Teknik Pemisahan

Yum Eryanti
HP 08127581951
Email ym.eryanti@gmail.com
 Introduction

Separation
– Anderson, 1987 “physical transfer of a particular
chemical substance from one phase or medium to
another, or the actual physical separation of the
components of a mixture into separate fractions.”
– Meloan, 1999 “is a process whereby compounds of
interest are removed from the other compounds in
the sample that may react similarly and interfere with
a quantitative determination.”
– Seader and Henley, 1998 “The separation of chemical
mixtures into their constituents. Separations
including enrichment, concentration, purification,
refining, and isolation.”
Pada umumnya sebelum suatu senyawa dapat di
identifikasi dan di ukur kadarnya, perlu dilakukan
pemisahan dari matriksnya. Oleh karena itu
pemisahan merupakan langkah penting dalam analisis
kualitatif maupun kuantitatif. Pengetahuan yang
cukup mengenai metode-metode pemisahan
merupakan suatu keharusan bagi mereka yang
berkecimpung dalam suatu bidang yang masih
berhubungan dengan kimia, seperti Farmasi, Kimia,
Biologi, Tehnik Kimia dan Tehnik Pertanian.
 Methods of Separating Mixtures
• Filter
• Evaporation
• Centrifuge
• Chromatography
• Distillation
 Filtration
• Filtration is the process of separating
suspended solid matter from a liquid, by
causing the latter to pass through the pores of
some substance, called a filter. The liquid
which has passed through the filter is called
the filtrate. The filter may be paper, cloth,
cotton-wool, asbestos, slag- or glass-wool,
unglazed earthenware (tembikar), sand, or
other porous material.
 Filtration organic chemistry
• Filtration is a technique used either to remove
solid impurities from an organic solution or to
isolate an organic solid. The two types of
filtration commonly used in organic chemistry
laboratories are gravity filtration and vacuum
or suction (penghisap) filtration.
 Gravity Filtration

• Gravity filtration is the method of choice to remove solid impurities from

an organic liquid. The impurity can be a drying agent or an undesired
(yang tak diinginkan) side product or leftover (sisa) reactant. Gravity
filtration can be used to collect solid product, although generally vacuum
filtration is used for this purpose (maksud) because it is faster.
• A filtration procedure called "hot gravity filtration" is used to separate
insoluble impurities from a hot solution. Hot filtrations require
(memerlukan) fluted filter paper and careful attention to the procedure
to keep the apparatus warm but covered so that solvent does not
evaporate. Hot gravity filtrations are no longer included in the routine
procedures for the experiments in the organic chemistry teaching labs.
• To perform a standard gravity filtration, first select the size of filter
paper that, when folded (dilipat), will be a few millimeters below the rim
of your stemmed funnel. Fold the paper into a cone by first folding it in
half, and then in half again, as shown.
Cara melipat kertas saring
 Vacuum Filtration

• Vacuum filtration is used primarily to collect a desired

solid, for instance, the collection of crystals in a
recrystallization procedure. Vacuum filtration uses a 
Buchner funnel and a side-arm flask. Vacuum filtration is
faster than gravity filtration, because the solvent or
solution and air is forced through the filter paper by the
application of reduced pressure. Do not use vacuum
filtration to filter a solid from a liquid if it is the liquid that
you want, and if the liquid is low boiling. Any solvent
which boils at about 125 degrees or lower will boil off
under the reduced pressure in the vacuum flask.
Vacuum Filtration
Vacuum Filtration
 Filtration
Mixture of
solid and
Stirring
liquid
rod

