chapter 8 isolation and characterization of...

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CHAPTER 8

ISOLATION AND CHARACTERIZATION OF

PHYTOCONSTITUENTS BY COLUMN

CHROMATOGRAPHY

8.1 INTRODUCTION

Column chromatography is an isolation technique in which the

phytoconstituents are being eluted by adsorption. The principle involved in

this separation of constituents is adsorption at the interface between solid and

liquid. The component must have various degree of affinity towards adsorbent

and also reversible interaction to achieve successful separation. No two

compounds are alike in the above aspect. Low affinity compounds will elute

first. The columns of different sizes were used for the present studies.

Since the methanolic extract was found to possess significant

pharmacological activity when compared to other extracts an attempt was

made to fractionate the methanol extract by column chromatography.

The elution was done by using solvents of different polarity like n-

hexane, ethyl acetate and methanol.

8.2 MATERIALS AND METHODS

Type of extract : Methanol extract

Method : Dry packing method

Packing material : Silica gel G 70 -325

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Procedure

The methanol extract was subjected to Silica gel column

chromatography for the isolation of the phytoconstituents.

An appropriate column sized 5cm diameter and 50cm length was

used. It was washed with water and rinsed with acetone and then dried

completely. Little of pure cotton was placed at the bottom of column with the

help of a big glass rod. Solvent hexane was poured into the column upto ¾th

level. Methanol extract was mixed with equal amount of graded silica gel

until it became free flowing powder. When it reached a defined state it was

slowly poured into the column containing hexane solvent with slight

movement of stirring by glass rod to avoid clogging. Little cotton was placed

on top of silica gel- extract mixture pack to get neat column pack. The knob at

the bottom was slowly opened to release the solvent. The elution was done

using hexane, ethyl acetate and methanol in different ratios like Hexane

(100%- broad fraction 1), Hexane: Ethyl acetate (50:50- broad fraction 2),

Ethyl acetate (100%- broad fraction 3), Ethyl acetate: Methanol (50:50 –

broad fraction 4) and methanol (100%- broad fraction 5).

All the five broad fractions were collected separately and subjected

to TLC. The solvents were evaporated by rotary vacuum evaporator. Since

there was no yield in the Hexane fraction (100%) and very less yield in broad

fractions 2 and 3, fractions 4, 5 were selected and again subjected to sub

column fractionation.

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Isolation of Compound I and II

The 5th fraction (methanol 100%) on concentrating on a rotary

vacuum evaporator yielded light yellow sedimentation. It was then filtered

and the light yellow powder was dried. The filtrate was concentrated and a

brown semisolid was obtained. Both the fractions were subjected to thin layer

chromatographic studies. The yellow coloured powder was named as

compound I and the brown semisolid was named as compound II.

Isolation of compound III

The broad fraction 4 (Ethyl acetate: Methanol 50:50) was again

subjected to sub column fractionation using different ratios of Iso propyl

alcohol(IPA) and ethyl acetate. The fractions were collected and subjected to

thin layer chromatographic studies. The similar fractions were mixed together

and evaporated using rotary vacuum evaporator. The fractions 10 -60 (5%

IPA and 95% Ethyl acetate) on evaporation yielded a light brown solid which

was named as compound III.

The pure compounds obtained were then subjected to spectral

analysis for the determination of the structure of the compound.

8.3 TLC

As soon as the fractions were eluted, it was analysed by using ready

made TLC plate with suitable mobile solvent according to the polarity of

elute. The developed chromatogram was observed under UV and also

derivatized with detecting agent.

8.4 SPECTRAL ANALYSIS[128-130]

The isolated compounds were taken to determine the structure by

instrumental spectral analysis such as

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IR spectroscopy

NMR - 1H and 13 C

Mass spectroscopy

8.4.1 IR

IR spectrum is considered as vibrational-rotational spectra. KBr

pellet technique is used for solid compound, for liquid compound Nujol mull

method is followed. It is very helpful record which would give information

about functional group present in the organic compounds. Mechanism of bond

stretching and bending is happened when electromagnetic radiation ranging

from 500cm 1 to 4000 cm 1 passed through sample. Instrument used was

ABD BOWMEN Spectrometer.

8.4.2 NMR

Nuclear magnetic spectrum is the most powerful spectral technique

used to detect chemical structure of the molecules. The differences in the

chemical environment around the different nuclei are exploited to obtain this

information and is expressed in terms of chemical shifts in parts per million.

