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 HQHigh 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