Viswanadha institute of pharmaceutical sciences, Anandapuram, Visakhapatnam
Ultra-High-Performance Liquid Chromatography (UHPLC) and Ultra Performance Liquid Chromatography (UPLC) are two of the most advanced liquid chromatographic techniques, offering improved separation, sensitivity, and speed over traditional High-Performance Liquid Chromatography (HPLC). While both technologies share similar principles, they differ in system configurations, column technologies, and applications. UHPLC generally refers to any liquid chromatography system capable of using sub-2 µm particles, while UPLC is a proprietary technology developed by Waters Corporation that optimizes chromatographic performance specifically with sub-2 µm particles. This review explores the principles, advancements, differences, and applications of UHPLC and UPLC, discussing their unique advantages and limitations, and providing insights into their roles in industries such as pharmaceuticals, food safety, environmental analysis, clinical diagnostics, and forensics.
Liquid chromatography (LC) has become a cornerstone technique in analytical chemistry for separating, identifying, and quantifying compounds in complex mixtures. Over the past few decades, significant improvements in chromatographic technology have led to the development of Ultra-High-Performance Liquid Chromatography (UHPLC) and Ultra Performance Liquid Chromatography (UPLC). These technologies, which provide higher resolution, speed, and sensitivity compared to traditional HPLC, have revolutionized various fields of analysis. While both UHPLC and UPLC utilize sub-2 µm particle columns to achieve high-resolution separations, there are key differences in their system design, operating conditions, and specific applications. UHPLC is a more generalized term for advanced liquid chromatography systems, whereas UPLC, developed by Waters Corporation, is a specific brand optimized for ultra-fast and high-resolution separations using sub-2 µm particles. Understanding the advantages, limitations, and differences between these two technologies is essential for selecting the appropriate system for various analytical applications. This review aims to compare and contrast UHPLC and UPLC, focusing on their technological advancements, system configurations, performance characteristics, and practical applications across diverse industries.
Principles of UHPLC and UPLC
UPLC: UPLC- UPLC (ultra performance liquid chromatography) systems were first introduced in 2004. By almost doubling the overall operating pressure (to 15,000 psi) in order to obtain more rapid flow rates, UPLC developers were able to achieve equal or better resolution LC separations in much shorter time frames. Comment: Rather than “doubling the overall operating pressure in order to obtain more rapid flow rates” it’s better to say … “In order to take advantage of 2-micron particles higher pressures are required.” The typical ID of a UPLC column is 2.1 mm and in general flow rates are lower than HPLC, but due to the efficiency increase, explained above, the overall separation time is reduced. In terms of efficiency, accuracy and productivity, this was good news for labs the world over. UPLC is a variant of HPLC, also using columns and pumps. Comment: Whether we are talking about HPLC, UPLC or UHPLC, each of these techniques employs columns and pumps.
Principle: The basic principle of UPLC for the separation of components in a matrix is same as HPLC, the main difference is in the particle size of sorbent of the column, which is less than 2 ?m. The small particles in UPLC require a high pressure (6000 psi) to work with.This is also based on van deemeter equation which describes the relationship between flow rate and HETP or column efficiency.This review explores the underlying principles, development, key differences, and practical applications of UHPLC and UPLC. By analyzing their capabilities and limitations, the article aims to provide a detailed comparative overview of these advanced chromatographic techniques.
H = A + B/v + Cv
A - Eddy diffusion
B - Longitudinal diffusion
C - Equilibrium mass transfer
v - Flow rate
UHPLC
Ultra-High-Performance Liquid Chromatography (UHPLC) is a high-resolution separation technique that typically uses columns packed with smaller particles (ranging from 1.7 µm to 2.5 µm). The reduction in particle size leads to improved separation efficiency and reduced analysis times. UHPLC systems can typically operate at higher pressures, up to 15,000 psi or 1034 bar, allowing the mobile phase to pass through tightly packed columns.
Key Features:
Differences between UHPLC and UPLC
Although UHPLC and UPLC share many similarities in terms of their ability to use sub-2 µm particles and operate at high pressures, several differences set these two techniques apart. The primary differences lie in the system design, column technology, and flexibility.
