Kalliope: Input Parameters and Result View

Explained
Litesizer Series

Table of Contents
1 Input Parameters and results view Litesizer .................................................................................................... 1
1.1 Input parameters for DLS ............................................................................................................................................................................. 1
1.2 Acquired data and results for DLS ............................................................................................................................................................... 3
1.3 Input parameters for ELS ............................................................................................................................................................................. 4
1.4 Acquired data and results for ELS ............................................................................................................................................................... 6
1.5 Input parameters for MM .............................................................................................................................................................................. 7
1.6 Acquired data and results for MM ................................................................................................................................................................ 8
1.7 Input parameters for RI ................................................................................................................................................................................ 9
1.8 Acquired data and results for RI ................................................................................................................................................................ 10
1.9 Input parameters for Transmittance .......................................................................................................................................................... 11
1.10 Acquired data and results for Transmittance ............................................................................................................................................ 11
1.11 Input parameters for Dosing system Main menu ...................................................................................................................................... 12
1.12 Input parameters for My Settings ............................................................................................................................................................... 12

1 Input Parameters and results view Litesizer

The respective input parameter or result view is marked in the images. The related description is always listed in
the table below. Please note that images and descriptions always refer to series measurements if available,
however, these parameters will not be visible in single measurement screens.

1.1 Input parameters for DLS

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Parameter / Result Text + Image

Defines the measurement angle.
• Automatic: the optimum angle is automatically found for the sample, based on the transmittance,
which is continuously measured.
• Back scatter (175˚): suitable for strongly scattering samples, including large particles, highly
1 Measurement angle concentrated or turbid samples. Also suitable for weaker scatters and at low concentrations
• Side scatter (90˚): suitable for weakly scattering samples, including small particles and
transparent samples. We recommend side scattering for particles ranging from 0.3 nm up to 1 μm.
• Forward scatter (15˚): suitable for detection of large particles at low concentration; e.g., protein
aggregates, infusion solutions.
2 Target temperature Defines the targeted measurement temperature.
Defines the waiting time after the measurement temperature is reached. For measurements close to ambient
3 Equilibration time temperature, the equilibration time should be set at two minutes. Add one minute for every ˚C different from
ambient temperature, based on a 1 mL sample. For the Univette we recommend 4 minutes.
The analysis model is required for determination of the particle size distribution chart, and does not have any
influence on the Hydrodynamic diameter and Polydispersity Index value (determined via the Cumulant
model). Possible models are:
4 Analysis model • General: suitable if the sample is not well known, or if a single (broad) peak is expected.
• Narrow: Suitable if one or more narrow peaks is expected.
• Contin: an alternative algorithm that contains a non-negativity parameter and uses a regularization
parameter.
The cumulant model is required for determination of hydrodynamic diameter and Polydispersity Index, and
does not have any influence on the particle size distribution chart and peak analysis (determined via the
Analysis model). Possible models are:
5 Cumulant model
• ISO 22412: default model according to the ISO norm
• Advanced: Updated model to suppress data from possible sample containments. An elevated
baseline is used.
Defines the duration of a measurement and processed number or runs.
• Automatic: time for each run (measurement time) will be 10 s, and the measurements will
continue until the threshold number of counts has been accumulated (10 x 106) or until 60 runs
6 Quality mode
have been made.
• Quick: the threshold number of counts is 3 x 106, with maximum 30 runs
• Manual: Number of runs and Time for each run must be manually selected.
Defines the position of the grey wedge attenuator. The filter should be set to avoid detector saturation, but
have sufficient scattering signal. A filter value of 1 will decrease the laser intensity by an order of magnitude.
• Automatic: the optical filter density will be automatically optimized based on the detected
7 Filter mode scattered intensity.
• Manual: the optical filter density must be manually selected. In rare cases results might slightly
vary between two instruments using different detectors due to minor variations in the count rate
that influences the filter optical density used.
Defines the position of the focusing lens before the cuvette, which defines the actual measurement position
in the cell. It varies from -6 mm to +1 mm, where 0 is the center of the cuvette.
8 Focus mode
• Automatic: the focus position will be optimized automatically.
• Manual: the focus position must be manually selected.
Defines the sample material with its optical properties. The material must be selected from the user’s
database. New materials can be entered by clicking on the <Ka> icon. The material’s refractive index and
9 Material
absorption must also be entered before they can be selected for a measurement.
Is required for recalculation to volume or number weighting.
Defines the carrier liquid (solvent) with its optical properties. Once the solvent is selected, then the refractive
10 Solvent index and viscosity will be automatically filled. New solvents can be entered by clicking on the <Ka> icon in
the submenu “Solvents”.
D-Values are calculated using the size distribution data. A D-Value represents the point in the particle
11 D-values
diameter distribution up to which a defined percentage p of the total volume of material is contained.
This section defines the series parameter.
• Temperature: Temperatures between the measurements can be defined
• Repetition: Measurements are repeated by using the same parameters.
• Delay: Delay times between the measurements can be defined
12 Series • Concentration: Sample concentrations of single measurements can be defined
• pH value: pH values of single measurements can be defined
• Angle: The measurement angle of single measurements can be defined
• Measurement focus: The measurement focus position of single measurements can be defined

