Chromatography. writing in color

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Winfred Holland
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1 Chromatography writing in color

2 Outlines of Lecture Chromatographic analysis» Principles and theory.» Definition.» Mechanism.» Types of chromatography.» Uses of Chromatography.

3 In 1906 Mikhail Tswett used to chromatography to separate plant pigments as chlorophylls, xanthophylls and carotenoids by using calcium carbonate as adsorbent and petrol ether/ethanol mixtures as eluent. He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column Chroma means color and graphein means to write Mikhail Tswett Russian Botanist ( )

4 Definition: Is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components, by distribution of its components between a mobile and stationary phase over time Analyze Mixture Separate Components Identify Purify Quantify

5 Concepts: Mobile phase (Solvent or Eluent or carrier): The mobile phase can be a gas (GC), a liquid, or a supercritical fluid (SCFC), that carries the components. Stationary phase (Adsorbent): It is a part that does not move with the sample

6 Concepts: The sample (solute): The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples that interact greatly, then appear to move more slowly. Samples that interact weakly, then appear to move more quickly. Because of this difference in rates, the samples can then be separated into their components.

7 How Does Chromatography Work? Stationary Phase Separation Mixture Mobile Phase Components

8 Types of Chromatography Classification of chromatography according to mobile phase: Liquid chromatography (LC): mobile phase is a liquid (water or organic solvents). Gas chromatography (GC): a gas. mobile phase is Supercritical fluid (SCFC): mobile phase is a supercritical fluid (CO 2 (

9 Types of Chromatography Classification according to the packing of the stationary phase: 1.Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminum plates. 2.Column chromatography (CC): stationary phase is packed in a glass column. 3.Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert support.

10 Types of Chromatography Classification according to the force of separation: 1.Adsorption chromatography: Separation based on the relative differences in adsorption of components to the solid stationary phase. Stationary phase is solid: applies to only solid-liquid or solid-gas chromatography For polar non-ionic compounds Ex; CC, TLC.

11 Types of Chromatography Classification according to the force of separation: 2. Partition chromatography: Solute are separated based on their partition between a liquid mobile phase and a liquid stationary phase coated on a solid support. Stationary phase and mobile phase are liquid in nature. Normal analyte is nonpolar organic; stationary phase MORE polar than the mobile phase Reverse analyte is polar organic; stationary phase LESS polar than the mobile phase Ex : PC, HPLC, HPTLC, GC Phase 2 Phase 1 Phase 2 Phase 1

12 Types of Chromatography Classification according to the force of separation: 3. Ion exchange chromatography: Use ionic stationary phase ions separated on the basis of their tendency to displace counter ions adsorbed on stationary phase (Depends on charge, hydration, solubility ) Stationary phase: is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups Mobile phase: buffer, ph and salt concentration-opposite charged solute ions attracted to the stationary phage by electrostatic force For ionic compounds: large proteins, small nucleotides and amino acids Ex : CC, HPLC

Quaternary amine; and DEAE resin, DiEthylAminoEthane), used for cation separation Cationic exchange

13 Types of Chromatography Classification according to the force of separation: 3. Ion exchange chromatography: Anionic exchange chromatography: stationary phase is anion (Q-resin, a Quaternary amine; and DEAE resin, DiEthylAminoEthane), used for cation separation Cationic exchange chromatography: stationary phase is cation (S-resin, sulfate derivatives; and CM resins, carboxylate derived ions), used for anion separation

14 Types of Chromatography Classification according to the force of separation: 4. Size Exclusion Chromatography: Separation is a result of trapping of molecules in the pores of the packing material Very large molecules can t get into the pores unretained Very small molecules get hung up in to pores for a long time - most retained longest retention time stationary phase is a porous matrix Ex: CC, HPLC

15 Uses of 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

16 Uses of Chromatography Real-life uses of 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 (forensic): 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

17 Thin layer chromatography (TLC) Definition of TLC: TLC: is a method for identifying substances and testing the purity of compounds. The stationary phase: is a thin layer of adsorbent (usually silica gel) coated on a plate (glass, metal, or plastic). The mobile phase: is a developing liquid (A solvent of varying polarity), which travels up the stationary phase, carrying the samples with it.

18 Thin layer chromatography (TLC) Cover TLC sheet Adsorbant Glass tank Sample spot Base line Eluent

19 Thin layer chromatography (TLC)

20 Thin layer chromatography (TLC)

Thin layer chromatography (TLC) Interpreting the Data: If the

plate with visualizing agents: Alkaloids: Dragendorff s reagent,

21 Thin layer chromatography (TLC) Interpreting the Data: If the spots can be seen, outline them with a pencil. If no spots are obvious, use a UV lamp. If no spots are obvious with UV, use iodine solution or Spray plate with visualizing agents: Alkaloids: Dragendorff s reagent, Cardiac glycosides: Antimony trichloride, Sugar: Aniline phthalate, Amino acids: Ninhydrin, flavonoids: Aluminium chloride

22 Thin layer chromatography (TLC) Interpreting the Data: Once visible, the R f (relative flow OR retention factor) value of each spot can be determined. The R f is defined as the distance from the center of the spot moved divided by the distance the solvent front moved (both measured from the origin) R f value is constant for a particular compound, solvent system and insoluble matrix.