When sample absorbs radiation at different radio frequency region which

causes to excite type of proton or certain nuclei contained in the sample

against magnetic field.

The most commonly used NMR technique are the 1H and 13C. The

instrument used was for detection is PROBHD Nuclear Magnetic Resonance

spectrometer.

1H NMR

Most commonly used NMR is Proton NMR, because of the

sensitivity and wide range of characteristic information. Range of chemical

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shift ( ) from 0 – 14 ppm. Chemical shift of the test unknown compound was

compared with TMS protons which are attributed at 0 ppm. But, the shift

extends for the organic compound range 0 – 14 for the component.

13C – NMR

It is new technique but natural abundance is very low 1.1%. So, this

further reduces the sensitivity of the absorption. The range of chemical shift

) from 0 -180 ppm with use of TMS internal reference. An advantage is one

can directly observe the functional group contained carbon atom.

8.4.3 Mass Spectroscopy

It is an accurate method to determine the molecular mass of the

compound. The main advantage is very small amount of sample is required

for analysis than any other spectral methods. The mass spectroscopy uses the

electric and magnetic fields to produce electrically charged ions of chemical

substance under analysis.

Instrument used to determine the spectrum was JOEL Gcmate Mass

spectrometer. The record spectrum tells about the mass, relative abundance of

the molecular ions and positively charged fragments formed by electronic

bombardment. Sample was dissolved in CDCl3 and injected through direct

probe inlet. Electronic impact ionization method was used.

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8.5 RESULTS

8.5.1 Column Chromatography

Table 8.1 Broad fractions from methanol extract

Eluent Solvent Ratio Compound

n-Hexane 100% -

n-Hexane: Ethyl acetate 50:50 -

Ethyl acetate 100 -

Ethyl acetate: Methanol 50:50 -

Methanol 100 Compound-I

Compound- II

Table 8.2 Sub fractions from Ethyl acetate: Methanol(5:5) broad

fraction

Eluent Solvent Ratio Compound

Ethyl acetate 100% -

Ethyl acetate : IPA 95:5 Compound III

Ethyl acetate : IPA 10:90 -

Ethyl acetate: IPA 20:80 -

Ethyl acetate: IPA 40: 60 -



Ethyl acetate 100 -

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SPECTRAL ANALYSIS

The compounds (C-I, II and III) obtained with the methanol extract

have identified and spectral data’s were depicted in the Figures 8.1 to 8.12

8.5.2 Characterization of Compound – I

Light yellow solid (1.5gm) was obtained which is soluble in

methanol and water, the melting point was found to be 190-1950C. The

isolated compound answered the test for sugars. The various spectral data’s

obtained were given in Figures 8.1, 8.2, 8.3 and 8.4.

Ir cm-1: Nujol mull

Wave Numbers (cm-1) Type

3391 OH

2922 CH3, CH2, CH

1695 C=C

1415 (C-O)

1246 (C-O)

1196 (C-O)

1052 (C-O)

897 (CH)

732 (CH)

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Bruker NMR: 500MHz (solvent: D2O, ,ppm) , ppm: Parts per million

Atom Carbon, , ppm Hydrogen, , ppm

1 165.47 -

2 75.89 3.40 (d, 1H, J = 2.50 Hz)

3 95.85 3.92 (d, 1H, J = 2.50 Hz)

4 74.29 3.13 (t, 1H, J = 9.00 Hz)

5 71.08 3.48 (t, 1H, J = 9.50 Hz)

6 72.35 3.38 (m, 1H)

Properties

Name Glucuronic acid

Molecular formula C6H10O7

Molecular weight 194

Melting point 190-195 °C

Mass (m/z) 194[M+1]

Colour Yellow

Nature Solid

Solubility Soluble in methanol and water

TLC studies Methanol: Ethyl acetate:water:aceticacid(2:6:1:1)

Structure

O OH

OH

OH

HO

O

OH

12 3

4

56

154

Figure 8.1 IR Spectrum of compound I

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Figure 8.2 1H NMR Spectrum of compound I

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Figure 8.3 13C NMR Spectrum of compound I

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Figure 8.4 Mass spectrum of compound I

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8.5.3 Characterization of Compound – II

Yellowish brown semisolid mass (2gm) was obtained which is

soluble in methanol and water, the melting point was found to be 190-1950C.

