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Institute of Chemical Technology, Dept.

of Analytical Chemistry,
Technická 5, 166 28 Praha 6

HPLC ANALYSIS OF PHENOLIC


COMPOUNDS IN NORWAY SPRUCE
WITH DIODE ARRAY AND MASS
SPECTROMETRIC DETECTION

Gloria Cosco Salguero, Jan Fähnrich


Subdivision of Ultraviolet Radiation
1. UV-A 320 - 400 nm
Penetrates atmosphere at nearly full intensity
Probably only weakly harmful

2. UV-B 280 - 315 nm


Only partially absorbed in stratosphere
Very harmful - probably forest decline disease

3. UV-C 200 - 280 nm


Completely absorbed by ozone layer and oxygen (O2)
Hazardous

According to International Commission on Illumination


OZONE (O3) ABSORPTION
SPECTRA

(a) Spectrum from 200 to 300 nm


(b) Spectrum from 295 to 325 nm

Ozone concentration reduction


allows more UV light to penetrate
to earth’s surface.
Norway spruce needles
UV-B Radiation Effects on Plants

- Decrease in photosynthetic activity.


- Susceptibility to disease.
- Changes in plant structure and
pigmentation.
- Growth retardation.
Aim of study:
Identify and quantify phenolic compounds in
Norway spruce needles that absorb UV radiation
and may contribute to UV light screening.
HPLC ANALYSES SYSTEMS
IDENTIFICATION
A) DAD - LC system Waters
B) DAD - Spectra Monitor 500 (Watrex). Software
SM5000.
C) LC system HP 1100 Series - MS ion trap detector
with electrospray ionization.

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

BENZOIC ACID p-HYDROXY- 3,4-DIHYDROXY- VANILLIC ACID


BENZOIC ACID BENZOIC ACID
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 + +
(+) 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)

Phenolic compounds in different extract (μg/g)

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

-- Compounds studied : Phenolic acids (e.g. hydroxybenzoic,


- p- coumaric acids) and neutral compounds (catechin,
- p-hydroxyacetophenone).
-- HPLC system consisted of an isocratic pump LCP 4000. Diode
- array detector Spectro Monitor 500. Software SM5000.
-- Column Separon SGX C18 7µm, 3 x 150 mm.
-- Mobile phase, methanol: water 80:20 (v/v). Aqueous phase was
- proved with various concentrations of sodium dodecylsulfate
- from 0.01 mM to 30 mM.
Phenolic compound retention behaviour under ion-exclusion conditions
Column 150 x 3.2 mm, Separon SGX C18 7µm, mobile phase methanol-aqueous
sodium dodecylsulfate (SDS) 0.1 to 30 mmol l-1, 0.4 ml min-1
3.5

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)

2. Separation using principle of hydrophilic


interaction

- Compounds studied: Phenolic acids (e.g. hydroxybenzoic,


- p-coumaric acids) and neutral compounds (catechin,
- p-hydroxyacetophenone).
-- HPLC system consisted of an isocratic pump LCP 4000. Diode
- array detector Spectro Monitor 500. Software SM5000.
- Column Lichrosorb Si-60, 7µm, 3 x 150 mm.
- Mobile phase, acetonitrile:water (v/v). Organic phase in
percentage from 50 to 100%.
Phenolic compound retention behaviour under hydrophilic interaction conditions
Column Lichrosorb Si-60, 7µm, 3 x 150 mm, mobile phase acetonotrile-aqueous,
0.4 ml min-1

8.0
p-Hydroxybenzoic acid

7.0 p-Coumaric 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

50 60 70 80 90 100 110 Ethylenglycol

Benzyl alcohol
% acetonitrile
CONCLUSIONS

1. Phenolic compounds occur in Norway spruce needles in the


free form and as conjugates, probably glycosides or esters.
2. In extracts p-hydroxyacetophenone, vanillic, 3,4-
dihydroxybenzoic, ferulic and p-coumaric acids were identified
as UV-B radiation absorbing substances.
3. Sample requires clean up and fractionation in order to simplify
chromatograms and eliminate interfering components.
CONCLUSIONS

5. Systems operating on the principle of ionic exclusion can


separate the phenolic acids and neutral compounds like
catechin and p-hydroxyacetophenone. They are not directly
applicable to samples containing a large amount of acids or
salts like hydrolyzed samples.
6. Hydrophilic chromatography can separate phenolic acids from
compounds without carboxyl groups. Sample must be
extracted with acetonitrile (or solvent with similar properties) or
transferred to this solvent.
ACKNOWLEDGEMENTS

Financial support of the Ministry of Environment of


the Czech Republic (grant VaV340/1/01)
and of the Ministry of Education, Youth and Sports
of the Czech Republic (grant MSM 223400008)
is gratefully acknowledged.
Chromatographic conditions for
LC system HP 1100 Series

 - Column Luna LC-18, 3μm, 150 x 2.0 mm I.D. from Phenomenex


 (USA), and a security guard C-18 (2.0 x 4 mm I.D.).
 - Flow = 0,2 ml/min.
- Lineal gradient from 5:95 to 100:0 (v/v) water:acetonitrile in 40 min.
 - Injection volume 3 µl.
 - Temperature 20 °C
 - MS ion trap detector with electrospray ionization (Finnigan,Deca).
 - Capillary temperature 350 °C.
 - MS operated in negative-ion. [M-H]-
Chromatograms of phenolic acids
18,96 min
m/z= 192,9-193,9
100
F: - c ESI Full ms
193,37 [ 60,00-1000,00]
ferulová
50

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

 a = Compound eluted in greater times of 15 minutes

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