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CHROMATOGRAPHICAL
APPLICATIONS
Dr. S. TURE
UCL, Imperial College, Ph.D.
University of London
Chromatography
Chromatography basically involves the
separation of mixtures due to differences in
the distribution coefficient of sample components
between 2 different phases.
One of these phases is a mobile phase and
the other is a stationary phase.
Distribution Coefficient
Definition:
Concentration of component A in stationary phase
Concentration of component A in mobile phase
Different affinity of these 2 components to stationary
phase causes the separation.
Definition of Chromatography
Simplified Definition:
Chromatography separates the components of a
mixture by their distinctive attraction to the mobile
phase and the stationary phase.
Explanation:
•
•
•
•
Compound is placed on stationary phase
Mobile phase passes through the stationary phase
Mobile phase solubilizes the components
Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the
phases
Chromatography
Detector Signal
Chromatogram - Detector signal vs.
retention time or volume
1
2
time or volume
Types
Types of
of Chromatography
Chromatography
• Liquid Chromatography – separates liquid samples
with a liquid solvent (mobile phase) and a column
composed of solid beads (stationary phase)
• Gas Chromatography – separates vaporized samples
with a carrier gas (mobile phase) and a column
composed of a liquid or of solid beads (stationary
phase)
• Paper Chromatography – separates dried liquid
samples with a liquid solvent (mobile phase) and a
paper strip (stationary phase)
• Thin-Layer Chromatography – separates dried liquid
samples with a liquid solvent (mobile phase) and a glass
plate covered with a thin layer of alumina or silica gel
(stationary phase)
Types of Chromatography
LIQUID
MOBILE PHASE
Liquid-Liquid
Chromatography (Partition)
FORMAT
STATIONARY PHASE
Normal Phase
Liquid-Solid
Chromatography (Adsorption)
Solid
Liquid
Reverse Phase
Normal Phase
Mobile Phase - Nonpolar
Mobile Phase - Polar
Stationary phase - Polar
Stationary phase - Nonpolar
Reverse Phase
Sifat Fisika kimia kertas
untuk Kromatografi
•
•
•
•
Kertas terdiri dari 98 -99 %  selulose, 0,3-10 % 
selulose, dan 0,4-0,8 % pentosan. Juga mempunyai
gugus karboksilat yang dapat menimbulkan muatan
negatif pada kertas.
Kertas kromatografi terdapat kontaminan asam amino
yang mempunyai kadar Nitrogen 15 mg/kg
kertas.Senyawa lipofilik 25 mg/kg. dan senyawa an
organik (kadar abu) 0,04-0,07%,
Senyawa kontaminan tidak mengganggu dalam
pemisahan sampel pada kromatografi.Yang penting
kemampuan absorbsi dan kenaikkan kapileritas masingmasing kertas.
Whatman no.1 sebagai kertas standard yang digunakan,
no. 3MM digunakan untuk preparatif. Sedangkan no. 4
untuk elusi yang cepat, dan 33 ET untuk elusi sangat
cepat.
Paper
Chromatography
Paper chromatography. Molecules separate as they move up the paper. The
distance that the molecules travel depends on their size and solubility in the solvent.
Paper Chromatography
• Similar to TLC
• Stationary phase = H2O adsorbed by
cellulose
• Mobile phase = solvent
• Frequently used to polar compounds
– Amino acids, carbohydrates, etc.
Stationary phase:
Papers (cellulose), mechanism of separation is
through partition.
Mobile phase:
As TLC but more polar mixtures are usually used.
Buffers can also be used.
Sample application:
A line drawn by pencil, spot places
determined as dots. Apply sample as in TLC.
are
Paper Chromatography
Types of Paper Chromatography
• Radial chromatography
• Ascending chromatography
• Descending chromatography
Radial Chromatography
• In this type of chromatography, as the
pigment separates, the different colours
move outwards.
Radial Chromatogram
Ascending Chromatography
• The solvent moves
upwards on the
separating media
\
Development Type of Ascending:
1- Single development:
The solvent system is allowed to move through the
stationary phase one time only against gravity.
2- Repeated developments:
a- Multiple developments:
The plated are developed more than one time using
the same solvent system. The plates must be
completely dried after each development.
b- Stepwise developments:
The plated are developed more than one time using
different solvent systems.
