Agilent Technologies 1260 Infinity II User manual

Agilent Technologies
Agilent 1260Infinity II
High Temperature
Dual Angle LSD
User Manual

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G7822-91000
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Agilent 1260 Infinity II HT Dual Angle LSD User Manual

Agilent 1260 Infinity II HT Dual Angle LSD User Manual 3
In This Guide...
In This Guide...
This manual covers the
•G7822B Agilent 1260 Infinity II High Temperature Dual Angle Light
Scattering detector
1 Introduction to the High Temperature Dual Angle LSD
This chapter gives an introduction to the HT Dual Angle Light Scattering
Detector.
2 Site Requirements and Specifications
This chapter provides information on environmental requirements, physical
and performance specifications.
4 Troubleshooting and Diagnostics
This chapter gives an overview about the troubleshooting and diagnostic features and the
different user interfaces..
5 Maintenance and Repair
This chapter provides general information on maintenance and repair of the
detector.
6 Appendix
This chapter provides safetey and other general information
3 Optimizing Performance
This chapter gives hints on how to optimize the performance or use additional
devices.

4Agilent 1260 Infinity II HT Dual Angle LSD User Manual

5
Agilent 1260 Infinity II High Temperature Dual Angle LSD User Manual
Agilent Technologies
1
Introduction to the High
Temperature Dual Angle LSD
Introduction to the High Temperature Dual Angle LSD 6
Module Components 6
7
7
How the Detector Operates
Static Light Scaterring
Dynamic Light Scattering
Physical Basis of Light Scattering
Static Light Scattering 9
Measuring Molecular Weight 9
Using Refractive Index to Measure Concentration 12
The Form Factor 13
Characteristics of Low-Angle Light Scattering 13
Characteristics of High-Angle Light Scattering 14
Basics of Dynamic Light Scattering 14
Using Rh to Determine the Diffusion Constant 15
Using the Module 16
7
8

6Agilent 1260 Infinity II HT Dual Angle LSD User Manual
Introduction to the High Temperature Dual Angle LSD
The G7822B Light Scattering Detector is a static light-scattering system with
detectors positioned at both 15º and 90º to the incident laser beam. The detector is
designed to be installed inside the temperature chamber of the Agilent High-
Temperature GPC system. Alternatively, it can be installed inside a custom
temperature chamber. This configuration is used to determine the molecular weight,
radius of gyration and other parameters for a wide range of molecular sizes, typically
from 100 to 10,000,000 daltons and radii from 10 nm to 150 nm.
Installation of the detector within the detector chamber of the GPC system allows for
the determination of the light scattering at the elevated temperatures that are
commonly used to effect the separation. Locating the flow cell inside a constant
temperature oven can provide additional accuracy and precision to the
measurements than use of a stand alone detector. An additional benefit is that the
distance between the end of the column and the flow cell can be minimized, thus
reducing post column band broadening effects.
Module Components
The G7822B Light Scattering Detector consists of three components:
1. Laser/Flow Cell/Detector Module, which includes the flow cell, laser and
photodiode. This module is installed inside the column oven
2. PD Connect Module, which includes the analog to digital converter and is
interfaced to the personal computer.
3. Laser Control Module.
1Introduction to the Hight Temperature Dual Angle LSD
Introduction to the High Temperature Dual Angle LSD

Introduction to the High Temperature Dual Angle LSD
Light Scattering refers to the process in which light from an incident polarized
laser beam is scattered in all directions when it interacts with a molecule or
particle. Light scattering is an everyday occurrence and was first described by
Lord Rayleigh in the late 1800’s. An example of light scattering is the scattering
of sunlight by particles in the atmosphere: the sky is blue because shorter visible
wavelength radiation (blue light) is scattered more strongly by the gas molecules
in air than light of longer wavelengths (red light). There are two general
techniques for the measurement of physical properties of polymers (synthetic
polymers and natural polymers such as proteins and polysaccharides), virons,
liposomes and particles.
How the Detector Operates
Agilent 1260 Infinity II HT Dual Angle LSD User Manual 7
Static Light Scattering
The intensity of the scattered light from the sample dissolved in the solvent. This
difference between the two measurements can be used to determine the average
molecular weight and Rg (radius of gyration). To complete the molecular weight
determination, the concentration of the compound of interest is required
(typically obtained from a refractive index detector or an absorbance detector).
Static Light Scattering is described in section 5.1.2.
The fluctuations of the intensity of the scattered light is used to determine the
diffusion coefficient of the molecules as they move in solution (Brownian motion).
The Stokes-Einstein equation is used to determine Rh (the hydrodynamic radius of
the molecule). Dynamic Light Scattering is described in section 0.
Dynamic Light Scattering
1
How the Detector Operates

