analytical methods for analyse secondary...

Technische Universität München

Analytical methods for analyse secondary

metabolites

Susanne Rühmann

bioactive compounds in plants(polyphenols: flavonoids)

O

OH

HO

OH

OH

OH

OH

OHO

OH

HO

OH

O

OH

HO

OH

OH

OH

4

8

4

8

OHO

OH O

O

OH

OH

OH

O

O

O

OH

O

OH

OH

HO

OH

OH

O


Polyphenols (flavonoids)

Catechins

Enzymes

Enzymes

Enzymes

Enzymes

Enzymes


Functions in plants

• Colour component (Insects)


Why polyphenols?


Insects like bees and butterflies are

able to notice light till into UV-spectra

area

Functions of flavonoids at the interface plant and

environment (TREUTTER, 2007).

Enemies

Stress factors

Protection against sunlight

Leaf profile


UV-light affect

DNA in nucleus of

plant cells

Secondary plantmetabolites

for example

Polyphenols / Flavonoids




Polyphenols = suncreme of plants

Plant-Pathogen/ElicitorInteraction

Receptor

Nucleus

Pathogen

Plant cell

Nucleus

Pathogen

Accumulation of phenolics Plant cell

Plant-Pathogen/ElicitorInteraction


Functions in plants


• UV-protection (anthocyanidins, flavonols)

• Against pathogens

• Against stress factors (elicitors)


Elicitor

=> inductor

• UV-light

• Enzymes of microorganisms

• Dry-stress

• Saline stress

• High or low temperature

• Acids / alkalins

• Heavy metal

• Ozon (free radicals)

• Toxins (pesticides)

• Phytohormones


Functions in plants


• UV-protection (anthocyanidins, flavonols)

• Against pathogens

• Against stress factors (elicitors)

• Hormonal influence

• DNA influence


Why analysis (identification and quantification) of

secondary metabolites?

• Functions in plants

• Polyphenols: a marker for food-quality

• Understanding of mechanisms for human health


human health

In fruits and vegetables are:

• Mineral nutrients

• Vitamins

• Dietary fibres (Ballaststoffe)

• Bioactive compounds ????


Bioactive compounds:

Polyphenols for human health

• Well-known

since 1936 known as human health protector “Vitamin P”

Szent-Györgii



Polyphenols for human health

• New evaluate

”Bioactive plant compounds”Food against

cancer



Industry react with products



Dietary supplement



New juice creations



MOGUER, Cuna de Platero S.C.A., Camino de Montmayor, s/n., R.S. 21.0001352/H; R.I.A.21/4086/, HUELVA


Possible mechanisms of polyphenols in human health

• Cardio protective activity


Possible mechanism of cardio protective activity of polyphenols

Endothelzelle

Membran

O2-

Superoxid

Peroxynitrit

Aldini G., M. Carini, E. Bombardelli, R. Maffei Facino:

XXI International Conference on Polyphenols, Marrakech 2002



Endothelzelle

Membran

O2-

Superoxid

Peroxynitrit

Aldini G., M. Carini, E. Bombardelli, R. Maffei Facino:

XXI International Conference on Polyphenols, Marrakech 2002

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OHOH

OH

O

OHOH

OH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

OH

OHOH

OH

OH

OH

OH

OHOH

OH

OH

OHOH

OH

O

OHOH

OH

OH

OH

OH

OHOH

OH

OH

OH

OHOH

OH

Protection against

radicals



Inhibition of carcinoma-development in mouse caused by Apigenin (flavon)

Birt et al. (1997).

Carcinoma-

development under

UV-light

Protection by

polyphenols

Inflammation and developmemt of tumor

normal Epithel

StromaFibroblast

Inflammation

FibroblastOH-O2

- H2O2

NOcankered cells

Tumor-tissue

Tumor-cells

Inhibition of immun-regulationCankered, inflammation protective signals

Inh

ibit

ion

of

infl

amm

atio

nce

nte

r

Different reactors of inflammation

Prof. D. Treutter

Different signal-chains => Inflammation

Catalysts are wounds, oxidative stress, UV-light …

RTK

Signalacceptor

PLC

JAK

STAT3

Transcriptions-factors

CytosolSTAT3

TranslocationInflammation genes

Nucleus

Cankered inflammation protection signalsiNOS, COX-2, IL-6, IL-1β, TNF-α, VEGF …

Transcription

Pan et al. 2009, J. Agr. Food Chem.

