Ultra-high performance liquid chromatography (UHPLC) or simply ultra-performance liquid chromatography (UPLC) is a modern and one of the most advanced versions of liquid chromatography. It offers chromatographic analytical separations with high efficiency, superior resolution, and faster speed of analysis.
This article brings to you all that you would like to know about ultra-high performance liquid chromatography. Additionally, we will address the oft-asked question that is how UHPLC is different from high-performance liquid chromatography (HPLC).
What is ultra-high performance liquid chromatography
Ultra-high performance liquid chromatography (UHPLC) is a type of liquid column chromatography. It works on the principle of applying an extremely high i.e., an ultra-high pressure, about 15,000 Psi (100 MPa) pressure for chromatographic separation. The stationary phase in UHPLC is compactly packed into a column. Very small particle size (2µm) stationary phase material allows a compact column packing. A high pressure applied against this stationary phase enables a rapid analytical separation with an excellent resolution and a higher sensitivity for quantification purposes.
UHPLC is relatively a new chromatographic technique. It was introduced in the mid-2000s, but it has gained maturity and popularity over the last ten years.
What is the stationary phase in UHPLC
2 µm, fully porous, or 3 µm core-shell particles are used as stationary phase in ultra-high performance liquid chromatography. Silica is the most common stationary phase choice for UHPLC as well, like all other column chromatographies. Other than silica, hybrid silica and non-silica-based materials such as metal oxides and organic polymers can also be used for stationary phase development in UHPLC. These alternative options can help to overcome pH and thermal instability restrictions associated with a silica stationary phase.
Metal oxides suitable for UHPLC stationary phase are alumina (Al2O3), zirconia (ZrO2), titania (TiO2), etc. The stationary phase can be developed in normal-phase, reverse-phase, or hydrophilic interaction (HILIC) mode depending upon the analysis requirement. The polar silica gel can be chemically treated and converted to C8 or C18 octadecyl silane (ODS) for performing UHPLC in reverse-phase mode.
What is the mobile phase in UHPLC
Conventional organic solvents are used as mobile phases in ultra-high liquid chromatography, for example, methanol, acetonitrile, acetic acid, formic acid, etc. Aqueous buffer solutions such as ammonium hydroxide/ammonia solution and/or ammonium acetate can also be employed as UHPLC mobile phases.
Components of ultra-high performance liquid chromatography
An ultra-high performance liquid chromatography system is based on the following components:
- Solvent reservoir: The mobile phase solvents are stored in and supplied through external reservoirs.
- Pressure pumps: A high pressure, close to 15000 Psi or 100 MPa is applied, maintaining a flow rate between 0.05 to 8 mL/min.
- Sample injector: 1 to 100 µL sample volume can be injected into the column, with the help of a special needle injector. The injection valves used for UHPLC may be manual or automatically programmed to protect the analytical column from crucial pressure instabilities.
- Analytical column: A compactly packed column is used. The column dimensions are usually 150 x 2.1 mm. The column compartment temperature is maintained between 5 to 110°C.
- Detector: A mass spectrometric (MS), an ultraviolet (UV), an evaporative light scattering detector (ELSD), or a fluorescence detector is usually coupled with a UHPLC column, as per sample specialty.
How is UHPLC different from HPLC
There are many similarities present between UHPLC and HPLC which is why often the two chromatographic techniques are perceived as identical. Both are types of liquid chromatography that work on high-pressure application. A liquid mobile phase is used against a stationary phase tightly packed into the column. Both techniques offer similar levels of accuracy and precision. The principles of both high-performance liquid chromatography and ultra-high-performance liquid chromatography are the same, but there exists a significant difference between their performance.
