Resolving A Multi Component Mixture Of Trace Biology Essay

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Chromatography is a non-destructive procedure for resolving a multi-component mixture of trace, minor or major constituent into its individual fraction. Chromatography is essentially a group of technique for the separating a mixture of component through equilibrium distribution between two phases, one of which is moving the other. The main principle of separation may be partition or adsorption. The chromatography studies used for the separation and identification of their component.

Thin layer chromatography is an important analytical tool in the separation, identification and estimation of different classes of natural products. The techniques of paper chromatography are similar in that they are both 'open bed' techniques in which substances are separated by the differential migration that occurs when a solvent flows along a thin layer of paper (paper chromatography) or fine powder spread on a glass or plastic plate (TLC). Separation may be achieved on the basis of partition or a combination of partition and adsorption, depending on the particular type of stationary phase, its preparation and use with different solvents. The advantage of TLC is that the samples do not have to undergo the extensive cleanup steps, and the ability to detect a wide range of compounds, using reactive spray reagents. Non-destructive detection (fluorescent indicators in the plates, examination under UV lamp) also makes it possible for purified samples to be scraped off the plate and be analyzed by other techniques [Instrumental methods of Chemical Analysis. 1979].

PROCEDURE:

Preparation of plate:

The silica gel G was mixed in a glass mortar to smooth consistency with a requisite amount of distilled water. The slurry was quickly transferred on the TLC glass plates by spreading technique, and uniform silica gel later was adjusted to 0.25mm thickness. The coated plates were allowed to dry in an air for 30 minutes and it was activated by heated in hot air oven at 100-105°C for 1 hour, cooled and protected from moisture. The plates were stored in dry atmosphere, before using the plates were activated at 110°C for 10 minutes.

Development of chromatography:

About 2mm of adsorbent from the edge of the plate has been removed to give sharply defined edges. The Chloroform and Methanol extract was dissolved in respective solvent and made up to 10ml. Then with the help of capillary tube extract was spotted on TLC plate which was developed in TLC chamber, previously saturated with different solvent systems. The solvent front was allowed to rise to distance of about 12 cm from the base line and the plate was removed from the chamber and allowed to dry in the air. By trial and error method the Chloroform and Methanol extract of Clerodendrum Inerme (L) Gaertn showed better resolution of spot with following solvent systems with some modification which was selected on the basis of phytochemical screening and presence of plant constituent [Stahl. E. A laboratory Hand Book. 1969].

Detection:

Spraying with a reagent solution is the most widely used technique for visualization of colorless substances on chromatograms. The antimony chloride reagent has been often mentioned as especially suitable for detection of terpenoids [Wagner. H. Ins Plant Drug Analysis. 1996].

Selected solvent system for the present study as follows:

1. Hexane : Ethyl acetate (8.5 : 1.5)

2. N-butanol : Acetic acid : Water (4 : 1 : 5)

Table No: 8

TLC profile of aerial part extract of Clerodendrum Inerme (L) Gaertn

Extract

Solvent system

No

Of

spot

Colour

Of spot

Detector

Rf value

Chloroform Extract of

Clerodendrum Inerme(L) Gaertn

Hexane : Ethyl acetate

2

Green

Antimony chloride

0.76

0.32

N-butanol : Acetic acid : Water

1

Brown

Antimony chloride

0.88

Fig 11: TLC OF CHLOROFORM EXTRACT OF CLERODENDRUM INERME (L) GAERTN

COLUMN CHROMATOGRAPHY

The column chromatography technique is widely used for separation, isolation and purification of the natural products. The principle underlying the compounds is adsorption at the solid liquid interface. For successful separation, the compounds of a mixture must show different degrees of affinity for the solid support and the adsorbent and component must be reversible. As the adsorbent is washed with fresh solvent the various components will therefore move down the column until, ultimately, they are arranged in order of their affinity for the adsorbent. Those with least affinity move down the column at a faster rate than, and are eluted from the end of the column before, those with greatest affinity for the adsorbent. By changing the polarity of the mobile phase, the separation can be achieved by column chromatography. Characterization of the isolated compounds can be carried out by analytical techniques, like UV, IR, NMR and Mass spectroscopy.

