LS Spectrometer

User’s Manual

Version 5.5

August 2019

LS Instruments AG
Switzerland
www.lsinstruments.ch
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.1 LS Spectrometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2 Introduction to theory of light scattering . . . . . . . . . . . . . . . . . . . . . 5
2.1 Static light scattering (SLS) . . . . . . . . . . . . . . . . . . . . . . . 5
2.2 Dynamic light scattering (DLS) . . . . . . . . . . . . . . . . . . . . . . 5
2.3 Modulated 3D cross-correlation . . . . . . . . . . . . . . . . . . . . . . 6
3 Basic operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.1 Hardware . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
3.2 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3.3 Startup procedure and shut down procedure . . . . . . . . . . . . . . . 9
3.4 Detector count rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.5 Switching between 2D and 3D configuration . . . . . . . . . . . . . . . 10
3.6 Switching between polarized and depolarized scattering . . . . . . . . . 10
3.7 Adjustment of the Sample Goniometer . . . . . . . . . . . . . . . . . . 11
3.8 Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
3.9 Safe Operation at high temperature . . . . . . . . . . . . . . . . . . . 13
3.10 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
3.10.1 Alignment check . . . . . . . . . . . . . . . . . . . . . . . . 14
3.10.2 Adding index-matching fluid . . . . . . . . . . . . . . . . . . 15
3.10.3 Replacing index-matching fluid . . . . . . . . . . . . . . . . . 15
3.10.4 Index-matching vat removal and cleaning . . . . . . . . . . . 16
3.10.5 Temperature calibration . . . . . . . . . . . . . . . . . . . . 16
3.10.6 Realignment of the spectrometer . . . . . . . . . . . . . . . . 16
4 LS Spectrometer software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
4.1 Structure of the main program . . . . . . . . . . . . . . . . . . . . . . 17
4.1.1 Measurement settings . . . . . . . . . . . . . . . . . . . . . 18
4.1.2 Goniometer settings . . . . . . . . . . . . . . . . . . . . . . . 20
4.1.3 Sample properties . . . . . . . . . . . . . . . . . . . . . . . . 21
4.1.4 Thermostat settings . . . . . . . . . . . . . . . . . . . . . . 21
4.1.5 Laser attenuator (+Shutter actuation) . . . . . . . . . . . . . 21
4.1.6 Focal length compensation . . . . . . . . . . . . . . . . . . . 22
4.1.7 Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . 22
4.1.8 DLS Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2 Structure of the saved files . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2.1 Data files . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
4.2.2 Analysis files . . . . . . . . . . . . . . . . . . . . . . . . . . 27

A DLS analysis 29
1 Cumulant analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2 CONTIN analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

B Instrument connections 31

C FAQ and troubleshooting 33

1
2 Manual 3D LS Spectrometer

D Health and Safety 43

E Certificates 47

Bibliography 49
1 Introduction 3

1 Introduction
Before the first use of the instrument, the user must read the section ”Health and Safety”.

1.1 LS Spectrometer
The LS Spectrometer by LS Instruments is among the most sophisticated instruments for
colloidal system characterization. It performs both static and dynamic light scattering (SLS &
DLS). The modular design allows a high degree of customization adapted to your specific
requirements, without compromising either precision or quality. In its basic version the LS
Spectrometer is a goniometer system with a single laser beam at 633 nm or 660 nm wavelength.
The following options can be added depending to your requirements:

1. 3D Cross-Correlation: DLS and SLS experiments are based on the assumption that only
singly scattered light is detected. Already a small amount of multiple scattering can result
in significant measurement errors. This is why DLS and SLS frequently require dilution of
the sample to avoid multiple scattering. With the 3D Cross-Correlation upgrade, multiple
light scattering is suppressed efficiently, thus allowing measurements of samples in their
natural undisturbed state, without any need for dilution. Two simultaneous light scattering
experiments are performed at the same scattering vector on the same sample volume in
order to extract only the single scattering information common to both.
Furthermore, all PMT (Photo-Multiplier Tube) and APD-based(Avalanche Photo Diode)
photon detectors have a certain probability to produce a second electronic pulse after they
detect a photon (the so-called ”after-pulsing effect”). Although the after-pulsing probability
is less than 2% with the high sensitivity detectors of the LS Spectrometer, this can result
in significant errors for lag times shorter than approximately 1 µs. Pseudo cross-correlation
of the signal eliminates the after-pulsing effect, thus allowing measurements down to 12.5
ns lag time. The 3D upgrade automatically eliminates the after-pulsing-effect due to the
cross-correlation approach.

2. 3D Modulation: Although the 3D Cross-Correlation technique offers significant

advantages to regular DLS and SLS, it also comes with a drawback. Because of cross-talk
between the two detectors the maximum theoretical intercept is decreased from 1 to 0.25,
effectively reducing the signal-to-noise ratio. The 3D Modulation Upgrade eliminates this
disadvantage. The two scattering experiments are temporally separated by modulating
the incident laser beams and gating the detector outputs at frequencies exceeding the
timescale of the system dynamics. This robust modulation scheme eliminates cross-talk
between the two beam detector pairs and leads to a four-fold improvement in the cross-
correlation intercept.

3. Correlation Mode Switch: This allows switching from 3D Cross-Correlation mode to

Pseudo Cross-Correlation mode. A custom three-input two-output fiber system makes
switching between pseudo cross and 3D cross-correlation modes as simple as moving a
mechanical shutter. The beamsplitter for 3D operation is mounted on a robust flip mount.
When it is moved out of the beam-path it enables full-power on-axis illumination with no
loss of laser intensity. Such on-axis illumination provides a more robust alignment that is
less sensitive to sample and temperature changes and is preferable whenever the sample
can be diluted.

4. Sample Goniometer (Pro Upgrade): An upper sample goniometer is available for

performing DLS and SLS experiments on extremely turbid samples which are placed in a
special square measurement cell. Using a θ − 2θ detection scheme in conjunction with the
4 Manual 3D LS Spectrometer

square cells enables the optical path length in the sample to be significantly reduced (as
low as 0.2 mm). The sample goniometer is also necessary for measurements of non-ergodic
systems (e.g. gels, glasses, foams) which must be placed under continuous rotation during
measurement.

5. Automatic Laser Intensity Regulation: This option automatically regulates the intensity
of the laser to the ideal intensity and manual setting is no longer required. Automated
control of the sensitive APD detector(s) is also included such that it(they) is(are) only
active under safe illumination conditions.

6. Interference Filter, 90% Transmission: This laser line filter can be used in front of
the detector so as to suppress all wavelengths (outside a 10 nm passband) other than the
input wavelength.

7. High Performance Polarizers for Depolarized Light Scattering: One Glan-Thompson

polarizer with an extinction ratio of 10−6 for the incoming side and one Glan-Thompson
polarizer with an extinction ratio of 10−6 for the detection side. This allows measurements
of translational and rotational diffusion coefficients of weakly optically anisotropic particles
(DLS) and full characterization of this anisotropy (SLS).

8. Focal Length Compensation: In a 3D cross-correlation or modulation experiment the

precise focal position (and hence the crossing of the two laser beams) is essential for a
good performance. The focal position depends on the refractive indexes along the optical
beam path. Thus the performance depends on the solvent, the size of the scattering cell
and the temperature of the index-matching fluid. The focal length compensation system
can correct for this change, hence preserving optimal performance at various conditions.

The setup enables you to characterize particles from 0.15 nm to 5 µm. This characterization
includes the hydrodynamic radius, the radius of gyration, the form and the structure factors,
the size distribution as well as the molecular weight.
2 Introduction to theory of light scattering 5

2 Introduction to theory of light scattering

Light scattering is a powerful non-destructive tool for material characterization. Here, a brief
introduction to light scattering techniques is presented. For a more detailed discussion, we
recommend to visit our website www.lsinstruments.ch. We also recommend the following
literature: for dynamic light scattering [1], for static light scattering [2] and for advanced readers
for both subjects [3].

2.1 Static light scattering (SLS)

In static light scattering experiments the intensity of light scattered due to the presence of
objects (scatterers) with refractive index different from the surrounding medium is measured
as a function of the scattering angle, θ. This angle is directly related to the modulus of the
scattering vector q = 4π/λ sin(θ/2), by the wavelength λ. The average scattered intensity Is (q)
is proportional to the particle form factor P (q), the static structure factor S(q) and the number
of particles. The form factor conveys information about the shape and size of the particles,
whereas the static structure factor describes the interference due to the spatial correlation
among the scatterers.
A typical application is the determination of the weight average molecular weight Mw of
a macromolecule such as a polymer or a protein. Measurement of the scattered intensity
at different angles allows calculation of the root mean square radius, also called the radius
of gyration Rg . By measuring the scattered intensity for one macromolecule at various
concentrations, the second virial coefficient A2 , can be calculated.

2.2 Dynamic light scattering (DLS)

In a colloidal dispersion suspended particles undergo Brownian motion. The motion results in
fluctuations of the distances between the particles and hence also in fluctuations of the phase
relations of the scattered light. Additionally the number of particles within the scattering volume
varies in time. The end result is a fluctuating scattered intensity Is (q, t). The corresponding
measured intensity correlation function is written as:

hIs (q, t)Is (q, t + τ )i

g2 (q, τ ) = (2.2.1)
h|Is (q, t)|2 i

with τ being the time delay. By means of the Siegert relation g2 (q, τ ) can be related to the field
correlation function g1 (q, τ ):

g2 (q, τ ) = 1 + β|g1 (q, τ )|2 (2.2.2)

hEs (q, t)Es∗ (q, t + τ )i
g1 (q, τ ) = (2.2.3)
h|Es (q, t)|2 i

β being the so-called intercept. The field correlation function may be used to determine the
diffusion coefficient D of the scatterers. In the simplest approach g1 (q, τ ) is fitted to an
exponential function

g1 (q, τ ) = exp(−Γτ ) (2.2.4)

yielding the decay rate, Γ. From its definition

Γ = q2D (2.2.5)
6 Manual 3D LS Spectrometer

one obtains the diffusion coefficient. By using the Stokes-Einstein equation one can obtain the
hydrodynamic radius as
kT
R= (2.2.6)
6πηD
with k the Boltzmann, T the temperature, η the viscosity of the suspending medium and R the
hydrodynamic radius of the particle.

