Kromatografipcdantlc-130704081354-phpapp02.pdf

Kromatografi : Dasar Teori,PC dan TLC Kimia Analitik Semester Genap 2012/2013 Esti Widowati,S.Si.,M.P

07/04/13

S1-Ilmu dan Teknologi Pangan

1

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. • Analyze Separate

• Identify • Purify

Mixture 07/04/13

Components S1-Ilmu dan Teknologi Pangan

• Quantify 2

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

07/04/13

S1-Ilmu dan Teknologi Pangan

3

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

07/04/13

S1-Ilmu dan Teknologi Pangan

4

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)

07/04/13

S1-Ilmu dan Teknologi Pangan

5

Definition of Chromatography Simplified Definition: Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase.

Explanation: • • • •

07/04/13

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 S1-Ilmu dan Teknologi Pangan

6

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 07/04/13

S1-Ilmu dan Teknologi Pangan

7

Jenis-Jenis Kromatografi Berdasarkan

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

07/04/13

S1-Ilmu dan Teknologi Pangan

8

Kromatografi di dalam bentuk tempat Komatografi

Kolom : Kromatografi kolom merupakan teknik pemisahan dimana tempat stasioner dalam tabung.

Kromatografi

Planar

 Kromatografi

Kertas  Kromatografi Lapisan Tipis 07/04/13

S1-Ilmu dan Teknologi Pangan

9

Kromatografi

Gas

Cair

Plat

Kolom

07/04/13

Fasa FasaDiam Diam

Pertukaran Ion

Padat

Cair

Anion

Fasa Gerak

Kation

S1-Ilmu dan Teknologi Pangan

Gel

Cair

GPC 10

Types of Chromatography • Liquid Chromatography – separates liquid samples with a liquid solvent (mobile phase) and a column of solid beads (stationary phase)

composed

• 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 (stationary phase)

paper strip

• 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)

07/04/13

S1-Ilmu dan Teknologi Pangan

11

Contoh Chromatography 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

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. 07/04/13

S1-Ilmu dan Teknologi Pangan

Paper Chromatography Dapat digunakan untuk memisahkan komponen-komponen tinta, pewarna, senyawa tumbuhan (klorofil), make-up, dan banyak zat lain 12

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 07/04/13

S1-Ilmu dan Teknologi Pangan

13

Materials List           

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

07/04/13

S1-Ilmu dan Teknologi Pangan

14

Preparing the Isopropanol Solutions • Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100%

07/04/13

S1-Ilmu dan Teknologi Pangan

15

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

07/04/13

S1-Ilmu dan Teknologi Pangan

16

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

07/04/13

S1-Ilmu dan Teknologi Pangan

17

07/04/13

S1-Ilmu dan Teknologi Pangan

18

Developing the Chromatograms

07/04/13

S1-Ilmu dan Teknologi Pangan

19

Developing the Chromatograms

07/04/13

S1-Ilmu dan Teknologi Pangan

20

Observing the Chromatograms

0% 07/04/13

20%

50%

70%

Concentration Isopropanol S1-Ilmu danof Teknologi Pangan

100% 21

Black Dye 1. Dyes separated – purple and black 2. Not soluble in low concentrations of isopropanol 3. Partially soluble in concentrations of isopropanol >20%

0% 07/04/13

20%

50%

70%

S1-Ilmu dan Teknologi Pangan

Concentration of Isopropanol

100% 22

Illustration of Chromatography Stationary Phase

Separation

Mobile Phase

Mixture

07/04/13

Components

Components

Affinity to Stationary Phase

Affinity to Mobile Phase

Blue

----------------

Insoluble in Mobile Phase

Black





Red





Yellow

Teknologi Pangan S1-Ilmu dan

        

23

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.

