Phytochemical screening Herbs

Extraction Method

Galenic Extracts
TLC monitoring of extracts

Selected extract
Fractionation Method (I )

Fractions
TLC monitoring of fractions

Selected fractions
Fractionation Method (II)

Subfractions
TLC/PC monitoring of subfractions

Selected subfractions
Purification

Characterization
ISOLATE Purity test
Identification
 Kuliah Fitokimia
• Pendahuluan (1 x) 21/9/17
• Skrining Fitokimia (1 x) 28/9/17
• Ekstraksi & Fraksinasi (2 x) 5 & 12/10/17
• Pemurnian & Uji kemurnian (1 x) 19/10/17
• Elusidasi struktur isolat (2 x) 26/10 & 2/11/17
• UTS 6-18 Nopember 2017
• Alkaloid (2 x) 23 & 30/11/17
• Flavonoid (2 x) 7 & 14/12/17
• Terpenoid (2 x) 21 & 28/12/17
• Review (1 x) 4/1/18
• UAS 8-20 Januari 2018
2
 PEMURNIAN
10/11/2017 2:00:18 PM 3
 Separation Techniques
• How would you separate…..?

− Two liquids that boil at different temperatures

− Sand and water
− Oil and iron filings
− Oil and paper
− Stones and peanuts
− A pile of sticks and dry leaves
− A mixture of dogs and cats
Summary: The Organization of Matter
 PEMURNIAN

 Metode memperoleh isolat murni

(hanya satu senyawa dalam satu wadah)

A pure substance contains only one type of

substance, and is not mixed with any other
substance.

Isolat murni : jika kemurniannya > 90%

 7

Sinensetin ( Flavonoid )
3’,4’,5,6,7 penta-metoksi flavon

OCH3

OCH3

H3CO O

H3CO

OCH3 O
 2 3
1

6
5 7
4

8 9
10
 Kepolaran senyawa tergantung pada :
1. Gugus fungsi yang paling dominan
* Gugus fungsi polar : -OH, -COOH, -SH, -NH2, dll
* Gugus fungsi semi polar : -RCOOR, -ROR, dll
* Gugus fungsi non-polar : -C-C, -C-H, benzen

2. Jumlah atom C (panjang rantai C)

* H2O, CH3OH, C2H5OH, C10H21OH
* HCOOH, CH3COOH, C2H5COOH, C10H21COOH
* Jika hanya ada 1 gugus polar (-OH, -COOH, dll)
C < 5  polar,
C = 5 – 10  semi polar
C > 10  non polar
 -NR2 -RCONR2
-RCH3 -COOR -RNH2
-C6H6 -COR -COOH

-RX -CO -OH

-RCN -S(O)2NH2
-RNO2 -RSO2R
-RSH

Non-polar Semi polar Polar

 Pelarut Td C/ Tetapan Viskositas
pengembang* 760 dielektrik pada 20C
torr pada 20C
n- heksana 68.7 1.89 0.33
Non polar

heptana 98.4 1.92 0.41

sikloheksana 81.4 2.02 1.02
karbontetraklorida 76.8 2.24 0.97
benzena 80.1 2.28 0.65
kloroform 61.3 4.81 0.58
Semi polar

eter (dietil eter) 34.6 4.34 0.23

etil asetat 77.1 6.02+ 0.45
piridina 115.1 12.3+ 0.97
aseton 56.5 20.7+ 0.37
etanol 78.5 24.30 1.2
Polar

metanol 64.6 33.62 0.59

air 100.0 80.37 1.01

untuk pelarut pengembang tambahan, dan berbagai sifatnya, lihat Biochemistry

Handbook (Berlin-Gottingen-Heidelberg; Springer Verlag, 1964) dan Handbook of
Chemistry and physics (40th ed. FA
Clevelanf, Ohio;dan
3213 Kromatografi Chemical Rubber Co.,1959)
10/11/2017 2:00:18 PM 13
+ pada 25C, http://www.pan-biotech.de/de/Produkte/R_560-Zusammenst.htm
Elektroforesis
Non polar
Semi polar
Polar

FA 3213 Kromatografi dan

10/11/2017 2:00:18 PM 14
Elektroforesis
DETEKSI
Pereaksi-pereaksi untuk mendeteksi alkaloid:
Pereaksi semprot pada pelat KLT:
• Pereaksi Dragendorf: larutan senyawa kompleks Bi(NO3)3/KI dalam asam
Positif  noda berwarna jingga kemerahan
 Chemical content of Curcuma domestica (kunyit)
and C. xanthorriza (temulawak)
C.domestica

C.xanthorrhiza O O
H3CO OCH3

HO OH

Curcumin
Xanthorrhizol
O O
H3CO

HO OH
Curcumin
Demetoxycurcumin
Demetoxy O O
curcumin

Bis
demetoxy HO OH
curcumin
Bisdemetoxycurcumin
ISOLASI: skema umum

Pelarutan dengan
cara perendaman
EKSTRAKSI

Partisi asam-basa/
Pengambilan Kromatografi Cair Vakum (KCV)
semua FRAKSI
komponen
yang terlarut
• Partisi (ECC)
Pengambilan • Kromatografi kolom
hanya FRAKSI
komponen LANJUTAN
alkaloid
• Kromatografi
Penyederhana • Kristalisasi
an jumlah PEMURNIAN
komponen

