- Chromatography is a technique that is used for the separation of constituents in a mixture. This technique involves 2 phases stationary and mobile phases. The separation of these constituents is based on the difference between the partition coefficients of the two phases.
- The different types of chromatography are paper chromatography, thin-layer chromatography (TLC), column chromatography, partition chromatography, high-performance liquid chromatography, and gas chromatography are commonly used for qualitative as well as quantitative determination of molecules.
- High-Performance Liquid Chromatography is an analytical technique for the separation, identification, and quantification of constituents in a mixture. HPLC is an advanced technique of column liquid chromatography.
What is HPLC?
- HPLC stands for high-performance liquid chromatography, it is an analytical technique used to separate, identify, and quantify components in a sample mixture. It is the most common chromatography technique used in most laboratories across the world. HPLC system can withstand pressure up to 6000 PSI. The most commonly used form of HPLC is reversed-phase (RP-HPLC).
What is UPLC?
- UPLC stands for ultra-performance liquid chromatography, it becomes the modern standard HPLC platform. Because it offers increased efficiency, sampling throughput, sensitivity, and reduced run time making it more powerful and time and cost-effective.
Use of UPLC system
- In this Elevated – temperature chromatography, it allows high flow rates by lowering the viscosity of the mobile phase, which reduces the column back pressure. Monolithic columns contain a porous support (polymerized) structure which provides lower flow resistance than any conventional particle-packed columns.
- Ultra Pressure Liquid chromatography, it improves in three areas:
- Resolution,
- Speed,
- Sensitivity.
Advantages of UPLC
- Decreased run time and increased sensitivity.
- More selectivity, sensitivity, and dynamic range of LC analysis.
- Higher resolution performance.
- Expands scope of Multiresidue Methods.
- UPLC’s fast resolving power quickly quantifies related and unrelated compounds.
- Faster analysis through the use of very fine particle size.
- Operation cost is minimized.
- Decreases consumption of solvent.
- Quicker analysis.
Disadvantages of UPLC
- High back pressure compared to HPLC which reduces the life of the column
- So far performance similar or even higher has been demonstrated by using stationary phases of size around 2 μm without the adverse effects of high pressure.
- Particle size of less than 2 μm is generally non - regenerable.
Both high-performance liquid chromatography and ultra-performance liquid chromatography are the separation techniques use to separate different components found in a complex mixture. UPLC works on high pressures and the particle size of columns is less, Whereas, whereas HPLC works under low pressure, hence UPLC offers better detection and separation, shorter run times, and consumption of less solvent.
HPLC vs UPLC
Sr. No | Characteristics | HPLC | UPLC |
1 | Particle size | 3 to 10 µm | Less than 2 μm |
2 | Maximum back pressure | 35 to 40 Mpa | 103.5 MPa |
3 | Analytical column | Alltima C18 | Acquity UPLC BEH C18 |
4 | Column dimensions | 150 ×3.2 mm | 150 X 2.1 mm |
5 | Column temperature | 30°C | 65°C |
6 | Sample throughput | Less | More |
7 | Sample preparation | Simple | Tedious |
8 | Column coagulation | Does not take place | Take place |
9 | Analysis time | More | Less |
10 | Sensitivity | Less | Higher |
11 | Plate count | More than 2000 | More than 7500 |
Difference between HPLC and UPLC
- The principles of both (HPLC and UPLC) are the same, the only difference is in the design of column material particle size which is less than 2 μm. This makes a big difference in performance and maximizes the advantages of a column, creating a powerful, robust and reliable solution
- The major difference between HPLC and UPLC is that due to particle size HPLC works at lower pressures, while UPLC works at higher pressures, which allows UPLC a reduced run time, lower solvent consumption, and better analysis separation and detection by the detector.
- UPLC works on about max 15000 psi pressure, while HPLC works on max up to 6000 psi pressure.
- UPLC the particle size of the column stationary phase is less than 2 μm, whereas the particle size of the column stationary phase in HPLC is 3 to 5 μm.
- One more difference between HPLC and UPLC is they can analyze the sample ofUPLC very quickly, which is about 3-10 minutes which can reduce the cost of operation; while HPLC can take about 10-20 minutes to separate the analytes.
- UPLC analysis required less solvent and increases sample throughput than HPLC, while high-performance liquid chromatography analysis required more solvent or mobile phase and less sample throughput.
- UPLC is more selective and sensitive than HPLC, while HPLC is less selective and sensitive than UPLC.
- UPLC has high resolving power compared to HPLC, while HPLC has less resolving power compared to UPLC.
- Because the packing material particles are less than 2 μm, UPLC columns are often non-regenerable and have a narrow use, whereas HPLC columns have more use.
- Sample injection necessitates high precision in UPLC, while HPLC has lower compared to UPLC.
- A small injection volume is required for UPLC (generally 2 microlitres), while a large injection volume is required for HPLC (generally 5 to 20 microlitres).
- UPLC system works with higher pressures and requires more efficient pumps than HPLC systems.
- UPLC achieves a higher theoretical plate count than HPLC, while HPLC achieves a lower plate count.
- Higher back pressure shortens column life in UPLC, while lower back pressure increases column life in conventional HPLC.
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HPLC
