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HIGH EFFICIENCY AQUEOUSGEL PERMEATIONCh u_..j J:
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By Rick Niel son _ _ "_-._.
Mr. Nielson is Polymer ApplicationsChemist, Industrial o
Waters ChromatographyDivision, MiIlipore Corporation.
The new line of Waters UltrahydrogelTM high-efficiencyaqueous gel
permeationchromatography(GPC) columnswill allow an analyst to perform size
separationsof water-solublepolymers ranging in molecularweight from a few
hundredto severalmillion.
These new columns are packed with a hydroxylatedpolymethacrylatebased
gel, with pore sizes ranging from 120 Angstroms (for the Ultrahydrogel120
column)to 2000 Angstroms (for the Ultrahydrogel2000 column). The
correspondingchain length exclusionlimits, as determinedwith poly(ethylene
oxide) (PEO) standards,vary from 5 x lO3 to an estimated 2 x lO7. Table
l summarizessome of the importantcharacteristicsof 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
greatermobile phase flexibility,and minimal non-sizeexclusioneffects. In
addition,these columns are considerablymore efficient (much narrower peak
widths and higher plate counts)than conventionalaqueous GPC columns.
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MATERIALSAND METHODS:,/
Each column was evaluated using poly(ethyleneoxide) (PEO) standardsfrom
18,000 to 996,000 molecularweight (availablefrom Waters), polysaccharide
(pullulan)standardsfrom 5,800 to 853,000 molecular weight, and poly(ethylene
glycol) (PEG) standards,from 440 to 12,600 molecularweight.
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. Detectionwas accomplishedwith the Waters
410 DifferentialRefractometer. Data reduction was carried out by the Waters
840 Data and ChromatographyControl Stationwith Waters ExpertTM GPC
software.
SAMPLE/STANDARDSPREPARATION:
In addition to the poly(ethyleneoxide), polysaccharideand poly(ethylene
glycol) standards,samplesof dextrans, gelatin, agar, poly(vinylalcohol),
carrageenans,hyaluronicacid, sugars and polyacrylamidewere
chromatographed. The concentrations(w/v) of the standardsand samplesvaried
from 0.02% to 0.10%, dependingon the molecular weight. All solutionswere
filtered through a 0.45 micron MilliporeMillexR-Hv Filter prior to
injection.
APPLICATIONS:
The Ultrahydrogelcolumns offer excellent resolution for such variedwater
soluble polymersas methyl-cellulose,poly(vinyl alcohol), polyacrylamide,
polyvinylpyrrolidone,in addition to the poly(ethyleneoxide), poly(ethylene
glycol)and polysaccharidediscussed previously. The Ultrahydrogelcolumns
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have also been successfullyused to determinethe molecularweight
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-vinylpyridinium) 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
analyzinganionicor cationic polymers,the chromatographerhas to be
concernedwith ionic effects (such as ion exchange, inclusion,exclusion,
etc.). Dependingon the ionic nature of the polymer (andwhether the polymer
is hydrophilicor hydrophobic),the mobile phase has to be carefullychosen to
minimize non-size exclusioneffects. Adding acetonitrileat a 20% level to
O.IM sodium nitrate, for example, will allow the successfulseparationof
anionic and non-ionichydrophobicpolymers.
RESULTS AND DISCUSSION:
All the poly(ethyleneoxide), poly(ethyleneglycol) and polysaccharide
standardswere 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 nitratemobile phase. The
poly(ethyleneoxide) and poly(ethyleneglycol) standardsfit the same
calibrationcurve for all of the columns tested (See Figure l). The
polysaccharidestandard curves (Figure2) shifted to a slightlyhigher elution
volume, indicatinga smalleroverall molecularsize. This was not observed
for the Ultrahydrogel250 column,which has the lowest pore size of the
columns tested. Note the excellent linearityof 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).
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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(ethyleneoxide) 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 polysaccharidestandardswith 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(Figure7) consistsof _n overlay of two
separate injections of polyethyleneoxides. The column used is a single
UltrahydrogelLinear column using O.IM NaNO3. It was possible virtuallytosuperimposethe poly(ethyleneoxide)/poly(ethylenegl_col) calibrationcurve
on the polysaccharidecurve with the O.IM NaNO3. There is an approximate0.3 mL retentiondifference in the phosphatebuffer mobile phase.
