Chemistry of Separation

1. Chemistry of Separation

2. Outline • Introduction • Types • Extraction • Phase changes • Electric Fields • Flotation • Membranes • Other • Chromatographic

3. Sampling Instrumentation Processing Sample Calibration Measurement Interpretation Pretreatment Extraction Separation Clean up Concentration Derivatization Analytical Process Food

4. Homework • Using the flow diagram for the analytical process, fit your research project into an analytical process. • Food, biological tissue or fluid • Sampling • Extraction – what is the analyte? • Instrumentation – what will you use to measure the analyte? How will you calibrate? • Processing and interpretation

5. Introduction • Separation • Anderson, 1987 “physical transfer of a particular chemical substance from one phase or medium to another, or the actual physical separation of the components of a mixture into separate fractions.” • Meloan, 1999 “is a process whereby compounds of interest are removed from the other compounds in the sample that may react similarly and interfere with a quantitative determination.” • Seader and Henley, 1998 “The separation of chemical mixtures into their constituents. Separations including enrichment, concentration, purification, refining, and isolation.”

6. Introduction

7. Gas Chromtography Phase • Volatilization • Conversion of all or part of a solid or liquid into a gas • What are ways that support this conversion? • Heat • Strong acids • Oxidation • Reduction • What analytical instrument uses this same principle?

8. Phase • Distillation • The production of a vapor from liquid by heating, condensing the vapor, and collecting in a separate vessel • Vapor pressure – the pressure exerted by molecules that have escaped the liquid’s surface • Molecules in the gas state are in constant motion • Usually several hundred miles per hour • Size, shape, and chemical properties • This relates to surface tension • Examples: simple, fractional

9. Fractional Distillation Toluene + Benzene

10. Fractional Distillation • Plates • Theoretical plates • Represent each equilibrium step in the refluxing system • HETP (Height Equivalent to a Theoretical Plate) • Takes into account the distance from surface of liquid to the top of the column • Measures the efficiency of distillation

11. Fractional Distillation • Continuous Refluxing • Total • Partial

12. Homework • Ethyl isobutyrate (b.p. = 111C) and ethyl isovalerate (b.p. = 135C) are used for flavors and essences. • Briefly explain how fractional distillation works? • Can these be separated using this technique? • Explain you answer? • Think about theoretical plates?

13. Azeotropic & Extractive Distillations • Azeotrope • Liquid mixture characterized by a maximum or minimum boiling pt. (bp) which is lower or higher than bp for any of the components and that distills without change in composition • Distillation – form an azeotrope

14. Azeotropic & Extractive Distillations From: Meloan, 1999. Chemical Separations: Principles, Techniques, and Experiments, John Wiley & Sons, Inc., New York.

15. Azeotropic & Extractive Distillations • Extractive • A third component is added to extract one of the major components • Other interactions • Hydrogen, dipole-dipole, ion-dipole, pi bonds Solvent

16. Steam & VacuumDistillations • Used for components that decompose at or near its bp • Steam • Limited to those components that are immiscible with water • Problem – Emulsion form • Usually forms when densities of 2 liquids are similar • Breaking emulsions • Glass wool • Centrifuge • Salts • Acids • Phase separation paper (Whatman PS-1)

17. Steam & VacuumDistillations • Vacuum • Any distillation below atmospheric pressure • Advantage boiling pt differences increase at reduced pressures

18. Sublimation • Process which converts a solid to a gas bypassing the liquid phase • A solid will sublime if its vapor pressure reaches atmospheric pressure below its melting point

19. Sublimation • Lyophilization

20. Electrical Field Separations • Gel Matrix • Electrophoresis • Disc • Isoelectric Focusing • Immuno • Capillary Electrophoresis

21. Electrical Field Separations • Electrophoresis • Charged molecules in solution are separated based on differences in size and charge when a high voltage is applied

22. Electrical Field Separations • Electrophoresis • Theory • Mobility (U) – requires a net electrostatic charge • Can neutral particles be separated electrophoretically? • Charging processes: acids and bases, dissociation into ions by polar solvents, hydrogen bonding, chemical reactions, polarization, ion pair formation Fs F - + + Fs=6prhu r, radius of the particle (cm), h, viscosity of the medium (poises), u, electrophoretic velocity (cm/sec) F=QE Q, charge on the particle E, field strength

23. Electrical Field Separations • Electrophoresis • Theory Fs F - + + Fs=6prhu r, radius of the particle (cm), h, viscosity of the medium (poises), u, electrophoretic velocity (cm/sec) F=QE Q, charge on the particle E, field strength Thus, Fs=QE=6prhu and U=Q/6prh