Column chromatography is advantageous over most other chromatographic techniques because it can be used in both analytical and preparative applications. Not only can column chromatography be used to determine the number of components of a mixture, but it can also be used to separate and purify substantial quantities of those components for subsequent analysis. This is in contrast to paper chromatography, which is solely an analytical method [Dyer. J.R. App Absorp Spec Organic comp. 1994].

For the isolation of active marker compound from the Chloroform and Methonal extract of Clerodendrum Inerme (L) Gaertn, here used the solvent system which was selected on the basis of phytochemical screening and thin layer chromatography study.

Details of column chromatography:

Adsorbent - Silica gel (60 - 120 mesh)

Solvent system - Hexane : Ethyl acetate (8 : 2)

Length of column - 100 cm

Diameter of column - 2.5 cm

Chloroform extract of

Clerodendrum inerme (L) Gaertn used - 2 gm

Rate of elution - 30 drops per minute

Fraction collected - 70 fractions, each of 30 ml

Method of column packing - Wet packing

Technique employed - Isocratic elution

Procedure for preparation of column:

1. The silica gel 60-120 mesh size was made into slurry with selected solvent system (Hexane : ethyl acetate). The silica gel was previously activated by heating in hot air oven at 100°C for 1 hr.

2. The bottom of the column was plugged by cotton and then the silica gel slurry was poured into the column which was filled with solvent system up to 40 cm height, after that it was set aside for 10 minutes and allowed to settle.

3. Individually Chloroform and Methanol extract of Clerodendrum Inerme (L) Gaertn was mixed with small amount of silica gel and wetted with solvent system and mixed well and allowed to evaporate the solvent to get the dry residue.

4. Then the dry residue was charged on column with the help of solvent system, after that Whatmann filter paper of suitable size was placed over it, in order to avoid the disturbance of the top layer of the adsorbent as fresh mobile phase was added to the column.

5. The column was eluted with the selected solvent system by Isocratic method and the fractions were collected in clean 100 ml beaker up to with the speed of drops was 30 drops per minutes [Furniss. B.S. Practical Organic chem.. 2005].

Each collected fraction was tested for the presence of various constituent by TLC for the number of types of constituents and similar fractions were pooled together.

Table no: 9

Data showing details of Column Chromatography of Chloroform

Extract of clerodendrum Inerme (L) Gaertn

Fraction

No

Eluates

Nature of Residue

Analysis by TLC

Rf value

1 - 10

Petroleum ether

100

No Residue

_

_

11 - 15

Pet ether : Hexane

80 : 20

No Residue

_

_

16 - 20

Pet ether : Hexane

60 : 40

Yellow waxy

No distinct spot

_

21 - 25

Pet ether : Hexane

20 : 80

Greenish Yellow color

Mixture of spot

0.52

0.50

26 - 30

Hexane

100

Greenish Yellow color

Mixture of spot

0.60

0.57

31 - 35

Hexane : chloroform

80 : 20

Green color

Two spots

0.76

0.32

36 - 40

Hexane : chloroform

60 : 40

Greenish Yellow color

Mixture of spot

0.73

0.70

41 - 45

Hexane : chloroform

20 : 80

Light green color

No distinct spot

_

46 - 50

Chloroform

100

Light green color

No distinct spot

_

51 - 55

Chloroform : Ethyl acetate

80 : 20

Greenish Yellow color

Could not be processed further

_

56 - 60

Chloroform : Ethyl acetate

60 : 40

Greenish Yellow color

Mixture of spot

0.83

0.80

0.78

61 - 65

Chloroform : Ethyl acetate

20 : 80

Greenish Yellow color

Three spots

0.85

0.81

0.79

66 - 70

Ethyl acetate

100

No Residue

_

_

Table No: 10

Rechromatography of Fraction 31 - 35

The Chloroform extract fraction 31 -35 were mixed and rechromatographed over the silica gel (100 - 200 mesh).