The isolated compound answered the test for flavanoids. The various spectral

data’s obtained were given in the Figures 8.5, 8.6, 8.7 and 8.8.

Ir cm-1: Nujol mull

WAVE NUMBERS (cm-1) TYPE3369 OH2930 CH3, CH2, CH1686 C=C1412 (C-O)1276 (C-O)1075 (C-O)913 (C-O)634 (CH)

Bruker NMR (500MHz, ,ppm in values):ppm:parts per million

Atom Carbon, , ppm Hydrogen, , ppm1 73.41 3.36 – 4.04 (m, 1H)2 73.62 3.36 – 4.04 (m, 1H)3 101.78 4.91 – 5.17 (m, 1H)4 64.36 3.36 – 4.04 (m, 2H)5 75.25 3.36 – 4.04 (m, 1H)6 71.82 3.36 – 4.04 (m, 1H)7 72.40 3.36 – 4.04 (m, 1H)8 76.46 3.36 – 4.04 (m, 1H)9 97.98 4.91 – 5.17 (m, 1H)10 71.62 3.36 – 4.04 (m, 1H)11 76.52 3.36 – 4.04 (m, 1H)12 61.35 3.36 – 4.04 (m, 2H)

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Properties

Name Rhamnetin-3-O- -D-Galactosyl-4-O- -D-galactopyranoside



Melting point 210-2150C

Mass (m/z) 639( M )+

Colour Yellowish brown

Solubility Soluble in methanol and water

TLC studies Methanol: Ethyl acetate:water:aceticacid(2:6:1:1)

Nature Semi-solid

Structure

O

O

O

O

OH

OH

HO

HO

HO

OH

OH

O

OH O

OH

OH

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Figure 8.5 IR Spectrum of compound II

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Figure 8.6 1H NMR Spectrum of compound II

162

Figure 8.7 13C NMR Spectrum of compound II

163

Figure 8.8 Mass spectrum of compound II

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8.5.4 Characterization of Compound – III

The compound III was obtained as brown solid and the melting

point was found to be 225-2280C. The isolated compound answered the test

for triterpenoids. The various spectral data’s obtained were given in the

Figures 8.9, 8.10, 8.11 and 8.12.

Ir cm-1: Nujol mull

WAVE NUMBERS(cm-1) TYPE

3392 OH

2926 CH3, CH2, CH

1726 -C=O

1646 (C=C)

1367 (C-O)

1076 (C-O)

1035 (-CH)

824 (C-O)

775 (C-H)

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Bruker NMR (500MHz, , ppm in values)


1 33.77 1.18 – 1.38

2 25.52 1.18 – 1.38

3 82.94 3.20

4 39.98 -

5 56.81 1.18 – 1.38

6 20.83 1.18 – 1.38

7 34.87 1.18 – 1.38

8 40.52 1.18 – 1.38

9 47.84 1.18 – 1.38

10 37.70 1.18 – 1.38

11 25.35 2.01

12 123.60 5.16

13 144.64 -

14 39.98 -

15 29.91 1.18 – 1.38

16 25.04 1.18 – 1.38

17 56.81 -

18 32.87 -

19 42.74 1.18 – 1.38

20 28.44 -

21 42.40 1.18 – 1.38

22 26.78 1.18 – 1.38

23 25.20 1.18 – 1.38

24 25.20 1.18 – 1.38

25 21.61 1.18 – 1.38

26 21.96 1.18 – 1.38

27 16.94 1.18 – 1.38

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28 21.96 1.18 – 1.38

29 17.62 1.18 – 1.38

30 17.50 1.18 – 1.38

31 177.98 -

Glycosides Linkages

1’ 97.87 4.93

2’ 72.79 4.02

3’ 73.52 3.50

4’ 71.46 3.40

5’ 77.70 3.76

6’ 62.43 3.79

7’ 102.96 4.93

8’ 74.64 3.73

9’ 70.97 3.73

10’ 77.78 3.35

11’ 76.02 4.53

12’ 175.31 -

13’ 105.77 4.93

14’ 71.60 3.73

15’ 76.02 3.49

16’ 69.18 3.41

17’ 64.66 3.89

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Properties

Name 3-O-[ -l-Arabinopyranosyl-(1,4)-O- -D-glucuronopyranosyl)]-31-O-( -D-glucopyranosyl) oleanolic acid