• Filtration separates
a liquid from a
solid Funnel

Filter paper
traps solid

Filtrate (liquid
component
of the mixture)
 Filtration and Evaporation
Filtration is a method for
the separation of the parts
of a heterogeneous mixture.
When students mixed two
common substances, a
heterogeneous mixture was
formed.
There was a new precipitate
formed in a solution.
• In preparation for the
filtration, the filter
paper must be folded
into a cone (kerucut).
• The students may be
measuring the mass of
the filter paper so that
they can determine the
mass of the precipitate
after the experiment.
• The cloudy (keruh),
heterogeneous mixture
is carefully poured
(menuangkan) into a
funnel that has been
set up with filter paper.
• Notice that the filtrate
is being collected in a
beaker.
• 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.
• Evaporation is a method for
separating the components
of a homogeneous mixture.
• After filtration, the students
evaporated the filtrate
solution to separate it from
any dissolved solute.
• The liquid (solvent) from the
filtrate solution will
evaporate leaving behind any
dissolved solute.
Mean while, the insoluble
solid (original precipitate!)
that was collected on the
filter paper was dried in an
oven.
By taking the mass of the
filter paper before the
filtration and the mass of the
paper and solid after it dries…
…the mass of precipitate can
be calculated
 Centrifugation

• Spin sample very rapidly: AFTER

Before
denser materials go to bottom
(outside)
• Separate blood into serum and
plasma Serum
– Serum (clear)
Blood
– Plasma (contains red blood cells
‘RBCs’) RBC’s

• Check for anemia (lack of iron)

A B C
 Highlights from this issue
• Centrifugation is one of the most basic of laboratory
applications and is used by a wide range of clinical and
research personnel. However, information on
centrifugation theory and separation techniques are
usually only found in centrifuge instrument manuals or
by contacting the manufacturers of density gradient
media directly. This issue of Biofiles is intended to
provide a basic understanding of how biological
particles behave in a centrifugal field and the types of
density gradient media used to separate them.
 Highlights from this issue
• Differential Centrifugation The simplest form of
separation by centrifugation is differential centrifugation,
sometimes called differential pelleting (butiran). Particles
of different densities or sizes in a suspension will
sediment at different rates, with the larger and denser
particles sedimenting faster. These sedimentation rates
can be increased by using centrifugal force. A suspension
of cells subjected to a series of increasing centrifugal force
cycles will yield a series of pellets containing cells of
decreasing sedimentation rate. Centrifugation Time
Centrifugal Force A. B. C
Centrifuge
 Pengertian Kromatografi
Kromatografi secara umum.

Teknik Suatu
pemisahan campuran Komponen

Kromatografi adalah teknik untuk

memisahkan campuran menjadi
komponennya dengan bantuan
perbedaan sifat fisik masing-masing
komponen.
 Paper chromatography
• Paper chromatography is an analytical
 method that is used to separate coloured
chemicals or substances, especially pigments.
This can also be used in secondary or primary
colours in ink experiments. This method has
been largely replaced by 
thin layer chromatography, but is still a
powerful teaching tool
 Rƒ value

The retention factor (Rƒ) may be defined as the ratio of the distance

traveled by the substance to the distance traveled by the solvent.
Rƒ values are usually expressed as a fraction of two decimal places. If
Rƒ value of a solution is zero, the solute remains in the stationary phase
and thus it is immobile. If Rƒ value = 1 then the solute has no affinity for
the stationary phase and travels with the solvent front. To calculate the
Rƒ value, take the distance traveled by the substance divided by the
distance traveled by the solvent (as mentioned earlier in terms of
ratios). For example, if a compound travels 2.1 cm and the solvent front
travels 2.8 cm, (2.1/2.8) the Rƒ value = 0.75. Rƒ value depends on
temperature and the solvent used in experiment, so several solvents
offer several Rƒ values for the same mixture of compound.
 Pigments and Polarity

Paper chromatography is one method for testing the purity of

compounds and identifying substances. Paper chromatography is a
useful technique because it is relatively quick and requires small
quantities of material. Separations in paper chromatography involve
the same principles as those in thin layer chromatography. In paper
chromatography, like thin layer chromatography, substances are
distributed between a stationary phase and a mobile phase. The
stationary phase is usually a piece of high quality filter paper. The
mobile phase is a developing solution that travels up the stationary
phase, carrying the samples with it. Components of the sample will
separate readily according to how strongly they adsorb onto the
stationary phase versus how readily they dissolve in the mobile phase.
 Paper Chromatography
Prinsip
Pelarut bergerak
lambat pada kertas,
komponen-komponen
bergerak pada laju
yang berbeda dan
campuran dipisahkan
berdasarkan pada
perbedaan bercak
warna.
Paper Chromatography
 Thin Layer Chromatography
Thin-layer chromatography (TLC) is a chromatography
 technique used to separate non-volatile mixtures. Thin-layer
chromatography is performed on a sheet of glass, plastic, or
aluminium foil, which is coated with a thin layer of adsorbent
 material, usually silica gel, aluminium oxide, or cellulose.
This layer of adsorbent is known as the stationary phase.