Prof. D. Treutter



Signalacceptor


Cytosol


Nucleus


Transcription

PLC

JAK

STAT3

STAT3, NFκB, NFAT,

DAG

PKC

NIK

IKK

NFκBIκBα /

Prof. D. Treutter




Signalacceptor


Cytosol


Nucleus


Transcription

RTK

MAPK

PLC

JAK

STAT3

STAT3, NFκB, NFAT, AP-1, C/EBP,

DAG

PKC

NIK

IKK

NFκBIκBα /

TLR

MAP3K

MAP2K

Prof. D. Treutter




Signalacceptor


Cytosol


Nucleus


Transcription

RTK

MAPK (p38, ERK1/2, JNK ½)

PLC

JAK

STAT3

STAT3, NFκB, NFAT, AP-1, C/EBP, CBP/p300, ATF …

DAG

PKC

NIK

IKK

NFκBIκBα /

TLR

MAP3K

MAP2K

G protein

Raf

Prof. D. Treutter




Signalacceptor


Cytosol


Nucleus


Transcription

RTKCytokinreceptor

MAPK (p38, ERK1/2, JNK ½)

PLC

JAK

STAT3

STAT3, NFκB, NFAT, AP-1, C/EBP, CBP/p300, ATF …

DAG

PKC

NIK

IKK

NFκBIκBα /

TLR

MAP3K

MAP2K

G protein

Raf

Ras

PI3K

Akt

Prof. D. Treutter




Nucleus


Transcription

RTKDAG

TLR

G protein

Cytokinreceptor

Ras

NIK

IKK

NFκB

PI3K

Akt

MAP3K

MAP2K

MAPK (p38, JNK ½)

JAK

STAT3

OO

O

O O

CH3

CH3

CH3

O

OCH3

CH3

OOH

OH

OH O

OH

OH

OH

OOH

OH

OH O

X

Prof. D. Treutter



Biological activity of secondary plant metabolites

• Interaction with enzymes

– Reversible/irreversible binding on proteins

– Chelatisation of metal-ions (Fe-III, Al-III, Cu-II)






• Antioxidative activity and radical interceptor potential

– Protection of DNA, nucleotids, enzymes, membrans, lipids, vitamins






• Antioxidative activity and radical interceptor potential

– Protection of DNA, nucleotids, enzymes, membrans, lipids, vitamins

• Modulation of gene activity

– Interaction of transkriptions-factors



• Antimicrobial activity

– Inhibition of microbial enzymes (lyases)

– Protection of substrates (Glucans, Proteins, Nucleic acids)

– Cell-toxicity



Human health activity of natural phenolic

compounds

Scientific papers

An

tho

cya

nid

ins

Ch

loro

gen

ic

acid

Ell

ag

icacid

Iso

fla

vo

no

ids

OP

C a

nd

Ca

tec

hin

s

Ph

lori

dzin

Re

sve

ratr

ol

Ru

tin

/ F

lavo

no

ls

Chlorogenic acid Rutin (flavonol)

Anthocyanidin

OPC (oligomeric

procyanidin)