2.3 Modulated 3D cross-correlation

The models used to extract meaningful physical information about the samples from DLS or SLS
measurements require singly scattered photons. The dilution of a sample represents a way to
achieve singly scattered light as it reduces the probability that a scattered photon be scattered
again. Dilution however inherently alters the original sample. Additionally, it is not obvious
how to recognize the limit of turbidity for single scattering. The impossibility of measuring
reliably and accurately concentrated samples in their original state is indeed one of the main
limitations of conventional 2D light scattering techniques. Modulated 3D cross-correlation is
a highly effective measurement technique that suppresses the influence of multiply scattered
photons in turbid samples while keeping high signal-to-noise ratio [4]. The 3D arrangement uses
two simultaneous light scattering experiments at the same scattering vector in order to extract
only single scattering information common to both. Two light sources and detectors are placed
symmetrically out of the scattering plane while the signal of both detectors is used to calculate
the cross correlation function [5–8].
In this configuration, the signal contains a non-negligible contribution of a second undesired
scattering vector [9], being a result of the other light source. The intercept as described in
equation 2.2.2 is hence reduced by a factor 4. The correlation intercept strongly influences
measurement accuracy and precision due to its pivotal role in accurately fitting models to the
measured data. However, this loss of intercept can almost be fully compensated by temporal
modulation of the radiation source and detectors(optional). The modulation is hereby exceeding
the timescale of the system dynamics [4]. Thus multiple scattering is suppressed with only
marginal loss of the intercept. A scheme of the modulation is shown in Fig. 2.1.

Figure 2.1: Schematic of modulated 3D cross-correlation light scattering instrument

showing the two states (a& b) wherein one of the modulators is activated
and one detector is gated [4].

Fig. 2.1 shows the two states of the setup. The beam path and the detection corresponds to
2 Introduction to theory of light scattering 7

the one of a conventional 3D setup. A beam is split into two beams which are then focused
in the sample, defining two overlapping scattering volumes. The scattered light at a specific
scattering vector is collected with two independent photon detectors. Additionally to the 3D
setup, two intensity modulators are inserted in the beam path such that each of the two beams
can be modulated in intensity. The signal transmission from the detectors to the correlator can
be also interrupted for each detector. Hence the scattering signal for each of the two scattering
experiments in the 3D configuration can be recorded while the other one is switched off. The
two possible configurations are shown in (a) and (b). By switching fast in between those two
configurations dynamics up to 300 kHz can be recorded. The temporal limit is given by the
switching frequency.
8 Manual 3D LS Spectrometer

3 Basic operation
This section aims at giving the user basic information about the steps required to perform a
measurement on the LS Spectrometer. Hardware, software, sample preparation, and maintenance
are discussed in separate sections.

3.1 Hardware
A scheme of the LS Spectrometer is presented in Fig. 3.1. It depicts the spectrometer from two
points of view. Available options are marked in blue. A continuous wave (CW) laser is deflected
by 180 degrees in the deflection unit. An automated (or manual) attenuator wheel adjusts the
intensity of the laser beam used for the scattering experiment. On the same support, a beam
sampler is installed, to monitor the intensity of the attenuated beam and a beam block lets the
user block the laser beam when no measurement is running. The 3D configuration (optional) is
achieved by means of a beam splitter, which can be flipped up (3D configuration) and down (2D
configuration) depending on the type of measurement. This is a very delicate optical element,
which strongly influences the quality of the alignment. In order to keep a high quality alignment
over time, the flip-mount should always be operated as carefully as possible.
The modulator unit (optional) consists of 2 acousto-optical modulators which alternatively
deflect light laterally. The lens mount is optionally equipped with an auto-focus correction
system. The back side of the lens contains an anodized plate with pinholes to reduce reflections
in the system.

Figure 3.1: Scheme of the LS Spectrometer shown from the front side. Available options
are marked in blue.

The main goniometer allows measurements in the angular range between 8◦ and 153◦ (valid
for new instruments). The position of the goniometer arm is read with a precision of 0.01◦ .
An additional sample goniometer (optional) enables the rotation and horizontal translation of
the sample. The sample is held inside an index-matching liquid (decalin) contained in a high
3 Basic operation 9

precision quartz vat. Its temperature is controlled by an external thermostat. The detection
system includes a lens, a fiber system and two detectors (second detector is optional). Fibers
are an extremely sensitive component of the detection unit. In order to preserve an optimal
alignment quality avoid touching the detection housing.

Note:

• If for any reason the fibers must be unplugged from the APD, pay attention to cap their
connectors and that the instrument is powered off.

• Since the goniometer arm moves over a wide range of angles, do not place obstacles within
the accessible range of the arm avoiding damage to the system.

3.2 Preparation
Samples can be loaded in round cells (5 mm or 10 mm) or in square cells (10 mm). For each cell
a corresponding sample holder is available. The most recent version of the sample holder has
a fixed orientation relative to the housing of the LS Spectrometer, to increase the centricity of
the cell.1 Cells should be filled such that the sample meniscus is higher than the laser beam but
lower than the level of the index-matching liquid (Please see Fig. 3.8 ). This is to allow proper
thermal equilibration between the sample and the thermostated index-matching liquid. When
inserting a cell into the LS Spectrometer make sure that the surface is clean and dust free to
avoid contamination of the index-matching liquid. A suggested procedure is that of cleaning the
cell with optical tissues (made for delicate tasks) and ethanol.

3.3 Startup procedure and shut down procedure

Thermal equilibration of the setup requires approximately 30 minutes before stable conditions
are reached. Before starting up the LS Spectrometer verify that the manual beam block, if
present, is closed. For recent instruments (2018 and up), an automated shutter is in place. The
beam is blocked when the software starts and until the user manually opens it, or launches
a measurement. The manual beam is blocked when the marked line on the turning wheel

Figure 3.2: Closing the beam block. The line on top indicates whether the beam can pass
or not. As shown in the picture the beam is blocked. If the marking is parallel
to the beam (turned by 90 deg regarding the shown position), the beam block
is open. This item is replaced by an automatic shutter on recent instruments.2

1
The fixed orientation of the sample holder (all types) applies only to the most recent models. Older models
can be rotated around the main axes of the goniometer.
10 Manual 3D LS Spectrometer

is orthogonal to the optical beam path (cf. Fig. 3.2).2 Turn on the laser, the electronic
controller of the LS Spectrometer, the thermostat (optional) and the PC. (If you are using
other models of Julabo Thermostat than the CF31 please switch on both the circulator and
the refrigerator. Failing to do so might result in damage of the thermostat). Open the Program
LS Spectrometer located on the taskbar and wait for initialization of the components. The
progress of the initialization is shown in the lower panel of the program. Correctly initialized
hardware modules are indicated by a green led to the right of their name, as shown in Fig. 4.1.
Trials for measurements can already be performed at this stage.3 Note: The thermostat is
software-controlled and thus equilibrates only after start of the software.
To shut down of the instrument, first block the beam by turning the manual beam block, if
present. Exit the software by using the exit button in the upper right part of the main software
panel.4 Turn off the laser, the electronic controller box of the LS Spectrometer, the thermostat
and the PC.

3.4 Detector count rate

Ideally, the count rate should be within the range of 25 kHz to 500 kHz (10 kHz to 200 kHz for
Modulated 3D experiments). The lower limit is set by the dark count rate of the APD, which is
below 250 Hz. The upper limit is given by the appearing non-linearity of the APD.
The easiest and most reliable way to optimize the count rate is to use the Automated Intensity
Attenuator (optional). If the laser intensity is manually controlled, start with very low laser
intensity and increase it until you reach optimum count-rate range. Be aware of the angular
dependence of the count-rate!
An electronic gating protection for the detectors is embedded in the LS Spectrometer. In case the
counts reach a set threshold, the detectors are switched OFF so as to prevent any damage. The
threshold may be adjusted in case of difficult measurements, such as sample where aggregates
are present. Please contact LS Instruments for advice on the safest way to proceed.

3.5 Switching between 2D and 3D configuration

Switching between the 2D and the 3D scattering geometry requires software and hardware
settings changes. In the 2D configuration, the beam splitter (optional) should be flipped down
and only the central detector aperture should be open (if the sliding beam shutter is installed,
see “Beam selection” in Fig. 3.1). Attention: please operate/flip the beam splitter with care
as shown in Fig. 3.3! Careless handling might result in a loss of alignment. The change of the
scattering geometry needs to be set in the software as well, since in 2D and 3D configurations
the scattering angle corresponds to different goniometer arm angles.

3.6 Switching between polarized and depolarized scattering

The entrance polarizer, placed at the exit of the deflection housing, is always in a vertical position
and should not be manipulated.
The detection polarizer can be easily switched between a vertical or horizontal position enabling
measurements in VV or VH configuration respectively. The position is labeled both by a mark on
the polarizer (Figure 3.4(a) and (b)), and by a sticker onto the polarizer holder (Figure 3.4(c)).
2
Old versions of the Spectrometer might have a marker orthogonal to the shown model. This implies that for
those models the beam block is closed if the marker is parallel to the beam. Please check at low laser intensity,
which model you have.
3
The He-Ne laser will however require 30 min to stabilize fully.
4
In older software versions (before 6.5), do not use the cross in the top right corner of the program to quit the
software.
3 Basic operation 11

Figure 3.3: Beam splitter operation. Please manipulate this beam splitter carefully. Shocks
might result in a loss of alignment.

Figure 3.4: Vertical (a) and horizontal (b) position of the detection polarizer. (c) Labels
on the detection polarizer holder

3.7 Adjustment of the Sample Goniometer

The sample goniometer may be manually set in two different positions by adjusting two
micrometric screws (Figure 3.5):
12 Manual 3D LS Spectrometer

Figure 3.5: Center (a) and corner (b) positions of a square cell inserted in the sample
goniometer.

1. Center position: in this scheme, the center of the scattering cell is aligned with the laser
beam. Measurements should be performed in this position by default. Only cylindrical cells
may be used for non-ergodic measurements (Please see Section 4).

2. Corner position: a square cell is translated along two axis to reduce the optical path
length, which in turns reduces multiple scattering. You may use this option to further
increase the highest concentration measurable with your instrument.

The center and corner positions are marked on each micrometric screw (Figure 3.6), and their
values (in mm) are referenced onto your computer’s desktop, as well as on the technical report
provided by LS Instruments. The alignment of the square cell is extremely precise, and it is
likely that you will disturb it when adjusting the micrometrics screws. Use the ”Change Sample”
option in the LS Spectrometer software (Section 4).

Figure 3.6: (a) Overview of the Sample Goniometer. (b) Parallel and (b) perpendicular
micrometric screw for square cell position adjustment.

3.8 Measurement
After following the instructions for the sample preparation and the start-up procedure (sections
3.2 & 3.3) you are ready to start the first measurement on the LS Spectrometer. Insert the
clean scattering cell in the corresponding conical holder. Unscrew the top centric cover (Fig. 3.7
3 Basic operation 13

a)) and insert the sample with the conical sample holder (Fig. 3.7 b)). Note: when the sample
goniometer is available, the sample holder is dented for square cell alignment purposes. Be sure
to check for the position of the dents when inserting the sample holder.