3- Two-dimensional development:
Is used to verify if a given spot on TLC using
the above methods of development (one
Dimensional) is one pure compound or mixture
of two closely related compounds. The spots
are applied to one corner and the plate
developed as usual. The plate is then rotated 90
˚C and then developed again. This method allow
better separation of related compounds.
.
.
One compound
.
Two closely related compounds
.
Descending Chromatography
• The solvent moves
downwards on the
separating media.
• Pada kromatografi kertas lebih banyak digunakan
sistem menurun sehingga lebih cepat perambatan
nya. Keuntungan yang lain kromatografi kertas
dapat digunakan lembaran kertas yang lebih
panjang sehingga dapat dipisahkan campuran
yang lebih kompleks.
• Pemisahan yang terjadi berdasar atas peristiwa
partisi, karena fase gerak yang digunakan adalah
pelarut organik yang semi polar.
• Dan umumnya pelarut yang digunakan mengandung air sehingga air akan mudah terikat oleh
selulosa, dan selulosa dapat mengembang
menyerap air, maka air akan berfungsi sebagai
fase diam.
• Komposisi Fase gerak yang dikenal dengan nama
BAW (Butanol, Acetic Acid Water). Banyak
digunakan untuk pemisahan flavanoid.
 Fase gerak yang berupa pelarut organik akan
berkompetisi melarutkan sampel yang dianalisis
 Kromatografi kertas dapat diubah polaritasnya
dengan cara inpregnasi atau pembaceman,
antara lain dengan asetilasi, foforilasi, fomilasi.
Atau dengan senyawa yang bersifat lifofilik
seperti parafin, vaselin, undekan.
 Pembaceman sistemnya seperti pada KLT,
hanya pada kromatografi kertas dengan arah
yang menurun atau desenden.
 Dengan cara tersebut kromatografi kertaspun
dapat digunakan sebagai kromatografi fase
terbalik.
 Arah elusi dari kertas untuk kromatografi biasa
nya ditunjukkan oleh panah, kalau tak ada,
digunakan arah yang memanjang dari kertas.
Gambaran fase diam selulose
24
PENGUBAHAN GUGUS HIDROKSIL
 Asetilasi (CH3COOH)
 (C6H12O6)n + n x 4 (CH3COOH) 
O-CO- CH3
O OH
HH
O-CO- CH3 CH2 O-CO- CH
H
3
HO
H
O-CO- CH3
25
PENGUBAHAN GUGUS HIDROKSIL

OH
 Fosforilisasi (HO-P=O = H3PO4
OH
OH
O OH
HH
OH H
CH2-O- PO4
HO
(OH)2
O- PO4
H
(OH)2
26
Principles of Paper
Chromatography
• Capillary Action – the movement of liquid within the spaces of a
porous material due to the forces of adhesion, cohesion, and
surface tension. The liquid is able to move up the filter paper
because its attraction to itself is stronger than the force of gravity.
• Solubility – the degree to which a material (solute) dissolves into
a solvent. Solutes dissolve into solvents that have similar
properties. (Like dissolves like) This allows different solutes to be
separated by different combinations of solvents.
Separation of components depends on both their solubility in the
mobile phase and their differential affinity to the mobile phase and
the stationary phase.
Visualization (Detection of spots):
A- Universal methods:
1- Destructive methods:
The plated are sprayed with corrosive reagents and then
heated in oven where organic compounds will give charred
spots. After this treatment the materials can not be
recovered.
e.g. Anisaldehyde / H2SO4
Vanillin / H2SO4
2- Non – Destructive methods:
In these methods the materials can be
recovered.
–
–
–
–
Day light for colour compounds.
UV
light
for
fluorescent
compounds
(conjugated double bonds).
I2 vapour for any compounds contain at least
one double bond
Spray with water where organic compounds
appear as white opaque spots.
B- Specific Methods:
•
These reagents are used for the detection of
certain classes of compounds. They are usually
destructive.
•
Dragendorff΄s reagent for Alkaloids.
•
Ferric Chloride (FeCl3) for phenolic compounds.
•
Aniline phthalate for sugars.