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Kalliope: Input Parameters and Result View
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1.2 Acquired data and results for DLS

Parameter / Result Text + Image

Shows the attenuation level used by the instrument. For automatic measurements, a value of 0 suggests that
1 Filter optical density the sample concentration is low, although the results may still be meaningful if the count rate is sufficiently
high.
2 Focus position Indicates the position of the optical focus used in the measurement.
Indicates the proportion of light that travels through the whole sample, as picked up by the dedicated photo
diode situated after the cuvette directly opposite the laser beam.
• A value of 100 % indicates that the sample is perfectly transparent while 0 % indicates that the
3 Transmittance
sample is completely opaque.
• Transmittance values lower than 70 % will prompt the instrument to choose the backscatter
measurement angle.
4 Processed runs Displays the number of runs measured.
5 Mean intensity Displays mean detected light intensity in kcount/s.
Displays the absolute detected light intensity in kcount/s, using the mean intensity and the filter optical
6 Absolute intensity density (OD) to calculate. I(absolute) = I(mean) * 10^(OD). We do not recommend to compare absolute
intensities measured by different Litesizer instruments due to slight instrument specific variations.
The auto run criteria displays the percentage of the threshold counts accumulated. Depending on the chosen
Quality mode, the applied criteria differs:
• Automatic mode: 106 counts or min 6, maximum 60
7 Auto run criteria • In Quick mode 3 x 106 counts or min 3 runs, max 30 runs.
• In Manual mode this value will stay 0%.
If the value is > 100% it means that the criteria in terms of kcount are already met before the minimum
number of runs is reached.
Contains the information regarding the diffusion of the particles within the sample being measured. An
Correlation function optimal correlation function is smooth, has a single exponential decay function, a flat baseline at 1.0 and a
8
graph height larger than 1.4. By fitting this curve to a function, the diffusion coefficient can be calculated and then
this value can be converted into hydrodynamic diameter using the Stokes-Einstein equation.

• Size distribution: It shows the probability of finding a particle in a size bin from the whole
populations.
• Undersize: Shows the relative amount of particles at or below a particular size. Hence, a value of
Particle size n % read for a diameter of D µm indicates that n % of the particles in the distribution have a
9
distribution graph diameter smaller than or equal to D µm.
• Size distribution- histogram: Representation is identical to the size distribution representation
except that it is not a curve but a bar-type graph showing the size of the particles in the different
size bins.

• The intensity distribution weights the particles by their contribution to the overall scattering
intensity. This is the native result obtained by a DLS measurement, therefore most commonly
10 Weighting model used.
• The volume distribution shows the total volume of particles in the different size bin. It is only
visible if the sample material is known.

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• The number distribution shows the number of particles in the different size bins. It is only visible
if the sample material is known.
Mean hydrodynamic
11 Indicates the mean hydrodynamic diameter of all particles detected in the sample, according to ISO norm
diameter
Indicates the width of the size distribution according to the ISO norm. A value of 10% or less indicates that
12 Polydispersity index the sample is monodisperse, according to ISO 22412:2008(E) for 100 nm latex. If the value is larger than
20% always evaluate the particle size distribution as well.
The intercept g12 is calculated as g2 - 1 (where g2 is the correlation function intercept). It refers to the
intersection of the correlation function curve and the y-axis. It is a data quality parameter and it evaluates the
signal-to-noise ratio of a sample:
13 Intercept g12 • Good quality measurements: g12 = 0.7 - 0.95, acceptable until 0.4
• g12 < 0.2 indicates the presence of weakly scattering particles or turbid samples.
• g12 > 1.0 indicates that there are dust particles present or that it contains large sedimenting
particles that cause fluctuations
The baseline should ideally be 1.000. If the measured baseline deviates by more than 0.01, then according
14 Baseline
to ISO 22412:2008(E), the measurement is unreliable.
The cumulant fit error is typically below 1e-5 for samples with narrow size distributions (e.g. latex size
15 Fit error standards) and is a measure of how well the fit curves align to the measured data. For samples with broader
size distribution, the error can be higher.
The velocity of the Brownian motion is defined by a property known as the translational diffusion coefficient.
16 Diffusion Coefficient The particle diameter is calculated from the translational diffusion coefficient using the Stokes–Einstein
equation.

