Hydrophilic Polymers
Pullulan standards were analyzed using OHpak SB-804 HQ (a column for aqueous SEC (GFC)).
Sample : Pullulan
1. P-800
2. P-200
3. P-50
4. P-10
5. P-1
6. Glucose
Columns : Shodex OHpak SB-804 HQ (8.0mmID*300mm) x 2 Eluent : 0.02% NaN3 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 30deg-C
Pullulan standards were analyzed using Asahipak GF-310 HQ (a column for aqueous/organic SEC).
Sample :
Pullulan P-20 (MW: 23,000)
PEG, Poly(ethylene glycol) 4,000 (MW: 3,000)
PEG, Poly(ethylene glycol) 1,000 (MW: 1,000)
EG, Ethylene glycol (MW: 62)
Column : Shodex Asahipak GF-310 HQ (7.5mmID*300mm) Eluent : H2O Flow rate : 0.5mL/min Detector : Shodex RI Column temp. : 30deg-C
Pullulan standards were analyzed using Asahipak GF-710 HQ (a column for aqueous/organic SEC).
Sample :
1. Pullulan P-800
2. Pullulan P-200
3. Pullulan P-50
4. Pullulan P-10
5. EG, Ethylene glycol
Column : Shodex Asahipak GF-710 HQ (7.5mmID*300mm) Eluent : H2O Flow rate : 0.5mL/min Detector : Shodex RI Column temp. : 50deg-C
Poly(ethylene glycol) standard which is in the area of oligomers was analyzed using RSpak DE-613. Oligomers from Dp-3 to 14 were separated.
Sample : PEG, Poly(ethylene glycol)
Column : Shodex RSpak DE-613 (6.0mmID*150mm) Eluent : H2O/CH3OH=95/5 Flow rate : 1.5mL/min Detector : Shodex RI Column temp. : 27deg-C
Poly(ethylene glycol) standards were analyzed using OHpak SB-2002.5 (a column for aqueous SEC (GFC)).
Sample :
1. PEG, Poly(ethylene glycol) 20K
2. PEG, Poly(ethylene glycol) 3K
3. PEG, Poly(ethylene glycol) 400
4. EG, Ethylene glycol
Column : Shodex OHpak SB-2002.5 (20.0mmID*300mm) Eluent : 0.02% NaNO3 aq. Flow rate : 3.0mL/min Detector : Shodex RI Column temp. : Room temp.
Poly(ethylene glycol) standards were analyzed using OHpak SB-802.5 HQ (a column for aqueous SEC (GFC)).
Sample :
Poly(ethylene glycol)
EG, Ethylene glycol
Column : Shodex OHpak SB-802.5 HQ (8.0mmID*300mm) Eluent : H2O Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : Room temp.
Poly(ethylene glycol) standards were analyzed using Asahipak GS-220 HQ (a column for multimode).
Sample : 20micro-L
1. 0.1% PEG, Poly(ethylene glycol) (MW: 4,000)
2. 0.1% PEG, Poly(ethylene glycol) (MW: 1,000)
3. 0.2% PEG, Poly(ethylene glycol) (MW: 400)
4. 0.1% EG, Ethylene glycol (MW: 62)
Columns : Shodex Asahipak GS-220 HQ (7.5mmID*300mm) x 2 Eluent : H2O Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 60deg-C
Poly(ethylene oxid) and poly(ethylene glycol) standards were analyzed using OHpak SB-804 HQ (a column for aqueous SEC (GFC)).
