Analysis of Lutein in Spinach using HPLC

Summary

This video provides a detailed overview of the process for analyzing rutin in spinach samples using Shimadzu HPLC. The sample preparation involves blending spinach, adding ethanol solvent, and heating with potassium hydroxide, followed by centrifugation and evaporation of the organic solvent. The final sample is filtered and analyzed via HPLC. The procedure includes creating a calibration curve with rutin standards and running both standard and real spinach samples. Results show that spinach contains 5 to 8 milligrams of lutein per 100 grams of sample. The video highlights precision and excellence in scientific analysis using Shimadzu technology.

Takeaways

• 😀 Sample preparation involves measuring the weight of spinach, cutting off the root, and blending the remaining spinach with a solvent mixture.
• 😀 The spinach sample is blended at 5000 RPM for 20 seconds before being transferred to a centrifuge tube for further processing.
• 😀 A mixture of ethanol solvent and potassium hydroxide is added to the sample, which is then heated at 70°C for 30 minutes.
• 😀 After cooling, sodium chloride and hexane ethyl acetate are added, followed by vortexing and centrifugation to separate the components.
• 😀 The supernatant is transferred into a flask and the organic solvent is removed using a rotary evaporator.
• 😀 The residue is reconstituted with BHT ethanol solution and transferred into a 10 ml volumetric flask for further processing.
• 😀 The solution is filtered through a PTFE filter, and the filtrate is collected in an amber vial, preparing the sample for HPLC analysis.
• 😀 The HPLC process starts with opening the auto sampler rack and loading the samples, followed by checking the status of the instrument and mobile phase connections.
• 😀 In HPLC analysis, a master file for routine is loaded, and both standard and real spinach samples are processed in batches for analysis.
• 😀 The baseline is established, with peaks indicating the routine standard and spinach sample, creating a calibration curve for accurate measurement.
• 😀 Nine spinach samples, selected from 27 batches, show an average of 5 to 8 milligrams of lutein per 100 grams of spinach, as per the results from the analysis.

Q & A

What is the main topic of the video?

The main topic of the video is the analysis of rutin in spinach samples using Shimadzu HPLC (High-Performance Liquid Chromatography).

How is the spinach sample prepared for HPLC analysis?

The spinach sample is prepared by weighing, cutting, blending with ethanol, and then centrifuging. The sample is further treated with potassium hydroxide and heated, followed by the addition of sodium chloride and hexane ethyl acetic acid before being filtered and reconstituted with BHT ethanol solution.

What solvent mixture is used during the sample preparation?

A per gallon ethanol solvent mixture is used during the sample preparation.

What is the purpose of using a rotary evaporator in the sample preparation process?

The rotary evaporator is used to remove the organic solvent from the sample, leaving behind the residue for further analysis.

What equipment is used to filter the final solution?

A PTFE (Polytetrafluoroethylene) filter is used to filter the final solution before transferring it into a 1.5 ml amber vial.

What is the next step after preparing the sample?

After preparing the sample, it is transferred into the auto-sampler of the HPLC system for analysis.

What is the role of the HPLC in this analysis?

HPLC is used to separate and quantify rutin in the spinach samples by analyzing the peaks in the chromatogram.

How is the calibration curve for rutin established?

The calibration curve is established by using rutin standards, which are analyzed in the HPLC to compare and measure rutin in the spinach samples.

How many spinach batches were tested in the analysis?

A total of 27 batches of spinach were analyzed, from which 9 samples were selected for testing.

What is the average amount of lutein found in the spinach samples?

The final results showed that the spinach samples contained an average of 5 to 8 milligrams of lutein per 100 grams.