UNIVERSITY OF VETERINARY AND
PHARMACEUTICAL SCIENCES BRNO
Faculty of Pharmacy
Department of Natural Drugs
Phytochemistry
Instructions for laboratory practice
PharmDr. Markéta Gazdová, Ph.D.
PharmDr. Zuzana Hanáková, Ph.D.
1.Isolation of content compounds – extraction, purification
Content compounds in plants:
 Primary metabolites (carbohydrates, aminoacids, purins and pyrimidins of nucleic
acids, fatty acids, lipids, plant pigments)
 Secondary metabolites (terpenoids, nitric compounds, phenolics, acetic acid
derivatives)
Content compounds division:
 Major
 Minor
 Ballast (chlorophyll, celulose, lignin)
Content compounds ar attached to plant material:
 Chemically
 Physically
Methods for obtaing content compounds:
EXTRACTION
Method using two immiscible solvents occuring in various state (solid/liquid; liquid/liquid)
Types of media for extraction:
 Liquid
 Gas
 Solid
Liquid extractions:
 Total
 Selective
Types of liquid extraction according to process
 Periodical
 Maceration
 Digestion
o Infusion
o Concoction
 Semicontinuous
 Continuous
MICRO-SUBLIMATION
The change of solids into gases followed by the desublimation is the principle of
microsublimation.
PURIFICATION
Proper method of extraction leads to acquisition of content compounds in certain state of
purity. For the further purification we can use these methods
 Liquid-liquid extraction
 Lyofilization
 Precipitation
 Crystallisation
Excercise No. 1:
Preparation of plant material for phytochemical analysis,
maceration
The easiest method for obtaining content compounds is maceration. Main advantages of this
method are simplicity of execution, cheapness (instrumental and financial), efectivness and
selectivity.
Maceration is performed in dark, well-closed vessels filled with plant material and suitable
solvent for 24 hours under normal conditions (temperature, pressure).
The aim of this practice is to perform maceration with Cudrania tricuspidata wood. The crude
extract will be then used for the further excercises.
Procedure:
Fill in the vessel with approx. 100 g of dried wood of Cudrania tricuspidata and wash them
over with 500-1000 ml of ethanol (the plant material must be fully submerged) and mix with
glass stick. Close and label the vessel and leave till next laboratory practice.
Scheme:
Conclusion:
Excercise No. 2:
Isolation of plant pigments from Urtica dioica by Soxhlet
extraction
Urtica dioica (Urticaceae), stinking nettle, contains a high amount of plant pigments in leaves,
especially chlorophyll, carotenoids, flavonoids and xanthophylls. The aim of this excercise is
to isolate these pigments with acetone. Later on, the pigments will be identified using thin layer
chromatography (TLC) and isolated using column chromatography (CC).
Procedure:
10 g of fresh plant leaves are cut and extracted in 300 ml of acetone in Soxhlet apparatus for 4
hours. After that, the extract is dried used anhydrous sodium sulphate, filtered and the solvent
is removed under vacuum to a volume of approx. 10 ml.
Scheme of Soxhlet apparatus:
Conclusion:
Excercise No. 3:
Isolation of compounds from Species urologicae
Species urologicae is a medicinal plant mixture in the form of tea with urinary disinfection and
antiseptic properties. Methanol is used for the extraction to obtain phenolic glycosides and
flavonoids.
Procedure:
1g of the tea mixture is extracted by boiling for 15 minutes with 10 ml of water and 10 ml of
methanol under reflux in a water bath. The mixture is then filtered immediately through a cotton
wool. Once cooled, 5 ml of lead acetate solution is added and the solution is then filtered again
(using a filter paper). The filtrate is collected for further use.
Scheme:
Conclusion:
Excercise No. 4:
Micro-sublimation of caffeine
Cautious heating of coffee leads to the sublimation of caffeine at the temperature of 140-150
ºC. Caffeine can be observed as small colorless needles under the microscope.