Figure 3.7: Inserting a sample. a) unscrew the cover b) insert the scattering cell and its
holder, c+d) unscrew the sample removal tool which is helpful for inserting
the cell. Finally screw the cover as in a)

Unscrew the sample removal tool from the sample holder (Fig. 3.7 c & d) and slightly press
the sample holder in, until the scattering cell is well fixed. Watch from the side through the
vat to check if the sample level inside the cell is sufficiently high to let the laser beam pass
through the sample. Also ensure that the round lower part of the scattering cell is below the
laser beam. For this purpose you might unblock the laser beam by the mechanical shutter on
the attenuation stage and increase the laser power until you see the beam passing through the
cell. Attention: reflections of the laser light might damage your eyes! Examples of a too low,
too high and correctly placed cell are shown in Fig. 3.8.
After adjusting the height of the cell, reduce the laser power and fix the position of the scattering
cell by screwing in the top centric cover (Fig. 3.7 a). Do not over tighten the centric cover
otherwise you might have trouble removing the cell.
Now adjust all the parameters in the software according to your needs in the “settings” panel.
A detailed explanation of the software parameters can be found in section 4. After setting
the software parameters you can unblock the laser beam (Fig. 3.2). However, before doing
so, ensure the power is at its lowest level. If manual intensity regulation is applied, start a
measurement at this low power by clicking “Start Measurement” on the “Measurement” panel
while observing the count rate (Fig.4.4). Adjust the laser intensity to the suggested detector
count rates (section 3.4). Note: In case of angle dependent measurements the count rate might
strongly vary with angle, depending on the sample. Once the laser intensity is set, you can start
with your measurement.

3.9 Safe Operation at high temperature

The operation of the instrument with decalin is not safe above its flashpoint of 57o C. For
operation above 57o C and up to 90o C, a special index-matching fluid is needed. The user must
contact LS Instruments to determine the type of fluid to be used. The alignment is preserved
14 Manual 3D LS Spectrometer

Figure 3.8: Position of the cell inside the index-matching liquid. a) The level of the liquid
is too low. The scattering beam is deviated. b) good position. c) the position
of the cell is too high. It modifies the beam path due to the curvature of the
cell.

up to 90o C. However, prolonged high temperature operation and repeated temperature cycling
might result in the need of more frequent servicing. The use of toluene is strongly advised against,
as it is a flammable liquid with a flashpoint of 6o C. See the FAQ section of this manual for
more information about the use of decalin as a refractive index-matching fluid, and performing
accurate measurements at high temperature.
Ensure that the solvents used in the sample do not evaporate or inflame at the chosen sample
temperature.

3.10 Maintenance
In the following section necessary maintenance work for the proper functioning of the
LS Spectrometer is described in the Table 3.1

Table 3.1: Maintenance scheduler

Task Section Interval

Alignment check 3.10.1 monthly
Adding index matching fluid 3.10.2 weekly-monthly (depending on usage)
Replacing index matching fluid 3.10.3 2-4 months (depending on usage)
Temperature calibration 3.10.5 yearly
Realignment 3.10.6 according to alignment check
Index-matching vat removal and cleaning 3.10.4 only by service personal

3.10.1 Alignment check

We suggest to perform regular checks of the alignment of the LS Spectrometer. Such checks
are important both for DLS and SLS measurements and they help tracking changes of the
alignment. It is also advisable to store the results of the alignment checks and submit them to an
3 Basic operation 15

LSI technician before a realignment service is ordered. Take a sample of pure and filtered toluene
and perform an SLS measurement over the whole range of angles, in 2D Pseudo-Cross and 3D
Cross when available. Perform a similar script on a sample of dilute monodisperse particles with
a diameter < 100nm, in all geometries available. We suggest the following parameters for a
script as described in the Table 3.2. The comparison to the alignment check performed during

Table 3.2: Alignment check script.

lower angle upper angle step number of measurements duration (s) wait
20 140 10 5 5 (Toluene) 30 (Standard) 0

the installation allows you (and the LS Instruments technicians) to evaluate the performance of
the LS Spectrometer.

3.10.2 Adding index-matching fluid

Due to evaporation, the index-matching fluid will periodically need to be refilled. Typical refill
intervals greatly depend on usage and working temperatures, but may lie in the range between
several weeks and several months. In any case, the level of the fluid must always be above the
window visible from the side of the instrument. You can safely add 20 mL of filtered (0.22 µm
mildly hydrophilic PTFE filter) mixed trans-cis decalin using a glass syringe when the decalin
level is just at the top of the visible window. Use care not to overfill the level as excess fluid will
spill over the sides of the vat, potentially requiring careful cleaning of the outer surface of the
vat (with optical wipes and a few drops of butanone). The most convenient way of checking
the level of decalin is to carefully slide a metal ruler into the sample compartment, as shown
in Fig. 3.9. Dip the ruler into the decalin until reaching the metal bottom. Tilting of the ruler
will result in slight deviation of the result. Take the ruler out and watch for the wet section of
the ruler. The minimum fill level is 20 mm and the maximum fill level is 30 mm. Remember to
always place back the screw cap onto the sample holder in order to avoid decalin evaporation.

Figure 3.9: Measuring the level of decalin in the vat.

3.10.3 Replacing index-matching fluid

Due to dust and other contaminants, the index-matching fluid (mixed trans-cis decalin) should
be replaced periodically. The recommended replacement interval varies depending on usage and
adherence to good practices. Generally, we suggest an interval of 2 to 4 months between decalin
replacements.
16 Manual 3D LS Spectrometer

The recommended fill volume of the vat is 80 mL. Use care not to overfill the level as excess
fluid will spill over the sides of the vat, potentially requiring careful cleaning of the outer surface
(with optical wipes and a few drops of isopropanol).
The best procedure is to first remove the old fluid by means of a glass syringe inserted into
the opening for the sample holder. Then add ∼25 mL of new fluid to re-disperse any remaining
dust, remove this fluid, and repeat this several times. Finally, add 80 mL of filtered (0.22 µm
mildly hydrophilic PTFE filter) mixed trans-cis decalin using a glass syringe and wait 15 minutes
before making measurements in order to allow any bubbles to clear.

3.10.4 Index-matching vat removal and cleaning

In general we recommend that users avoid removing the index-matching vat in the
LS Spectrometer . Removing the vat requires partial dismounting of the instrument, which in
rare cases could lead to the necessity of a full realignment. Major contamination (for example
due to breaking a cell in the vat) may however require vat removal and cleaning. In this case,
please contact LS Instruments to obtain a copy of instructions for vat removal and cleaning or
to schedule an on-site service by a qualified technician.

3.10.5 Temperature calibration

For a detailed description of the Temperature calibration ask the LS Instruments customer
service.

3.10.6 Realignment of the spectrometer

We suggest to have your LS Spectrometer serviced by LS Instruments every 18 months.
During the maintenance service a full realignment will be conducted to ensure optimum
performance of your LS Spectrometer. Furthermore the latest software version will be installed
during the service. Please contact [email protected] to arrange a maintenance service
approximately 3 months in advance. Realignment can also be performed by experienced
scientists, if they have participated in a certified alignment training by LS Instruments. Please
contact [email protected] to arrange alignment training either in Fribourg or on site at your
laboratory.
4 LS Spectrometer software 17

4 LS Spectrometer software
Version: 6.3 to 8.0
If an older version is installed on your system you might ask LS Instruments for an update.

4.1 Structure of the main program

This section explains in detail the structure and functionality of the LS Spectrometer software
provided with the instrument. The graphical user interface is organized into 3 different sections
as shown in Fig. 4.1.

Figure 4.1: Overview of the LS Spectrometer software.

The upper section (A) informs about the current status of the measurement and setup:
The upper section shows on the left the temperature of the sample. This temperature is based
on the temperature readout of a thermometer close to the sample and a calibration which
relates the temperature of the sample to the readout of the thermometer. The steady state
of this temperature is indicated above with a colored led (grey=unavailable, green=steady
state, red=steady state not reached). The two fields below inform about the set values for
the sample viscosity and the refractive index of the sample. In the second column, the status
of the detector(s) is shown by an indicator (green=on, red=off, grey=unavailable). Below the
intensity of the laser is shown, as well as the actual value of the scattering angle and the
18 Manual 3D LS Spectrometer

actual mode of the instrument. A precise description of the different modes can be found
below. In the middle of the upper panel, the actual count rate, its average and standard
deviation are shown for both channels separately (if second detector is installed). On the right
the timing of the current measurements are indicated. The main exit button is also located here.5

The middle section (B) is divided into three tabs, Settings, Measurement and DLS Analysis:
This section contains all the software-controlled settings of the LS Spectrometer. Depending
on the hardware configuration and options installed, certain parts are grayed out. The settings
are divided into three columns. On the left, measurement settings, in the center the goniometer
settings and on the right the laser attenuator settings can be adjusted.

The lower section (C) informs about the initialization and status of the hardware and the
software:
When the software starts it initializes sequentially all the available options, the corresponding
status is indicated by a colored led for each option and details are provided in the status text
field on the right. Options which are not available remain grayed-out. A correctly functioning
option turns to green after the initialization, while options that fail to initialize are indicated by
a red led. When the initialization process is finished, Ready is displayed in the text field on the
right.

4.1.1 Measurement settings

Experiment type Two types of experiments can be conducted with the LS Spectrometer
software: in a ”Generic” experiment, the results will be displayed in terms of the decay rate,
Gamma, expressed in units of the inverse of a second. This is especially useful for depolarized
DLS where the decay rate contains a translational and rotational component. In a ”Sizing”
experiment, the results will be expressed in terms of the particle diameter, in nanometers.

Scattering geometry Here one can select the scattering geometry, which can be either 2D or
3D. In section 3.6 the switching between scattering geometries is explained. Upon the setting
change, the software will show a short animation illustrating the required corresponding hardware
operations. The different options of the selected scattering geometry can be selected in the pull
down menu below. For each scattering geometry, there are different correlation types available.
The available correlation types depend on the options you have installed and the actual selected
scattering geometry.

Scattering geometry and correlation type

1. auto: The auto-correlation is the standard correlation mode. In this mode a single APD is
used and the correlation function is calculated auto correlating the corresponding signal.

2. pseudo (optional requiring two APD): In this mode of measurement, the signals of both
APDs are correlated. It is only available in the 2D scattering geometry. The pseudo-cross
mode suppresses after pulsing which has a particularly negative effect on the measurement
of small particles. For more information study section 1.

3. 3D Cross (optional requiring 3D option): The 3D cross correlation mode is required for
turbid samples to suppress multiple scattering. For more information study sections 1 and
2.
5
Please use only the exit button and not the cross button in the top right corner of the program to exit.
4 LS Spectrometer software 19

4. Mod3D Cross (optional requiring the 3D and the modulation option): Further
improvement of the 3D cross-correlation mode. For more information study sections 1
and 2.

