ITP UNS SEMESTER 2 Kromatografi pc dan tlc

07/04/13 S1-Ilmu dan Teknologi Pangan 1
Kromatografi :
Dasar Teori,PC dan TLC
Kimia Analitik
Semester Genap 2012/2013
Esti Widowati,S.Si.,M.P

What is Chromatography?
Chromatography is a technique for
separating mixtures into their components
in order to analyze, identify, purify,
and/or quantify the mixture or
components.
Separate
• Analyze
• Identify
• Purify
• QuantifyComponentsMixture

Uses for Chromatography
Chromatography is used by scientists to:
• Analyze – examine a mixture, its components,
and their relations to one another
• Identify – determine the identity of a mixture or
components based on known components
• Purify – separate components in order to isolate
one of interest for further study
• Quantify – determine the amount of the a mixture
and/or the components present in the sample

Dasar Kromatografi
Kromatografi adalah teknik pemisahan
campuran didasarkan atas perbedaan
distribusi dari komponen-komponen
campuran tersebut diantara dua fase,

Definition of Chromatography
Detailed Definition:
Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a mobile medium
and for a stationary adsorbing medium through which they
pass.
Terminology:
• Differential – showing a difference, distinctive
• Affinity – natural attraction or force between things
• Mobile Medium – gas or liquid that carries the components
(mobile phase)
• Stationary Medium – the part of the apparatus that does not
move with the sample (stationary phase)

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
Definition of Chromatography

Uses for Chromatography
Real-life examples of uses for
chromatography:
• Pharmaceutical Company – determine amount of
each chemical found in new product
• Hospital – detect blood or alcohol levels in a
patient’s blood stream
• Law Enforcement – to compare a sample found at
a crime scene to samples from suspects
• Environmental Agency – determine the level of
pollutants in the water supply
• Manufacturing Plant – to purify a chemical
needed to make a product

Jenis-Jenis Kromatografi
Berdasarkan fase gerak yang digunakan,
kromatografi dibedakan menjadi dua
golongan besar yaitu gas chromatography
dan liquid chromatography.
Kromatografi adsorpsi dan partisi

Kromatografi di dalam
bentuk tempat
Komatografi Kolom : Kromatografi kolom
merupakan teknik pemisahan dimana
tempat stasioner dalam tabung.
Kromatografi Planar
Kromatografi Kertas
Kromatografi Lapisan Tipis

Fasa Gerak
Fasa DiamFasa Diam
Kromatografi
Padat GelPertukaran Ion
Gas Cair
Plat Kolom Anion
Cair Cair
GPCKation

• 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

Gas Chromatography
Digunakan untuk menentukan komposisi kimia
zat-zat yang tidak diketahui, seperti senyawa
berbeda dalam bensin yang ditunjukkan oleh
tiap-tiap puncak dalam grafik di bawah ini.
Paper Chromatography
Dapat digunakan untuk memisahkan
komponen-komponen tinta,
pewarna, senyawa tumbuhan
(klorofil), make-up, dan banyak zat
lain
Liquid Chromatography
digunakan untuk identifikasi pigmen
tumbuhan atau komponen lain
Thin-Layer Chromatography
Menggunakan lapisan tipis atau gelas
kaca untuk memisahkan komponen
kimia dan bahan lainnya
Contoh Chromatography

Kromatografi Kertas (PC)
Fase diam kertas serap→
Fase gerak pelarut atau campuran pelarut yang→
sesuai.
Dilakukan secara Ascending , Descending, Horizontal
Rf (Retordation Factor). Jarak relatif pada pelarut
disebut sebagai nilai Rf. Untuk setiap senyawa berlaku
rumus sebagai berikut:
Rf=jarak yang ditempuh oleh senyawa
jarak yang ditempuh oleh pelarut

 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
Materials List

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

Observing the Chromatograms
Concentration of Isopropanol
0% 20% 50% 70% 100%

Black Dye
Concentration of Isopropanol
0% 20% 50% 70% 100%
1. Dyes separated – purple and black
2. Not soluble in low concentrations of
isopropanol
3. Partially soluble in concentrations of
isopropanol >20%

Illustration of Chromatography
Components Affinity to Stationary Phase Affinity to Mobile Phase
Blue ---------------- Insoluble in Mobile Phase
Black        
Red       
Yellow          
Mixture Components
Separation
Stationary Phase
Mobile Phase

Kromatografi Kertas
Dua Arah
Digunakan dalam menyelesaikan masalah
pemisahan substansi yang memiliki nilai Rf yang
sangat serupa.
Menggunakan dua pelarut yang berbeda. Pelarut
pertama harus kering dahulu.

Kromatografi Lapis Tipis
Menggunakan sebuah lapis tipis silika atau
alumina yang seragam pada sebuah lempeng gelas
atau logam atau plastik yang keras.
Fase diam Gel silika→ (SiO(SiO22) atau alumina (Al) atau alumina (Al22OO33),),
atau substansi yang dapat berpendarflour dalam
sinar ultra violet.
 Fase gerak pelarut atau campuran pelarut→
yang sesuai.