Procedure:
Approx. 0,1 g of the powdered test drug (coffee) is placed on the middle of a watch glass with
a diameter of 7 cm. This glass is covered with a glass plate. The margins of the glass plate
should exceed those of the watch glass. The upper glass is cooled (using a small beaker filled
with cold water) to improve the condensation of caffeine. The system is heated for approx. 15
minutes. The upper glass is then removed, left to cool and then examined using the microscope.
Scheme of apparatus:
Scheme of caffeine crystals:
Conclusion:
Excercise No. 5:
Purification of Cudrania tricuspidata extract by liquid-liquid
extraction
Liquid liquid extraction or shaking out is a method where the limited miscibility of two solvents
is used. Solvents are chosen from the opposite sides of eluotropic line. The sample dissolved
in one phase is placed into separatory funnel and the second immiscible solvent is added (the
ratio of solvents is usually 1:1). The main principle of the shaking out is the diffusion of
dissolved compounds between two immiscible solvents and the set of equilibrium concentration
according to the solubility of the substances in solvents. After separation process, the individual
phases are evaporated on a rotary evaporator.
Procedure:
Crude extract is dissolved in 200 ml of 80 % methanol. 100 ml of hexane is added and the
mixture is placed into separatory funnel where two separate layers can be observed. The
separatory funnel is removed from the rack and is shaked carefully while the vapor is
continuously released through the tap. Place back in the rack, let settle down and then separate
both layers into flasks and evaporate using rotavapor. Methanolic portion is then dissolved in
200 ml of chloroform and 200 ml of water and the shaking out process is repeated. Place back
in the rack, let settle down, separate the chloroform layer and evaporate on rotary evaporator.
The residual water is mixed with 200 ml of ethylacetate, similarly shaked out and both layers
are separated into flasks. Ethylacetate portion is evaporated using rotary evaporator, water is
removed by lyophilization. Residues will be used for chromatographic separation in the
following practices.
Scheme:
Conclusion:
2.Chromatographic analysis and separation of plant extracts
Analytical method using two immiscible phases (mobile and stationary) for the separation of
the molecules of analyzed sample. Each molecule has different affinity to individual phases.
 Mobile phase
 Stationary phase
Chromatographic methods can be divided into groups according to these criteria:
1) According to the character of stationary phase (SP)
 Stationary phase is flat
 Stationary phase is placed in chromatographic column
2) According to the character of mobile phase (MP)
 Liquid
 Gas
3) According to the forces causing the separation between SP a MP
 Adsorptive
 Distributive
 Ion exchange
 Exclusive
 Affinity
4) According to supposed utilization
 Analytical
 Preparative
TLC: thin layer chromatography. Widespread in secondary metabolites analysis for the
separation of non-volatile mixtures. Performed in planar arrangement on a sheet of glas, plastic
or aluminium foil, which is coated with a thin layer of adsorbent material (stationary phase),
usually silica gel, aluminium oxide (alumina) or cellulose.
To quantify the results we use the retardation factor (Rf)
Retardation factor:
Dimensionless, characteristic for each compound, changes depending on the exact conditions
of the mobile and stationary phase
Calculation:
The distance traveled by the substance is divided by the total distance traveled by the mobile
phase.
Methods of detection TLC:
 Non-destructive
 Destructive
 Non-specific
 Specific
CC: column chromatography. Type of chromatography using a column (usualy glass) filled
with stationary phase. For the wet method, a slurry is prepared of the eluent (mobile phase) with
the stationary phase powder and then carefully poured into the column. The molecules of the
sample are divided according to the affinity to each phase.
HPLC: high performance liquid chromatography is a technique in analytical chemistry used to
separate, identify and quantify each component in a mixture. It relies on pumps to pass a
pressurized liquid solvent containing the sample mixture through a column filled with a solid
adsorbent material. Each component in the sample interacts slightly differently with the
adsorbent material, causing different flow rates for the different components and leading to the
separation of the components as they flow out the column.
