Aqueous SEC (GFC) Columns
For analyzing substances using aqueous SEC (GFC) mode, there are two kinds of columns: silica-based columns and polymer-based columns.
Generally, silica gel provides better resolution because the pore size distribution is sharper and the pore volume is larger than polymer-based columns. However, as silica gel with large pore size cannot be used for GFC analysis because of it brittleness, polymer-based column is suitable for the separation of macromolecules.
In general, silica-based columns are recommended for protein separations of low molecular weight from a few thousand to approximately one million. And polymer-based columns would be best used for the analysis of proteins, lipoproteins or DNA with molecular weights of more than one million. OHpak SB-800 HQ series and SB400-4E series packed with polyhydroxymethacrylate gel allow the analysis of proteins of low molecular weight range of less than 10,000, for which silica-based columns would show low resolution.
Silica-based columns cannot be used with the eluent whose pH is 7.5 or more because silica gel is dissolved in such eluent. In contrast, polymer-based columns have a wider usable eluent pH range and these columns can be washed with alkaline solution, resulting in longer column life. The usable pH range for OHpak SB-800 HQ series is from 3 to 10, whereas the usable pH range for Asahipak GF-HQ series is form 2 to 9.
The packing material of Asahipak GF-HQ or GS-HQ series is a highly cross-linked polyvinyl alcohol gel. Their packing material delays the elution of substances such as human and bovine serum albumins which show strong affinity with the molecular structure of the packing material due to interaction with the molecular structure of the packing material. Making use of this characteristics, Asahipak GS-520 HQ is especially used for the analysis of serum albumin.
Column Specifications
| KW-800 | · Silica-based packedcolumns for aqueous SEC (GFC) analysis · Suitable for the analysis of proteins and enzymes |
(Standard Columns)
| Product code | Product name | Plate number (TPN/column) |
Exclusion limit | Particle size (micron) |
Size(mm) ID x Length |
|
|---|---|---|---|---|---|---|
| (Pullulan) | (Proteins) | |||||
| F6989000 F6989103 F6989104 F6700131 |
PROTEIN KW-802.5 PROTEIN KW-803 PROTEIN KW-804 PROTEIN KW-G |
> 21,000
> 21,000 > 16,000 Guard column |
60,000 170,000 500,000 – |
150,000 700,000 *(1,000,000) – |
5 5 7 7 |
8.0 x 300 8.0 x 300 8.0 x 300 6.0 x 50 |
| Product name |
Max. usable pressure (MPa) |
Recommended flow rate (mL/min) |
Max. usable flow rate (mL/min) |
Max. usable temp. (deg-C) |
Usable pH range |
Usable salt conc. |
Shipping solvent |
|---|---|---|---|---|---|---|---|
| KW-802.5 KW-803 KW-804 KW-G |
5.0
5.0 5.0 – |
1.0
1.0 1.0 – |
1.5
1.5 1.5 – |
45
45 45 45 |
3.0 to 7.5
3.0 to 7.5 3.0 to 7.5 3.0 to 7.5 |
> 0.3M
> 0.3M > 0.3M > 0.3M |
H2O
H2O H2O H2O |
*( ): EStimated value
Usable organic solvent concentration: Up to 100% for methanol and acetonitrile.
