ITP UNS SEMESTER 2 Kromatografi pc dan tlc

3,649 views

Published on

Published in: Technology
0 Comments
5 Likes
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
3,649
On SlideShare
0
From Embeds
0
Number of Embeds
3
Actions
Shares
0
Downloads
213
Comments
0
Likes
5
Embeds 0
No embeds

No notes for slide

ITP UNS SEMESTER 2 Kromatografi pc dan tlc

  1. 1. 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
  2. 2. 07/04/13 S1-Ilmu dan Teknologi Pangan 2 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
  3. 3. 07/04/13 S1-Ilmu dan Teknologi Pangan 3 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
  4. 4. 07/04/13 S1-Ilmu dan Teknologi Pangan 4 Dasar Kromatografi Kromatografi adalah teknik pemisahan campuran didasarkan atas perbedaan distribusi dari komponen-komponen campuran tersebut diantara dua fase,
  5. 5. 07/04/13 S1-Ilmu dan Teknologi Pangan 5 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)
  6. 6. 07/04/13 S1-Ilmu dan Teknologi Pangan 6 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
  7. 7. 07/04/13 S1-Ilmu dan Teknologi Pangan 7 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
  8. 8. 07/04/13 S1-Ilmu dan Teknologi Pangan 8 Jenis-Jenis Kromatografi Berdasarkan fase gerak yang digunakan, kromatografi dibedakan menjadi dua golongan besar yaitu gas chromatography dan liquid chromatography. Kromatografi adsorpsi dan partisi
  9. 9. 07/04/13 S1-Ilmu dan Teknologi Pangan 9 Kromatografi di dalam bentuk tempat Komatografi Kolom : Kromatografi kolom merupakan teknik pemisahan dimana tempat stasioner dalam tabung. Kromatografi Planar Kromatografi Kertas Kromatografi Lapisan Tipis
  10. 10. 07/04/13 S1-Ilmu dan Teknologi Pangan 10 Fasa Gerak Fasa DiamFasa Diam Kromatografi Padat GelPertukaran Ion Gas Cair Plat Kolom Anion Cair Cair GPCKation
  11. 11. 07/04/13 S1-Ilmu dan Teknologi Pangan 11 • 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
  12. 12. 07/04/13 S1-Ilmu dan Teknologi Pangan 12 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
  13. 13. 07/04/13 S1-Ilmu dan Teknologi Pangan 13 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
  14. 14. 07/04/13 S1-Ilmu dan Teknologi Pangan 14  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
  15. 15. 07/04/13 S1-Ilmu dan Teknologi Pangan 15 Preparing the Isopropanol Solutions • Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100%
  16. 16. 07/04/13 S1-Ilmu dan Teknologi Pangan 16 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
  17. 17. 07/04/13 S1-Ilmu dan Teknologi Pangan 17 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
  18. 18. 07/04/13 S1-Ilmu dan Teknologi Pangan 18
  19. 19. 07/04/13 S1-Ilmu dan Teknologi Pangan 19 Developing the Chromatograms
  20. 20. 07/04/13 S1-Ilmu dan Teknologi Pangan 20 Developing the Chromatograms
  21. 21. 07/04/13 S1-Ilmu dan Teknologi Pangan 21 Observing the Chromatograms Concentration of Isopropanol 0% 20% 50% 70% 100%
  22. 22. 07/04/13 S1-Ilmu dan Teknologi Pangan 22 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%
  23. 23. 07/04/13 S1-Ilmu dan Teknologi Pangan 23 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
  24. 24. 07/04/13 S1-Ilmu dan Teknologi Pangan 24 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.
  25. 25. 07/04/13 S1-Ilmu dan Teknologi Pangan 25
  26. 26. 07/04/13 S1-Ilmu dan Teknologi Pangan 26 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.
  27. 27. 07/04/13 S1-Ilmu dan Teknologi Pangan 27 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.
  28. 28. 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
  29. 29. TLC  The two most common classes of TLC are:  Normal phase (Fase normal)  Reversed phase (Fase terbalik) 07/04/13 29S1-Ilmu dan Teknologi Pangan
  30. 30.  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. 07/04/13 30S1-Ilmu dan Teknologi Pangan
  31. 31. Adsorbents for TLC  Silica gel  Silica gel-F (Fluorescing indicator added)  Magnesium Silicate (Florisil)  Polyamides  Starch  Alumina 07/04/13 31S1-Ilmu dan Teknologi Pangan
  32. 32. 07/04/13 S1-Ilmu dan Teknologi Pangan 32 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.
  33. 33. 07/04/13 S1-Ilmu dan Teknologi Pangan 33 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
  34. 34. 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 07/04/13 34S1-Ilmu dan Teknologi Pangan
  35. 35. 07/04/13 S1-Ilmu dan Teknologi Pangan 35 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
  36. 36. 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 07/04/13 36S1-Ilmu dan Teknologi Pangan
  37. 37. 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 07/04/13 37S1-Ilmu dan Teknologi Pangan
  38. 38. 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 38S1-Ilmu dan Teknologi Pangan
  39. 39. 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 39S1-Ilmu dan Teknologi Pangan
  40. 40. 07/04/13 S1-Ilmu dan Teknologi Pangan 40 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)
  41. 41. 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 41S1-Ilmu dan Teknologi Pangan
  42. 42. 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) 07/04/13 42S1-Ilmu dan Teknologi Pangan
  43. 43. 07/04/13 S1-Ilmu dan Teknologi Pangan 43 Development
  44. 44. 07/04/13 S1-Ilmu dan Teknologi Pangan 44 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.
  45. 45. 07/04/13 S1-Ilmu dan Teknologi Pangan 45 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:
  46. 46. 07/04/13 S1-Ilmu dan Teknologi Pangan 46 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.
  47. 47. 07/04/13 S1-Ilmu dan Teknologi Pangan 47 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.
  48. 48. 07/04/13 S1-Ilmu dan Teknologi Pangan 48 Visualization
  49. 49. 07/04/13 S1-Ilmu dan Teknologi Pangan 49 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
  50. 50. 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 07/04/13 50S1-Ilmu dan Teknologi Pangan
  51. 51. 07/04/13 S1-Ilmu dan Teknologi Pangan 51 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 ?

×