| UHPLC | HPLC |
| A relatively newer technique, introduced in 2004 by Waters Corporation | One of the oldest and most popular chromatographic techniques, HPLC is in use since 1967 |
| Ultra-high pressure (15000 Psi) is applied | Relatively lower pressure (6000 Psi) than UHPLC is applied |
| Smaller particle size (2 µm) of stationary phase as compared to HPLC | Larger particle size (up to 4 µm) of stationary phase as compared to UHPLC |
| Lower flow rate, usually 0.6 mL/min | Comparatively higher flow rate (5 mL/min) |
| Higher back pressure inside the column requires a more mechanically stable stationary phase | Lower back pressure inside the column as compared to UHPLC |
| High pressure applied allows higher sensitivity for component detection and identification | Lower sensitivity than UHPLC |
| Higher column efficiency (Number of theoretical plates, N=7500) |
Relatively lower column efficiency than UHPLC (N= 3000) |
| High priced technique | Less expensive than UHPLC |
| Lack of experience in handling | Full experience gained in its handling |
As a result of all these considerations, now we know that UHPLC is an upgraded version of HPLC that offers higher sensitivity and greater reproducibility. The time spent in HPLC method development and validation can be saved by a quicker UHPLC analysis.
Having said that, HPLC is still a more commonly used chromatographic technique because it is readily accessible everywhere around the world. The switch to UHPLC is gradual but it is gaining momentum in the scientific world for more complicated applications.
Applications of ultra-high performance liquid chromatography
- UHPLC is an extremely sensitive chromatographic technique. It has a low LOQ and LOD value as compared to traditional HPLC. Thus, UHPLC can be applied for analyzing small quantities of target compounds present in complicated biological samples and dietary supplements.
- UHPLC finds useful applications in metabolomics i.e., the assessment of metabolites of a biological system that can be used as biomarkers for disease diagnosis. These metabolites include amino acids, organic acids, nucleosides, lipids, etc.
- Ultra-high performance liquid chromatography finds special applications in food safety. Typical food contaminants such as pesticides can be efficiently detected via UHPLC. Allergens, antibiotics, hormones, food additives, and mycotoxins present in animal-based foods can be readily identified and quantified by UHPLC-MS.
- Low concentration of analytes present in complex environmental matrices such as wastewater, sludge, soil, and aerosols can be analyzed via ultra-high performance liquid chromatography.
- UHPLC is used in the pharmaceutical industry for quality control and assurance. Pollutants present in trace quantities in the drugs can be easily separated in a high-pressure UHPLC column.
For more extensive applications of ultra-high performance liquid chromatography, you may consult the following two sources:
- UPLC: a novel technique of liquid chromatography.
- Ultra-high performance liquid chromatography and its applications.
Limitations of ultra-high performance liquid chromatography
- UHPLC due to its high-pressure conditions is not suitable for separating sensitive inorganic ions and polysaccharides
- Aggressive non-polar solvents are not UHPLC compatible
- There is no ideal universal detector yet available for UHPLC
- High-pressure application requires greater column maintenance in UHPLC
You may like to read about different types of detectors in chromatography for additional reference.
Conclusion
An important advantage of ultra-high performance liquid chromatography is that it provides a rapid analyte separation. A quicker response means less organic solvent consumption. This makes UHPLC an environment-friendly alternative to traditional liquid chromatographic techniques. Therefore, UHPLC is gaining an unprecedented popularity in the world of chromatography which will continue to rise with time.
The theoretical aspects of UHPLC chromatographic separation and performance are the same as those discussed in our article: Theories of column chromatography.
Do you know!
As opposed to ultra-high pressure applied in UHPLC, there is a lower pressure variant of HPLC, specifically important for protein purification in bulk quantities called fast-protein liquid chromatography (FPLC).
References
1.Nováková, L., P. Svoboda and J. Pavlík (2017). Chapter 29 – Ultra-high performance liquid chromatography. Liquid Chromatography (Second Edition). S. Fanali, P. R. Haddad, C. F. Poole and M.-L. Riekkola, Elsevier: 719-769.
2. Perez de Souza, L., S. Alseekh, F. Scossa and A. R. Fernie (2021). “Ultra-high-performance liquid chromatography high-resolution mass spectrometry variants for metabolomics research.” Nature Methods 18(7): 733-746.
3. Rathod, R. H., S. R. Chaudhari, A. S. Patil and A. A. Shirkhedkar (2019). “Ultra-high performance liquid chromatography-MS/MS (UHPLC-MS/MS) in practice: analysis of drugs and pharmaceutical formulations.” Future Journal of Pharmaceutical Sciences 5(1): 6.