Sample :
1. PEO, Poly(ethylene oxide) (MW: 594,000)
2. PEO, Poly(ethylene oxide) (MW: 145,000)
3. PEO, Poly(ethylene oxide) (MW: 73,000)
4. PEO, Poly(ethylene oxide) (MW: 40,000)
5. PEO, Poly(ethylene oxide) (MW: 18,000)
6. PEG, Poly(ethylene glycol) (MW: 3,000)
7. PEG, Poly(ethylene glycol) (MW: 600)
8. EG, Ethylene glycol (MW: 62)
Columns : Shodex OHpak SB-804 HQ (8.0mmID*300mm) x 2 Eluent : 0.02% NaN3 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 30deg-C
Since Asahipak GF-HQ series can be used with both aqueous and organic solvents, they are suited to the analysis of hydrophilic polymers such as sodium poly(styrene sulfonate) and sodium polyacrylate. Several kinds of pydrophilic polymer standard are commercially available. When analyzing a sample, a standard which resembles it in structure should be selected to obtain the calibration curve for the analysis.
Sample : Sodium poly(styrene sulfonate) standard
Columns : Shodex Asahipak GF-HQ series (7.5mmID*300mm each) Eluent : 50mM LiCl aq./CH3CN=60/40 Flow rate : 0.4mL/min Detector : Shodex RI Column temp. : 30deg-C
Since Asahipak GF-HQ series can be used with both aqueous and organic solvents, they are suited to the analysis of hydrophilic polymers such as sodium poly(styrene sulfonate) and sodium polyacrylate. Several kinds of pydrophilic polymer standard are commercially available. When analyzing a sample, a standard which resembles it in structure should be selected to obtain the calibration curve for the analysis.
Sample : Sodium polyacrylate standard
Columns : Shodex Asahipak GF-HQ series (7.5mmID*300mm each) Eluent : 50mM Na2HPO4 aq./CH3CN=90/10 Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 40deg-C
Since Asahipak GF-HQ series can be used with both aqueous and organic solvents, they are suited to the analysis of hydrophilic polymers such as sodium poly(styrene sulfonate) and sodium polyacrylate. Several kinds of pydrophilic polymer standard are commercially available. When analyzing a sample, a standard which resembles it in structure should be selected to obtain the calibration curve for the analysis.
Sample : Poly(sodium methacrylate) standard
Columns : Shodex Asahipak GF-HQ series (7.5mmID*300mm each) Eluent : 50mM Na2HPO4 aq./CH3CN=90/10 Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 40deg-C
Calibration curves of Asahipak GF-7M HQ (packed with poly(vinyl alcohol)) and OHpak SB-806M HQ (packed with poly(hydroxy methacrylate)) are compared using a hydrophilic polymer standerd, sodium poly(styrene sulfonate). The comparison shows that the calibration curve of Asahipak GF-7M HQ is more linear than that of OHpak SB-806M HQ.
Sample : Sodium poly(styrene sulfonate) standard
Columns : Shodex Asahipak GF-7M HQ (7.5mmID*300mm), OHpak SB-806M HQ (8.0mmID*300mm) Eluent : 50mM Na2HPO4 aq./CH3CN=90/10 Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 40deg-C
Calibration curves of Asahipak GF-7M HQ(packed with poly(vinyl alcohol)) and OHpak SB-806M HQ (packed with poly(hydroxy methacrylate)) are compared using a hydrophilic polymer standerd, sodium polyacrylate. The comparison shows that the calibration curve of Asahipak GF-7M HQ is more linear than that of OHpak SB-806M HQ.
Sample : Sodium polyacrylate standard
Columns : Shodex Asahipak GF-7M HQ (7.5mmID*300mm), OHpak SB-806M HQ (8.0mmID*300mm) Eluent : 50mM Na2HPO4 aq./CH3CN=90/10 Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 40deg-C
Calibration curves of Asahipak GF-7M HQ (packed with poly(vinyl alcohol)) and OHpak SB-806M HQ (packed with poly(hydroxy methacrylate)) are compared using a hydrophilic polymer standerd, poly(sodium methacrylate). The comparison shows that the calibration curve of Asahipak GF-7M HQ is more linear than that of OHpak SB-806M HQ.
