High Pressure Liquid Chromatography (UPLC-UV) Laboratory

UPLC and HPLC are both liquid chromatography techniques used for separating the components of a compound or mixture.The primary distinction lies in the particle size packed into the column. Particles of ≤ 4 µm are commonly employed for UPLC, while particles ranging from 3 µm to 5 µm are typically used for classical analytical HPLC. Additionally, UPLC operates at higher pressures (15,000 psi) and allows for lower particle sizes in columns, while HPLC operates at lower pressures (max <6000 psi). This makes it possible to increase throughput, and therefore analysis speed, without compromising chromatographic performance.

Key Advantages of UPLC:

• High Efficiency: UPLC uses smaller particles in columns, resulting in higher efficiency and resolution of peaks.
• Speed: It operates at higher pressures and shorter column lengths, leading to faster separations.
• Sensitivity: UPLC can detect compounds at lower concentrations due to its improved resolution.
• Reduced Solvent Usage: Smaller column dimensions and faster separations lead to reduced solvent consumption.
• Broad Application: UPLC is suitable for a wide range of analytical applications, including pharmaceuticals, environmental analysis, food and beverage testing, and more.

Here are the steps for a UPLC analysis:

1. Sample Injection: The process begins with the injection of a small volume of the liquid sample into the UPLC system. This sample typically contains the compounds you want to separate and analyze.
2. Column Selection: UPLC employs columns with smaller particle sizes and narrower diameters than those used in HPLC. These columns are packed with high-quality stationary phases (usually silica-based) that are designed to interact with the sample components.
3. Mobile Phase: A liquid called the "mobile phase" is used to carry the sample through the column. The composition of the mobile phase can be varied to optimize separation. It typically consists of a solvent or a mixture of solvents.
4. High-Pressure Pump: UPLC systems use high-pressure pumps capable of generating pressures of up to 15,000 psi (pounds per square inch) or more. This high pressure is crucial for forcing the mobile phase through the narrow columns filled with small particles, resulting in faster and more efficient separations.
5. Column Separation: As the sample travels through the column, the individual components interact with the stationary phase. Compounds that interact more strongly with the stationary phase will move through the column more slowly, while those with weaker interactions will move more quickly. This differential interaction leads to separation.
6. Detection: After separation, the individual components of the sample pass through a detector, which can be a UV-Vis spectrophotometer, fluorescence detector, mass spectrometer, or other types of detectors depending on the nature of the analytes. The detector records signals as the separated compounds elute from the column.
7. Data Analysis: The signals obtained from the detector are processed and analyzed by a computer system. The resulting chromatogram provides information about the retention times, peak areas, and peak heights, which can be used to identify and quantify the components in the sample.

Ultra-Performance Liquid Chromatography (UPLC) is a versatile analytical technique widely used in various fields for separating, identifying, and quantifying compounds in complex mixtures. Some of the common applications of UPLC include:

• Pharmaceutical Analysis:
  • Drug formulation analysis.
  • Quantification of active pharmaceutical ingredients (APIs) and impurities.
  • Pharmacokinetic studies.
  • Quality control and batch testing in pharmaceutical manufacturing.
• Environmental Analysis:
  • Detection of environmental contaminants such as pesticides, herbicides, and industrial pollutants.
  • Analysis of water and soil samples for regulatory compliance.
• Food and Beverage Testing:
  • Determination of food additives, preservatives, and flavor compounds.
  • Detection of contaminants and adulterants in food and beverages.
  • Analysis of vitamins and nutrients in dietary supplements.
• Clinical Chemistry:
  • Quantification of biomarkers in blood, urine, and other biological fluids.
  • Drug monitoring in clinical samples.
  • Analysis of metabolites and small molecules in clinical research.
• Life Sciences and Proteomics:
  • Separation and identification of proteins, peptides, and amino acids.
  • Analysis of nucleic acids, including DNA and RNA fragments.
  • Metabolomics studies for understanding metabolic pathways.
• Chemical Research:
  • Characterization of organic compounds in synthetic chemistry.
  • Analysis of natural product extracts and chemical reactions.
• Polymer Analysis:
  • Determination of molecular weight distribution in polymers.
  • Analysis of additives and degradation products in polymer materials.
• Petroleum and Petrochemical Industry:
  • Analysis of hydrocarbons and impurities in crude oil and refined products.
  • Monitoring of process streams in petrochemical plants.
• Biopharmaceuticals:
  • Characterization of biologics, including monoclonal antibodies and proteins.
  • Analysis of gene therapies and cell-based products.
• Material Science:
  • Examination of material properties and composition.
  • Identification of contaminants or additives in materials.
• Academic and Research:
  • UPLC is used in various research disciplines to separate and analyze compounds of interest, contributing to advancements in chemistry, biology, and related fields.