Difference Between HPLC and UHPLC
HPLC (High-Performance Liquid Chromatography) and UHPLC (Ultra-High Performance Liquid Chromatography) are both powerful techniques used in chemical analysis, but there are some key differences between them:
Pressure and Particle Size:
- HPLC typically operates at pressures up to 6000 psi and uses particles in the column ranging from 3-5 microns in size.
- UHPLC operates at much higher pressures, often up to 15,000 psi or more, and uses smaller particles (sub-2 micron) which lead to higher resolution and faster analysis times.
Speed and Efficiency:
- HPLC can be slower because of the larger particle size and lower pressure, which means lower efficiency and longer run times.
- UHPLC, thanks to smaller particle sizes and higher pressure, provides much faster separations, greater resolution, and enhanced sensitivity.
Applications:
- HPLC is often sufficient for many analytical tasks, especially for routine testing.
- UHPLC is typically used for more complex analyses, offering higher sensitivity, resolution, and speed.
Cost and Maintenance:
- HPLC instruments are generally less expensive and easier to maintain compared to UHPLC, which, due to the high pressure and small particle size, often requires more frequent maintenance.
Personal Experience with Waters and Agilent Systems
I’ve worked with three systems so far: Waters, Agilent 1290, and Agilent 1260 II Infinity. Throughout my experience, I encountered a few challenges:
Pre-column (Guard) and C18 Column Contamination:
One of the recurring issues I’ve faced is contamination of the pre-column and the main C18 column, which affects the accuracy and precision of the results. Cleaning and proper maintenance are critical, but contamination still seems to be a persistent challenge.
Air Bubbles Trapping:
Another issue I’ve come across is air bubbles getting trapped in the system, which leads to inconsistency in flow and pressure, affecting the quality of results and even causing occasional system errors.
Pump Failures:
Pump malfunction, especially when it’s not delivering the mobile phase at a consistent pressure, has caused some setbacks. This issue often requires thorough troubleshooting to ensure the pumps are functioning optimally.
Baseline Noise, Peak Shape Issues, and Peak Shifting:
I’ve also experienced baseline noise, irregular peak shapes, and even peak shifting. These can be linked to factors like unstable flow rates, improper solvent preparation, or even column problems.
Question for the Audience:
I’d love to hear from others who have used similar systems. Have you encountered any of the issues I’ve mentioned? Specifically:
- Pre-column or C18 column contamination
- Air bubbles in the system
- Pump failures
- Baseline noise, peak shape issues, or peak shifting
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Resource Person: Hamideh Mousavi
