Handbook of Size Exclusion Chromatography and Related ...

gas lasers as the solid state lasers are more efficient, produce higher power levels,
and are far more compact. They do have one serious problem and that is stability.
Despitetheir normalizationcapabilitybymeansofaninternalbeammonitor,they
generallysufferfromso-called“modehopping”andthiscanresultinlargeoutput
power fluctuations, irrespective of the efficiency of power normalization or
feedback control. Such mode hopping depends critically upon laser temperature
andage.Becausethesefluctuationscanbeveryrapid,theyareoftenunsusceptible
to monitoring correction. Solid-state laser sources are now available over arange
ofwavelengthsfrom bluethroughinfrared. Most commonly,awavelengtharound
680nmisused.Inrecentyears,eliminationofmodehoppinghasbeenachievedby
some vendors without relying on temperature stabilization attempts.
The detectors shown are generally selected as high-gain transimpedance
photodiodes or even small CCD arrays. The former span afar greater dynamic
range. An important characteristic of all detectors is their collimation and angular
resolution. Very large macromolecules produce scattering patterns exhibiting
considerable curvature. With detectors subtending large solid angles, the derived
results can be compromised by this unnecessary smoothing of the angular
variation of the scattered light. In addition, if detectors accept toogreat arange of
angles, it becomes difficult to separate noise contributions within the collected
data.
Detectors should be capable of being fitted with narrow band pass
interference filters for the measurement of fluorescent materials, such as lignins
and asphaltines. The depolarizing effects of some molecules are best studied with
thefittingofpolarizationanalysers,anapplicationof increasingimportanceinthe
field of nanoparticle characterization.
Other elements useful for light scattering detectors include temperature and
(as needed) humidity control of the optics. Indeed, agreat amount of SEC work
relates to the high temperature environment (100–2208C). Not only must the
optical train be able to withstand such temperatures without distortion, but the
detectors must often be shielded from long-wavelength radiation as theyare often
very sensitive well into the infrared region. Without such filtering, the noise
contributions arising from black body radiation may overwhelm the sample
signatures themselves.
Figure 2shows atypical configuration for collecting MALS data from the
sample following separation in the columns shown. Note several key elements: the
mobile phase is both degassed and filtered, the latter generally through 0.1mm
filters. Either UV or DRI detectors may be used to determine concentration
[needed to solve Eq. (1)], an essential element of the measurement. The UV
detector is generally placed before the MALS detector, the DRI after it. For most
SEC separations, a DRI detector is preferred. This is particularly true for proteins
whose refractive index increment is about 0.175 within 5% for most proteins. With
UV detection, the protein extinction coefficients must be known before the
© 2004 by Marcel Dekker, Inc.