Course Name: Pharmacognosy ITopic: Chromatography and Isolation Techniques

Instructor: Dr. Javed AhamadAssistant Professor

PhD Pharmacognosy & Phytochemistry

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TISHK INTERNATIONAL UNIVERSITY

PHARMACY FACULTY

PHARMACY DEPARTMENT

3rd Grade- Fall Semester 2020-2021

Contents

• Introduction to chromatography

• Isolation of Natural Compounds

Column chromatography (CC)

Prep-TLC

Flash chromatography (FC)

Prep-HPLC

Prep-GC

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• Chromatography represents a group of methods for separating

molecular mixtures that depend on the differential affinities of the

solute between two immiscible phases.

• Among the various methods of separating plant constituents, the

chromatographic methods originated by Tswett is one of the most

commonly used techniques of general application.

• In 1944, Consden, Gordon and Martin introduced a method of

partition chromatography using strip of filter paper for the analysis

of amino acids.

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Chromatography

Principles of Chromatography

1. Adsorption chromatography

• Adsorption chromatography is a process of separation of components

in a mixture.

• It introduced into chromatography system based on the relative

differences in adsorption of components to the stationary phase

present in the chromatography column.

2. Partition chromatography

• Partition chromatography is process of separation whereby the

components of the mixture get distributed into two liquid phases due

to differences in partition coefficients during the flow of mobile

phase in the chromatography column.

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1. Column chromatography (CC)

2. Preparative-Thin layer chromatography (prep-TLC),

3. Flash chromatography (FC)

4. Preparative-High performance liquid chromatography (prep-

HPLC)

5. Preparative-Gas chromatography (prep-GC)

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Isolation of Natural Compounds

Column chromatography (CC)

• Basically it is a liquid chromatography in which mobile phase in the

form of liquid passes over the stationary phase packed in a column.

• The column is either a glass or metallic column.

• In column adsorption chromatography, large number of adsorbents

are used like silica gel, starch, calcium carbonate, lime, alumina etc.

• To optimize the resolution various mobile phases are used either

singly or in combination like petroleum ether, cyclohexane,

chloroform, acetone, ethyl acetate, methanol etc.

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7Fig. Column chromatography (CC)

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Preparative-Thin Layer Chromatography (prep-TLC)

• Thin layer chromatography is planer chromatography and it is one

of the most popular and simple chromatographic technique used

qualitative analysis of natural products.

• Prep-TLC is a chromatographic technique performed with the aim of

isolating milligram to gram quantities of compounds for structural

elucidation, for different other analytical purposes, or for

determination of biological activity.

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• In TLC fingerprinting, the data is presented as:

• The number of spots

• Retention factor (Rf) values

• The color of the separated bands

• In the phytochemical evaluation of herbal drugs, TLC is being employed

extensively for the following reasons:

• It enables rapid analysis of herbal extracts with minimum sample

clean-up requirement.

• It provides qualitative and semi quantitative information of the

resolved compounds.

• It enables the quantification of chemical constituents in the form of

HPTLC.

• Prep-TLC is applied for isolation of phytochemicals.11

High Performance Thin Layer Chromatography

HPTLC is an enhanced form of thin layer chromatography (TLC).

HPTLC commonly applied for the identification, the assay and the

testing for purity, stability, dissolution or content uniformity of raw

materials and formulated products.

This is a flexible and cost-effective techniques and present the

advantage of the simultaneous processing of standards and samples

with versatile detection possibilities, including a great variety of post-

chromatographic derivatization reagents.

It also provides the means for flexible screening procedure, qualitative

analysis and quantitative determination.

HPTLC Instrumentation CAMAG WinCATS software

TLC SPRAYERTwin Trough TLC ChambersSample Applicator:

(Camag Linomat 5)

TLC SCANNER (Densitometer)

CHROMATOGRAPHY

VISUALISATION – (UV

Cabinet)

O

O

O

O

HO

O

OH

CH2OH

HO OH

H

Equipment: CAMAG HPTLC (Muttenz, Switzerland), Linomat IV sample applicator equipped

with a 100 μL Hamilton (USA) syringe and winCAT software.

HPTLC plate: Silica gel 60 F254

Mobile phase: Ethyl acetate-methanol-water (80:15:5 v/v)

Scanning: 246 nm

Figure: HPTLC plate showing bands of standard swertiamarin (track 1-6) and extracts of

Enicostemma littorale (track 7-23). The plate was developed with ethyl acetate-methanol-water

(80:15:5 v/v) and visualized under UV light at 246 nm.

