What Is the Difference Between UHPLC and HPLC?

The two main differences between UHPLC and HPLC are:

•        Maximum System Pressure Rating
•        Typical Particle Size Used in the HPLC Column Stationary Phase

Before diving into these details, let’s first address some basic questions.

What is HPLC?

HPLC stands for High-Performance Liquid Chromatography. It is a separation technique used to isolate a complex mixture of known or unknown compounds and determine the quantity of each compound in the sample. The separation process begins by introducing the sample into a liquid stream (mobile phase), which carries it through the column. The movement of the mobile phase through the system is driven by the HPLC pump. HPLC columns are packed with small particles (stationary phase), and separation occurs based on how the different compounds interact with the mobile phase and stationary phase.

Some compounds/analytes spend a long time interacting with the stationary phase and are more retentive, while some compounds do not interact with the stationary phase and flow through the column at the same rate as the mobile phase (less retentive or no retention). When compounds exit the column, they flow through a detector, which sends a signal to a data system, and a chromatogram is displayed (graphical plot showing the separation).

Over the past 50 years, significant improvements have been made to the critical components of the HPLC system. HPLC fittings and HPLC connectors have been engineered to withstand higher pressures. HPLC column manufacturers can reproducibly make smaller particles (stationary phase). With the popularity of smaller HPLC column particle sizes due to the decrease in analysis time, HPLC system pressures have increased significantly. These smaller particles led to the development of HPLC systems that can perform at higher pressures.

Advances in analytical instrumentation and stationary phase chemistry have had a profound effect on chromatographic methods. These innovations have prompted a re-evaluation of the systems and techniques employed by chromatographers. The development of more robust HPLC components and the ability to manufacture smaller stationary phase particles not only enhances the efficiency of the analyses but also challenges traditional methods, pushing the boundaries of what can be achieved in the field.

As a result, chromatographers are now able to explore a wider range of applications and achieve faster, more accurate results. This ongoing evolution in technology and methodology is reshaping the landscape, requiring practitioners to continuously adapt and refine their approaches to stay at the forefront of chromatography.

What is UHPLC?

UHPLC is an acronym for Ultra High-Performance Liquid Chromatography (or sometimes referred to as Ultra High-Pressure Chromatography). UHPLC systems can withstand pressures up to 20,000 psi or 1,300 bar. Traditional HPLC Pressures are typically limited to 6,000 psi or 400 bar.

In UHPLC, it is common to use HPLC columns with a stationary phase that uses sub 2µm particle size. These smaller particles result in added back pressure to the system. With the added back pressure, it is critical that HPLC connections are made properly and the appropriate fittings and connectors are selected to withstand the higher pressures.

Understanding UHPLC and How It Differs from HPLC

Ultra High-Performance Liquid Chromatography (UHPLC) represents an advanced approach in liquid chromatography, engineered to enhance speed, sensitivity, and resolution. UHPLC systems can operate at pressures around 20,000 psi, although the exact range may vary based on the specific equipment used.

Key Characteristics of UHPLC

• Pressure Range: While UHPLC is known for high-pressure operation, not all systems achieve maximum capacity. Each system’s design influences its pressure capabilities.
• Efficiency: These systems provide remarkable efficiency due to their ability to work with very fine particles, typically below 2 microns.
• System Volume: UHPLC generally features lower extra-column volumes, minimizing dispersion and improving performance.

Comparing UHPLC with HPLC

High-Performance Liquid Chromatography (HPLC) is another innovative technique that focuses on optimizing chromatographic performance. Here’s how they compare:

1. Trademarked Technology: HPLC is a branded technology, whereas UHPLC is a generic term used by various manufacturers.

2. Volume and Dispersion: Both technologies aim to reduce system volume and dispersion, but UHPLC may offer a broader range of customization due to non-specific branding.

3. Pressure Capabilities: While UHPLC systems can achieve very high pressures, not all systems utilize the maximum potential. HPLC systems are similarly high-pressure but may have different limitations dependent on the manufacturer.

4. Clogging Propensity: Both UHPLC and HPLC systems are susceptible to blockages if samples are not properly prepared. This is largely due to the fine particles used in columns, not the system design itself.

Both UHPLC and HPLC aim to advance liquid chromatography performance, with unique attributes and operational capacities that cater to specific analytical needs. Understanding these differences helps in choosing the right system for your laboratory requirements.

How Do UHPLC and HPLC Techniques Enable the Separation of Components in a Mixture?

Ultra High-Pressure Liquid Chromatography (UHPLC) and High Performance Liquid Chromatography (HPLC) are advanced techniques that enhance the separation of components in a mixture, making them indispensable in the field of analytical chemistry.

Key Components and Fundamentals

Both UHPLC and HPLC are designed to improve the efficiency and resolution of liquid chromatography through the use of high-pressure systems and unique column technologies:

• High Pressure Systems: These techniques employ significantly higher pressures than traditional liquid chromatography. This increased pressure shortens the time it takes for the mixture components to travel through the column, thus expediting the entire process.

• Column Technology: The columns used in UHPLC and HPLC contain sub-2 micron particles. These tiny particles provide a larger surface area which facilitates better interaction between the mixture's components (analytes) and the stationary phase. This larger surface area enhances separation by reducing the diffusion path of analytes.

