Fast Ion Exchange Analysis
Non-porous gel has high mechanical strength, which enables the separation at high flow rates for fast analysis.
The differences between porous and non-porous gel can be summarized as follows:
1) Max. working flow rate: DEAE-825 and SP-825 (packed with porous gel) : 1.5mL/min.
DEAE3N-4T and SP-420N (pack with non-porous gel) : 2.0mL/min.
2)With the non-porous gel, the sample does not diffuse inside the pores of the packing material and the salt concentration of eluent equilibrates quickly, therefore sharp peaks can be obtained. However the maximum sample load is lower:
DEAE-825 and SP-825 (packed with porous gel) : 1,000micro-g/column
DEAE3N-4T and SP-420N (pack with non-porous gel) : 20micro-g/column
3) In conclusion, columns packed with non-porous gel is suited for fast analysis of proteins and peptides, especially for the analysis for tracing the amount of samples.
Recovery of proteins was measured using columns for ion exchange chromatography packed with non-purous gel.
DEAE-420N is an old type of DEAE3N-4T.
| Recovery of protein by IEC DEAE-420N |
|
|---|---|
| Protein | Recovery(%) |
| Transferrin | 94 |
| Ovalbumin | 94 |
| Trypsin inhibitor | 106 |
| BSA | 92 |
| alpha-Lactalbumin | 93 |
| beta-Lactoglobulin | 88 |
| Recovery of protein by IEC SP-420N |
|
|---|---|
| Protein | Recovery(%) |
| alpha Chymotrypsin | 95 |
| alpha Chymotrypsinogen A | 91 |
| Myoglobin | 92 |
| Lysozyme | 99 |
| Ribonuclease A | 91 |
| Cytochrome c | 96 |
| gamma-Globulin | 82 |
Sample load : 100 micro-g each
Effect of sample load on peak width for IEC DEAE-420N (a column for weak anion exchange chromatography packed with non-porous gel) is shown.
DEAE-420N is an old type of DEAE3N-4T.
Sample : Ovalbumin
Column : Shodex IEC DEAE-420N (4.6mmID*35mm)
Eluent : (A); 25mM Piperazine-HCl(pH6.0)
(B); (A) + 1M NaCl
Linear gradient: 0min to 20min, (A) to (B)
Flow rate : 1.0mL/min
Detector : UV(280nm)
Column temp. : Room temp.
Effect of pH on elution pattern IEC DEAE-420N (a column for weak anion exchange chromatography packed with non-porous gel) is shown.
DEAE-420N is an old type of DEAE3N-4T.
Sample :
1. Conalbumin
2. Transferrin
3. Ovalbumin
4. Trypsin inhibitor
Column : Shodex IEC DEAE-420N (4.6mmID*35mm)
Eluent : 25mM Piperazine-HCl buffer(pH6.0)
25mM Piperazine-HCl buffer(pH6.5)
25mM Tris-HCl buffer(pH7.5)
25mM Ethanolamine-HCl buffer(pH9.0)
Linear gradient: 0min to 10min, 0M to 0.5M NaCl
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
Three kinds of protein standards were analyzed using IEC DEAE3N-4T (a column for weak anion exchange chromatography packed with non-porous gel). Fast analysis is possible using DEAE-420N compared with using IEC DEAE-825 (a column for weak anion exchange chromatography packed with porous gel).
Sample :
1. Conalbumin 0.4mg/mL
2. Ovalbumin 1.2mg/mL
3. Trypsin inhibitor 1.0mg/mL
Columns : Shodex IEC DEAE-825 (8.0mmID*75mm) : Shodex IEC DEAE3N-4T (4.6mmID*35mm)
Eluent : (A); 20mM Piperazine-HCl buffer(pH6.0) : (A); 25mM Piperazine-HCl buffer(pH6.0)
(B); (A) + 0.5M NaCl (B); (A) + 0.5M NaCl
Linear gradient: 0min to 60min, (A) to (B) Linear gradient: 0min to 10min, (A) to (B)
Flow rate : 1.0mL/min : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
Five kinds of protein standards were analyzed using IEC SP-420N ( a column for strong cation exchange chromatography packed with non-porous gel ).
Sample :
1. Ovalbumin
2. Trypsinogen
3. Ribonuclease A
4. Cytochrome c
5. Lysozyme
Column : Shodex IEC SP-420N (4.6mmID*35mm)
Eluent : (A); 20mM MES* buffer(pH6.0)
(B); (A) + 0.5M Na2SO4
Linear gradient: 0min to 15min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
*MES: 2-(N-Morpholino)ethanesulfonic acid
Six kinds of protein standards were analyzed using IEC SP-420N (a column for strong cation exchange chromatography packed with non-porous gel).
Sample :
1. Myoglobin
2. Trypsinogen
3. Ribonuclease A
4. alpha Chymotrypsinogen A
5. Cytochrome c
6. Lysozyme
Column : Shodex IEC SP-420N (4.6mmID*35mm)
Eluent : (A); 20mM Sodium phosphate buffer(pH7.0)
(B); (A) + 0.5M NaCl
Linear gradient: 0min to 10min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
Hexokinase was analyzed using IEC DEAE-420N (a column for weak anion exchange chromatography packed with non-porous gel). Fast analysis is possible using DEAE-420N compared with using IEC DEAE-825 (a column for weak anion exchange chromatography packed with porous gel).
