O UTLINE Introduction Types Extraction Phase changes Electric Fields Flotation Membranes Other Chromatographic
A NALYTICAL P ROCESS Processing Interpretation Instrumentation CalibrationMeasurement Food Sample Sampling Pretreatment Extraction Separation Clean up Concentration Derivatization
H OMEWORK 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
I NTRODUCTION 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.”
P HASE 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? Gas Chromtography
P HASE 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
F RACTIONAL D ISTILLATION 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
F RACTIONAL D ISTILLATION Continuous Refluxing Total Partial
H OMEWORK 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?
A ZEOTROPIC & E XTRACTIVE D ISTILLATIONS 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
A ZEOTROPIC & E XTRACTIVE D ISTILLATIONS From: Meloan, 1999. Chemical Separations: Principles, Techniques, and Experiments, John Wiley & Sons, Inc., New York.
A ZEOTROPIC & E XTRACTIVE D ISTILLATIONS Extractive A third component is added to extract one of the major components Other interactions Hydrogen, dipole-dipole, ion-dipole, pi bonds Solvent
S TEAM & V ACUUM D ISTILLATIONS 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)
S TEAM & V ACUUM D ISTILLATIONS Vacuum Any distillation below atmospheric pressure Advantage boiling pt differences increase at reduced pressures
S UBLIMATION 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
E LECTRICAL F IELD S EPARATIONS Gel Matrix Electrophoresis Disc Isoelectric Focusing Immuno Capillary Electrophoresis
E LECTRICAL F IELD S EPARATIONS Electrophoresis Charged molecules in solution are separated based on differences in size and charge when a high voltage is applied
E LECTRICAL F IELD S EPARATIONS + -+ F F=QE Q, charge on the particle E, field strength F s =6 r r, radius of the particle (cm), , viscosity of the medium (poises), , electrophoretic velocity (cm/sec) FsFs 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
E LECTRICAL F IELD S EPARATIONS + -+ F F=QE Q, charge on the particle E, field strength F s =6 r r, radius of the particle (cm), , viscosity of the medium (poises), , electrophoretic velocity (cm/sec) FsFs Thus, Fs=QE=6 r and U=Q/6 r Electrophoresis Theory
E LECTRICAL F IELD S EPARATIONS 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 = I 2 R W, watts,R, resistance, I, current Smiling http://www.mnstate.edu/marasing/CHEM480/Handouts/Chapters/Capillary%20Electrophoresis.pdf
F LOTATION Purge and Trap Foam fractionation Gas-solid flotation Liquid-solid flotation
F LOTATION 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.
F LOTATION 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
F LOTATION 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
F LOTATION Foam fractionation Purge and Trap Purging system Trapping System
F LOTATION 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
F LOTATION Foam fractionation Purge and Trap Traps Factors for a good trap 1. Retain analytes of interest 2. Allow gases to pass readily 3. Release analyte easily 4. Stability – don’t release volatiles or cause side reactions 5. Reasonably priced
H OMEWORK 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?
M EMBRANES 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
M EMBRANES 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 1/4 th 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
M EMBRANES Osmosis & Reverse Osmosis 2 nd Law of Thermodynamics – systems tend toward disorder High concentration goes to low concentration Osmosis involves solvent Dialysis involves solute
M EMBRANES 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.
M EMBRANES 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
M EMBRANES 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?
O THER T ECHNIQUES 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 N 2 where r, radial distance of a particle from axis of rotation in cm; N, speed of rotation in rpm
H OMEWORK Why is it not appropriate when describing centrifugation protocols to list the conditions of centrifugation in rpm’s?