Technical Note
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concentration is too high for DLS. At extremely high
concentrations, this phenomenom usually reverses.
When the solution is ready for analysis, it should
be inspected for particles at the bottom of the cuvette. If
there are particles at the bottom of the cuvette, the
sample distribution will not be accurate, as the large
settled particles will not be measured, resulting in an
inaccurate size distribution. Samples with large particles
settling are either not dispersed correctly (wrong pH, not
enough sonication, not enough dilution time, etc.) or are
not suitable for DLS. For low density particles, samples
may exhibit the opposite behavior. Creaming is the term
used for large particles of lower density than the solvent
reaching the top of the sample. If the sample is
creaming, there is either a dispersion problem or the
particles are too big to be measured by DLS.
When the solution is ready for analysis and
placed in the cuvette, care should be taken to avoid
bubbles that may form on the walls of the cuvette.
Slowly tilting or tapping the cuvette on a hard surface
may help also.
Colored solutions
In the case of colored solutions, as long as the
laser light is not absorbed completely by the solution, the
sample can be measured. Colored samples and
fluorescing samples may be harder to measure. Often
the sample is available without the fluorescent dye or the
absorbing chromophore. In this case it will be easier to
measure the sample without the dye/chromophore
present. To distinguish the scattering from the inherent
color of a sample, try reading text through the sample. If
the text can be read, then the scattering is not creating
most of the opacity.
Filtering the solution
If the solution is to be filtered, keep in mind that
the size distribution may be changed if the particles are
removed by the filter. A good rule of thumb is to use a
pore size (filter size) 3 times larger than the largest size
to be measured. For DLS a 5 micrometer filter can be
used in most cases. Always verify that the largest size
measured is smaller than the filter pore size by a factor of
3. Always rinse the filter prior to use. These
recommendations are valid for all filters except the
Whatman Anotop series filters. For Whatman Anotop
series filters, pass the first drop of sample to waste.
Measurements
Once the solution is homogenous and ready for
DLS measurement, the solution can be placed in the
instrument. For the novice, there are two ways of
checking that the concentration is not too high and that
the DLS measurement will be valid.
1) Count rate check: For an instrument
equipped with an APD, the count rate for the scattering
intensity of the dilute solution should be less than 2 Mcps
(2,000,000 count per seconds) with the intensity
maximized. Note that measurements SHOULD NOT be
made at this high count rate. The maximum count rate
for the measurement should be 500-600 kcps. The
attenuator will need to be adjusted after measuring the
scattering intensity on maximum intensity in most cases,
especially if the count rate is greater than 600 kcps.
2) Dilution check: The second way to verify that
the concentration is suitable for DLS measurements is to
dilute your sample by 50% after the first measurement.
If the size of the dilution is the same as the size of the
more concentrated measurement and the count rate is
reduced by a factor of 2, then the first measurement
concentration was low enough.
After many measurements it will be easy to
assess the concentration of the solution to be measured.
For best practices for measuring samples size,
please refer to the document “Guide for DLS
measurements”.
Nanoparticle, Protein, & Polymer Characterization