Mendapatkan masing-
masing komponen murni
Phytochemical screening Herbs
Extraction Method

Galenic Extracts
TLC monitoring of extracts

Selected extract
Fractionation Method (I )

Fractions
TLC monitoring of fractions

Selected fractions
Fractionation Method (II)

Subfractions
TLC/PC monitoring of subfractions

Selected subfractions
Purification

Characterization
ISOLATE Purity test
Identification
 Bioactivity guided fractionation
Untuk memperoleh senyawa aktif dari suatu
bahan alam, dapat dilakukan dengan proses
isolasi yang dipandu dengan uji aktivitas.
Setiap tahapan isolasi (mulai dari ekstraksi,
fraksinasi, dan pemurnian isolat) selalu
dipantau dengan pengujian aktivitas.
Fraksi yang aktif yang diproses untuk diisolasi
lebih lanjut, hingga diperoleh isolat aktifnya.
Some terms :
– Solute : the solid that dissolves
– Solvent : the liquid that does the dissolving
– Solution : solid + solvent
– Residue : the insoluble solid trapped in the filter paper
– Filtrate : the liquid that passes through the filter paper

– Sublimate : the condensed solid produced during

sublimation
Penapisan fitokimia Simplisia
Skema
Metode ekstraksi

Sediaan galenik Ekstrak Uji aktivitas

Pemantauan ekstrak

Ekstrak yg dipilih

Metode fraksinasi ke 1

Fraksi Uji aktivitas

Pemantauan fraksi

Fraksi yg dipilih

Metode fraksinasi ke 2

Subfraksi Uji aktivitas

Pemantauan subfraksi

Subfraksi yg dipilih
Pemurnian

Karakterisasi Uji kemurnian

ISOLAT
Identifikasi Uji aktivitas
 PEMURNIAN

 Metode memperoleh isolat murni

(hanya satu senyawa dalam satu wadah)

A pure substance contains only one type of

substance, and is not mixed with any other
substance.

Isolat murni : jika kemurniannya > 90%

 7

Sinensetin ( Flavonoid )
3’,4’,5,6,7 penta-metoksi flavon

OCH3

OCH3

H3CO O

H3CO

OCH3 O
Beberapa metode pemurnian
1. Sublimasi  spesifik (unt senyawa tertentu)
2. Destilasi  spesifik (unt senyawa tertentu)
3. Kristalisasi  spesifik (unt senyawa tertentu)
4. Pengendapan
5. KLT preparatif Umum
6. KKt preparatif (dapat untuk banyak
7. Kolom kromatografi senyawa)
8. dll
 1. Sublimation
• Sublimation differs from ordinary distillation
because the vapour condenses to a solid
instead of a liquid.
• Usually, the pressure in the heated system is
diminished by pumping, and the vapour is
condensed (after travelling a relatively short
distance) on to a cold finger or some other
cooled surface.
 Sublimation
Sublimation
used when one of the
solid sublimes
Camphor or
Eg. Caffeine
Camphor or caffeine
with other substances Mixture of camphor
or caffeine with
other substances
 2. Distillation
• Some Terms Used :
– Distillate - the liquid that distils over
– Miscible liquids - liquids that mix completely to
form a single layer
– Immiscible liquids - liquids that do not mix

• A nicotine can be separated and collected by

distillation.
 ALKALOIDS PURIFICATION BY DESTILLATION

• Alkaloids with low molecular weight or volatile liquid

For example:
nicotine , sparteine and coniine Distillation

An aqueous extract of plant is made alkaline

with caustic soda or sodium carbonate and
alkaloid is distilled off with steam.
The distillate is extracted with solvent to isolate
the desired alkaloid.
 Destillation of Alkaloids

Volatile Alkaloids
• The best way for their extraction and purification is steam
distillation
• Plant material + water + Fixed alkali
Heat steam contain alkaloids
received in acidic solution.

• Fractional Distillation:
e.g. Separation of Nicotine and Anabasine
 Principle of Distillation
A liquid boils and turns into vapour at its boiling
point.
When the vapour is condensed, the (pure) liquid is
obtained again.
 Simple Distillation
3. the condenser is cold,
2. .. vapourise. so the vapour condenses
The vapour rises to liquid water.
thermometer
up the flask

flask
sea water condenser

Boiling chips
4. Pure water
drips into the
beaker. It is
1. Solution is heated, distillate distilled water.
causing the solvent to
…
 Simple Distillation

thermometer

Water out
flask
sea water condenser

Boiling chips

To maintain even
boiling, with not too
much bumping Water in
 Liebig Condenser

Condensed vapour in liquid

Vapour enters form (distillate) leaves

Direction of water flow

Water Cold
out water in
Water flows in anti-current to the flow of vapour.
 Liebig Condenser

Condensed vapour in liquid

Vapour enters form (distillate) leaves

Direction of water flow

Water Cold
Thisout
is to make sure the coldest part of the
water in
condenser is just before the vapour escapes.
 Simple Distillation
Qns. : Where is the thermometer placed?
What is the reason for this?