Introduction to the High Temperature Dual Angle LSD
Light consists of perpendicular electric and magnetic fields that oscillate in a
direction that is perpendicular to the direction of propagation of the
light as shown in Figure2. When light strikes a molecule, the electrons
will experience a force due to the electric field and will move slightly.
This movement will induce an oscillating dipole moment that will radiate
light in all directions at the oscillating frequency. This radiated light is the
scattered light that is detected and processed as described below.
Static and Dynamic measurements can be made on a sample in a cuvette or in
a flowing stream such as that found in high performance liquid
chromatography (HPLC), size exclusion chromatography (SEC) or gel
permeation chromatography (GPC). In flowing streams, measurements are
made on each elution slice using a unique light scattering design and very fast
digital signal processors (which are essentially special purpose computers).
Figure 2: Light scattered by a molecule
Physical Basis of Light Scattering
Figure 2 describes the spatial arrangement of the incident light and scattered light
in the light scattering experiment. The light is polarized in the vertical direction,
thus the electric field will oscillate in the Z direction and the magnetic field will
oscillate in the X direction.
A diode laser, which is a monochromatic source of light that can be focused to a
very small point in the centre of the sample cell, is used in the light scattering
system. Typical sources include a semiconductor laser that can provide 20 to
30mW at 685nm and another, which provides 100 to 150mW at 800nm. The
scattered light is collected at a given angle and orientation (e.g. 15° or 90° ) from
the incident radiation, and is used to deduce the desired molecular properties.
8Agilent 1260 Infinity II HT Dual Angle LSD User Manual
How the Detector Operates
1

The electric dipole moment that is induced is shown in equation 1.
where: p is the dipole moment
A is the polarizability
E is the electric field
The polarizability can be related to measurable parameters via equation 2.
Static Light Scattering
Measuring Molecular Weight
where: Mw is the dipole moment
NA is the Avogadro Number (6.02 x 1023 molecules/mol)
dn/dc is the change in the index of refraction as a function of the
change in concentration. It is considered to be a constant for any
specified solvent-solute pair under constant operating conditions
The oscillating dipole will radiate light in all directions at the oscillating
frequency. This is the origin of scattered light. If a single molecule has
dimensions that are small with respect to the wavelength of the incident
light, the intensity of the light can be defined by equation 3.
Agilent 1260 Infinity II HT Dual Angle LSD User Manual 9
Introduction to the High Temperature Dual Angle LSD
1
Static Light Scattering

10
where: Is is the intensity of the radiated light ls
I0 is the intensity of the incident light
λ0 is the wavelength of light in a vacuum
Φ and R are as defined in Figure 2
If we collect light from a volume V of a solution with a concentration c
(gm/ml), the intensity of scattered light can be found by multiplying
equation 3 by the number of molecules in the volume V.
The number of molecules can be expressed by
Now if we solve for W M , we obtain equation 4
Collecting all the constants and instrumental parameters into an overall
instrumental constant, A, we obtain equation 5.
Equation 5 can be used to measure the molecular weight Mw of small
molecules at any scattering angle Θ . It should be noted, however, that
larger molecules scatter less light at high values of Θ than at low
angles because of interference effects caused by the fact that light
scattered from one part of the molecule travels a different distance
from another part of the molecule, and is not in phase with light
scattered. This phenomenon can be quantified by defining the light
scattering form factor (equation 6). A more detailed discussion of the
form factor is presented in The Form Factor sub section.
It should be noted that P(Θ) can be written as a series as shown in equation 7.
Agilent 1260 Infinity II HT Dual Angle LSD User Manual
1Introduction to the High Temperature Dual Angle LSD
Static Light Scattering

