DynaPro-MSXTC

Operation:
An Overview

A step by step guide to operating the DynaPro

MSXTC

Assumes the operator is familiar with the Start-up Guide,

Operator’s Manual, and Data Interpretation Guide.

Nov. 12, 2004

中国科学技术大学生命科学学院
 Outline of Operation
 Introduction
 Power on DynaPro and launch Dynamics software
 Open the preset file Or Select and modify the experimental parameters
(solvent, temperature)
 Load the sample into the cuvette
 Place cuvette into the microsampler
 Wait 5 minutes for equilibration to temperature
 Launch event scheduler or Acquire data manually
 Monitor datalog for indications of acceptable data
 Monitor trace view for preliminary indications of homogeneity and
heterogeneity
 Upon completion of data acquisition, review the ‘goodness’ of data, and take
appropriate action.
 If the data is ‘good’, accept and save the data into a file
 Preliminary analysis of size distributions
 If the data is ‘not good’, or if acquisition is interrupted, troubleshoot the
problem
 When complete, remove the cuvette and recover sample
 Clean the cuvette for next sample or for storage
 Load the next sample into the cuvette
 Decide if include next sample中国科学技术大学生命科学学院
in same data file or if start new data file
Introduction
 The purpose of the DynaPro Operation Overview is to provide
step-by-step guidance for obtaining ‘good’ data.

 The manual assumes the reader is familiar with the DynaPro

Start-up Guide (e.g. the DynaPro is already installed in your
laboratory), the Operator’s Manual (has been reviewed at least
one time and have attempted to collect data); and familiar with
the Data Interpretation Guide (e.g. some attempt to understand
size distributions and their interpretation, and some qualification
of the raw data).

 The manual will not cover the basic theories of light scattering,
however it will provide you with references which do cover the
theories in detail.

中国科学技术大学生命科学学院
 System Block Diagram
Electrical

Analog/
Digital
Correlator
I/O DynaPro MSXTC
I/O

Detector
TTL
USB
Optical fibers
Port
Laser

DynaPro Host

USB RS232
Cable Serial
Personal Computer

中国科学技术大学生命科学学院
Set-up

 Refer to the start-up guide provided with your

DynaPro.
 Remove Host Unit and Plate Reader Optics from
Box
 Connect optical fibers
 Connect Power cords, USB Cable, and RS232
Cable to the back of the Plate Reader
 Install Dynamics Software CD and Floppy Disk
 Power-on DynaPro
 Start software
中国科学技术大学生命科学学院
Power on and Launch
Dynamics 1. Turn power on to both
components

2. Double click the

Dynamics V6 Icon

Dynamics V6 .lnk

中国科学技术大学生命科学学院
Open Preset File or Edit
Parameters
1. Open a Preset file (File,
Open Preset) that contains
previously defined
experiment parameters and
software settings.

2. Alternatively, begin a new

experiment file (File, New) and
manually select and edit the
parameters (particularly the
Solvent)

中国科学技术大学生命科学学院
Load Sample into Cuvette

Proterion recommends:
2. first load the sample unfiltered, with a pipettor or syringe needle; at
minimum load 12 microliters into the 12 microliter cuvette. Place
the tip onto the bottom of the cuvette and then slowly inject into the
sample chamber.
3. If the data indicate aggregation or presence of large particles, it
may be necessary to spin the sample (10,000 rpm for 20-30 minutes

中国科学技术大学生命科学学院
Insert Cuvette into
Microsampler

 With the frosty side of

the cuvette to the left,
insert the cuvette into
the optical chamber.
 Press the cuvette
down until it no longer
moves.
 Then close the lid of
the DynaPro.

中国科学技术大学生命科学学院
Wait Five Minutes for
equilibration
 Proterion recommends
waiting approximately
five minutes for the
sample to reach the
temperature of the
optical chamber,
particularly when the
chamber is temperature
controlled.
 While waiting, make sure
the count rates are more
than the pure solvent
count rate by examining
the count rate monitor,
as shown to the left.

中国科学技术大学生命科学学院
Measure Manually

 When the cuvette is inserted press the green“Go”

button to acquire data.
 If not green, you must Connect the software to the
DynaPro (refer to start up guide or manual)
 After approximately 100 seconds of data have
been acquired, press the “Stop” Button – the same
button, which is now the color red.
中国科学技术大学生命科学学院
Measure Automatically
 The Event Scheduler is
opened by placing the cursor
on the ‘Tree’ (e.g.
Measurements), right clicking,
and choosing Event
Scheduler.