thermometer

flask
sea water condenser

Boiling chips

distillate
 Simple Distillation
Thermometer placed at the side arm of the flask so
that it records the temperature of the vapour as
it enters the condenser.

thermometer

flask
sea water condenser

Boiling chips

distillate
 Separating miscible liquids -
Fractional distillation

• miscible liquids can only be separated

by fractional distillation if they have
different boiling points .
• Eg. mixture of essential oil components.
 Separating miscible liquids -
Fractional distillation
thermometer
Fractionating Water out
column
condenser

flask distillate
Water in

Boiling chips mixture of esential

oil components
 Separating miscible liquids -
Fractional distillation
2. The fractionating column 3. Eventually, the
is packed with glass beads liquid with the lower
to increase its surface area. boiling point reaches
Vaporisation followed by the top and distils
condensation takes place over.
many times as the vapour is
swept upwards.

1. When heated, the

liquid with the lower
boiling point will
vaporize more
readily.
 Separating miscible liquids -
Fractional distillation

4. The temperature
stays constant at 78°C.
When all the
component A has
distilled over, the
temperature reading
rises above 78°C. At
100°C, component B
starts to distil over.

5. The receiver is changed

to collect each distillate
separately.
 Separating miscible liquids -
Fractional distillation
Sketch a graph of temperature versus time to show the
changes in temperature readings throughout the distillation.

temperature

Compound B
100°C
Compound A
78°C

time
 Separating miscible liquids -
Fractional distillation

Note : glass
• The glass beads in the beads
fractionating column
provides a large surface
area so that condensation
occurs more readily.
• The liquid with the lower
boiling point distils over first,
followed by the liquid with
the next higher boiling point.
 Separating miscible liquids -
Fractional distillation

Note : glass
• If the liquids in the mixture beads
have the same boiling point,
fractional distillation is not
possible.
• If the difference in boiling
point is great, fractional
distillation occurs readily.
Fractional distillation of crude oil
 Separating the Solvent from the Solution

Evaporation Crystallisation Distillation

to dryness
or
Precipitation solute solvent

solution
 3. Crystallisation
• used to recover a soluble solid from its solution
• for solids that decompose on heating
• e.g. alkaloids, steroids, etc.

Steps :
– The solution is heated (evaporated) to saturation
point OR „heated to remove most of the solvent’
– The saturated solution is left to cool; crystals are
formed.
– The crystals are removed by filtration. To purify the
crystals, they can then be washed with cold
distilled water and dried between filter papers.
 Crystallisation - the Principle behind

• Substances are usually more soluble in hot water than

cold water e.g. more caffeine.

• When the hot saturated solution is cooled, the cooled

solution is unable to hold as much solute as when it was
hot. The extra solute that cannot remain dissolved
appears as crystals.
 Teknik Kristalisasi
Beberapa senyawa mudah membentuk kristal

1. Rekristalisasi
Larutkan sejumlah kristal/serbuk dalam pelarut tertentu
Biarkan pelarut menguap dan terbentuk kristal

2. Pencucian
a. Tambahkan sejumlah kristal dengan sedikit pelarut tertentu,
sehingga kristal larut, sedangkan pengotor tidak larut, atau
b. Tambahkan sejumlah kristal dengan sedikit pelarut tertentu,
sehingga pengotor larut, sedangkan kristal tidak larut.

3. Pengendapan kristal
a. Larutkan kristal dalam pelarut tertentu (I)
b. Ubah kepolaran kristal dengan menambahkan sejumlah pelarut lain
(II) dengan kepolaran yang sangat berbeda dengan pelarut
c. Kristal akan mengendap
10/11/2017 2:00:18 PM Phytochemistry 69
SUPERSATURATION
Nucleation

Two different nucleation mechanisms can be distinguished:

primary and secondary nucleation.

Primary nucleation is new phase formation from a clear liquid

or solution. It can be subdivided into homogeneous and
heterogeneous nucleation.

In the latter case a foreign substrate of tiny invisible particles,

e.g. dust or dirt particles, is present in the solution on which
nucleation starts.

In homogeneous nucleation such a substrate is absent and

nuclei are formed by statistical Suctuations of solute entities
that cluster together.
Secondary nucleation is the breeding of nuclei from crystals of
the crystallizing material that are already present in the
solution.

These nuclei are in general attrition fragments, and result from

collisions of the larger crystals with the hardware of the
crystallizer, in particular with the blades of impellers and
pumps.