07/04/13

S1-Ilmu dan Teknologi Pangan

24

07/04/13

S1-Ilmu dan Teknologi Pangan

25

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 (SiO2) atau alumina (Al2O3), atau substansi yang dapat berpendarflour dalam sinar ultra violet.  Fase gerak → pelarut atau campuran pelarut yang sesuai. 07/04/13

S1-Ilmu dan Teknologi Pangan

26

Thin-Layer Chromatography (TLC) TLC is a fast, simple, and inexpensive analytical technique used to determine or monitor: - The # of components in a mixture. - The identity of two substances. - The effectiveness of a purification. - The appropriate conditions for a column chromatographic separation. - The progress of a reaction. - Column chromatography effectiveness. 07/04/13

S1-Ilmu dan Teknologi Pangan

27

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

S1-Ilmu dan Teknologi Pangan

28

TLC  The

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

07/04/13

S1-Ilmu dan Teknologi Pangan

29





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 C18 and the mobile phase is a mixture of water and organic solvent which is more polar than the stationary phase.

07/04/13

S1-Ilmu dan Teknologi Pangan

30

Adsorbents for TLC  Silica

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

07/04/13

S1-Ilmu dan Teknologi Pangan

31

Thin-Layer Chromatography (TLC) 



A polar solvent will carry a polar compound farther while a non-polar solvent will carry a non-polar compound farther. Rf value is the ratio of the distance the spot travels from the origin to the distance the solvent travels.

07/04/13

S1-Ilmu dan Teknologi Pangan

32

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: .

07/04/13

distance traveled by substance distance traveled by solvent front .

S1-Ilmu dan Teknologi Pangan

33

Advantages of TLC           

07/04/13

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 S1-Ilmu dan Teknologi Pangan

34

Applications of TLC TLC has several important uses in organic chemistry. Some examples are:



1.

2.

3.

4. 07/04/13

To establish that two compounds are identical To determine the number of components in a mixture To determine the appropriate solvent for a column-chromatographic separation To monitor the progress of a reaction S1-Ilmu dan Teknologi Pangan

35

Steps in TLC Analysis  The

following are the important components of a typical TLC system:     

07/04/13

Apparatus (developing chamber) Stationary phase layer and mobile phase Application of sample Development of the plate Detection of analyte

S1-Ilmu dan Teknologi Pangan

36

General Procedure (1)       

07/04/13

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 S1-Ilmu dan Teknologi Pangan

37

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

07/04/13

S1-Ilmu dan Teknologi Pangan

38

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)

07/04/13

S1-Ilmu dan Teknologi Pangan

39

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) 07/04/13

(Less Polar) S1-Ilmu dan Teknologi Pangan

40

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)

07/04/13

S1-Ilmu dan Teknologi Pangan

41

Procedure: TLC Plate Development  



  

07/04/13

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)

S1-Ilmu dan Teknologi Pangan

42

Development

07/04/13

S1-Ilmu dan Teknologi Pangan

43

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.

07/04/13

S1-Ilmu dan Teknologi Pangan

44

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:

07/04/13

S1-Ilmu dan Teknologi Pangan

45

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 bercakbercak 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.

07/04/13

S1-Ilmu dan Teknologi Pangan

–Ultraviolet light at 254 nm (shortwave UV). –Long wave UV (340 nm) is used less commonly. 46

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 senyawasenyawa 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.

07/04/13

S1-Ilmu dan Teknologi Pangan

47

Visualization

07/04/13

S1-Ilmu dan Teknologi Pangan

48

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

07/04/13

S1-Ilmu dan Teknologi Pangan

49

TLC Problems: Troubleshooting           

Over migration Developer too polar Under migration Developer too non-polar Distorted solvent front Developer not equilibrated Distorted spots Wrong adsorbent Distorted spots Spotted too much No separation Wrong developer No separation Wrong adsorbent Tailing Spot overloading Tailing Component is basic Tailing Component is acidic Tailing/no separation Decomposition 07/04/13

S1-Ilmu dan Teknologi Pangan

Reduce polarity Increase polarity Equilibrate Change plates Change concentration Change developer Change plate type Reduce concentration Increase acidity Increase basicity Developer/plate 50

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 ?

07/04/13

S1-Ilmu dan Teknologi Pangan

51