Praktikum Instrumen: Kromatografi Cair Kinerja Tinggi

Summary

This video provides a comprehensive introduction to High-Performance Liquid Chromatography (HPLC), a key technique used in pharmaceutical analysis. It covers the preparation steps for both the sample and mobile phase, including filtration and sonication, and the setup of the HPLC system. The process includes configuring the pump, mobile phase composition, and UV detector for caffeine analysis. The video walks through injecting the sample, ensuring no air bubbles, and observing the resulting chromatogram. The analysis concludes with the interpretation of the chromatogram, showing retention time and peak area, essential for determining sample concentration and identification.

Takeaways

• 😀 HPLC (High-Performance Liquid Chromatography) is an essential method for pharmaceutical analysis, used to determine the concentration and purity of compounds.
• 😀 Sample preparation is crucial before HPLC analysis; it involves weighing the sample and dissolving it into a mobile phase.
• 😀 The mobile phase must be filtered using PTFE or nylon filters with a pore size of 0.45 µm before injection into the HPLC system.
• 😀 Sonication is used to remove air bubbles from the mobile phase, ensuring it is free from impurities before injection.
• 😀 The HPLC system needs to be properly conditioned before use, including setting the pump flow rate and adjusting the solvent composition.
• 😀 In this demonstration, the mobile phase consists of 30% methanol and 70% glacial acetic acid, with a flow rate of 1.5 mL/min.
• 😀 The UV detector is set to detect caffeine at a wavelength of 254 nm to ensure accurate analysis of the sample.
• 😀 Before injecting the sample, the system needs to be properly configured, including selecting the sample ID and the injection volume.
• 😀 Sample injection must be done carefully to avoid introducing air bubbles, which could affect the accuracy of the results.
• 😀 The resulting chromatogram shows the caffeine peak, with a retention time of 3.5 minutes, which is used for identifying and quantifying the substance.
• 😀 The chromatogram provides critical data, such as peak height and area, which are essential for determining the concentration of caffeine in the sample.

Q & A

What is High-Performance Liquid Chromatography (HPLC) used for?

HPLC is used for determining the concentration, identifying compounds, and measuring the purity of mixtures, especially in pharmaceutical analysis.

What are the key steps in preparing the sample for HPLC analysis?

The key steps include weighing the sample, dissolving it in the mobile phase, filtering the mobile phase, and sonicating to remove air bubbles before injecting the sample into the HPLC system.

Why is it necessary to filter the mobile phase before use?

The mobile phase must be filtered to remove any impurities or particles that could interfere with the analysis and clog the HPLC system.

What is the purpose of using a sonicator in the HPLC preparation process?

A sonicator is used to remove air bubbles from the mobile phase, ensuring accurate flow and preventing issues during the analysis.

What are the flow rate and composition settings for the mobile phase in this experiment?

The flow rate of the mobile phase is set to 1.5 mL per minute. The composition is 30% methanol and 70% glacial acetic acid.

What wavelength is the UV detector set to for caffeine analysis?

The UV detector is set to a wavelength of 254 nm for detecting caffeine.

What is the role of the UV detector in the HPLC process?

The UV detector identifies and measures the absorption of the sample as it passes through, helping to detect the compounds present, such as caffeine in this case.

What steps should be taken before injecting the sample into the HPLC system?

Before injection, label the sample, prepare the syringe, and ensure no air bubbles are present in the syringe. The injection volume should be set to 20 µL.

What does the chromatogram show in the caffeine analysis?

The chromatogram shows a peak for caffeine with a retention time of approximately 3.47 minutes. The peak area is about 359,000 and the peak height is around 400 units.

How can the chromatogram data be used?

The chromatogram data, including retention time and peak area, can be used to calculate the concentration and purity of caffeine in the sample.