After the sample has been applied on the plate, a solvent or

solvent mixture (known as the mobile phase) is drawn up
the plate viacapillary action. Because different analytes
 ascend the TLC plate at different rates, separation is
achieved
 Thin Layer Chromatography
• Thin-layer chromatography can be used to monitor
the progress of a reaction, identify compounds
present in a given mixture, and determine the
purity of a substance. Specific examples of these
applications include: analyzing ceramides and 
fatty acids, detection of pesticides or insecticides in
food and water, analyzing the dye composition of
fibers in forensics, assaying the radiochemical purity
of radiopharmaceuticals, or identification of 
medicinal plants and their constituents
 Plate preparation

• TLC plates are usually commercially available, with

standard particle size ranges to improve reproducibility.
They are prepared by mixing the adsorbent, such as 
silica gel, with a small amount of inert binder like 
calcium sulfate (gypsum) and water. This mixture is spread
as a thick slurry on an unreactive carrier sheet, usually 
glass, thick aluminum foil, or plastic. The resultant plate is
dried and activated by heating in an oven for thirty
minutes at 110 °C. The thickness of the absorbent layer is
typically around 0.1 – 0.25 mm for analytical purposes
and around 0.5 – 2.0 mm for preparative TLC
 Technique

• The process is similar to 

paper chromatography with the advantage of
faster runs, better separations, and the choice
between different stationary phases. Because
of its simplicity and speed TLC is often used for
monitoring chemical reactions and for the
qualitative analysis of reaction products.
 Separation Process and Principle

• Different compounds in the sample mixture travel at

different rates due to the differences in their attraction to
the stationary phase and because of differences in
solubility in the solvent. By changing the solvent, or
perhaps using a mixture, the separation of components
(measured by the Rf value) can be adjusted. Also, the
separation achieved with a TLC plate can be used to
estimate the separation of a flash chromatography
 column
• Separation of compounds is based on the competition of
the solute and the mobile phase for binding places on the
stationary phase
Thin Layer Chromatography
Thin Layer Chromatography

Chromatogram of 10 essential oils coloured with vanillin reagent.

 Prinsip kerja Kromatografi Lapis Tipis

• KLT menggunakan sebuah lapis tipis silika atau

alumina yang seragam pada sebuah lempeng
gelas atau logam atau plastik yang keras.
• Jel silika (atau alumina) merupakan fase diam.
• Fase gerak merupakan pelarut atau campuran
pelarut yang sesuai.
• Pelaksanaan ini biasanya dalam pemisahan
warna yang merupakan gabungan dari
beberapa zat pewarna.
 Column chromatography
• Column chromatography in chemistry is a method
used to purify individual chemical compounds from
mixtures of compounds. It is often used for
preparative applications on scales from micrograms
up to kilograms. The main advantage of column
chromatography is the relatively low cost and
disposability of the stationary phase used in the
process. The latter prevents cross-contamination
and stationary phase degradation due to recycling.
 Column chromatography
• The classical preparative chromatography
column is a glass tube with a diameter from
5 mm to 50 mm and a height of 5 cm to 1 m
with a tap and some kind of a filter (a glass frit
or glass wool plug – to prevent the loss of the
stationary phase) at the bottom. Two methods
are generally used to prepare a column: the
dry method and the wet method.
 Column chromatography
• For the dry method, the column is first filled with dry
stationary phase powder, followed by the addition of mobile
phase, which is flushed through the column until it is
completely wet, and from this point is never allowed to run
dry.
• For the wet method, a slurry is prepared of the eluent with
the stationary phase powder and then carefully poured into
the column. Care must be taken to avoid air bubbles. A
solution of the organic material is pipetted on top of the
stationary phase. This layer is usually topped with a small
layer of sand or with cotton or glass wool to protect the shape
of the organic layer from the velocity (kecepatan) of newly
added eluent. Eluent is slowly passed through the column to
advance the organic material. Often a spherical eluent
reservoir or an eluent-filled and stoppered separating funnel
 is put on top of the column.
 Stationary phase