Auto save data The “auto save data” option, if checked, allows automated saving of the
recorded data while measurements are performed. Click on the folder icon and chose the folder
path where you want your data to be saved and a base file name. Consecutive measurements
will be saved in different files having the specified base name followed by running number. The
first number of this running number can be specified on the main panel of the measurement
settings.

Duration and number of repetitions per measurement The length of a measurement can
be defined by two numbers. One is the duration per repetition and the other is the number
of repetitions per measurement. The total duration of one measurement is thus the number
of repetitions times the duration per repetition. The splitting of one measurement into several
repetitions has the advantage of allowing to assess the stability of each measurement.

Setup script The script window is very useful if several measurements in a row should be
conducted. A screenshot of the script window is shown in Fig. 4.2. The upper line,“New Set”, is
the line which can be edited. It contains the lowest angle and the highest angle of the series of
measurements, the angular step size , the number of measurements and their duration as well as
the waiting time in between each run and the temperature. Note that depending on the angular
step size the last measurement may be at a lower angle than the highest specified angle. For
example: lowest angle 20◦ , highest angle 135◦ and step size 10◦ would lead to an highest angle
in this series of 130◦ . Once the parameters are set, you may click on ”Insert line” to include the
line just prepared in the script.
All sets of repetitions are shown as a list in the middle of the window. If you would like to delete
a line click on it and select ”Delete line”. It is not possible to directly edit a line in the main
window. On the right panel, the estimated time for the script to be completed is shown.
If this time is unexpectedly large, check the number of repetitions per measurement as this
influences the total script time.
The buttons ”Load script”and ”Save script”enable you to save and load scripts. The lower panel
in this window shows the file name and location as well as the start number added to the files
for auto saved data. Once the script is configured, you may click on the OK button to return
to the main window.

Figure 4.2: Overview of scripted window.

20 Manual 3D LS Spectrometer

CONTIN settings Within a scripted measurement, CONTIN [10] analysis can be performed
automatically. If ticked, the settings of the CONTIN analysis can be adapted. The according
window is shown in Fig. 4.3. As explained in section 4.1.8 the scattering model, the type of
boundary selection, and the number of nodes can be selected. Additionally, the definition of
the baseline can be adjusted. This is adjusted in terms of number of channels starting from
the highest available channel. For more information on the other parameters please study
section 4.1.8.

Figure 4.3: Overview of scripted CONTIN analysis settings.

Channel scaling method When performing measurements on the same sample but at different
angles, the measured correlation function shifts along the lag time abscissa. This can cause
problems for the cumulant analysis fit. To avoid this issue you can apply lag time scaling. Upon
an angle change, the software will try to guess an optimal channel interval for the new angle
depending on the chosen channel scaling method.
1. No scaling: No scaling is applied. The maximum lag time for the fit is constant for all
measurements.
2. Decay factor: The adjustment of the maximum lag time is based on the decay of the
g 2 function. The maximum lag time is thus set at the lag time where the correlation
function decreases from the minimum set lag time by a given percentage which can be
fixed by the user. Details on setting the decay factor are described in section 4.1.7.

Total counts threshold This value is used for the cumulant analysis performed during the
measurement. A minimum number of counts is required to perform a cumulant analysis with a
meaningful result. The total counts threshold fixes this threshold.

Manual input temperature If no software-controlled thermostat is connected to the system,

this input option appears in the program. Use it to manually set the temperature of the sample.

4.1.2 Goniometer settings

The goniometer settings control the movement of the main goniometer and sample goniometer
(optional). For the main goniometer chose a scattering angle and move the arm by clicking
4 LS Spectrometer software 21

“Go”.
The sample goniometer has a drop-down menu with four entries.
1. Off: Switches the Sample Goniometer off.

2. Square Cell: For usage of square cells. The angle of the square cell is adjusted according
to the angle of the main goniometer following the θ − 2θ scheme [6].

3. Non-Ergodic DLS: Sample rotation speed is optimized for best results of a non-ergodic
sample in a DLS measurement.

4. Non-Ergodic SLS: Sample rotation speed is optimized for best results of a non-ergodic
sample in a SLS measurement.
An additional ”Change Sample” option releases the sample goniometer while a manipulation is
done (insertion of a sample, or adjustment of the micrometric screws) and then commands a
new reference search. This ensures that the alignment is always maintained when using a square
cell.

4.1.3 Sample properties

This section describes the relevant physical properties of the solvent used for the sample. The
solvent can be chosen from a list of standard solvents. This list contains: Water, Methanol,
Ethanol, Toluene, Glycerol.
For any other solvent the viscosity and the refractive index can be defined by the user. For this
purpose either choose “other” or “load file” from the list. The option “other” allows the user to
directly enter the viscosity and the refractive index. The option “load file”, enables specification
of an arbitrary temperature dependence for the solvent refractive index and viscosity by means
of a text file. The corresponding file format is illustrated in the following example. The sign tab
corresponds to a tab and eol corresponds to a line break:

Temperature tab Refractive Index tab Viscosity (cP) eol

eol
280 tab tab 1.32 tab tab 0.8 eol
285 tab tab 1.33 tab tab 0.89 eol
290 tab tab 1.34 tab tab 0.78 eol
295 tab tab 1.53 tab tab 0.76 eol
300 tab tab 1.23 tab tab 0.75 eol

This text can be saved as a text file. Respecting the precise syntax is important for a correct
readout. Several advanced text editors, such as Notepad++, allow the user to display tabs and
line breaks, this greatly facilitates the creation of the file above.

4.1.4 Thermostat settings

The temperature of the sample can be controlled via an external thermostat. The temperature
of the sample can be set here as well as the accuracy of a temperature. The options “low
accuracy”and“high accuracy”define when the temperature will be considered sufficiently stable
for a measurement. Higher accuracy of course implies larger equilibration times.

4.1.5 Laser attenuator (+Shutter actuation)

This panel controls the automated laser intensity attenuator (optional) as well as the shutter
position. The automated intensity attenuator can be controlled in three different ways:
22 Manual 3D LS Spectrometer

1. Auto: In the auto mode the incident laser intensity is set automatically to yield an optimal
scattered count rate based on the best range of the detector. Detectors are automatically
powered on and off. Before and after a measurement, the beam path into the scattering
cell is automatically closed.

2. Set Scattered Intensity: This option allows the user to perform an experiment yielding
a defined scattered count rate in the experiment. In the field “Target Scattered Intensity
(kHz)” you can define the desired scattered count rate. Upon measurement start, the
software will find the right laser attenuation. You can also define the precision by which
the software should try to obtain the desired scattered count rate. The requested precision
should be chosen depending on the sample. As a general rule monodisperse samples will
allow the use of higher precision settings, whereas for more polydisperse systems higher
precision settings might cause the automatic attenuation algorithm to fail. In such cases
the user should choose a lower precision setting. Detectors are powered whenever they are
needed and switched off in the standby. The laser is fully attenuated whenever it is not
used.

3. Set Laser Intensity: This option gives the user full manual control over the laser
attenuation. You can either type in a value in terms of percentage of the full laser power or
use the slider on a logarithmic scale and then click “Go”. If this option is used,the shutter
must be actuated on manually by using the tick box right next to “Laser shutter”. Make
sure that you tick this button only if you cannot damage the detectors.

If the automated intensity attenuator is not installed, you can control the position of the Laser
shutter via the tick box.

4.1.6 Focal length compensation

For the auto focus system (optional) you need to enter the type of scattering cell used in the
measurement. For square cells only the optical path length needs to be entered. After a change,
the button “adjust” needs to be clicked. Starting a measurement will adjust the focal system
according to the current settings.

4.1.7 Measurement
The measurement panel (Fig.4.4) shows all relevant information during a measurement. After
defining the parameters in the settings panel, click on the button “Start Measurement”. In case
you have chosen a scripted measurement this button is replaced by the button“Start Script”. The
buttons to the right allow you to stop or pause a measurement. The buttons below enable you
to save recorded data, if not already saved automatically and to load previous measurements. A
summary of the measurement conditions which will be also saved in the file is presented below
the control buttons.

Count rate trace: On the upper left side are the count rate traces for both (or one) detector.
On the right side of this graph the distribution of the count rate is shown. This latter graph
is useful for assessing the quality of a measurement. If, for example, a dust particle enters the
scattering volume the distribution will clearly indicate this by displaying a long tail towards large
intensities.

Correlation function: The graph displays the measured correlation function. From the drop
down menu you may chose between logarithmic and linear scaling of this plot. Scaling of the
axis can either be performed by the autoscale button or by clicking on the numeric values of
4 LS Spectrometer software 23

Figure 4.4: Overview of the software during a measurement.

the axis. The total counts threshold is defined on the settings panel. When said threshold is
reached during a measurement, additional black, blue and red lines appear on the graph. Those
lines define the lowest and highest channels defining the correlation function subset taken into
account in the cumulant analysis fit. The red line is used for the lower limit and the black and
blue ones are used for the upper limit. Depending on the channel scaling method you may either
move the vertical or the horizontal line, which is indicated by its dark blue color. The limits of
the cumulant analysis can be alternatively defined by the numeric fields below the figure. This
can be performed in terms of channels of the correlator or in time steps.
If the scripted CONTIN analysis is performed, an additional light blue line appears in the plot
indicating the limit of the baseline the analysis.

Measurement trend / angle plots /time trends (from vs 6.6): The lower figure on the
right side can show the angular trends of the intercept as used for SLS. The time trend of the
hydrodynamic radius and the intensity can for example be used to observe aggregation and the
panel of the 2nd order radius and the intercept as a function of the measuring time can be used
for determining the quality of the measurement. This panel also indicates the number of total
counts and information of the cumulant analysis as the first, second and third order of the radius
and its width.
The appearance of the graphs can be edited by a right click on the figure.
24 Manual 3D LS Spectrometer

Figure 4.5: View of the time trends

4.1.8 DLS Analysis

The DLS Analysis offers you cumulant and CONTIN [10] analysis of single DLS measurements
(Fig. 4.6). A theoretical overview of those analyses are given in the Appendix A.
The panel is divided into two sections. On the left are the entries of the analysis as the plot of
the data, the limits and settings of the cumulant and the CONTIN analysis. On the right part,
the results are presented.
As in the plot of the correlation function on the measurement panel, the plot of the measured
g 2 function is shown. The limits for the cumulant analysis can be modified by the blue and the
red bar in the figure by simple drag or via numeric values in the panel below. Additionally, the
resulting simulations (CONTIN and cumulant) can be shown in the figure. In the case of the
CONTIN analysis the simulation appears only after it was calculated by clicking on the button
CONTIN analysis (on the bottom right side).
Parameters for the CONTIN analysis can be set in the CONTIN Settings box. As scattering
model either a RGD sphere or point scatterer model can be chosen. The latter assumes the
scatterers to be significantly smaller in size than the incident laser wavelength while the former
relaxes this assumption. The number of nodes (discrete radii) calculated can be specified, too.
This number defines the grid of the calculation. The more points are taken for the grid the
higher is the resolution of the result. Finer grids result, however, in longer calculation times. It
is also important to choose the grid size accordingly to the quality of the measurement. If not
ticked, the boundaries of the radius are defined automatically according to cumulant analysis
radius. By ticking “Set Bounds Manually” you may define the smallest and the largest radius of
the simulation manually.
On the right top part, the results of the cumulant analysis are shown which can be saved with
the“Save Fit”button. On the lower part of this panel, the CONTIN simulation results are shown
either as a graphical distribution or as a list of peaks within the distribution.