11 Agilent 1260 Infinity II High Temperature Dual Angle LSD User Manual
where: q = 4 π .n.sin(Θ/2)/λλ0
Rg is the radius of gyration of the molecule
n is the index of refraction of the liquid
λ0 is the wavelength of light in a vacuum
For scattering at 15° and 90°, equation 7 can be expressed as
equations 8 and 9, respectively.
We present a table of P values for 15 and 90 degrees as a function of
molecular weight (for molecules with molecular weight from 104 to
108 Daltons) in Table 1. These values assume that the molecules are
random coils.
Table 1 V Value of P(90˚) and P(15˚) for λ0 = 685nm, n = 1.5
Mw Rg (approx) nm P (15°)
5 x 1032.3
7
P (90°)
1.0000
5 x 104
0.9993
0.9998
0.9993
0.9976
0.9993
0.9780
*1
5 x 105
5 x 106
23
70
5 x 1070.7622
*1
230
*1 values depend on shape
1
Static Light Scattering
Introduction to the High Temperature Dual Angle LSD

12
If the value of P(Θ) found in equation 7 is below 0.7, higher order
components become important. In this case, depends on Rg and
also on the shape of the molecule as shown in figure 1
Figure 1: Values of P-1(Θ)as a function of q2<Rg2> for various particle shapes:
1 = Sphere, 2 = Gaussian Coils, 3 = Rods
It is clear that all three molecular shapes yield the same value of
P-1(Θ) when q2<Rg2> is less than approximately 1. As the value of
P-1(Θ) increases, the shape of the molecule clearly influences the light
scattering intensity.
Using Refractive Index to Measure Concentration
Refractive index measurements can be used in conjunction with
static light scattering measurements to determine the concentration
of the compound in the chromatographic slice.
The difference in refractive index between the solution under study
and the pure solvent is measured by passing a light beam through two
cells. One of the cells contains pure solvent and the other contains
the eluent as it passes through the cell after the column. The cell with
the pure solvent should be filled with the solvent prior to the
separation and should be at the same temperature as the eluent.
The observed signal (RI), which corresponds to the deviation of the
light beam, is proportional to the difference in the refractive index of
the fluid in the two cells as shown in equation 10
Agilent 1260 Infinity II HT Dual Angle LSD User Manual
1Introduction to the High Temperature Dual Angle LSD
Static Light Scattering

13 Agilent 1200 Infinity II Series RID User Manual
where: B is an instrumental constant
c is the concentration of the sample in the solvent
dn/ dc is the change in the index of refraction of the solution
as a function ofconcentration. It is considered as a constant
for any specified solvent-solute pair under constant operating
conditions
Characteristics of Low-Angle (15°) Light Scattering
The form factor
The form factor at a particular angle is the ratio of the signal at that
angle when compared to the signal expected at the theoretical angle
of 0° (where there is not form factor) as indicated by equation 6. The
importance of the form factor is that small molecules (e.g. those
which have a radius that is < 10nm, which is small when compared to
the wavelength of the incident light) are studied, generate comparable
signals at all angles, while large molecules generate signals that are
smaller at higher angles and larger at small angles.
Low-angle light scattering data is collected at a 15° angle to the
incident beam and is typically used for determination of the molecular
weights of large molecules. This measurement angle is especially
useful for the study of proteins with molecular weight of
greater than 1,000,000 Daltons and for random coils with molecular
weight between 200,000 Daltons and about 10,000,000 Daltons.
In addition, 15° data is also used with 90° data to measure Rg, the
radius of gyration of molecules over a limited range of sizes
(12-150nm) using static light scattering analysis.
1
Static Light Scattering
Introduction to the High Temperature Dual Angle LSD

