1Research Scholar, Department of Pharmaceutics, Delight College of Pharmacy, Koregaon Bhima, Pune, Maharashtra, India-412216.
2Assistant Professor, Department of Quality Assurance Techniques, Delight College of Pharmacy, Koregaon Bhima, Pune, Maharashtra, India-412216
Ultra Performance Liquid Chromatography (UPLC) is a modern analytical technique widely utilized in analytical chemistry and the pharmaceutical sector. It operates on the principles of column chromatography, which is typically employed to analyze mixtures. UPLC offers innovative advancements in liquid chromatography, allowing for shorter columns, reduced analysis time, and lower solvent usage. By utilizing particles smaller than 2µm, it significantly improves the speed, resolution, and sensitivity of analyses. Ultra Performance Liquid Chromatography (UPLC) leverages advancements in particle chemistry, system optimization, detector technology, and data processing and control to enhance performance. The quality control assessments of different pharmaceutical formulations are being moved from an HPLC system to a UPLC system. The separation in UPLC is carried out at extremely high pressures (up to 100 MPa), yet this does not adversely affect the analytical column or any other parts of the chromatography system. In fact, the efficiency of separation is either preserved or even enhanced by UPLC.
Chromatography is a technique used to separate the components of a mixture, known as solutes, by examining how well each solute distributes itself between a stationary phase and a mobile phase, which is a flowing fluid. The stationary phase may be either solid or liquid, while the mobile phase can be a liquid or gas. Separation is influenced by factors such as the molecular properties related to adsorption, affinity, and partitioning, as well as differences in molecular weights. Consequently, some mixture components move quickly through the mobile phase and exit the chromatography system, while others move more slowly through the stationary phase and remain longer. This method consists of three key components: Chromatography is a technique employed to separate mixtures into their individual components. The term comes from Greek, with "chromo"meaning "color"and"graphic" meaning "writing." All types of chromatography operate on the same fundamental principle, utilizing both a stationary phase (which can be a solid or a liquid supported on a solid) and a mobile phase (either a liquid or a gas). The process involves the transfer of mass between these two phases. Stationary phase: This consists of either a solid phase or a layer of liquid that is absorbed on a solid support Separated molecules. Mobile phase: This typically involves a liquid or gas component Many HPLC methods can be carried out on UPLC for its less time consumption and high sensitivity. These methods could be optimized to get better results. In this respect, an HPLC method for quality control (QC) was optimized for UPLC.
Table 1: Comparison of Original HPLC and Optimized UPLC Parameters
|
Characteristics |
HPLC |
UPLC |
|
Column |
Xterra, C18, 50 x 4.6 mm, 4 µm particles |
AQUITY UPLC BEH C18, 50 x 2.1 mm, 1.7 µm particles |
|
Flow Rate |
3.0 ml/min |
0.6 ml/min |
|
Injection Volume |
20 µl |
3 µl (partial loop fill) or 5 µl (full loop fill) |
|
Gradient (time in min) |
ACN T0 (25:75), T6.5 (25:75), T7.5 (95:5), T9 (25:75), T10 (25:75) |
T0 (36:64), T1.1 (95:5), T1.3 (36:64) |
|
Total Run Time |
10 min |
1.5 min |
|
Total Solvent Consumption |
Acetonitrile: 10.5 ml, Water: 21.0 m |
Acetonitrile: 0.53 ml, Water: 0.66 ml |
|
Plate Count |
2000 |
7500 |
|
USP Resolution |
3.2 |
3.4 |
|
Lower Limit of Quantization (LOQ) |
~ 0.2 µg/m |
~ 0.054 µg/ml |
|
Delay Volume |
~ 720 µl |
~ 110 µl |
While the underlying principles of UPLC and HPLC are the same, their performance differs significantly. UPLC utilizes column materials with particle sizes of less than 2 μm, which enhances its efficiency. To fully leverage the benefits of these columns, UPLC offers apowerful,robust, and reliable solution. The familiar design of the UPLC H-Class's Quaternary Solvent Manager (QSM) and Sample Manager (SMFTN), featuring a flow-through needle design, provides the flexibility and usability similar to traditional HPLC, while achieving the superior separations unique to UPLC
Principle of UPLC:
UPLC operates on the principle of using stationary phases made up of particles smaller than 2 μm. This advancement is influenced by the Van Deemter equation, an empirical formula that outlines the relationship between linear flow rate and plate height (HETP or column efficiency). The equation is represented as follows:
H=A+B/V+CV
Where:
A, B, and C are constants
H = Height Equivalent to a Theoretical Plate (HETP)
A = Eddy diffusion
B = Longitudinal diffusion
C = Equilibrium mass transfer
V = flow rate
Eddy diffusion:
refers to the movement of the mobile phase through a column filled with stationary phase, where solute molecules follow various random paths. This randomness leads to band broadening.
