HPLC vs UHPLC – What are the differences?

When discussing liquid chromatography, two techniques are commonly compared, High-Performance Liquid Chromatography (HPLC) and Ultra-High-Performance Liquid Chromatography (UHPLC). Both are essential for separating compounds in various industries.

However, UHPLC offers distinct advantages in terms of performance, speed and efficiency over traditional HPLC.

Below, we explore the key differences and why you might choose UHPLC for your laboratory.

What is UHPLC?

Ultra-High-Performance Liquid Chromatography (UHPLC) is an advanced form of liquid chromatography that operates under higher pressures, enabling faster and more precise separations.

Additionally, with reduced column dimensions and smaller particle sizes, UHPLC offers enhanced resolution and sensitivity, making it ideal for applications that demand high throughput and accuracy.

To learn more about liquid chromatography, visit our blog here.

Applications of UHPLC

UHPLC is used across a range of fields, including:

• Pharmaceuticals: For drug development and quality control.
• Environmental analysis: For detecting pollutants in air, water, and soil.
• Food and beverage: Ensuring product safety and quality control.
• Clinical research: Analysing biomarkers and metabolites.

Key differences between HPLC and UHPLC

Particle sizes

Particles in HPLC are typically larger, ranging from 3 to 5 microns, while UHPLC utilises much smaller particles, generally under 2 microns. These smaller particles enhance the system’s separation power, delivering sharper peaks and improved resolution.

Column dimensions

HPLC uses larger columns, often with an internal diameter of 4.6 mm and a length of 250 mm, while UHPLC columns are more compact, typically with diameters of 2.1 mm or smaller and a length of about 100 mm.

Backpressure

The larger particle size and column dimensions in HPLC result in lower backpressure, typically up to 600 bar, whereas the reduced particle sizes and smaller columns in UHPLC generate higher backpressure, with modern systems capable of handling pressures up to 1,500 bar.

Flow rates

HPLC operates at flow rates of around 1-2 mL/min. UHPLC uses lower flow rates, typically between 0.2 and 0.7 mL/min, which improves efficiency and reduces solvent usage.

Detection parameters

While HPLC is suitable for many routine applications, it may struggle with very complex samples. UHPLC, with its greater sensitivity, is capable of detecting even trace amounts of substances, making it ideal for complex analyses.

Why choose UHPLC?

UHPLC provides numerous benefits, making it an attractive choice for many laboratories:

• Increased speed and throughput: Faster separations allow more samples to be processed in less time, boosting productivity.
• Better resolution: Smaller particle sizes and compact columns deliver superior separation, even for closely related compounds.
• Reduced solvent costs: Lower flow rates and shorter analysis times mean less solvent usage, resulting in considerable cost savings.
• Higher sensitivity: Enhanced resolution improves detection limits, making UHPLC ideal for trace-level analyses.

However, there are a few challenges to keep in mind, such as:

• Initial investment: UHPLC systems and columns are generally more expensive than HPLC systems, which may be a factor for some laboratories.
• Maintenance: Operating under higher pressures can increase wear on components, requiring more frequent maintenance.
• Method transfer: Transitioning from HPLC to UHPLC involves optimising existing methods, which may take time and resources.

How to transition from HPLC to UHPLC

Switching from HPLC to UHPLC doesn’t have to be complicated. Here are a few steps to help you make the transition smoothly:

1. Evaluate your methods: Review current methods to see if they can be adapted for UHPLC. Many methods can be easily scaled down.
2. Adjust parameters: You may need to modify flow rates, gradient times and injection volumes to meet the specifications of UHPLC.
3. Test and validate: Once the adjustments are made, test your methods to ensure they deliver comparable or improved results.
4. Upgrade equipment: Ensure your system can handle the higher pressures required for UHPLC. Modern systems are designed to handle both HPLC and UHPLC methods, providing flexibility.

How LC Services can help

Whether you’re looking to upgrade to UHPLC or optimise your existing HPLC system, we can help. Our range of reconditioned chromatography equipment and parts offers a cost-effective solution to enhance your lab’s capabilities without compromising on quality.

If you’re considering transitioning to UHPLC, we can assist you with everything from method development to sourcing the necessary equipment.

Contact us today