MACROIMS™

MACROION MOBILITY SPECTROMETER

MOLECULAR WEIGHT AND
PARTICLE SIZE DETERMINATION OF
POLYVINYLPYRROLIDONE (PVP)
APPLICATION NOTE CHEMC-009 (A4)

Polyvinylpyrrolidone (PVP) Polymer

Polyvinylpyrrolidone (PVP) is a water-soluble polymer made from the
monomer N-vinylpyrrolidone and is used in pharmaceutical and chemical
applications. Typical molecular weights of PVP products are between 2 kDa
and over 2 MDa.

This application note shows measurements with the TSI MacroIMS™ Macroion
Mobility Spectrometer (MacroIMS) System as described below. Two PVP
powders were purchased from Sigma: PVP10 with approximate mean
molecular weight of 10 kDa, and PVP360, with approx. mean molecular weight
of 360 kDa.

Macroion Mobility Spectrometry for Molecular Weight and Size

Determination
The MacroIMS System is a versatile ion mobility
spectrometry (IMS) system that relies on first-
principle size analysis to determine the molecular
weight of macromolecules and complexes between
8 kDa and > 100 MDa. This technique reduces the
breakdown of non-covalent interactions which
usually accompany electrospray ionization
through charge neutralization, allowing for
analysis of macromolecular complexes. Separation
of the intact, high mass macromolecules and
complexes takes place in an ion mobility drift cell.
The number of macromolecules is determined in
each fraction by a single-macromolecule counter.
The technique has been extensively reviewed in
numerous citations, and a new development has
enabled automated, high-throughput analysis and
hyphenation options, making it applicable for
routine laboratory applications.

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Sample Preparation
PVP10
PVP10 powder (Sigma Aldrich, approx. average molecular weight 10 kDa) was dissolved into
20 mM ammonium acetate buffer of pH approx. 6.8, resulting in a 3.39 mg/mL solution. The
solution was vortexed until the PVP was completely dissolved. A dilution series was prepared
using the same buffer, covering a concentration range of 42 nM to 3390 nM (0.42 µg/mL to 33.9
µg/mL, respectively).

PVP360
PVP360 powder (Sigma Aldrich, approx. average molecular weight 360 kDa) was dissolved into
20 mM ammonium acetate buffer of pH approx. 6.8 resulting in a 3.8 mg/mL solution. The
solution was vortexed until the PVP was completely dissolved. A dilution series was prepared
using the same buffer, covering a concentration range of 26 nM to 1056 nM (9.5 µg/mL to 380
µg/mL, respectively).

Results
In summary, molecular weight, polydispersity, and particle diameter have been determined for
PVP10 and PVP360 for several concentrations. The signal response was found to be linear
within a 95% confidence interval for the assessed concentrations.

Table 1: Summary of basic polymer properties found for

PVP10 and PVP360 measured with MacroIMS spectrometer.

PVP10 PVP360
Mp (kDa) 12.1 259.5
Mn (kDa) 16.8 153.6
Mw (kDa) 36.4 532.4
Polydispersity 2.2 3.5
Diameter (Dp) (nm) 4.2 11.5
Mp: Molecular weight in peak; Mn: number average molecular weight;
Mw: weight average molecular weight; Dp: Diameter in peak

The molecular weight found for PVP360 using the MacroIMS system would appear to be
surprisingly low, and there are various factors that should be taken into account. Table 1 shows
an Mp of 259.5 kDa as the molecular weight in the peak of a count (number)-based distribution.
The mass average molecular weight based on the measured distribution was determined to be
approx. 532.4 kDa. The size distribution of the PVP360 is very broad with a large tail preceding
the main peak. Thus, when using a number-based measurement method like MacroIMS, the
resulting molecular weight values are smaller than those derived from analytical methods that
rely on separation interactions, optical properties, or mass dependent detection.

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Figure 1: Molecular weight distribution of PVP10 measured with the MacroIMS System
at concentrations from 42 nM to 3390 nM. The scan range covered approx. 2 kDa to 28
MDa. The molecular weight at the peak (Mp) was determined to be 12.1 kDa.
Polydispersity was calculated as approx. 2.

Figure 2: Polymer size distribution of PVP10 measured with the MacroIMS System at
concentrations from 0.42 µg/mL to 33.9 µg/mL. The scan range covered 2 nm to 55 nm.
The particle diameter at the peak was determined to be 4.2 nm.

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Figure 3: Peak area integral of the PVP10 signal plotted versus the prepared
concentration. Graph shows the 95% confidence interval.

Figure 4: Molecular weight distribution of PVP360 measured with the MacroIMS System
at concentrations from 26 nM to 528 nM. The scan range covered approx. 2 kDa to 28
MDa. The molecular weight at the peak (Mp) was determined as 259.5 kDa.
Polydispersity was calculated to be approx. 3.5.

-4-
Figure 5: Polymer size distribution of PVP360 measured with the MacroIMS System at
concentrations from 10 µg/mL to 190 µg/mL. The scan range covered 2 nm to 55 nm.
The particle diameter at the peak was determined to be 11.5 nm.

Figure 6: Peak area integral of the PVP360 signal plotted versus the prepared
concentration. Graph shows the 95% confidence interval.

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Discussion
Knowing the size and molecular weight of polymers is of importance for developing
pharmaceutical, chemical, cosmetic or other products with regards to bioavailability, drug
stabilization, product stability, etc. Methods used to determine size and molecular weight may
be biased by the performance of the separation technique used to fractionate the polydisperse
polymers, and the detection method. The MacroIMS System performs separation of sizes in the
gas-phase under atmospheric pressure, and therefore has no analyte-column material
interaction dependence. The primary measurement of particle size is based on a first-order
principle and requires no calibration. The detector delivers counts of polymer particles in the
size fractions, and as such provides a truly number-based concentration determination. The size
distribution obtained is independent of elution volumes. Thus the number-weighted size
distribution may differ from calculated data using conventional methods.

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CHEMC-009-A4 (9/3/2014) ©2014 TSI Incorporated Printed in U.S.A.