Longitudinal diffusion:
occurs because the concentration of analytes is lower at the edges of the band compared to the center. As a result, analytes diffuse from the center toward the edges, contributing to band broadening. Eddy diffusion (A) is at its lowest when the particles in a packed column are small and uniform. the B term, which reflects longitudinal diffusion or the natural tendency of molecules to diffuse, decreases at higher flow rates, so it’s divided by B. The C term represents equilibrium mass transfer, which is affected by kinetic resistance during the separation process. This resistance is the delay experienced by molecules moving from the gas phase to the stationary phase and back. As gas flow increases, molecules in the packing tend to lag behind those in the mobile phase
Chemistry of Small Particles:
The design and development of sub-2 µm particles is a significant challenge, and researchers have been very active in this area to capitalise on their advantages [2, 3]. Although high efficiency nonporous 1.5 µm particles are commercially available, they suffer from low surface area, leading to poor loading capacity and retention. To maintain retention and capacity similar to HPLC, UPLC must use a novel porous particle that can withstand high pressures. Silica based particles have good mechanical strength, but suffer from a number of disadvantages. These include tailing of basic analytes and a limited pH range. Another alternative, polymeric columns, can overcome pH limitations, but they have their own issues, including low efficiencies and limited capacities. In 2000, Waters introduced a first generation hybrid chemistry, called XTerra®, which combines the advantageous properties of both silica and polymeric columns - they are mechanically strong, with high efficiency, and operate over an extended pH range. XTerra columns are produced using a classical solgel synthesis that incorporates carbon in the form of methyl groups. However, in order to provide the kind of enhanced mechanical stability UPLC requires, a second generation hybrid technology was developed [4], called ACQUITY UPLCTM. ACQUITY UPLC 1.7 µm particles bridge the methyl groups in the silica matrix, as shown in Figure 3, which enhances their mechanical stability. Packing a 1.7µm particle in reproducible and rugged columns was also a challenge that needed to be overcome. The column hardware required a smoother interior surface and the end frits were re-designed to retain the small particles and resist clogging. Packed bed uniformity is also critical, especially if shorter columns are to maintain resolution while accomplishing the goal of faster separations. All ACQUITY UPLC columns also include the eCordTM microchip technology that captures the manufacturing information for each column, including the quality control tests and certificates of analysis. When used in the Waters ACQUITY UPLC system, the eCord database can also be updated with real time method information, such as the number of injections, or pressure information, to maintain a complete column history pler, detector, data system, and service diagnostics was required. The ACQUITY UPLC system has been holistically designed for low system and dwell volume to take full advantage of low dispersion and small particle technology. Achieving small particle, high peak capacity separations requires a greater pressure range than that achievable by today's HPLC instrumentation. The calculated pressure drop at the optimum flow rate for maximum efficiency across a 15 cm long column packed with 1.7 µm particles is about 15,000 psi. Therefore a pump capable of delivering solvent smoothly and reproducibly at these pressures, which can compensate for solvent compressibility and operate in both the gradient and isocratic separation modes, is required. Sample introduction is also critical. Conventional injection valves, either automated or manual, are not designed and hardened to work at extreme pressure. To protect the column from experiencing extreme pressure fluctuations, the injection process must be relatively pulsefree. The swept volume of the device also needs to be minimal to reduce potential band spreading. A fast injection cycle time is needed to fully capitalise on the speed afforded by UPLC, which in turn requires a high sample capacity. Low volume injections with minimal carryover are also required to realise the increased sensitivity benefits. With 1.7 µm particles, half-height peak widths of less than one second are obtained, posing significant challenges for the detector. In order to accurately and reproducibly integrate an analyte peak, the detector sampling rate must be high enough to capture enough data points across the peak. In addition, the detector cell must have minimal dispersion (volume) to preserve separation efficiency. Conceptually, the sensitivity increase for UPLC detection should be 2-3 times higher than HPLC separations, depending on the detection technique. MS detection is significantly enhanced by UPLC; increased peak concentrations with reduced chromatographic dispersion at lower flow rates (no flow splitting) promotes increased source ionisation efficiencies.
