School of Pharmacy-ITB

School of Pharmacy- ITB

CHROMATOGRAPHY

Physicochemical method to separate compounds based on difference of residence time of each component

Component A goes outside earlier than B, because the residence time of A < B

Stahl


Physicochemical method to separate compounds based on difference of movement of each component

Component A goes outside earlier than B, because A moves faster than B

Martin


Chromatography Classification

A. Based on mobile phase

Liquid chromatography Gas chromatography


B. Based on stationary phase

Liquid solid chromatography Liquid liquid chromatography


C. Based on process/mechanism

Partition chromatography Adsorption chromatography


D. Based on place of stationary phase

Planar liquid chrom Column liquid chrom


silica gel

Adsorbent

cellulose

alumina

kieselguhr


SILICA GEL

Silica gel S

Silica gel G


Silica gel GF254

Silica gel H

SILICA GEL

Silica gel S

Silica gel G

contain binding agent: gypsum

(CaSO4) 5 -15 %

contain binding agent: starch


Silica gel GF254

contain:

1. Binding agent: gypsum

2. Fluorescent indicator, that fluorescen at 254 nm

Silica gel H

without binding agent


Alumina G, F, H and P

ALUMINA

(Al2O3)

Base, neutral, and acid

alumina

Less polar than silica gel


contain

SiO2 > 89 %

Al2O3, Fe2O3, TiO2, CaO,

MgO, Na2O, etc

KISELGHUR

low activity



References

1. Shriner R.L., et.al., (2004), The

Systematic Identification of Organic

Compounds, 8th ed., John Wiley &

Sons, Singapore.

2. Cannell, R.J.P, (1998), Natural

Products Isolation, Humana Press,

New Jerse.

3. Gritter, R.J., Schwarting, A.E., (1991),

Pengantar Kromatografi, Penerbit ITB,

Bandung.

4. Geiss, F., (1987), Fundamental of TLC,

Huthig Verlag, Heidelberg.



Supporting of thin layer

Adsorbent

cellulose

Aluminium



Adsorption

Mechanism of TLC Silica Gel

Elution

Desorption


Pre coated plate (Merck)

Adsorbent/Stationary phase

* Plate is prepared using

Stahl-Desaga apparatus

powder


Single solvent

Mobile Phase/ Developing system

Mixture solvent

based on polarity

of substances


SPRAY REAGENT

Ce2(SO4)3

universal

Dragendorff

specific


Silica gel G

uv 254 nm


Silica gel G

H2SO4 10%


Silica gel GF254

uv 254 nm


The compounds

have conjugated

double bond The compounds have no

conjugated double bond

Silica gel GF254

H2SO4 10%


Direction of TLC development

Ascending development

Centrifugal development






CHROMATOTRON



CHROMATOTRON



CHROMATOTRON




1. Single development

KINDS OF TLC DEVELOPMENT

2. Multiple development

3. Two-dimensional development

Purity test

Extract monitoring

Chromatogram pattern

Marker content

Extract monitoring

Purity test


Single development TLC using

3 different mobile phases

Purity test by TLC silica gel

Two-dimensional development TLC

1.

2.


Visualization of spot in purity test

TLC silica gel G

Universal spray

reagent

H2SO4 5-10%

Ce2(SO4)3

TLC silica gel GF254

Universal spray

reagent

H2SO4 5-10%

Ce2(SO4)3

UV light 254 nm


Purity test by single development TLC

using 3 different mobile phases

A-B (8:2) B-C (9:1) E-F (11:2)

Less polar Semi-polar More polar


SP: silica gel G

Visualization:

H2SO4 10%

MP:

I 2 3

Purity test by single development TLC

using 3 different mobile phases

A-B (8:2) B-C (9:1) E-F (11:2)

Less polar Semi-polar More polar


SP: silica gel GF254

Visualization:

UV 254 nm

MP:

I 2 3

Purity test by two-dimensional TLC

A-B (8:3)

Less polar


SP: silica gel GF254 Visualization: UV 254 nm

MP:

A B

CD

After spotting

A B

CD

I

1 2


A-B (8:3)

Less polar



MP:

A B

CD

B C

DA

After turn right

90°

I

2 3


E-F (10:1)

More polar



B C

DA

After turn right

90°

B C

DA

MP:

II

3 4

Multiple development

After 1st development, dry it, then do the 2nd development

Direction of 1st development = 2nd development

The 1st mobile phase = 2nd mobile phase

Rf 0.1-0.2 by 1st mobile phase

Then perform 3rd, 4st or 5st development, until get the

good chromatogram pattern


• Saturation of chamber

• Adsorbent

• Temperature

• Humidity

• Pre coated plate or not

• Thickness of thin layer

• Method in activating plate

• The distance of spot from

plate side

FACTORS THAT INFLUENCE Rf VALUE


• Total of plates in chamber

• The distance that is taken

on by developm system

• The size of spot

• Composition of developm

system

• The purity of developm syst

• Concentration of sample

(big, tailing)


The usage of TLC

Analysis TLC

to get pure compound

Preparative TLC

Qualitative

Quantitative

Plate 0.25 mm

Plate 1-1.5 mm


• Result: bands form

TLC PREPARATIVE

• Sample is spotted 1-5 mm in bands form

• Thickness optimum of plate 1-1.5 mm

• Using pre coated plate for preparative

or prepare using by Stahl-Desaga

apparatus



band X

• Extraction with solvent

• Scrape off the bands

Bands visualization by:

UV light spray reagent

(at the plate side)


Determine Rf and max

TLC QUANTITATIVE

SPECTROPHOTODENSITOMETRY METHOD

Without or using spray reagent

Sample and standard are spotted

Standard: minimum 6 concentrations


Calculate concentration of sample using the

calibration curve!

Make a calibration curve!

The results:

- area under curve (AUC)

- energy that is absorbed (A)

Sample & standard are measured at the same



Mechanism in TLC cellulose = paper chromatograhy

Solute partitionbetween stationary phase

and mobile phase

School of Pharmacy- ITBSchool of Pharmacy- ITB

thin liquid layer on thesupporting agent

Cellulose (Whatman paper)

Stationary phase

supporting agent


Mobile phase in TLC cellulose

BAW

Forestal

Acetic acid 2-50 %

Water

butanol-acetic acid-water (4:1:5)

HCl-acetic acid-water (3:30:10)


Position of spot

Polarity of

Stationary

Phase

Mobile

Phase

Depend on


The usage of PC

Preparative AnalysisQualitative Analysis

Paper Whatman No.1

Thickness of paper: 0.16 mm

Paper Whatman No.3

Thickness of paper: 0.31 mm


Direction of development


Descending development





Descending development


Development time: 30’ – 12 hrs

PC QUALITATIVE

Chamber saturation: 24 hrs

Using Whatman paper No. 1


Extraction using solvent

PC PREPARATIVE

Bands are cut

Result: bands

Sample is spotted in band form

Using Whatman paper No. 3


TLC SILICA GEL AND PC

TLC silica gel: better separation than PC

TLC silica gel : shorter time than PC

TLC silica gel : can use corrosive spray reagent

TLC silica gel : can be used for orientation in CCC

TLC silica gel: bigger capacity than PC


TLC cellulose = PC, can’t use sulfuric acid conc.as spray reagent

TLC CELLULOSE and PC

TLC cellulose shorter time than PC

TLC cellulose spot smaller than PC

Mechanism TLC cellulose = PC


Thank you


Documents

School of Pharmacy-ITB