Itwas decided to use the O.IM NaNO3 mobile phase for the majority ofaqueous polymers,especiallywhen some non-size exclusion (ionic)effectsare
suspected to be likely to occur. Figure 8 shows separate and overlaid
chromatogramsof broad distributiondextran standards. These were separated
on two UltrahydrogelLinear columnsand had weight-averagemolecularweights
(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
averagemolecularweight 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 molecularweight and the
calibrationcurve was extrapolatedto higher molecularweights. 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.
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Figure lO illustratesthe 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 samplesof 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 molecularweight
distributions.
The next chromatogram(Figure12) is that of a gelatin sample,separated
on a set of two Linearsplus one 250 Ultrahydrogelcolumn. The distribution
appears to be almost bimodal,with a shoulder being observed on the high
molecularweight end of the curve.
Figure 13 illustratesthe GPC chromatogramsof three different
carrageenans. Carrageenansare used extensivelyin the food industry as
thickeners,or gelling compounds. These samples are different not only in the
MWD, but also in the presenceof a low molecularweight component for the #1
sample. One would expect these three samples to have markedly different
gelIing characteristics.
The next chromatogram(Figure14) is that of a sample of hyaluronicacid.
Hyaluronic acid is used extensivelyin ophthalmic surgery,and also in
treatmentof inflammatoryand degenerativebone diseases. In most cases the
higher the molecularweight, the more positive the therapeuticresponse. We
were able to correlatethe molecularweights of hyaluronicacid sampleswith
their respective intrinsicviscosities. The GPC procedure,however, has much
better reproducibilityand can be done much faster.
The last chromatograms(Figure15) are those of simple sugars. They were
separatedon two DP columns in*20 minutes. A single DP column can be used to
separate the mono, di, and trisaccharidesin less than lO minutes.
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v
CONCLUSION:
The Ultrahydrogelcolumnsafford highly efficientseparationsof water
solublepolymers by GPC. For hydrophilicpolymers (anionicor non-ionic),
O.IM sodium nitrate solution is recommendedas the eluent. For some anionic
and non-ionichydrophobicpolymers the additionof up to 20% (by volume)
acetonitrilewill prevent non-size exclusioneffects. Cationic hydrophilic
polymers (such as DEAE-dextranand glycol chitosan)requirea mobile phaseof
O.8M sodium nitrate in order to prevent extra column effects. Cationic
hydrophobicpolymers are the most difficultto chromatograph,requiringa
mobile phase of O.SM acetic acid plus O.3M sodium sulfate. As long as an
analystunderstandsthe chemistryof the polymer, choosing the correcteluent
and obtaininggood results should be easy. Minimumcolumn efficiency
specificationsare conservative,and most columns exceed the minimum by a
significantmargin.
There are numerous aqueous polymers in the industrytoday which can be
separatedand characterizedon these columns. The author has mentioned justa
few for the purposeof demonstratingthe mobile phase chemistriesneeded to
characterizeanionic, cationic and neutral aqueous solublepolymers.
Additionalmethods developmentprojects currently are in progress.
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TABLE 1
Column Pore Minimum Exclusion
Size Efficiency Limit
(A) (plates/col) (PEO)
Ultrahydroge] 120 14,000 5,000
Ultrahydrogel250 250 14,000 80,000
Ultrahydrogel500 500 lO,O00 400,000
UltrahydrogellO00 lO00 l0,000 l,000,000
Ultrahydrogel2000 2000 7,000 20,000,000
UltrahydrogelLinear Blend 7,000 20,000,000
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FIGURE I
PEO * PEG
STANDARDS
500 ¢eI000 _LINF_AR
,oo_ I \'_.,,
¢, o,,
•\ -. \
IOK 1_ ®
\
\ \
6 8 I0 12 14, 16MINUTES
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FIGURE 2
POLYSACC HARIDE
STANDARDS
500 _00 INEARIM
lOOK
%
IOK \
R,\ \1000
25O
IK
6 8 I0 12 14 I$
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F IGURE 3
POLY(ETHYLE NE GLYCOLS)
1,580/_I
I I
ELUENT: HzO-pH:7 ii,z5o j ,FLOW RATE" ' , '
'I 4.,820 j0.8 m_/min
i ICOLUMN 250 , 630i I
J L I
TEMP'45c ' ,J I 440
II
I II
II |
! !