(Preparative Columns)(Remark)
| Product code | Product name | Plate number (TPN/column) |
Particle ize (micron) |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|---|---|
| PROTEIN KW-2002.5 PROTEIN KW-2003 PROTEIN KW-2004 PROTEIN KW-LG |
> 17,000
> 17,000 > 14,000 Guard column |
5 5 7 7 |
20.0 x 300 20.0 x 300 20.0 x 300 8.0 x 50 |
KW-802.5
KW-803 KW-804 Guard column |
| KW400 | · Reducing particle size of the packing materials ebhances column performance · Three- or four- fold higher sensitivity than KW-800 columns · KW405-4F is applicable to samples with a molecular weight higher than 1 million |
(High Performance Semi-microcolumns)
| Product code | Product name | Plate number (TPN/column) |
Exclusion limit | Particle size (micron) |
Size(mm) ID x Length |
|
|---|---|---|---|---|---|---|
| (Pullulan) | (Proteins) | |||||
| F6989201 F6989202 F6989203 F6989204 F6700132 |
KW402.5-4F KW403-4F KW404-4F KW405-4F KW400G-4A |
> 35,000
> 35,000 > 25,000 > 25,000 Guard column |
60,000 150,000 500,000 1,300,000 – |
150,000 600,000 *(1,000,000) *(20,000,000) – |
3 3 5 5 5 |
4.6 x 300 4.6 x 300 4.6 x 300 4.6 x 300 4.6 x 10 |
| Product name |
Max. usable pressure (MPa) |
Recommended flow rate (mL/min) |
Max. usable flow rate (mL/min) |
Usable temp. range (deg-C) |
Usable pH range |
Usable salt conc. |
Shipping solvent |
|---|---|---|---|---|---|---|---|
| KW402.5-4F KW403-4F KW404-4F KW405-4F KW400G-4A |
10
10 10 7 – |
0.35
0.35 0.35 0.35 – |
0.5
0.5 0.5 0.5 – |
5 to 45
5 to 45 5 to 45 5 to 45 5 to 45 |
3.0 to 7.5
3.0 to 7.5 3.0 to 7.5 3.0 to 7.5 3.0 to 7.5 |
> 0.3M
> 0.3M > 0.3M > 0.3M > 0.3M |
H2O
H2O H2O H2O H2O |
*( ): Estimated value
Usable organic solvent concentration: Up to 100% for methanol and acetonitrile.
(Micro column and Semimicro columns)(Remark)
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| KW402.5-4D KW402.5-4B KW402.5-2E KW402.5-2D KW402.5-2B KW402.5-1E KW402.5-1D KW402.5-1B |
4.6 x 150
4.6 x 50 2.0 x 250 2.0 x 150 2.0 x 50 1.0 x 250 1.0 x 150 1.0 x 50 |
KW402.5
|
|
| KW402.5-M8E KW402.5-M8D KW402.5-M8B KW402.5-M5E KW402.5-M5D KW402.5-M5B KW402.5-M3E KW402.5-M3D KW402.5-M3B |
0.8 x 250
0.8 x 150 0.8 x 50 0.5 x 250 0.5 x 150 0.5 x 50 0.3 x 250 0.3 x 150 0.3 x 50 |
KW402.5
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| KW403-4D KW403-4B KW403-2E KW403-2D KW403-2B KW403-1E KW403-1D KW403-1B |
4.6 x 150
4.6 x 50 2.0 x 250 2.0 x 150 2.0 x 50 1.0 x 250 1.0 x 150 1.0 x 50 |
KW403
|
|
| KW403-M8E KW403-M8D KW403-M8B KW403-M5E KW403-M5D KW403-M5B KW403-M3E KW403-M3D KW403-M3B |
0.8 x 250
0.8 x 150 0.8 x 50 0.5 x 250 0.5 x 150 0.5 x 50 0.3 x 250 0.3 x 150 0.3 x 50 |
KW403
