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Chapter 12. Light scattering (determination of MW without calibration)

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Title: Chapter 12. Light scattering (determination of MW without calibration)


1
Chapter 12. Light scattering (determination of
MW without calibration)
Electromagnetic radiation ? ???? ????? ??
  • ? ?? ??
  • transmission transmitted radiation passes
    through the medium unaltered.
  • absorption energy from the incident beam is
    taken up, resulting in (1)heating, (2)
    re-emitting at another wavelength (fluorescence,
    phosphorescence), (3)supporting chemical
    reactions. In this discussion, we assume that
    radiation heating is negligible. Other
    absorption effects are specific to the particular
    medium, and will also not be considered here.
  • scattering scattering is non-specific, meaning
    the incident radiation is entirely re-emitted in
    all direction with essentially no change in
    wavelength. Scattering results simply from the
    optical inhomogeneity of the medium.
  • reflection scattering at the surface of a matter
    (not considered here)

2
  • Now we focus on the light scattering.
  • Application of Light Scattering for Analysis
  • Classical Light Scattering (CLS) or Static Light
    Scattering (SLS)
  • Dynamic Light Scattering (DLS, QELS, PCS)
  • CLS
  • ?? Scattering center small volumes of material
    that scatters light. ? individual molecule in
    a gas.
  • Consequences of the interaction of the beam with
    the scattering center depends, among other
    things, on the ratio of the size of the
    scattering center to the incident wavelength
    (?o). Our primary interest is the case where
    the radius of the scattering center, a, is much
    smaller than the wavelength of the incident light
    (a lt 0.05?o, less than 5 of ?o). This
    condition is satisfied by dissolved polymer coils
    of moderate molar mass radiated by VISIBLE light.
    When the oscillating electric field of the
    incident beam interacts with the scattering
    center, it induces a synchronous oscillating
    dipole, which re-emits the electromagnetic energy
    in all directions. Scattering under these
    circumstances is called Rayleigh scattering.
    The light which is not scattered is transmitted
    , where Is and It are the
    intensity of the scattered and transmitted light,
    respectively.

3
  • Oscillating electric field of incident beam
    interacts with scattering center, induces a
    synchronous oscillating dipole, which re-emits
    electromagnetic energy in all directions.

Rayleigh scattering? ?? ???? ??? ?? ??? ?? ???
(1cos2?)? ????, scattering center? observer???
??(r)? ??? ???.
  • 1944, Debye
  • Rearrange

Constant, K
?o ?????, dn/dc refractive index
increment no ??? refractive index, p???,
c????g/mL
4
I? is inversely proportional to ?o. Shorter
wavelength scatters more than longer
wavelength Assume system is dilute, the net
signal at the point of observation is sum of all
scattering intensities from individual scatterer
- no multiple scattering (scattered light from
one center strike another center causing
re-scattering, etc.).
  • Two ways to access the light scattering
    information experimentally
  • Turbidimeter (or spectrophotometer)
  • Light scattering

5
1. Turbidimeter experiment (Transmitted light
intensity, It is measured)
Solution is dilute, so higher order concentration
terms can be ignored.
6
Procedure Measure t at various conc. ? Plot Hc/T
vs. c (straight line) ? Determine M from
intercept, 2nd virial coeff., B from slope
7
2. Light Scattering experiment (measure I? at
certain ? and r)
?6? ?4? ??
??? ??? ? 5 (?/20) ??? ??? ??? Rayleigh
limit
8
?-condition?? ???0.
9
lt??gt For polydisperse sample, Turbidity (?? light
scattering) is contributed by molecules of
different MW. Define ti ??? Mi? ?? ???? ??
turbidity ?
??? turbidity? light scattering???? ?? ????
weight-average MW??.

10
Rayleigh-Gans-Debye (RGD scattering) when the
scattering centers are larger than Rayleigh limit
Different part of more extended domain (B)
produce scattered light which interferes with
that produced by other part (A) - constructive or
destructive
11
Distribution is symmetrical for small particles
(lt?/20). For larger particles, intensity is
reduced at all angles except zero.
Contributions from two scattering centers can be
summed to give the net scattering intensity.
The result is a net reduction of the scattered
intensity
P? "shape factor" or "form factor"
Always P? lt 1, function of size and shape of
scattering volume. Now we start seeing the
angle dependence of the scattered light !
12
  • p(?) decreases with ?.
  • p(?) decreases more for higher MW.

13
Effect of MW and Chain Conformation on P?, and on
measured MW at 90o.
Conformation MW (g/mol) RG (nm) P(90o) MW(90o)
Random coil
Polystyrene 51K 8 0.98 51K
Polystyrene( ? condition) 420K 19 0.95 400K
PMMA 680K 36 0.70 480K
Polyisoprene(70 cis) 940K 48 0.56 530K
Spherical
Bovine serum albumin 66K 3 1.00 66K
Bushy stunt virus 10700K 12 0.98 10500K
Rod shaped
Poly- -benzyl-L-glutamate 130K 26 0.91 118K
Myosin 493K 47 0.74 365K
DNA 4000K 117 0.35 1400K
14
Final Rayleigh equation for random coil polymer
? ?? ?? ??
Case 1 ??0
Plot Kc/R? vs. c y-??1/M, ???2A2
Case 2 c?0
Plot Kc/R? vs. sin2(?/2) y-??1/M, ???
(16p2/3M?2) rg2
Three information!
15
??? ?? ?? (1) ??? ??? ???? R???. (2) Kc/R? vs.
c, Kc/R? vs. sin2(? /2) plot ??. (3) ? 0 ? c
0 ? extrapolate.
Kc/R? vs. sin2(? /2)
Kc/R? vs. c
Zimm plot
??? ? ?? ???. ? ? extrapolated points
16
Cases
1. Small polymers ????? ??. (Horizontal line)
Zimm plot for PMMA in butanone ?o546 nm, 25?, no
1.348, dn/dc 0.112 cm3/g
- ?? ???? ??? ???. - Mw ? A2 ?? ?? - ???? ?? ???.
2. Small polymers in ?-solvent ?? ? ?? ??? ??.
Zimm plot of poly(2-hydroxyethyl methacrylate) in
isopropanol ?o436 nm, 25?, no 1.391, dn/dc
0.125 cm3/g
?-solvent A20? ?? ??, ???-???, ???-????? ?????
???? ??, ????? ?? ??.
  • Calculated values Mw 66,000 g/mol
  • A2 0 mol
    cm3/g2
  • - Kc/R? at small angles fall mostly below the
    horizontal line plotted through the points from
    medium and large angles.

17
3. Larger polymers in good solvent ?? ? ??? ??.
Zimm plot of polystyrene in toluene ?o546 nm,
25?, no 1.498, dn/dc 0.110 cm3/g
- ??? ? 2x105 ??? ??, Kc/R? ? ?? ??? (A2??)?
???. - Athermal Condition - No effect of
temperature on polymer structure
4. Polymers in poor solvent A2 ? ??? ? (? ???
? ? ??. ? ?? ?? ?? ??)
Zimm plot of polybutadiene in dioxane ?o546 nm,
25?, no 1.422, dn/dc 0.110 cm3/g
  • - ?????? ??? ?? (nonlinear).
  • - ?? microgel, ??, aggregate? ?? ? ?? ??.
  • Curve-fitting? ??? ??.
  • ???? ??? ???? ??.
  • ??? ??? good solvent???
  • ???? ??? ? ??.

18
Stand-alone vs. On-line MALS
  • ltStand-alone modegt
  • Stand-alone mode LS instrument is used itself.
  • Zimm plot ? ?? M, A2, R?? ??
  • ltOn-line modegt
  • LS instrument is used as a detector for a
    separator.
  • c0 ?? ??.
  • ? slice? ?? Kc/R? vs. sin2(?/2) ???? ??,
    y-?????? ??? (M), ???????? rg? ??. y-??1/M,
    ????? (16p2/3M?2) rg2
  • ? slice? monodisperse??? ???? ?????? ????? ??.
    ??? ?? ???? ??? (?????? ? ?????? ???).
  • Average Molecular Weights
  • No-average Mn(Sci)/(S(ci/Mi))
  • Wt-average MwS(ci Mi)/ S(ci)
  • Z-average Mz S(ci Mi2)/S(ci/Mi)
  • Average Sizes (mean square radii)
  • No-average ltrg2gtn S(ci/Mi)ltrg2gti/S(ci/Mi)
  • Wt-average ltrg2gtw S(ciltrg2gti)/Sci
  • Z-average ltrg2gtzS(ciMiltrg2gti)/S(ciMi)

19
Light scattering instruments MALLS (Multi Angle
Laser Light Scattering) I? is measured at 15
angles (1) Stand-alone mode Measure scattered
light at different angles for different
concentrations ? Make a Zimm plot ? Determine M,
B, Rg
Assuming each slice is narrow distribution, Mw ?
Mi Average M can be calculated. It is therefore
very important to have a good resolution.
20

21
Angular Dependence of Kc / R?(?? high molecular
weight DNA)
22
Effect of Particles/Gels on Light Scattering
Measurement Note the delicacy of extrapolation to
zero angle from larger distances.
23
  • DALLS (Dual Angle) I?? is measured at 15o and
    90o
  • LALLS (Low Angle) I? is measured at one low
    angle (assume ?? 0)
  • Static mode measure LS at a few c ? Plot Kc/R?
    vs. c ? Determine M and B from intercept and
    slope.
  • On-line mode determine Kc/R? for each slice (
    calculate M). Considering each slice is
    narrow distribution, let Mw ( Mi, from which
    average MW's can be calculated (as learned in
    chapter 1). It is therefore again very
    important to have a good resolution.
  • RALLS (Right Angle)
  • I? is measured at 90o.
  • Simple design
  • Higher S/N ratio, Application is limited to
    cases where P? is close to 1 (e.g., less than
    200K of linear random polymer)
  • RALLS combined with differential viscometer
    (commercially available from Viscotek, "TRISEC")

24
ltTRISEC ?? ??gt Assume P? 1 and A2 0.
Determine Mest.
? is determined by differential viscometer, and
M determined in step 2.
Calculate new MW by
Go to step 2. Repeat until Mest does not change.
25
ltLight scattering experiment? ??? ???gt
?? K? B? ??? ?? parameter? ?? ?? ??. ???
??? ?? ? ?? ??? ??.
  1. n ??? refractive index
  2. dn/dc Specific refractive index increment
  3. B 2nd virial coefficient (Static mode??? B? ???
    ?? ??? ? ?? ??? Static mode? ??).

26
1. ??? Refractive Index ?? ?? ??? ?? RI ??? ???
??.
?? ??? ??? (R?? ???? ?)
Solvent RI R? x 106 cm-1
Carbon disulfide 1.6207 57.5
a-chloronaphthalene (140 oC) 1.5323 52.8
1,2,4-Trichlorobenzene (135 oC) 1.502 35.7
Chlorobenzene 1.5187 18.6
o-Xylene (35 oC) 1.50 15.5
Toluene 1.49 14.1
Benzene 1.50 12.6
Chloroform 1.444 6.9
Methylene chloride 1.4223 6.3
Carbon tetrachloride 1.46 6.2
Dimethyl formamide 1.43 (589 nm) 5.6
Cyclohexane 1.425 5.1
Cyclohexanone 1.4466 4.7
Methyl ethyl ketone 1.38 4.5
Ethyle acetate 1.37 4.4
THF 1.41 4.4
Acetone 1.36 4.3
Dimethyl sulfoxide 1.478 (589 nm) 4.1
Methanol 1.33 2.9
Water 1.33 1.2
  • Except where otherwise noted, all measurements
    made at ? 632.8 nm and T23 oC. RI at 632.8 nm
    calculated by extrapolation from values measured
    at other wavelengths.
  • Extrapolation? ?? reference Johnson, B. L.
    Smith, J. "Light Scattering from Polymer
    solutions" Huglin, M. B. ed., Academic press, New
    York, 1972, pp 27

27
2. Specific refractive Index, dn/dc
  • ???? ?? ? ?? (Polymer Handbook, Huglin, ed.,
    Light Scattering from Polymer Solutions, Academic
    Press, 1972)
  • ???? ?? ? ?? ?? ??? ?? ??
  • Conventional method
  • DRI? ??
  • ? ?? ?? ???? (n2-n1)? ?? (recommended conc. 2,
    3, 4, 5 x 10-3 g/mL) ? (n2-n1)/c2 vs. vs. c2?
    plot ? zero concentration?? extrapolate ? dn/dc?
    intercept? ? ? ???.

28
For concentration ranges generally used, the
refractive index difference, n2-n1, is a linear
function of concentration. In other words,
(n2-n1)/c2 is constant. ? (n2-n1)/c2 vs. c2 ????
???0.
This means that (n2-n1) needs to be measured for
only one or two different concentrations. If
(n2-n1)/c2 shows no significant dependence on c,
then dn/dc can be obtained by averaging
(n2-n1)/c2 values
29
  • SEC/RI? ??
  • ?? ?? ?? ??

?? dn/dc? ?? ????? ???? kR? ??
  • ???? ??? ?? ?? ? ?? ?? estimate? ? ?? ??.
  • extrapolate to desired wavelength

?? 2) polymer? ??? refractive index? ??
estimate
???? n2? polymer? partial specific volume
mL/g??. ?? n2 ? 1.
  • lt????gt
  • dn/dc ? ??? ????? light scattering ??? ?? ??? ???
    ??? ?? ???? ???? ??.
  • Dn/dc ? ??? ????? ???? ??? ??. Dn/dc ? ???? ??.
  • ??? dn/dc ?? ??. ???? ???? ?? ??? ??.

30
3. Virial Coefficient, B or A2
  • ???? ?? ? ?? (? Polymer Handbook). ???? ?? ? ??
    ?? ??? ?? ?? (stand-alone Light scattering)
  • 2nd Virial Coefficient? Solute-Solvent
    interaction? ??.
  • Polymer-solvent interaction, good solvent (the
    higher, the better solvent).
  • 0 Unperturbed system
  • - Polymer-polymer interaction, poor solvent.
  • A2? ???? ?? A2 b M-a ? log A2 vs. log M?
    ??. ?? ???? ??, ? ???? ???.
  • dn/dc? A2? ???? ?? ???? S. Lee, O.-S. Kwon,
    "Determination of Molecular Weight and Size of
    Ultrahigh Molecular Weight PMMA Using Thermal
    Field-Flow Fractionation/Light Scattering" In
    Chromatographic Characterization of Polymers.
    Hyphenated and Multidimensional Techniques,
    Provder, T., Barth, H. G., and Urban, M. W. Ed.
    Advances in Chemistry Ser. No. 247 ACS
    Washington, D. C., 1995 pp93.

31
  • Light scattering ??? ? ? ?? ?? ? ?? (concerns)
  • ??? dn/dc, RI constant, A2? ??.
  • As dn/dc increases, calculated MW decrease,
    calculated mass decrease, and no effect on
    calculated RG.
  • As RI constant increases, calculated MW
    decreases, calculated mass increases, and no
    effect on RG .
  • As A2 increases, calculated MW increases, no
    effect on calculated mass, RG slightly increases.
  • Refractive Index Detector Calibration ? ????? ?
    ??
  • RI Calibration constant inversely proportional
    to the detector sensitivity.
  • Sensitivity of most RI detector is
    solvent-dependent.
  • A calibration constant measured in a solvent may
    not be accurate for other solvents. It is
    recommended to use a solvent that will be used
    most often (e.g., THF or toluene).
  • For RI calibration, only the RI signal is used.
    Light scattering instrument calibration is not
    needed.
  • Concentration of standards should be such that
    the output of RI detector varies between about
    0.1 - 1.0 V and should correspond to normal peak
    heights of samples (For a Waters 410 RI at
    sensitivity setting of 64, this corresponds
    roughly to concentrations of 0.1 - 1.0 mg/mL. RI
    output can be usually monitored by light
    scattering instrument (e.g., channel 26 of DAWN).
  • Use NaCl in water as a standard for aqueous
    system.
  • The RI calibration constant will change if you
    change the sensitivity setting of the detector
    So it is important to use the same sensitivity
    setting of RI detector as that used when the
    detector was calibrated.

32
  • RI calibration preparation One Manual injector
    with at least 2 mL loop, Five or more known
    concentrations (0.1 - 1 mg/mL) of about 200 K
    polystyrene in THF.
  • RI calibration Procedure
  • Remove columns. Place manual injector with loop.
  • Pump THF through a RI detector at normal flow
    rate (about 1 mL/min). Purge both reference and
    sample cells of detector until baseline becomes
    flat stable.
  • Stop purging and wait till baseline becomes
    stable.
  • Set up the light scattering data collection
    software (enter filename, dn/dc, etc.) Enter 1 x
    10-4 for RI constant (light scattering instrument
    usually requires the RI constants to be entered).
    Set about 60 mL for Duration of Collect .
  • Begin collecting data with ASTRA.
  • Inject pure solvent first followed by stds from
    low to high conc, and finish with pure solvent.
  • Repeat the measurements if you want.
  • Data Analysis (1)set baseline using signals from
    pure solvent at the beginning and the end
    (2)calculate each concentration as a separate
    peak by marking exactly 1 mL as peak width (or 30
    slices at 1 mL/min, 2 seconds of collection
    interval).(3)calculate the mass of the peak
    (4)plot the injected mass (y-axis) vs. calculated
    mass (x-axis) (5)do linear regression on data by
    forcing the intercept be zero (6)calculate RI
    constant using RI constant slope x 1x10-4

33
  • Chemical heterogeneity within each slice leads to
    non-defined dn/dc ? Quantitation of chemical
    heterogeneous samples is very difficult.
  • Limited sensitivity to low MW components.
    Mn(exp)gtMn(true). The same concern with
    differential viscometer experiments.
  • Limited Sensitivity of Light Scattering and RI
    Detector
  • g' values may be in error if each peak slice
    contains both linear and branched polymer or
    different types of long-chain branching g' will
    be overestimated.
  • Quality of data is highly affected by the
    presence of particles.
  • Lower limit of RG with MALS? ? 10 nm (about 100K
    MW)
  • Inter-detector volume must be known accurately.

34
Comparison of online LS vs. viscometer
LS Viscometer
MWD Absolute Relative
need precise n and dn/dc Universal calibration must be valid or need M-H coefficient
independent of separation mechanism Independent of separation mechanism if M-H coefficients are used. Dependent on separation mechanism if universal calibration is used.
? distribution indirect from universal calibration direct, independent of separation mechanism
RG direct from MALS (limited to gt10 nm) indirect from universal cal. and Flory-Fox eqn. applicable to linear molecules only
Chain conformation MALLS RG vs. M plot ? vs. M plot (M-H coefficients can be obtained) RG vs. M plot.
Branching g obtained directly from MALS, indirectly from LALLS universal calibration g' obtained directly
heterogeneous samples limited because of dn/dc uncertainty directly applicable with univ. calib., but the change in dn/dc will affect DRI responses
Lower MW detectability 2K. depends on dn/dc and polydispersity as low as 300-400 has been reported
Response to particle contamination LALLS highly sensitive, MALLS less sensitive Insensitive
35
Information Content
Primary Secondary
LALLS M
MALLS M RG
PCS D Rh, M
Viscometer ? M, RG
Primary information high precision and accuracy,
insensitive to SEC variables, requires no SEC
column calibration.
36
ltSEC-VISC-LS instrumentgt
  • Features
  • MWD measured by LS
  • IVD measured by Viscometer

37
  • Both Viscometer and LS are insensitive to
    experimental conditions and separation mechanism
  • No band broadening corrections are needed for Mw,
    ? , a, k, and g
  • Precise and accurate calculation of hydrodynamic
    radius distribution, M-H constants, and Branching
    distribution

38
  • Dynamic light scattering (DLS, QELS, PCS)
  • Classical light scattering "time-averaged
    scattering intensity"? ?? ???? ??? ? scattering
    center??? ?? ?? ?? ??? ? (algebraic summation).
  • ??? algebraic summation? ??? ? ???? random??
    array????, ?? phase relationship? scattering
    volume dimension? ??? ?? ?? ??? ?????? ??
    interference effect ?? average-out?? ??? ????
    ???.
  • Scattering volume dimension? ?? ???, ???? ??? ?
    scattering center? ?? ?? ?? ?? ?? ??? interfere
    (constructive or destructive) ???? ?? ???? ???
    ???? ???? ??? ?? ????.
  • ? ???? Brownian motion (diffusion) ? ?? ?? ?????
    ???? ???? ?? ?? ?? ????. ??? ???? ???? ??? ???
    ?? fluctuate??.
  • Fluctuate?? ??? ???? diffusion rate? ??
    (diffusion rate? ???? ??? fluctuate).
  • nanometer ?? micron??? ??? ??? ???? ?? viscosity?
    ??? viscosity? ??? media? disperse?? ?? ?, ????
    ??? ?? ?? (fluctuation)? microsecond ??
    millisecond??.

39
  • A vertically polarized laser beam is scattered
    from a colloidal dispersion. The
    photomultiplier detects single photons scattered
    in the horizontal plane at an angle ? from the
    incident beam, and the technique is referred to
    as "photon correlation spectroscopy (PCS)
  • Because the particles are undergoing Brownian
    motion, there is a time fluctuation of the
    scattered light intensity, as seen by the
    detector. The particles are continually
    diffusing about their equilibrium positions.
    Analyzing the intensity fluctuations with a
    correlator yields the effect diffusivity of the
    particles.
  • Measured intensity, I vector sum of scattering
    from each particle
  • Brownian motion motion caused by thermal
    agitation, that is, the random collision of
    particles in solution with solvent molecules.
    These collisions result in random movement that
    causes suspended particles to diffuse through the
    solution. For a solution of given viscosity, ?,
    at a constant temperature, T, the rate of
    diffusion (diffusion coefficient) D is given by
    the Stokes-Einstein equation, D(kT)/(6p?d),
    where k Boltzman's constant, d equivalent
    spherical hydrodynamic diameter. ??? diffusion
    coefficient (D)? ?????? ?? ?? (?? ???)? ??? ? ??.
  • DLS??? ? ??? ??? ?? ?? ???? ??? ?? ??(t time
    interval)?? ???? ??? ????. ???? ??? ???? ???
    ??? t? ?? ?, I(0)? I(t)? ??. ?? ?? ?? interval?
    ?? ???? I(0)? I(t)? ??? ? intensity product,
    I(0)I(t)? ???? ltI2(0)gt, ? average of the square
    of the instantaneous intensity ? ???? - ?? "I(0)?
    I(t)? correlate????"?? ??. ???? ??? ???? ???
    ??? t? ? ?, I(0)? I(t)? ??? ??? ?? ??? - "I(0)?
    I(t)? correlate???? ??" ?? "I(0)? I(t)?
    un-correlate ????" ?? ??. ???? intensity
    product, I(0)I(t)? ???? ??? ltI2gt, ? square of the
    long-time averaged intensity? ??. ???? ??? ????
    ??? ??? t? ??? ??? ?? ?, "I(0)? I(t)? ?????
    correlate????".

40
  • Measured intensity, I vector sum of scattering
    from each particle
  • Measure I at various time interval, ?,
  • I(0) I(t) for short t ? correlated,
    correlation decreases as ? increases.
  • I(0)? I(t)? ?????? Correlation? ??? ??? ? ??.
    correlation? ??? ???? ?? average of the intensity
    product, G(t)? ????.
  • ?? G(t)Anto correlation function
    ltI(t)I(tt)gt average of the intensity product.
  • ?? ???? t ? ???? ?? G(t)? ??.
  • G(t) is high for high correlation, and is low for
    low correlation.
  • High correlation means that particles have not
    diffused very far during t. Thus G(t)
    remaining high for a long time interval indicates
    large, slowly moving particles.
  • The time scale of fluctuation is called "decay
    time
  • Decay time is directly related with the particle
    size. The inverse of decay time is the decay
    constant, ?.
  • Usefulness of G(t) directly relatable to the
    particle diffusivity
  • For monodisperse samples,

41
?? ?? ??? ?? ??? interval?? autocorrelation
function, G(t)? ???
G(t) vs. t? ???? ??? Exponential function? ????
G(t)? fit??.
Rh? ??, ??? ?? ???? Measure I(t) at various ? ?
G(t) ?
?? DLS ? ??? ???? diffusion? ?? ??? ?? ?? ??
dispersion ( ? 0.03)? ??? ???. ? volume
fraction of suspended spheres.
, where N Avogadro's no., M MW, Vh
hydrodynamic vol.). Infinite dilution D?? ??
???? ?? ? 0.005? ?? ??? ??.
42
???? f(a) distribution function, I(a,?)
scattering intensity function for RGD spheres.
PC? ??, normal?? log-normal distribution
function? G(t)? fit??.
?? Narrow, mono-modal distribution ??? ??,
"method of cumulant"? ??, ??? ?? ??? ? ??.
  • ???? an nth moment of f(a).
  • We see that DLS yields a somewhat unusual
    average radius (the inverse "z-average", and one
    which is quite highly sensitive to the presence
    of outsized particles.
  • DLS uses a single exponential decay function, and
    thus it does not give information on sample
    polydispersity.

43
  • ??
  • RI values of medium and sample are needed for DLS
    experiments.
  • RI 1.333 for water, and 1.5 - 1.55 for typical
    polymers and proteins.
  • RI of sample is needed only when the intensity
    weight needs to be converted to the volume weight
    (e.g., for samples having broad distributions).
  • Theory to convert the intensity to the volume
    is only for solid particles. So the conversion
    will not be accurate for samples such as
    liposomes which are hollow inside.
  • For samples such as liposome, a value between 1.5
    - 1.55 can be used as it is typical values for
    polymers and proteins.
  • For samples having narrow distributions, only the
    unimodal analysis is performed, and thus there is
    no need to convert the intensity to the volume
    .
  • RI value will not make any difference in the
    average size data because only the RI of medium
    is need for unimodal analysis.

DLS summary
  • D depends on MW and conformation
  • Diffusion coefficient distribution can be
    obtained
  • D is independent on chemical composition. ?D can
    be obtained without knowing chemical composition.
  • Concentration is not needed to determine D
  • Input parameters (T, n, ? ) are easily measured.
  • Concerns sensitivity, interference from
    particulates, inconsistency, not very useful
    for polydispersed or multi-modal distributions.

44
lt??gt Particle Size Conversion Table
Mesh size Approximate ?µ size
4 4760
6 3360
8 2380
12 1680
16 1190
20 840
30 590
40 420
50 297
60 250
70 210
80 177
100 149
140 105
200 74
230 62
270 53
325 44
400 37
625 20
1250 10
2500 5
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