Calibration Curves

A column, which has the most appropriated calibration curve for the sample molecular weight range should be selected. For example, regarding the two columns (1) and (2), whose calibration curves are shown in the figure, column (2) is best suited for the analysis of the mixture of A, B and C. In other words, among the column calibration curves which covers all the sample molecular weight range, a column whose calibration curve has the smallest inclination should be selected to obtain a good separation.

THF Eluent Generally, polystyrene (PS) standards are used as calibration standards for organic SEC (GPC) with THF eluent.
However, when there is a large difference between the structure of the polymer sample and that of the polystyrene, other standards may be used, if the structure is close to that of the polymer. Poly(ethylene glycol) (PEG) or poly(methyl methacrylate) (PMMA) can be used for this purpose.
Chloroform Eluent PS standards are commonly used for organic SEC (GPC) analysis with chloroform eluent.
DMF Eluent Polyethylene oxide (PEO) and PEG are used for the calibration standards of organic SEC (GPC) analysis with DMF eluent, when using KD-800 columns. If PS is used for the standard, PS elutes slower because PS is adsorbed by the packing material of KD-800 columns.
Different from KD-800 columns, PS as well as PEG and PEO can be used as calibration standards for the SB-800 HQ series with DMF eluent. For samples, which cannot be analyzed by the KD-800 columns because of the adsorption by the packing material, the SB-800 HQ series can be employed.
Toluene Eluent PS is used for the standard for organic SEC (GPC) analysis with toluene eluent.
Quinoline Eluent PEG is used for the standard for organic SEC (GPC) analysis with guinoline eluent.
Methanol Eluent PEO and PEG is used for the standard for organic SEC (GPC) analysis with methanol eluent because they are easily dissolved in methanol.
Aqueous Eluent Pullulan, PEO and PEG are used for Aqueous SEC (GFC) analysis with aqueous eluents, such as pure water or aqueous salt solution.

Calibration curves for GPC K-800 series are shown.

Sample : PS, Polystyrene
100micro-L each

Columns      : Shodex GPC K-800 series (8.0mmID*300mm each)
Eluent       : CHCl3
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Calibration curves for GPC K-800L series, linear type columns are shown.

Sample : PS, Polystyrene

Columns : Shodex GPC K-800L series (8.0mmID*300mm each)
Eluent  : CHCl3

Calibration curves for GPC KF-800 series are shown.

Sample : PS, Polystyrene

Columns  : Shodex GPC KF-800 series (8.0mmID*300mm each)
Eluent   : THF

Calibration curves for GPC KF-800L series, linear type columns are shown.

Sample : PS, Polystyrene

Columns : Shodex GPC KF-800L series (8.0mmID*300mm each)
Eluent  : THF

GPC KF-806M and KF-806L are both packed with mixed type gel. However, compared with KF-806M, KF-806L which was developed recently has better linearity in the calibration curve.

Sample : PS, Polystyrene

Columns : Shodex GPC KF-806M, KF-806L (8.0mmID*300mm each)
Eluent  : THF

Around the range of molecular weight 100, a solvent peak and/or other peaks coming from polymer additives may appear. For improving the separation in the low molecular weight range without affecting the separation in the high molecular weight range, it is recommended to use a solvent-peak separation column such as GPC KF-800DKF-600DKF-400D (for THF) or K-800D (for chloroform). The solvent-peak separation column should be used with linear type columns.

Sample : PS, Polystyrene

Calibration curves for GPC KF-600 series are shown.

Sample : PS, Polystyrene

Column : Shodex GPC KF-600 series (6.0mmID*150mm each)
Eluent : THF

These are calibration curves of GPC KF-401HQ to KF-404HQ.

Sample : PS, Polystyrene

Columns      : Shodex GPC KF-401HQ , KF-402HQ, KF-403HQ, KF-404HQ (4.6mmID*250mm each)
Eluent       : THF
Flow rate    : 0.3mL/min

These are calibration curves of GPC KF-405LHQ andKF-406LHQ.

Sample : PS, Polystyrene

Columns      : Shodex GPC KF-405LHQ x 2, KF-406LHQ x 2 (4.6mmID*250mm each)
Eluent       : THF
Flow rate    : 0.3mL/min
Column temp. : 40deg-C

Calibration curves are shown for the GPC KF-806L with the following three different eluents. The difference of the calibration curves can be explained by the difference of molecular sizes of polystyrene. The three dimensional size of polystyrene is considered to be different depending on the kind of solvent in which the polystyrene is dissolved.

