Lineout • Motivation • Precipitation • Sedimentation • Solvent-controlled precipitation • Density gradient centrifugation • Solvent extraction • Dialysis • Filtration • Microfiltration • Ultrafiltration • Gel electrophoresis • Chromatography • Size-exclusion chromatography (SEC), gel filtration • Preparative SEC • Analytical SEC • Field-flow fractionation
Motivation • Separation of a mixture: • different nanoparticles (small ones, large ones → size) • impurities (excess reagents, unwanted reaction products, aggregates) • solvent replacement • Goals: • Purification • preparation of pure samples • (Analysis) • Sometimes by-product of preparative method
Sedimentation & Precipitation • Sample is dispersed in solvent • Molecules as “solution” • Particles as “suspension” → colloids • “solubility” / colloidal stability depends on sample and solvent: • polar samples are well soluble in polar solvents (e.g. water) • non-polar (hydrophobic) samples are soluble in non-polar (organic) solvents • Changing the properties of the solvent can cause precipitation. • Samples: • Small particles are more stable (stronger diffusion) • Interaction between particles: • Electrostatic or steric repulsion • Attraction by van-der-Waals forces, • External force field (e.g. gravity, centrifugal force, electric force) • In solution: Thermal energy (Brownian movement), solvent drag
Centrifugation Sedimentation by gravity or centrifugal force: • density of particles P > S density of solvent • Particles that are larger or more dense sediment faster → separation by size and density Fg • Application: Purification by repeated centrifugation and dilution: After one dilution step: Concentration c1= c0 * v0 / v1 remove supernatant add fresh solvent centrifugation v0 v1 S = 0 • Density gradient, e.g. sucrose in water:* • Sedimentation stops in a zone where S = P • high speed and long centrifugation time is no problem • Preparative approach: Centrifuge tubes • Analytical approach: Disc centrifuge** with detector S = 1 > 0 * e.g.  R. D. Corato, … , T. Pellegrino, Chemistry of Materials 2007. ** I. Laidlaw, M. Steinmetz, in Analytical Ultracentrifugation: Techniques and Methods, Royal Society of Chemistry, 2006, pp. 270-290.
Solvent-controlled precipitation • Example: Au nanoparticles with excess surfactant molecules: • Nanoparticles are hydrophobic, well „soluble“ in non-polar solvents: toluene • Variation:Size-selective precipitation • Solvent is gradually changed by addition of second solvent • Big particles precipitate first, smaller particles stay in solution • Fractionation: Different fractions of different sizes Methanol or acetone (polar) Aggregates can be precipitated in centrifuge Supernatant taken off Fresh non-polar solvent is added Methanol isadded Mixture is more polar,NP aggregate NP dispersedin tolune
Soxhlet extraction • Solvent is heated in flask • Vapor is condensed in cooler • The liquid is collected in tube • Solvent flows back to flask cooler • Sample in filter paper is placed into extractor • Only one component is soluble • The insoluble component remains in extractor • The soluble component is accumulated in flask • Example: Au NP in methanol or acetone • NP are not soluble • Aggregates stay in extractor • Sample is continously rinsed with fresh solvent • NP are thoroughly washed from unbound molecules* * : C. A. Waters, A. J. Mills, K. A. Johnson, D. J. Schiffrin: "Purification of dodecanethiol derivatised gold nanoparticles", Chem. Commun. 2003, 540-541.
