Analysis of Lutein in Spinach using HPLC

Summary

This video demonstrates the process of analyzing rutin levels in spinach samples using Shimadzu HPLC. The procedure includes sample preparation, blending spinach with ethanol, and centrifuging the mixture with potassium hydroxide. After extracting the supernatant, the solvent is evaporated, and the residue is reconstituted for HPLC analysis. The spinach samples are then loaded into an HPLC auto-sampler for analysis, with a calibration curve established using leucine standards. Results show that spinach contains an average of 5 to 8 milligrams of rutin per 100 grams.

Takeaways

• 😀 Sample preparation begins with weighing spinach, cutting off the root, and blending it with an ethanol solvent mixture.
• 😀 The blended spinach is transferred into a centrifuge tube, with potassium hydroxide added before heating at 70°C for 30 minutes.
• 😀 After cooling, sodium chloride and hexane ethyl acetate are added to the centrifuge tube, which is then vortexed and centrifuged.
• 😀 The supernatant is transferred into a flask, and the organic solvent is evaporated using a rotary evaporator.
• 😀 The residue is reconstituted with PhD ethanol solution, transferred to a 10 mL volumetric flask, and filtered through a PTFE filter.
• 😀 The filtered solution is collected in a 1.5 mL amber vial and is now ready for HPLC analysis.
• 😀 Samples are loaded into the Shimadzu HPLC auto sampler rack, with the instrument status and mobile phase being checked.
• 😀 The HPLC analysis involves loading the master file for rutin and running standard and real spinach samples in batches.
• 😀 Peaks from the spinach sample and rutin standard are observed in the chromatogram, with the rutin standard peak at the top.
• 😀 A calibration curve for rutin is created using leucine standards for accurate analysis.
• 😀 Final results show that the average rutin content in 100 grams of spinach ranges from 5 to 8 milligrams.

Q & A

What is the first step in preparing the spinach sample for analysis?

The first step is to measure the weight of each sample and cut off the root of the spinach. The remaining spinach is then cut into small pieces.

What solvent mixture is used during the blending process of spinach?

A per-gallon ethanol solvent mixture is added into the blender cup along with the spinach for blending.

How long and at what speed should the spinach be blended?

The spinach should be blended at 5000 RPM for 20 seconds.

After blending the spinach, what is the next step in the preparation process?

One gram of the blended spinach is transferred into a centrifuge tube, and additional ethanol solvent mixture and potassium hydroxide are added.

Why is the centrifuge tube heated in a water bath, and at what temperature?

The centrifuge tube is heated at 70°C for 30 minutes to facilitate the extraction of compounds from the spinach.

What is added to the centrifuge tube after heating and before centrifuging?

Sodium chloride solution (10 mL) and hexane ethyl acetate (6 mL) are added to the centrifuge tube before it is vortexed and centrifuged.

What happens after the centrifugation step?

The supernatant is transferred into a flask, and the organic solvent is evaporated using a rotary evaporator.

How is the residue reconstituted after evaporation of the organic solvent?

The residue is reconstituted with PhD ethanol solution and transferred into a 10 mL volumetric flask.

What is the final step in the preparation of the sample before analysis?

The solution is filtered with a PTFE filter, and the filtrate is collected in a 1.5 mL amber vial.

What type of equipment is used for the actual analysis of the spinach sample?

The spinach sample is analyzed using an HPLC (High-Performance Liquid Chromatography) system.

What is the purpose of loading a master file and using leucine standards in the HPLC analysis?

The master file for rutin is loaded in the HPLC analysis, and leucine standards are used to establish a calibration curve for quantifying rutin in the spinach samples.

How many spinach samples were selected for analysis, and what did the results show?

Nine samples were selected from 27 batches of spinach, and the results showed an average of 5 to 8 milligrams of rutin in 100 grams of spinach.