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| KW404-4D KW404-4B KW404-2E KW404-2D KW404-2B KW404-1E KW404-1D KW404-1B |
4.6 x 150
4.6 x 50 2.0 x 250 2.0 x 150 2.0 x 50 1.0 x 250 1.0 x 150 1.0 x 50 |
KW404
|
|
| KW404-M8E KW404-M8D KW404-M8B KW404-M5E KW404-M5D KW404-M5B KW404-M3E KW404-M3D KW404-M3B |
0.8 x 250
0.8 x 150 0.8 x 50 0.5 x 250 0.5 x 150 0.5 x 50 0.3 x 250 0.3 x 150 0.3 x 50 |
KW404
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| KW405-4D KW405-4B KW405-2E KW405-2D KW405-2B KW405-1E KW405-1D KW405-1B |
4.6 x 150
4.6 x 50 2.0 x 250 2.0 x 150 2.0 x 50 1.0 x 250 1.0 x 150 1.0 x 50 |
KW405
|
|
| KW405-M8E KW405-M8D KW405-M8B KW405-M5E KW405-M5D KW405-M5B KW405-M3E KW405-M3D KW405-M3B |
0.8 x 250
0.8 x 150 0.8 x 50 0.5 x 250 0.5 x 150 0.5 x 50 0.3 x 250 0.3 x 150 0.3 x 50 |
KW405
|
| SB-800 HQ | · Polymer-based packed columns for aqueous SEC (GFC) analysis · Supports a wide range of molecular weights · The eluent can be substituted with DMF (in columns other tha SB-802 HQ and SB-807 HQ), enabling the analysis of polar polymers · SB-804 HQ and SB-805 HQ are capable of determining the mean molecular weight od the gelatin compliant with PAGI method (ver. 10, Japan) |
| SB-807 HQ | · Column for the analysis of water -soluble ultra-high molecular polymer · Packing materials with a large particle size are packed in order to prevent the breakage of molecular chains |
(Standard Columns)
| Product code | Product name | Plate number (TPN/column) |
Exclusion limit (Pullulan) |
Particle size (micron) |
Size(mm) ID x Length |
|---|---|---|---|---|---|
| F6429100 F6429101 F6429102 F6429103 F6429104 F6429105 F6429106 F6709430 |
OHpak SB-802 HQ OHpak SB-802.5 HQ OHpak SB-803 HQ OHpak SB-804 HQ OHpak SB-805 HQ OHpak SB-806 HQ OHpak SB-806M HQ OHpak SB-G |
> 12,000
> 16,000 > 16,000 > 16,000 > 12,000 > 12,000 > 12,000 Guard column |
4,000 10,000 100,000 1,000,000 *(4,000,000) *(20,000,000) *(20,000,000) – |
8 6 6 10 13 13 13 10 |
8.0 x 300 8.0 x 300 8.0 x 300 8.0 x 300 8.0 x 300 8.0 x 300 8.0 x 300 6.0 x 50 |
| Product name |
Max. usable organic solvents(%) |
Max. usable presure (MPa) |
Max. usable flow rate (mL/min) |
Max. usable temp. (deg-C) |
Usable pH range |
Shipping solvent |
||
|---|---|---|---|---|---|---|---|---|
| CH3OH | CH3CN | DMF | ||||||
| SB-802 HQ SB-802.5 HQ SB-803 HQ SB-804 HQ SB-805 HQ SB-806 HQ SB-806M HQ SB-G |
0 100 100 75 75 75 75 75 |
0 75 75 75 75 75 75 75 |
0 100 100 100 100 100 100 100 |
5.0 5.0 5.0 3.0 3.0 3.0 3.0 – |
1.2 1.2 1.2 1.2 1.2 1.2 1.2 – |
70 70 70 70 70 70 70 70 |
3 to 10 3 to 10 3 to 10 3 to 10 3 to 10 3 to 10 3 to 10 3 to 10 |
0.02% NaN3 aq.