Excercise No. 6:
TLC of Species urologicae extract– identification of arbutin
Tested solution: The extract of Species urologicae prepared in Excercise No.2
Standart solution: Methanolic solution of arbutin (2 mg/10 ml)
Mobile phase: Ethyl acetate: methanol: H2O (100:17:13)
Detection I: Diazotized sulfanilic acid (freshly prepared – to 5 ml of sulfanilic acid add sodium
nitrite until pale yellow solution appears)
Detection II: Ethanolic solution of KOH
Procedure:
Apply cca 10 μl of the standart and 10 μl of the sample solution separately on the TLC plate.
Develop the TLC plate to a distance of approx. 10 cm. Aftery drying with a hair dryer, apply
detection I and II consequently by spraying. Observe red spots located at the top of the TLC
plate (=arbutin)
Calculate the Rf values of standard and sample spots.
Calculation of retardation factor of the standard and sample solution:
Conclusion:
Excercise No. 7:
TLC of Urtica dioica extract – plant pigments identification
Tested solution: Extract of Urtica dioica prepared in Excercise No.2
Mobile phase: Diethyl ether: petroleum ether (1:1)
Procedure:
Apply a suitable amount of the sample solution on the TLC plate (the application must be well
visible as a spot on a plate). Develop the TLC plate to a distance of approx. 10 cm. After drying
at room temperature, separated spots of plant pigments are visible using UV/Vis lamp.
RF = 1,0 Carotenoids (α-,β-carotene) yellow
RF = 0,48 Pheophytin green-brown
RF = 0,35 Chlorophyll A green-blue
RF = 0,20 Chlorophyll B green-yellow
RF = 0,11-0 Xanthophylls bright green, yellow
Calculate the retardation factor values of spots.
Calculation of Rf values of sample spots:
Conclusion:
Excercise No. 8:
Preparative TLC of Paulownia tomentosa extract– isolation of
flavonoids
Chromatographic methods can be used for both analytical and preparative purposes. The
analytical TLC is used for quantification or identification of the constituents (comparison with
standart solution), the aim of preparative TLC is to isolate the entity from the mixture.
The aim of this practice is to isolate flavonoid constituents from the mixture (fraction obtained
by CC separation) using preparative TLC. Isolated compounds will be used for further HPLC
analysis.
Tested solution: 10 mg of fraction obtained by CC separation, diluted in 0,5 ml of methanol
Mobile phase: Chloroform: ethyl acetate (9:1)
Procedure:
Apply the sample solution as a „strip“ on the start of the TLC plate using pasteur pipette. After
drying, put the TLC plate into a beaker with methanol and develop for a while to settle the strip
of the sample at the start. Dry the plate again and then develop to a distance of approx. 10 cm.
After drying in room temperature, detect by using UV/Vis lamp and mark the separated strips
with a crayon. Scratch the defined area of the most visible strip using the kopist and transfer
the silica to the eppendorf with 0,5 ml of methanol. After centrifugation transfer the liquid with
care to the vial with insert and analyze using HPLC.
Conclusion:
Excercise No. 9:
Isolation of carotenoid pigments from Urticae folium extract using
column chromatography (CC)
Extract from Folium urticae dioicae contains chlorophyll, which is a mixture of two
compounds: blue-green chlorophyll A nad bright green chlorophyll B. Furthermore, extract
contains yellow pigments – xanthophylls and carotenoids. Single pigments can be separated
using column chromatography on silica gel.
The task of this excercise is to isolate yellow carotenoid pigments using column
chromatography. Isolated pigments will be used for further spectrometric analysis.
Tested solution: Extract Urticae folium previously prepared in excercise No.2
Stationary phase: silica gel
Mobile phase: Diethyl ether : petroleum ether (1:1)
Procedure:
Column preparation:
Close the tap of the column, use a piece of cotton wool to close the conus of column. Fill column
with approx. 10 mL of mobile phase to replace air from the cotton wool. Put approx 30 g (60
mL) of silica gel into a beaker and add approx. 50 mL of mobile phase to make a bubble-less
slurry. Then our the suspension carefully without bubbling into the column. The particles of
suspension should homogenously settle. Place carefully a disk of filtration paper on the top of
the column (the paper should have the same diameter as the diameter of the column).