At high solid densities in the crystallizer collisions between the

larger crystals can create fragments that act as secondary
nuclei.
IMPURITY
IMPURITY
IMPURITY
10/11/2017 2:00:18 PM Phytochemistry 77
10/11/2017 2:00:18 PM Phytochemistry 78
 Recrystallisation
The most commonly used procedure for purification
of a solid material by recrystallisation from a solution
involves the following steps :
1. The impure material is dissolved in a suitable solvent
to form a near-saturated solution
2. The hot solution is filtered to remove any insoluble
particles
3. It is then allowed to cool so that the dissolved
substance crystallises out
4. The crystals are separated from the mother liquor
5. They are washed free from mother liquor with a little
fresh cold solvent, then dried.
 Guide to the selection of a suitable solvent

1. Substances usually dissolve best in solvents to

which they are most closely related in chemical
and physical characteristics.
Hydroxylic compounds are likely to be most soluble in water,
methanol, ethanol, acetic acid or acetone.
Petroleum ether might be used with water-insoluble
substances.
2. Higher members of homologous series
approximate more and more closely to their
parent hydrocarbon.
3. Polar substances are more soluble in polar, than
in non-polar, solvents.
10/11/2017 2:00:18 PM Phytochemistry 82
 Mixed solvents
Where a substance is too soluble in one solvent
and too insoluble in another, it is often possible
to use them as mixed solvent.
The technique of recrystallisation from a mixed
solvent is as follows :
1. The material is dissolved in the solvent in which it is more solube,
then the other solvent is added cautiously to the hot solution
until a slight turbidity persists or crystallisation begins.
2. This is cleared by adding several drops of the first solvent, and the
solution is allowed to cool and crystallise in the usual way.

10/11/2017 2:00:18 PM Phytochemistry 83

 Choice of solvents
The best solvent for recrystallisation have these
properties :
1. The material is much more soluble at higher
temperatures than it is at room temperature or
below
2. Well-formed crystals are produced
3. Impurities are either very soluble or only sparingly
soluble
4. The solvent must no reaction between the solvent
and the substance being purified
5. There must be not be inconveniently volatile or too
highly flammable
 4. Pengendapan serbuk amorf
Beberapa senyawa mudah mengendap berbentuk amorf

1. Penguapan pelarut
Larutkan sejumlah serbuk dalam pelarut tertentu
Biarkan pelarut menguap dan terbentuk endapan amorf

2. Pencucian
a. Tambahkan sejumlah serbuk dengan sedikit pelarut tertentu,
sehingga larut, sedangkan pengotor tidak larut, atau
b. Tambahkan sejumlah serbuk dengan sedikit pelarut tertentu,
sehingga pengotor larut, sedangkan serbuk tidak larut.

3. Pengendapan serbuk
Biarkan beberapa waktu atau sentrifuse sampai terbentuk endapan
serbuk yang terpisah. Kemudian dilakukan dekantasi atau jika
perlu dilakukan penyaringan.
• CENTRIFUGATION
Involves the use of the
centrifugal force. More
dense components
migrate away. Solid
particles remain on the
bottom.
• DECANTING
Used to separate a liquid from an insoluble solid.
The solid stays in the bottom.
 Filtration
• In preparation for the
filtration, the filter
paper must be folded
into a cone.
• The students may be
measuring the mass of
the filter paper so that
they can determine the
mass of the precipitate
after the experiment.
 Filtration
• The cloudy,
heterogeneous mixture
is carefully poured into
a funnel that has been
set up with filter paper.
• Notice that the filtrate
is being collected in a
beaker.
 Filtration
• The precipitate in the
original heterogeneous
mixture beaker must be
washed out using the
stirring rod and the
wash bottle.
• We have collected all
the precipitate in the
filter paper now.
10/11/2017 2:00:18 PM Phytochemistry 92
 Preparative Chromatography

Preparative elution chromatography is generally

carried out under mass and/or volume
overloaded conditions in order to increase the
product throughput.
In volume overloading, the sample
concentration is maintained in the linear region
of the isotherm and the volume is increased
until the throughput is optimized.

10/11/2017 2:00:18 PM Phytochemistry 93

10/11/2017 2:00:18 PM Phytochemistry 94
 Preparative Chromatography
Paper and Thin Layer Chromatography

Larger TLC plates, called a Preparative Plates, can be used for

separations of milligram quantities of materials because they are
coated with thick layers (1-3 mm) of stationary phase. Once the
plate is developed, the spots are scraped off the plate along with
the absorbent.

Preparative elution paper and thin layer chromatography

is carried out under mass and/or volume overloaded conditions
in order to increase the product throughput.
Each separate component is then extracted from
the stationary phase with a polar solvent.
Extract stained spot with solvent

10/11/2017 2:00:18 PM Phytochemistry 95

 Kualitatif vs. Preparatif
Plate size 20 x 20 20 x 20

Layer thickness 100 - 250 m 0.5- 2 mm

Starting line from 1.5 cm 2-3 cm
bottom
Sample application Spot Line
and concentration 200 mg/ml
1 ng/l
Front migration 10 – 15 cm 20 cm
length
Detection
- Fluorescence ~ 0.006 ng 0.01 ng
- Spray reagent
whole of plate edge of plate
 KLT Preparatif

Isolat utama
Hasil KLT preparatif fraksi VII KK fraksi etil asetat
 KLT Preparatif

