(In the name of GOD) HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY(HPTLC) Dr. A.R.Bekhradnia

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1 (In the name of GOD) HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY(HPTLC) 1

2 HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY (HPTLC) 2

3 THIN LAYER CHROMATOGRAPHY (TLC) 3

4 Chromatography is a physical process of separation in which the components to be separated are distributed between 2 immiscible phases a stationary phase which has a large surface area and mobile phase which is in constant motion through the stationary phase. 4

5 THIN LAYER CHROMATOGRAPHY (TLC) 5

6 Mikhail Tsvet Born 14 May 1872 Asti, Italy Died 26 June 1919 (age 47) Nationality Fields botany Russia Mikhail Semyonovich Tsvet (Михаи л Семёнович Цвет, also spelled Tsvett, Tswett, Tswet, Zwet, and Cvet) ( ) was a Russian-Italian botanist who invented adsorption chromatography. 6

7 Invention of Chromatography by M. Tswett Ether Chromatography Chlorophyll Colors CaCO 3 LAAQ-B-LC001B 7

8 Comparing Chromatography to the Flow of a River... Light leaf Heavy stone Water flow Base LAAQ-B-LC001B 8

9 Chromato-graphy / -graph / -gram / -grapher Chromatography: Chromatograph: Chromatogram: Chromatographer: Analytical technique Instrument Obtained picture Person LAAQ-B-LC001B 9

10 Three States of Matter and Chromatography Types Mobile phase Gas Liquid Solid Gas Stationary phase Liquid Solid Gas chromatography Liquid chromatography LAAQ-B-LC001B 10

11 Liquid Chromatography Chromatography in which the mobile phase is a liquid. The liquid used as the mobile phase is called the eluent. The stationary phase is usually a solid or a liquid. In general, it is possible to analyze any substance that can be stably dissolved in the mobile phase. LAAQ-B-LC001B 11

12 Interaction Between Solutes, Stationary Phase, and Mobile Phase Differences in the interactions between the solutes and stationary and mobile phases enable separation. Solute Degree of adsorption, solubility, ionicity, etc. Stationary phase Mobile phase LAAQ-B-LC001B 12

13 Classification According to the force of separation: Adsorption chromatography Partition chromatography Ion exchange chromatography Gel filtration chromatography Affinity chromatography LAAQ-B-LC001B 13

14 Column Chromatography and Planar Chromatography Separation column Paper or a substrate coated with particles Packing material Column Chromatography Paper Chromatography Thin Layer Chromatography (TLC) LAAQ-B-LC001B 14

15 Output concentration Chromatogram Time 15

16 Intensity of detector signal t 0 t R h Peak A t R : Retention time t 0 : Non-retention time A : Peak area h : Peak height Time 16

17 Separation Process and Chromatogram for Column Chromatography Output concentration Chromatogram Time LAAQ-B-LC001B 17

18 THIN LAYER CHROMATOGRAPHY Once the solvent is within ~1-2 cm of the top of the TLC sheet, the TLC is removed from the developing chamber and the farthest extent of the solvent (the solvent front) is marked with a pencil. The solvent is allowed to evaporate from the TLC sheet in the hood. The spots are visualized using a UV lamp. A fluorescent compound, usually Manganeseactivated Zinc Silicate, is added to the adsorbent that allows the visualization of spots under a blacklight (UV254). The adsorbent layer will fluoresce light green by itself, but spots of analyte quench this fluorescence and appear as a dark spot. 18

THIN LAYER CHROMATOGRAPHY - Visualization As the chemicals being separated may be colorless, several methods exist to visualize the spots: Visualization of spots under a UV 254 lamp.

19 THIN LAYER CHROMATOGRAPHY - Visualization As the chemicals being separated may be colorless, several methods exist to visualize the spots: Visualization of spots under a UV 254 lamp. The adsorbent layer will thus fluoresce light green by itself, but spots of analyte quench this fluorescence. Iodine vapors are a general unspecific color. Chromatogram of 10 essential oils, Stained with vanillin reagent. Specific color reagents exist into which the TLC plate is dipped or which are sprayed onto the plate. Once visible, the R f value of each spot can be determined 19

