HPLC is an indispensable tool in analytical laboratories, playing a crucial role in separating, identifying, and quantifying analytes. At the heart of this system lies the HPLC pump, demanding precision and care to ensure its efficacy and reliability. Delve into the intricate science and methodology behind HPLC pump priming.
Why HPLC Pump Priming Is Important
Before learning more about the priming process, it's necessary to recognize the significance of its role in the broader HPLC system.
- Removes air bubbles: Bubbles can lead to pressure fluctuations, affecting the chromatographic baseline and the reliability of results.
- Ensures consistent flow: Proper priming ensures a consistent solvent flow, foundational for reproducible chromatograms.
- Improves instrument longevity: Regular maintenance, including priming, extends the lifespan of your pump and reduces the likelihood of breakdowns.
Priming might seem like an inconvenience, but it can save a lot of time and wasted resources over the long term.
Indications for Pump Priming
So, how do you know when the pump should be primed? While there will be times when you need to exercise judgment to determine when the pump needs to be primed, there are some helpful guidelines you can follow. Typically, the HPLC pump should be primed:
- After instrumental maintenance or significant servicing.
- After prolonged periods of HPLC inactivity (including overnight).
- When transitioning between solvents.
- When pressure anomalies or erratic chromatographic patterns are observed.
As a general rule, the pump should also be primed any time air bubbles are observed.
A Systematic Approach to Priming Your HPLC Pump
The following steps provide general guidelines for priming the pump, as advised by John Dolan, one of the world’s foremost HPLC troubleshooting authorities. However, it’s always advisable to consult your manufacturer’s manual. Here, you can acquaint yourself with any model-specific guidelines or nuances.
- Prepare the solvent: Begin by ensuring that the solvent is filtered and degassed. Using a solvent filtration kit or an online degasser will remove any dissolved gases, preventing bubble formation.
- Open the purge valve: Before you begin, open the purge valve located at the pump outlet. As Dolan notes, with some pumps, this will mean filling the tubing between the reservoir(s) and pump with the aid of a syringe.
- Run at high flow: Next, Dolan advises to run the pump for several minutes at high flow (e.g., 10 mL/min) to clear all the air from the pump and connecting tubing.
- Focus on the column's inlet: After the initial flush, reduce the flow rate to 1 mL/min or so and close the purge valve until no more bubbles emerge. Dolan advises that this will clear the tubing to the inlet of the column.
- Employ low viscosity solvents: Before concluding the priming process, using solvents like methanol (MeOH) or acetonitrile (ACN) can be advantageous. Dolan notes that these solvents will help to remove any air bubbles from the column due to their low viscosities.
Priming the pump is not just a routine task; it's an imperative step that safeguards the accuracy, repeatability, and longevity of the equipment. Mastering HPLC pump priming is an investment in ensuring consistent, high-quality outputs from your chromatographic processes. It mitigates challenges like air bubbles and inconsistent solvent flows, which could compromise the integrity of results. Recognizing the signs for pump priming, such as after maintenance or observed anomalies, is crucial. While the provided steps offer a general roadmap to the priming process, it's essential always to refer to the manufacturer's instructions to account for model-specific intricacies.
This article has been reviewed by Tom Jupille, founder and moderator of Chromatography Forum. Tom has been a practicing chromatographer for more than 40 years, during which he has written more than 30 papers on chromatography and related subjects. His career has focused on instrument and column development and user support, providing a broad foundation of practical experience to call on as an instructor. Over the past 25 years, Tom Jupille has presented courses and seminars in the field of chromatography to more than 3,000 students.