Fraction

No

Eluates

Nature of Residue

Analysis by TLC

Rf value

1 - 5

Hexane

100

No Residue

_

_

6 - 10

Hexane : chloroform

90 : 10

Greenish Yellow color

No distinct spot

_

11-15

Hexane : chloroform

75 : 25

Green color

Single spot

0.65

16 -20

Hexane : chloroform

50 : 50

No Residue

No distinct spot

_

21- 25

Hexane : chloroform

25 : 75

Greenish Yellow color

No distinct spot

_

26- 30

Chloroform

100

Yellow color

Single spot

0.46

31 - 35

Chloroform : Ethyl acetate

90 : 10

Greenish Yellow color

Could not be processed further

_

36 - 40

Chloroform : Ethyl acetate

75 : 25

Greenish Yellow color

No distinct spot

_

41 - 45

Chloroform : Ethyl acetate

50 : 50

Greenish Yellow color

No distinct spot

_

46 - 50

Chloroform : Ethyl acetate

25 : 75

No Residue

_

_

51 - 55

Ethyl acetate

100

No Residue

_

_

SPECTRA CHARACTERIZATION OF THE ISOLATED COMPOUNDS OF CLERODENDRUM INERME (L) GAERTN

A) Chloroform extract of Clerodendrum Inerme (L) Gaertn compound I:

Colour of the compound : Green colour

Solubility : Methanol, Chloroform

TLC pattern : Single spot

Mobile phase : Hexane : Ethyl acetate (8 : 2)

Rf value : 0.65

Melting point : 190-195°C

UV Spectra of compound : λ max - 277 nm [Harborne. J.b. Phytochemical

Method].

IR Spectra of compound : IR γ κвr Max Cm⁻¹ 2959.51, 1731.03,

1452.45, 1376.18, 1020.99, 887.03 [William

Kemp. Org spectroscopy].

NMR Spectra of compound : ¹H NMR (CDCL3) 1.38δ, 2.3δ, 2.8δ,

0.88δ, 5.3δ, 4.19δ[William

Kemp. Org spectroscopy].

Table No: 11

Spectral data of the isolated compounds from Chloroform

Extract of Clerodendrum inerme (L) Gaertn

compound

UV

IR Spectral Data

NMR Spectral Data

Chloroform

Extract of

Clerodendrum

Inerme (L) Gaertn compound I

λ max - 277 nm

Solvent used Hexane

C -H Stretch, aliphatic at

2959.51, 2916. 08 and

2849.12 Cm-1

C=O Stretch, aldehyde at

1731.03 Cm-1

C=C Stretch, aromatic at

1452.45 Cm-1

C-H Stretch , alkyl at 1376.18,

1245.84 Cm-1

C-H Stretch, alkane at

1020.99 Cm-1

C-H Meta di Subsitution at

887.03 Cm-1

δ(0.0072, 1.38) - Methine proton,

δ(2.3, 2.0) - acetate groups proton,

δ(2.8) - carbon bearing oxygen,

δ(0.88,0.95,0.96) - methylene groups proton,

δ(5.3) - double bond in Pentacyclic carbon framework

δ(4.19) - existence of hydroxyl group pf proton in carbon atom

UV SPECTROSCOPY

Fig 12 : CHLOROFORM EXTRACT OF CLERODENDRUM INERME (L) GAERTN COMPOUND I

Fig 13 :IR SPECTROSCOPY

Instrument Nam : SHIMADZU No. of Scans : 20 Date : 09.10.2009

Sample Name : Chloroform Extract of Clerodendrum Inerme (L) Gaertn compound I Time : 1:17:41 PM

Fig 14: NMR SPECTROSCOPY

CHLOROFORM EXTRACT OF CLERODENDRUM INERME (L) GAERTN COMPOUND I

B) Chloroform extract of Clerodendrum Inerme (L) Gaertn compound II:

Colour of the compound : Brown colour

Solubility : Methanol, water

TLC pattern : Single spot

Mobile phase : Hexane : Ethyl acetate (6 : 4)

Rf value : 0.46

Melting point : 205-207°C

UV Spectra of compound : λ max - 533 nm [Harborne. J.b. Phytochemical

Method].