DEAE-420N is an old type of DEAE3N-4T.
Sample :
Hexokinase
(Left) (Right)
Column : Shodex IEC DEAE-825 (8.0mmID*75mm) : Shodex IEC DEAE-420N (4.6mmID*35mm)
Eluent : (A); 20mM Tris-HCl buffer(pH7.0) : (A); 25mM Tris-HCl buffer(pH8.0)
(B);(A) + 0.5M NaCl (B);(A) + 0.5M NaCl
Linear gradient: 0min to 60min, (A) to (B) Linear gradient: 0min to 10min, (A) to (B)
Flow rate : 1.0mL/min : 1.5mL/min
Detector : UV(280nm) : UV(280nm)
Column temp. : Room temp. : Room temp.
Hemoglobins were analyzed using IEC SP-420N ( a column for strong cation exchange chromatography packed with non-porous gel ).
Sample : Hemogrobin A, F, S and C
Column : Shodex IEC SP-420N (4.6mmID*35mm)
Eluent : (A); 20mM MES* buffer(pH5.6)
(B); (A) + 0.5M Na2SO4
Linear gradient: 0min to 30min, 5% (B) to 100% (B)
Flow rate : 1.5mL/min
Detector : VIS(415nm)
Column temp. : Room temp.
*MES : 2-(N-Morpholino) ethanesulfonic acid
Glycohemoglobin control I was analyzed using IEC SP-420N ( a column for ion exchange chromatography packed with non-porous gel ).
Sample : Glycohemoglobin control I
Column : Shodex IEC SP-420N (4.6mmID*35mm)
Eluent : (A); 20mM MES* buffer(pH5.6)
(B); (A) + 0.5M Na2SO4
Linear gradient: 0min to 30min, 5% (B) to 100% (B)
Flow rate : 1.5mL/min
Detector : VIS(415nm)
Column temp. : Room temp.
*MES : 2-(N-Morpholino) ethanesulfonic acid
Lipoxidase was analyzed using IEC DEAE-420N (a column for weak anion exchange chromatography packed with non-porous gel).
DEAE-420N is an old type of DEAE3N-4T.
Sample : Lipoxidase
Column : Shodex IEC DEAE-420N (4.6mmID*35mm)
Eluent : (A); 20mM Tris-HCl (buffer(pH8.0)
(B); (A) + 0.5M NaCl
Linear gradient: 0min to 10min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
Urease was analyzed using IEC DEAE-420N (a column for weak anion exchange chromatography packed with non-porous gel).
DEAE-420N is an old type of DEAE3N-4T.
Sample : Urease
Column : Shodex IEC DEAE-420N (4.6mmID*35mm)
Eluent : (A); 20mM Tris-HCl buffer(pH8.0)
(B); (A) + 0.5M NaCl
Linear gradient: 0min to 10min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
Human serum was analyzed using IEC DEAE-420N (a column for weak anion exchange chromatography packed with non-porous gel).
DEAE-420N is an old type of DEAE3N-4T.
Sample : Human serum
Column : Shodex IEC DEAE-420N (4.6mmID*35mm)
Eluent : (A); 20mM Tris-HCl buffer(pH8.0)
(B); (A) + 0.5M NaCl
Linear gradient: 0min to 10min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
Trypsinogen was analyzed using IEC SP-420N ( a column for strong cation exchange chromatography packed with non-porous gel ).
Sample : Trypsinogen
Column : Shodex IEC SP-420N (4.6mmID*35mm)
Eluent : (A); 20mM MES* buffer(pH6.0)
(B); (A) + 0.5M Na2SO4
Linear gradient: 0min to 15min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
*MES: 2-(N-Morpholino)-ethanesulfaonic acid
Lipoxidase was analyzed using IEC SP-420N (a column for strong cation exchange chromatography packed with non-porous gel).
Sample : Lipoxidase
Column : Shodex IEC SP-420N (4.6mmID*35mm)
Eluent : (A); 20mM MES* buffer(pH6.0)
(B); (A) + 0.5M Na2SO4
Linear gradient: 0min to 15min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
MES: 2-(N-Morpholino) ethanesulfonic acid
Alpha chymotrypsinogen A was analyzed using IEC SP-420N (a column for strong cation exchange chromatography packed with non-porous gel).
Sample : Alpha Chymotrypsinogen A
Column : Shodex IEC SP-420N (4.6mmID*35mm)
Eluent : (A); 20mM MES buffer(pH6.0)
(B); (A) + 0.5M Na2SO4
Linear gradient: 0min to 15min, (A) to (B)
Flow rate : 1.5mL/min
Detector : UV(280nm)
Column temp. : Room temp.
*MES: 2-(N-Morpholino)-ethanesulfaonic acid