optical sensor that activates a timer.
- When the meniscus reaches a second sensor, the timer stops, a valve opens and the tube drains.
- Readings are displayed on the meter’s four-digit LCD panel and recorded to a six-digit printer or computer.
- The process repeats itself.
Measurement accuracy is within ±1% of the reading over a flow rate range of 0.05 to 25 mL/min. Consistent readings mean the HPLC pump system is operating correctly. Otherwise the HPLC system requires attention.
Automated Testing Improves Productivity
Because measurements are performed automatically, technicians no longer need to operate stopwatches, monitor graduated cylinders, or manually record results.
The FlowCal 5000 can be programmed to collect multiple consecutive readings and transfer the data directly to a computer or printer. This not only improves repeatability but also frees laboratory personnel to perform other tasks while testing is underway.
Installing a Digital Flowmeter
While the FlowCal 5000 is portable, wherever it is used in the laboratory requires careful placement. For accurate operation, the instrument should be mounted within approximately 10 degrees of vertical with a suitable waste container positioned beneath the discharge outlet.
Options accommodated by the kit include wall-mounting; freestanding; stack system-mounting for positioning it on the front, left or right side of a stacked pump/detector system; and a bottle top adapter kit for mounting onto standard wide-necked reagent bottles.
Calibration Ensures Measurement Accuracy
As with any precision instrument such as analytical balances and moisture analyzers, the accuracy of HPLC flow meters must be confirmed at the outset and checked periodically to insure their accuracy is maintained.
The FlowCal 5000 meters are factory calibrated and are shipped with a certificate documenting calibration at a flow rate of 1.0 ml/min. Customers can request additional flow rates. The calibration certificate verifies that the displayed flow rate is within ±1% of the actual flow rate at each calibration point.
Factory Calibration
Calibration points recorded on the certificate result from an extensive process aimed at achieving statistically accurate data by taking, for example, 30+ readings at 0.5ml/min, 40+ readings at 1.0ml/min and 100 readings at 5.0ml/min.
Data are captured and averaged via an RS 232 interface to a PC. The certificate shows the average of these readings at each flow rate. The percent error and percent standard deviation from the actual flow rates are also calculated from these averages and shown on the certificate.
The actual flow rate is determined gravimetrically by collecting a mass of pure water passing through the flow meter at a constant flow rate over a recorded period of time. The mass of water is converted to a volume of water by correcting for density based on the water temperature.
The actual volume over time measurements are compared to the displayed readings on the digital flowmeter. The firmware of the flow meter is adjusted so that the displayed rate matches the actual flow.
Annual Recalibration
Good Laboratory Practice (GLP) and many quality systems recommend recalibrating digital flowmeters annually or whenever measurement accuracy is questioned.
Recalibration follows the same procedures used during factory calibration and requires certified reference equipment capable of producing highly stable flow rates. Because firmware adjustments are required, recalibration cannot be performed by the user.
Tovatech recommends returning the FlowCal 5000 for annual recalibration. The process typically requires approximately two weeks, after which the instrument is returned with an updated calibration certificate.
Here is a brief description of the recalibration process:
The flow meter is first flushed with water. Then “as found” (or “before”) readings are taken at 1.0 ml/min to measure the accuracy of the flow meter in the state in which it was received. Customers may request that calibration certificates show “before” data at all the calibration points rather than exclusively at 1.0 ml/min.
The flow tube is cleaned, sensors are checked, any repairs undertaken, and necessary adjustments are made to bring the digital flowmeter into specification. The flow meter is then calibrated to all the customer’s required flow rates. "As left" (or "after") data are recorded on the new calibration certificate to confirm that the returned flowmeter is performing according to specification.
4 Steps to Maintain a Digital Flow Meter
Good laboratory practices call for annual flowmeter recalibration as noted above. But improper use of these precision instruments can soon render them inaccurate, thereby compromising fast, accurate calibration of HPLC pumping system flow rates.
Maintenance instructions are usually included in user manuals and cleaning kits shipped with the flowmeters. Here are four tips:
- For flow meters in constant use clean them every two weeks in a process that should take about 15 minutes.
- Do not introduce two immiscible liquids into the digital flow meter without either allowing the first to fully evaporate or by introducing a third solvent in which the others are miscible.
- A dirty sensing tube may trap bubbles and create falsely high readings. If bubbles are observed, a quick fix is to turn the unit off, wait approximately two seconds to allow it to drain, then switch it on. Regular use of the cleaning kit will avoid this problem. Do not disassemble the unit.
- Flush the flowmeter with ethanol if it will not be used for a week or more. This prevents algae growth in the tube.
These digital flow meters have been performance-proven in the field as a way to save time and improve accuracy when checking HPLC system pump rates. To quote a comment by a UCLA graduate researcher in chemical engineering "we use the FlowCal 5000 for its accuracy at low volumetric flow rates, reliability and ease of use."
Contact the scientists at Tovatech for complete details on using and maintaining these precision instruments.