24. Smiling Electrical Field Separations • Electrophoresis • Major problem • Heating • An increased rate of diffusion of sample and buffer ions leading to broadening of the separated samples. • The formation of convection currents, which leads to mixing of separated samples. • Thermal instability of samples that are rather sensitive to heat. This may include denaturation of proteins or loss of activity of enzymes. • A decrease of buffer viscosity, and hence a reduction in the resistance of the medium. R = V / I R, resistance, V, voltage, I, current W = I2 R W, watts,R, resistance, I, current http://www.mnstate.edu/marasing/CHEM480/Handouts/Chapters/Capillary%20Electrophoresis.pdf

25. Flotation • Purge and Trap • Foam fractionation • Gas-solid flotation • Liquid-solid flotation

26. Flotation • Foam fractionation • Based on transferring one or more components in a liquid to the surface of gas bubbles passing through it and collecting the separated components in a foam at the top of the liquid.

27. Flotation • Foam fractionation • Factors • Foamers – use material of opposite charge to the sample to make a good foam • Defoamers – benzene, quanternary amines, silicones • Chain Length – chain length of nonpolar end of surfactant increases, its absorption and separation increases • Surfactant concentration – separation increases as concentration increases up to a point • pH – alters ionic species

28. Flotation • Foam fractionation • Purge and Trap • Removal and collection of volatile compounds from a liquid by diffusion of the volatiles into a stream of gas bubbles passing through it and trapping the expelled particles. • Purpose - concentration

29. Flotation • Foam fractionation • Purge and Trap Trapping System Purging system

30. Flotation • Foam fractionation • Purge and Trap • Purge Efficiency • Vapor pressure – higher vapor pressure, higher purge efficiency • Solubility – greater solubility in the sample matrix, harder to remove • Temperature – increase in temperature always increases purge efficiency • Sample size – increase sample size requires increase in purge volume • Purge volume – increase in purge volume improves efficiency • Purge method – given same purge volume, fine bubble dispersion better than large bubbles

31. Flotation • Foam fractionation • Purge and Trap • Traps • Factors for a good trap • Retain analytes of interest • Allow gases to pass readily • Release analyte easily • Stability – don’t release volatiles or cause side reactions • Reasonably priced

32. Homework • Explain the technique of purge and trap? • Include in your explanation • What is meant by purging and trapping? • What factors influence purge efficiency? • What factors influence trap efficiency?

33. Membranes • Filtering and Sieving • Selectively remove a portion of a mixture by passing through a semi-porous material • Material if porous with small pore holes – filtering • Material is a screen with large pore holes – screening • There is a slew of filtering papers for the analytical chemist to use • Filters with phases bonded which allows the filter to behave like a column in HPLC or GLC

34. Membranes • Filtering and Sieving

35. Membranes • Filtering and Sieving • Proper filtering 1. Use proper grade filter; 2. Decant; 3. Use long stem funnel; 4. Use narrow diameter stem rather than long one; 5. Use fluted funnel if possible; 6. Fold paper with 1/8 to 1/4th inch offset; 7. Tear paper at top of fold to prevent air intake; 8. Keep stem full of solution; 9. Touch end of stem to side of beaker

36. Membranes • Osmosis & Reverse Osmosis • 2nd Law of Thermodynamics – systems tend toward disorder • High concentration goes to low concentration • Osmosis involves solvent • Dialysis involves solute

37. Membranes • Osmosis & Reverse Osmosis • Difference in thermodynamic potential – Gibbs Free energy • Higher in pure solvent than solution • Tendency for system to reach equilibrium – free energy equal – the difference is the driving force and therefore osmosis.

38. Membranes • Osmosis & Reverse Osmosis • Application of pressure to force the solvent back to the other side – Reverse osmosis • Parameters • Diffusion coefficient D; permeability coefficient P; solubility constant S; filtration coefficient Lp; solute permeability coefficient ; reflection coefficient 

39. Membranes • Dialysis • Removal of low molecular weight solute molecules from a solution by passing through a semi-permeable membrane driven by a concentration gradient • Ultrafiltration • Combination of reverse osmosis and dialysis?

40. Other Techniques • Density • Use density gradients • Principle – object placed in a fluid will sink if density is greater than the fluid, will float if density less than fluid or will stay suspended if densities of object and fluid are the same. • Centrifugation • Separates based on density and amplified by applying a rotational force • RCF = 1.118 x 10-5 r N2 where r, radial distance of a particle from axis of rotation in cm; N, speed of rotation in rpm

41. Homework • Why is it not appropriate when describing centrifugation protocols to list the conditions of centrifugation in rpm’s?

42. Solubility • Extraction • Solvent • Chromatography