•
Ninhydrine for nitrogenous
Amines, Amino acids.
compounds
as
Tabel Beberapa penggunaan pelacak bercak
pada kromatografi kertas
Nama
pereaksi
l.Sinar UV254 nm
Analit
Senyawa flouresen
Seny.Amin
2 lodoplatinat
ter/kuaterner
•
3.Pereaksi furfural Turunan Karbamat
Nama
pereaksi
Analit
9. Pereaksi
Marquis
Tur. morfin
Fenol, aril
10. Peraksi Millon amin Ikatan
.KMn04 +
tak jenuh
Sebyawa fiuoresen Amin 11
ter/kuater.
Turunan
as.sulfet
4.Pereaksi
Simon HeterosikUk
12 .Ninhidrin
karbamat
kanabinol,As. amino
Heterosiklik amin amin
13. Nitroso-naftol primer
sulfonamida Alkaloid/Amin
kuar
Ergot
5.DABdlm etanol
Karbinol/Sulfonamid 14. Pereaksi
alkaloid
Mandelin
Turunan
a
seny.organik
6.Dragendorff
ajmalin
Alkaloid/amin
kuater,
•
15. Vanilinas.
Barbiturat
Ter.Heksa/penta
Sulfat
fenetoin,
7. Uap iodium
klorfenol
8. UapNO2
S
Ikatan rangkap/
senyawa organik
31
Ter.Heksa(penta klorfenol) Ikatan rangkap,
31
Rate of flow (Rf Value):
Distance traveled by the spots
Rf = ----------------------------------------Distance traveled by the solvent
The Rf of any compound must be less than one.
Solvent front
Distance travelled by
the solvent
Distance travelled by
the spot
Start line
• Tailing in Paper Chromatography:
In some cases instead of getting round spots a Tailed or
comet like spots are obtained leading to overlapping of the
spots and poor resolution.
Tailed or comet like spot
Reasons and solution for tailing problem:
1-Ionic characters of acids and bases when they are
chromatographed under neutral conditions.
Solution: add acids or bases to the developing system.
2-Application of large amounts of material.
Solution: decrease conc. of material.
3-Unproper choice of solvent system.
Solution: change the solvent system.
Application:
1- Qualitative:
 Identification through comparison of the Rf value with that
of Reference material.
 Determination of Complexity of mixtures. That will be
indicated from number of spots.
 Determination the purity of materials.
 Monitoring the progress of Chemical reactions.
 Monitoring of column chromatography.
 Development of finger print TLC for extracts, volatile oils or
pharmaceutical preparation for future identification and
comparison.
In this application plates 5×5, 5×10 cm with thin film of
coating material are usually used.
•
2- Quantitative:
In this case an accurate volume of samples are applied
using syringes. The dimensions of plates range from 5x10 to
20x20 according to the number pf spots used. The plates
are developed as usual in the chromatographic tanks. After
development the concentration of material can be
determined by:



Spot area measurement: Which is directly proportional to the
conc. of materials.
Photodensitometry: Measure transmittance, reflection or
fluorescence of spots.
Radioactivity: For radioactive material.
These measurements are done using TLC Scanner
connected to computer that perform all calculations.
Paper Chromatography
Experiment
What Color is that Sharpie?
Overview of the Experiment
Purpose:
To introduce students to the principles
and terminology of chromatography and
demonstrate separation of the dyes in
Sharpie Pens with paper
chromatography.