Resveratrol




compounds

Scientific papers

An

tho

cya

nid

ins

Ch

loro

gen

ic

acid

Ell

ag

icacid

Iso

fla

vo

no

ids

OP

C a

nd

Ca

tec

hin

s

Ph

lori

dzin

Re

sve

ratr

ol

Ru

tin

/ F

lavo

no

ls

anti-oxidative capacity x x x x x x x x

Potential of radical protection x x x x x x x

Anti-cancer-causing effect x x x x x

Protection of blood vessels x x x x x

Protection of blood plates x x x x x

Regulation of blood sugar x x x x x

Protection of neuronal cells x x x x x

Anti-mutagen potential x x x




compounds

Scientific papers

An

tho

cya

nid

ins

Ch

loro

gen

ic

acid

Ell

ag

icacid

Iso

fla

vo

no

ids

OP

C a

nd

Ca

tec

hin

s

Ph

lori

dzin

Re

sve

ratr

ol

Ru

tin

/ F

lavo

no

ls

Anti-viral effect x x x x

Anti-microbial effect x x x x

Protection of cell membranes x x x

Affect to fatty acid metabolism x x x

Affect NO-level in plasma x x

Anti-allergenic effect x x

Immunmodulation x x

Anti-tumor activity x x




compounds

Scientific papers

An

tho

cya

nid

ins

Ch

loro

gen

ic

acid

Ell

ag

icacid

Iso

fla

vo

no

ids

OP

C a

nd

Ca

tec

hin

s

Ph

lori

dzin

Re

sve

ratr

ol

Ru

tin

/ F

lavo

no

ls

arteriosclerosis x x x

Estrogenic effect x x

Influence of cholesterol level x x

Protection of vitamin C x x

Regulation of blood preasure x

Protection of DNA x

Dissolving of anxiety state (psychic) x

Against climacteric trouble x


Polyphenols: a marker for food-quality

• Controlling right description of a product

• Evaluating inner quality of food

• Capturing influence of handling procession in food

industry (heating, cooking, washing, …) on oxidation

processes of polyphenols in basis products


Controlling right description of a product

• Creation of polyphenol-profiles (pattern)

profile cranberry juice


Controlling right description of a product

• Creation of polyphenol-profiles (pattern)

profile cranberry juice

profile apple juice

profile mixed juice


Processions in food industry

• Any thermic procedures =>

degradation of some polyphenols

(creation of cereals)

• Other thermic procedures =>

little degradation of polyphenols

(backing of bread)


Secondary metabolites (polyphenols) in plants


Influence factors of polyphenol concentration in plants

• Primary metabolism

• Cultivar

• Crop cultivation methods

• Nutrients

• Time of harvest

• Storage


Influence factors of polyphenol concentration in plants

Cultivar

• Different plant organs show

– Different compound concentration (mostly)

– Different phenol profiles (sometimes)


Chl

orog

ensä

ure

Phl

orid

zin

Hyp

erin

Isoq

uerc

itri

nR

utin

Avi

cula

rin

Que

rcet

in

Hyd

roxy

zim

tsäu

re-D

eriv

at

Hyd

roxy

zim

tsäu

re-D

eriv

at

Hyd

roxy

zim

tsäu

re-D

eriv

at

Hyd

roxy

zim

tsäu

re-D

eriv

at

Fla

vano

l

Fla

vano

l

Fla

vano

l

Fla

vano

l

Käm

pfer

ol-D

eriv

ate

Phl

oret

in-2‘-

xylo

gluc

osid

Apple phenolics „Golden Delicious“

Apple leaf

Apple fruit skin


Crop cultivation methods

Change of polyphenol content caused by

• Cutting of the trees => changing in hormone content

• Condition of soil

• Climate conditions

• Light condition (UV-B light)

• Plant protection management (pesticides)

• Level of pathogen attack


Fertilization

High N-fertilization indice mostly

• Increasing of primary metabolism (plant growth)

• Decreasing of secondary metabolism (plant resistance)


Time of harvest / changes during storage

• Red pigments (anthocyanidins) increase during ripening

• Catechins decrease during ripening (strawberry)


Analytical methods for secondary metabolites

• Gas chromatography (volatile compounds)

• Liquid Chromatography (low- or not-volatile compounds)

– Thin layer chromatography

– HPLC (High-performance-liquid-chromatography; High-pressure-liquid-chromatography)


Liquid Chromatography

Definition of physical method:

Separation of compounds caused by:

• An inactive (stationary), hard phase

And

• A moving (mobile), liquid phase

Preconditions:

• Mixture of compounds is dissolved in a solvent

• Compounds are low-volatile or not volatile



Separation processes:

Types of separation mechanisms differ in interaction between stationary

phase, mobile phase and compound

• Absorption

• Allocation

• Ion-change

• Exclusion

• Affinity

How to quantify and identify plant metabolites



• Thin layer chromatography

• HPLC (High-performance-liquid-chromatography;

High-pressure-liquid-chromatography)


HPLC (high-performance-liquid-chromatography)

Separation processes of HPLC:

Types of separation mechanisms differ in interaction between stationary

phase, mobile phase and compound

• Absorption

• Allocation




Types of separation mechanisms

• Absorption

– Sample molecule is bound reversible on stationary phase by dipol-

dipol-interactions

– Exposer time depends on different intensity of interaction




Types of separation mechanisms

• Allocation: Normal-phase-chromatography:

– Stationary phase is more polar than mobile phase

• Allocation: Reverse-phase-chromatography:

– Mobile phase is more polar than stationary phase



Field of application:

• Is used for identifying, quantifying and purification the individual

components of a mixture

• Is used in:

– Analytical chemistry

– Biochemistry

– Medicine and pharmaceutical industry

» Vitamin D in blood serum

» Drugs in urine

» Purification of substances from a complex biological sample

» Manufacturing pharmaceutical (control)



Column:

• Separation column is filled with solid particles

– Silica gel

– Polymers

– sorbents

• Sample mixtures is separated into single compounds as it interacts with

the column particles (absorbance)



Column:

• Analytic HPLC columns => analysis of extract

• inside diameter (ID) 2 - 4.6 mm

• length till 250 mm

• max. flow 2 ml/min



Column: Analytic HPLC columns (institute of fruit science)

• C-18-column

• Chemical modified silica gel C-18-chains are bonded at surface



Column:

• Semipräparative column => cleaning of extract

• ID 10 - 50 mm

• length 250 mm

• max. flow 100 ml/min



Column:

• Präparative column => getting clean single compounds

• ID more than 75 mm

• Flow more than 100 ml/min



Column:

• Pre-columns => before head column: protection against

waste in extract (same material)

Method:

Sample mixture is carried to a

separation columncolumn

sample-loop

Injection outlet

hand injection by syringe

Auto-sampler


Method:

mechanical pumps transport

liquid solvent (eluent) through

a separation column

column

outlet


Method:

Single compounds remove the solid

particles at different moments and cross

the column with different speeds

column

eluent


column

eluent


Method:

Column leaving compound is transported

to detector

detector


column

eluent

detector



lamp vessel photo cell Data collector

(computer)

cover



(computer)

cover

Light (e.g. 280

nm)


100

%100

%

100

%


(computer)

cover

Light (e.g. 280

nm)


compound


(computer)

cover


Compound absorbs light

100

%20 % 80 %

80 %


(computer)

cover


100

%50 % 50 %

50 %


(computer)

cover

Higher concentration =>

more absorbtion


Absorption

concentr

ation

lamp Vessel photo cell Data collector

(computer)

cover

column

eluent

detector

chromatogram

absorption

Retention

time


column

eluent

detector

chromatogram

absorption

Retention

time

only eluent for 10 minutes

0

10


Retention

time

First molecules of a separated

compound leaves the column

0

10


column

eluent

detector

chromatogram

absorption

Retention

time

Information:

little absorption => software

0

10


column

eluent

detector

chromatogram

absorption

Retention

time

0

10

More molecules of the separated

compound leaves the column


column

eluent

detector

chromatogram

absorption

Retention

time

0

10

Information:

More absorption => software


column

eluent

detector

chromatogram

absorption

Retention

time

0

10

Rest of molecules leave the column


column

eluent

detector

chromatogram

absorption

Retention

time

0

10

Information:

Again lesser absorption => software

13


column

eluent

detector

chromatogram

absorption

Retention

time

0

10

All molecules of compound

have leave the column

13


column

eluent

detector

chromatogram

absorption

Retention

time

0

10

Information:

No absorption => software

13


column

eluent

detector

chromatogram

absorption

Retention

time

0

10

Same with the next compound

13 20


column

eluent

detector

chromatogram

absorption

Retention

time

0

10 13 20


column

eluent

detector

chromatogram

absorption


Retention

time

0

10 13 20 22


column

eluent

detector

chromatogram

absorption


Retention

time

0

10

First compound: Ret. Time max: 11.5 min (10 – 13 min); Abs. : 30 (mAU)

Second compound: Ret. Time max: 21 min (20 – 22 min); Abs.: 20 (mAU)

13 20 22

20

3011.5

21


chromatogram

absorption

Retention

time

0

10

System calculate from peak high and peak duration => area of peak

13

3011.5


chromatogram

absorption

Retention

time

0

10

System calculate from peak high and peak duration => peak area

We calculate later from peak area => real concentration

13

3011.5


chromatogram

absorption

Method:

Most digital analyte detector can measure at

different selectable wavelength (channels)

Channels in fruit science lab:

640 nm (catechins after post-column-derivatization)

280 nm (simple phenolics, catechins, …)

320 nm (hydroxycinnamic acids)

350 nm (flavonols)

540 nm (anthocyanidins)

UV-vis (spectra)

detector


Method:

digital analyte detector =>

quantitative analysis of compounds

detector


Diodenarray-detector

Method:

Diodenarray-detector =>

qualitative analysis of compounds

and

Identification of compounds


Light for identification

UV: 100 – 380 nm

Visible light: 380 - 780 nm

wavelength

Infra red

Absorbed colorAbsorbed wavelength[nm]

Reflected wavelength (visible color)

blue 446 yellow

green 500 red

yellow 562 blue

orange 595 Blue-green

red 660-668 green

complementary colors

leaf

Leaf absorb red light for photosynthesis > 600 nm


Leaf reflects green light (short wavlength) < 550 nm

leaf


Green and blue receptors in human eyes are activated

=> Green leaf

leaf

eye


Absorption green light < 550 nm Reflection red light > 600 nm


Absorption Reflection



prism

lampvessel photo cell Data collector

(computer)

cover

wavelength

abso

rpti

on

prism


(computer)

cover cover


wavelength

abso

rpti

on

prism


(computer)

cover cover

In our lab: 250 – 600 nm


prism


(computer)

cover cover

wavelength

abso

rpti

on

550 – 600 nm


prism


(computer)

cover cover

wavelength

abso

rpti

on

480 – 550 nm


prism


(computer)

cover cover

wavelength

abso

rpti

on

400 – 480 nm


prism


(computer)

cover cover

wavelength

abso

rpti

on

380 – 400 nm


prism


(computer)

cover cover

wavelength

abso

rpti

on

250 – 380 nm


wavelength

abso

rpti

on

Anthocyanidin 278, 515 nm

250 nm 500 nm

UV-Vis-spectrum

abso

rpti

on

250 nm 500 nm

Flavonol 258, 354 nm

R1

R2A C

B

wavelength

UV-Vis-spectrum

abso

rpti

on

250 nm 500 nm

Hydroxycinnamic acids 309 - 329 nm

wavelength

UV-Vis-spectrum

Method relies on:

High-performance liquid chromatography (HPLC)

Diodenarray

detector

(DAD)

column

sample-loop

Injection outlet

autosampler

outlet

Eluent pumpEluent

HPLC

software

regulate

C:/Users/Secondadmin/Desktop/Analytik englisch/Analytikmethoden.pptx#43. HPLC (high-performance-liquid-chromatography)

C:/Users/Secondadmin/Desktop/Analytik englisch/Analytikmethoden.pptx#43. HPLC (high-performance-liquid-chromatography)



HPLC separation is influenced by:

• the liquid solvent’s condition

• Pressure

• temperature

• chemical interactions between the sample mixture and the

liquid solvent

• Hydrophobicity

• Protonation, …

• chemical interactions between the sample mixture and the solid

particles packed inside of the separation column

• Ligand affinity

• Ion exchange, …


Many different types of columns

varying in:

• Size

• Type (i.e. chemistry) of solid packed particle types


Eluent (solvent) process in HPLC

• Isokratic solvent process

• One solvent (eluent): interaction between stationary phase and

mobile phase is the same all the time of separation

• Good separation of similar compounds

Method relies on:


Diodenarray

detctor (DAD)

column

sample-loop

ventile

Eluent pumpEluent

HPLC

software

regulate


Isokratic Elution

No changing of mobile phase composition during separation process

100 min

100

%

Solvent A (80%H2O; 20% MeOH)


Eluent (solvent) process in HPLC

• Isokratic solvent process

• One solvent (eluent): interaction between stationary phase and

mobile phase is the same all the time of separation

• Good separation of similar compounds

• Gradient between different eluents

• Composition of different eluents (mostly 2 or 3) is changed

continued about the sepatation period: interaction between

stationary phase and mobile phase change all the time

• Good separation of compounds with different polarity

Method relies on:


Diodenarray

detctor (DAD)

column

sample-loop

Eluent pumpEluent B

HPLC

software

regulate

Eluent A

HPLC-gradient

column

Column particles(unpolare surface)

Extract (mixture of different compounds)=> On the column top

HPLC-gradient

column


Compounds are fixed on column particle bypolarity power (like magnetic effect)

HPLC-gradient

column


column


HPLC-gradient

Eluent 1 (polar) transfer column (e.g. water)

% Solvent A (H2O)

column


HPLC-gradient

Compounds with similar polarity like water (green) => solve from particles and transfer the column .

Compounds with more unpolar character => stayon partcles

Eluent 1 (polar) (e.g. water) was combinated withEluent 2 (more unpolar) (e.g. methanol)(1:1; v:v) (50% MeOH)=> Mixture is more unpolar than water

column


HPLC-gradient

% Solvent A (H2O)

% Solvent B (MeOH)

column


HPLC-gradient

Compounds with similar polarity 1:1 (water : methanol) (blue) => solve from particles and transfer the column .

Compounds with more unpolar character => stay on partcles

column


HPLC-gradient

Eluent 1 (polar) (e.g. water) was combinated withEluent 2 (more unpolar) (e.g. methanol)(2:8; v:v) (80% MeOH)=> Mixture is more unpolar than (water:methanol) (1:1)

% Solvent A (H2O)

% Solvent B (MeOH)

column


HPLC-gradient

Yellow coumpounds leave the column


Gradient-method

changing of mobile phase composition during separation process

0 2

0

40

60

80

100 % % Solvent A (H2O)

% Solvent B (MeOH)


Begining of HPLC-work:

•Creating of a data base of polyphenol standards

•Different compounds

•Different concentrations

•Analyse at different wavelength

What about linearity

Where is the concentration limit of HPLC


Extraction methods

• Fresh-extraction

• Dry-extraction


Dry-extraction of polyphenols

Ground freeze-dried

plant material

Swing mill

Mortar



Ground freeze-dried

plant material

fill powder [x mg]

in 2 ml Eppendorf tubes



Ground freeze-dried

plant material

fill powder [x mg]


extract with methanol

containing an internal

standard

+





standard

MeOH

Plant powder

complex with

anthocyanidins in

vacuoles





standard

MeOH

Fresh methanol

transport

anthocyanisins out of

plant material (high

concentration gradient:

inside much outside

nothing))





standard

MeOH

anthocyanisins

accumulate around the

plant complex.

Concentration gradient

is smaller => extraction

of anthocyanidins

inside is during longer





standard

MeOH

Better extraction when

concentration gradient

stay at high level

=>



Ground freeze-dried

plant material

fill powder [x mg]




standard

Shake the extract!