Column Technology and Particle Size
System Configuration and Optimization
Speed and Resolution
Advancements in UHPLC and UPLC:
Ultra-High-Performance Liquid Chromatography (UHPLC) and Ultra Performance Liquid Chromatography (UPLC) have undergone significant advancements over the past decade. These improvements have contributed to their widespread adoption in various fields, such as pharmaceuticals, environmental analysis, clinical diagnostics, and food safety. The advancements in both techniques are primarily driven by innovations in instrumentation, column technology, data processing, and method development. Below is a detailed overview of the key advancements in UHPLC and UPLC.
1. Column Technology Advancements
Sub-2 µm Particles
Novel Stationary Phases
Column Selectivity
2. High Pressure and Flow Optimization
Enhanced Pressure Capabilities
Improved Flow Control
3. Miniaturization and High-Throughput Capabilities
Microfluidic Systems
High-Throughput Screening
4. Coupling with Advanced Detectors
Mass Spectrometry (MS) Coupling
Diode-Array Detectors (DAD) and Fluorescence Detectors
5. Improved Software and Data Analysis
Advanced Data Processing Algorithms
Automation and Integration
6. Green Chemistry and Sustainability
Solvent and Mobile Phase Innovations
Applications of UHPLC and UPLC
1. Pharmaceutical Industry
Both UHPLC and UPLC are extensively used in pharmaceutical analysis for quality control, method development, and pharmacokinetic studies. The high resolution and sensitivity provided by both techniques are essential for the detection of impurities and the analysis of complex formulations.
UHPLC: Used for method development, impurity profiling, stability testing, and bioanalytical applications.
UPLC: Ideal for high-throughput analysis, routine quality control, and the analysis of small molecules, proteins, and peptides.
2. Environmental Analysis
In environmental monitoring, UHPLC and UPLC are used for the detection of contaminants such as pesticides, herbicides, heavy metals, and other pollutants in water, soil, and air.
UHPLC: Suitable for detecting low concentrations of a wide range of environmental pollutants.
UPLC: Preferred for fast and sensitive analysis, especially in high-throughput environmental screening.
3. Food Safety and Quality Control
In food analysis, UHPLC and UPLC are used to detect contaminants, such as pesticides, preservatives, and toxins, and to verify the authenticity of food products.
UHPLC: Preferred for method development in complex food matrices.
UPLC: Useful for rapid, high-throughput testing of food products for contaminants.
4. Clinical Diagnostics
In clinical applications, both UHPLC and UPLC are used for the analysis of biomarkers, drugs, and metabolites in biological fluids such as blood and urine.
UHPLC: Applied for detailed clinical assays and the analysis of complex biological samples.
UPLC: Ideal for high-throughput clinical testing, providing faster and more sensitive results.
5. Forensic Toxicology
Both UHPLC and UPLC are used in forensic toxicology for the analysis of drugs, alcohol, and poisons in biological samples.
UHPLC: Suitable for more complex forensic applications that require method development.
UPLC: Used for rapid analysis and high-throughput toxicology screening in forensic laboratories.
CONCLUSION
Both Ultra-High-Performance Liquid Chromatography (UHPLC) and Ultra Performance Liquid Chromatography (UPLC) represent the forefront of liquid chromatography technology, offering significant improvements over traditional HPLC in terms of resolution, speed, and sensitivity. While UHPLC is more versatile and flexible in terms of column chemistries and system configurations, UPLC is optimized for high-speed, high-resolution separations with sub-2 µm columns and specific hardware configurations. Each technique has its strengths, and the choice between UHPLC and UPLC depends on the specific application and requirements of the analysis. For high-throughput, routine testing and ultra-fast separations, UPLC offers unmatched performance, particularly in industries such as pharmaceuticals, food safety, and clinical diagnostics. UHPLC, on the other hand, offers broader flexibility and is suitable for more diverse applications, making it an ideal choice for method development and complex analyses. Overall, both technologies represent the evolution of liquid chromatography and continue to shape the future of analytical chemistry across multiple industries.
REFERENCE
Manda Swapna*, Rongali Indu, Dr. P.V. Madhavi Latha, B. Rama Madhuri, A Comprehensive Review on UHPLC and UPLC: Advancements, Comparison, and Applications, Int. J. Sci. R. Tech., 2024, 1 (11), 87-91. https://doi.org/10.5281/zenodo.14173528