Praktikum Kimia Farmasi Analisis - KCKT (2)

Work From Lab

28 Nov 202104:21

Summary

TLDRThis video explains the process of High-Performance Liquid Chromatography (HPLC) used to measure and separate compounds based on their polarity. The technique involves injecting a sample into a mixture of water and methanol, pumped through a stationary phase column. The compounds are separated and analyzed using a computer system to record retention times. Key elements discussed include the use of Luna 18 columns and the importance of precise solvent ratios and flow rates. The video serves as an introduction to the HPLC measurement process and its application in chemical analysis.

Takeaways

😀 High-Performance Liquid Chromatography (HPLC) separates compounds based on their polarity using a mobile phase and a stationary phase.
😀 The mobile phase in the HPLC setup is typically a solvent mixture, such as water and methanol, which helps move the sample through the system.
😀 A pump regulates the flow of the mobile phase, and in this case, it operates at a flow rate of 1 mL per minute.
😀 The stationary phase, contained in the column, interacts with the sample to cause the separation of different components based on their polarity.
😀 The HPLC column used in this tutorial is a Luna 18 column, sized at 250 x 4.6 mm, which is designed for efficient separation of compounds.
😀 A syringe is used to inject the sample into the HPLC system, initiating the separation process as it moves through the column.
😀 After the sample passes through the column, the separated compounds are detected and analyzed using a computer system.
😀 The data collected by the computer includes retention times, which help identify the separated compounds based on how long they take to pass through the column.
😀 The HPLC method provides precise and reliable data for chemical analysis, helping identify compounds in complex mixtures.
😀 The video demonstrates a step-by-step process of setting up and running an HPLC analysis, from preparing the mobile phase to interpreting the results.
😀 Understanding HPLC components such as the pump, column, and mobile phase is essential for successful analysis and accurate results.

Q & A

What is High-Performance Liquid Chromatography (HPLC)?
-HPLC is a technique used to separate compounds in a sample based on their polarity, aided by high pressure. It allows for precise analysis of various substances in a sample.
What role does the mobile phase play in HPLC?
-The mobile phase is the solvent or mixture that moves through the system, carrying the sample. Common mixtures include water and methanol, and its flow rate is controlled by a pump to ensure accurate separation.
Why is pressure used in HPLC?
-High pressure is applied in HPLC to accelerate the separation process, making it faster and more efficient.
What is the function of the stationary phase in HPLC?
-The stationary phase is a column through which the mobile phase and sample pass. It interacts with the compounds in the sample to separate them based on their chemical properties.
What type of column is used in this HPLC setup?
-The column used in this setup is a Luna 18 column, with dimensions of 250 mm in length and 4.6 mm in diameter.
How is the sample injected into the HPLC system?
-The sample is injected into the system through an injector, where it enters the column to begin the separation process.
What happens to the sample once it enters the column?
-Once the sample enters the column, its compounds are separated based on their interaction with the stationary phase, and the results are analyzed as they elute from the column.
What is a chromatogram, and how is it used in HPLC analysis?
-A chromatogram is a graph that shows the results of HPLC analysis, displaying the retention times of different compounds in the sample. The peaks in the chromatogram represent different compounds, and their area correlates with the quantity of the compound.
What is meant by retention time in HPLC?
-Retention time refers to the time it takes for a particular compound to travel through the column and be detected. It helps identify different compounds in the sample.
How is the data from HPLC analyzed?
-The data collected by the HPLC system is processed by specialized software, which generates a chromatogram that provides insights into the identity and quantity of the compounds in the sample.