By hand



Ground freeze-dried

plant material

fill powder [x mg]




standard

Cool (4°C) ultrasonic

bath for 30 min



Ground freeze-dried

plant material

fill powder [x mg]




standard

centrifuge for 10 min

at 10000 rd/min


bath for 30 min



Ground freeze-dried

plant material

fill powder [x mg]




standard


at 10000 rd/min

Fill supernatant in a new

Eppendorf tube =>

Extract is used for HPLC

analysis (soluble phenolics)


bath for 30 min

Storage extract

at -20°C


at 10000 rd/min

before filling extract

in sample tubes



Volume of plant material:

fill powder [x mg]


plant organ Fresh

weight

[mg]

Dry

weight

[mg]

apple leaves 1000 100

grape flowers 2000 200



Best extraction-solvent and best concentration

compounds Optimal solvent

flavonoids Methanol 100 %

anthocyanidins Methanol +

Formic acid

95% + 5% (v/v)

carotenoids Aceton

Tetrahydrofuran

Diethylether

Ethylacetat

Hexan

Petrolether

100 %

xanthophylls Ethanol 100 %



Best extraction-solvent and best concentration

• For extraction of anthocyanidins the formic acid was used

for stabilization of pH and so for protection anthocyanidins

compounds Optimal solvent

anthocyanidins Methanol +

Formic acid

95% + 5% (v/v)



Best relation between plant material and solvent

plant organ Fresh

weight

[mg]

Dry

weight

[mg]

Volume

MeOH +

Std. [µl]

apple skin 1000 100 1000

apple leaves 1000 100 500

grape flowers 2000 200 500



Best internal Standard-compound

=> Place in a chromatogram

Retention time [min]



Best concentration of

internal Standard-compound

y = 348.5x + 2.1166R² = 0.9996

0

50

100

150

200

250

300

350

400

0 0.2 0.4 0.6 0.8 1 1.2

Are

a(m

AU

*min

)

Concentration (mg/ml)

Optimal linearity between 0,02 und 1 mg/ml



Best concentration of internal Standard-compound

• Take concentration in the middle of linear area => you can dilute or

concentrate sample with results in linear area of standard

y = 348.5x + 2.1166R² = 0.9996

0

50

100

150

200

250

300

350

400

0 0.2 0.4 0.6 0.8 1 1.2

Are

a(m

AU

*min

)

Concentration (mg/ml)



Best concentration of internal Standard-compound

• Take concentration in the middle of linear area => you can dilute or

concentrate sample with results in linear area of standard

• Take concentration nearly with the same high of the peaks like the biggest

peaks in the sample



When peaks are too small

• concentrate extract by evaporation (drying under vacuum => protect phenolic

compounds against oxidation)

• Resuspend dried extract in smaller volume of methanol (500 µl initial extract=>

250 µl concentrate extract)



When peaks are too big

• Dilute extract by addition of more solvent (methanol without standard

compound)



Best time of extraction

• Extract sample for different time (10, 20, 30, 40, 50, 60 min)





• Remove extract after centrifugation






• Extract staying plant material again with the same volume of solvent






• Extract staying plant material again with the same volume of solvent

HPLC-

analysis


We will begin practical work with:

•Creating of a data base of polyphenol standards

•Different compounds

•Different concentrations

•Analyse at different wavelength

What about linearity

Where is the concentration limit of HPLC


Development of Standard-database

Susanne Rühmann


Different concentrations of standard compounds

(0,001; 0,005; 0,01; 0,025; 0,05; 0,25; 0,5; 1,0 mg/ml)

• Catechin

• Epicatechin

• P-coumaric acid

• Chlorogenic acid

• Quercetin

• Rutin

• Kämpferol


Rutin (Croma; Nucleosil; gradient: isokratic 80% MeOH)

260, 351 nm

280 nm320 nm

350 nm

Absortion

mAU*min

Concent

ration

[mg/ml]