Isolat utama
Hasil KLT preparatif fraksi VII KK fraksi etil asetat
Pemantauan isolat hasil KLT preparatif beberapa fraksi hasil kromatografi
kolom dari fraksi etil asetat dan n-butanol.
 5. KLT preparatif
1. Siapkan bejana (chamber)
2. Lapisi bejana dengan kertas saring
3. Siapkan eluen/fase gerak (pelarut tunggal atau campuran)
4. Masukkan eluen ke dalam bejana dan tutup rapat. Biarkan bejana
jenuh dengan uap eluen.
5. Sejumlah fraksi/subfraksi kental dilarutkan dalam beberapa mL pelarut,
sampai diperoleh ekstrak yang tidak terlalu kental dan tidak terlalu
encer .
6. Totolkan fraksi/subfraksi secara tidak terputus pada pelat KLT
preparatif dengan menggunakan pipa kapiler, sehingga berbentuk pita
(tebal pita maksimum 5 mm) .
7. Biarkan totolan fraksi/subfraksi mengering (pelarut menguap)
8. Masukkan pelat yang sudah ditotolkan ke dalam bejana. (Perhatian:
tinggi permukaan eluen dalam bejana harus lebih rendah dari pada
totolan bercak)
9. Biarkan eluen/fase gerak naik sampai sekitar 2 cm sebelum pinggir
pelat.
10.Angkat pelat, biarkan pelat mengering (pelarut menguap)
KLT preparatif lanjutan
11. Lihat warna-warna pita secara visual atau di bawah lampu
ultraviolet. Jika pita tidak berwarna atau berfluorosensi di bawah
lampu ultraviolet, semprot bagian pinggir kiri dan kanan pelat
dengan penampak bercak universal asam sulfat 10 % dalam
metanol . (Pada waktu menyemprot bagian pinggir kiri dan kanan
pelat, tutup bagian tengah pelat)
12. Kerok pita senyawa yang akan diisolasi, kumpulkan pada suatu
wadah
13. Masukkan pelarut tertentu ke dalam wadah yang berisi kerokan
pita, aduk, sehingga senyawa yang akan diisolasi larut
sedangkan serbuk silika gel tidak larut.
14. Saring dengan kertas saring, sehingga sebagian besar serbuk
silika gel akan tertahan di kertas saring.
15. Ulangi penyaringan berkali-kali dengan kapas, sehingga tidak
terdapat partikel serbuk silika gel dalam larutan senyawa.
 6. Kromatografi kertas preparatif
1. Siapkan eluen
2. Siapkan bejana (chamber)
3. Lapisi bejana dengan kertas saring
4. Masukkan eluen ke dalam bejana dan tutup rapat. Biarkan bejana
jenuh dengan uap eluen sekitar 24 jam.
5. Sejumlah fraksi/subfraksi kental dilarutkan dalam beberapa mL
pelarut., sampai diperoleh ekstrak yang tidak terlalu kental dan tidak
terlalu encer
6. Totolkan fraksi/subfraksi secara tidak terputus pada kertas Whatman
No 3 dengan menggunakan pipa kapiler sehingga berbentuk pita
(tebal pita maksimum 5 mm) .
7. Biarkan totolan fraksi/subfraksi mengering (pelarut menguap)
Masukkan kertas Whatman yang sudah ditotolkan ke dalam bejana.
(Perhatian: tinggi permukaan eluen dalam bejana harus lebih rendah
dari pada totolan bercak.
8. Biarkan eluen/fase gerak naik sampai sekitar 2 cm sebelum pinggir
kertas
9. Angkat kertas, biarkan kertas mengering (pelarut sudah menguap)
Kromatografi kertas preparatif (lanjutan)
10. Lihat warna-warna pita secara visual atau di bawah lampu ultraviolet.
Jika pita tidak berwarna atau berfluorosensi di bawah lampu ultraviolet,
semprot bagian pinggir kiri dan kanan kertas dengan penampak bercak
khusus (Pada waktu menyemprot bagian pinggir kiri dan kanan kertas,
tutup bagian tengah kertas

11. Gunting pita senyawa yang akan diisolasi menjadi bagian-bagian kecil,
kumpulkan pada suatu wadah

12. Masukkan pelarut tertentu ke dalam wadah yang berisi guntingan pita,
aduk, sehingga senyawa yang akan diisolasi larut sedangkan kertas
tidak larut.

13. Saring dengan kertas saring, sehingga guntingan kertas akan tertahan
di kertas saring.

14. Ulangi penyaringan berkali-kali, sehingga tidak terdapat partikel

selulosa dalam larutan senyawa.
7. Kromatografi kolom
 Preparative Chromatography

Preparative elution chromatography is generally

carried out under mass and/or volume
overloaded conditions in order to increase the
product throughput.
In volume overloading, the sample
concentration is maintained in the linear region
of the isotherm and the volume is increased
until the throughput is optimized.
 Disadvantages of Column
Chromatography
• Properly setting up the column requires some
technical skill and manual dexterity, and takes
some time.
• Column chromatography is less foolproof than
paper chromatography and requires constant
attention while the experiment is being
performed: collection vessels must be
frequently switched and solvent levels need to
be topped up.
Preparative analysis using Column Chromatography

leaf pigments color

carotenes golden
pheophytin olive green
chlorophyll a blue green
yellow
chlorophyll b
green
lutein yellow
violaxanthin yellow
neoxanthin yellow
Column after Step 3 Step 5 Column after Step 9
 Packing a (silica gel) column:
Quickly but carefully pour the slurry into the column. Stir and pour
immediately to maximize the amount of silica that goes into the column
instead of remaining behind in the beaker. You may find a clean spatula or
glass rod helpful in transfering the silica.

