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Physical and Chemical Properties. DensityBoiling PointMelting PointIndex of RefractionSolubility PolarityViscosityColor, Odor, Phase. Steps to Identify an Organic Compound. Isolate compoundFrom reaction mixtureFrom natural sourcePurify compoundChromatographyPhysical PropertiesIdentify

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1. Chromatography Chemistry 223 Fall 2008

2. Physical and Chemical Properties Density Boiling Point Melting Point Index of Refraction Solubility – Polarity Viscosity Color, Odor, Phase

3. Steps to Identify an Organic Compound Isolate compound From reaction mixture From natural source Purify compound Chromatography Physical Properties Identify pure compound Spectroscopy

4. History of Chromatography 1903 Tswett - plant pigments separated on chalk columns (radial chromatography) 1931 Lederer & Kuhn - LC of carotenoids 1938 TLC and ion exchange 1950 Reverse phase LC 1954 Martin & Synge (Chemistry, 1952) 1959 Gel permeation 1965 Instrumental LC (Waters)

5. Early Separation Techniques Liquid-Liquid extraction Two non-miscible liquids “Like dissolves like” Fractional recrystallization Fractional distillation Use distillation columns with high surface areas (glass helices, metal turnings, beads) Filtration methods Adsorption methods (activated charcoal)

6. Liquid Chromatography The first separations were achieved by using gravity to drive the separations: Gravity flow column chromatography Planar chromatography (TLC) Descending paper chromatography Radial paper chromatography Gravity separations could be done in two dimensions with planar methods

7. Thin Layer Chromatography Process occurs on a thin (<100 µm) of support placed on glass or aluminum sheets Sample spotted on silica, alumina, cellulose surface (1-10 µL) Plate placed in solvent to develop the plate, allowing the solvent to move by capillary action Plate dried and separated compounds visualized with specific reagents Coeluting samples can be resolved by using 2-d methods with two solvent systems

8. Chromatographic Materials TLC: silicic acid (H4SiO4), silica gel, alumina (Al2O3), cellulose Mess size important (5-400 mesh) Binders used to hold the silica gel on the substrate (aluminum, glass, plastic) Polymeric binders Calcium sulfate Plate thickness also important Thicker surfaces can be used preparatively to purify large amounts of an analyte

9. Modern Techniques Gas-Liquid Chromatography Gas-Solid Chromatography Gravity Column Chromatography High Performance Chromatography Electrophoresis Gel, Paper, Cellulose acetate Capillary Electrophoresis

10. Key Ingredient In… Chromatography Based Instruments are essential to: Genomics Capillary Electrophoresis Lipidomics GC-MS Systems Proteomics Gel, Capillary Electrophoresis Metabolomics

11. Key Terminology Tswett separated plant pigments on chalk columns with a solvent mixture. He also used radial chromatography and coined the term chroma (color) graphy (writing) Column: support Solvent: eluent Fractions: effluent fractions

12. The Chromatographic Process Critical defining properties of the process Immiscible stationary and mobile phases Arrangement whereby a mixture is deposited at one end of the stationary phase Flow of the mobile phase toward the other end of the stationary phase Different rates or ratios of partitioning for each component of the mixture, and many cycles of this process during elution A means of visualizing bands of separated components on or adjacent to the stationary phase, or of detecting eluting bands as peaks in the mobile phase effluent

13. Types of Chromatography

14. Chromatographic Detectors Mass detectors (amount detectors) give signals proportional to the total amount of molecules exiting the column – mass spectrometer total ion current (TIC) Concentration detectors measure the total number of molecules exiting the column in a given volume (µg/mL) at any given time. If the flow rate is faster, the peak will be narrower, if the flow rate slows, the peaks will be broader. The longer the residence time on the column the broader the peaks will be

15. Rtx-5 Diphenyl Polysiloxane Stationary Phase

16. GC Columns (1) GC first used metal columns (1/4 SS or copper) and packed columns with the liquid phase coating inert particles made of silica or crushed firebrick. Glass (race track, coiled Ľ inch columns) were used with packed column GC to avoid the active surfaces on the metal which caused oxygenated compounds to decompose. The next development was capillary columns (0.52 inch OD) with the liquid phase (support) coating the inner surface (SCOT columns)

17. GC Columns (2) Capillary columns have been created from 0.52 to 0.22 in in diameter and lengths up to 100 meters. They are coated on the outside with a polyamide cladding to protect them from breaking. The inner surface has a chemically bonded liquid phase that prevents or reduces “bleeding” of the liquid phase at higher temperatures

18. Packed Column GC Materials Early liquid phases were silicone stopcock grease (polar) and Apiezon (high vacuum grease) nonpolar with many silicone polymers in between. The support materials were substances like deactivated Kiesulghur (diatomaceous earth) and crushed firebrick. The liquid loading was from 1-30% depending on the porosity of the materials.

19. GC - Common Industrial Solvents

20. Spectroscopy FT-Infrared Spectroscopy UV-Vis Spectrophotometry Proton NMR Spectrometry Carbon NMR Spectrometry Raman Spectrometry Mass Spectrometry

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