Sample preparation:
20g of fresh leaves were extracted in acetone in Soxhlet apparatus for 4 hours. Extract was dried
using anhydrous natrium sulphate, filtered and the volume was reduced into 10 mL.
Sample application:
Open the column tap and let the level of mobile phase to reach the top of the sorbent and close
the tap. Draw approx. 0,2 mL of sample solution into pipette or pasteur pipette and apply
carefully on the top and let it soak into the sorbent. Use 1 mL of mobile phase to overlay the
top and let it soak into the sorbent again. Repeat this twice while the tap column is open and
closed again. Be aware of drying of the surface of the sorbent. Carefully fill the column with
the mobile phase and open the valve to flow rate of mobile phase approx. 40 drops/min.
Isolation of carotenoid pigments:
Mobile phase is gathered into test-tubes as 10 mL fractions. Carotenoid pigments move with
the top of the mobile phase, therefor we can combine several fractions at the beginning into
beaker and re-use them as a mobile phase again. At the moment when the yellow band moves
to the end of the column (3 cm) we can start with the collection of the fractions containing
carotenoids. Purity of isolates can be checked via TLC on silica, mobile phase diethyl etherpetroleum
ether 1:1.
Spots on TLC and column
Rf = 1,0 Carotenoids Yellow
Rf = 0,48 Pheophytine Green-brown
Rf = 0,35 Chlorophyll A Green-blue
Rf = 0,20 Chlorophyll B Green-yellow
Rf = 0,11-0 Xanthophylls Bright green, yellow
Scheme of CC apparatus:
Conclusion:
Excercise No. 10:
HPLC analysis of C. tricuspidata extracts
Instrument: HPLC system Agilent 1100 series with DAD detector
Tested solution: hexane, chloroform, and ethylacetate portions of ethanolic extract of C.
tricuspidata obtained by liquid-liquid extraction in previous excercises
Mobile phase: methanol (MeOH), 0,2% HCOOH, acetonitrile (ACN); gradient elution
Stationary phase: HPLC column Ascentis®
Express RP-amide 100 × 2,1 mm, particle size 2,7
µm
Temperature: 40 °C
Pressure: maximum 400 bar
Flow: 0,3 ml/min
Injection: 1 µl
Gradient elution: 0. minute 10 % ACN, 90 % 0,2% HCOOH
36. minute 100 % ACN
36.–40. minute 100 % ACN
Detection: DAD, observing wavelengths 254 and 280 nm.
Procedure: Put 1 mL of each sample (concentration approx. 1 mg/mL) into the vials using
automatic pipette, mark the vials and place them into the HPLC sampler. According to the
teacher's instructions turn on all the units of the HPLC system and fill in all necesary acquisition
parameters (name of the sequence, total number of vials, name of the sample, method etc.).
Once all these steps are ready, start the acquisition.
Scheme of HPLC apparatus:
Basic HPLC characteristics:
Conclusion:
3.Identification of content compounds- spectroscopic
techinques (UV, IR, NMR, CD)
Excercise No. 11:
Determination of carotenoid pigments in Urticae folium using
UV/VIS spectrophotometry
Carotenoid pigments (mixture of α-carotene and β-carotene) were isolated from the Urticae
folium extract. Their content in the sample can be easily determined by using spectrophotometry
in the area of the visible light. Sample absorption is directly related to the concentration of
carotenoid pigments according to the Lambert- Beer law: (A = Ɛ×b×c), where A is absorbance,
Ɛ is molar extinction coefficient , b is the path of light and c is concentration.
The aim of this excercise is to determine the carotenoid content in the sample
Procedure:
Sample prepared by column chromatography is transferred quantitatively into 25 ml volumetric
flask and the mixture of diethy lether-petroleum ether (1:1) is added. Absorbance of the sample
is then measured at λ=460 nm. Carotenoid content is deduced from the calibration curve and
re-calculated to carotenoid content in the drug (%).
Calculation:
Conclusion: