Post on 21-Mar-2022
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
School of Pharmacy- ITB
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
School of Pharmacy- ITB
Chromatography Classification
A. Based on mobile phase
Liquid chromatography Gas chromatography
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B. Based on stationary phase
Liquid solid chromatography Liquid liquid chromatography
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C. Based on process/mechanism
Partition chromatography Adsorption chromatography
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D. Based on place of stationary phase
Planar liquid chrom Column liquid chrom
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SILICA GEL
Silica gel S
Silica gel G
contain binding agent: gypsum
(CaSO4) 5 -15 %
contain binding agent: starch
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Silica gel GF254
contain:
1. Binding agent: gypsum
2. Fluorescent indicator, that fluorescen at 254 nm
Silica gel H
without binding agent
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Alumina G, F, H and P
ALUMINA
(Al2O3)
Base, neutral, and acid
alumina
Less polar than silica gel
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contain
SiO2 > 89 %
Al2O3, Fe2O3, TiO2, CaO,
MgO, Na2O, etc
KISELGHUR
low activity
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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.
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Pre coated plate (Merck)
Adsorbent/Stationary phase
* Plate is prepared using
Stahl-Desaga apparatus
powder
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Single solvent
Mobile Phase/ Developing system
Mixture solvent
based on polarity
of substances
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Silica gel GF254
uv 254 nm
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The compounds
have conjugated
double bond The compounds have no
conjugated double bond
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
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Single development TLC using
3 different mobile phases
Purity test by TLC silica gel
Two-dimensional development TLC
1.
2.
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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
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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
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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
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SP: silica gel GF254
Visualization:
UV 254 nm
MP:
I 2 3
Purity test by two-dimensional TLC
A-B (8:3)
Less polar
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SP: silica gel GF254 Visualization: UV 254 nm
MP:
A B
CD
After spotting
A B
CD
I
1 2
Purity test by two-dimensional TLC
A-B (8:3)
Less polar
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SP: silica gel GF254 Visualization: UV 254 nm
MP:
A B
CD
B C
DA
After turn right
90°
I
2 3
Purity test by two-dimensional TLC
E-F (10:1)
More polar
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SP: silica gel GF254 Visualization: UV 254 nm
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
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• 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
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• 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)
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The usage of TLC
Analysis TLC
to get pure compound
Preparative TLC
Qualitative
Quantitative
Plate 0.25 mm
Plate 1-1.5 mm
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• 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
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• Extraction with solvent
• Scrape off the bands
Bands visualization by:
UV light spray reagent
(at the plate side)
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Determine Rf and max
TLC QUANTITATIVE
SPECTROPHOTODENSITOMETRY METHOD
Without or using spray reagent
Sample and standard are spotted
Standard: minimum 6 concentrations
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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
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Mechanism in TLC cellulose = paper chromatograhy
Solute partitionbetween stationary phase
and mobile phase
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thin liquid layer on thesupporting agent
Cellulose (Whatman paper)
Stationary phase
supporting agent
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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)
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Position of spot
Polarity of
Stationary
Phase
Mobile
Phase
Depend on
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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
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Development time: 30’ – 12 hrs
PC QUALITATIVE
Chamber saturation: 24 hrs
Using Whatman paper No. 1
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Extraction using solvent
PC PREPARATIVE
Bands are cut
Result: bands
Sample is spotted in band form
Using Whatman paper No. 3
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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
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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
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