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  Section: Plant Lab Protocols
 
 
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Methodology for Phenolics

 
     
 
Chlorogenic acid
 
Phenolic compounds are widely distributed in plants. They contribute to the color and flavor of plant parts. The presence of phenolic compounds in oilseeds and grains pose some nutritional problems. Among the phenolic compounds, chlorogenic acid, found in the range 2-4g/100g of defatted sunflower meal is a matter of immediate concern. Breeding for low chlorogenic acid containing sunflower and/or technology to remove chlorogenic acid from the defatted meal are, today, the major areas of sunflower research. A rapid method to estimate chlorogenic acid is described.
 
 



Principle

Chlorogenic acid is extracted with alcohol, dried and dissolved in acetone. It is reacted with titanium ion to form a colored complex which is measured at 450nm.
 
 
Materials
Titanium Reagent: 20% TiCl4 in con. HCl
Standard 25-200mg/mL Chlorogenic Acid in Acetone
Acetone
80% Ethanol
2.5NHCl
 
 
Procedure
1.
Reflux twice a known quantity of defatted sunflower meal in 80% ethanol (adjusted to pH 4.0 with 2.5 N HCl) for 30 min (125mL to 1g meal).
2.
Discard the precipitate and collect 250mL of the extract.
3.
Remove 0.5mL samples and dry in a vacuum oven at 50°C and 700mm pressure for 2h.
4.
Dissolve dried extract in 4.75ml of acetone.
5.
Add 0.25ml of TiCl4.
6.
Read the color at 450nm against a reagent blank (acetone plus TiCl4).
7.
Similarly treat the standards with TiCl4 and read the color intensity.
8.
Draw a standard curve and find out the chlorogenic acid content in the sample.
 
 
Calculation
Express chlorogenic acid content as g per 100g sample.
 
 



Notes

1. If titanium chloride is not available, this estimation can be performed with Folin-Ciocalteau reagent. Dissolve the dried extract in 5mL ethanol and proceed for Folin-Ciocalteau method (Refer phenol estimation). Similarly, prepare the standard in 80% ethanol and develop the color with Folin-Ciocalteau reagent. In this method, there is a possibility of interference of tyrosine and phenylalanine.
2. The titanium chloride method can be followed for other phenols, too. But the wavelength of maximum absorbance differs. Total phenols can be measured at 410nm, catechol and catechin at 430nm.
 
 



References

1. Michael Eskin, M A, Hochn, E and Frenkel, C (1978) J Agric Food Chem 26 973.
 
 
     
 
 
     




     
 
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