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HIGH EFFICIENCY AQUEOUS GEL PERMEATIONCh
By Rick Niel son
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Mr. Nielson is Polymer ApplicationsChemist, Industrial
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Waters ChromatographyDivision, MiIlipore Corporation.
The new line of Waters Ultrahydrogel
TM high-efficiencyaqueous gel
permeationchromatography (GPC) columns will allow an analyst to perform size
separationsof water-solublepolymers ranging in molecular weight from a few
hundred to several million.
These new columns are packed with a hydroxylated polymethacrylatebased
gel, with pore sizes ranging from 120 Angstroms (for the Ultrahydrogel120
column) to 2000 Angstroms (for the Ultrahydrogel2000 column). The
correspondingchain length exclusion limits, as determinedwith poly(ethylene
oxide) (PEO) standards,vary from 5 x lO3 to an estimated 2 x lO7. Table
l summarizessome of the important characteristicsof these columns.
These columns offer many advantagesover conventionalaqueous GPC columns,
such as a wide pH range (2-12),compatabilitywith high organic aqueous eluent
concentration(up to 20% organic; 50% organic if introducedby gradient),or
greater mobile phase flexibility,and minimal non-size exclusion effects. In
addition, these columns are considerablymore efficient (much narrower peak
widths and higher plate counts) than conventionalaqueous GPC columns.
-455-
,/
MATERIALS AND METHODS:
Each column was evaluated using poly(ethyleneoxide) (PEO) standards from
18,000 to 996,000 molecular weight (availablefrom Waters), polysaccharide
(pullulan)standards from 5,800 to 853,000 molecular weight, and poly(ethylene
glycol) (PEG) standards,from 440 to 12,600 molecular weight.
The eluents used for this work were distilled water (phosphatebuffered to
pH 7.0) and O.IM sodium nitrate, with column temperaturesof 30° and 45" being
evaluated. The two eluents could be used interchangeablyfor non-ionic
polymers. The O.IM sodium nitrate should be used for some of the anionic
polymers.
The flow rate in most cases was 0.8 ml/min. The Waters 590 Programmable
Solvent Delivery Module was used. Detection was accomplishedwith the Waters
410 DifferentialRefractometer. Data reduction was carried out by the Waters
840 Data and ChromatographyControl Station with Waters ExpertTM GPC
software.
SAMPLE/STANDARDSPREPARATION:
In addition to the poly(ethylene oxide), polysaccharideand poly(ethylene
glycol) standards,samples of dextrans, gelatin, agar, poly(vinylalcohol),
carrageenans,hyaluronicacid, sugars and polyacrylamidewere
chromatographed. The concentrations (w/v) of the standards and samples varied
from 0.02% to 0.10%, depending on the molecular weight. All solutionswere
filtered through a 0.45 micron Millipore MillexR-Hv Filter prior to
injection.
APPLICATIONS:
The Ultrahydrogelcolumns offer excellent resolution for such varied water
soluble polymers as methyl-cellulose,poly(vinyl alcohol), polyacrylamide,
polyvinylpyrrolidone,in addition to the poly(ethyleneoxide), poly(ethylene
glycol) and polysaccharidediscussed previously. The Ultrahydrogelcolumns
-456-
have also been successfullyused to determine the molecular weight
distributionsof anionic polymers such as alginic acid sodium salt,
polyacrylicacid sodium salt and sodium polystyrenesulfonate,and also
cationic polymers such as glycol chitosan, DEAE dextran and
poly(N-methyl-2-vinyl
pyridinium) iodide salt.
The methacrylate-basedgel packing in these columns has a slight negative
charge due to a small amount of residual carboxyl groups. Therefore,in
analyzing anionic or cationic polymers, the chromatographerhas to be
concerned with ionic effects (such as ion exchange, inclusion,exclusion,
etc.). Dependingon the ionic nature of the polymer (and whether the polymer
is hydrophilic or hydrophobic),the mobile phase has to be carefully chosen to
minimize non-size exclusion effects. Adding acetonitrileat a 20% level to
O.IM sodium nitrate, for example, will allow the successfulseparationof
anionic and non-ionic hydrophobic polymers.
RESULTS AND DISCUSSION:
All the poly(ethyleneoxide), poly(ethyleneglycol) and polysaccharide
standards were chromatographedat the 30°C and 45°C temperaturesusing the
phosphate-bufferedwater (pH = 7) mobile phase. The standardswere also
chromatographedat 45°C using the O.IM sodium nitrate mobile phase. The
poly(ethyleneoxide) and poly(ethyleneglycol) standards fit the same
calibration curve for all of the columns tested (See Figure l).