• Peak: Up to three peaks from the size distribution graph will be listed indicating the most prevalent
17 diameter of the particles. Note that it differs from the hydrodynamic diameter result in that it
represents the harmonic mean of the peak area. It is not the mode of the peak.
Peak Analysis
18 • Peak area under the curve in %.

• Standard deviation: Refers to the broadness of the peak. The standard deviation around the peak
19
value gives the full width half maximum.

1.3 Input parameters for ELS

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Parameter / Result Text + Image

• Omega cuvette Mat.No. 225288: made of polycarbonate, can thus only be used with aqueous
samples. Sample volume: 650-900 µL, maximum sample temperature: +70 °C.
• Univette: suitable for water-based and organic solvents, as well as for high conductivity and/or high
temperature measurements. Min. volume: 650 µL, maximum sample temperature: +90 °C.
• Univette Low-Volume: same as Univette, but for sample volumes between 50 µL and 70 µL.
Maximum sample temperature: +70 °C.
1 Measurement cell • Omega cuvette Mat.No. 155765: made of polycarbonate, can thus only be used with aqueous
samples. Sample volume: 350 µL, maximum sample temperature: +70 °C. Discontinued - this
cuvette is no longer available for purchase.
• Omega cuvette Z Mat.No. 189417: made of polycarbonate, can thus only be used with aqueous
samples. Sample volume: 370 µL, maximum sample temperature: +70 °C. Discontinued - this
cuvette is no longer available for purchase.

2 Target temperature Defines the target measurement temperature.

Defines the waiting time after the measurement temperature is reached. For measurements close to ambient
3 Equilibration time temperature, the equilibration time should be set at two minutes. Add one minute for every ˚C different from
ambient temperature, based on a 1 mL sample. For the Univette we recommend 4 minutes.
• Smoluchowski: corresponds to the Smoluchowski approximation of the Henry factor. Sets the
Henry factor to a value of 1.5. It is suitable for water-based samples.
• Hueckel: corresponds to the Hückel approximation of the Henry factor. Sets the Henry factor to a
4 Approximation
value of 1.0. It is suitable for samples dispersed in non-polar solvents.
• Other: Henry factor can be set manually between 1.0 and 1.5. Using the Calculator in the Kalliope
menu you can calculate the Henry factor for your experimental conditions.

The Henry factor f(Κa), also termed Henry’s function, is necessary to calculate the zeta potential. The zeta
potential of particles in suspension is calculated from the measured electrophoretic mobility of particles using
the Henry equation
5 Henry factor

Defines the applied voltage between the cuvette electrodes.

• Automatic: The voltage is increased in increments until the maximum voltage is reached, for
6 Power adjustments
which the maximum power of 1 W is not exceeded.
• Manual: The voltage can be set from 1 to 200 V
Defines the duration of a measurement and processed umber or runs.
7 Quality • Automatic: the experiment will stop when the standard deviation reaches the threshold value.
• Manual: The number of runs can be set from 20 to 1000.
Please select a solvent from the database. New solvents can be entered by clicking on the <Ka> icon in the
8 Solvent submenu “Solvents”. For ELS measurement the Refractive index, the viscosity and the relative permittivity is
required.
This section defines the series parameter.
• Temperature: Temperatures between the measurements can be defined and varied.
• Repetition: Measurements are repeated by using the same parameters.
9 Series • Delay: Delay times between the measurements can be defined and varied.
• Concentration: Sample concentrations of single measurements can be defined and varied.
• pH value: pH values of single measurements can be defined and varied.