14 Agilent 1200 Infinity II Series RID User Manual
Characteristics of High-Angle (90°) Light Scattering
High-angle light scattering data is collected at a 90° angle to the
incident beam and is typically used with static light scattering analysis
to measure the molecular weight of smaller molecules such as proteins
with a molecular weight below 1,000,000 Daltons, random coils with
molecular weight below 200,000 Daltons and for lower molecular
weight non-spherical coil polymers. In addition, it is used with
dynamic light scattering analysis to measure Rh, the hydrodynamic
radius of molecules and particles from 1 to 1,000nm.
Basics of Dynamic Light Scattering
The fundamental measurement for dynamic light scattering is the
fluctuation of the intensity of the scattered light. This data is analyzed
as described to yield the diffusion of the molecules or particles moving
under Brownian movement.
When polarized laser light is scattered, the scattered light is at the
same wavelength as the incident beam. However, it should be noted
that when the light is monitored over extremely short time increments
(in the microsecond time regime), a Doppler shift occurs and the
frequency of the light appears to be changing. As the particle moves
toward the detector, the frequency increases, and as it moves away the
frequency decreases. The amount of the increase (decrease) of the
change in the frequency is related to the diffusion rate of the scattering
molecule in the solvent.
While the diffusion coefficient could be obtained from the frequency
spectrum, it is easier to measure the small intensity fluctuations and
then compute the autocorrelation function. The autocorrelation
function is related to the frequency spectrum by the Fourier transform
(i.e. the data processing is performed in the time domain rather than
the frequency domain).
1Introduction to the High Temperature Dual Angle LSD
Static Light Scattering

15 Agilent 1200 Infinity II Series RID User Manual
From an experimental perspective, the intensity of the scattered light
is detected by counting the photons scattered via an avalanche
photodiode, an electronic device that emits a pulse every time a photon
passes through its detector. This high sensitivity detector sees a time
varying signal rather than a relatively constant (in batch mode) or
slowly varying “total intensity“ signal. It should be noted that even
when measurements are taken in flow mode (SEC or GPC), the sample
can be considered as slowly varying. This is due to the time frame of
the scattered light measurement, relative to the rate of elution of the
light scattering molecules from the column.
The photon counting measurement is performed over a very short
period of time to record the very rapid diffusion that is taking place.
Small particles or biomolecules diffuse quickly with the scattered light
showing rapid small fluctuations while larger particles such as protein
aggregates, nanoparticles, polymers, etc., diffuse more slowly, resulting
in lower frequency fluctuations
Using Rh to Determine the Diffusion Constant
D, the diffusion constant of the compound of interest can be
determined using the Stokes-Einstein equation.
where: k is Boltzman’s constant
T is the temperature of the eluent
v is the viscosity of the sample
D is the Diffusion coefficient calculated from the
autocorrelation function
Before running solvent through the device make sure that the solvent
within the detector is completely miscible with the intended solvent
to be used. If this is not the case, the flow cell must be rinsed through
with an intermediate solvent (e.g. Acetone), which is miscible with
both solvents.
1
Static Light Scattering
Introduction to the High Temperature Dual Angle LSD

The differential pressure transducer monitors the pressure drop across the bridge,
ΔP, and the inlet pressure transducer measures the pressure drop through the
bridge, Pi. When the sample is introduced across capillary 4 (C4) of the bridge, an
imbalance is caused due to the hold-up columns. This change in pressure, ΔPis
proportional to the specific viscosity, η sp, according to the following equation.
Where ΔPis the differential pressure across the bridge and
Pi
,is the pressure of the
flow through the capillary bridge. The instrument makes a true differential
measurement directly, making it very sensitive to dilute solution viscosity
measurements.
Using the Module
The fiber optic cables must be securely in position in both the Detector heads and the
Laser Control Module before the power is turned on.
The power on switch for the Laser Control Module is on the rear panel and
the power on switch for the PD Connect Module is on the front panel. The
green light on the face panel of each module should illuminate when the
power is turned on.
Allow the system to warm up and equilibrate for a short period of time (ca 30
minutes) and monitor the signal, which should be close to zero. If necessary,
you can reposition the signal to zero using the potentiometers on the top of
the rear panel of the Laser Control Module.
Do not remove the cables while the system is powered up. This can cause irreversible
damage to the detector.
CAUTION
CAUTION
Agilent 1260 Infinity II HT Dual Angle LSD User Manual
16
1Introduction to the High Temperature Dual Angle LSD
Using the Module

17
Agilent Technologies
Agilent 1260 High Temperature Dual Angle LSD User Manual
2
Site Requirements and Specifications
Site Requirements 18
Physical Specifications 21
Performance Specifications 22
Module Overview 23
This chapter provides information on environmental requirements, physical and
performance specifications.