Dialysis • Semi-permeable membrane with molecular-weight cut-off (MWCO) • Molecules or particles smaller than MWCO can penetrate membrane Repeat with fresh buffer v0 ,c1 v0 ,c0 diffusion (time) In equilibrium: Concentration c1= c0 * v0 / (v0+v1) v1 ,c1 v1 • + easy to do, gentle treatment • - time consuming, no good control of efficiency
Filtration • A filter is a sieve: • Small particles can pass, • Bigger particles are hold back. • Filter has a certain (nominal) pore size • Usual classification: • Macrofiltration (sieve, filter paper) • Microfiltration (cut-off ~ 0.1 – 20 µm) • Ultrafiltration (cut-off ~ 1 – 100 nm)
Microfiltration (~ 0.1 – 20 µm) • Syringe filters • Centrifuge filters • (Vacuum and pressure filters) • Nanoparticles and any smaller molecules can pass. • Removes big objects like aggregates, bacteria, dust: • yields optically clear samples • routinely used for buffer filtration • Different membrane materials available • PVDF (polyvinylidenedifluoride), (CH2CF2)n • PES (polyethersulfone), (Ph-SO2-Ph-O) n • PTFE (polytetrafluorethylene), (CF2CF2)n • Nylon, CME, regen. cellulose, ... • compatible with different solvents • nonspecific (unwanted) binding of sample Manufacturers: e.g. http://www.sartorius.com :: http://www.millipore.com :: http://www.whatman.com :: http://www.corning.com
Ultrafiltration (~ 1 – 100 nm) • Pore size specified in molecular weight cut-off (MWCO), originally used for protein purification • Nanoparticles are retained, smaller molecules pass the membrane Cross-section of porous UF membrane • Used for concentration and buffer exchange • Repeated concentration and dilution: “Diafiltration” c0 v0, c0 v1, c1 dilution centrifugation After one dilution step: Concentration c1= c0 * v0 / v1
Gel electrophoresis • Electrophoresis: Displacement in electric field. • Particles in aqeuous solution are usually negatively charged • stabilization by electrostatic repulsion • Electric field: particles will move to electrode with opposite charge. - + - - - + - - - + - - - + - - - + - - + - - + Fe - - - + - - - + - - - - + - + Negatively charged particle V = • Mobility depends on particle size and charge • Electric force is compensated by friction force r v, F = 0  Á. V. Delgado, F. J. Arroyo, in Interfacial electrokinetics and electrophoresis, Dekker, New York, 2002, pp. 1-54.
Gel electrophoresis • Gel = random network of fibers in solvent (like gelatin, pudding) • Gel improves separation, particles are sieved by pores between fibers • Agarose: Natural polysaccharide, extracted from red algae. • The powder is cooked up with buffer solution, becomes solid. Horizontalagarose gel Nanoparticlesamples - electrode + electrode Electrode reservoirs (buffer) • The gel provides a porous separation matrix, inert to samples. • pore size is controlled by agarose concentration (~ 0.2 – 5 % gels)
Gel electrophoresis of nanoparticles • If size-distribution is good, NP show a sharp band. • Binding of molecules observed by increased particle size • Single molecules can be resolved • Preparative scale: • Particles can be extracted from gel: – + – +  R. Westermeier, S. Gronau, P. Beckett, in Electrophoresis in Practice, Wiley-VCH, 2005, pp. 1-32.
Size-exclusion chromatography (SEC) aka gel filtration aka gel permeation chrom. (GPC) Column with porous gel beads Sample: Large and small particles (or molecules). • Separation mechanism: • Small particles can enter pores • Large particles are excluded from pores • Large particles come out first, • Small particles take longer to diffuse into and out of pores, come out later. Elution: Particles or molecules fractionated by size. • Gel beads have to be inert (no sticking) • Pore size has to fit to species in sample • group separation (desalting) or • high resolution of similar species • Mostly for aqueous applications, e.g. protein purification  L. Hagel, in Protein Purification. Principles, High Resolution Methods and Applications, John Wiley & Sons, New York, 1998, pp. 79-143.  Gel filtration - Principles and Methods, Amersham Pharmacia Biotech, 2001.
Preparative SEC • Column connected to HPLC apparatus (high performance liquid chromatography) • HPLC is a large field • Different separation principles • reverse phase (RP) • Ion exchange (IE) • Affinity chromatography (AC) • Automation: • Autosampler • Detectors • fraction collectors • ... • SEC: Analysis of elution time te (better: elution volume ve) Polymer micelles Small molecules Polymer-coated nanoparticles Polymer-coated Au nanoparticles Au nanoparticles saturated with PEG of different MW
Analytical SEC • Analysis by elution time te (better: elution volume ve) • Column characterized by void volume v0 and total liquid volume vt • Normalized coefficient , values between 0 and 1. pore size Largest molecules Smallest molecules • KSEC allows comparison of different column materials
Analytical SEC • Standards with known radius rh • From KSEC (standards), the diameter of samples can be derived • Calibration for different • columns necessary: • Calibration curve has to be extrapolated • realistic results compared to other methods * *  Journal of Physical Chemistry C 2007, 111, 11552 -11559.
Field-flow fractionation • Flow field-flow fractionation (FFFF) • Sample in flat flow channel • Accumulation on membrane by perpendicular external flow field • Small particles diffuse back faster: elute first • And many more variations: • Continuous operation • Sedimentation • Thermophoresis • Electrophoresis, HPLC ... Commercial devices: http://www.wyatt.com :: http://www.consenxus.com :: http://www.postnova.com