Sample : PS, Polystyrene

Column       : Shodex GPC KF-806L (8.0mmID*300mm)
Eluent       : 1; DMF, 2; DMAc, 3; NMP
Column temp. : 40deg-C

DMF: Dimethylformamide
DMAc: Dimethylacetamide
NMP: N-Methylpyrrolidone

Calibration curves for GPC LF-804 is shown. The good linear line can be obtained in the range of molecular weights 300-2,000,000.

Sample : PS, Polystyrene

Columns      : Shodex GPC LF-804 (8.0mmID*300mm) x 3
Eluent       : THF
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Different kinds of pullulan were separated using organic SEC (GPC) column GPC LF-804. DMSO/DMF eluent is sometimes necessary to analyse starch. In this case, linear type LF-804 is effective.

Sample : 100micro-L
Pullulan (MW:1,600,000) 0.05%
Pullulan (MW:788000) 0.1%
Pullulan (MW:404,000) 0.1%
Pullulan (MW:212,000) 0.1%
Pullulan (MW:112,000) 0.1%
Pullulan (MW:47,300) 0.1%
Pullulan (MW:22,800) 0.1%
Pullulan (MW:11,800) 0.1%
Pullulan (MW:5,900) 0.1%
Maltotriose (MW:504) 0.1%
Maltose (MW:342) 0.1%
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

The calibration curve of PEG/PEO standards using organic SEC (GPC) column LF-804 with DMAc as an eluent is shown.

Sample : 200micro-L
PEO, Poly(ethylene oxide)
PEG, Poly(ethylene glycol)

Columns      : Shodex GPC LF-804 (8.0mmID*300mm) x 2
Eluent       : 30mM LiBr in DMAc
Flow rate    : 0.7mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

The calibration curve of polystyrene standards using organic SEC (GPC) column GPC LF-804 with DMAc as an eluent is shown.

Sample : 200micro-L
PS, Polystyrene

Columns      : Shodex GPC LF-804 (8.0mmID*300mm) x 2
Eluent       : 30mM LiBr in DMAc
Flow rate    : 0.7mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Poly(methyl methacrylate) (PMMA) standards were analyzed using organic SEC (GPC) column GPC LF-804.

Sample : Poly(methyl methacrylate) (PMMA)
100micro-L
(MW: 1,520,000) 0.05%
(MW: 772,000) 0.1%
(MW: 211,000) 0.1%
(MW:55,900) 0.1%
(MW:21,700) 0.1%
(MW:6,900) 0.1%
(MW:2,220) 0.1%

Columns      : Shodex GPC LF-804 (8.0mmID*300mm) x 2
Eluent       : Methyl ethyl ketone
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

GPC LF-404 and LF-604 are downsized columns of LF-804. Comparison of calibration curves for GPC LF-404 and LF-604 are shown. They show good linear lines as same as LF-804.

Sample : PS, Polystyrene

Columns      : Shodex GPC LF-404 (4.6mmID*250mm) x 2, Shodex GPC LF-604 (6.0mmID*150mm) x 2
Eluent       : THF
Flow rate    : 0.3mL/min(LF-404), 0.6mL/min(LF-604)
Detector     : Shodex RI(small cell volume)
Column temp. : 40deg-C

Calibration curve for GPC LF-604 is shown.

Sample : 20micro-L
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns      : Shodex GPC LF-604 (6.0mmID*150mm) x 2
Eluent       : DMF
Flow rate    : 0.5mL/min
Detector     : Shodex RI(small cell volume)
Column temp. : 50deg-C

Calibration curve for GPC LF-604 is shown.

Sample : 20micro-L
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns     : Shodex GPC LF-604 (6.0mmID*150mm) x 2
Eluent      : NMP
Flow rate   : 0.3mL/min
Detector    : Shodex RI(small cell volume)
Column temp.: 50deg-C

Calibration curve for GPC LF-604 is shown.

Sample : PS, Polystyrene, 20micro-L

Columns     : Shodex GPC LF-604 (6.0mmID*150mm) x 2
Eluent      : NMP
Flow rate   : 0.3mL/min
Detector    : Shodex RI(small cell volume)
Column temp.: 50deg-C

Calibration curves for GPC KD-800 series are shown.