Sample : Poly(sodium methacrylate) standard
Columns : Shodex Asahipak GF-7M HQ (7.5mmID*300mm), OHpak SB-806M HQ (8.0mmID*300mm) Eluent : 50mM Na2HPO4 aq./CH3CN=90/10 Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 40deg-C
Hydroxyethyl cellulose was analyzed using OHpak SB-805 HQ (a column for aqueous SEC (GFC)).
Sample : Hydroxyethyl cellulose
Columns : Shodex OHpak SB-805 HQ (8.0mmID*300mm) x 2 Eluent : 0.1M NaNO3 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 30deg-C
In-column solvent of GPC KF-806M was replaced from THF to DMSO and dextran standards of MW 2,000,000 and MW 500,000 were analyzed. The molecular weight distribution of dextran, MW 2,000,000 was rather wide and two peaks were observed.
Sample : 0.5% each, 100micro-L
Dextran T2000
Dextran T500
Column : Shodex GPC KF-806M (8.0mmID*300mm) Eluent : DMSO Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 60deg-C
Dextran was analyzed using OHpak SB-806M HQ (a column for aqueous SEC (GFC)).
Sample : 0.1% Dextran Industrial grade, 200micro-L
Columns : Shodex OHpak SB-806M HQ (8.0mmID*300mm) x 2 Eluent : 0.1M NaCl aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Dextran sulfate was analyzed using OHpak SB-806M HQ. (a column for aqueous SEC (GFC))
Sample : 0.1% Dextran sulfate, 200micro-L
Columns : Shodex OHpak SB-806M HQ (8.0mmID*300mm) x 2 Eluent : 0.1M NaCl aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Poly(ethylene imine) was analyzed using OHpak SB-804 HQ and SB-803 HQ (columns for aqueous SEC (GFC)).
Sample : Poly(ethylene imine)
Columns : Shodex OHpak SB-G (6.0mmID*50mm) + SB-804 HQ + SB-803 HQ (8.0mmID*300mm each) Eluent : 0.5M Acetic acid + 0.5M Sodium acetate aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : Room temp.
Polyacrylamide was analyzed using OHpak SB-805 HQ (a column for aqueous SEC (GFC)).
Sample : Polyacrylamide
1. Mw 74,000
2. Mw 2,000,000
Columns : Shodex OHpak SB-805 HQ (8.0mmID*300mm) x 2 Eluent : 0.1M NaNO3 aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 30deg-C
Poly(vinyl alcohol) was analyzed using OHpak SB-803 HQ and SB-806 HQ (columns for aqueous SEC (GFC)).
Sample :
1. PVA 1,500, Poly(vinyl alcohol)
2. PVA 500, Poly(vinyl alcohol)
Columns : Shodex OHpak SB-806 HQ + SB-803 HQ (8.0mmID*300mm each) Eluent : 0.1M NaCl aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C
Poly(vinylpyrrolidone) was analyzed using OHpak SB-805 HQ. When a sample has both hydrophilic and hydrophobic parts, hydrophobic interaction sometimes exists between the sample and the packing material. To cancel out such hydrophobic interaction and realize SEC, an addition of organic solvent to the eluent is necessary.
Sample : Poly(vinylpyrrolidone)
60 K(Mw: 160,000)
30 K(Mw: 40,000)
15 K(Mw: 10,000)
Columns : Shodex OHpak SB-805 HQ (8.0mmID*300mm) x 2 Eluent : 0.1M NaNO3 aq./CH3CN=90/10 Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 30deg-C
Sodium polyacrylate was analyzed using OHpak SB-802 HQ and SB-803 HQ (columns for aqueous SEC (GFC)).
Sample : Sodium polyacrylate
Columns : Shodex OHpak SB-G (6.0mmID*50mm) + SB-803 HQ + SB-802 HQ (8.0mmID*300mm each) Eluent : 0.1M NaCl aq. Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : Room temp.
Sodium polyacrylate was analyzed using Asahipak GSM-700 7E.