20 THIN LAYER CHROMATOGRAPHY Calculation of Rf s R f (A) = 2.0 cm 5.0 cm = 0.40 Solvent Front Distance solvent migrated = 5.0 cm Distance A migrated = 3.0 cm 4.0 cm R f (B) = R f (C) = 3.0 cm 5.0 cm 0.8 cm 5.0 cm = 0.60 = 0.16 Distance B migrated = 2.0 cm 3.0 cm R f (D) = 4.0 cm = cm Origen Distance C migrated = 0.8 cm x x x x A B U C The R f is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin) x D 0.8 cm R f (U 1 ) = 3.0 cm 5.0 cm 0.8 cm R f (U 2 ) = 5.0 cm = 0.60 =

21 THIN LAYER CHROMATOGRAPHY Calculation of Rf s R f (A) = 2.0 cm 5.0 cm = 0.40 Solvent Front Distance solvent migrated = 5.0 cm Distance A migrated = 3.0 cm 4.0 cm R f (B) = R f (C) = 3.0 cm 5.0 cm 0.8 cm 5.0 cm = 0.60 = 0.16 Distance B migrated = 2.0 cm 3.0 cm R f (D) = 4.0 cm = cm Origen Distance C migrated = 0.8 cm x x x x A B U C x D The R f is defined as the distance the center of the spot moved divided by the distance the solvent front moved (both measured from the origin) 0.8 cm R f (U 1 ) = 3.0 cm 5.0 cm 0.8 cm R f (U 2 ) = 5.0 cm = 0.60 =

22 THIN LAYER CHROMATOGRAPHY R f s R f values can be used to aid in the identification of a substance by comparison to standards. The R f value is not a physical constant, and comparison should be made only between spots on the same sheet, run at the same time. Two substances that have the same R f value may be identical; those with different R f values are not identical. 22

23 Absorption of Solutes THIN LAYER CHROMATOGRAPHY R f s The adsorption strength of compounds increases with increasing polarity of functional groups, as shown below: -CH=CH 2, -X, -OR, -CHO, -CO 2 R, -NR 2, -NH 2, -OH, -CONR 2, -CO 2 H. (weakly adsorbed) (strongly adsorbed) (nonpolar) (more polar) Elution Strength of Mobile Phase (ε) Elution strength is generally considered to be equivalent to polarity. A solvents elution strength depends on Intermolecular Forces between the solvent and the analytes and between the solvent and the stationary phase. A more polar (or more strongly eluting solvent) will move all of the analytes to a greater extent, than a less polar, weakly elution solvent. For example, the elution strength of hexane is very low; ε = the elution strength of ethyl acetate is higher; ε = 0.45 the elution strength of ethanol is even higher; ε =

24 Solvent Properties and Elution Strengths Solvent Hexane CH 3 (CH 2 ) 4 CH 3 MF MW C 6 H Bp ( o C) Density (g/ml) Hazards* Dipole Elution Stength (ε) Flammable Toxic Toluene C 6 H 5 CH 3 C 7 H Flammable Toxic Diethyl ether CH 3 CH 2 OCH 2 CH 3 C 4 H 10 O Flammable Toxic, CNS Depressant Dichloromethane CH 2 Cl 2 CH 2 Cl Toxic, Irritant Cancer suspect Ethyl Acetate CH 3 CO 2 CH 2 CH 3 C 4 H 8 O Flammable Irritant Acetone CH 3 COCH 3 C 3 H 6 O Flammable Irritant Butanone CH 3 CH 2 COCH 3 C 4 H 8 O Flammable Irritant Butanol CH 3 CH 2 CH 2 CH 2 OH C 4 H 10 O Flammable Irritant Propanol CH 3 CH 2 CH 2 OH C 3 H 8 O Flammable Irritant Ethanol CH 3 CH 2 OH C 2 H 6 O Flammable Irritant Methanol CH 3 OH Water HOH CH 4 O H 2 O Flammable Toxic >1 24

25 Elution Strength of Mixed Solvents The elution strength of the mixture is assumed to be the weighted average of the elution strengths of the components: ε o net = ε o A (mole % A) + ε o B (mole % B) where: mole % A = (moles A) / (moles A + moles B) Thus, to determine the ε o net of a solvent mixture, the molar ratio of the solvents must first be calculated. For example, the ε o net of a solvent mixture prepared from 1.0 ml of ethyl acetate plus 9.0 ml of hexanes is calculated as shown below: ε o net = ε o EtOAc [(moles EtOAc)/(moles EtOAc+moles hexane)] + ε o hexane [(moles hexane)/(moles EtOAc+moles hexane)] where: moles EtOAc = [(volume EtOAc) (density EtOAc)] / [molecular weight of EtOAc] thus: ε o net = {0.45[(1.0mLEtOAc)(0.902g/mL)/(88.11g/mole)]+0.01[(9.0mLhexane)(0.659g/mL)/86.18g/mole)]} {(1.0 mletoac)(0.902g/ml)/88.11g/mole) + (9.0 mlhexane)(0.659g/ml)/86.18g/mole)} and ε o net =