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Preparative-High-performance liquid chromatography (prep-

HPLC)

• Prep-HPLC is a sophisticated, relatively fast, accurate, versatile and

robust technique for isolation of phytochemicals from complex

mixtures.

• Over the past decades, HPLC has most extensive application in the

analysis of herbal medicines.

• Reversed-phase (RP) columns may be the most popular columns

used in the analytical separation of herbal medicines.

• HPLC is a chromatographic technique that can separate a mixture of

compounds and is used in herbal industry and analytical chemistry to

identify, quantify and purify the individual components of the mixture.

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• The main difference between prep-HPLC and other ‘‘lower

pressure’’ column chromatographic system is the consistency and

size of the particles (3 and 10 mm) in the stationary phase.

• The small particle size results in having to use high pressures (up to

3-4000 psi) to push the mobile phase through the system which

helps better and efficient separation of phytocompounds.

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• In preparative-HPLC (pressure >20 bar), larger stainless steel

columns and packing materials (particle size 10-30 μm) are needed.

The examples of normal phase silica columns are:

• Kromasil 10 μm, Kromasil 16 μm and Chiralcel AS 20 μm

The examples of reverse phase columns are:

• Chromasil C18 and Chromasil C8

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HPLC as compared with the classical LC technique:

High resolution

Small diameter (4.6 mm), stainless steel, glass or titanium

columns

Column packing with very small (3, 5 and 10 μm) particles

Relatively high inlet pressures and controlled flow of the mobile

phase

Continuous flow detectors capable of handling small flow rates

and detecting very small amounts

Rapid analysis.

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Equipment: A Shimadzu model HPLC equipped with quaternary LC-10A VP pumps, variable

wavelength programmable UV/VIS detector SPD-10AVP column oven (Shimadzu), SCL 10AVP

system controller (Shimadzu), and Class-VP 5.032 software. Hamilton 25μL HPLC syringe.

Chromatographic column: Reverse phase C18, 250 X 4.6 mm, 5 μm Zorax RP-HPLC.

Mobile phase: Methanol-water (80:20)

Flow rate : 1.0 mL/min

Scanning : 238 nm

Figure: HPLC Chromatogram (A) standard swertiamarin and

(B) extract of E. littorale showing Rt at 3.517 min.

A

B

Determination of swertiamarin by HPLC method

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Medium pressure liquid chromatography (MPLC)

• MPLC is a type of chromatographic separation procedure most

appropriate for extensive research centre for the isolation of pure

compounds from partially purified fractions.

• The procedure is accordingly complimentary to the flash

chromatography; pressure range between 5-20 bars with appropriate

flow rate, flash chromatography can be used to increase the speed

without lowering the quality of the separation.

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Flash chromatography (FC)

• It provides a rapid and inexpensive general method for the

preparative separation of mixtures requiring only moderate

resolution.

• It can be applied to normal-phase and Reverse Phase separations.

• FC can endure relatively high flow rate with low pressure, offering

good separation in a short time under appropriate chromatographic

conditions.

• Depending on the applied pressure, one refers to FC (0.1-5 or 10

bars) or medium-pressure LC (MPLC; 5 or 10-50 bars).

• FC has been used extensively for rapid and easy purification of

natural product.22

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Flash chromatography (FC)

Gas Chromatography (GC-MS)

• It is well-known that many pharmacologically active components in

herbal medicines are volatile chemical compounds.

• Thus, the analysis of volatile compounds by gas chromatography is very

important in the analysis of herbal medicines.

• The GC analysis of the volatile oils has a number of advantages.

• Firstly, the GC of the volatile oil gives a reasonable “fingerprint” which

can be used to identify the plant.

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Preparative Gas Chromatography (Prep-GC)

• Gas chromatography (GC) with high separation efficiency and fast

separation and analysis makes it potentially ideal method for separation of

volatile constituents.

• The injection port, column, split device and trap device of GC equipment

must be modified for preparative separation due to lack of commercial

prep-GC.

• Prep-GC becomes an important separation technique for separation of

essential oil compounds; however, a heavier sample load and the large-

diameter preparative column employed decreased the efficiency of prep-

GC.

• Especially, the hyphenation with MS provides reliable information

for the qualitative analysis of the complex constituents.

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Fig. Gas Chromatography (GC-MS)

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Figure. GC/MS chromatogram of oil from drupes of Olive

Thank you for your kind attention

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