Thin-Layer Chromatography (TLC)Thin-Layer Chromatography (TLC)
TLC is a fast, simple, and inexpensiveTLC is a fast, simple, and inexpensive
analytical technique used to determine oranalytical technique used to determine or
monitor:monitor:
-- The # of components in a mixture.The # of components in a mixture.
-- The identity of two substances.The identity of two substances.
-- The effectiveness of a purification.The effectiveness of a purification.
-- The appropriate conditions for a columnThe appropriate conditions for a column
chromatographic separation.chromatographic separation.
-- The progress of a reaction.The progress of a reaction.
-- Column chromatography effectiveness.Column chromatography effectiveness.

Principles of TLC
 TLC is one of the simplest, fastest, easiest
and least expensive of several
chromatographic techniques used in
qualitative and quantitative analysis to
separate organic compounds
 Michael Tswett is credited as being the father
of liquid chromatography. Tswett developed
his ideas in the early 1900’s.
07/04/13 28S1-Ilmu dan Teknologi Pangan

TLC
 The two most common classes of TLC are:
 Normal phase (Fase normal)
 Reversed phase (Fase terbalik)

 Normal phase is the terminology used when the
stationary phase is polar; for example silica gel, and
the mobile phase is an organic solvent or a mixture
of organic solvents which is less polar than the
stationary phase.
 Reversed phase is the terminology used when the
stationary phase is a silica bonded with an organic
substrate such as a long chain aliphatic acid like C-
18 and the mobile phase is a mixture of water and
organic solvent which is more polar than the
stationary phase.

Adsorbents for TLC
 Silica gel
 Silica gel-F (Fluorescing indicator added)
 Magnesium Silicate (Florisil)
 Polyamides
 Starch
 Alumina

Thin-Layer Chromatography (TLC)Thin-Layer Chromatography (TLC)
 A polar solvent will carry a polar compoundA polar solvent will carry a polar compound
farther while a non-polar solvent will carry afarther while a non-polar solvent will carry a
non-polar compound farther.non-polar compound farther.
 RRff value is the ratio of the distance the spotvalue is the ratio of the distance the spot
travels from the origin to the distance thetravels from the origin to the distance the
solvent travels.solvent travels.

Overview of TLC—The Rf Value
 A given compound will always travel a fixed distance
relative to the distance the solvent travels
 This ratio is called the Rf value and is calculated in the
following manner:
. distance traveled by substance .
distance traveled by solvent front

Advantages of TLC
 Low cost
 Short analysis time
 Ease of sample preparation
 All spots can be visualized
 Sample cleanup is seldom necessary
 Adaptable to most pharmaceuticals
 Uses small quantities of solvents
 Requires minimal training
 Reliable and quick
 Minimal amount of equipment is needed
 Densitometers can be used to increase accuracy of spot
concentration

Applications of TLC
 TLC has several important uses in
organic chemistry. Some examples are:
1. To establish that two compounds are
identical
2. To determine the number of components in
a mixture
3. To determine the appropriate solvent for a
column-chromatographic separation
4. To monitor the progress of a reaction

Steps in TLC Analysis
 The following are the important components
of a typical TLC system:
 Apparatus (developing chamber)
 Stationary phase layer and mobile phase
 Application of sample
 Development of the plate
 Detection of analyte

General Procedure (1)
 Decide if you are going to do Normal or
Reversed phase chromatography
 Prepare a plate or select a plate with the
proper sorbent material
 Prepare the mobile phase
 Mark the plate
 Apply the sample
 Develop the plate
 Detect the analytes

General Procedure (2)
 Silica gel with or without an added fluorescing indicator is the
most commonly used and is classified as Normal phase
chromatography
 The mobile phase is generally a non-polar solvent such as
hexane. The hexane can be modified to a more polar solvent by
the addition of or organic type solvents such as methanol, diethyl
ether, ethyl acetate, toluene, dimethyl-formamide, etc. to achieve
the required retention.
 The mobile phase can be further modified by the addition of
acids or bases such as acetic acid or triethylamine to reduce
tailing

Polarity
 Polarity of solutes; Polar and non-Polar
 Polar solutes: alcohols (ROH), acids (RCOOH),
amines (RNH2)
 Polar solvents: Methanol, ethanol, acetic acid
 Non-Polar solutes: hydrocarbons, ketones
(compared to methanol)
 Non-Polar solvents: hexane, toluene (compared to
methanol)

Overview of TLC
 This technique manipulates POLARITY
 More polar substances bind strongly to the
adsorbent and elute SLOWER
 Less polar substances bind weakly to the
adsorbent and elute FASTER
 The strength of interactions between the adsorbent
and eluting components vary approximately in this
order:
Salt formation > coordination > H-bonding > dipole-dipole > van der Waals
(More Polar) (Less Polar)

Procedure:
TLC Plates
 The plates can be pre-marked for origin and
development finish line as well as for sample
zones
 Generally a distance of approximately 10 cm is
used as the development of a plate so as to
make the calculation of the Rf value easy.
 Rf is defined as the movement of the sample
zone (x) divided by the movement of the
developing solvent (= x/ 10 cm)