 The DynaPro can also be operated in an automatic fashion

with the Event Scheduler (access by right clicking mouse on
‘measurement’ tree, left hand side). The Event Scheduler can
perform time delays, acquire data, save data, set temperatures
and more. 中国科学技术大学生命科学学院
Save the Data
 Before proceeding, Proterion recommends saving the data to avoid
potential data loss due to uncontrollable events such as power
failure, PC malfunction, etc.
 Press File, Save or the ‘save file’ icon, and insert the name of the
file.

中国科学技术大学生命科学学院
Good or Bad: Judging the Goodness
of the Data
 The DynaPro software, Dynamics, does
provide basic data analyses that
indicate if the data are in ‘acceptable’
ranges. The analyses are based upon
simple numerical data filters or
qualifiers. Yet these data filters do not
always capture or allow for good and
bad raw data.

 We will explain what is good or bad by

commenting on various examples of
raw data. The name applied to the raw
data is an autocorrelation function. An
autocorrelation function is a collection
of correlation coefficients – unitless
values indicating the level of similarity
among sets of data. At this time we will
not concern ourselves with the
underlying theory and physical meaning
of the autocorrelation function. We will
examine only how to interpret these
functions.
中国科学技术大学生命科学学院
 The Autocorrelation Function
The DynaPro determines the size of particles in
solution by exploiting the physical process of
Brownian Motion: the particles are moving in solution
as a function of time, and their rate of motion is
related to their size. The rate of motion is measured
by illuminating the particles with laser light and
determining the rate at which light scattered or
reflected by the particles changes with time.

The technique of autocorrelation determines the rate

of these time intensity fluctuations, expressed as an
autocorrelation function (shown here).

An autocorrelation function is an exponential function

comprised of correlation coefficients (y-axis)
dependent upon the ‘delay time’ (x-axis), the time-
value separating the sets of data. The function can be
mathematically described by one or more decays. The
rate of decay is related to particle size. A faster decay
indicates a smaller particle, a slower decay a larger
particle.
Autocorrelation functions are determined during each
acquisition comprising a measurement, as described
earlier.
Numerical algorithms are applied to determine the rates of
decay or size distributions of the exponential autocorrelation
functions. The DynaPro utilizes a proprietary ‘non-negative
least squares’ algorithm, a method that finds the size
distribution producing the smoothest distribution with the least
amount of error. The error is the difference between the
measured autocorrelation function and the fitted autocorrelation
function.

中国科学技术大学生命科学学院
Evaluate Autocorrelation
Function
Proceed Caution Stop

Spin or filter sample

Increase acquisition time

Increase acquisition time If not pure solvent…

Increase acquisitions Increase acquisition time
Increase laser power Increase acquisitions
Increase concentration Increase laser power
Increase concentration
中国科学技术大学生命科学学院
 Take Action
Proceed
If the Autocorrelation function
is in the Proceed Category,
continue with the interpretation
of the size distribution analysis,
which follows next.
Caution
Before proceeding, attempt to
improve the quality of the data
by following all or some of the
recommended changes to the
experiment. Leave the sample
in the cuvette and follow these
steps. Increase acquisition time
(double the current value as a
rule of thumb); and/or increase
the # of acquisitions (double);
Increase laser power (to
maximum value of 100%); if
none of the above steps lead to
functions shown in ‘Proceed’, it
may be necessary to increase
the concentration of the analyte.
Ultimately, one may accept the
imperfect data from this
category and continue.
中国科学技术大学生命科学学院
Stop
Do not proceed with data
interpretation if the
autocorrelation function appears
as shown. If the data appear as
in the top figure, the sample
probably contains large
particles, and should be spun
(10,000 rpm for 20-30 minutes)
or filtered using syringe filters
(.1 micron). If the data appear
as shown in the lower figure,
make sure the cuvette is inserted
properly, the lid is closed, and
that the sample is not pure
solvent. If all of these items
check, follow the
recommendations under
“Caution”. If these steps fail,
contact technical support.
Physical Interpretations of Size
Distributions
Monomodal Monomodal Bimodal Bimodal
Monodisperse Polydisperse
Radius = R1
Radius = 1.5*R1 The samples contains two
types of particles, monomer
and trimer. The radius of the
trimer is less than twice the
radius of monomer so only
one peak is resolved, the
distribution is monomodal.
However the population
consists of two species and
Radius = 5*R1 this increase in size
heterogeneity causes an
increase in measured
polydispersity compared to the
samples containing 100%
monomer and 100% trimer.
Also, the mean radius of the
peak will be larger than R1
but smaller than 1.5 R1.
中国科学技术大学生命科学学院
Monodisperse
The sample contains two types
of particles, the monomer and
a large aggregate. The large
particle is more than twice the
radius of the monomer and in
sufficient quantities to be
measured, so two peaks are
resolved by the DynaPro. The
mean radius of Peak 1 will be
R1 and Peak 2 will be equal to
5*R1. Both species are
homogeneous so measured
polydispersity is low.
Monodisperse
Polydisperse
The sample contains three types
of particles monomer, trimer,
and larger aggregate. In this
case the DynaPro resolves only
two peaks. The monomer and
trimer are not resolved from
each other and form only one
peak, a polydisperse peak. The
second peak is formed by the
larger particle, which is
resolvable from both monomer
and trimer. The second peak is
monodisperse.
Hydrodynamic Radius: The Physical
Interpretation of ‘Size’
The DynaPro measures the size
distribution of the particles in the
sample. The size, previously
defined as the radius or diameter of
the particle, is represented in this
figure as Rh. Rh, or
Hydrodynamic Radius, is a particle
radius that embodies a ‘hard
sphere’ particle which is in fact
aspherical and typically
surrounded or covered by solvent.
Please refer to PSI Books or the
article for a more thorough
explanation of the physical
interpretation of particle size as
measured by the DynaPro.

