Light Scattering
Light scattering in different forms has been used to sense the presence of high molecular weight materials eluted from a liquid chromatographic column. There are three forms of light scattering detectors that have been reported, evaporative light scattering, low angle light scattering (LALLS) and multiple angle light scattering (MALLS). The evaporative light scattering detector is, in fact, a type of transport detector, where the transport medium is a gas. Its successful use depends on the solute to be detected being completely involatile. The mobile phase leaving the column is atomized, the dust suspension so formed, is exposed to a strong beam of light and the scattered light at right angles to the incident beam is sensed by a photo cell. The light dispersion is largely due to Raleigh scattering and, thus, the output from the photo cell is proportional to the mass of solute present. Light is also scattered by large molecular weigh materials such as polymers, and both (LALLS) and (MALLS). employ a laser light source in their sensor system. In both devices the light dispersion is largely due to Raleigh scattering and thus, the intensity of the scattered light at a low angles will give a value for the molecular weight of the solute. However, it is usual to calibrate the sensor using a polymer of known molecular weight. In multi angle light scattering, the light scattered at three different angles (at least three) is sensed and from the three scattered light intensity values both molecular weight and the rms molecular radius of the solute can be assessed. The various constants used in the equations for (MALLS) are also usually determined by prior calibration.
Author: RPW Scott
Book:Liquid Chromatography Detectors
Section:HPLC-Detectors Liquid-Light-Scattering
Liquid Light Scattering Detectors
Light
scattering detectors differ from evaporative light scattering detectors in that
they respond to the light scattered by a polymer or large molecular weight
substance present in the column eluent itself. The scattering is measured as it
passes through an appropriate sensor cell while illuminated by a high intensity
beam of light. The high intensity light source is achieved by the use of a laser
(light amplification by the stimulated emission of radiation) that also
generates the light at the appropriate wavelength for measurement. There are
two forms of the detector: the low angle laser light scattering (LALLS)
detector and the multiple angle laser light scattering (MALLS) detector.
Both devices are commonly used but the multiple angle laser light scattering
detector is more versatile as it provides molecular dimensions as well as the
molecular weight of the eluted solute.
HPLC-Detectors Liquid-Light-Scattering
Author: RPW Scott
Book:Liquid Chromatography Detectors
Section:HPLC-Detectors Evaporative-Light-Scattering
The Evaporative Light Scattering Detector
The
evaporative light scattering detector, as its name implies,
utilizes a spray that continuously atomizes the column eluent into
small droplets. These droplets are allowed to evaporate, leaving the solutes as
fine particulate matter suspended in the atomizing gas.
Courtesy of Polymer Laboratories Inc.
Figure 48.
A Diagram of the Evaporative Light Scattering Detector
The atomizing
gas can be air or, if necessary, an inert gas. The suspended particles pass
through a light beam and the scattered light viewed at 45o to the incident
light beam by means of a pair of optical fibers. The scattered light
transmitted through the fibers is sensed by a photomultiplier and the output
electronically processed and passed either to a computer data acquisition
system or to a potentiometric recorder. A diagram of the light scattering
detector is
HPLC-Detectors Evaporative-Light-Scattering
Author: RPW Scott
Book:Liquid Chromatography Detectors
Section:HPLC-Detectors Liquid-Light-Scattering Multiple-Angle-Laser
Figure 52
Determination of Molecular Weight from Low Angle Light Scattering Measurements
The detector
sensitivity appears to be similar to that of the refractive index detector and
with about the same linearity. However, the greatest advantage of this detector
is that it can provide molecular weight data for extremely large molecules.
The Multiple Angle Laser Light Scattering (MALLS) Detector
The multiple
angle laser light scattering detector differs from the low angle device, in that scattering measurements are made at a number
of different angles, none of which are close to the incident light. This
significantly reduces problems associated with light scattering from
particulate contaminants. Data taken at different angles to the incident light
allows the root-mean-square (rms) of the molecular radius to
be calculated in addition to the molecular weight of the substance. The
relationship that is used is as
HPLC-Detectors Liquid-Light-Scattering Multiple-Angle-Laser
Author: RPW Scott
Book:Liquid Chromatography Detectors
Section:HPLC-Detectors Liquid-Light-Scattering Low-Angle-Laser
;
Now (c), (K), and (Rf) are either known or can all be
calculated from known data and light scattering measurements; thus, by plotting
against
(c) a straight line will be produced with the intercept being .
The Low Angle Laser Light Scattering Detector
The optical
system of the low angle laser light scattering detector produced by LDC
Analytical of the Thermo Instruments Corporation is shown diagramatically in
figure 51. To conserve space, a folding prism is used that allows the device to
be contained to a reasonable size yet accommodate the length of the laser
generator. Light from the laser passes through a diverging lens, through a
chopper and then through the folding prism. On leaving the prism the beam
passes
HPLC-Detectors Liquid-Light-Scattering Low-Angle-Laser
Author: RPW Scott
Book:Liquid Chromatography Detectors
Section:HPLC-Detectors Refractive-Index-Based Christiansen-Effect
(14,15). Consider a cell packed with particulate material having the
same refractive index as the mobile phase passing through. If a beam of light
passes through the cell there will be little of no refraction or scattering.
However, if the refractive index of the mobile phase changes, there will now be
a refractive index difference between the mobile phase and that of the packing.
As a consequence some light will be refracted away from the incident beam and
the intensity of the transmitted light will be attenuated. Thus, if the
transmitted light is focused onto an appropriate photocell, then any change in
refractive index caused by the elution of a solute will produce scattering and
a consequent change in electrical output.
In practice,
he optical dispersions of the media are likely to differ, and consequently the
refractive index will only match at one particular wavelength. As a result the
fully transmitted light will be largely monochromatic. Light of different
wavelengths
HPLC-Detectors Refractive-Index-Based Christiansen-Effect
Author: RPW Scott
Book:Liquid Chromatography Detectors
Section:HPLC-Detectors Liquid-Light-Scattering Multiple-Angle-Laser
for their different responses.
The number of
different angles of measurement differs with different instruments, and some
measure the scattered light intensity at 16 different angles. In general, the
more data points taken at different angles, the more precise the results will
be. A diagram of a (MALLS) detector system which measures the light scattered
at three different angles is shown in figure 53.
Courtesy of Wyatt Technology Corporation
Figure 53 The Multiple Angle
Laser Light Scattering Detector (miniDawn)
This device
(the miniDawn), manufactured by Wyatt Technology Corporation, contains no
mirrors, prisms or moving parts and the light paths are direct and not "folded".
Light passes from the laser (wavelength 690 nm) directly through the sensor
cell. Light scattered from the center of the cell passes through three narrow
channels to three different photocells, set at 45o and 90o
and 135o to the incident
light. Thus scattered light is
HPLC-Detectors Liquid-Light-Scattering Multiple-Angle-Laser