The stationary phase or adsorbent in column chromatography is

a solid. The most common stationary phase for column
chromatography is silica gel, followed by alumina. Cellulose
 powder has often been used in the past. Also possible are ion
exchange chromatography, reversed-phase chromatography
 (RP), affinity chromatography or expanded bed adsorption(EBA).
The stationary phases are usually finely ground powders or gels
and/or are microporous for an increased surface, though in EBA
a fluidized bed is used. There is an important ratio between the
stationary phase weight and the dry weight of the analyte
mixture that can be applied onto the column. For silica column
chromatography, this ratio lies within 20:1 to 100:1, depending
on how close to each other the analyte components are being
eluted.
 Mobile phase (eluent)

• The mobile phase or eluent is either a pure solvent or

a mixture of different solvents. It is chosen so that
the retention factor value of the compound of
interest is roughly around 0.2 - 0.3 in order to
minimize the time and the amount of eluent to run
the chromatography. The eluent has also been
chosen so that the different compounds can be
separated effectively. The eluent is optimized in small
scale pretests, often using thin layer chromatography
 (TLC) with the same stationary phase.
 Adsorbent

• Silica gel (SiO2) and alumina (Al2O3) are two

adsorbents commonly used by the organic chemist
for column chromatography. An example of each of
these adsorbents is shown below.
 Solvent

• The polarity of the solvent which is passed through the column

affects the relative rates at which compounds move through the
column. Polar solvents can more effectively compete with the
polar molecules of a mixture for the polar sites on the adsorbent
surface and will also better solvate the polar constituents.
Consequently, a highly polar solvent will move even highly polar
molecules rapidly through the column. If a solvent is too polar,
movement becomes too rapid, and little or no separation of the
components of a mixture will result. If a solvent is not polar
enough, no compounds will elute from the column. Proper
choice of an eluting solvent is thus crucial to the successful
application of column chromatography as a separation
technique. Thin-Layer Chromatography (TLC) is generally used to
determine the system for a column chromatography separation.
 Analysis of Column Eluents

• 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 fraction is analyzed by TLC.
(Other methods of analysis are available; this is the
most common method and the one used in the
organic chemistry teaching labs.)
Dry column chromatography
Dry column chromatography (Kromatografi
kolom kering)

Disebut juga kromatografi vakum cair

(KVC / VLC)
Teknik pemisahan menggunakan pompa
penghisap untuk mempercepat proses
elusi.
Proses elusi: eluen ditambahkan secara
bertahap dalam volume tertentu dan
elusi dilakukan hingga kering sebelum
penambahan eluen berikutnya.
 Separation by Chromatography

sample
mixture

a chromatographic column

stationary phase mobile phase detector

selectively absorbs sweeps sample
components down column

http://antoine.frostburg.edu/chem/senese/101/matter/slides/sld006.htm
Chromatotron
Chromatotron

Pemishan secara preperatif dg

Chromatotron yg bekerja
lebih cepat dg adanya gaya
Sentrifugal yg dapat
menggantikan plat TLC
Diameter plat 24 cm, fasa diam
dengan ketebalan 2 mm
Biasanya menggunakan
Siliga gel 60GF254 untuk TLC
Plat sebelum digunakan
dikeringkan dg oven pd suhu
60-70oC selama 1 jam
Kecepatan perputaran
motornya 800 rpm dengan
kecepatan elusi 1-10 ml/menit
 Chromatotron

Keunggulan Ketebalan fasa

diam 1-4 mm

sederhana cepat pelarut sedikit

dapat dipisahkan
Sebanyak 0,1 – 1 g
dg perbedaan
Rf : 0,2-0,5.
Flash Chromatography
 Ukuran kolom pada Flash Chromatography

