Agilent Technologies 1260 Infinity II Manual de usuario

Agilent Technologies

Agilent 1260Infinity II

High Temperature

Dual Angle LSD

User Manual

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G7822-91000

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07/2017

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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...

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

Agilent 1260 Infinity II High Temperature Dual Angle LSD User Manual

Agilent Technologies

Introduction to the High

Temperature Dual Angle LSD

Introduction to the High Temperature Dual Angle LSD 6

Module Components 6

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

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

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

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

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

Static Light Scattering

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

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