4.2 Structure of the saved files

Several files can be produced as result of either a measurement or an analysis.

4.2.1 Data files

The output format of all files is pure text, which can be imported into your preferred
data-treatment program. A single measurement (here for example made in Sizing and Pseudo
4 LS Spectrometer software 25

Figure 4.6: Overview of the Software for DLS analysis.

cross-correlation) produces an output as shown below, whereas tab corresponds to a tab and
eol corresponds to a line break:

10/29/2013 tab 12:01 PM eol

Experiment Type:tab Sizing eol
Pseudo Cross Correlation eol
Scattering angle: tab 15.0 eol
Duration (s): tab 40 eol
Wavelength (nm): tab 632.8 eol
Refractive index: tab 1.330 eol
Viscosity (mPas): tab 0.896 eol
Temperature (K): tab 297.9 eol
Laser intensity (mW): tab 0.029722 eol
Average Count rate A (kHz): tab 334.5 eol
Average Count rate B (kHz): tab 294.0 eol
Intercept: tab 0.9316 eol
Cumulant 1st tab 295.04 eol
Cumulant 2nd tab 303.97 tab 80.62 eol
Cumulant 3rd tab 307.54 tab 117.01 eol
eol
26 Manual 3D LS Spectrometer

Lag time (s) g2-1 eol

1.250000E-8 tab 0.917454 eol
2.500000E-8 tab 0.928283 eol
3.750000E-8 tab 0.935385 eol
...
..

Count Rate History (KHz) CR CHA / CR CHB eol

0.000000 tab 194.520950 tab 184.640884 eol
0.104858 tab 459.136963tab 411.806107 eol
0.209715tab 651.416779tab 559.101105 eol
0.314573tab 204.992294tab 183.868408 eol
...
..

The first 14 lines are used to summarize the experimental conditions as well as the result of
the cumulant analysis. Below the summary values of the g 2 (t) − 1 function are printed in a
tab separated format. At the end of the g 2 (t) − 1 function an under sampled history of the
count rate is shown. The first few lines of such history are also printed above for the sake of
illustration.
If a script was performed, an additional file is saved. While each separate measurement in a
script is saved with a running number, the summary file does not have a running number. Its
structure is shown below for a Sizing experiment:

Scattering angle Mean CR ∗ sin(angle) / Laser Intensity (kHz/mW) g2(t=0)-1 CR CHA

(kHz) CR CHB (kHz) Temperature (K) Laser intensity (mW) Diameter(nm) PDI(-) eol
15.004272 tab 2735.121749 tab 0.931612 tab 334.522076 tab 293.984703 tab 297.860000 tab
0.029684 tab 40.81 tab 0.045 eol
15.004272 tab 3363.434631 tab 1.007546 tab 413.088534 tab 360.682696 tab 297.890000 tab
0.029711 tab 38.52 tab 0.042 eol
19.997803 tab 2650.292570 tab 0.863937 tab 409.287551 tab 358.341338 tab 297.890000 tab
0.049417 tab 37.12 tab 0.034 eol
...
..

and for a Generic experiment:

Scattering angle Mean CR ∗ sin(angle) / Laser Intensity (kHz/mW) g2(t=0)-1 CR CHA (kHz)
CR CHB (kHz) Temperature (K) Laser intensity (mW) Gamma(1/s) PDI(-) eol
15.004272 tab 2735.121749 tab 0.931612 tab 334.522076 tab 293.984703 tab 297.860000 tab
0.029684 tab 0.00321 tab 0.045 eol
15.004272 tab 3363.434631 tab 1.007546 tab 413.088534 tab 360.682696 tab 297.890000 tab
0.029711 tab 0.00314 tab 0.042 eol
19.997803 tab 2650.292570 tab 0.863937 tab 409.287551 tab 358.341338 tab 297.890000 tab
0.049417 tab 0.0029 tab 0.034 eol
...
..

It contains thus the scattering angle, the count rate corrected by the scattering angle and the
4 LS Spectrometer software 27

intensity, the g 2 − 1 function, count rates for both channels, the temperature, the laser intensity,
the decay rate or diameter (depending on the experiment type) and the PDI. Note: Scaling
of the intensity by measured laser intensity is only performed in case of an installed automatic
intensity attenuator (optional).

4.2.2 Analysis files

The file created by the button ”Save fit” in the cumulant analysis section will output the same
file structure as shown above for a single measurement.
The button ”Save Fit”in the CONTIN section outputs a ”.fit”file which is also a pure text based
file containing the calculated size distribution. Its structure is described below by an example
recorded in Sizing and Pseudo cross-correlation. (tab corresponds to a tab and eol corresponds
to a line break.)

10/29/2013 tab 12:03 PM eol

Experiment Type: tab Sizing eol
Pseudo Cross Correlation eol
Scattering angle: tab 20.0 eol
Duration (s): tab 40 eol
Wavelength (nm): tab 632.8 eol
Refractive index: tab 1.330 eol
Viscosity (mPas): tab 0.896 eol
Temperature (K): tab 297.9 eol
Laser intensity (mW): tab 0.049960 eol
Average Count rate A (kHz) :tab 409.3 eol
Average Count rate B (kHz): tab 358.3 eol
Intercept: tab 0.8639 eol
Cumulant 1st tab 338.67 eol
Cumulant 2nd tab 316.84 tab 120.52 eol
Cumulant 3rd tab 317.35 tab 116.95 eol
eol
radius (nm) rel. intensity eol
31.700000 tab 0.000000 eol
32.700000 tab 0.000000 eol
33.700000 tab 0.000000 eol
...
..

and for an experiment of type Generic:

10/29/2013 tab 12:03 PM eol

Experiment Type: tab Generic eol
Pseudo Cross Correlation eol
Scattering angle: tab 20.0 eol
Duration (s): tab 40 eol
Wavelength (nm): tab 632.8 eol
Refractive index: tab 1.330 eol
Viscosity (mPas): tab 0.896 eol
Temperature (K): tab 297.9 eol
Laser intensity (mW): tab 0.049960 eol
Average Count rate A (kHz) :tab 409.3 eol
28 Manual 3D LS Spectrometer

Average Count rate B (kHz): tab 358.3 eol

Intercept: tab 0.8639 eol
Cumulant 1st tab 338.67 eol
Cumulant 2nd tab 316.84 tab 120.52 eol
Cumulant 3rd tab 317.35 tab 116.95 eol
eol
Decay Rate (1/s) rel. intensity eol
0.000234 tab 0.000000 eol
0.000215 tab 0.000000 eol
0.000198 tab 0.000000 eol
...
..
A DLS analysis
In this Appendix a short description of the cumulant and the CONTIN [10] analysis is given.
The measured quantity in a DLS measurement is the so-called normalized intensity correlation
function, g2 (τ, q):

hI(0, q)I(τ, q)i

g2 (τ, q) = (0.0.1)
hI(0, q)2 i

(To simplify the equation in comparison to section 2.2 we set t=0)

The physically interesting quantity in terms of data analysis is however the normalized electric
field correlation function, g1 (τ, q):

hE(0, q)E ∗ (τ, q)i

g1 (τ, q) = (0.0.2)
hI(0, q)i

It allows us to extract useful information about the physical system at hand. Indeed, we can
express g1 (τ, q) as follows:
R
dΓf (Γ)I(Γ, q) exp(−Γτ )
g1 (τ, q) = R (0.0.3)
dΓf (Γ)I(Γ, q)

where Γ = q 2 D is the decay rate, f (Γ) the decay rate distribution normalized to 1, and I(Γ, q)
the intensity scattered by the class of particles whose decay rate is Γ. In order to obtain g1 (τ, q)
from the measured g2 (τ, q), we make use of the so-called Siegert relationship:
2
g2 (τ, q) = 1 + β g (1) (τ, q) (0.0.4)

1 Cumulant analysis
The cumulant analysis is an expansion of the normalized electric field correlation function,
g1 (τ, q):
cv
ln [g1 (τ, q)] = −D̄0 q 2 τ + (D̄0 q 2 τ )2 + ... (1.0.1)
2
D̄0 is the intensity weighted diffusion coefficient. Using the Stokes-Einstein equation and the
viscosity of the sourrounding medium the radius can be calculated:
kT
R̄ = (1.0.2)
6πη D̄0

2 CONTIN analysis
Once g1 (τ, q) is obtained the purpose of the CONTIN [10] analysis is that of extracting f (Γ)
from it by means of Eq.0.0.3. The first step to be taken is the introduction of a numerical
discretization for the integral which is accompanied by introducing a discrete set of Γ, Γ = {Γi }
values for which the corresponding discretized values of f (Γi ), f = {fi } are calculated. Taking

29
30 Manual 3D LS Spectrometer

into account the discretization and understanding that the denominator at right hand side of Eq.
0.0.3 is included in f (Γ) itself, the correlation function measured at the delay time τj belonging
to the discrete set of delay times τ = {τj } where experimental data are available is expressed
as
X
gj = g2 (τj , q) = Aij fi (2.0.1)
i

where Aji arises from the numerical discretization scheme adopted for the integral. In matrix
notation the previous equation takes the following form:

g = Af (2.0.2)

The simpler way to obtain the desired result is to simply invert the previous matrix equation as:

f = A−1 g (2.0.3)

The result of this is not well defined as Eq. 0.0.3 is said to be ill-posed; whichever the
discretization we adopt and whatever the experimental accuracy we achieve, the discretized
matrix equation will have a poor condition number and its inversion will not give a reasonable
result.
The CONTIN algorithm is an optimal smoothing strategy where among all the f fitting our
experimental data the most suitable is chosen. This smoothing algorithm is based on the
following: First of all Eq. 2.0.3 can be recasted in the equivalent least square form:

f = min
0
||g − Af 0 ||2 (2.0.4)
f

The equivalence can be proven by substituting f = A−1 g into the previous equation. Once
in this form, we can add to Eq. 2.0.4 a term that is inversely proportional to a measure of
smoothness of f :

||g − Af 0 ||2 + λBf 0

 
f = min
0
(2.0.5)
f

could be, for example, the result of the discretization of the following curvature modulus:
Z
 00 2
f (Γ) dΓ (2.0.6)

Clearly in this way noisy solutions are penalized and thus we regularize the problem. However
the amount of regularization depends on the so-called regularization parameter λ. To close the
problem we have to state an algorithm for the choice of λ, which leads to the optimal value
that retains a parsimonious amount of information about the physical system in question and
compatible with the noise present in the experimental data. This algorithm is the gist of the
CONTIN procedure, however we cannot go into further detail due to its complexity. Please refer
to [10] for more information.
B Instrument connections
Before unplugging cables from the controller please assure that the controller is
switched off. The controller box should only be opened by trained personnel.