中国科学技术大学生命科学学院
Size Distribution Results
Results are shown in
graphical as well as
tabular form. The table
located below the size
distribution histogram
describes the number of
peaks and their mean
value (Radius),
polydispersity (Pd), %
polydispersity (%Pd),
molecular weight
estimated from the
measured radius (MW-
R), relative amount of
light scattered by each
population (%Int), and
estimated relative amount
of mass (concentration)
of each peak or species
(%Mass).

中国科学技术大学生命科学学院
 Monomodal Size Distribution
Histogram
Y-axis
Relative amount of
This histogram has one peak so we
call it a monomodal size distribution.
light scattered by The peak is defined by the mean value
each bin, % and polydispersity.
Intensity (% of
Total Light
Scattered).
Represents the
probability of
existence of the The ‘width’ of the peak is the standard
species.
deviation of the weighted bin values, also
known as the Polydispersity.

The mean value of the peak is defined

X-axis
Discrete particle sizes,
by a weighted average of the number of
in nanometers
bins comprising the histogram, in this
case three. The bins by themselves do
not represent real, distinct, physical
particles however their mean and
standard deviation do.
中国科学技术大学生命科学学院
 Multimodal Size Distribution
What causes Modality?
The presence of different and resolvable
species in the sample cause modes in the
size distribution. To be resolved as a This histogram has more than one
separate peak, a species must have a size peak so we call it a multimodal
(radius) larger than another species by a
size distribution. Specifically this
factor of two or more, and be detectable
(produce sufficient scattered light for histogram is trimodal. The
detection by the DynaPro). Roughly DynaPro determined three distinct
speaking a factor of two in radius is populations exist in this sample.
equivalent to a factor of eight (octamer) in
MW. When the sizes of the species are
below this factor, a separate peak will not
be resolved for each species.

By definition, a multimodal size distribution is heterogeneous: the sample contains

distinct populations of particles that are not the same size. The DynaPro can
resolve up to four or five modes in a size distribution. For each mode, the DynaPro
estimates the relative amount of light scattered and the relative amount of mass
based upon one of several possible particle scattering properties. Often times the
relative amount of mass of a peak is quite small e.g. less than .1 % and is
considered to be negligible.

中国科学技术大学生命科学学院
 Polydispersity
Each peak has a unique mean value
and width or Polydispersity. It is
Polydispersity refers to the level of homogeneity useful to normalize Polydispersity to
of the sizes of the particles. When the level of
homogeneity is high, the particles can be
the mean size of the peak, also
considered to be virtually identical in their size, or known as percent polydispersity.
monodisperse. The level of homogeneity is
considered high when the percent polydispersity is
less than 15%. When the level of homogeneity is
low (percent polydispersity greater than 30%), the
particle population can be considered to contain
significantly different sizes, or ‘polydisperse’.

What causes Polydispersity? Heterogeneity is caused by the presence of different species that can
not be resolved by the technique of dynamic light scattering (species with sizes less than a factor of
two relative to other species exist in solution can not be resolved). A peak containing 100%
monomer will have a smaller polydispersity than peak containing a mixture of monomer:octamer.
The peaks shown here all have % Polydispersity greater than 30%.
Note: refer to appendix for an alternate cause of polydispersity.