Diameter Volume Jumlah sampel Ukuran fraksi

kolom eluen (mg) tampungan
(mm) (ml) Δ Rf ≥ Δ Rf ≥ (ml)
0,2 0,1

10 100 100 40 5

20 200 400 160 10

30 400 900 360 20

40 600 1.600 600 30

50 1.000 2.500 1.000 50

Gas Chromatography
 Gas Chromatography
• Gas pembawa (biasanya menggunakan
helium, argon / nitrogen) dengan tekanan
tertentun dialirkan secara konstan melalui
kolom yang berisi fase diam.
• Komponen sampel akan terabsorbsi oleh fase
diam dengan kecepatan berbeda.
 Distillation

Distillation is the process of heating a liquid until it

boils, then condensing and collecting the resultant hot
vapors.
The boiling point of a compound is one of the physical
properties used to identify it. Distillation is used
to purify a compound by separating it from a non-
volatile or less-volatile material. When different
compounds in a mixture have different boiling points,
they separate into individual components when the
mixture is carefully distilled.
 Distillation for Boiling Point Determination

• The organic teaching labs employ distillation routinely,

both for the identification and the purification of
organic compounds. The boiling point of a compound,
determined by distillation, is well-defined and thus is
one of the physical properties of a compound by which
it can be identified. Distillation is used to purify a
compound by separating it from a non-volatile or less-
volatile material. Because different compounds often
have different boiling points, the components often
separate from a mixture when the mixture is distilled.
 Distillation for Compound Purification

Simple distillations are used frequently in the

organic chemistry teaching labs. They are useful in
the following circumstances (dalam keadaan):
• the liquid is relatively pure to begin with (e.g., no
more than 10% liquid contaminants)
• the liquid has a non-volatile component, for
example, a solid contaminant
• the liquid is contaminated by a liquid with a
boiling point that differs by at least 70°C
 Fractional Distillations

• Mixtures of liquids whose boiling points are

similar (separated by less than 70°C) cannot
be separated by a single simple distillation. In
these situations, a fractional distillation is
used. You can see 
photos of a fractional distillation set-up here.
 Destilasi bertingkat
• Destilasi bertingkat atau destilasi fraksinasi merupakan proses pemurnian zat/senyawa cair
dimana zat pencampurnya berupa senyawa cair yang titik didihnya rendah dan tidak
berbeda jauh dengan titik didih senyawa yang akan dimurnikan. Dengan perkataan lain,
destilasi ini bertujuan untuk memisahkan cairan dari suatu campuran yang komponen-
komponennya memiliki perbedaan titik didih relatif kecil.
• Secara prinsip destilasi bertingkat sama dengan destilasi sederhana perbedaan ada pada
perbedaan titik didih antar komponen, dimana untuk destilasi sederhana perbedaan titik
didih lebih besar dari 30 derajat Celsius, sedangkan destilasi bertingkat perbedaan titik
didih <30 derajat Celsius, keadaan ini karena pada destilasi bertingkat adanya kolom
fraksinasi.
• Kolom fraksionasi berfungsi agar kontak antara cairan dengan uap terjadi lebih lama.
Sehingga komponen yang lebih ringan dengan titik didih yang lebih rendah akan terus
menguap dan masuk ke kondensor. Sedangkan komponen yang lebih besar akan kembali
kedalam labu destilasi. Di kolom ini terjadi pemanasan secara bertahap dengan suhu yang
berbeda-beda pada setiap platnya. Pemanasan yang berbeda-beda ini bertujuan untuk
pemurnian distilat yang lebih dari plat-plat di bawahnya. Semakin ke atas, semakin tidak
volatif cairannya. Kolom fraksionasi digunakan untuk memberikan luas permukaan yang
besar agar uap yang berjalan naik dan cairan yang turun dapat bersentuhan.
Fractional Distillations
 Vacuum Distillations

• Vacuum distillation is distillation at a reduced pressure.