Sodium polyacrylate is a polymer whcih has weak ionic groups (carboxyl groups). The effect of eluent pH on GFC analysis of sodium polyacrylate is shown below :
Eluent of pH3——Dissociation of carboxyl groups is suppressed and mainly -COOH exists in the state of equilibrium. Therefore, the polymer is hydrophobic and adsorbed by the packing material and will not elute.
Eluent of pH7——In the low salt solution around 50mM, the polymer is partly dissociated and both -COO– and -COOH exist in the state of equilibrium. When -COOH is major, the adsorption mode with packing materials exists together with GFC mode. In this case, addition of organic solvents around 10 percent can remove adsorption mode and realize pure GFC mode.
Eluent of pH10—–Carboxyl groups are dissociated and mainly -COO– exists in the state of equilibrium. Therefore, the polymer is hydrophillic and recovery is very high. In this case, pure GFC mode can be realized without the addition of organic solvents.
As described in the avobe, the eluent pH is very important factor in the GFC analysis of weak ionic polymers.
GSM-700 7E is an old type of GF-7M HQ.
Sample : Sodium polyacrylate
Column : Shodex Asahipak GSM-700 7E (7.5mmID*250mm) Eluent : 50mM Phosphate buffer/CH3CN=90/10 Flow rate : 0.5mL/min Detector : Shodex RI Column temp. : Room temp.
Poly(sodium methacrylate) was analyzed using Asahipak GF-710 HQ (a column for aqueous/organic SEC).
Sample : Poly(sodium methacrylate)
Column : Shodex Asahipak GF-710 HQ (7.5mmID*300mm) Eluent : 50mM Na2HPO4 aq./CH3CN=90/10 Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 50deg-C
When a sample has both hydrophilic and hydrophobic parts, hydrophobic interaction sometimes exists between the sample and the packing material. To cancel out such hydrophobic interaction and realize SEC, an addition of organic solvent to the eluent is necessary. Specially, with Asahipak GF-510 HQ column, since many kinds of organic solvents can be added to the eluent, the column is useful for SEC analysis with mixed-solvent as eluents.
Sample : Sodium poly(styrene sulfonate)
Column : Shodex Asahipak GF-510 HQ (7.5mmID*300mm) Eluent : 50mM LiCl aq./CH3CN=60/40 Flow rate : 0.6mL/min Detector : UV(254nm) Column temp. : 30deg-C
In case of polymers which have both hydrophobic and hydrophillic groups, hydrophobic interaction with packing material may exist. For such polymers, the addition of organic solvents in the eluent can remove hydrohorbic interatction and realize pure SEC mode. Since (a aqueous/organic SEC column, Asahipak GF-7M HQ can be used with many kinds organic solvents, it is suited to be used with mixed solvents.
Poly(vinyl alcohol) gel with different hydrophobicity are packed into Asahipak GF-310 HQ and GS-320 HQ columns. The hydrophobicity of the gel of the two columns is going stronger according to the temperature rising. Such influence is observed stronger with GF-310 than GS-320. Then under using water as the eluent at 50 deg-C, PEG is not eluted according to its molecular weight as shown the figure. In order to avoid such influence of hydrophobicity, it is applicable to make close the hydrophobicity of gel and eluent. For example the addition of organic solvent, such as Methanol, to eluent is in effect.
Please refer to Hydrophobicity of Columns.
Sample :
1. PEG, Poly(ethylene glycol) (MW: 20,000)
2. PEG, Poly(ethylene glycol) (MW: 4,000)
3. PEG, Poly(ethylene glycol) (MW: 1,000)
4. PEG, Poly(ethylene glycol) (MW: 400)
5. EG, Ethylene glycol (MW: 62)
Columns : Shodex Asahipak GF-310 HQ or GS-320 HQ (7.5mmID*300mm each) Eluent : H2O Flow rate : 0.5mL/min Detector : Shodex RI Column temp. : 30deg-C or 40deg-C or 50deg-C
If water solution containing salt is used as eluent, PEG will be absorbed to Asahipak GF-310 HQ column, because hydrophobic interaction of PEG will be strong. This influence will becomel stronger if column temperature is made high.
