Professional Documents
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of Analytical Chemistry,
Technická 5, 166 28 Praha 6
QUANTIFICATION
D) UV Detection - LC system Dionex. Software
Chromeleon.
SAMPLE PREPARATION
1. Hot water extraction:
50 g sample was mixed with 400 ml of water boiled in a round
bottom flask under reflux for 1 hour. Samples were cooled to
room temperature; aqueous layer was separated, centrifuged,
filtered and analysed by HPLC.
2. Methanolic extraction:
Needles about 2.7 g were cut with a pair of scissors, then
extracted three times in ultrasonic bath for 45 min with 8 ml of
80% methanol in water. Combined extracts were collected and
the volume was adjusted to 25 ml. The supernatant was
centrifuged, filtered and analysed by HPLC.
ANALYSED COMPOUNDS :
OH Me O
COOH COOH
OH
H
HO O
OMe H
OH OH OH OH
OH
d
FERULIC ACID p-COUMARIC ACID CATECHIN p-HYDROXY-
ACETOPHENONE
COOH COOH COOH COOH
OH OMe
OH OH OH
AQUEOUS METHANOLIC
PHENOLIC COMPOUNDS EXTRACT EXTRACT
(A,B) (A,B)
p -Hydroxyacetophenone + +
Catechin + +
p -Hydroxybenzoic acid - +
Vanillic acid - -
3,4-Dihydroxybenzoic acid - -
Benzoic acid - -
Ferulic acid - -
p -Coumaric acid + +
(+) IDENTIFIED COMPOUNDS
Phenolic compound identification (A,B,C)
(A)Solvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 min
Column Separon SGX C-18, 7µm, 3 x 150 mm
(B) Phosphoric acid 10mM/Methanol (70:30) Isocratic elution
Column Separon SGX C-18, 7µm, 3 x 150 mm
(C) Solvent A (Water) / Solvent B (Acetonitrile)
Linear Gradient from 5 to 100%B in 40 min
ANALYSIS BY
AQUEOUS METHANOLIC
SPECTROMETRIC MASS (C)
PHENOLIC COMPOUNDS EXTRACT EXTRACT
AQUEOUS METHANOLIC
(A,B) (A,B)
EXTRACT EXTRACT
p -Hydroxyacetophenone + + + +
Catechin + + + +
p -Hydroxybenzoic acid - + + +
Vanillic acid - - + +
3,4-Dihydroxybenzoic acid - - + +
Benzoic acid - - + +
Ferulic acid - - + +
p -Coumaric acid + + + -
Chromatogram
of methanolic
extract
SAMPLE PREPARATION
3. Acid hydrolysis:
Aliquots of water and methanolic extraction were filtered,
then mixed with HCl 6N. Final solution had concentration
of 1.7N. Solution was heated at 70oC at temperature bath
for 2 hours. Then solutions were cooled, centrifuged,
filtered and analysed by HPLC.