Melting point 225-228°C

Mass (m/z) 825 ( M )+

Colour Brown

Nature Sticky mass

Solubility Soluble in water

Structure

OO

O

HO

OOH

OH

O

O

O

HOOH

OH

OH

O

OH

OH

OH

1

2

3 45

67

8

9

10

1112

13

14

1516

17

18

1920 21

22

23 24

25

26

2728

29 30

31

1' 2'

3'

4'5'

6'

7'

8'

9'10 '

11'12'

13'

14'

15 '16 '17'

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Figure 8.9 IR Spectrum of compound III

169

Figure 8.10 1H NMR Spectrum of compound III

170

Figure 8.11 13C NMR Spetrum of compound III

171

Figure 8.12 Mass spectrum of compound III

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8.6 DISCUSSION

Methanolic extract was found to possess significant pharmacological

activity when compared to other extracts it was subjected to column

chromatography for the isolation of phytoconstituents. Three compounds

were isolated from the extract and their structures were identified by spectral

studies.

The structure of the isolated compound I was analysed by

spectroscopic techniques like IR, NMR and mass spectroscopy. The melting

point of the compound was found to be 190-195 0C and it answered the test

for sugars. It had IR absorptions at 3391(hydroxyl), 2922 (C-H stretching of

alkanes), 1686 (carbonyl group of acid), 1415, 1246, 1196 and 1052 (C-O

vibrations of alcohols).

The 1H NMR spectrum showed the presence of 5 protons on

saturated carbon atoms. The value at 3.40 (d, 1H, J = 2.50Hz) and 3,92(d, 1H, J

= 2.50Hz) showed the doublet protons . The other two protons were present as

triplet with the value of 3.13(d, 1H, J = 9.00 Hz) and 3.48 (d, 1H, J = 2.50Hz).

The value at 3.38 (M,1H) showed the presence of multiplet.

The 13C NMR spectrum showed peaks at 75.89, 95.85, 74.29,

71.08 and 72.35 corresponding to carbon connected to oxygen linkage. The

peak at 165.47 corresponds to the carbonyl carbon. Mass spectrum of the

compound showed the molecular ion peak at m/z 194. From the spectral

studies the possible structure of compound I may be glucoronic acid.

The structure of the isolated compound II was analysed by

spectroscopic techniques and the melting point of the compound was found to

be 210-2150C. It answered the test for flavanoids. It had IR absorptions at

3369(hydroxyl), 2930 (C-H stretching of alkanes), 1412,1276, 1075 and 913

(C-O vibrations of alcohols) and 634 (C-H bending vibration of alkanes.

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The 1H NMR spectrum showed the presence of 14 protons on

saturated carbon atoms. The protons are multiplet with the value of 3.36- 4.04

(M,H) in up field and the value at 4.19- 5.17 (M,H) showed the protons in the

down field. The peak appeared in the range between 3.36-4.04 ppm corresponds

to carbon connected to oxygen linkage. 1H and 13C NMR spectrum of the

compound showed the peaks at 73.41, 73.62, 75.25, 71.82, 72.40, 76.46,

71.62 and 76.52 indicated the carbon connected to oxygen linkage.

Mass spectrum of the compound showed the molecular ion peak at

m/z 639. From the spectral studies the possible structure of compound II may

be Rhamnetin-3-O- -D-Galactosyl-4- O- -D-galactopyranoside.

The melting point of the compound III was found to be 225-228 0C

and it answered the test for triterpenoids. It had IR absorptions at

3392(hydroxyl), 2926 (C-H stretching of alkanes), 1726 (C=O stretching

vibration of ketones), 1646 (C=C stretching vibration of alkanes), 1367, 1076

(C-O stretching vibraton of alcohols), 1035 (C-H bending vibration of

alkenes), 824(C-O bending vibration of alcohols) and 725 (C-H bending

vibration of alkanes).

The 1H NMR spectrum showed the presence of 50 protons in which

8 methyl protons were present with value of 1.18- 1.38. the 1H NMR signal

at 3.20 was found to be secondary hydroxyl group. It showed the presence

of olefinic proton with 5.16ppm.

13C NMR spectrum showed peaks at 82.94, 171.98 corresponds to

carbon connected to oxygen. The peak at 177.98 showed the presence of

carbonyl carbon and at 123.60 , 144.63 showed the presence of double bond.

Mass spectrum of the compound III showed the molecular ion peak

at m/z 825. From the spectral studies the possible structure of compound II

may be oleanolic acid derivative.

chapter 8 isolation and characterization of...

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