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1.4 Acquired data and results for ELS

Parameter / Result Text + Image

Shows the attenuation level used by the instrument. For automatic measurements, a value of 0 suggests that
1 Filter optical density the sample concentration is low, although the results may still be meaningful if the count rate is sufficiently
high.
2 Mean Intensity Displays the mean detected light intensity in kcount/s.
3 Adjusted voltage The voltage applied in the measurement.
Indicates the proportion of light that travels through the whole sample, as picked up by the dedicated photo
4 Transmittance diode situated after the cuvette directly opposite the laser beam. A value of 100 % indicates that the sample
is perfectly transparent while 0 % indicates that the sample is completely opaque.
Shows the interference between the modulated and unmodulated reference beams. A good measurement
5 Monitor trace
will show a stable signal with constant amplitude.
Shows interference between scattered light from the sample and the modulated reference beam. The mean
6 Detector trace
value must be at least 1000 kcount for meaningful results.
Plots the phase analysis light scattering (PALS), which shows the phase difference between the detector
7 Phase plot
trace and the monitor trace in white. The blue line shows the fit of the PALS to the data.
Zeta potential The Fourier transform of the detector trace is used to generate the zeta potential distribution as a function of
8
distribution frequency.
9 Mean zeta potential The mean zeta potential is calculated from all particles detected in the sample
10 Standard deviation The standard deviation of the mean zeta potential
Electrophoretic
11 Related electrophoretic mobility to the given mean zeta potential value
mobility
Conductivity The conductivity should ideally be less than 1 mS/cm. The Litesizer 500 can measure zeta potential of
12 samples with conductivities up to 200 mS/cm; however, such samples may be degraded by the voltage
applied.
Distribution peak The distribution peak is the maximum of the zeta potential distribution. The value should be similar to the
13
mean zeta potential value
14 Processed runs Number of runs carried out in the measurement.
15 Auto run criteria "Reached" indicates that the threshold standard deviation was reached.

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1.5 Input parameters for MM

Parameter / Result Text + Image

1 Measurement cell Defines the optical parameters of the cuvette.
2 Target temperature Defines the measurement temperature.
Defines the waiting time after reaching the measurement temperature. For measurements close to ambient
3 Equilibration time temperature, the equilibration time should be set at two minutes. Add one minute for every ˚C different from
ambient temperature, based on a 1 mL sample.
The refractive index increment is defined as the change in refractive index as a function of the change in
4 dn/dc
concentration. This is a sample specific parameter.
5 Quality Defines the length of the signal gathering for each sample.
Defines the optical parameters of the solvent. New solvents can be entered by clicking on the <Ka> icon in
6 Solvent
the submenu “Solvents”.
Is a solvent with known refractive index and Rayleigh ratio (e.g. Toluene). It is necessary to calculate the
7 Reference scattering intensity on an absolute scale, which is required to calculate the Debye-plot. New references can
be entered in the solvent database.
A shape correction should be selected if the particles do not scatter isotropically. The hydrodynamic radius
8 Shape correction
must be inserted if known, or it must first be measured by performing a particle size measurement.
Concentration Defines concentrations to be use. At least three concentrations are required to make a measurement. They
9
can be selected between 0.001 and 500 mg/mL. Additional concentrations can be entered.

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1.6 Acquired data and results for MM

Parameter / Result Text + Image

Shows the attenuation level used by the instrument. For automatic measurements, a value of 0 suggests that
1 Filter optical density the sample concentration is low, although the results may still be meaningful if the count rate is sufficiently
high.
2 Focus position Indicates the position of the optical focus used in the measurement.
3 Intensity trace The intensity trace displays the current measurement as soon as it begins.
It displays the median detected light intensity in kcount/s. If the median intensity is less than 20 kcount/s,
4 Median Intensity
then increasing the concentration should give better results.
The Debye plot relates the intensity of the scattered light (KC/R) to the particle concentration.

5 Debye plot

6 Molecular mass It is calculated as the inverse of the Y-intercept of the Debye plot.
The 2nd virial coefficient is calculated from the slope of the Debye plot. It describes the relation between
particle-solvent and particle-particle interactions. A positive gradient indicates that the particle–solvent
7 2nd virial coefficient interactions are stronger than the particle–particle interactions; therefore the dispersion or solution is stable.
Vice versa, a negative gradient indicates that the particle–particle interactions are stronger than the particle–
solvent interactions, so the particles will tend to aggregate.

