itp uns semester 2 kromatografi pc dan tlc

04/12/23 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 ChromatographyReal-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 PlanarKromatografi KertasKromatografi Lapisan Tipis


Fasa Gerak

Fasa Diam Fasa 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 ChromatographyDigunakan 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 ChromatographyDapat digunakan untuk memisahkan komponen-komponen tinta, pewarna, senyawa tumbuhan (klorofil), make-up, dan banyak zat lain

Liquid Chromatographydigunakan untuk identifikasi pigmen tumbuhan atau komponen lain

Thin-Layer ChromatographyMenggunakan lapisan tipis atau gelas kaca untuk memisahkan komponen kimia dan bahan lainnya

Contoh Chromatography


Kromatografi Kertas (PC)

Fase diam → kertas serapFase 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 black2. Not soluble in low concentrations of

isopropanol3. 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 ArahDigunakan dalam menyelesaikan masalah

pemisahan substansi yang memiliki nilai Rf yang sangat serupa.

Menggunakan dua pelarut yang berbeda. Pelarut pertama harus kering dahulu.


Kromatografi Lapis TipisMenggunakan 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 inexpensive TLC is a fast, simple, and inexpensive analytical technique used to determine or analytical 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 The appropriate conditions for a columncolumn

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.

04/12/23 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 compound A polar solvent will carry a polar compound farther while a non-polar solvent will carry a farther while a non-polar solvent will carry a non-polar compound farther.non-polar compound farther.

RRf f value is the ratio of the distance the spot value is the ratio of the distance the spot

travels from the origin to the distance the travels 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 Berwarna1.Menggunakan pendarflourFase 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

Technology

chromatography chromatography

s1ilmu dan teknologi

liquid solvent mobile

sample stationary phase

carrier gas mobile phase

paper strip stationary

dan banyak zat

separate components