中国科学技术大学生命科学学院
Size Distribution Interpretations
Monomodal Monomodal Multimodal
Monodisperse ‘Polydisperse’ Polydisperse

BLGA, 4 mg/ml, PBS, T = 25 C BLGA, 4 mg/ml, PBS, T = 5 C BSA, 2 mg/ml, PBS, T = 25 C

Peaks: 1 Peaks: 1 Peaks: 2
Mean Radius: 2.8 nm Mean Radius: 3.4 nm Peak 1:
% Poly: 13.8 % % Poly: 22.1 % • Mean Radius: 4.3 nm
Majority monomer Increasing amounts of Dimer • % Poly: 32.1 %
• Monomer, Dimer, Trimer

Peak 2:
• Mean Radius: 130.9
• % Poly: 34.5 %
• Various non-specific aggregates

中国科学技术大学生命科学学院
Manage and Analyze Data
 Dynamics V6, the state-of-the-
art DynaPro application
software, provides several data
management and analysis
tools specifically designed for
large amounts of data.

 The Trace View is an X-Y based graphical system, with

customer selectable X-axis and Y-axis parameters (left and
right hand axes, multiple parameters), for data analysis and
presentations. The View is easily exported to other MS
applications for professional reports and presentations.

 The Datalog View is a customer configurable Table designed

to support database management tools available in Excel or
中国科学技术大学生命科学学院
 Trace View
 Select the Trace View to Present large
quantities of data in graphical form, as line or
symbols, with customer selectable X-axis and Y-
axis parameters.

 The
control
 Add panel
data to provides
the ‘file’, lists of
create a variables
databas for X, Y
e for axes as
your well as
protein scaling,
or zooming,
polymer, and
at any legend
time. control

中国科学技术大学生命科学学院
Datalog
 Select the Datalog View to access customer
selected and designed parameters, and export
to database management and analysis
programs.

中国科学技术大学生命科学学院
Excel
 Copy and paste from the Datalog View into Excel. With the
Excel “AutoFilter” and other data analysis tools sort,
organize, search, graph, manipulate the data. The data
shown below have been sorted by Radius, increasing
Item Time (s) Temp (C) Intensity (Cnt/s)
value. R (nm) %Pd MW-R (kDa)
Amp Baseline SOS
C1 822 24.9 206606 4.3 22.8 100 0.383 1 1.633
E9 1731.2 25 195739 4.3 24.6 104 0.345 1 1.907
L13 4554.2 25.2 214885 4.3 31.2 101 0.353 1 2.081
D9 1340 25.1 196897 4.4 25.9 110 0.355 1 2.939
G5 2450 24.9 201713 4.4 26 106 0.311 1 3.591
I13 3362.1 24.9 225963 4.4 37.7 111 0.378 1 4.038
N22 5495.1 25.2 161876 4.5 13.9 112 0.349 1 3.297
F6 2075.2 24.8 161549 4.5 22.5 113 0.291 1 2.71
K22 4304 25 166371 4.5 22.6 112 0.34 1 6.024
M14 4969 24.9 177285 4.5 23.1 116 0.312 1 5.31
D1 1211 25 234376 4.5 25.9 112 0.35 1 3.582
M13 4952.3 24.9 224175 4.5 27.9 113 0.336 1 3.863
D6 1292.2 25 161660 4.5 29.5 113 0.376 1 3.75
H6 2858.1 25.2 161148 4.5 30.4 113 0.392 1 5.349
D5 1276.1 25 216812 4.5 31.3 115 0.346 1 3.323
G9 2515.3 25 200602 4.5 35.7 114 0.36 1 3.721
L17 4620.2 25.1 233250 4.5 37.4 114 0.3 1.001 6.082

中国科学技术大学生命科学学院
Remove cuvette,
recover sample

Open lid and extract


cuvette.
 Insert the pipettor tip or
syringe needle into the
cuvette and recover the
sample, if required.
 Then rinse thoroughly
with water and dry with
air or nitrogen.
 Insert next sample and
continue.
 If finished for the day,
Proterion recommends
placing the cuvette into a
detergent solution
overnight.
中国科学技术大学生命科学学院
Repeat and add data to
file
 If continuing with
experiments, it is possible
to continue to add data to
the file.
 If currently connected,
insert cuvette and press
the green button.
 If not connected, perhaps
because it is the next day,
press the connect button
and then proceed.

中国科学技术大学生命科学学院
Ending

 If not continuing with

experiments,
 save the data

 Print and summarize

results
 and then exit the

software (File Exit).

 Power down the

DynaPro

中国科学技术大学生命科学学院