Procedure:
TLC Plate Development
 The development of the plate is linear and ascending
 The developing chamber is usually glass to prevent any
interaction with the developing solvent and capable of
holding the size plate you will be using
 The chamber may or may not be pre-saturated with the
developing solvent
 Development may be with multiple solvents
 Development may be continuous (seldom used)
 Development may be two-directional (right angles)

Development

Sebuah garis menggunakan pensil digambar
dekat bagian bawah lempengan dan setetes
pelarut dari campuran pewarna ditempatkan
pada garis itu.
Ketika bercak dari campuran itu mengering,
lempengan ditempatkan dalam sebuah gelas
kimia bertutup berisi pelarut dalam jumlah yang
tidak terlalu banyak. Perlu diperhatikan bahwa
batas pelarut berada di bawah garis dimana
posisi bercak berada.
Menutup gelas kimia untuk meyakinkan bawah
kondisi dalam gelas kimia terjenuhkan oleh uap
dari pelarut. Untuk mendapatkan kondisi ini,
dalam gelas kimia biasanya ditempatkan
beberapa kertas saring yang terbasahi oleh
pelarut. Kondisi jenuh dalam gelas kimia dengan
uap mencegah penguapan pelarut.

Perhitungan nilai Rf
Nilai Rf untuk setiap warna dihitung
dengan rumus sebagai berikut:
Sebagai contoh, jika komponen
berwarna merah bergerak dari 1.7
cm dari garis awal, sementara
pelarut berjarak 5.0 cm, sehingga
nilai Rf untuk komponen berwarna
merah menjadi:

Analisis Sampel yang Tidak Berwarna
1.Menggunakan pendarflour
Fase diam pada sebuah lempengan lapis tipis
seringkali memiliki substansi yang ditambahkan
kedalamnya, supaya menghasilkan pendaran
flour ketika diberikan sinar ultraviolet (UV).
Pendaran ini ditutupi pada posisi dimana bercak
pada kromatogram berada, meskipun bercak-
bercak itu tidak tampak berwarna jika dilihat
dengan mata. Ketika sinar UV diberikan pada
lempengan, akan timbul pendaran dari posisi
yang berbeda dengan posisi bercak-bercak.
Bercak tampak sebagai bidang kecil yang gelap. –Ultraviolet light at 254 nm
(shortwave UV).
–Long wave UV (340 nm) is
used less commonly.

2. Penunjukkan bercak secara kimia
Dalam beberapa kasus, dimungkinkan untuk membuat bercak-bercak
menjadi tampak dengan jalan mereaksikannya dengan zat kimia sehingga
menghasilkan produk yang berwarna. Sebuah contoh yang baik adalah
kromatogram yang dihasilkan dari campuran asam amino.
Kromatogram dapat dikeringkan dan disemprotkan dengan larutan
ninhidrin. Ninhidrin bereaksi dengan asam amino menghasilkan senyawa-
senyawa berwarna, umumnya coklat atau ungu.
Dalam metode lain, kromatogram dikeringkan kembali dan kemudian
ditempatkan pada wadah bertutup (seperti gelas kimia dengan tutupan
gelas arloji) bersama dengan kristal iodium.
Uap iodium dalam wadah dapat berekasi dengan bercak pada
kromatogram, atau dapat dilekatkan lebih dekat pada bercak daripada
lempengan. Substansi yang dianalisis tampak sebagai bercak-bercak
kecoklatan.

Visualization

TLC Visualization Methods
 Ultraviolet Light—some organic compounds
illuminate or fluoresce under short-wave UV light
 Iodine Vapor—forms brown/ yellow complexes with
organic compounds
 Fluorescent Indicators—compounds fluoresce when
placed under UV light
 Silver Nitrate Spray (for Alkyl Halides)—dark spots
form upon exposure to light
 Sulfuric Acid Spray + Heat—permanent charred
spots are produced

TLC Problems: Troubleshooting
 Over migration Developer too polar Reduce polarity
 Under migration Developer too non-polar Increase polarity
 Distorted solvent front Developer not equilibrated Equilibrate
 Distorted spots Wrong adsorbent Change plates
 Distorted spots Spotted too much Change concentration
 No separation Wrong developer Change developer
 No separation Wrong adsorbent Change plate type
 Tailing Spot overloading Reduce concentration
 Tailing Component is basic Increase acidity
 Tailing Component is acidic Increase basicity
 Tailing/no separation Decomposition Developer/plate

Tugas PC dan TLC
 Menurut anda bagaimana mengidentifikasi suatu zat
asing/tidak diketahui dengan PC dan/atau TLC ?
 Jika campuran yang anda pisahkan adalah senyawa
yang tidak volatil apakah dapat dipisahkan dengan
PC atau TLC ? Berdasarkan prinsip apa pemisahan
itu dilakukan ?

ITP UNS SEMESTER 2 Kromatografi pc dan tlc

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