Sample : PEO, Poly(ethylene oxide)

Columns : Shodex GPC KD-800 series (8.0mmID*300mm)
Eluent  : DMF

Calibration curves for GPC HFIP-800 series are shown.

Sample :
PMMA, Poly(methyl methacrylate)
Ethyl methacrylate

Columns      : Shodex GPC HFIP-800 series (8.0mmID*300mm each)
Eluent       : HFIP
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : Room temp.

Calibration curves for HFIP-600 series are shown.

Sample : PMMA, Poly(methyl methacrylate)

Columns      : Shodex GPC HFIP-600 series (6.0mmID*150mm)
Eluent       : 5mM CF3COONa in HFIP
Detctor      : Shodex RI(small cell volume)
Column temp. : 40deg-C

Calibration curves for GPC KD-800 series are shown.

Sample : PEO, Poly(ethylene oxide)

Columns : Shodex GPC KD-800 series (8.0mmID*300mm)
Eluent  : DMF

Calibration curves for GPC HFIP-800 series are shown.

Sample :
PMMA, Poly(methyl methacrylate)
Ethyl methacrylate

Columns      : Shodex GPC HFIP-800 series (8.0mmID*300mm each)
Eluent       : HFIP
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : Room temp.

Calibration curves for PROTEIN KW-800 series (columns for aqueous SEC (GFC)) are shown.

Sample :
1. Thyroglobulin (MW: 669,000)
2. Aldolase (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. Adenylate kinase (MW: 32,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Columns      : Shodex PROTEIN KW-800 series (8.0mmID*300mm each)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Calibration curves for PROTEIN KW-802.5 (a column for Aqueous SEC (GFC)) are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)
Proteins

Column       : Shodex PROTEIN KW-802.5 (8.0mmID*300mm)
Eluent       : (Proteins); 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
               (Pullulan, PEG, PEO); H2O
Flow rate    : 1.0mL/min
Detector     : (Proteins); UV(220nm), (Pullulan, PEG, PEO); Shodex RI 
Column temp. : Room temp.

Calibration curves for PROTEIN KW-803 (a column for Aqueous SEC (GFC)) are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)
Proteins

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : (Proteins); 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
               (Pullulan, PEG, PEO); H2O
Flow rate    : 1.0mL/min
Detector     : (Proteins); UV(220nm), (Pullulan, PEG, PEO); Shodex RI 
Column temp. : Room temp.

Calibration curves for PROTEIN KW-804 (a column for Aqueous SEC (GFC)) are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)
Proteins

Column       : Shodex PROTEIN KW-804 (8.0mmID*300mm)
Eluent       : (Proteins); 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
               (Pullulan, PEG, PEO); H2O
Flow rate    : 1.0mL/min
Detector     : (Proteins); UV(220nm), (Pullulan, PEG, PEO); Shodex RI 
Column temp. : Room temp.

KW400-4F series is aqueous SEC (GFC) columns and downsized KW-800 series. Figure is calibration curves of KW400-4F series columns with pullulan. KW405-4F is newly line-upped column, and it was designed having larger pore size than KW-804. KW405-4F can analyze high molecular weight substances more than 1,000,000 as protein like agglomerated protein. KW400-4F series can be applied wider range of molecular weight substances than KW-800 series.

Sample : Pullulan

Columns      : Shodex KW400-4F series (4.6mmID*300mm each)
Eluent       : H2O
Flow rate    : 0.33mL/min
Detector     : Shodex RI (small cell volume)
Column temp. : Room temp.

KW400-4F series is aqueous SEC (GFC) columns and downsized KW-800 series. Figure is calibration curves of KW400-4F series columns with pullulan. KW405-4F newly line-upped column, and it was designed having larger pore size than KW-804. KW405-4F can analyze high molecular weight substances more than 1,000,000 as protein like agglomerated protein. KW400-4F series can be applied wider range of molecular weight substances than KW-800 series.

Sample : Proteins

Columns      : Shodex KW400-4F series (4.6mmID*300mm each)
Eluent       : 50mM Sodium phosphate buffer (pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

KW400-4F series is aqueous SEC (GFC) columns and downsized KW-800 series. Figure is calibration curves of KW400-4F series columns with pullulan. KW405-4F newly line-upped column, and it was designed having larger pore size than KW-804. KW405-4F can analyze high molecular weight substances more than 1,000,000 as protein like agglomerated protein. KW400-4F series can be applied wider range of molecular weight substances than KW-800 series.