The
polysaccharidestandard curves (Figure 2) shifted to a slightly higher elution
volume, indicatinga smaller overall molecular size. This was not observed
for the Ultrahydrogel250 column, which has the lowest pore size of the
columns tested. Note the excellent linearity of the two UltrahydrogelLinear
column curves. The UltrahydrogelLinear and 2000 column calibrationcurves
are linear up to the highest standard (just under l million). Although the
minimum plate count for the UltrahydrogelLinear column is 7,000 plates, plate
counts between I0,000 and II,500 p/ft were obtained for the columns that were
evaluated. Using three UltrahydrogelLinears in series at 45°C will provide
an analyst with tremendousefficiency (over 30,000 theoreticalplates).
-457-
Figure 3 shows the chromatograms (two overlays) for poly(ethyleneglycol)
standards,separating the molecular weights from II,250 down to 440 in
approximately13 min. Three poly(ethylene oxide) standards,594,000, 86,000
and 39,000, were separated in less than 10 min. on an Ultrahydrogel500
column, as shown in Figure 4. The plate count for this column averaged ll,o00
p/ft. Figure 5 illustratestwo different injectionsof poly(ethyleneoxide)
standards. The column used in this case was a single Ultrahydrogel1000, and
the chromatogramshows separationof molecular weights ranging from 18,000 to
996,000. Three polysaccharidestandards with molecularweight of 23,700,
186,000 and 853,000 separatedon an Ultrahydrogel2000 column are shown in
Figure 6.
Notice that in all cases the chromatogramsfor these standardsshow
peak shapes that are narrow and symmetrical.
The last standard chromatogram (Figure 7) consists of _n overlay of two
separate injections of polyethyleneoxides. The column used is a single
UltrahydrogelLinear column using O.IM NaNO3. It was possible virtually to
superimposethe poly(ethyleneoxide)/poly(ethylenegl_col) calibrationcurve
on the polysaccharidecurve with the O.IM NaNO3. There is an approximate
0.3 mL retention difference in the phosphate buffer mobile phase.
It was decided to use the O.IM NaNO3 mobile phase for the majority of
aqueous polymers, especiallywhen some non-size exclusion (ionic) effects are
suspected to be likely to occur. Figure 8 shows separate and overlaid
chromatogramsof broad distributiondextran standards. These were separated
on two Ultrahydrogel Linear columns and had weight-averagemolecular weights
(Mw, determined by light scattering)of 10,000, 42,000 and 71,000. From the
PEO/PEG calibrationcurve, Mw values of 15,000, 47,000 and 71,000,
respectively,were obtained. The dispersivitieswere approximately1.5 for
all three dextrans.
For a polyacrylamide(See Figure 9), which was said to have a viscosity
average molecular weight of 4 million, the author obtained a peak molecular
weight of just under 3 million. The accuracy of this value is somewhat
questionable,since the highest standardwas 996,000 molecular weight and the
calibration curve was extrapolatedto higher molecular weights. Because the
concentrationof this sample was only 0.03% (w/v) due to the high molecular
weight, there is a slight increase in the baseline noise on the chromatogram.
-458-
Figure lO illustrates the GPC chromatogramof a broad MWD poly(vinyl
alcohol) (PVA) sample. This sample was said to have been "pure" and "fairly
monodisperse,"but one can readily observe that there is a significantlow-end
tail, as well as a componenteluting at MW,-1200. The peak molecularweight
for this poly(vinylalcohol) was 60,000.
The chromatogramsof two different samples of agar are shown in Figure
If. They were from different sources, but thought to be of the same molecular
weight. The overlay clearly shows a difference in the molecular weight
distributions.
The next chromatogram (Figure 12) is that of a gelatin sample, separated
on a set of two Linears plus one 250 Ultrahydrogelcolumn. The distribution
appears to be almost bimodal, with a shoulder being observed on the high
molecular weight end of the curve.
Figure 13 illustratesthe GPC chromatogramsof three different
carrageenans. Carrageenansare used extensively in the food industry as
thickeners,or gelling compounds. These samples are different not only in the
MWD, but also in the presence of a low molecular weight component for the #1
sample. One would expect these three samples to have markedly different
gelIing characteristics.
The next chromatogram (Figure 14) is that of a sample of hyaluronicacid.
