Spectrophotometric and colorimetric

Method for the Determination of

total polyphenol content in olive oil

Friday 03/4/2020
Rapid Ultraviolet Spectrophotometric and Liquid Chromatographic
Methods for the Determination of Natamycin in Lactoserum Matrix
JOURNAL OF AOAC INTERNATIONAL VOL. 83, NO. 4, 2000
Overview:

Olive oil is a source of antioxidants and poses positive effects on human

health, in particular to exert some of the cardio protective properties
(Carrióna et al., 2016). A wide range of phenolic compounds, belonging to
many classes, including phenolic acids, alcohols, flavonoids…
Phenolics are important minor components in olive oil which, the powerful
antioxidant effect, contribute to shelf life stability and sensory properties of
olive oil.
Detrimental effects of factors such as oxygen, light, heat and time on phenol
compounds of olive oil have been reported in a number of studies. After a short
term or long term storage, significant decreases in total polyphenols were
reported for mono-cultivar and commercial olive oils
Regulations and Norms:
Commission Implementing Regulation (EU) No 29/2012 of 13 January 2012 on
marketing standards for olive oil.

of the legislation Regulation (EEC) 2568/91 in conjunction with Regulation (EC)

1019/2002 lay down the relevant market access requirements for olive oil.

Polyphenol content classification

0.05-0.1/kg Low
0.1-0.15g/kg Medium
>0.15g/kg High
Common methods of analysis:

1- UV VIS spectrophotometer- Folincieau calteau method

2- HPLC high performance liquid chromatography

1- Chemicals:
 Gallic acid standard high purity
 Folin-ciocalteau reagent
 Na2CO3 sodium carbonate anhydrous
 Hexane
 Methanol/water solution (70/30; v/v)

2- Equipment's:
Spectrophotometer UV vis with quartz cuvette
50 mL conical centrifuge tubes
Balance
Pipettes
Vortex
Centrifuge
Extraction and Analysis:
Extraction of Phenolic Compounds
Prior analysis samples were left to thaw at room temperature. Meanwhile, an
internal standard solution was prepared by dissolving 15 mg of syringic acid in 10
mL of 60:40 v/v methanol–water. Then, 1 mL from this solution was diluted in a
25 mL volumetric flask with 60:40 v/v methanol–water.
The phenolic compounds in the olive oil were extracted using a modification of
the procedure described by Montedoro et al.

An amount of 3 g of olive oil was shaken manually with 2 mL of n-hexane for 15

s. Then, a volume of 1.75 mL of 60:40 (v/v) methanol–water mixture was added
together with 0.25 mL of the internal standard solution and shaken for 2 min to
undergo the first extraction. For the second extraction, 2 mL of methanol/water
(60/40) was added and shaken for 2 min. The extracts from both extractions
were combined and placed in the dark at −20°C for further determinations.
Preparation of Methanol/water solution:
1. Using a graduated burette measure 30 mL of water (for HPLC) and 70 mL of methanol
2. Mix in a 100 mL graduated flask.

Liquid-liquid extraction:
1. Weight 5 g of olive oil in 50 mL conical centrifuge tube, write the exact weight that has
been measured of each sample
2. Add 2.5 mL of n-hexane and mix by shaking
3. Add 5 mL of mix methanol/water (70:30) and shake by vortex for 5 mins
4. Centrifuge at 3000 rpm for 10 mins and collect the lower phase as much as we can
without taking from the supernatant using pipette or syringe
5. Repeat steps ( 3 and 4 ) and combine the two extracts
6. Each sample is repeated three times, triplicate n=3
7. Cover all of the tubes in aluminium foil
5g oil sample+2.5ml hexane Shake for 5min vortex
Extraction with
5 ml water/methanol(3/7)

Centrifuge for 10min at 3000rpm

Repeat the extraction procedure

Lower phase transferred to a flask for derivatization

step
Spectrophotometric Estimation of Total Phenols
The total phenols (TP) content of the oil extracts was determined according to
the Folin-Ciocalteu spectrophotometric method. 200 μL of the sample (with
prior 1:50 dilution with water) was, in this order, mixed with 1.58 mL of water,
0.3 mL of 20% (w/v) Na2CO3 aqueous solution, and 0.2 mL of F–C reagent.
Then, the resulting solution was allowed to stand for 30 min. The reaction
product was spectrophotometrically monitored at 740 nm by a UV/Vis
spectrophotometer.