0.02% NaN3 aq. 0.02% NaN3 aq. 0.02% NaN3 aq. 0.02% NaN3 aq. 0.02% NaN3 aq. 0.02% NaN3 aq. 0.02% NaN3 aq. |
*( ): Estimated value
CH3OH: Methanol, CH3CN: Acetonitrile, DMF: Dimethyformamide
High performance semi-micro column
| Product code | Product name | Plate number (TPN/column) |
Exclusion limit (Pullulan) |
Particle size (micron) |
Size(mm) ID x Length |
|---|---|---|---|---|---|
| F6429108 F6709431 |
OHpak SB-807 HQ OHpak SB-807G |
> 1,500
Guard column |
*(500,000,000) – |
35 35 |
8.0 x 300 8.0 x 50 |
| Product name |
Max. usable organic solvents(%) |
Max. usable presure (MPa) |
Max. usable flow rate (mL/min) |
Max. usable temp. (deg-C) |
Usable pH range |
Shipping solvent |
||
|---|---|---|---|---|---|---|---|---|
| CH3OH | CH3CN | DMF | ||||||
| SB-807 HQ SB-807G |
30 30 |
30 30 |
0 0 |
0.5 – |
1.5 – |
60 60 |
3 to 10 3 to 10 |
H2O
H2O |
*( ): Estimated value
CH3OH: Methanol, CH3CN: Acetonitrile, DMF: Dimethyformamide
(Micro column and Semi-micro columns)(Remark)
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| SB802-4E SB802-4D SB802-4B SB802-2E SB802-2D SB802-2B SB802-1E SB802-1D SB802-1B |
4.6×250
4.6×150 4.6×50 2.0×250 2.0×150 2.0×50 1.0×250 1.0×150 1.0×50 |
SB-802 HQ
|
|
| SB802-M8E SB802-M8D SB802-M8B SB802-M5E SB802-M5D SB802-M5B SB802-M3E SB802-M3D SB802-M3B |
0.8×250
0.8×150 0.8×50 0.5×250 0.5×150 0.5×50 0.3×250 0.3×150 0.3×50 |
SB-802 HQ
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| SB802.5-4E SB802.5-4D SB802.5-4B SB802.5-2E SB802.5-2D SB802.5-2B SB802.5-1E SB802.5-1D SB802.5-1B |
4.6×250
4.6×150 4.6×50 2.0×250 2.0×150 2.0×50 1.0×250 1.0×150 1.0×50 |
SB-802.5 HQ
|
|
| SB802.5-M8E SB802.5-M8D SB802.5-M8B SB802.5-M5E SB802.5-M5D SB802.5-M5B SB802.5-M3E SB802.5-M3D SB802.5-M3B |
0.8×250
0.8×150 0.8×50 0.5×250 0.5×150 0.5×50 0.3×250 0.3×150 0.3×50 |
SB-802.5 HQ
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| SB803-4E SB803-4D SB803-4B SB803-2E SB803-2D SB803-2B SB803-1E SB803-1D SB803-1B |
4.6×250
4.6×150 4.6×50 2.0×250 2.0×150 2.0×50 1.0×250 1.0×150 1.0×50 |
SB-803 HQ
|
|
| SB803-M8E SB803-M8D SB803-M8B SB803-M5E SB803-M5D SB803-M5B SB803-M3E SB803-M3D SB803-M3B |
0.8×250
0.8×150 0.8×50 0.5×250 0.5×150 0.5×50 0.3×250 0.3×150 0.3×50 |
SB-803 HQ
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| SB804-4E SB804-4D SB804-4B SB804-2E SB804-2D SB804-2B SB804-1E SB804-1D SB804-1B |
4.6×250
4.6×150 4.6×50 2.0×250 2.0×150 2.0×50 1.0×250 1.0×150 1.0×50 |
SB-804 HQ
|
|
| SB804-M8E SB804-M8D SB804-M8B SB804-M5E SB804-M5D SB804-M5B SB804-M3E SB804-M3D SB804-M3B |
0.8×250
0.8×150 0.8×50 0.5×250 0.5×150 0.5×50 0.3×250 0.3×150 0.3×50 |
SB-804 HQ
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| SB805-4E SB805-4D SB805-4B SB805-2E SB805-2D SB805-2B SB805-1E SB805-1D SB805-1B |
4.6×250
4.6×150 4.6×50 2.0×250 2.0×150 2.0×50 1.0×250 1.0×150 1.0×50 |
SB-805 HQ
|
|
