Real-Time Monitoring of HPLC Pump PerformanceHigh Performance Liquid Chromatography (HPLC) is nowadays one of the mostwidely used techniques in analytical chemistry. The range of applications using
HPLC covers a vast range of sample types, concentrations, and compositions. This
extreme flexibility is achieved by using specific columns carefully designed for eachparticular investigation. However, all HPLC separations have one detail in common:
they are reliant on a stable and constant flow rate of solvent and by this they all require constant performance of the HPLC pump. This technical note describes how anovel method of continuous monitoring of flow rate can provide real-time validationof the total HPLC system performance and as such, improve reliability of results. .
Traditional Methods
Up to now, direct determination of flow rate has been limited to system validation using volumetricflowmeters, or, when monitoring was required, by use of Coriolis force flowmeters.
While Volumetric flowmeters are known to be accurate, the method they use is relatively slow as
measurement requires that a known volume must be filled and time difference must be detected
in order to obtain a single data value. As such, volumetric flowmeters integrate over a relatively
large volume (often several hundred microlitres) and are not suitable for delivering “real time”
flow information.
Coriolis Force flowmeter are well proven for providing very accurate flow measurement in HPLC
systems but unfortunately are quite expensive, therefore again a limited solution.

A New Technological Approach
Introduced in 2021, the new Biotech Liquid Flowmeter is based around a thermal Flow Sensor(Fig. 1). In the Liquid Flowmeter – the solvent flows
through a quartz tube. Temperature sensors (T1& T2) are positioned before and after a heating
element which elevates the solvent temperatureslightly. It can be demonstrated that the differencein temperature measured by the two sensors isfunction of the flow rate in the quartz tube. Usinga proprietary algorithm this temperature differenceis then converted into absolute flow rate units. Not
only is this flow measurement technique extremely accurate and sensitive – but it also offers theadvantage of being non-invasive i.e. flow is notinterrupted in any way.
Consequently, using this flow measurement method, the Biotech Liquid Flowmeter has thepotential to be an ideal tool for monitoring of solvent flow rates in HPLC Systems as it does notinterfere with the measurement itself.

Pump Monitoring Deasibility Study
Feasibility of continuous monitoring of a relatively simple HPLC System, consisting of isocratic
pump, sample injector and differential refractive index (DRI) detector was carried out using a Biotech Liquid Flowmeter. No column was used to enable the experiment to focus strictly on HPLC
pump performance and avoid any flow variation due to the column (such as column bleeding).
Pure deionized water was used as the solvent for the experiment, flow rate was set to a nominal
1.000 mL/min. The Liquid Flowmeter was connected to the outlet of the DRI Detector and theoutlet of the flowmeter was then led to a waste bottle. Data acquisition of flow rate values wasrealized using the Flowmeter USB Port, which also provides power supply to the device, and aPC. Data rate was approximately 15 Hz or 15 values per second. The pump flow was then started
along with data acquisition. The experiment was run for several days, day and night, with daily
replenishment with fresh deionized water.

Results
The complete flow stability data collected over several days is far too large to be displayed inthis technical note, we therefore picked a representative 6000 seconds segment (see Fig. 2) toillustrate typical data output using the Chromatography flowmeter to monitor an HPLC pump.

The above Figure 2 plot shows the flow rate to be close to the nominal 1.000 mL/min andappearing to be very stable over time. However, if this data is studied in high-resolution (seeFig 3 below) we can clearly see term variation of flow from the HPLC system.

Conclusions        This experiment clearly demonstrates the ability of the Biotech Liquid Flowmeter in combination
with the Flow App to collect in real-time flow data from an HPLC pump and therefore determine
total HPLC system performance.
The high-resolution, real-time flow data that can be obtained using a Liquid Flowmeter opens
the gate to deeper analysis of HPLC Pump performance parameters such as pulsation thereby
allowing a stricter definition of required parameters for reliable HPLC separation results. Furthermore, this technology offers a completely new tool for troubleshooting of HPLC Systems. Thiswill be object of future investigations.
Furthermore there is potential for using this new flow meter in many other applications where itis necessary to have a close control of the flow rate. Other scientific analytical instrumentationsuch as Surface Plasmon Resonance (SPR), different types of imaging systems and DNA Analyzers are examples on high performance systems where it is important to have the flow rate wellcontrolled.
Dispensing systems is another area where anomalies of flow rate could cause severe deviations
when used in for example a production application. Flow chemistry applications where additionof small volumes of reagents could have big impact on the final result is also dependent on
accurate flow rates.