Hyaluronic acid is used extensively in ophthalmic surgery, and also in
treatment of inflammatoryand degenerativebone diseases. In most cases the
higher the molecular weight, the more positive the therapeutic response. We
were able to correlate the molecular weights of hyaluronicacid samples with
their respective intrinsicviscosities. The GPC procedure,however, has much
better reproducibilityand can be done much faster.
The last chromatograms (Figure 15) are those of simple sugars. They were
separated on two DP columns in*20 minutes. A single DP column can be used to
separate the mono, di, and trisaccharidesin less than lO minutes.
"459-
v
CONCLUSION:
The Ultrahydrogelcolumns afford highly efficient separationsof water
soluble polymers by GPC. For hydrophilicpolymers (anionic or non-ionic),
O.IM sodium nitrate solution is recommendedas the eluent. For some anionic
and non-ionic hydrophobicpolymers the addition of up to 20% (by volume)
acetonitrilewill prevent non-size exclusion effects. Cationic hydrophilic
polymers (such as DEAE-dextranand glycol chitosan)require a mobile phaseof
O.8M sodium nitrate in order to prevent extra column effects. Cationic
hydrophobicpolymers are the most difficult to chromatograph,requiring a
mobile phase of O.SM acetic acid plus O.3M sodium sulfate. As long as an
analyst understandsthe chemistry of the polymer, choosing the correct eluent
and obtaining good results should be easy. Minimum column efficiency
specificationsare conservative,and most columns exceed the minimum by a
significantmargin.
There are numerous aqueous polymers in the industry today which can be
separatedand characterizedon these columns. The author has mentioned justa
few for the purpose of demonstratingthe mobile phase chemistriesneeded to
characterizeanionic, cationic and neutral aqueous soluble polymers.
Additionalmethods developmentprojects currently are in progress.
-a60-
TABLE 1
Column
Pore
Minimum
Exclusion
Size
Efficiency
Limit
(A)
(plates/col)
(PEO)
Ultrahydroge]
120
14,000
5,000
Ultrahydrogel 250
250
14,000
80,000
Ultrahydrogel500
500
lO,O00
400,000
Ultrahydrogel lO00
lO00
l0,000
l,000,000
Ultrahydrogel2000
2000
7,000
20,000,000
Ultrahydrogel Linear
Blend
7,000
20,000,000
-461-
FIGURE I
PEO * PEG
STANDARDS
500
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-462-
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FIGURE 2
POLYSACC HARIDE
STANDARDS
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F IGURE
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POLY(ETHYLE NE GLYCOLS)
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FIGURE 4
POLY(ETHYLENE
OXIDES)
ELUENT
FLOW
H20- pH--7
RATE'O.8m#io
594K
COLUMN" 500
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TEMP.
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FIGURE5
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F IGURE 6
POLYSACCHARIDES
IBBK
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FLOW RATE" 0.8 ml/min
COLUMN"
2000
2 3.7K
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pH- 7
FIGURE 7
POLY('ETHYLENE
OXIDES)
ELUENT. O.IMNAN03
FLOW RATE: Q8 rnyrnin
COLUMN:LINEAR
39K
IBK
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FIGURE
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DEXTRANS
Mw=71K
ELUENTQIMNANO
Mv_IOK
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F LOW RATE'
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FIGURE 9
POLYACRYLAMIDE
M_,_O00,O00
ELUENT'QIM
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FLOW RATE:0,Sinai,
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MINUTES
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FIGURE
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POLY (VINYL ALCOHOL)
MW:SO,
OO0
ELUENT" 0.1M NANO3
F LOW RATE' 0.Sin#it,
COLUMNS " 2LINEARS
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TEMP. "45c
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MINUTES
FIGUREII
AGARS
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FLOW RATE" O.B ml/min
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FIGURE
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GELATIN
ELUENT'
O.iM NANO 3
FLOW RATE'.:ImVmin
COLUMN S:2 LI NEARS.i"
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DETECTION
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MINUTES
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CARRAGEENANS
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Mw
ELUF..NT; O.IN NaNO 3
F LOW RATE:O.8ml/t'nin
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9: I.EIMIglw
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COLUMNS: 2 LINEARS
DETECTION"
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FIG. 14
HYALURONI C ACI D
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Mw--2.,674,0O0
ELUENT: O.IN NAN03 i
FLOW RATE: l.Oml/min
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COLUMNS: 2 2000+!
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