Instrumentation
The fundamental principles and instrumentation of a UPLC system are similar to those of HPLC, with enhancements in the equipment and hardware. The UPLC system includes a binary solvent system, a sample manager, a column manager, and a detector. The solvent manager features two flow pumps that create a parallel binary gradient under high pressure. A degassing system removes dissolved gases from the mobile phase, which can be selected from up to four solvents via a valve. UPLC systems can handle pressures of approximately 15,000 psi (around 1000 bar), allowing them to utilize sub-2-mm particles effectively. Additionally, the sample manager employs advanced technology, enabling sample temperatures to be lowered to 0°C, while the column manager can regulate temperatures up to 90°C. This high-temperature capability in ulta performance liquid chromatography (UPLC) significantly reduces analysis time without compromising efficiency
Components of instrumentation
1. Pumping system
2. Sample introduction device
3. UPLC columns
4. Detection instruments
Figure 1: Schematic diagram of UPLC
Pumping System:
To achieve effective separations of small particles with high peak capacity, a wider pressure range is necessary than what current HPLC instruments can provide. For optimal efficiency with a 15 cm column packed with 1.7 μm particles, the estimated pressure drop at the best flow rate is approximately 15,000 psi. Thus, a pump that can deliver solvent consistently and smoothly at these pressures—while accounting for solvent compressibility and functioning in both gradient and isocratic modes—is essential. The binary solvent manager utilizes two serial flow pumps to create a parallel binary gradient and features built-in solvent selection valves for up to four different solvents. It has a pressure limit of 15,000 psi (around 1000 bar) to fully leverage the benefits of sub-2 μm particles.
There are two types of pumps used:
1. Reciprocating pump
2. Pneumatic pump.
Figure 2: Piston Pump
1) Reciprocating Pump: Reciprocating pumps function by utilizing a piston or diaphragm that moves back and forth. Liquid enters the pumping chamber through an inlet valve and is expelled via an outlet valve as the piston moves. These pumps are highly efficient and suitable for very high flow rates.
There are two main categories of reciprocating pumps:
A) Piston Pump
B) Diaphragm Pump
Diaphragm pumps can be further divided into two types: Hydraulically Operated Diaphragm Metering Pumps: These pumps can handle toxic and explosive fluids, delivering pressures of up to 700 bars and flow rates of up to 20 m³/hr. Air-Actuated Diaphragm Pumps: The capacity of these pumps is constrained by the available air pressure (typically around 7 bar) and the diaphragm's design. A maximum flow rate of about 40 m³/hr is achievable with larger models.
2) Pneumatic Pump: Originally used for standard liquid chromatography separations, pneumatic pumps were found to be noisy and generated significant flow pulses, which destabilized detectors. Today, they are primarily used for slurry packing in liquid chromatography columns. These pumps are the simplest type and can be designed to achieve very high pressure.
Sample Injection:
Figure 3: Sample injector
In UPLC, the process of introducing samples is crucial. Traditional injection valves, whether automated or manual, are not built to withstand high pressures. To safeguard the column from pressure variations, the injection should be as pulse-free as possible, and the device’s swept volume should be kept to a minimum to limit band spreading. A quick injection cycle is essential to maximize the speed benefits of UPLC, necessitating a high sample capacity. Additionally, low-volume injections with minimal carryover are important to enhance sensitivity
UPLC Columns:
The resolution improves with a column packed with 1.7 μm particles due to enhanced efficiency. To effectively separate the components of a sample, a bonded phase that offers both retention and selectivity is necessary. There are four types of bonded phases available for UPLC separations: 1. ACQUITY UPLCTM BEH C18 and C8 (straight-chain alkyl columns) 2. ACQUITY UPLC BEH Shield RP18 (columns with embedded polar groups)3. ACQUITY UPLC BEH Phenyl (with phenyl groups attached to a silyl functionality and a C6 alkyl chain). Each column type presents a unique combination of hydrophobicity, silanol activity, hydrolytic stability, and interaction with analyte
Figure 4: Acquity beh column
Detector:
For UPLC detection, a tunable UV/Visible detector is utilized. Among analysts, spectro photometric detectors in the UV-visible range are the most commonly used for HPLC, making them relatively affordable and often the first option available to lipid analysts. It is advisable to use detectors designed specifically for HPLC, featuring a cell volume of approximately 8 microliters, rather than standard UV spectrophotometers that offer a flow-cell as an optional addition. Additionally, detectors that provide continuously variable wavelengths are particularly beneficial for lipid analysis.
