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