Phenolic compound identification (A,B)
(A)Solvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 min
Column Separon SGX C-18, 7µm, 3 x 150 mm
(B) Phosphoric acid 10mM/Methanol (70:30) Isocratic elution
Column Separon SGX C-18, 7µm, 3 x 150 mm
AQUEOUS METHANOLIC
PHENOLIC COMPOUNDS EXTRACT EXTRACT
(A,B) (A,B)
p -Hydroxyacetophenone + +
Catechin - -
p -Hydroxybenzoic acid - +
Vanillic acid - -
3,4-Dihydroxybenzoic acid - -
Benzoic acid - -
Ferulic acid - +
p -Coumaric acid - +
Phenolic compound identification (A,B,C)
(A)Solvent A (Trichlorocetic ac.0.1%) Solvent B (Methanol)Linear gradient from 0% B to 100% B in 25 min
Column Separon SGX C-18, 7µm, 3 x 150 mm
(B) Phosphoric acid 10mM/Methanol (70:30) Isocratic elution
Column Separon SGX C-18, 7µm, 3 x 150 mm
(C) Solvent A (Water) / Solvent B (Acetonitrile)
Linear Gradient from 5 to 100%B in 40 min
ANALYSIS BY
AQUEOUS METHANOLIC
SPECTROMETRIC MASS (C)
PHENOLIC COMPOUNDS EXTRACT EXTRACT
AQUEOUS METHANOLIC
(A,B) (A,B)
EXTRACT EXTRACT
p -Hydroxyacetophenone + + + +
Catechin - - + +
p -Hydroxybenzoic acid - + + +
Vanillic acid - - + +
3,4-Dihydroxybenzoic acid - - + +
Benzoic acid - - + +
Ferulic acid - + + +
p -Coumaric acid - + + -
Chromatogram
of methanolic
extract after
hydrolysis
Phenolic compound quantification(D)
Solvent A (Phosphoric acid 0.01mol/l) Solvent B (Methanol)
Linear Isocratic (70:30)
ANALYSIS AFTER
AQUEOUS METHANOLIC HYDROLYSIS
PHENOLIC COMPOUNDS
EXTRACT EXTRACT AQUEOUS METHANOLIC
EXTRACT EXTRACT
p -Hydroxyacetophenone 44.4 164.5 564.5 1497.6
Catechin - - - -
p -Hydroxybenzoic acid - - 45 56
Vanillic acid - - - -
3,4-Dihydroxybenzoic acid - - - -
Benzoic acid 5.3 - - -
Ferulic acid - 63 12.2 -
p -Coumaric acid 12.4 67.3 36.4 102.3
SAMPLE FRACTIONATION (1)
-1. Separation using principle of ionic exclusion
tR , min
3.0
p-Hydroxyacetophenone
P hloroglucinol
Catechin
p-Cinnamic acid
2.5 Ferulic acid
p-Hydroxybenzoic acid
P rotocatechuic acid
Benzoic acid
p-Coumaric acid
2.0 Vainillic acid
m-Coumaric acid
1.5
0 1 2 3 4 5 6
1 1
−
c(SDS) , mmol 2
L 2
SAMPLE FRACTIONATION (2)
8.0
p-Hydroxybenzoic acid
Benzoic acid
6.0
Catechin
5.0 p-Hydroxyacetophenone
tR, min
4.0 Fenol
Floroglucin
3.0
Pyrocatechin
2.0 Resorcin
1.0 Hydrochinon
Benzyl alcohol
% acetonitrile
CONCLUSIONS
NL: 3,81E5
15,19 min
m/z= 136,9-137,9
Relative Abundance
100
F: - c ESI Full ms
137,46
[ 60,00-1000,00]
50 p-hydroxybenzoic acid
0
NL: 9,24E5
15,84 min
m/z= 166,9-167,9
100 167,39 F: - c ESI Full ms
[ 60,00-1000,00]
vanillic acid
50
0
0 5 10 15 Time (min)
20 25 30 35 40
Behaviour of several phenolic acids and neutral
compounds
Time retention ACN : 50% ACN : 80% ACN : 95% ACN : 98%
ACN : 100%
(min) H2O : 50% H2O : 20% H2O : 5% H2O : 2%
p -Hydroxybenzoic acid 1.27 1.58 2.81 8.59 a
p -Coumaric acid 1.22 1.53 3.01 9.57 a
p -ferulic acid - - - 10.88 -
Vainillic acid - - - 12.52 -
Benzoic acid - - 8.4 9.94 a
Quevercetin a - a - a
Rutin a - a - a
Catechin 1.82 1.92 2.09 - a
p -Hydroxyacetophenone 1.83 1.89 2.04 2.10 2.30
Fenol 1.88 - 1.98 - 2.05
Floroglucin 1.86 - 2.05 - 2.23
Pyrocatechin 2.03 - 2.17 - 2.35
Resorcin 1.86 - 1.99 - 2.11
Hydrochinon 1.96 - 2.02 - 2.17
Glycerol 1.98 - - - -
Ethylenglycol 1.94 - 3.66 - 7.58
Benzyl alcohol 1.93 - 2.00 - 2.33