I I
s I
I i
Ii I I i I I I ' I
7 8 9 I0 II 12 13 I._
MINUTES
FIGURE 4
POLY(ETHYLENE OXIDES)
ELUENT H20- pH--7
FLOW RATE'O.8m#io594K
COLUMN" 500
eeK TEMP. "45c
I
I
j# ! !
5 I0 15
MINUTES
FIGURE5
POLY_ETHYLENE OXIDES)145K 86K r_
i i /_39K594K/,^ i _ i
,', , , ,, ELUENT:H20_pH=7I L I I I t
I t " _ f II
' ' ' ,' ' FLOW RATE'O.Smk996K I ' l _ ,
!
' COLUMN" I000t I iI
! I I
o, ' ' leK TEMP:45co_ | I "I I , I
I iII I
I|
t t
f
7 8 9 I0 II 12 13 I_MINUTES
F IGURE 6
POLYSACCHARIDES
IBBK
E LUENT" H20 pH- 7
853K FLOW RATE" 0.8 ml/min
COLUMN" 20002 3.7K
i TEMR:45c,,,.II
t- �4
5 I0 15
MINUTES
FIGURE 7
POLY('ETHYLENEOXIDES)
ELUENT. O.IMNAN03
FLOW RATE: Q8 rnyrnin
COLUMN:LINEAR39K IBK
594K /q
TEMR'45c //_ ,_SK , ,I I
' / i:'(3h I I(3o II _ I
I I
I # I
i t I
8 9 JoII
12MINuTEs
FIGURE 8
DEXTRANS
Mw=71K Mv_IOK
ELUENTQIMNANO 3
FLOW RATE'0.8_io
'-- COLUMNS0
,-, 2 LINEARSI ._1_1
4_ i-,i
'_ tEMP. "45cI
I ! I I I I I
19 20 2t 22 23 24 25
MINUTES
FIGURE 9
POLYACRYLAMIDE
M_,_O00,O00
ELUENT'QIM NANO_
FLOW RATE:0,Sinai,COI--
" COLUMNS' 2 LINEARS0
Hd
I ....I
'-' TEMP. 45c-,4'0
I
0
16 17 18 19 20 21 22 23 24MINUTES
FIGURE I0
POLY (VINYL ALCOHOL)
MW:SO,OO0 ELUENT" 0.1M NANO3
F LOW RATE' 0.Sin#it,
COLUMNS " 2LINEARS
I,-..-J TEMP. "45c0
I1,,-II d
4_ d
I
"-1200
# $ ! # # # # I
t8 t9 20. 2 t 22 23 24 25 26
MINUTES
FIGUREII
AGARS
F_.LUENT: 0.1M NANO 3
FLOW RATE" O.B ml/min
COLUMNS" 2LINEARS
\
\kI
-,-11%)I
I
L i i !
15 20 25
MINUTES
FIGURE 12
GELATIN
ELUENT' O.iM NANO 3
FLOW RATE'.:ImVmin
COLUMN S:2 LI NEARS.i"
I 25O
DETECTION dRlrt'}
.._Io
H_J_.I
,,..]L_I
L O J I | | 0 0 e O O 0 O e I !
t6 t7 t8 t9 20 2i 22 23 24 25 26 27 28 29 30 3i
MINUTES
riG.13CARRAGEENANS
9 I: I.OM Mw ELUF..NT;O.IN NaNO 3
F LOW RATE:O.8ml/t'nin
I( 9: I.EIMIglw COLUMNS: 2 LINEARS
I0: 1.3M Iglw DETECTION" dRIZ0HI.-.,it11:t-Z
I Iii4_ u,,4 Z
=" ° '10! U
LOG MOLECULAR WGT.
FIG. 14HYALURONI C ACI D
lot 3821
Mw--2.,674,0O0
ELUENT: O.IN NAN03 i
FLOW RATE: l.Oml/minICOLUMNS: 2 2000+!
7- I 25OOH
I.----_ DETECTION " RItri--zi,i
i u
•..4 oi..,i
i
LOG [MOLECULAR WEIGHT]