Sample :
1. PEG, Poly(ethylene glycol) (MW: 4,000)
2. PEG, Poly(ethylene glycol) (MW: 1,000)
3. EG, Ethylene glycol (MW: 62)
Column : Shodex Asahipak GF-310 HQ (7.5mmID*300mm) Eluent : H2O or 0.1M NaCl aq. Flow rate : 0.5mL/min Detector : Shodex RI Column temp. : 30deg-C or 40deg-C
The polymer-based OHpak SB-803 HQ column has a large distribution of pore size. So, SB-803 HQ is suitable for the measurement of the synthetic polymer with large molecular weight distribution. The silica-based PROTEIN KW-803 and KW-802.5 columns are suitable for the analysis of protein, because a distribution of pore size of them is comparatively narrow.
Sample :
1. Poly(ethylene glycol) (MW: 20,000)
2. Poly(ethylene glycol) (MW: 4,000)
3. Poly(ethylene glycol) (MW: 1,000)
4. Poly(ethylene glycol) (MW: 400)
5. EG, Ethylene glycol (MW: 62)
Columns : Shodex OHpak SB-803 HQ or PROTEIN KW-802.5 or KW-803 (8.0mmID*300mm each) Eluent : H2O Flow rate : 0.5mL/min Detector : Shodex RI Column temp. : 30deg-C
The comparison of chromatograms of mixture of PEG(MW: 20000, 4000, 1000, 400) and Ethyrene glycol (EG) with Asahipak GF-310 HQ column depending on the composition ratio of water and methanol is shown fig.1. When 100% of water is used for eluent, as compared with the case where methanol/water is used for eluent, PEG (MW: 4000, 1000, 400) elute behind time. This phenomenon is related to hydrophobicity of gel of GF-310 HQ column. The hydrophobic interaction may araise between the hydrophobic portion of PEG and the hydrophobic portion of gel, as the result, reverse phase mode may work and a higher hydrophobic sample is eluted later. Since molecule size of PEG MW: 20000 is so large that it cannot go into the pore of gel, PEG MW: 20000 is excluded from the pore of gel. Therefore, the area of PEG in contact with gel is small, and hydrophobic interaction does not work. So, PEG MW: 2000 is not retained. If methanol is added to eluent, the polarity of eluent will turn into the polarity of gel closely. In this case, hydrophbic interaction of PEG and gel is lost and PEG is separated with SEC mode. Furthermore, when the eluent is 100% methanol, the polarity of gel and eluent is reversed and normal phase mode works. An interaction between the polar portion of a sample, and the polar portion of gel works and it becomes later to elute a sample as polarity of a sample becomes high.
Please refer to Influence of Analysis of PEG Mixtures with GS-320 HQ by Composition of Eluent.
Sample : 0.1% each, 100micro-L
1. Poly(ethylene glycol) (MW: 20,000)
2. Poly(ethylene glycol) (MW: 4,000)
3. Poly(ethylene glycol) (MW: 1,000)
4. Poly(ethylene glycol) (MW: 400)
5. EG, Ethylene glycol
Column : Shodex Asahipak GF-310 HQ (7.5mmID*300mm) Detector : Shodex RI Column temp. : 30deg-C
The comparison of chromatograms of mixture of PEG(MW: 20000, 4000, 1000, 400) and Ethyrene glycol(EG) with Asahipak GS-320 HQ column depending on the composition ratio of water and methanol is shown Fig.1. Since GS-320 HQ has higher polarity than GF-310 HQ, with GS-320 HQ, even if it uses 100% of water for eluent, hydrophobic adsorption does not take place and PEG are separated in SEC mode.
Please refer to Influence of Analysis of PEG Mixtures with GF-310 HQ by Composition of Eluent.