Sample :
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns      : Shodex KW400-4F series (4.6mmID*300mm each)
Eluent       : H2O
Flow rate    : 0.33mL/min
Detector     : Shodex RI
Column temp. : 25deg-C

Calibration curves for OHpak SB-800 HQ series are shown.

Sample :
Pullulan
Maltotriose
Maltose
Glucose

Columns      : Shodex OHpak SB-800 HQ series (8.0mmID*300mm each)
Eluent       : H2O
Detector     : Shodex RI 
Column temp. : Room temp.

Calibration curves for OHpak SB-800 HQ series are shown.

Sample :
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns : Shodex OHpak SB-800 HQ series (8.0mmID*300mm each)
Eluent  : H2O

Calibration curves for OHpak SB-800 HQ series are shown.

Sample : PS, Polystyrene

Columns : Shodex OHpak SB-800 HQ series (8.0mmID*300mm each)
Eluent  : DMF

Calibration curves for OHpak SB-800 HQ series are shown.

Sample :
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns      : SHodex OHpak SB-800 HQ series (8.0mmID*300mm each)      
Eluent       : 20mM LiBr in DMF
Flow rate    : 0.8mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Calibration curves for OHpak SB-800 HQ series are shown.

Sample : PMMA, Poly(methyl methacrylate)

Columns      : Shodex OHpak SB-800 HQ series (8.0mmID*300mm each)
Eluent       : 20mM LiBr in DMF
Flow rate    : 0.8mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Calibration curves for OHpak SB-806M HQ are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns      : Shodex OHpak SB-G + SB-806M HQ (8.0mmID*300mm) x 2
Eluent       : 50mM NaNO3 aq.
Flow rate    : 1.0mL/min
Detector     : Shodex RI
Column temp. : 35deg-C

Calibration curves for OHpak SB-806M HQ are shown. For samples, which cannot be analyzed using the GPC KD-800 columns because of the adsorption by the packing material, the OHpak SB-800 HQ columns can be employed.
However, frequent replacement of in-column eluent is not recommended as it may shorten column life.

Sample :
PEO, Poly(ethylene oxide)
PEG, Poly(ethylene glycol)
PS, Polystyrene

Column       : Shodex OHpak SB-806M HQ (8.0mmID*300mm)
Eluent       : DMF
Column temp. : 40deg-C

SB400-4E series is aqueous SEC (GFC) type columns and downsized SB-800 HQ series. Figure is calibration curves of SB400-4E series columns with pullulan. SB401-4E is newly line-upped column, and it was designed having smaller pore size than SB-802 HQ.

Sample : Pullulan
Maltotriose
Maltose
Glucose

Columns      : Shodex SB400-4E series (4.6mmID*300mm each)
Eluent       : H2O
Flow rate    : 0.3mL/min
Detector     : Shodex RI(small cell volume)
Column temp. : Room temp.

Calibration curves for GPC KD-800 series and OHpak SB-800 HQseries are shown. Compared with the KD-800 columns, the SB-800 HQ columns perform better over a low molecular weight range.

Sample :
PEO, Poly(ethylene oxide)
PEG, Poly(ethylene glycol)

Columns      : Shodex OHpak SB-805 HQ + SB-802.5 HQ (8.0mmID*300mm each)
               Shodex GPC KD-805 + KD-802.5 (8.0mmID*300mm each)
Eluent       : DMF
Column temp. : 40deg-C

For the calibration curve for GPC KD-802.5 , PEG and PEO should be used as the standards. The calibration curve with PS is far different from that with PEG and PEO. For the calibration curve for OHpak SB-802.5 HQ, both PEG, PEO and PS can be used as the standards. The calibataion cuve with PS is almost as same as that with PEG and PEO.