A picture of the connection board is shown in Fig.B-1. The electronic connection between
the main controller and the different components are color coded. Not color-coded are the
BNC connectors for the modulators and the detectors. If not labeled, please label them before
disconnection to avoid mis-connections. This does not only refer to accidental switching of the
connection to the modulators or the detectors but also of channel A and B. Channel A and B
are not inter convertible.
The fuse can be found on the left side (seen from the front) of the controller underneath the
panel. It can be replaced by unscrewing the black cap.

Figure B-1: Overview of the electronic connections.

31
C FAQ and troubleshooting
• Which laser safety eyewear should I choose?
It is recommended to wear laser safety eyewear when using and particularly when aligning
the LS Spectrometer. For general operation of the instrument, laser safety goggles with
a high OD (optical density) at the specific instrument laser wavelength should be used
since vision of the laser light is not necessary. For alignment purposes, it is convenient to
attenuate the laser only partially, such that the laser light is still visible. For a case-specific
recommendation, we advise you to contact LS Instruments directly.

• Do I need to measure in a completely dark room?

The answer to this question depends on the sample, measurement type, and ambient
environment. The best starting point is to measure the scattering intensity in your
laboratory (with preferred lighting) with the laser beam blocked. Dark or very dim rooms
will lead to measurement of only the inherent detector dark count which according to
specification is <250Hz. Very bright rooms could yield as much as 15 kHz of dark signal.
In general, you should keep the counts measured as above to less than 1% of your measured
intensity. This rule of thumb implies that greater care in minimizing ambient light entry
into the detection system is important for weakly scattering samples. For extremely weakly
scattering and/or highly absorbing samples, LS Instruments offers high power laser options.
High relative ambient light contributions will reduce the correlation function intercept and
can confound SLS measurements. Note also that ambient light entry into the detection
system often displays an angular dependence.
In the event that your dark counts are significant compared to the signal measured
from your sample, you should make an effort to darken the room and then subtract the
remaining dark counts from your data. Dark counts arising from ambient light could also
be measured and subtracted from your data, but as it is difficult to measure these in a
reproducible way, it is instead recommendable that you minimize them. In order to make
a dark count measurement, you should simply turn the mechanical beam shutter so that
it is blocking the incident laser light, make a measurement, and save the file, which
will then contain the independent dark count rate for each detector. You should then
subtract the respective dark count rate of each detector from its count rate the next time
you acquire data. In addition to adjusting the count rates, the scattering angle corrected
mean count rate should also be recomputed. Note that these dark count measurements
are independent of the angle at which you are measuring, and are a feature of the detectors.

• What type of square cell can I use?

SLS and DLS measurements on highly turbid samples are possible using a 3D LS
Spectrometer equipped with the Sample Goniometer option. In this configuration, the
light scattering experiment is performed close to the corner of a square cell, thus reducing
the laser path length through the sample down to 0.2 mm.
The sample holder delivered for the square cells has been designed and manufactured to
fit best the dimensions of 10 mm Hellma square cells. External dimensions are: height

33
34 Manual 3D LS Spectrometer

46mm, width 12.5mm. Hellma square cells can be ordered from LS Instruments, or from
your local Hellma distributor (ordering number 111-10-20, type 111-OS).

• Why use decalin rather than toluene as an index-matching

fluid?
Toluene has traditionally been used as an index-matching liquid in light scattering
instrumentation due to its good refractive index match with optical glass. The refractive
index of toluene at 633nm is approximately 1.50. The refractive index of the ’vat’ used in
the LS Spectrometer is 1.46 at 633nm. The refractive index of typical optical glass used
in sample cells is 1.5 at 633nm. Another useful characteristic of toluene is its low density
(0.86 rel to water), thus helping contamination sediment out of solution
However, we strongly recommend using decalin rather than toluene due to its lower
volatility, lower toxicity and less acrid smell. The refractive index of decalin is approximately
1.47 at 633nm, and thus provides an essentially equivalent index match as does toluene,
yet being far more practical to use. Decalin has a very slightly higher density (0.9 rel
to water) and is mildly hygroscopic. In our opinion the common use of toluene in light
scattering instrumentation is mostly just a result of it being done that way for a long time.
The index matching vat is not fully sealed from the ambient environment, thus it is
normal that you will smell the solvent to some extent. The vat cannot be easily sealed
due to required compatibility with the sample goniometer system which provides variable
offset of the sample cuvette about the scattering volume axis. Thus it is likely the 3D
LS Spectrometer may exhibit higher evaporation rates than with other light scattering
instrumentation, and therefore a less volatile index-matching fluid like decalin can be
advantageous.
The desire to run the 3D LS Spectrometer with toluene as the index matching liquid for
closer comparison to other instrumentation using toluene is a good tendency but likely
not so well founded. The optical properties of the two liquids are so close that it is unlikely
one would ever see a difference when measuring the same sample on the same instrument
except perhaps in some extreme case. There will be far greater differences between two
different instruments resulting from their designs (the 3D geometry in particular) than
from the use of a different solvent. Static light scattering measurements must in any case
be properly referenced in order to remove any instrumentation-dependent quantities, and
thus any differences arising from the index matching fluid should be normalized out.

• Why is the intercept lower in 3D Cross correlation mode?

In the presence of multiple scattering, only a portion of the photons detected correspond
to a single scattering event. The rest of the photons resulting from multiple scattering
events constitutes the noise of the signal. The 3D technology eliminates this noise
by cross correlating the signals obtained from the two channels. However, if there are
statistically very few single scattering photons because the concentration is too high,
and/or the optical path length is too long, the intercept of the correlation function,
which is equivalent to the signal to noise ratio, will be very low. In 2D mode this
does not happen since the multiple scattering photons are not removed: you will get
a high intercept, but with a wrong intensity correlation function, resulting in wrong
measurements. The 2D mode should only be used for very dilute suspensions in which
there is no multiple scattering.

• Which correlation mode should I use?

 35

The best correlation mode to choose for your measurement depends on 3 parameters:
1. Sample turbidity.
For samples displaying any multiple scattering, you must measure in either 3D cross-
correlation or Modulated 3D cross-correlation. If the cross-correlation intercept measured
for this sample (at all scattering angles of interest) is less than that for a dilute reference
sample (same refractive index solvent as in sample) then multiple scattering is present.
You must be careful to measure across all angles of interest as the presence of multiple
scattering can have a great angular dependence. If the intercept is the same as that of
the dilute reference sample across all angles of interest, then you can safely measure in
pseudocross-correlation or autocorrelation (you cannot use autocorrelation if you have a
sample that displays fast dynamics - see below).
2. Sample dynamics.
If you have for example a small particle dispersion (say 30nm radius) then you will notice
that the correlation function decays very quickly. This is because the particles are moving
very quickly in the solvent by way of Brownian motion. However, if you have a large
particle dispersion or a viscous solvent you will notice that the correlation function decays
very slowly. Similarly, the slow decay is caused by slow motion of the scatterers in the
sample.
3. Scattering angle.
Due to the fact that the length scales being probed by a light scattering experiment
depend upon scattering angle, you will notice that the correlation function measured of
a sample at high angle (eg 140-deg) will decorrelate much faster than that measured for
the exact same sample at low scattering angle (say 25-deg).
For very fast decays of the correlation function (caused usually by the combination of
fast dynamics and high scattering angles) you will notice that the Mod3D mode may be
insufficient because it fails to capture very short lag times of the correlation function.
In such a case you should use 3D rather than Mod3D since the standard 3D mode
can measure at timescales 2 orders of magnitude smaller than for Mod3D. If you can
verify that the sample does not exhibit multiple scattering (again by comparing the
cross-correlation intercept of your sample to a dilute reference sample), then you can also
measure in ’pseudo cross-correlation’ mode. Autocorrelation measurements are not valid
for samples displaying fast dynamics due to detector afterpulsing which causing a sharp
rise of the correlation function at short lag times.

• How is the average intensity computed into the summary file?

The average intensity is computed as the geometric mean of the intensities of the two
detection channels multipled by sin(Θscattering ) where Θscattering is the scattering angle.
In a perfectly aligned system the angular dependence of the count rate for an isotropically
scattering sample can be approximated by a sin(theta) dependence to first order. For this
reason we (and most other SLS instrument providers) present scattered intensity data as
normalized by the quantity Θscattering when it is collected and summarized as a function
of scattering angle. The reason for this approximate sin(theta) dependence is due to the
angular-dependent size of the scattering volume formed by the overlap of the incident
beam and the focal volume of the detection optics.

• How should I optimize the detector count rate?

The easiest and most reliable way to optimize the measurement count rate is to use
the Automated Intensity Regulation option available for your LS Spectrometer. Ideally,
36 Manual 3D LS Spectrometer

measurements should in general be made below about 500 kHz and above approximately
100x the detector dark count (<250 Hz). The APDs have a strong non-linearity that
affect both the count rate and the intercept. At 5MHz, the actual intensity is nearly 1.5x
what the APD measures. At 1 MHz the correction is on the order of 3%. In the range of
200-500 kHz, this non-linearity is negligible.
Count rate optimization in 2D and 3D-Cross modes should be performed in the same way.
For both modes, we advise setting the intensity in the range of 200 - 500 kHz. If you
exceed 500 kHz, the intercept will start to slowly decrease and you begin to slowly lose
linearity of the signal.
Mod3D cuts the total amount of light entering the scattering volume by about a factor
of 2. In Mod3D, each of the two beams is on 50% of the time. Thus, the net effect is
that the incident intensity in Mod3D and 2D modes is approximately equal. The further
complication in Mod3D is that we are throwing away some of the detected light. These
photons that get discarded do not show up in the count rate trace in the instrument
software and do not affect the correlation function. So for example, the APD itself may
be ’seeing’ 500 kHz of light, but the amount of light actually reported in the software
(after throwing away unwanted photons) is maybe only 200 kHz. So if you were to run
your measurement in Mod3D at 500kHz, the actual intensity measured by the APDs is
much higher, and already into the non-linear regime. To figure out how much light is
actually arriving to the APDs, the easiest technique is to quickly switch to ”Auto” mode
in which no photons are being thrown away but yet the light incident on the scattering
volume is approximately the same. This is in some ways a limitation of the Mod3D
technique. However, you must remember that 75% of the photons in 3D cross mode
are just noise. Thus, the effective photon statistics of our measurement for Mod3D are
approximately equal to that for 3D cross.