280 nm 320 nm 350 nm

0,001 4.8208 5.9552 5.4592

0.005 4.3318 4.6422 4.8743

0.01 6.0476 6.8603 8.1012

0.025 11.4039 15.1045 22.0569

0.05 20.7677 27.508 43.4618

0.25 50.5845 69.7975 115.2529

0.5 113.268 150.7571 236.7213

1 202.579 287.9368 460.4141

Biggest

area



260, 351 nm

280 nm320 nm

350 nm

Absortion

mAU*min

Concent

ration

[mg/ml]

280 nm 320 nm 350 nm

0,001 4.8208 5.9552 5.4592

0.005 4.3318 4.6422 4.8743

0.01 6.0476 6.8603 8.1012

0.025 11.4039 15.1045 22.0569

0.05 20.7677 27.508 43.4618

0.25 50.5845 69.7975 115.2529

0.5 113.268 150.7571 236.7213

1 202.579 287.9368 460.4141

Overview-

chromatogram



260, 351 nm

280 nm

350 nm

Absortion

mAU*min

Concent

ration

[mg/ml]

280 nm 350 nm

0,001 4.8208 5.4592

0.005 4.3318 4.8743

0.01 6.0476 8.1012

0.025 11.4039 22.0569

0.05 20.7677 43.4618

0.25 50.5845 115.2529

0.5 113.268 236.7213

1 202.579 460.4141



260, 351 nm

280 nm

350 nm

Absortion

mAU*min

Concent

ration

[mg/ml]

280 nm 350 nm

0,001 4.8208 5.4592

0.005 4.3318 4.8743

0.01 6.0476 8.1012

0.025 11.4039 22.0569

0.05 20.7677 43.4618

0.25 50.5845 115.2529

0.5 113.268 236.7213

1 202.579 460.4141

Detection limit

(upper, lower)



260, 351 nm

280 nm

350 nm

Absortion

mAU*min

Concent

ration

[mg/ml]

280 nm 350 nm

0,001 4.8208 5.4592

0.005 4.3318 4.8743

0.01 6.0476 8.1012

0.025 11.4039 22.0569

0.05 20.7677 43.4618

0.25 50.5845 115.2529

0.5 113.268 236.7213

1 202.579 460.4141


Croma; Nucleosil; gradient: isokratic 80% MeOH

HPLC Column gradient RT [min]wavelenght

[nm]Compound-group Compound

conc. [mg/ml]

Area [mAU*min] RF

linear (from .. To)

[mg/ml]

linear (from .. To) [mAU/min] UV max

croma nucleosil 80% MeOH 280 flavonols Rutin 0.25 50.58 4.76E-05 0.25 - 1 50 - 202 256; 358

croma nucleosil 80% MeOH 350 flavonols Rutin 0.25 115.2529 2.15E-05 0.25 - 1 115 - 460 256; 358

croma nucleosil 80% MeOH 280Hydroxy cinnamic acids Chlorogenic acid 0.1 37.5499 2.63E-05 0.005 - 1 1.9 - 350 328

croma nucleosil 80% MeOH 320Hydroxy cinnamic acids Chlorogenic acid 0.1 76.2917 1.31E-05 0.005 - 1 3.6 - 825 328

croma nucleosil 80% MeOH 280 flavan-3-ols Eopicatechin 0.1 21.1954 4.70E-05 0.1 - 1 21 - 208 279

croma nucleosil 80% MeOH 280Hydroxy cinnamic acids p-coumaric acid 0.1 124.5077 8.37E-06 0.025 - 0.25 28 - 299 309

croma nucleosil 80% MeOH 320Hydroxy cinnamic acids p-coumaric acid 0.1 165.4809 6.35E-06 0.05 - 0.25 75 - 393 309

croma nucleosil 80% MeOH 280 flavonols Quercetin 0.1 30.647 3.29E-05 0.01 - 1 3 - 306 255; 372

croma nucleosil 80% MeOH 350 flavonols Quercetin 0.1 63.059 1.62E-05 0.01 - 1 6 - 639 255; 372

croma nucleosil 80% MeOH 280 flavan-3-ols Catechin 0.1 24.2558 4.51E-05 0.1 - 1 24 - 244 279

analytical methods for analyse secondary...

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