Sample :
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)
PS, Polystyrene

Columns      : Shodex GPC KD-802.5, OHpak SB-802.5 HQ (8.0mmID*300mm each)
Eluent       : DMF
Column temp. : 40deg-C

Calibration curves for Asahipak GS-HQ series are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)

Columns      : Shodex Asahipak GS-HQ series (7.5mmID*300mm each)
Eluent       : H2O
Flow rate    : 0.6mL/min
Detector     : RI
Column temp. : 30deg-C

A calibration curve for Asahipak GS-520 7G (a multimode column) is shown. When an eluent with a relatively high salt concentration is used for Asahipak GS series, substances are eluted in decreasing order of molecular weight as it is the GFC mode which function mainly. GS-520, which has a large pore size, can analyze proteins with molecular weight up to 1,000,000. Samples such as human and bovine serum albumins which exhibit strong affinity with the molecular structure of the packing material are eluted belatedly. Making use of this characteristics, GS-520 is used exclusively for the analysis of serum albumin.
GS-520 7G is an old type of GS-520 HQ.

Sample :
1. IgM
2. IgA
3. IgG
4. Transferrin
5. BSA
6. Ovalbumin
7. Chymotrypsinogen A
8. Myoglobin
9. Ribonuclease
10. Cytochrome c

Column       : Shodex Asahipak GS-520 7G (7.5mmID*500mm)
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.3M NaCl 
Flow rate    : 1.0mL/min
Detector     : UV(210nm)
Column temp. : Room temp.

Calibration curves for Asahipak GF-510 HQ and GF-7M HQ (columns for GFC separation) are shown.

Sample:
1. IgM
2. IgA
3. IgG
4. Transferrin
5. Conalbumin
6. BSA
7. Ovalbumin
8. Myoglobin
9. Ribonuclease A
10. Cytochrome c
11. L-Phenylalanin
12. Creatinine

Columns      : Shodex Asahipak GF-510 HQ (7.5mmID*300mm)       : GF-7M HQ (7.5mmID*300mm) 
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.1M NaCl : 0.2M Sodium phosphate buffer(pH7.0)
Flow rate    : 0.5mL/min                                       : 0.5mL/min
Detector     : UV(280nm)                                : UV(280nm)
Column temp. : 30deg-C                                         : 30deg-C

Calibration curves for Asahipak GF-HQ series are shown.

Sample : PS, Polystyrene

Columns      : Shodex Asahipak GF-HQ series (7.5mmID*300mm each)
Eluent       : CHCl3
Flow rate    : 0.6mL/min
Detector     : UV(254nm)
Column temp. : 30deg-C

Calibration curves for Asahipak GF-HQ series are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)

Columns      : Shodex Asahipak GF-HQ series (7.5mmID*300mm each)
Eluent       : H2O
Flow rate    : 0.6mL/min
Detector     : RI
Column temp. : 30deg-C

Calibration curves for Asahipak GF-HQ seires are shown.

Sample : PS, Polystyrene

Columns      : Shodex Asahipak GF-HQ series (7.5mmID*300mm)
Eluent       : 20mM LiBr in DMF
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Calibration curves for Asahipak GF-HQ series are shown.

Sample :
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns      : Shodex Asahipak GF-HQ series (7.5mmID*300mm each)
Eluent       : 20mM LiBr in DMF
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Calibration curves for Asahipak GF-HQ series are shown.

Sample : PMMA, Poly(methyl methacrylate)

Columns      : Shodex Asahipak GF-HQ series (7.5mmID*300mm each)
Eluent       : 20mM LiBr in DMF
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

Calibration curves for Asahipak GF-HQ series are shown using acetone eluent for PMMA.

Sample :
PMMA, Poly(methylmethacrylate) (MW:772,000)
PMMA, Poly(methyl methacrylate) (MW:211,000)
PMMA, Poly(methyl methacrylate) (MW:55,900)
PMMA, Poly(methyl methacrylate) (MW:21,700)
PMMA, Poly(methyl methacrylate) (MW:6,900)
PMMA, Poly(methyl methacrylate) (MW:2,220)

Column       : Shodex Asahipak GF-310 HQ, GF-510 HQ, GF-710 HQ, GF-7M HQ (7.5mmID*300mm each)
Eluent       : Acetone
Flow rate    : 0.6mL/min
Detector     : Shodex RI
Column temp. : 40deg-C

A calibration curve for Asahipak GF-510 HQ + GF-310 HQ is shown.

Sample : PS, Polystyrene

Column       : Shodex Asahipak GF-510 HQ + GF-310 HQ (7.5mmID*300mm each)
Eluent       : THF
Flow rate    : 0.5mL/min
Detetctor    : UV(254nm)
Column temp. : 30deg-C