18
2Site Requirements and Specifications
Site Requirements
Site Requirements
A suitable environment is important to ensure optimal performance of the
instrument.
Power Considerations
The module power supply has wide ranging capability. It accepts any
line voltage in the range described in Table 1. Consequently there is no
voltage selector in the rear of the module. There are also no externally
accessible fuses, because automatic electronic fuses are implemented in
the power supply.
WARNING
Hazard of electrical shock or damage of your instrumentation
can result, if the devices are connected to a line voltage higher than specified.
➔Connect your instrument to the specified line voltage only.
WARNING
The module is partially energized when switched off, as long as the power cord is
plugged in.
Repair work at the module can lead to personal injuries, e.g. electrical shock, when
the cover is opened and the module is connected to power.
➔Always unplug the power cable before opening the cover.
➔Do not connect the power cable to the instrument while the covers are removed.
WARNING
Inaccessible power plug.
In case of emergency it must be possible to disconnect the instrument from the
power line at any time.
➔Make sure the power connector of the instrument can be easily reached and
unplugged.
➔Provide sufficient space behind the power socket of the instrument to unplug the
cable.
Agilent 1260 Infinity II HT Dual Angle LSD User Manual

Site Requirements and Specifications 2
Site Requirements
Power Cords
Country- specific power cords are available for the module. The female end
of all power cords is identical. It plugs into the power- input socket at the
rear. The male end of each power cord is different and designed to match
the wall socket of a particular country or region.
Agilent makes sure that your instrument is shipped with the power cord
that is suitable for your particular country or region.
WARNING
Absence of ground connection
The absence of ground connection can lead to electric shock or short circuit.
➔Never operate your instrumentation from a power outlet that has no ground
connection.
WARNING
Unintended use of supplied power cords
Using power cords for unintended purposes can lead to personal injury or damage of
electronic equipment.
➔Never use a power cord other than the one that Agilent shipped with this
instrument.
➔Never use the power cords that Agilent Technologies supplies with this instrument
for any other equipment.
➔Never use cables other than the ones supplied by Agilent Technologies to ensure
proper functionality and compliance with safety or EMC regulations.
WARNING
Power cords
Solvents may damage electrical cables.
➔Prevent electrical cables from getting in contact with solvents.
➔Exchange electrical cables after contact with solvents.
Agilent 1260 Infinity II HT Dual Angle LSD User Manual 19

20
2Site Requirements and Specifications
Site Requirements
Bench Space
The detector should be placed in a position so that the distance between the end of
the column and the flow cell of the detector is minimized. This will reduce post column
band broadening effects and optimize chromatographic resolution. Typically, the
Laser/Flow Cell/Detector Module is installed within the oven of the 1260 Infinity II
High Temperature GPC system.
The Control electronics modules at the back of the back of the 1260 Infinity II High
Temperature GPC system placed in an area that is free from drafts and/or significant
temperature changes. Avoid placing the modules near air conditioning vents, windows
ovens, etc.
The system should be placed on a sturdy bench top that can support the units, and
provides access to all components.
Environment
CAUTION
Your module will work within the specifications at ambient temperatures and
relative humidity described in Table 2.
ASTM drift tests require a temperature change below 2 °C/hour
(3.6 F/hour) over one hour period. Our published drift specification (refer also to
“Performance Specifications” on page 32) is based on these conditions. Larger
ambient temperature changes will result in larger drift.
Better drift performance depends on better control of the temperature fluctuations.
To realize the highest performance, minimize the frequency and the amplitude of
the temperature changes to below 1 °C/hour
(1.8 F/hour). Turbulences around one minute or less can be ignored.
Condensation within the module
Condensation can damage the system electronics.
➔Do not store, ship or use your module under conditions where temperature
fluctuations could cause condensation within the module.
➔If your module was shipped in cold weather, leave it in its box and allow it to warm
slowly to room temperature to avoid condensation.
NOTE
This module is designed to operate in a typical electromagnetic environment, i.e. where RF
transmitters such as mobile telephones may not be used in close proximity.
Agilent 1260 Infinity II HT Dual Angle LSD User Manual