Since the boiling point of a compound is lower at a lower
external pressure, the compound will not have to be
heated to as high a temperature in order for it to boil.
Vacuum distillation is used to distill compounds that have a
high boiling point or any compound which might undergo
decomposition on heating at atmospheric pressure. The
vacuum is provided either by a water aspirator or by a
mechanical pump. You can see photos of a fractional
distillation set-up here. Always check for star cracks (retak)
in the flasks before beginning a vacuum distillation.
 Distilasi vakum
• Distilasi vakum biasanya digunakan jika senyawa yang
ingin didistilasi tidak stabil, dengan pengertian dapat 
terdekomposisi sebelum atau mendekati titik didihnya
atau campuran yang memiliki titik didih di atas
150 °C. Metode distilasi ini tidak dapat digunakan pada
pelarut dengan titik didih yang rendah jika kondensornya
 menggunakan air dingin, karena komponen yang
menguap tidak dapat dikondensasi oleh air. Untuk
mengurangi tekanan digunakan pompa vakum atau 
aspirator. Aspirator berfungsi sebagai penurun tekanan
pada sistem distilasi ini
Vacuum Distillations
 Distilasi vakum
• Untuk senyawa cair dengan titik didih tinggi
Untuk senyawa cair yang mudah mengurai
Untuk senyawa cair yang larut atau mudah
tercampur dengan air, Titik didih akan turun bila
tekanan di atas senyawa tersebut diperkecil.
Untuk menurunkan Titik didih senyawa tsb, agar
tidak mengurai, maka tekanan di dalam labu
diturunkan dengan jalan memompa udara ke
dalam labu, sehingga perlu alat manometer.
 Destilasi Uap
Untuk senyawa cair dengan titik didih tinggi
Untuk senyawa cair yang mudah mengurai
Untuk senyawa cair tidak larut dalam air
Labu yang berisi senyawa yg akan dimurnikan
dihubungkan dengan labu pembangkit uap
Uap air dialirkan ke dalam labu yang berisi senyawa
yg dimurnikan untuk menurunkan Titik didih
senyawa tsb, karena Titik didih campuran lebih
rendah dp Titik didih komponen-komponennya.
 Destilasi Uap
• Destilasi uap adalah istilah secara umum
digunakan untuk destilasi campuran air
dengan senyawa yang tidak larut dalam air,
dengan cara mengalirkan uap air ke dalam
campuran sehingga bagian yang dapat
menguap berubah menjadi uap pada
temperatur yang lebih rendah.
 Destilasi uap
• Untuk memurnikan zat/senyawa cair yang tidak larut dalam air, dan titik didihnya
cukup tinggi, sedangkan sebelum zat cair tersebut mencapai titik didihnya, zat cair
sudah terurai, teroksidasi atau mengalami reaksi pengubahan (rearranagement),
maka zat cair tersebut tidak dapat dimurnikan secara destilasi sederhana atau
destilasi bertingkat, melainkan harus didestilasi dengan destilasi uap.
• Destilasi uap adalah istilah yang secara umum digunakan untuk destilasi campuran
air dengan senyawa yang tidak larut dalam air, dengan cara mengalirkan uap air ke
dalam campuran sehingga bagian yang dapat menguap berubah menjadi uap pada
temperatur yang lebih rendah dari pada dengan pemanasan langsung. Untuk
destilasi uap, labu yang berisi senyawa yang akan dimurnikan dihubungkan dengan
labu pembangkit uap (lihat gambar alat destilasi uap).
• Uap air yang dialirkan ke dalam labu yang berisi senyawa yang akan dimurnikan,
dimaksudkan untuk menurunkan titik didih senyawa tersebut, karena titik didih
suatu campuran lebih rendah dari pada titik didih komponen-komponennya.
Destilasi Uap
 A Distillation Apparatus
thermometer

liquid with a solid

dissolved in it condenser

tube
distilling
flask

receiving pure
hose connected to flask liquid
Dorin, Demmin, Gabel, Chemistry The Study of Matter , 3rd Edition, 1990, page 282 cold water faucet
 The solution is boiled and steam is driven off.

Zumdahl, Zumdahl, DeCoste, World of Chemistry 2002, page 39

 Salt remains after all water is boiled off.

Zumdahl, Zumdahl, DeCoste, World of Chemistry 2002, page 39

 No chemical change occurs when salt water
is distilled.

Distillation
(physical method)

Salt

Saltwater solution Pure water

(homogeneous mixture)

Zumdahl, Zumdahl, DeCoste, World of Chemistry 2002, page 40

 Separation of a sand-saltwater mixture.

Zumdahl, Zumdahl, DeCoste, World of Chemistry 2002, page 40