Figure 5: UPLC Detector
UV detectors, particularly for HPLC of lipids using evaporative light scattering detection, often provide high selectivity and, at times, sensitivity for analyzing specific compounds. They tend to be less affected by variations in ambient temperature or mobile phase flow rate. Although they can occasionally be used in gradient elution applications, baseline drift can be an issue. Additionally, the detector cell can become contaminated during use, which may not be immediately noticeable.
Photodiode Array (PDA) Detector:
This detector provides advanced optical detection across a range of 190 to 800 nm, allowing for exceptional detection and quantification of trace impurities with spectral analysis capabilities. It enables definitive identification of compounds and detection of co-elutions through simultaneous 2D and 3D operations. It is primarily utilized in drug discovery and pharmaceutical development
Fluorescence (FLR) Detector:
Designed for sensitivity and selectivity in fluorescence-based applications, this detector enhances UPLC technology for analyzing polynuclear aromatic hydrocarbons (PAHs), drugs of abuse, and vitamins—essentially any substances with chemiluminescent properties, such as fluorescence or phosphorescence.
Refractive Index (RI) Detector:
The refractive index (RI) detector is a versatile tool used when substances have little to no UV absorbance. This includes compounds such as alcohols, sugars, fatty acids, excipients, raw materials, and pharmaceutical products. Additionally, it can be used for the characterization of low molecular weight polymers in UPLC. However, a significant drawback of this detector is its low sensitivity.
Mass Detector (MS):
UPLC can be integrated with mass spectrometers (MS) and tandem mass spectrometers (MS/MS), which are applicable in various fields and serve to identify compounds Quantification and mass analysis of materials, as well as the structural identification of unknown molecules, can be achieved through fragmentation. Various mass analyzers are available depending on the specific application, including single quadrupole, triple quadrupoles (tandem), ion trap, and time-offlight (TOF) analyzers. These detectors offer high sensitivity, selectivity, and time resolution. In addition to these detectors, several others can be coupled with UPLC, such as infrared (IR), inductively coupled plasma mass spectrometry (ICP-MS), nuclear magnetic resonance (NMR), evaporative light scattering detector (ELSD), and electrochemical detectors (EC).12
Advantages of UPLC:
1. Faster Development: UPLC enables quicker laboratory method development while preserving resolution, selectivity, sensitivity, and dynamic range in liquid chromatography (LC) analysis.
2. Reduced Cycle Times: By shortening process cycle times, UPLC allows for the analysis of more products using the same resources.
3. Quality and Cost Efficiency: It ensures high-quality results with shorter run times and lower costs, while enhancing the sensitivity of sample analyses and reducing solvent usage.
4. Improved Sample Throughput: Utilizing a novel separation material with very fine particle size, UPLC increases sample throughput.
5. Consistent Product Quality: Manufacturers can produce more material that consistently meets or exceeds specifications, reducing variability and minimizing failed batches or the need for rework.
6. Rapid Compound Quantification: UPLC’s fast resolving power enables quick quantification of both related and unrelated compounds.
Disadvantages of UPLC:
1. UPLC performance can match or exceed that of stationary phases around 2 μm.
2. Using stationary phases smaller than 2 μm often results in nonregenerable columns.
3. Smaller particle sizes require higher pressures.
4. Increased maintenance needs.
5. Reduced column lifespan.
APPLICATION
1. Analysis of organic compounds and traditional herbal treatments.
"Analysis" conveys a systematic study, while "organic compounds" is a broader term that encompasses natural substances. "Traditional herbal treatments" maintains the original meaning but uses slightly different wording.
2. Identification of metabolic byproducts.
Identification" emphasizes recognizing specific substances, and "metabolic byproducts" provides a clearer definition of what metabolites.
3. Study of metabonomics and metabolomics.
Study" is a direct synonym for "investigation," simplifying the phrase while retaining its meaning.
4. Research on bioanalytical methods and bioequivalence.
This rephrasing clarifies that the focus is on research methods related to bioanalysis and bioequivalence
5. Evaluation of dissolution rates.
Evaluation" suggests a more thorough assessment than just "testing," which enhances the understanding of the process
6. Manufacturing, quality assurance, and quality control processes.
"Manufacturing" is a more specific term than "production," and adding "processes" emphasizes the series of actions involved.
7. Pharmaceutical development.
Pharmaceutical development encompasses the entire process of creating drugs, offering a broader perspective than just discovery.
8. High-throughput quantitative analysis:
UPLC combined with time-of-flight mass spectrometry enables metabolic stability testing.