IR Spectra of compound : IR γ κвr Max Cm⁻¹ 3744.92, 3414.12, 2950.51,

1730.15, 1456.30, 1385.19 [William Kemp. Org

spectroscopy].

NMR Spectra of compound : ¹H NMR (CDCL3) 2.3δ, 5.1δ, 0.87δ,

0.93δ,1.38δ[William Kemp.Org spectroscopy].

Table No: 12

Compound

UV

IR Spectral Data

NMR Spectral Data

Chloroform

Extract of

Clerodendrum

Inerme (L) Gaertn compound II

λ max -533 nm

solvent used Hexane

O-H Stretch at 3744.92 Cm-1

O-H Stretch, alcohol at

3414.12, 3476.81 Cm-1

C-H Stretch, aliphatic at

2950.51, 2924.18 Cm-1

C=O Stretch, aldehyde at

730.15 Cm-1

C=C Stretch, aromatic at

456.30 Cm-1

C-H Stretch , alkyl at

1385.19 Cm-1

C-O Stretch at 1175.65 Cm-1

δ(2.3,2.0) - acetate groups proton,

δ(5.1) - β substituent carrying hexahydro furofuran ring,

δ(0.87,0.88) - secondary methyl groups proton,

δ(0.93,0.95) - tertiary methyl groups proton,

δ(1.38) - methine protonSpectral data of the isolated compounds from Chloroform

Extract of Clerodendrum inerme (L) Gaertn

UV SPECTROSCOPY

Fig 15 : CHLOROFORM EXTRACT OF CLERODENDRUM INERME (L) GAERTN COMPOUND II

Fig 16 :IR SPECTROSCOPY

Instrument Name : SHIMADZU No. of Scans: 20 Date : 10.08.2009

Sample Name : Chloroform extract of Clerodendrum Inerme (L) Gaertn compound II Time : 2:51:46 PM

Fig 17: NMR SPECTROSCOPY

CHLOROFORM EXTRACT OF CLERODENDRUM INERME (L) GAERTN COMPOUNDII

Result:

Compound I:

The chemical characterization of the chloroform extract of Clerodendrum Inerme (L) Gaertn and the spectral data of the isolated compounds were summarized in table. 11 and Fig. 12, 13, 14 respectively. The λmax (277 nm) of the isolated compounds were taken to account of diterpenoid compound. The IR data shown characterized C-H stretching at 2959 Cm⁻¹, the carbonyl and C=C stretching at 1731 Cm⁻¹, 1452 Cm⁻¹ region respectively.

The isolated compound further assigned data from ¹H NMR spectra, signal of methane and methylene appeared in the region of δ 1.38 and δ 0.88. signal of acetate appeared in the region of δ 2.3. Further it indicated a single double bond at δ 5.3 accounted by pentacyclic carbon frame and the signal at δ 4.19 which showed the existence of OH group in carbon atom. Its NMR spectrum was consistent with proposed structure and clearly showed six quaternary methyl functionalities as three proton each singlet at 0.0072, 0.8594, 0.8732 respectively. Phytochemical screening and spectral data's suggest that the isolated compounds may be diterpenoid.

Compound II:

The chemical characterization of the chloroform extract of Clerodendrum Inerme (L) Gaertn and the spectral data of the isolated compounds were summarized in table. 12 and Fig. 15, 16, 17 respectively. The λmax (533 nm) of the isolated compounds were taken to account of diterpenoid compound. The IR data shown characterized O-H stretching at 3744 Cm⁻¹, O-H stretching alcohol at 3414 Cm⁻¹, the carbonyl and C=C stretching at 1730 Cm⁻¹, 1456 Cm⁻¹ region respectively.

The isolated compound further assigned data from ¹H NMR spectra, signal of methane and methylene appeared in the region of δ 1.38 and δ 0.87. Signal of acetate appeared in the region of δ 2.3. Further it indicated a signal at δ 5.1which showed the β substituent carrying hexahydrofurofuran ring . Its NMR spectrum was consistent with proposed structure and clearly showed six quaternary methyl functionalities as three proton each singlet at 0.9343, 0.9695, 0.8304 respectively. Phytochemical screening and spectral data's suggest that the isolated compounds may be diterpenoid.

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