ANALISIS KLORAMFENIKOL MENGGUNAKAN INSTRUMEN KROMATOGRAFI CAIR KINERJA TINGGI (KCKT)

Summary

In this video, viewers are guided through a practical chromatography experiment using High-Performance Liquid Chromatography (HPLC) to analyze chloramphenicol. The process involves preparing standard and sample solutions, including measuring and dissolving chloramphenicol, followed by filtration and transfer to vials. The HPLC system is set up with a fixed flow rate, and after 30 minutes of system stabilization, the samples are analyzed. The resulting chromatograms provide both qualitative and quantitative data, such as retention time and area under the curve (AUC), which help determine the presence and concentration of chloramphenicol in capsules. The experiment emphasizes factors influencing chromatography results and the importance of accurate analysis.

Takeaways

• 😀 The experiment focuses on the analysis of chloramphenicol using High Performance Liquid Chromatography (HPLC).
• 😀 HPLC works on the principles of absorption and partition for the separation and purity testing of compounds.
• 😀 The process involves preparing a stock solution of chloramphenicol by dissolving it in methanol to achieve a concentration of 1000 ppm.
• 😀 Different concentrations of the chloramphenicol solution (5 ppm, 10 ppm, 15 ppm, 20 ppm, and 25 ppm) are created for analysis.
• 😀 A sample is extracted from chloramphenicol capsules, homogenized, and dissolved in methanol to prepare a 1% solution.
• 😀 The sample and stock dilutions are filtered and transferred into HPLC vials for analysis.
• 😀 The HPLC system is calibrated by setting the flow rate at 1 mL per minute and the mobile phase at a 70:30 methanol to water ratio.
• 😀 The system is allowed to stabilize for 30 minutes before samples are injected into the HPLC for analysis.
• 😀 Chromatograms are generated, showing both qualitative and quantitative data, including retention time and area under the curve (AUC).
• 😀 The analysis allows for the determination of the presence and concentration of chloramphenicol in capsules, with data stored for reference.
• 😀 Key factors affecting chromatography include retention time, capacity factor, column selectivity, mobile phase velocity, and solution characteristics.

Q & A

What is the main principle behind High Performance Liquid Chromatography (HPLC)?

The main principle behind HPLC is absorption and partitioning, where the sample is separated based on these characteristics to analyze its composition or purity.

How is the standard solution of chloramphenicol prepared in the experiment?

A 1000 ppm stock solution of chloramphenicol is prepared by dissolving 0.01 grams of chloramphenicol into 10 ml of methanol. Then, this solution is further diluted to create concentrations of 5 ppm, 10 ppm, 15 ppm, 20 ppm, and 25 ppm.

Why is methanol used in preparing the chloramphenicol solutions?

Methanol is used as the solvent because it is a common, effective solvent for dissolving many organic compounds like chloramphenicol, allowing for accurate and consistent preparation of the solutions.

What steps are involved in preparing the sample for analysis from the chloramphenicol capsules?

To prepare the sample, 10 capsules are weighed and ground to achieve homogeneity. A 0.1g sample is then dissolved in methanol to create a 1% concentration and transferred into a 10 ml volumetric flask.

How does the HPLC system perform the analysis of the chloramphenicol sample?

The HPLC system is set up with a constant flow rate of 1 mL/min, and the mobile phase (70% methanol, 30% aquabides) is passed through the system. The sample is injected into the auto sampler, where the analysis occurs, and the results are recorded as a chromatogram.

What is the significance of the retention time in HPLC analysis?

The retention time is the time it takes for a compound to travel through the chromatography column and be detected. It is used to identify compounds based on how long they take to elute from the column.

What does the area under the curve (AUC) in a chromatogram represent?

The area under the curve (AUC) represents the quantity of the compound in the sample, and it is used for quantitative analysis to determine the concentration of chloramphenicol in the sample.

What role does the mobile phase play in HPLC?

The mobile phase is the solvent mixture that carries the sample through the column. In this experiment, it consists of 70% methanol and 30% aquabides, which helps in the separation of compounds based on their interaction with the stationary phase.

What factors can affect the outcome of HPLC chromatography?

Factors such as retention time, column selectivity, flow rate, and molecular movement in the mobile phase can all impact the results of the chromatography analysis.

What does the software connected to the HPLC system provide during analysis?

The software connected to the HPLC system provides real-time data, including chromatograms, retention times, and AUC values. It helps in both qualitative and quantitative analysis of the samples.