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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