Solid Core Particle Design

A prominent advancement in column technology is the use of solid core particles:

• Encapsulation with Small Particles: These solid core particles are surrounded by a layer of smaller particles. This design significantly minimizes the diffusion path, allowing for increased efficiency in separation.

• Layer Thickness: The thickness of the layer around the solid core plays a crucial role in determining separation efficiency. Thinner layers result in quicker and more efficient separations.

Enhanced Efficiency and Resolution

By implementing these technologies, UHPLC and HPLC systems achieve:

1. Faster Analysis Times: The enhanced pressure and improved column designs allow for quicker analysis without sacrificing accuracy.

2. Higher Resolution: The improved interaction between analytes and column results in clearer separation of components, enabling precise analysis.

3. Better Reproducibility: Consistent results across multiple runs are achievable due to the stable and efficient column technology.

UHPLC and HPLC leverage high pressure and innovative column designs to significantly enhance the separation of components in complex mixtures, providing rapid, accurate, and detailed analysis for researchers and industry professionals.

UHPLC and HPLC Fittings

In HPLC and UHPLC, it is critical to make proper HPLC connections to avoid system leaks, which lead to dreaded instrument down time. Prior to the pump, in both HPLC and UHPLC, low-pressure fittings can be used; we recommend Flangeless Fittings for ease of use. These Flangeless fittings are typically ¼-28 threaded flat bottom ferrule connections. After the pump, system pressure dramatically increases due to the back pressure caused by the HPLC column (the smaller the particle size, the higher the system back pressure).

It is imperative to make good connections in the high-pressure portion of the system, and we recommend Idex Fittings for HPLC and UHPLC connections. Idex Health and Science is the global authority in fluidics, and we recommend that all new chromatographers (all chromatographers, actually) download the Idex Fittings 101 Guide, A Practical Guide to Connections and Fittings in Your Laboratory. The Fittings 101 Guide was designed to give you the fundamentals of equipment fittings and accessories, as well as the basics about liquid chromatography as an analytical technique.

Uses of HPLC and UHPLC Systems Across Various Fields

HPLC (High Performance Liquid Chromatography) and UHPLC (Ultra High Performance Liquid Chromatography) are powerful analytical tools employed across a broad range of disciplines. Their versatility and precision make them invaluable in several key areas:

1. Legal and Regulatory Applications

HPLC and UHPLC are crucial in enforcing laws and regulations, particularly in drug testing and forensic analysis. These systems can swiftly and accurately detect the presence of substances, such as performance-enhancing drugs, ensuring compliance in competitive sports and upholding justice in legal investigations.

2. Pharmaceutical and Medical Fields

In the pharmaceutical industry, these systems are indispensable for drug development and quality control. They help in assessing the purity of compounds and in the discovery of new medications. In medical diagnostics, they are used to measure critical biomarkers, such as vitamin D levels in blood serum, delivering accurate results quickly.

3. Research and Development

HPLC and UHPLC drive innovation in scientific research and manufacture. They offer the sensitivity, speed, and resolution necessary to uncover new insights in fields like biochemistry and molecular biology. With their capacity to handle complex samples, researchers can conduct comprehensive studies efficiently.

4. Integration with Mass Spectrometry (MS)

The advanced performance characteristics of HPLC and UHPLC make them perfect partners for mass spectrometry (LC-MS). This combination amplifies the capabilities of analytical laboratories, allowing for the precise identification and quantification of molecules. The latest generation of MS instruments, which are increasingly affordable and user-friendly, enhance the practicality of LC-MS for a wider range of testing facilities.

HPLC and UHPLC systems are indispensable across many scientific and industrial domains, significantly improving the efficiency and accuracy of analysis in fields from legal investigations to cutting-edge research.

How Have UHPLC and HPLC Evolved Over The Years?

Over the past six years, both Ultra-High Performance Liquid Chromatography (UHPLC) and Ultra Performance Liquid Chromatography (HPLC) have significantly advanced in the scientific community. These technologies have revolutionized the way researchers work with smaller particles, offering remarkable speed and efficiency improvements compared to previous methods.

Key Differences Have Evolved:

• Particle Interaction: UHPLC and HPLC systems now allow scientists to interact with smaller particles more effectively, thus enhancing precision and accuracy in analyses.
• Technological Advancements: While both systems have made significant strides, they differ in certain technical specifications and applications, catering to a variety of research needs.
• Efficiency Gains: The evolution of these chromatographic techniques has accelerated experimental processes, delivering results faster than ever before.

Ultimately, the progression of UHPLC and HPLC has empowered researchers with enhanced analytical capabilities, driving innovation and discovery in various scientific fields.

Conclusion

The evolution of UHPLC and HPLC has propelled analytical chemistry into new realms of efficiency, sensitivity, and reliability. From their humble beginnings to today's cutting-edge technologies, these chromatographic techniques continue to redefine what's achievable in separation science.

As researchers and industries demand faster analyses, higher resolutions, and greater sensitivity, UHPLC and HPLC stand ready, offering indispensable tools for a wide range of applications—from pharmaceutical development to environmental monitoring. Embracing these advancements ensures that scientists remain at the forefront of discovery, paving the way for future innovations and deeper insights into complex mixtures and compounds.