Analytical Ultracentrifugation of s d r u s ω2 = t ln(r/r ) s 2 m ω = 1 step means 1 component...

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Transcript of Analytical Ultracentrifugation of s d r u s ω2 = t ln(r/r ) s 2 m ω = 1 step means 1 component...

  • Analytical Ultracentrifugation of Nanoparticles

    Analytical Ultracentrifugation Analytical Ultracentrifugation of of NanoparticlesNanoparticles

    Helmut Clfen

    Max-Planck-Institute of Colloids and Interfaces,Colloid Chemistry, Am Mhlenberg 2, D-14424 Potsdam

    Email: Coelfen@mpikg-golm.mpg.de

  • Why fractionating analytics in solution ?Why fractionating Why fractionating analytics in analytics in solutionsolution ??

    AUC / FFFAUC / FFFAUC / FFF

    Sensitive to bigger particles /

    dust asI r 66

    Problem:Problem:Big Big particlesparticlesAbsorptionAbsorption

    multiple multiple scatteringscattering

    Light ScatteringLight ScatteringLight Scattering

    EveryEvery particle isdetected,

    simultaneousmultiple detection

    possible

    MicroscopyMicroscopyMicroscopy

    Counting ofparticles

    Drying artifacts

    Problem: Problem: StatisticsStatistics

    NoNo fractionationfractionation FractionationFractionation

  • Svedbergs Colloid ResearchSvedbergs Colloid Svedbergs Colloid ResearchResearch T. Svedberg, J.B. Nichols, J.

    Amer. Chem. Soc. 45 (1923) 2910

    T. Svedberg, H. Rinde, J. Amer. Chem. Soc. 45 (1923) 943

    T. Svedberg, H. Rinde, J. Amer. Chem. Soc. 46 (1924) 2677

    T. Svedberg, Kolloid-Z. Zsigmondy Festschrift, Erg.-Bd. Zu 36 (1925) 53

    1926: First 1926: First protein workprotein work

    1925: Nobel 1925: Nobel prizeprize forforcolloidcolloid workwork

  • h

    rr r

    rt m

    VapourSolution

    b

    Radial Detection

    Rayleigh Interference optics UV/VIS Absorption opticsn

    Abs

    .

    r r

    Light

    Detector

    Principle of AUCPrinciple Principle of AUCof AUC

    Experiments very simple, evaluation not always

  • Sedimentation Sedimentation velocityvelocity

    Sedimentation Sedimentation equilibriumequilibrium

    Density gradientDensity gradient

    High centrifugal forceSedimentation stronger than back diffusion

    50000 RPMScaninterval10 min

    6.2 6.4 6.6 6.8 7.0 7.2

    0.0

    0.5

    1.0

    Abs

    orba

    nce

    Radius (cm)

    6.7 6.8 6.9 7.0 7.10.0

    0.5

    1.0

    1.5

    2.0

    Abs

    orba

    nce

    Radius (cm)

    Moderate centrifugal forceSedimentation in the order of back diffusion

    Sedimentation

    Diffusion

    10000 RPM

    6.2 6.4 6.6 6.8 7.0 7.20.0

    0.2

    0.4

    0.6

    0.8

    1.0A

    bsor

    banc

    e

    Radius (cm)

    50 % CH2Cl2 + 50 % CHCl360000 RPM

    Different AUC experimentsDifferent AUC Different AUC experimentsexperiments

    High centrifugal force for distributionof a low molecular salt or a second solvent

  • =

    43421321 termentationdimSe

    22

    termDiffusion

    crsdrdcDr

    drd

    r1

    tc

    Lamm equationLamm Lamm equationequation

    Experiment Effective term in the Lamm equation

    Characteristics

    Sedimentationvelocity

    Sedimentation term much bigger than diffusion term

    High rotational speed

    Synthetic boundary experiment for the determination of D

    Only diffusion term effective

    Synthetic boundary cell,low rotational speed

    Sedimentationvelocity

    Sedimentation and diffusion term effective / equilibrium between sedimentation and diffusion

    Moderate / low rotational speed

    Density gradient(special case ofsedimentation equilibrium)

    Sedimentation and diffusion term effective / equilibrium between sedimentation and diffusion

    Moderate / highrotational speed,locally dependent solution density

  • 50000 RPMScaninterval 10 min

    6.2 6.4 6.6 6.8 7.0 7.2

    0.0

    0.5

    1.0

    Abs

    orba

    nce

    Radius (cm)

    Basic evaluation important for nanoparticles

    Basic Basic evaluation important for evaluation important for nanoparticlesnanoparticles

    a) Determine distance travelled in given time intervalb) Calculate sedimentation coefficient s or its distributionc) Calculate particle size or distribution

    =2

    ii

    s18dr

    us 2=

    t)r/rln(s 2

    m

    =

    1 step means1 component

    Flat baseline indicates purity

  • ( )MsRT

    D v=

    1 f

    RTN DA

    =

    Sedimentation velocitySedimentation Sedimentation velocityvelocity

    =2

    ii

    s18d

    Sedimentation velocity depends on:

    Molar Molar mass mass / / particle sizeparticle sizeDensityDensityShape Shape / / FrictionFrictionChargeCharge

    for given exptl. parameters (temperature, solvent density & viscosity etc.)

