Absolute Molecular Weights
Quickly, easily, and accurately
determine absolute molecular weights of proteins and polymers. Eliminate
SEC/GPC column calibration and improve data quality. Rapidly reduce
uncertainty with no assumptions required and true extrapolation to zero
angle. Best of all, the BI-MwA
has the highest performance/price ratio of any light scattering detector used
for molecular weight determination.
The BI-MwA is simple to use, but
incorporates sophisticated features. Inject your sample into the low-volume,
7-angle flow cell. The sample is illuminated by a temperature stabilized,
precision power-controlled diode laser. The ultra-stable, high-sensitivity,
low-noise CCD detector automatically collects the scattered light. Then, the
software extrapolates the data to zero angle for the absolute molecular weight
determination.
Two Ways to Use the BI-MwA
There are two ways to use the BI-MwA: determine the molecular
weight distribution (flow mode); determine the average molecular weight
(batch-mode). Macromolecular samples often have a range of molecular weights.
In some cases, this distribution is quite narrow, and in other cases, it is
very broad and/or multimodal. Variations in the distribution can indicate the
presence of impurities or aggregation. However, for other applications, the
average molecular weight is sufficient to characterize your sample. Sometimes
the distribution is of interest, and sometimes the overall average is
sufficient. Either way, the BI-MwA
is the right tool for the job.
Molecular
Weight Distribution (Flow Mode)
When the distribution of
molecular weights in your sample is important, consider the BI-MwA as a chromatography detector
for your SEC/GPC system. In this way, you can readily determine the true
molecular weight distribution. Many average values unavailable from a batch
mode measurement can be calculated from these distributions. Better yet, the
molecular weight distribution is determined while the problems of column
calibration are avoided.
Using the BI-MwA with a
chromatography system to determine molecular weight distribution
The way it was:
Size exclusion chromatography
(SEC), also known as gel permeation chromatography (GPC), uses columns to
separate polymer samples. In the standard method, columns are calibrated to obtain
a relationship between molecular weight and elution volume. However, the
column-sample interaction depends on not just the size (molecular weight) but
also the chemistry of the sample. Therefore, an accurate column calibration
requires standards over a range of molecular weights with exactly the same
chemistry and structure (e.g., branching) as the sample. With a few
exceptions, such standards are difficult or impossible to obtain, especially
for new or unique materials.
A better approach:
The BI-MwA provides a method of
determining absolute molecular weights without resorting to any assumptions
about the sample or column calibration. Simply attach the BI-MwA and an appropriate
concentration detector such as the BI-DNDC to the end of the SEC column. The
column is then used to separate the species of interest and the BI-MwA gives full information on
the molecular weight of each fraction. Figures 1 and 2 show the results of a
measurement of the molecular weight distribution of a dextran sample by SEC.
Figure 1 Data from an SEC system equipped with a
BI-MwA. Sample
concentration and molecular weight of dextran are plotted as a function of
elution volume. No column calibration
was required to obtain this plot.
Figure 2 Molecular weight distribution of calculated from
the data in figure 1.
Average
Molecular Weight (Batch Mode)
When the average molecular
weight is sufficient to characterize your sample, use the BI-MwA in batch mode to easily
determine average properties. The BI-MwA
permits routine determination of the weight average molecular weight, Mw,
the z-average radius of gyration, Rg, and the second virial
coefficient, A2. Readily prepare Zimm, Berry, or Debye plots to
calculate these parameters with the optional BI-ZPMwA software.
Using the BI-MwA to determine
average molecular weight
Obtain molecular weights in
batch mode by preparing dilute solutions of known concentration. Follow the
steps in the software to enter concentration values and inject solutions. The
software will automatically evaluate the data. With the click of a mouse,
data are analyzed and presented. Data review and analysis are under user
control. Results are clearly presented on screen and in printed reports. See one example of batch mode data in Figure 3.
Figure 3 Zimm plot of a polystyrene sample in
toluene. The results are in excellent agreement with expectations. The weight
average molecular weight, Mw, radius of gyration. Rg,
and second virial coefficient, A2, are rapidly and easily
obtained.
Samples are unaffected by
measurement and can be recovered for future analysis.
User-Friendly Application
Software
The application software is easy-to-learn,
convenient to use, and maximizes user productivity.
The BI-MwA can be used with
industry-leading, SEC-system software, which allows rapid measurement setup,
user customizable output, and reliable data collection and analysis.
The batch-mode software can
generate Zimm, Debye, and Berry plots. Figure 3 is an example of a Zimm plot
obtained with the BI-MwA.
For complex samples, both linear and higher order polynomial fits can be
performed. All fitting is coupled with sophisticated statistical analysis.
Finally, the software incorporates algorithms for taking advantage of the
flow system to remove artifacts due to contaminants (dust). Thus, good
information can be recovered even from imperfect samples.
Why Seven Angles?
Absolute molecular weight is
determined from the scattered intensity at zero angle. Unfortunately, it is
impossible to measure scattered light at zero angle since transmitted light
will also be measured. Measurement at one or more nonzero angles and
extrapolation to zero angle is necessary for molecular weight determination.
A single-angle light scattering instrument relies on extrapolation based on
one data point; occasionally, it works. In addition, measurement of the
change in scattering with angle is necessary to determine radius of gyration.
Therefore, a multi-angle instrument is far better than a single-angle
instrument.
When comparing multi-angle
instruments, keep in mind that the precision of the light scattering results
is approximately proportional to the square root of the number of angles.
Thus, results from a seven-angle instrument such as a BI-MwA are significantly more
precise than results from a two- or three-angle instrument. The seven-angle
design offers the best performance/price ratio in the industry.
Optimal Design, Small
Footprint, Rugged Construction
The BI-MwA design eliminates the
pitfalls of similar instruments. In order to avoid artifacts and drift due to
trapped bubbles, the flow path is vertical, not horizontal. The patented cell
is more easily flushed clean when samples are changed.
The sample cell can withstand
pressures up to 3.5 MPa, a much higher pressure than other instruments of
this type. Therefore, the BI-MwA
is a perfect complement to an SEC system where high pressures often lead to
premature instrument failure.
In addition to the cell design,
the overall mechanical design is also optimized for maximum performance. The
footprint (21 cm wide x 38 cm deep) conserves valuable bench space. In
addition, the internal partition between the electronics and the liquids
minimizes the extent of damage and the cost of repair if a leak occurs.
Contact us for complete
assistance in setting up a customized SEC system.
Join a large and growing community of users by
contacting Brookhaven Instruments about your application.
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