26 Resolution The separation between two analytes on a chromatogram can be expressed as the resolution, Rs and can be determined using the following equation: Rs = (distance between center of spots) (average diameter of spots) In TLC, if the Rs value is greater than 1.0, the analytes are considered to be resolved. x x 26

27 Improving Resolution: For two closely migrating components, optimum resolutions are usually obtained when the R f s of both compounds are between 0.2 and 0.5 * To Improve Rs, change the elution strength of the solvent to optimize R f s change ε o net, all compounds will be effected similarly. Alter the composition of the solvent system so that the components affinity for the mobile phase vs. the solid phase are differentially changed (= change in selectivity). Changing the chemical nature of the solvent system, such as changing a hydrogen bonding solvent to a solvent which cannot hydrogen bond to the analyte, is often the most effective. ** Improve Rs by decreasing the diameter of the analyte spots. This can be achieved by applying smaller and less concentrated spots. TLC/TLCprocedure.html 27

28 HIGH PERFORMANCE THIN LAYER CHROMATOGARPHY (HPTLC) 28

29 Introduction of HPTLC HPTLC is the improved method of TLC which utilizes the conventional technique of TLC in more optimized way. HPTLC takes place in highspeed capillary flow range of the mobile phase. There are three main steps HPTLC procedure, they are 1] Sample preparation, volume precision and exact position are achieved by use of suitable instrument. 2] Solvent (mobile phase) migrates the planned distance in layer (stationary phase) by capillary action. In this process sample separated into it s components. 3] Separation tracks are scanned in densitometer with light beams in visible or uv region 29

30 Steps Involving in HPTLC Sample Preparation Application of sample Selection of chromatography layer Pre-washing Pre-conditioning Chromatography development Detection of spots Scanning & documentation 30

31 Sample preparation Normal phase chromatography: non polar solvent Reversed phase chromatography: polar solvent Selection of chromatography layer Depends on nature of material to be separated Commonly used(silica gel, alumina) 31

32 Pre-washing It is purification step Mainly methanol is used Essential for quantitative evaluation 32

33 Linomat lv applicator 33

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35 Selection of HPTLC plates Previously hand made plates is used in TLC for both qualitative and quantitative work. Certain drawbacks with that is nonuniform layer, formation of thick layer, paved for advent of precoated plates. Nowadays precoated plates are available in different format and thickness by various manufactures. Precaoted plates can be used for both qualitative and quantitative work in HPTLC, they are GLASS PLATES POLY ESTER/POLYETHYLYNE ALUMINIUM PLATES 35

Glass Plates: Offers superior flat and smooth surface. - fragile - high weight - higher production cost Polyester/polyethylene plates: Thickness of plate is 0.2mm.

36 Glass Plates: Offers superior flat and smooth surface. - fragile - high weight - higher production cost Polyester/polyethylene plates: Thickness of plate is 0.2mm. - It can be produced in roll forms. - Unbreakable. - Less packing material is required. - Development of plate cann t be above temperature c loses its shape. 36

37 Aluminium plates: Thickness of plate is 0.1mm. - It can be produced in roll forms. - Unbreakable. - Less packaging material is required. 37

38 SORBENTS USED IN HPTLC PLATES: sorbents which are used in convential TLC are also used in HPTLC with or without modification. - silica gel 65F - highly purified silicagel 60 - aluminium oxide - cellulose microcrystalline - silica gel - reversed stationary phase 38

The layer thickness in HPTLC is around 100-200cm,in conventional it is

39 The layer thickness in HPTLC is around cm,in conventional it is 250mm. Layer prewashing: Ascending method - Dipping method - Continuous method 39

40 The plates are activated by placing in an oven at C for 30 min, this step will removes water that has been physically absorbed on surface at solvent layer. Freshly opened box of HPTLC plates usually does not require activation. Activation at higher temp and for longer time is avoided which leads to very active layer and there is risk of sample being decomposed 40

41 - Methanol (commonly used) - Chloroform:methanol:ammonia(90:10:1) - Chloroform:methanol(1:1) - Methylene chloride:methanol(1:1) - Ammonia(1%)solution 41

42 Usual concentration range is 0.1-1µg / µl,above this causes poor separation. Linomat IV (automatic applicator) - nitrogen gas sprays sample and standard from syringe on TLC plates as bands. Band wise application - better separation - high response to densitometer. 42

Processes in the Developing Chamber The «classical» way of developing a chromatogram is to place the plate in a chamber, which contains a sufficient amount of developing solvent.