Remove the pinch clamp to allow solvent to drip into a clean flask. Tap on the
side of the column with a rubber stopper or tubing to help the silica settle
uniformly.

Use a Pasteur pipet to rinse any silica that is sticking to the sides of the
column. Allow the silica to settle while eluent continues to drip into the flask.

Once the silica has settled, carefully add sand to the top of the column. Sand
is heavier than silica. If the silica has not settled, the sand may sink into the
silica instead of forming a layer on top of it. (You may need to rinse down
sand that sticks to the side of the column.
 Pengemasan kolom
Pemasukan cuplikan
• Cuplikan disuspensikan dalam sesedikit mungkin pelarut yg
sama dg fase gerak yg dipakai, kemudian dituang perlahan-
lahan di atas permukaan adsorben sampai merata.
Pengelusian (pembilas lepas)
• Penambahan/penetesan fase gerak harus hati-hati, jangan
sampai mengganggu permukaan adsorben
Pengumpulan fraksi
• Pengumpulan fraksi dapat dilakukan dengan manual, dan dapat
juga dengan alat pengumpul fraksi otomatik.
• Volume setiap fraksi tergantung kepada banyaknya cuplikan
dan banyaknya jenis senyawa yang dipisah.
Applying mixture (loading)
 Loading a sample onto the column:
1. Drain eluent from the column until no solvent remains
above the surface of the sand.
2. Using a long Pasteur pipet, carefully add your sample
to the column.
3. Drain eluent from the column until no sample remains
above the surface of the sand.
4. Use ~ 1 mL of eluent to rinse your container and pipet.
Add this milliliter of sample to the sand. Drain eluent from the
column until no liquid remains above the surface of the sand.

5. Repeat step 2 two or three times to completely transfer

your sample onto the silica gel. If you do not do and repeat
step 2, your sample will remain in the sand instead of on the silica.
Sample remaining in the sand will dissolve in the eluent that you add
in step 4, ruining the possibility of good separation of components.
 Eluting the sample:

Once you have rinsed your sample onto the silica,

carefully add eluent to the top of the column. To avoid
disturbing the top of the column, it's a good idea to
carefully pipet an inch or two of solvent onto the column
instead of pouring solvent directly onto the sand.

Add more eluent as necessary. The eluent collected prior

to the elution of sample can be recycled. The composition
of the eluent can be changed as the column progresses. If
the eluent composition is to be changed, ALWAYS start
with least polar solvent/mixture and change to the more
polar solvent/ mixture.
 Other Comments:
• The success of your separation will be dependant on how well you pack
and load the column. It is important to have level sand and silica. It is also
important to carefully and evenly add your sample to the packed column.

• Do not allow the silica to dry out as the column progresses. Cracks will
form within the silica column if it dries, and compounds can fall down the
cracks instead of partitioning between mobile and stationary phases.

• Compounds pass through sand quickly and do not stick to it. Sand is used
at the bottom of the column to help ensure a level silica gel line. The bottom
of the column is typically cone shaped. If no sand were present at the
bottom of the column, molecules traveling down the center of the column
would encounter less silica gel than molecules traveling down the edge,
closer to the glass. As a result, a particular component would elute as a
broader band which is undesirable.

• Sand is used at the top of the column to aid even loading of the sample.
Sample diffuses evenly through the sand. Once the pinch clamp is removed
from the bottom of the column, sample loads evenly onto the silica. Without
sand, the sample would be added directly to the silica and would stick
whereever it is added, not evenly across the surface of the silica.
 Fractions can be
Components a, b,
Column after collected in test tubes,
and c separate as
Step 13 vials, beakers, or
column progresses.
Erlenmeyer flasks.
 Analysis of Column Eluants
If the compounds separated in a column chromatography
procedure are colored, the progress of the separation can
simply be monitored visually.

More commonly, the compounds to be isolated from column

chromatography are colorless. In this case, small fractions of
the eluent are collected sequentially in labeled tubes and the
composition of each fractions is analyzed by thin layer
chromatography.

(Other methods of analysis are available; this is the most

common method and the one used in the organic chemistry
teaching labs.)
 Analyzing the fractions:
Analyze the fractions by thin-layer chromatography to
determine
a) if the fraction contains more than one component and
b) if fractions can be combined without affecting the purity of those
fractions.
intial TLC TLC of fractions
 Thin-Layer Chromatography: A Two-
Component Mixture

s o l v en t f r o n t

c o m p o n en t B Less polar!
s o l v en t f r o n t

c o m p o n en t B

c o m p o n en t A More polar!
c o m p o n en t A

o r i g i n mixture ori gin origin

s o l v en t f r o n t

Increasing Development Time

 Visualization Method
• Most of the time, the spots won’t show
unless they are visualized!
• Vizualization is a method that is used to
render the TLC spots visible.
• A visualization method can be:
– Ultraviolet light
– Iodine vapors to stain spots
– Colored reagents to stain spots
– Reagents that selectively stain spots while leaving
others unaffected.
 UJI KEMURNIAN
10/11/2017 2:00:19 PM Phytochemistry 124
 Assessing the degree of purity
1. Examination of physical properties
1. Melting, freezing and boiling point 5. Density
2. Refractive index 6. Specific conductivity
3. Optical rotation 7. Mass spectroscopy
4. Absorption spectra (UV, IR, and NMR)
2. Empirical analysis (C,H,N, ash, etc)
3. Chemical testsfor particular types of impurities
4. Physical tests for particular types of impurities
1. Chromatography 4. Electron spin resonance
2. X-ray spectroscopy 5. Mass spectroscopy
3. Fluorometry 6. Emission and atomic
absorption spectroscopy
5. Electrochemical methods
6. Nuclear methods
•UJI KEMURNIAN
 memastikan bahwa isolat yang diperoleh adalah
memang satu senyawa tunggal