Time Required:
Prep. time: 10 minutes
Experiment time: 45 minutes
Materials List
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•
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•
•
•
•
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•
6 beakers or jars
6 covers or lids
Distilled H2O
Isopropanol
Graduated cylinder
6 strips of filter paper
Different colors of
Sharpie pens
Pencil
Ruler
Scissors
Tape
Preparing the Isopropanol
Solutions
• Prepare 15 ml of the following isopropanol solutions
in appropriately labeled beakers:
- 0%, 5%, 10%, 20%, 50%, and 100%
Preparing the
Chromatography Strips
• Cut 6 strips of filter paper
• Draw a line 1 cm above
the bottom edge of the
strip with the pencil
• Label each strip with its
corresponding solution
• Place a spot from each
pen on your starting line
Developing the
Chromatograms
• Place the strips in the beakers
• Make sure the solution does
not come above your start line
• Keep the beakers covered
• Let strips develop until the
ascending solution front is
about 2 cm from the top of the
strip
• Remove the strips and let them
dry
Developing the
Chromatograms
Developing the
Chromatograms
Observing the
Chromatograms
0%
20%
50%
70%
Concentration of Isopropanol
100%
Black Dye
1. Dyes separated – purple and black
2. Not soluble in low concentrations of
isopropanol
3. Partially soluble in concentrations of
isopropanol >20%
0%
20%
50%
70%
Concentration of Isopropanol
100%
Blue Dye
1. Dye separated – blue
2. Not very soluble in low
concentrations of isopropanol
3. Completely soluble in high
concentrations of isopropanol
0%
20%
50%
70%
Concentration of Isopropanol
100%
Green Dye
1. Dye separated – blue and yellow
2. Blue – Soluble in concentrations
of isopropanol >20%
3. Yellow – Soluble in concentrations
of isopropanol >0%
0%
20%
50%
70%
Concentration of Isopropanol
100%
Red Dye
1. Dyes separated – red and yellow
2. Yellow –soluble in low concentrations of isopropanol and
less soluble in high concentrations of isopropanol
3. Red – slightly
soluble in low
concentrations
of isopropanol,
and more
soluble in
concentrations
of isopropanol
>20%
0%
20%
50%
70%
Concentration of Isopropanol
100%
Case One
“Signed with a Kiss”
The Discovery…
• Before third period, Marie goes to her locker to
grab her chemistry book. She and Christopher
have shared this locker for the last semester
when they started going together
• As she pushes Christopher’s geometry books
aside, it falls to the floor and a note falls out.
– “…can’t wait to see you again, baby. Last
nite was so much fun! Call me on my cell
after school today.”
• Marie was already bugged out by the words of
the note, but on top of that, the girl had the
nerve to sign the note with nothing but a kiss!
The Plan
• After school, Marie calls Mark for support. Between sobs, Marie
explains to Mark, “I was so cold to Christopher after Chem class. I’m
afraid he thinks I know about the note.” Mark reassures her, “Naw,
he has no idea.
• “Okay,” she says, “let’s continue with our plan.”
• Just make sure you get a sample from each of those girls. Oh yeah,
and make sure that you have plenty of nail polish remover. I think
that will be the best solvent to separate the mixtures. When I come
over tomorrow, I’ll bring the coffee filters.”
• The next day, Marie and Mark meet up at her house to begin the
investigation phase of their plan.
– Begin lipstick chromatography lab
– Paper Chromatography.ppt
How to Catch Your Man Cheating!
•
Lipstick Chromatography Lab Protocol
Materials:
Chromatography paper or Coffee filters
Scissors
3 different Lipsticks similar in shade, but different brands
Acetone or Nail Polish remover with Acetone
Beaker or Cup large enough for 3 strips of paper (about 500 ml)
Tape
Procedure:
1. The teacher has smeared samples of the lipstick from the note and each of the
suspects onto filter paper. Each group will analyze one suspect or the sample from the
note 3 times.
2. Carefully pour 10 ml of solvent into the beaker.
3. Place all three strips of paper into the beaker so that the paper touches the solvent,
but that the level of the solvent does NOT reach the lipstick.
4. Secure the top of each paper strip to the beaker with tape if necessary to keep it from
slipping.
5. After 15 minutes remove all the samples from the beaker and place the papers flat on
the bench top.
6. Measure the distance the acetone traveled up each strip of paper. Also measure the
distance each component moved up the paper.
7. Make a data table with average Rf value for each component in your lipstick sample.
Epilogue
• After discovering that Christopher and her girl
Muhsinah were not the friends she thought they
were, Marie confronted them with the evidence.
Christopher and Muhsinah were shocked at her
use of chemistry to catch them. Marie recovered
from the nasty breakup with Christopher with the
help of Mark's comforting shoulder... ;)
•
This scene and laboratory protocol were adapted from, "Who's Lipstick?" in Crime Scene
Investigations by Pam Walker and Elaine Wood, 1998.
The images of the kiss and paper chromatograpy where found at http://www.ausetute.com.au/
chromato.html and http://www.consumerreports.com, respectively.