HPLC CHROMATOGRAPH6A -C QUESTION 701.pptx

definitions and components of HPLC

1. HPLC

2. WHAT IS HPLC & USES High-performance liquid chromatography  ( HPLC ), formerly referred to as  high-pressure liquid chromatography , is a technique in  analytical chemistry  used to separate, identify, and quantify specific components in mixtures. The mixtures canoriginatefrom   food ,  chemicals ,  pharmaceuticals ,   biological ,  environmental  and  agriculture , etc., which have been dissolved into liquid solutions . High performance liquid chromatography is a powerful tool in analysis, it yields high performance and high speed compared to traditional columns chromatography because of the forcibly pumped mobile phase. HPLC is a chromatographic technique that can separate a mixture of compounds. A type of liquid chromatography where the sample is forced through a column that is packed with a stationary phase composed of irregularly or spherically shaped particles, a porous monolithic layer , or a porous membrane by a liquid ( mobile phase ) at high pressure

3. HPLC APPLICATIONS Pharmaceutical Tablet dissolution of pharmaceutical dosages. Shelf life determinations of pharmaceutical products. Identification of counterfeit drug products. Pharmaceutical quality control. Forensics Identification of anabolic steroids in serum, urine, sweat and hair. Simultaneous quantification of psychotherapeutic drugs in human plasma. Clinical Therapeutic Drug Monitoring (TDM) for drugs with low therapeutic index Relates Plasma drug level with efficacy/toxicity Bioavailability/Bioequivalent studies

4. FIVE MAJOR COMPONENTS OF AN HPLC MACHINE Solvent reservoir Pump Injector Column Detector Solvent Reservoirs: Function: Store the mobile phase solvents (liquids used to carry the sample through the column). Details: Typically made of glass or inert plastic and often multiple reservoirs are used to allow gradient elution (varying solvent composition during the run ). The mobile phase, or solvent, in HPLC, is usually a mixture of polar and non-polar liquid components whose respective concentrations are varied depending on the composition of the sample. The type and composition of the mobile phase affect the separation of the components . For HPLC we use high-grade solvent. Different solvents are used for different types of HPLC. For normal-phase HPLC, the solvent is usually non-polar, and, in reverse-phase HPLC, the solvent is normally a mixture of water and a polar organic solvent. The most common solvent reservoirs are as simple as glass bottles with tubing connecting them to the pump inlet.

5. Pump: Function: Delivers the mobile phase through the system at a constant and precise flow rate. Details: Types include isocratic (single solvent) and gradient (multiple solvents mixed in varying ratios). High-pressure pumps are essential for consistent flow rates and pressure. The HPLC pump has to have ruggedness and at the same time should be able to provide reproducible flow characteristics run after run. The operational pressure limits have a vast range depending upon analysis requirements. In normal analytical operation the pressure can vary between 2000 – 5000 psi but in applications covered under UHPLC mode operating pressure can be as high as 15000 – 18000 psi. All HPLC systems include at least one pump to force the mobile phase through whose packing is fairly compact. A pump aspirates the mobile phase from the solvent reservoir and forces it through the system’s column and detector. The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatography at a specific flow rate, expressed in milliliters per min (mL/min). Normal flow rates in HPLC are in the 1-to 2-mL/min range.

6. Injector: Function: Introduces the sample into the mobile phase stream for analysis. Details: Manual or automated injectors are used. Auto-samplers allow for high-throughput analysis by handling multiple samples . . An injector for an HPLC system should provide an injection of the liquid sample within the range of 0.1-100 mL of volume with high reproducibility and under high pressure (up to 4000 psi). The injector must also be able to withstand the high pressures of the liquid system . For a manual injector, the knob is manually operated to deliver the sample to the column. The knob is set to LOAD position for sample injection using a syringe, the sample is injected into the sample loop , which is separated from the flow path. The knob is turned to INJECT position and the eluent travels through the loop from the pump and delivers the sample to the column

7. Column Oven: Function: Maintains the column at a constant temperature. Details: Temperature control is critical for reproducibility and can enhance separation efficiency. Consists of a cylindrical polished stainless steel of between 50 and 300 mm long, typically filled with small (usually 1.5 – 5 µm diameter) spherical particles. The particles are in most cases porous silica, with an individual pore as a cylinder of typically about 10 nm. The inside of each pore is covered with the stationary phase e.g C8, C18 groups that are attached to the silica particle. The types of columns are: Guard columns, Column temperature control.

8. Detector: Function: Identifies and quantifies the separated components as they elute from the column . Solutes are detected as soon as they are eluted, and quantitated with sensitive and specific or universal detectors. The detector can detect the individual molecules that elute from the column and convert the data into an electrical signal. It provides an output to a recorder or computer that results in the liquid chromatogram. Detector is selected based on the analyte or the sample under detection Details : Common types include UV-Vis absorbance detectors, fluorescence detectors, and mass spectrometers.

9. D escribe steps involved in analysis of a drug from a biological sample such as blood or plasma 1. Sample Collection: Blood or plasma samples are collected using sterile techniques to prevent contamination. Blood is typically drawn from a vein and then processed to obtain plasma or serum by centrifugation. 2. Sample Preparation: The biological sample is prepared to isolate the drug of interest and remove interfering substances. This often involves protein precipitation, liquid-liquid extraction, or solid-phase extraction (SPE). Protein Precipitation: Adding an organic solvent (e.g., acetonitrile) to precipitate proteins, followed by centrifugation to separate the supernatant. Liquid-Liquid Extraction: Mixing the sample with an organic solvent to transfer the drug from the aqueous phase to the organic phase, then separating the layers. Solid-Phase Extraction: Using SPE cartridges to selectively adsorb the drug, then eluting it with a suitable solvent.

10. 3. Sample Filtration: The prepared sample is filtered to remove any particulate matter. This is typically done using syringe filters (0.2 µm or 0.45 µm) to ensure that the sample does not clog the HPLC column. 4. Injection : The prepared sample is introduced into the HPLC system. This can be done manually or using an auto-sampler, which allows for precise and reproducible sample injection. 5. Chromatographic Separation: The sample is carried by the mobile phase through the HPLC column where separation occurs. The choice of column and mobile phase depends on the drug's chemical properties. Conditions such as flow rate, temperature, and gradient elution (if used) are optimized to achieve the best separation.

11. 6.Detection : The separated drug and any metabolites or impurities are detected as they elute from the column. The choice of detector (e.g., UV-Vis, fluorescence, mass spectrometry) depends on the drug's properties and required sensitivity. The detector sends a signal proportional to the concentration of the analyte . 7. Data Analysis: The detector signals are processed to identify and quantify the drug. Software integrates the peaks corresponding to the drug and compares them to a calibration curve generated from known standards to determine concentration.

12. 8. Quantification and Validation: Quantify the drug concentration in the sample and validate the results. Calibration curves, internal standards, and quality control samples are used to ensure accuracy and precision. Validation involves assessing parameters such as linearity, accuracy, precision, sensitivity, and specificity. Data Reporting: The final results are compiled and reported. A detailed report is prepared, including chromatograms, quantitative data, and any relevant observations or deviations.