| SB805-M8E SB805-M8D SB805-M8B SB805-M5E SB805-M5D SB805-M5B SB805-M3E SB805-M3D SB805-M3B |
0.8×250
0.8×150 0.8×50 0.5×250 0.5×150 0.5×50 0.3×250 0.3×150 0.3×50 |
SB-805 HQ
|
|
Product code |
Product name |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|
| SB806-4E SB806-4D SB806-4B SB806-2E SB806-2D SB806-2B SB806-1E SB806-1D SB806-1B |
4.6×250
4.6×150 4.6×50 2.0×250 2.0×150 2.0×50 1.0×250 1.0×150 1.0×50 |
SB-806 HQ
|
|
| SB806-M8E SB806-M8D SB806-M8B SB806-M5E SB806-M5D SB806-M5B SB806-M3E SB806-M3D SB806-M3B |
0.8×250
0.8×150 0.8×50 0.5×250 0.5×150 0.5×50 0.3×250 0.3×150 0.3×50 |
SB-806 HQ
|
(Preparative Columns)(Remark)
| Product code | Product name | Plate number (TPN/column) |
Particle size (micron) |
Size(mm) ID x Length |
Standard column |
|---|---|---|---|---|---|
| OHpak SB-2002 OHpak SB-2002.5 OHpak SB-2003 OHpak SB-2004 OHpak SB-2005 OHpak SB-2006 OHpak SB-2006M OHpak SB-LG |
> 9,000
> 12,000 > 12,000 > 12,000 > 12,000 > 12,000 > 12,000 Guard column |
15
10 10 18 20 20 20 18 |
20.0 x 300 20.0 x 300 20.0 x 300 20.0 x 300 20.0 x 300 20.0 x 300 20.0 x 300 8.0 x 50 |
SB-802 HQ
SB-802.5 HQ SB-803 HQ SB-804 HQ SB-805 HQ SB-806 HQ SB-806M HQ Guard column |
Exclusion limits and pore sizes are as same as those of corresponding standard columns.
For all products
Max. usable flow rate: 5.0mL/min
Max. usable pressure: 2.0 MPa
Max. usable temperature: 60deg-C
| SB400 | · Three- or four- fold higher sensitivity than the SB-800 HQ columns · SB401-4E can analyze small molecules with MW of under 1,000 |
(High perfonmance semi-micro Columns)
| Product code | Product name | Plate number (TPN/column) |
Exclusion limit (Pullulan) |
Particle size (micron) |
Size(mm) ID x Length |
|---|---|---|---|---|---|
| F6429111 F6429112 F6429113 F6429114 F6709432 |
SB401-4E SB402.5-4E SB403-4E SB404-4E SB400G-4A |
> 5,000
> 20,000 > 20,000 > 20,000 Guard column |
1,000 10,000 100,000 1,000,000 – |
10 6 6 7 7 |
4.6 x 250 4.6 x 250 4.6 x 250 4.6 x 250 4.6 x 50 |
| Product name |
Max. usable organic solvents(%) |
Max. usable presure (MPa) |
Max. usable flow rate (mL/min) |
Usable temp. range (deg-C) |
Usable pH range |
Shipping solvent | ||
|---|---|---|---|---|---|---|---|---|
| CH3OH | CH3CN | DMF | ||||||
| SB401-4E SB402.5-4E SB403-4E SB404-4E SB400G-4A |
0 100 100 75 75 |
10 75 75 75 75 |
0 100 100 100 100 |
1.8 8.0 8.0 5.0 – |
0.30 0.35 0.35 0.35 0.35 |
5 to 70 5 to 70 5 to 70 5 to 70 5 to 70 |
3 to 10 3 to 10 3 to 10 3 to 10 3 to 10 |
H2O
H2O H2O H2O H2O |
CH3OH: Methanol, CH3CN: Acetonitrile, DMF: Dimethyformamide
Column Information
Eluent conditions for SB-800 HQ series and SB400 series columns
Some nonionic sample can be analyzed with the use of deionized water as an eluent.
As the eluent, either a saltaqueous solution or buffer solution with or without a polar organic solvent added to it. The followings are how to select the eluent to be used.
