The purpose of the document is to establish a general and uniform method for the determination of Fiehe’s test and HMF content in honey.
2.0 SCOPE
This standard testing procedure is suitable for the determination of Fiehe’s test and HMF content in the honey sample.
3.0 RESPONSIBILITY
The expert and trained chemist are responsible for following this procedure. The technical manager is responsible for implementing this procedure.
4.0 REQUIREMENTS
4.1 Apparatus
4.1.1 Spectrophotometer
4.1.2 Mortar Pestle
4.1.3 Porcelain dish
4.1.4 Water bath
4.2 Glassware
4.2.1 Measuring Cylinder
4.2.2 Watch glass
4.3 Reagent for Fiehe Test
4.3.1 Resorcinol solution: Dissolve 1 gm of resublimed Resorcinol in 100 ml conc. HCl
4.3.2 Ether – Sulphuric ether
4.3.3 Hydrochloric Acid (Sp.gr.1.18 to 1.19)
5.0 PROCEDURE FOR FIEHE’S TEST
5.1 Weigh 5 g of honey sample into a mortar, and using a pestle mix it with 10 ml ether.
5.2 Decant the ether extract into a porcelain dish(or watch glass)
5.3 Repeat the extraction twice in the same manner
5.4 Collect the extracts in the same dish.
5.5 Allow the extracts to evaporate to dryness at room temp and add drop of freshly prepared Resorcinol solution.
5.6 The production of cherry red color appearing instantly indicates a positive reaction (+ve).
5.7 Faint pink color disappearing after a short time or yellow to salmon pink colors indicates a negative reaction (-ve).
5.8 RESULT
5.5.1 Appearance of cherry red color appearing instantly indicates a positive reaction.
5.5.2 Pink color disappearing after some time or yellow to salmon pink color indicates a negative reaction.
(When Fiehe’s test is positive then go for HMF content).
DETERMINATION OF HYDROXYMETHYL FURFURAL (HMF) CONTENT
5.9 Reagents for HMF Content
5.6.1 Barbituric Acid solution: (weighs 500 mg and makes up 100ml).
5.6.2 P-Toluidine Solution: (Weigh 10.0 g and dissolve in 50 ml isopropanol and add 10 ml glacial acetic acid and makeup to volume with isopropanol (keep in a dark place) in 100 ml volumetric.
5.6.3 Distilled water (oxygen-free): Nitrogen gas is passed through boiling distilled water and then cooled.
5.10 PROCEDURE FOR HMF CONTENT
5.7.1 Weigh 10.0g of honey sample
5.7.2 Dissolve without heating in 20 ml of oxygen-free water and make the final volume 50 ml in a volumetric flask.
5.7.3 Take 2.0 ml of honey solution is pipetted into each of the two test tubes and 5.0 ml p-toluidine is added to each.
5.7.4 Take 1 ml of water in one test tube for the blank and 1 ml of barbituric acid solution in the second test tube for the sample.
5.7.5 Mix both samples.
5.7.6 The addition of the reagents should be done without pause & should be finished in about 1 to 2 min.
5.7.7 The extinction of the sample is read against blank at 550 nm using a 1 cm cell, immediately after the maximum value is reached.
5.11 CALCULATION
Mg/100 g HMF = (Absorbance/ Thickness of layer ) X 19.2
6.0 REVISION/CHANGE HISTORY: NA
7.0 ATTACHMENTS
· Appendix-1: FLOW CHART FOR FIEHE’S TEST
· Appendix-2: FLOW CHART FOR HMF CONTENT
8.0 REFERENCES
· Annex F of IS 4941:1994;RA 2008; Amendment -1,2011
END OF THE DOCUMENT
You may like to read these links:
1. List of All SOPs and Documents for the Chemistry Laboratory
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3. List of All SOPs and Documents For Quality Assurance Dept.
4. List of SOPs and Documents Required to Achieve NABL Accreditation
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