9. Dosage form analysis:
This technique delivers rapid, accurate, and reproducible results for both isocratic and gradient analyses of drugs and their related substances, thereby reducing method development time.
10. Amino acid analysis
UPLC is utilized for precise, dependable, and consistent analysis of amino acids in applications such as protein characterization, cell culture monitoring, and nutritional assessment of foods.
11. Pesticide determination:
UPLC paired with triple quadrupole tandem mass spectrometry aids in detecting trace levels of pesticides in water. Ultra Performance Liquid Chromatography (UPLC) fingerprints can also be applied to identify Magnoliae officinalis cortex.
12. UPLC in Stress Degradation Studies:
Chemical stability is a crucial factor influencing the quality and safety of pharmaceuticals. The FDA and ICH guidelines offer stability testing data to assess how the quality of an active pharmaceutical ingredient (API) or drug product varies over time when subjected to forced degradation factors like heat, light, pressure, and moisture.
13. UPLC in Impurity Profiling:
Impurity profiling is a critical aspect of drug development and formulation, aiding in the detection, characterization, and quantification of drug substances and their impurities in both raw materials and final products.
14. PATROL™ UPLC Analyzer:
The PATROL™ UPLC analyzer automatically handles sample dilution when an operator introduces a sample into the system, utilizing a fully automated barcode system for at-line analysis. It monitors reaction progress and measures the concentrations of starting materials, intermediates, impurities, and final products. The analyzer halts the reaction once the residual starting material reaches the target level or impurities exceed a specified threshold.
15. ADME Screening:
ADME studies evaluate the absorption, distribution, metabolism, elimination, and toxicity of drugs targeting specific diseases. Combining tandem quadrupole mass spectrometry with UPLC enhances sensitivity and selectivity, enabling rapid analysis with minimal cleanup using multiple reaction monitoring (MRM) and automated optimization.17
RESULT AND DISCUSSION
1. Result Overview:
Retention Time (RT): The time taken for each component to elute. For example, the API might have a retention time of 3.5 minutes, while impurities might elute at 5.2 minutes.
Peak Area/Height:The peak area is proportional to the concentration of the compound. For example, the active ingredient (API) shows a higher peak area compared to impurities.
Resolution: UPLC offers high resolution, enabling excellent separation between peaks. For instance, a resolution value of 2.5 indicates effective separation of compounds.
DISCUSSION:
Faster Analysis: UPLC reduces analysis time by up to 90% compared to traditional HPLC, providing quicker results (e.g., analysis that would take 30 minutes in HPLC might take just 5-10 minutes in UPLC).
Better Sensitivity & Resolution: UPLC uses smaller particles (≤ 2 µm) and higher pressures, leading to better separation and sensitivity, allowing detection of trace levels of compounds (e.g., impurities at 0.1 ppm).
Cost and Complexity: UPLC equipment is more expensive than HPLC, but the faster throughput and increased sensitivity justify the cost, especially in industries like pharmaceuticals.
Applications: UPLC is widely used in pharmaceutical quality control, impurity profiling, and stability testing due to its efficiency, speed, and ability to detect low concentrations of compounds.
CONCLUSION:
UPLC offers faster, more efficient, and highly sensitive separations compared to traditional HPLC. Its ability to handle complex mixtures with high resolution and minimal sample volume makes it invaluable in industries like pharmaceuticals and food testing, despite its higher initial cost.
FUTURE SCOPE:
1. Enhanced Sensitivity and Detection: Future UPLC systems will offer even lower detection limits, enabling the analysis of trace compounds with high precision, especially in fields like pharmaceuticals and environmental monitoring.
2. Integration with Advanced Technologies: UPLC will be increasingly paired with mass spectrometry (UPLC-MS) and other hyphenated techniques for comprehensive analysis, improving its capabilities in drug development, biomarker detection, and environmental analysis.
3. Green Chemistry: With a focus on sustainability, UPLC will contribute to greener practices by using less solvent and energy, aligning with eco-friendly and sustainable analytical methods.
4. Pharmaceuticals and Biopharmaceuticals: UPLC will continue to play a key role in the development and quality control of drugs, especially biopharmaceuticals, by providing detailed impurity profiling and stability testing.
REFERENCE
Kiran Gaikwad*, Vishal Madankar, Ultra Performance Liquid Chromatography (Uplc): A New Trend in Analysis, Int. J. Sci. R. Tech., 2025, 2 (3), 458-467. https://doi.org/10.5281/zenodo.15082659
10.5281/zenodo.15082659