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1.7 Input parameters for RI

Parameter / Result Text + Image

1 Measurement cell Defines the optical parameters of the cuvette.
2 Target temperature Defines the measurement temperature.
Defines the waiting time after the measurement temperature is reached. For measurements close to ambient
3 Equilibration time temperature, the equilibration time should be set at two minutes. Add one minute for every ˚C different from
ambient temperature, based on a 1 mL sample.
Defines the length of gathered scattering signal at each focus position. If you experience large deviations, in
4 Time for each run
the measurement, increase the value. For clean solvents 2 sec is sufficient.
Solvents with known refractive index can be chosen from the drop-down menu and are used as a reference
5 First solvent
to generate the focus position over refractive index plot.

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1.8 Acquired data and results for RI

Parameter / Result Text + Image

1 Intensity trace The intensity trace displays the current measurement as soon as it begins.
2 Mean intensity Displays the mean detected light intensity in kcount/s.
3 Filter optical density Indicates the attenuation level used in the measurement.
4 Focus position Indicates the position of the optical focus used in the measurement.
An absolute intensity over focus position profile is created and used as a standard curve. For the sample, the
5 Refractive index focus position corresponding to the peak absolute intensity of the sample is determined and plotted onto the
standard curve. The sample’s Refractive Index is calculated as projection on the y-axis.

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1.9 Input parameters for Transmittance

Parameter / Result Text + Image

Measuring temperature must be manually entered and set between 0 and 90 ˚C (or 273 and 363 K). Note
1 Target temperature
that, for measurements above +70 ˚C, only the quartz cuvette, glass cuvette or the Univette might be used.
Defines the waiting time after the measurement temperature is reached. For measurements close to ambient
2 Equilibration time temperature, the equilibration time should be set at two minutes. Add one minute for every ˚C different from
ambient temperature, based on a 1 mL sample. For the Univette we recommend 4 minutes.
3 Measurement time The recommended initial time for each run is 10 s.

1.10 Acquired data and results for Transmittance

Parameter / Result Text + Image

Indicates the proportion of light that travels through the whole sample, as picked up by the dedicated photo
1 Mean transmittance diode situated after the cuvette directly opposite the laser beam. A value of 100 % indicates that the sample
is perfectly transparent while 0 % indicates that the sample is completely opaque.
2 Mean power Mean transmitted power of the current measurement, in watts.
Mean transmitted power of the reference measurement, in watts. If no extra reference measurement is
3 Reference power
made, then the reference will be taken as the power transmitted through an empty module.

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1.11 Input parameters for Dosing system Main menu

Parameter / Result Text + Image

This routine must be executed at least once a day (before the first measurement) or if the hoses are not free
from air bubbles:
• All hoses connecting a dosing unit to the titration vessel are filled with the corresponding liquids.
• The tubing from the transfer Dosino to the titration vessel (via the Omega cuvette) is filled with
dispersant and the tubings running from Dosinos 1 and 2 to the titration vessel are filled with the
corresponding additives (acid or base).
1 Prepare Dosinos
• As air bubbles very often get stuck in the cuvette during the filling of the tubing, a dialog box will
pop up to alert the operator to remove the air bubbles from the cuvette.
• To this end, open the module lid, remove and gently turn the cuvette up and down to move the air
bubbles close to the transfer Dosino outlet. During the workflow the air bubble will be transported
to the waste.
If necessary the "Prepare Dosino" workflow may be repeated.
This sequence is the opposite of the "Prepare Dosinos" routine (all hoses are emptied). This routine must be
2 Clean Dosinos
performed at the end of the experiment.
The tubing from the transfer Dosino to the titration vessel (via the Omega cuvette) is rinsed with the transfer
3 Clean sample unit
liquid.
4 Empty sample unit The tubing from the transfer Dosino to the titration vessel (via the Omega cuvette) is emptied.

1.12 Input parameters for My Settings

Parameter / Result Text + Image

Proteins are very sensitive and can easily be destroyed by the solvent’s temperature increase generated by
1 Protein mode the electrical current (Joule heating). When the protein mode is activated, an additional delay phase after
each ELS run is introduced, which reduces Joule heating and hence minimizes protein degradation.
2 Enable D-values Please tick this checkbox to enable user defined D-values in the particle size measurements.
Defines the illustrated peak order (Peak 1, Peak 2, Peak 3) in the analysis but also in the series
3 DLS: Peak order
measurement results. Selection between peak order by height, area or particle size.

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