• How can I make measurements at high count rates?

As mentioned above, due to the photon detectors non-linearity , LS Instruments does
not recommend making high count rate measurements (> 500 kHz in average) if it is
not necessary. Additionally, users are strongly advised against making measurements over
2MHz. However, operation in the range of 500 kHz - 2 MHz average count rates can be
very useful for measurement time reduction purposes.
While the detectors offered with the LS Spectrometer are specified for operation up to
10 MHz, it is strongly suggested to operate them under 2 MHz for two reasons. The first
is that, in particular for samples with slow dynamics, the instantaneous intensity at the
detector could reach close to the damage threshold of the device. Second, the detector
response non-linearity becomes relatively high and thus accurate correction of this becomes
difficult. Detector non-linearity arises from the short ”dead-time“ after a photon detection
event during which the detector is effectively turned off.
For DLS measurements, high count rates (500-2000 kHz) will lead to a small reduction
of the correlation intercept, but do not require correction for proper interpretation of the
correlation function. Static measurements conducted at high count rates are subject to
much greater complexities arising from the need to correct for the detector non-linearity.
The count rate, as well as the intercept must be corrected.
1. Count Rate Correction:
If you look at the output file of an angle-dependent script, the intensity of both channels
is given, as well as a ’mean CR * sin(θ)’. The intensity √ values are those actually
measured by the detectors. The mean value is defined as: I1 ∗ I2 ∗ sin(θ). The sin(θ)
 37

correction accounts for the angular dependence of the scattering volume. In order to
correct for detector non-linearity you must first correct each channel and then recompute
the geometric mean scaled by sin(θ). To measure the non-linearity, make measurements
of a strongly scattering sample for varying incident intensities. Construct a plot of count
rate versus incident intensity. Extrapolate the plot to zero, normalize the low-intensity
slope to 1, and then compute the deviation as a function of count-rate. Fit this deviation
vs count rate and use this function to correct the count rate data as described above. This
must be done separately for each detector - the non-linearity is specific to each detector.
2. Intercept Correction
The non-linearity of the detectors will cause a slight decrease in the cross-correlation
intercept at high count rates. While the cross-correlation intercept is angular-dependent,
the non-linear correction that is applied is equivalent for all angles (ie, β(θ) ∗ N LCF ,
where beta is the cross-correlation intercept and NLCF is the non-linear correction factor).
Compute the non-linear correction factor by measuring a dilute (non-multiply-scattering)
sample at any scattering angle at 6 to 9 different incident intensity levels such that the
detector count rates approach 2-3 MHz. Plot and fit the cross correlation
√ intercept as a
function of the geometric mean of the uncorrected count rates ie I1 ∗ I2 .

• How can I make accurate measurements at elevated

temperatures?
Due to the off-axis incidence angle of the 2 beams of the 3D geometry, it is inherently
more sensitive to changes in operating temperature (due to the temperature dependence
of the refractive index of the index matching fluid) and sample refractive index. Thus it is
normal to see gradual changes of the intercept with temperature or when moving between
solvents with greatly different refractive indices.
For single DLS measurements this is not a great concern unless working at temperature
extremes. If you often make measurements at more than 30o C above or below ambient,
we suggest considering the Auto Alignment Compensation option which automatically
corrects the alignment for temperature-dependent effects. Without this, high temperature
measurements can suffer from a greatly reduced cross-correlation intercept.
For SLS measurements this issue is slightly more problematic. A good set of reference
measurements is critical to achieving an accurate normalization of measured SLS data.
For best results, we therefore recommend making reference measurements at the sample
measurement temperature (within +/- 10deg at which the measurement is made) and
with a reference having the same solvent refractive index as that of the sample. Use of
the Auto Alignment Compensation module would be of great use here as well.
One further very important issue when working at temperatures far from ambient is the
possibility of convection within your sample. Sample cuvettes must always be filled to a
level lower than that of the decalin index matching fluid into which they are inserted.
Otherwise, part of the sample will be in strong thermal contact with the ambient air,
leading to temperature gradients within the sample. Temperature gradients can lead to
convection which will compete with the brownian motion of the particles which laser light
scattering is attempting to measure. At high temperatures, convection is nonetheless
possible in the sample even if it is properly filled due to the fact that there are unavoidable
temperature gradients (the magnitude of which scales with the difference in temperature
between ambient and the instrument setpoint) in the index-matching bath arising from
to the inability to mix this fluid. To minimize the possibility of convection, reduce your
sample volume to the absolute minimum necessary.
38 Manual 3D LS Spectrometer

• How is the temperature calibration done?

The temperature control system of the 3D LS Spectrometer consists of several elements.
The temperature is controlled by an external refrigerated/heated circulator. This device
circulates refrigerated/heated liquid through a custom heat-exchanging housing in the
3D which both holds and is immersed in the index-matching vat. A platinum resistance
temperature detector (RTD) probes the temperature in the index-matching vat and the
sample temperature is displayed in the LSI softwareu sing a custom calibration curve. This
calibration curve is necessary since the sample temperature deviates slightly from that
measured in the vat due to a position-dependence of the vat temperature resulting from an
inability to constantly mix the fluid. This calibration is performed upon installation of the
instrument and should be re-performed yearly. This calibration consists of measuring the
relationship between the temperature output by the software and the actual temperature
measured in the scattering volume (by measuring the viscosity of water using tracer
particles).
Compatible external circulators can be controlled remotely via the LSI software. Such
remote control enables temperature-dependent measurement scripts to be run and
furthermore allows the user to have confidence that the sample temperature (not just
the circulator temperature) has reached a steady state. An additional custom calibration
accounts for thermal losses between the circulator and the index matching vat. This
calibration is performed upon installation of remote temperature control and should be
re-performed yearly. Please contact LS Instruments to obtain the temperature calibration
procedure.
It should be noted that accurate light scattering measurements are only possible in the
absence of convection. Convection can occur in a sample that is not fully equilibrated or
is overfilled. Please see the question on making measurements at elevated temperatures
for more details.

• How to best perform the Cumulant Analysis?

The bounds for the cumulant analysis should be set such that the lower bound (at short
lag) is well up in the plateau of the correlation function but doesn’t include any low
channel artifact (after-pulse noise), and the upper bound (at long lag time) is at a lag
time for which the correlation value at that point on the curve is larger than the noise
in the baseline of the correlation function but not as large as to render the cumulant
expansion invalid. In practice, the last channel should be kept as high as to stay out
of the baseline and as low as to keep a good match between the fit and the measured
correlation function. This can be assessed by checking the box ”Show Cumulant Fit”in the
analysis tab. A general rule of thumb would be to set the ’decay factor’ (’channel scaling
method’ in settings tab must be set to ’decay factor’) to about 0.85-0.90, 0.60-0.70, and
0.40-0.50 for the third, second, and first order cumulant fits, respectively. For very turbid
samples where the intercept falls, or for short duration or low-count rate measurements
where there is substantial noise on the correlation function (which obviously you should
try to avoid), it would be safer to lower the decay factor ranges given above.

• Should I use the Cumulant or CONTIN Analysis?

The cumulant analysis is valid only for relatively low polydispersity samples. In a cumulant
analysis fit a subset of the correlation function, starting from the leftmost channels, is
fitted against a simplified model for the correlation function. Such simplified model is
asymptotically valid for narrow particle size distributions (PSD) and for short lag times
 39

and is expressed as follows:

p
g(τ ) − 1 = β exp(−K1τ + K2τ 2 − K3τ 3 + ...), (0.0.1)

where the parameters Ki are called cumulants, and β is the intercept. The wider the PSD
the more cumulants are to be included in the expansion, and the larger portion of the
correlation function must be included in the fit. However, a larger number of cumulants
leads to strong correlation between fitted parameters, hence making the fit meaningless.
The larger the intercept and the smaller the experimental noise on the correlation function
the larger the number of cumulants that can be used in the fit. In practice for noisy
low intercept experiments one chooses a first order cumulant fit, whereas for low noise,
large intercept experiments one can go as far as a third order cumulant fit. The first
cumulant divided by the square of the scattering vector gives the intensity weighted average
diffusion coefficient of the sample. The ratio of the second cumulant to the square of
the first gives the corresponding intensity weighted coefficient of variation of the diffusion
coefficient. The typical uncertainty on the latter is around 20%. Application of the Stokes-
Einstein relationship allows the estimation of the corresponding size parameters. The third
cumulant conveys very little information about the sample; its role is just that of improving
the estimation of the first two for relatively polydisperse samples (cf. discussion above).

The cumulant analysis can be applied to monomodal paucidisperse system. In all other
cases the following full model
p Z
g(τ ) − 1 = β f (D) exp(−q 2 D)dD, (0.0.2)

has to be numerically solved for the diffusion coefficient distribution function, f (D).
This in turn can be converted to the corresponding PSD by means of the Stokes-Einstein
relationship. Unfortunately such equation admits infinitely many solutions. The CONTIN
algorithm adds a-priori information that allows obtaining a single solution. For this
algorithm to yield a single solution that is also a good approximation of the true PSD,
the correlation data must have the least possible statistical noise, must not contain any
spurious systematic error, and, contrary to the cumulant analysis, the fitted correlation
data must contain as much as possible data without including any spurious noise. The
CONTIN analysis, making no simplificative assumptions on the model, doesn’t require
subjective choices like the selection of some model parameters, as the cumulant order in
the cumulant analysis requires, and the selections of the correlation data points to fit.
On the other hand the method is less robust than the cumulant analysis in that in many
situations, if the correlation function doesn’t perfectly meet the ideal requirements, the
obtained PSD can be far from the real one despite the fact has to be numerically solved
for the diffusion coefficient distribution function, f (D). This in turn can be converted
to the corresponding PSD by means of the Stokes-Einstein relationship. Unfortunately
such equation admits infinitely many solutions. The CONTIN algorithm adds a-priori
information that allows obtaining a single solution. For this algorithm to yield a single
solution that is also a good approximation of the true PSD, the correlation data must
have the least possible statistical noise, must not contain any spurious systematic error,
and, contrary to the cumulant analysis, the fitted correlation data must contain as much
as possible data without including any spurious noise. The CONTIN analysis, making
no simplificative assumptions on the model, doesn’t require subjective choices like the
selection of some model parameters, as the cumulant order in the cumulant analysis
requires, and the selections of the correlation data points to fit. On the other hand
the method is less robust than the cumulant analysis in that in many situations, if the
40 Manual 3D LS Spectrometer

correlation function doesn’t perfectly meet the ideal requirements, the obtained PSD can
be far from the real one despite the fact that its first moments are close to the real ones.
More details about the analysis method to be chosen can be found in their respective
best practices posts

• How are the Width and Polydispersity calculated in the

Cumulant Analysis?
The width that is referred to in the cumulant analysis tab in the software corresponds to
the zeta-averaged variance in the particle size distribution, σR , defined as:

2
p  6πηRave 
σR = K2 (0.0.3)
kB T q 2
4πns
q= λ sin 2θ , and kB is the Boltzmann’s constant.
The above can be rewritten by noting that the first Cumulant K1 , which is proportional
to the average hydrodynamic radius (particle size) is defined as:

kB T q 2
K1 = . (0.0.4)
6πηRave
Using this expression for K1 , we can express σR as
√
K2
σR = Rave . (0.0.5)
K1
The polydispersity, which is defined as the the ratio of σR to Ra ve is then simply
√
σR K2
= . (0.0.6)
Rave K1
In other words, to get an expression for the polydispersity, you must simply take the
width output by the software and divide it by Ra ve.

