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