23 Thin layer chromatography (TLC) Advantages of TLC: Low cost Short analysis time Ease of sample preparation All spots can be visualized Uses small quantities of solvents Wide choice of materials as sorbents

24 Paper chromatography (PC) Definition of PC: PC: A method of partition chromatography using filter paper as carrier or inert support. The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases. Stationary phase: usually water adsorbed on cellulose fibers in the paper (bound water). Mobile phase: is the organic solvent flows past the sample on the paper.

25 Paper chromatography (PC) Principles of PC: 1. Compound is placed on stationary phase 2. Mobile phase passes through the stationary phase 3. Mobile phase solubilizes the components and carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases

26 Paper chromatography (PC) Types of PC: 1. Mono-dimension PC: Ascending chromatography: The solvent travel in upward direction on the paper. Descending chromatography: The solvent travel in downward direction on the paper. Note: The solvent reservoir is at the top, The movement of solvent is assisted by gravity besides capillary action. Radial chromatography: The solvent travels from center towards periphery of paper

Paper chromatography (PC) Types of PC: 2. Two dimensional chromatography When large numbers of substances are to be separated on a single chromatogram.

27 Paper chromatography (PC) Types of PC: 2. Two dimensional chromatography When large numbers of substances are to be separated on a single chromatogram. The sample is applied on one corner of a square piece of paper and after development with the first solvent, the paper is dried, rotated 90 o and developed in the second direction. Usually, different types of solvents are used in each direction. It is essential that the first solvent be completely volatile

Paper chromatography (PC) Interpreting the Data: Spots in paper chromatograms can be detected in 4 different ways: 1. By their natural color. 2. By their fluorescence. 3. By their chemical reactions.

28 Paper chromatography (PC) Interpreting the Data: Spots in paper chromatograms can be detected in 4 different ways: 1. By their natural color. 2. By their fluorescence. 3. By their chemical reactions. 4. By radioactivity. The spots are usually identified by comparing of standards of known R f values. By comparing the R f values of components of a mixture with the R f values of known substances under identical conditions, the compounds present in a mixture can be identified

29 Column chromatography (CC) CC is an extremely valuable technique for purification of synthetic or natural products. The same mechanism as TLC. A variety of adsorbents can be used as the stationary phase (Organic: cellulose, polyamide, polyethylene or Inorganic: silica gel, aluminum oxide and magnesium silicate); silica gel (which is very polar) is most commonly used in organic chemistry. The mobile phase is a moving liquid or gas. There are two techniques based on column chromatography, high performance liquid chromatography (HPLC) and gas chromatography (GC).

30 Column chromatography (CC)

31 Gas chromatography (GC) This is the most sensitive chromatographic technique It is capable of detecting as little as 10-12g of a compound It is limited to compounds that can be easily vaporised without decomposing.

32 Gas chromatography (GC) Gas chromatography has the following features: The mobile phase is a gas, usually nitrogen, called the carrier gas A small amount of sample is injected into the top of the column through an injection port The injection port is heated to a temperature sufficient to instantly vaporise the sample, which is then swept into the column by the carrier gas. The column is in a loop, this is because of the fast moving gaseous phase. So the column must be longer than in HPLC to allow for effective interaction with the stationary phase The column is mounted in an oven and heated

33 Gas chromatography (GC) Interpreting Chromatograms: The time a component takes to pass through the column is called the retention time, Rt. The same compound will give the same retention time if the conditions (temp, mobile phase, stationary phase, flow rate, pressure etc) remain the same. Each component forms one peak, however it is possible for a number of peaks to coincide and be indistinguishable.

34 Gas chromatography (GC) Figure. Gas chromatogram of a petrol sample. Figure. Chromatogram of a reference sample containing a mixture of butane, 2-methylbutane, hexane, benzene and 2-methylhexane.

35 High performance liquid chromatography (HPLC) This method is used for pharmaceutical and industrial analysis. It allows extremely sensitive analysis of a wide range of compounds.

36 High performance liquid chromatography (HPLC) Advantages of HPLC: High separation capacity, enabling the batch analysis of multiple components Superior quantitative capability and reproducibility Moderate analytical conditions Unlike GC, the sample does not need to be vaporized. Generally high sensitivity Low sample consumption Easy preparative separation and purification of samples

37 High performance liquid chromatography (HPLC) What is HPLC used for? Food products: Vitamins, food additives, sugars, organic acids, amino acids, etc. Environmental samples: Inorganic ions, Hazardous organic substances, etc. Organic industrial products: Synthetic polymers, additives, surfactants, etc. Biogenic substances: Sugars, lipids, nucleic acids, amino acids, proteins, peptides, steroids, amines, etc. Medical products: Drugs, antibiotics, etc.

DEFINITION CHROMATOGRAPHY

Chromatography DEFINITION CHROMATOGRAPHY The separation of a mixture by distribution of its components between a mobile and stationary phase over time mobile phase = solvent stationary phase = column packing