• How should my SLS reference measurements look like?

In a perfectly aligned system the angular dependence of the count rate for an isotropically
scattering sample can be approximated by a sin(θ) dependence of first order. For this
reason we (and most other SLS instrument providers) present scattered intensity data
as normalized by the quantity sin(θ) when it is collected and summarized as a function
of scattering angle θ. The reason for this approximate sin(θ) dependence is due to the
angular-dependent size of the scattering volume formed by the overlap of the incident
beam and the focal volume of the detection optics.
In a typical SLS experiment the measured angular-dependent intensity data will be
normalized to a reference measurement, typically filtered toluene. For relative intensity
measurements the purpose of this reference step is to normalize out the instrument
response. The ideal sin(θ) dependence could be assumed, but it is more precise to simply
measure the actual instrument response. In this way the measured data is insensitive to
any imperfections in the alignment.
There is always going to be some non-ideal angular dependence to the count rate regardless
of what instrument you are using and what the state of the alignment. Clearly if the
instrument is better-aligned, then this correction will be closer to the ideal sin(θ) behavior.
 41

Due to the off-axis incidence angle of the 2 beams of the 3D geometry, it is inherently
more sensitive to changes in operating temperature (due to the temperature dependence
of the refractive index of the index matching fluid) and sample refractive index . It is
often also more difficult to achieve a ’perfect’ alignment for the 3D geometry. A good
reference measurement is therefore critical to achieving an accurate normalization of
measured SLS data. For best results, we recommend making reference measurements at
the sample measurement temperature (within +/- 10deg at which the measurement is
made) and with a reference having the same solvent refractive index as that of the sample.

• How to properly correct SLS measurements?

When making angle dependent intensity measurements, you should in general normalize
the data to a reference measurement. This reference is typically done with filtered toluene
due to its isotropic scattering profile as well as having a known Rayleigh Ratio, which
is important for absolute measurements including molecular weight determination. What
must be done is to divide the measured data by the normalized reference data on an
angle-by-angle basis. Typically such a reference is re-measured every couple of months to
check the instrument state and that single measurement is used to normalize all data. The
purpose of this normalization is to account for any alignment errors. Proper procedure
includes preparing a thoroughly filtered sample of toluene (using a 0.22 micron hydrophobic
PTFE filter) in the same geometry cuvette in which you will perform your measurement.
The above recommendations are typical for standard dilute light scattering measurements.
However, a number of special considerations exist when attempting to make statics
measurements on turbid samples. An exhaustive list of corrections to measured turbid
statics data include:
Alignment reference (typically toluene): As an alignment reference, an aqueous ultra-small
particle dispersion can be used in order to work with a reference with similar refractive index
to the sample to be measured. In many cases, particularly when working with sensitive
square cell measurements, this can be important for proper normalization.
Solvent reference (sample solvent): For turbid samples, a solvent reference is typically
unnecessary since any scattering of the solvent is overwhelmed.
Dilute cross-correlation intercept reference (dilute small particle dispersion): The intercept
reference can be any dilute particle dispersion, preferably with the same solvent refractive
index as the sample. It does not need to be a dilution of the actual sample - the purpose
is only to measure the angular-dependent cross-correlation intercept in the absence of
multiple scattering.
Turbidity reference (turbid small particle dispersion): A measurement of sample turbidity
is essential for static measurements made in a square cell, and is ideally also made for
round cell measurements. This measurement is typically made with the same dispersion
as used for the cross-correlation intercept reference, but at a much higher concentration
as to achieve an intercept of approximately half as that of the dilute case. Measuring the
actual angular dependence of the turbidity rather than assuming an ideal alignment is
preferable. Please see the upcoming application note on turbid statics measurements in a
square cell for more details.
Non-linear correction: see ”How can I make measurements at high count rates?”
It is important to note that measurements are in general dependent on temperature,
sample cell geometry, and sample refractive index. For best results, we recommend taking
all references for conditions as close as possible to the measurement conditions.
42 Manual 3D LS Spectrometer

Please refer to the LS Instruments online forum for further useful information on the
LS Spectrometer and light scattering topics:
http://www.lsinstruments.ch/services/online forum

For technical support please visit:

http://www.lsinstruments.ch/support/
The customer support center allows you to review the status of your request and start new
requests by opening a new ticket.

Optionally you may send an email to [email protected]

D Health and Safety
This chapter provides important health and safety information. Before the first use of the
instrument, the user must read this chapter.

General safety issues

• use the system only under conditions defined with LS Instruments. Refer to your signed
copy of the Laboratory Requirements.

• use only accessories that meet LS Instrument’s specifications

• do not attempt to open the housing of the instrument.

• do not operate the system if any part of the instrument is damaged.

• operate the instrument only in well ventilated environments, in particular when dealing
with hazardous samples.

• do not attempt to lift or displace the instrument. Please contact LS Instruments if the LS
Spectrometer must be moved to a different location.

• USB to be connected only to delivered PC

Laser safety

The Laser included in the LS Spectrometer belongs to the laser class IIIb (Laser power
5-500 mW). The classification implies that viewing of the direct or specularly reflected beam
during short duration (<0.25 s) may cause eye damage. Diffuse reflections from these lasers are
generally not hazardous, except for intentional staring at distances close to the diffuser.
Specific control measures covered in Class IIIb lasers shall be used in areas where entry by
unauthorized personnel can be controlled. Entry into the area by personnel untrained in laser
safety may be permitted by the laser operator if instructed in applicable safety requirements prior
to entry and provided with required protective eye wear. Suggestions for appropriate eyewear
can be found at our Web page.
Follow these guidelines at all times to insure the safety of all personnel working in the laboratory:

• Never look directly into the laser beam.

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44 Manual 3D LS Spectrometer

• Never look directly into specular or mirror-like reflections of the laser beam.

• Do not wear any objects on your hands and wrists that may cause reflections, such as
watches or jewelry.

• Keep all the beams in a plane parallel to the experiment table.

• Do not bring your eye level down to the plane of the laser beam.

• Close your eyes if you bend down to pick something up off the floor.

• Use beam stops and carefully plan the placement and movement of optical elements.

• Leave the room lights ON when possible. The eye’s pupils open wide in a dark room and
present a larger target for dangerous laser light.

Placing additional optics (e.g. a polarizer or a beam splitter) must be planned carefully in order
to minimize the possibility of stray reflections.
Always close the shutter or turn off the laser when no experiment is being performed.

Safe operation at high temperature

For operation at high temperature, special refractive index-matching fluid might be required.
The user must contact LS Instruments to determine the type of fluid to be used. Prolonged
high temperature operation and repeated temperature cycling might result in the need of more
frequent servicing.
The operation of the instrument with decalin is not safe above its flashpoint of 57o C. The use
of toluene is strongly advised against, as it is a flammable liquid with a flashpoint of 6o C. See
the FAQ section of this manual for more information about the use of decalin as a refractive
index-matching fluid, and performing accurate measurements at high temperature.
Ensure that the solvents used in the sample do not evaporate or inflame at the chosen sample
temperature.

Do not touch the sample cell housing, sample goniometer or sample cap
when the temperature is set to a value higher than 60◦ C. Care should be
taken when removing the sample cell after measurements at high
temperatures.

Environmental conditions
The site where the instrument is installed must conform to the following conditions:

• indoor use only

• protection conform to IP41 according to EN60529

• low level of mechanical vibrations

• away from strong heat sources

• storage temperature range: -10o C to 50o C

• maximal operational temperature range: dew point to 90o C

 45

• humidity below 80%

• altitude up to 2000m

• well ventilated environment, in particular when dealing with hazardous samples.

Set-up requirements
A minimum surface space of 100x160cm must be reserved for the LS Spectrometer. Note that
the instrument connections are located on the electronics unit, which requires additional space.
The instrument must not be positioned closer than 450mm to a wall. Allow at least 500mm
above the instrument for access to the sample housing. The total weight of the instrument is
above 50kg, ensure that the space onto which it is installed supports this weight.
The computer can be set up next to the instrument at a maximal distance of 1.5m.
At least one power outlet must be available near (maximum 2m away) the instrument.
For service and software updates, an Internet connection is required.
E Certificates

The LS Spectrometer is conform to the CE regulations.

47
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[2] C.F. Bohren and D.R. Huffman. Absorption and Scattering of Light by small particles.
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[3] P. Lindner, editor. Neutrons, X-rays and Light: Scattering Methods Applied to Soft
Condensed Matter. Elsevier B.V., Amsterdam, first edit edition, 2002.

[4] Ian D Block and Frank Scheffold. Modulated 3D cross-correlation light scattering:
improving turbid sample characterization. The Review of scientific instruments,
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[5] Klaus Schätzel. Suppression of Multiple Scattering by Photon Cross-correlation Techniques.

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[6] C Urban and P Schurtenberger. Characterization of Turbid Colloidal Suspensions Using

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[7] HJ J Mos, C Pathmamanoharan, J K G Dhont, and C G de Kruif. Scattered light intensity

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[8] Ekkehard Overbeck, Christian Sinn, Thomas Palberg, and Klaus Schätzel. Probing
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and Surfaces A: Physicochemical and Engineering Aspects, 122(1-3):83–87, April 1997.

[9] P.N. Pusey. Suppression of multiple scattering by photon cross-correlation techniques.

Current Opinion in Colloid & Interface Science, 4(3):177–185, June 1999.

[10] Stephen W. Provencher. A constrained regularization method for inverting data represented
by linear algebraic or integral equations. Computer Physics Communications, 27(3):213–
227, September 1982.

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