43 Processes in the Developing Chamber The «classical» way of developing a chromatogram is to place the plate in a chamber, which contains a sufficient amount of developing solvent. The lower end of the plate should be immersed several millimeters. Driven by capillary action the developing solvent moves up the layer until the desired running distance is reached and chromatography is stopped. The following considerations primarily concern silica gel as stationary phase and developments, which can be described as adsorption chromatography. 43

44 Provided the chamber is closed, four partially competing processes occur: Between the components of the developing solvent and their vapor, an equilibrium will be established eventually (1). This equilibrium is called chamber saturation. Depending on the vapor pressure of the individual components the composition of the gas phase can differ significantly from that of the developing solvent. While still dry, the stationary phase adsorbs molecules from the gas phase. This process, adsorptive saturation, is also approaching an equilibrium in which the polar components will be withdrawn from the gas phase and loaded onto the surface of the stationary phase (2). Simultaneously the part of the layer which is already wetted with mobile phase interacts with the gas phase. Thereby especially the less polar components of the liquid are released into in the gas phase (3). Unlike (1) this process is not as much governed by vapor pressure as by adsorption forces. During migration, the components of the mobile phase can be separated by the stationary phase under certain conditions, causing the formation of secondary fronts. 44

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50 Also called Chamber Saturation Low polarity mob. Phase:- no need High polar mob. Phase:- desirable For reverse phase saturate chamber with polar solvent 50

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52 LOW SOLVENT CONSUMPTION pre-equilibration with solvent vapor 52

53 CAMAG Twin Trough Chambers offer several ways to improve the results of TLC/HPTLC developing techniques. It allows low solvent consumption, reproducible pre-equilibration with solvent vapor, equilibration performed with any liquid and for any period of time, and development is started only when developing solvent is introduced into the trough with the plate. Twin Trough Chambers are available with stainless steel lid or as a Light-Weight Twin Trough Chamber made from highly transparent sheet glass with a glass lid. Start of development 53

54 After development, remove the plate and mobile phase is removed from the plate - to avoid contamination of lab atmosphere. Dry in vacuum desiccator - avoid hair drier because essential oil components may evaporate. 54

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56 Detection under UV light is first choice - non destructive. Spots of fluorescent compounds can be seen at 254 nm (short wave length) or at 366 nm (long wave length). Spots of non fluorescent compounds can be seen - fluorescent stationary phase is used - silica gel GF. 56

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60 CATS STANDARD PROGRAM. CATS PROGRAM OPTIONS 60

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62 Non UV absorbing compounds like ethambutol, dicylomine etc - dipping the plates in 0.1% iodine solution. When individual component does not respond to UV - derivatisation required for detection. 62

63 HPTLC 100µm High due to smaller particle size generated 3-5 cm Shorter migration distance and the analysis time is greatly reduced Wide choice of stationary phases like silica gel for normal phase and C8, C18 for reversed phase modes New type that require less amount of mobile phase Auto sampler Use of UV/ Visible/ Fluorescence scanner scans the entire chromatogram qualitatively and quantitatively and the scanner is an advanced type of densitometer TLC 250µm Less cm Slower Silica gel, Alumina More amount Manual spotting Not possible 63

64 Pharmaceutical Researches Biomedical Analysis Clinical Analysis Environmental Analysis Food Industry Therapeutic drug monitoring to determine concentration of drug and it s metabolite in blood, urine etc Analysis of environmental pollutions levels Quantitative determination of prostaglandin s and thromboxanes in plasma Analysis of nitrosoamines in food and body fluids Determination of sorbic acid in wine Characterization of hazards in industrial waste 64

Experiment 1: Thin Layer Chromatography

Experiment 1: Thin Layer Chromatography Part A: understanding R f values Part B: R f values & solvent polarity Part C: R f values & compound functionality Part D: identification of commercial food dye