Beberapa metode uji kemurnian:

1. KLT /KKt pengembangan tunggal
dengan 3 macam pengembang berbeda
2. KLT/Kkt dua dimensi
3. Penentuan Titik lebur/ leleh
4. HPLC/KCKT
5. GCMS/KGSM
4. dll
I. KLT /KKt pengembangan tunggal
(min. dng 3 pengembang berbeda)
1. Siapkan bejana (chamber)
2. Lapisi bejana dengan kertas saring
3. Siapkan eluen/fase gerak ke 1 (pelarut tunggal atau campuran)
4. Masukkan eluen ke 1 ke dalam bejana dan tutup rapat.
Biarkan bejana jenuh dengan uap eluen.
5. Sejumlah isolat dilarutkan dalam beberapa mL pelarut, sampai
diperoleh larutan yang tidak terlalu kental dan tidak terlalu encer
6. Totolkan larutan pada pelat/ kertas dengan menggunakan pipa
kapiler.
7. Biarkan totolan mengering (pelarut menguap)
8. Masukkan pelat/kertas yang sudah ditotolkan ke dalam bejana.
(Perhatian: tinggi permukaan eluen dlm bejana harus lebih
rendah dari totolan bercak
9. Biarkan eluen/fase gerak naik sampai sekitar 2 cm sebelum
pinggir pelat/kertas.
KLT /KKt pengembangan tunggal (lanjutan)

10.Angkat pelat/ kertas, biarkan pelat/ kertas mengering (pelarut

menguap)

11. Lihat warna bercak secara visual atau di bawah lampu ultraviolet. Jika
bercak tidak berwarna atau berfluorosensi di bawah lampu ultraviolet,
semprot dengan penampak bercak universal asam sulfat 10 %
dalam metanol .

12. Lakukan hal yang sama c – k dengan menggunakan pelat yang

berbeda dengan eluen ke 2 dan ke 3 (Eluen ke 2 lebih polar dari pada
eluen ke 1 dan eluen ke 3 lebih polar dari pada eluen ke 2). Bisa
diganti komposisi atau diganti dengan pelarut lain.

13. Isolat kemungkinan murni jika kromatogram pada ketiga macam

eluen/ fase gerak di atas hanya menunjukkan 1 bercak.
 UJI KEMURNIAN
• Kromatografi Lapis Tipis Pengembangan
Tunggal
Penampak Bercak H2SO4
UV 366
(a)n-heksana:etil asetat
(2:3)
(b)Toluen:etil asetat (1:3)
(c)Etil asetat:etanol (9:1)

(a) (b) (c)

 II. KLT/Kkt dua dimensi
1. Sejumlah isolat dilarutkan dalam beberapa mL pelarut,
sampai diperoleh larutan yang tidak terlalu kental dan tidak
terlalu encer.
2. Totolkan larutan pada pelat/ kertas dengan menggunakan
pipa kapiler.
3. Biarkan totolan mengering (pelarut menguap)
4. Masukkan pelat/kertas yang sudah ditotolkan ke dalam
bejana. (Perhatian: tinggi permukaan eluen dalam bejana
harus lebih rendah dari pada totolan bercak)
5. Biarkan eluen/fase gerak naik sampai sekitar 2 cm sebelum
pinggir pelat/kertas.
6. Angkat pelat/kertas, biarkan pelat/kertas mengering (pelarut
menguap)
7. Lihat warna bercak secara visual atau di bawah lampu
ultraviolet. Jika bercak tidak berwarna atau
berfluorosensi di bawah lampu ultraviolet.
 II. KLT/Kkt dua dimensi
8. Pelat diputar 90  ( tegak lurus)

9. Lakukan hal yang sama poin 4 s/d poin 7 dengan

menggunakan pelat yang berbeda dengan eluen ke 2
(Eluen ke 2 lebih polar dari pada eluen ke 1). Bisa
diganti komposisi atau diganti dengan pelarut lain.

10. Lihat warna bercak secara visual atau di bawah lampu

ultraviolet. Jika bercak tidak berwarna atau berfluorosensi
di bawah lampu ultraviolet, semprot dengan penampak
bercak universal asam sulfat 10 % dalam metanol .