A nine level calibration curve was prepared using gallic acid as a commercial
standard (r2 = 0.998), and the results were expressed as mg gallic acid
equivalent (GAE)/Kg of oil.
Calibration curve: absorbance vs concentration of working solutions
Linear equation with good correlation factor > 0.995
I. Procedure:
a) Folin-Ciocalteu assay:
1. Take the phenolic extract
2. For blank: in a vial add 200 µL of methanol/water mix then 200 µL of Folin-
Ciocalteu reagent and mix for 2 min then add 1.6 mL of sodium carbonate solution
3. For Gallic acid standards (calibration curve): in vials add 200 µL of Gallic acid
standards and then 200 µL of Folin-Ciocalteu reagent and mix for 2 min then add
1.6 mL of sodium carbonate
4. For samples: in vials add 200 µL of diluted extract then 200 µL of Folin-Ciocalteu
reagent and mix for 2 min then add 1.6 mL of sodium carbonate solution
5. Shake by vortex for 15 min
6. Allow reaction to reach stability by incubation for 90 min at room temperature
7. Vortex again before measurement
8. Measure absorbance at 740 nm using blank as reference
 Calibration curve Gallic acid
a) Preparation of Gallic Acid standards:
1. Prepare the solution at 1 mg/mL or 1000 ppm = 1000 mg/L:
a. Add 0.1 mg of Gallic Acid to a 100 mL graduated flask
b. Fill with methanol/water mix
c. Label and store at 4 degrees up to 2 weeks
2. Prepare working solution of 200 mg/L freshly:
a. Add 20 mL of 1000 ppm Gallic acid solution to a 100 mL graduated flask
b. Fill with methanol/water mix
3. Prepare 1 mL each of serial dilutions 0 mg/L for blank, 1, 5, 10, 25, 50, 100
mg/L in eppendorf tubes:
a. For 1 mg/L: add 5 µL of fresh working solution and 995 µL of
methanol/water mix
b. For 5 mg/L: add 25 µL of fresh working solution and 975 µL of
methanol/water mix
 Calibration curve Gallic acid
 The working solution should be prepared freshly
 The standards should be prepared freshly before the analysis
 On an Excel sheet, calculate the concentrations in mg/g and draw the calibration
curve using this concentration, example:
 for the 1 mg/L standard we calculate this way: 1 mg1000 mL x 0.2 mL and so
x=(1*0.2)/1000=0.0002 mg/g
Performance analytical method:

Analysis by UV VIS:
• Calibration curve linear withR2>0.992

 Extraction method: Recovery test

• The recovery is determined by Gallic acid internal standard

• Determine the concentration according calibration curve
𝑐𝑜𝑛𝑐 𝑜𝑏𝑡𝑎𝑖𝑛𝑒𝑑
• Calculate the recovery percentage = ∗ 100
𝑐𝑜𝑛𝑐 𝑠𝑝𝑖𝑘𝑒𝑑
Analysis of unknown samples:
 Extraction method
 Derivatization with the reagent Folin
 Analysis by visible Spectroscopy at 740 nm
 Determination for the concentration in extract according Gallic acid internal
standard
 Determination of the total polyphenol content equivalent Gallic acid in olive oil
sample As:

 TP(GAE) g/Kg=(Abs 740 * DF * 10)/(slope*0.2 * W)

DF = dilution factor = 10, slope for Gallic acid calibration curve
W = mass of the sample = the exact mass in g
 The total phenolic content is expressed as mg of Gallic Acid Equivalents
(GAE)/kg of oil.
Principle of UV vis spectrophotometer;

𝐴 = 𝑒𝑝𝑠𝑖𝑙𝑜𝑛 ∗ 𝐶 ∗ 𝑙

Select one wavelength

Absorbance value
Deuterium lamp for UV range
Tungsten lamp for visible range
 Absorbance
1
Concentration en mg/g Absorbance y = 46.394x + 0.002
0.9
R² = 0.9984
0.0002 0.002 0.8

0.001 0.047 0.7

0.002 0.084 0.6

0.005 0.262 0.5 Absorbance

Linear (Absorbance)
0.01 0.464 0.4

0.3

0.2

0.1

0
0 0.005 0.01 0.015 0.02 0.025

SLOPE=46.394