Sample : 0.1% each, 100micro-L
1. Poly(ethylene glycol) (Mw: 20,000)
2. Poly(ethylene glycol) (Mw: 4,000)
3. Poly(ethylene glycol) (Mw: 1,000)
4. Poly(ethylene glycol) (Mw: 400)
5. EG, Ethylene glycol
Column : Shodex Asahipak GS-320 HQ (7.5mmID*300mm) Detector : Shodex RI Column temp. : 30deg-C
The analytical results of polyacrylamide are shown. In the analysis with OHpak SB-806 HQ (exclusion limit: 2.0×107g/mol), polyacrylamide elutes around exclusion limit. On the other hand, in the analysis with SUGAR KS-807 (exclusion limit: 2.0×108g/mol), polyacrylamide elutes with SEC mode.
Sample : 100micro-L
0.02% Polyacrylamide (PAAm)
(Scientific Polymer Product)
Columns : Shodex SUGAR KS-807, OHpak SB-806 HQ (8.0mmID*300mm each) Eluent : 0.2M NaCl aq. Flow rate : 0.5mL/min Detector : Shodex RI and MALS(Multi angle laser light scattering detector) Column temp. : 30deg-C
Polyacrylamide was analyzed with OHpak SB-806 HQ (exclusion limit: 2.0×107g/mol) and SB-807 HQ (exclusion limit: 5.0×108g/mol). In the analysis with SB-806 HQ, polyacrylamide elutes around exclusion limit. On the other hand, in the analysis with SB-807 HQ, polyacrylamide elutes with SEC mode.
Sample : 100micro-L
Polyacrylamide (PAAm)
Columns : Shodex OHpak SB-807 HQ, SB-806 HQ (8.0mmID*300mm each) Eluent : 0.2M NaCl aq. Flow rate : 0.5mL/min Detector : Shodex RI and MALS(Multi angle laser light scattering detector) Column temp. : 30deg-C
Pullulan standards were analyzed using GPC LF-804. DMSO/DMF solvent was used as eluent.
Sample : 100micro-L
1. Pullulan (MW:1,600,000) 0.05%
2. Pullulan (MW:788000) 0.1%
3. Pullulan (MW:404,000) 0.1%
4. Pullulan (MW:212,000) 0.1%
5. Pullulan (MW:112,000) 0.1%
6. Pullulan (MW:47,300) 0.1%
7. Pullulan (MW:22,800) 0.1%
8. Pullulan (MW:11,800) 0.1%
9. Pullulan (MW:5,900) 0.1%
10. Maltotriose (MW:504) 0.1%
11. Maltose (MW:342) 0.1%
12. Glucose (MW:180) 0.1%
Column : Shodex GPC LF-804 (8.0mmID*300mm) Eluent : 10mM LiBr in (DMSO/DMF=75/25) Flow rate : 0.6mL/min Detector : Shodex RI Column temp. : 50deg-C
Lignosulfonic acid sodium salt was analyzed using Asahipak GS-520 HQ (a column for multimode).
Sample : 100micro-L
0.1% Lignosulfonic acid sodium salt
Columns : Shodex Asahipak GS-520 HQ (8.0mmID*300mm) x 2 Eluent : 20mM Na2HPO4 aq. Flow rate : 0.6mL/min Detector : UV(254nm) Column temp. : 40deg-C
Effect of Sample Load (Pullulan) The effect of sample load (concentration) using pullulan (a kind of polysaccharide) was investigated for aqueous SEC (GFC) OHpak SB-86M HQ. The higher the concentration is, the broader the peak width will be and its elution time tends to become later. The viscosity is high in high molecules, the effect of the pullulan concentration is significant even in a low concentration. Sample concentrations are very important in an analysis of molecular weight distribution.
Sample : 100micro-L
Pullulan (Mw:788000)
Pullulan (Mw:404,000)
Pullulan (Mw:112,000)
Glucose (Mw:180)
Column : Shodex OHpak SB-806M HQ (8.0mmID*300mm) Eluent : H2O Flow rate : 1.0mL/min Detector : Shodex RI Column temp. : 40deg-C