    Particle size distributions calculated Particle size distributions calculated on a on a hard sphere basishard sphere basis

  • Sedimentation velocitySedimentation Sedimentation velocityvelocity

    6.0 6.2 6.4 6.6 6.8 7.0 7.2

    0.2

    0.4

    0.6

    0.8

    1.0

    1.2

    1.4

    1.6

    a)

    Abs

    orba

    nce

    Radius [cm]4.00E+008 6.00E+008 8.00E+008

    1.820

    1.825

    1.830

    1.835

    1.840

    1.845

    1.850

    b)

    sw = 480 S

    Data for individual boundaries Regression line

    ln(r

    /r m)

    2t

    0 200 400 600 8000.0

    0.2

    0.4

    0.6

    0.8

    1.0

    1.2

    c) g*(s) c(s) diffusion corrected

    g*(s

    ) res

    p. c

    (s)

    sedimentation coefficient [S]

    6.0 6.2 6.4 6.6 6.8 7.0 7.20.0

    0.2

    0.4

    0.6

    0.8

    1.0 d)

    g*(d

    )

    Diameter [nm]

    t)r/rln(s 2

    m

    =

    =2

    ii

    s18d

    Gold in H2O at 5000 RPM, 25 C

    a) Raw data

    b) S-evaluation

    c) s-distribution

    d) d-distribution

  • Common ProblemsCommon ProblemsCommon Problems Extremely broad s-distributions, Big aggregates or small

    impurities are not detected Colloids aggregate or grow during centrifugation

    (concentration dependent aggregation) Density of hybrid colloids is unknown to access particle

    size Electrostatic stabilization complicates analysis due to

    charge contributions Particle polydispersity in size, shape, density and

    hydration High particle density often makes density gradients or

    density variation methods impossible Often multicomponent mixtures

  • Fractionation of heterogeneous samplesFractionation Fractionation of of heterogeneous samplesheterogeneous samples

    6,0 6,2 6,4 6,6 6,8 7,00

    1

    2

    3

    4

    5 Carrageenan+ iron oxyhydroxide

    Large aggregates

    Free

    Carrageenan

    Carrageenan + iron oxyhydroxide

    2.72 fringes

    0.55 fringes

    40000 RPM, 25 C

    Absorption optics

    Interference opticsA

    bsor

    banc

    e / F

    ring

    e sh

    ift

    Radius [cm]pH 2, -carrageenan

    pH > 5

    pH 2, Fe3+ crosslinking of -carrageenan

    Hollow space

    Protected partiallymobile FeOOH

    Hydrolized Fe3+

    + OH-+ Fe3+Fe3+

    Fe3+

    Fe3+

    F. Jones

  • 1 0 0 1 0 0 0

    0 .0

    0 .2

    0 .4

    0 .6

    0 .8

    1 .0

    d H [ m ]

    3 4 0 0 n m

    3 8 9 n m

    1 6 4 n m

    1 0 0 n m6 0 n m

    ci/c

    0

    given reproducedd [nm] % d [nm] %

    60 20 60 20103 20 100 20160 20 164 18347 20 389 202800 20 3400 22

    Latex mixtureLatex Latex mixturemixture

    LMP

    s18d

    =

    Particles analyzed over colloidal Particles analyzed over colloidal range range in in one experimentone experiment

    A. Vlkel

  • 1.5 2.0 2.5 3.0

    0.00

    0.07

    0.14

    0.21

    Species 1: 1.84 +/- 0.15 nm (48.7 %)Species 2: 1.95 +/- 0.20 nm (26.0 %)Species 3: 2.34 +/- 0.32 nm (25.3 %)

    Expt. Data Sum Gauss Fit Ind. Gauss Fit

    g(d H

    ) [A

    .U.]

    dH [nm]

    Au55[P(Phe)3]12Cl6AuAu5555[P([P(PhePhe))33]]1212ClCl66Reported Reported to to bebe definitedefinite cluster cluster withwith 55 Au, 55 Au, Transition betweenTransition between

    metal andmetal and moleculemolecule1530 counts

    Very small colloidsVery small colloidsVery small colloids

  • 6.0 6.2 6.4 6.6 6.8 7.0 7.20.0

    0.1

    0.2

    0.3

    0.4

    0.5

    0.6

    0.7

    0.8

    0.9

    1.0

    60000 RPM, 25 C, 380 nmScanint. 2 min

    Abso

    rban

    ce

    Radius (cm)

    0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.00.0

    0.2

    0.4

    0.6

    0.8

    1.0

    1.92

    nm

    1.77

    nm

    1.68

    nm

    1.59

    nm

    1.50

    nm

    1.40

    nm

    1.30

    nm

    0.83 nm

    Integral Differential

    Sum

    of m

    ass

    frac

    tions

    Particle diameter [nm]

    High resolution PSD

    High High resolutionresolution PSDPSDPt colloid in MeOH / HAc

    Baseline resolution > 1 Angstrm

    0.1 nm Baseline resolution

    Problem: Elimination of diffusion broadening by self-sharpening effectsis not common

  • Particles slightly bigger than from Particles slightly bigger than from AUC (AUC (DensityDensity))

    0.80.8 1.21.2 1.61.6 2.02.0 2.42.4 2.82.8 3.23.2 3.63.600

    55

    1010

    1515

    2020350350 countscounts

    particle diameterparticle diameter [nm][nm]

    Num

    ber f

    requ

    ency

    %TEMTEMTEM

  • Pt-Colloid growthPtPt--ColloidColloid growthgrowth

    Critical crystal

    nucleus

    Coalescence until particles are stabilized

    Further growth through reduction of PtCl4 at the

    crystal surfacedrdt

    const= . DifferencesDifferences in in thethe particleparticle sizesize afteraftercoalescence are conservedcoalescence are conserved

    Mechanism in G.A. Braun, Ph-D dissertation, Aachen 1997

    = Stabilizer = Pt-Particle

    CoalescenceC