11. Isolat kemungkinan murni jika kromatogram pada

kedua macam eluen/ fase gerak di atas hanya
menunjukkan 1 bercak.
 FA 3106 Kromatografi dan
10/11/2017 2:00:19 PM 132
Elektroforesis
133
 UJI KEMURNIAN
• Kromatografi Lapis Tipis Dua Dimensi

1. N-heksana:etil
asetat (2:3)
2. Etil aetat:etanol
(9:1)
2 Penampak bercak
H2SO4
UV 366

1
III. Penentuan Titik leleh/ lebur
(dengan alat Electrothermal, dll)
1. Masukkan sejumlah isolat ke dalam pipa kapiler
khusus untuk penentuan titik lebur.
2. Set angka pada alat Electrothermal 10 C di bawah
titik lebur isolat.
3. Alat Electrothermal dijalankan, amati suhu pada saat
isolat mulai melebur ( dicatat) sampai isolat melebur
semua (dicatat)
4. Jika isolat langsung melebur pada suhu leburnya atau
melebur pada jarak maksimum 2 C, maka isolat dapat
dinyatakan murni
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 Initial sample sintering First drop of Final crystals Liquid
Liquid visible disappear

10/11/2017 2:00:19 PM
Melting range 138
II. Melting point and freezing point determination
The melting point of compound is the temperature range at
which the solid phase change to liquid.

The freezing point of compound is the temperature range at

which the liquid change to crystals or solid phase.

If melting between :
0 – 25 C : analyzed by the freezing point
25 – 300 C : general procedure of melting point det.
300 – 500 C : use special equipment

10/11/2017 2:00:19 PM Phytochemistry 139

Freezing point determination
Place 5 – 10 ml of liquid in an ordinary test tube fitted with
a thermometer and a wire stirrer made of copper, nickel, or
platinum.

Fasten the tube in a slightly larger test tube by means of a

cork and cool them in an ice or ice – salt bath or an acetone
– dry ice bath.

Stir the liquid vigorously. As soon as crystals begin to

form, remove the tube from the bath. Continue the vigorous
stirring and read the temperature on the thermometer.

The freezing point is the temperature reached after initial

supercooling effect has disappeared.
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Melting point determination
Place a small amount of the sample, approximately one-half
of spatula, on a hard, clean surface such a watch glass.

Tap the open end of the capillary tube into the sample until
a few crystals are in the tube. Hold the capillary tube
vertically, open end up, and tap it gently on the counter so
that the crystals pack to the bottom. If necessary, rub it
with a file or a coin with a milled edge or drop it through a
glass tube to move the crystals to the bottom.

The capillary tube should contain 2-3 mm of sample.

10/11/2017 2:00:19 PM Phytochemistry 142

Mixture Melting points
The “mixed melting point” method provides a means of
testing for identity of two solids (which should, of course,
have identical melting points) by examination of the
melting point behavior of a mechanical mixture of the two.

In general, a mixture of samples of nonidentical

compounds shows a melting point depression.

A few pairs of substances when mixed show no melting

depression, but more frequently the failure to depress may
be observed only at certain composition.

A : B = 80% : 20 %
A : B = 50% : 50 %
A : B = 20% : 80 %
10/11/2017 2:00:19 PM Phytochemistry 143
Melting point standards

Mp (corr) Standards Mp (corr) Standards

C C

0 Ice 187 Hippuric acid

53 p-dichlorobenzene 200 Isatin
90 m-dinitrobenzene 216 Anthracene
114 Acetanilide 238 1,3-diphenil urea
121 Benzoic acid 257 Oxanilide
132 Urea 286 Anthraquinone
157 Salicylic acid 332 N,N’-diacetyl
benzidine

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Corrected Melting point
The thermometer should always be callibrated by observing the
melting points of several pure compounds.
If care is taken to use the same apparatus and thermometer in
all melting point determinations, it is convenient and timesaving
to prepare a calibration curve.
The observed melting point of the standard compound is plotted
against the corrected value, and a curve, DA, is drawn through
these points.
In subsequent determinations the observed value, B, is
projected horizontally to the curve and then vertically down to
give the corrected value, C.
The termometer should be callibrated by observing the melting
points of several pure compounds.
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10/11/2017 2:00:19 PM Phytochemistry 146
Melting point apparatus

- Thomas – Hoover melting point apparatus

- a Thiele tube
- a Mel-Temp melting point apparatus
- Melting block
- Fisher-John melting point apparatus
- Electrothermal apparatus

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10/11/2017 2:00:19 PM Phytochemistry 149
 IV. HPLC/ KCKT

Dapat untuk melihat % kemurnian

Kadar kemurnian senyawa dapat dihitung kuantitatif

Jumlah pengotor juga dapat diketahui

Disebut murni jika senyawa yang diinginkan (puncak

utama) > 90 % total berat senyawa

10/11/2017 2:00:19 PM Phytochemistry 150

 IV. HPLC/ KCKT

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 HPLC Columns:

• Stainless steel
• 10-30 cm long
• 4-10 mm internal diameter
• 1-10 mm particle size -
40,000-60,000 plates/m
• High Speed Isocratic
Separation :100,000
plates/m

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 V. GC/ Kromatografi Gas

Dapat untuk melihat % kemurnian

Kadar kemurnian senyawa dapat dihitung kuantitatif

Jumlah pengotor juga dapat diketahui

Disebut murni jika senyawa yang diinginkan (puncak

utama) > 90 % total berat senyawa

10/11/2017 2:00:19 PM Phytochemistry 163

 V. Kromatografi Gas

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