1) Ionic and nonionic hydrophilic samles.
Salt solutions or buffer solutions are generally used as the eluent. Typical aqueous salt solutions are as follows:
|
(Aqueous salt solutions)
|
(Buffer solutions)
|
|
|---|---|---|
|
Sodium chloride aqueous solution
Sodium nitrate aqueous solution Sodium sulfate aqueous solution Potasim sulfate aqueous solution Ammonium sulfate aqueous solution |
Phosphoric acid buffer solution
Trishydrochroric acid buffer solution Acetic acid buffer solution Citric acid buffer solution |
CAUTION
(1) Use of salt concentration in the range of 0.05M to 0.3M is recommended. Maxinium usable salt concentration is 0.5M.
(2) pH of eluent should be 3.0 to 10.0.
(3) pH of eluent should be higher than 6.0 when the eluent contains chloride ions.
(4) Boric acid buffer solution is not recommended because di-ol groups of packing material and boric acid make a complex.
2) Nonionic and ionic hydrophobic samples
Addition of polar organic solvents in the eluent is recommended to decrease the hydrophobic adosorption. Addition of salts in the eluent is also recomended when the sample is ionic.
Upper limit of concentration of polar organic solvents are as follows: Polymer-based GFC Columns: OHpak SB-800 HQ, High Perfonmance Semi-micro Polymer-based Aqueous SEC (GFC) Columns: SB400
CAUTION
Flow rate should be slower than 0.5mL/min when in-column solvent is changed to an eluent containing polar organic solvent.
In the case of DMSO, flow rate should be slower than 0.3mL/min and column temperature should be 50deg-C or higher.
3) Protein samples
Urea and 6M guanidine aqueous solutions, which are commnly used as protein modifier, can be used as the eluent. The eluent containing surfactants, such as SDS or Brij-35, is recoummended for the samples, such as membrane proteins, of which solubility in water is poor.
CAUTION
Frequent replacement of the in-column eluent from normal solvent to the above solvent will make the column life short. In such case, it is recommended to use one colunm specifically for the purpose.
Eluent conditions for KW400-4F series and KW-800 series columns
1) Typical eluent
The following solutions are generally used as the eluent.
Phosphate buffer solution
Tris hydrochloric solution
Acetate buffer solution
2) Salt to be added
Salts such as Na2SO4, K2SO4 and (NH4)2SO4 are usually added to the above mentioned buffer solutions. The suitable salt concentration is 0.1 to 0.3M.
CAUTlON
(1) The pH range of the eluent should be 3.0 to 7.5 range.
(2) Use of salts containing chlorine ions should preferably be avoided, as chlorine ions are corrosive to the column or piping of the instrument.
3) Use of urea or guanidlne hydrochloride
Urea or 6M guanidine hydrochloride solutions, which are often used as modifier of proteins, can be used as the eluent.
CAUTION
Since the replacement of the eluent takes a fairly long time, it is recommended to use one column specifically for such an eluent.
4) Use of surfactant
Aqueous solutions of surfactant such as SDS and Brij can also be used.
5) Use of polar organic solvent
Polar organic solvents, such as acetonitrile, methanol and ethanol can also be used as the eluent.
SEC is a method which uses the principle that a larger molecular size(three dimensional volume) sample elutes faster. In fact, the actual molecular size of the sample is different according to the solvent in which the sample is dissolved. In the case of polymers which have ionic functional groups, the ionic functional groups repel each other and the apparent molecular size of the polymers becomes larger. To prevent such ionic dissociation, the addition of salts such as NaNO3 or NaCl is known to be effective.
Especially, in the case of hydrophilic polymers, the existence of small amounts of ionic functional groups affect the peak pattern and measurement results. Addition of salts to the eluent is recommended even when it is considered that the sample polymer have no ionic functional groups.
(Left): Principle of SEC
(Right, above): Large apparent size because of repelling
(Right, below): Ordinary size