Ion Exchange Analysis

IEC QA-825 is packed with strong anion exchange resins bonded with quaternary ammonium groups. This column can be used over a wide range of eluent pH, as the pKa of the quaternary ammonium is 11.7.

IEC DEAE-825 and Asahipak ES-502N are packed with weak anion exchange resins bonded with diethylaminoethyl groups. The pKa of diethylaminoethyl groups is approximately 7.8, therefore DEAE-825 and ES-502N are generally recommended for the separation of acidic proteins and used most commonly for ion exchange chromatography of proteins. DEAE-420N packed with non-porous gel is suitable for fast analysis.

IEC SP-825 is packed with strong cation exchange resins bonded with sulfopropyl groups. As the pKa of ion exchange group is 2.3, the column can be used over a wide pH range. SP-420N packed with non porous gel is suitable for fast analysis.

IEC CM-825 and Asahipak ES-502C are packed with weak cation exchange resins bonded with carboxymethyl groups. These groups have a pKa value of 5.7, therefore these bonded columns are suitable for the analysis of basic proteins. The base packing material of IEC CM-825 is polyhydroxymethacrylate and that of Asahipak ES-502C a polyvinyl alcohol. In some case of high molecular weight samples such as proteins not only the ion exchange mode, but also the reversed phase mode contribute to some degree to the separation, therefore a difference of packing material may play an important role.

Recoveries of proteins and enzime activity for IEC QA-825 (a column forstrong anion exchange chromatography) are shown. When 0.5mg of each proteins was injected into the column by linear gradient elution, in which salt concentration increases linearly over one minute, sample recovery was found to be good. Then the same amount of enzyme was injected into the column in the same manner as the above protein and its recovery was also found to be good.

Recovery of proteins
Protein Recovery (%)
Ovalbumin 106
Conalbumin 100
beta- Lactoglobulin 99
Trypsin inhibitor 99
Recovery of enzyme activity
Enzyme Recovery (%)
Kallikrein 95
Lipoxidase 100
Catalase 96


Sample load : 0.5mg each

Column : Shodex IEC QA-825 (8.0mmID*75mm)
Eluent : (A); 20mM Tris-HCl buffer(pH7.5)
         (B); (A) + 0.5M NaCl
         Linear gradient: (A) to (B), 1min

The tables show good recovery of IEC DEAE-825 (a column for ion exchange chromatography) for proteins and enzyme activity.

Recovery of proteins
Protein Recovery (%)
Trypsin inhibitor 96
beta-Lactoglobulin 100
Ovalbumin 101
Myoglobin 100
gamma-Globulin 99
BSA 97
Thyroglobulin 90
Recovery of enzyme activity
Enzyme Recovery (%)
Kallikrein 92
Lipoxidase 96
Catalase 94


Sample load : 0.5mg each

Column : Shodex IEC DEAE-825 (8.0mmID*75mm)
Eluent : (A); 20mM Tris-HCl buffer(pH8.0)
         (B); (A) + 0.5M NaCl
         Linear gradient: 0min to 1min, (A) to (B)

The tables show good recovery of IEC SP-825 (a column for strong cation exchange chromatography) for proteins and enzyme activity.

Recovery of proteins
Protein Recovery (%)
Trypsinogen 93
alpha-Chymotrypsinogen A 99
alpha-Chymotrypsin 106
Myoglobin 96
Lysozyme 96
Ribonuclease A 99
Cytochrome c 100
gamma-Globulin 100
Recovery of enzyme activity
Enzyme Recovery (%)
Trypsin 90
alpha-Chymotrypsin 102


Sample load : 0.5mg each

Column : Shodex IEC SP-825 (8.0mmID*75mm)
Eluent : (A); 20mM Sodium phosphate buffer(pH6.0)
         (B); (A) + 0.5M NaCl
         Linear gradient: 0min to 1min, (A) to (B)

The tables show good recovery of IEC CM-825 ( a column for weak cation exchange chromatography ) for proteins and enzyme activity.

Recovery of proteins
Protein Recovery(%)
alpha Chymotrypsinogen A 90
Myoglobin 97
Ribonuclease A 100
Cytochrome c 101
Lysozyme 99
Recovery of enzyme activity
Enzyme Recovery(%)
alpha Chymotrypsin 97
Lysozyme 102


Sample load : 0.5mg each

Column : Shodex IEC CM-825 (8.0mmID*75mm)

Eluent : (A); 20mM Sodium phosphate buffer(pH7.0)

         (B); (A) + 0.5M NaCl

         Linear gradient: 0min to 1min, (A) to (B)

A mixture of transferrin and ovalbumin was injected into IEC QA-825 (a column for ion exchange chromatography) to obtain the relation between sample load and resolution(Rs). With up to 2mg of sample load, the Rs remained at approximately 3 with no decline. Rs was 2.5 when the sample load was 5mg. Even when the sample load was increased to 10 mg, Rs was approximately 2 with satisfactory resolution.

Sample :
Transferrin and Ovalbumin

Column       : Shodex IEC QA-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Piperazine-HCl buffer(pH6.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 20min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

When IEC DEAE-825 (a column for weak anion exchange chromatography) is used, the peak does not widen until the sample load of ovalbumin reaches 1mg. When the quantity is increased to 4mg, the peak approximately doubles its width.

Sample : Ovalbumin

Column       : Shodex IEC DEAE-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Piperazine-HCl buffer(pH6.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 20min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp .: Room temp.

When IEC SP-825 (a column for strong cation exchange chromatography) is used, the peak does not widen until the sample load of ribonuclease A reaches 1mg. When the quantity is increased to 5mg, the peak widths approximately 1.5 times.

Sample : Ribonuclease A

Column       : Shodex IEC SP-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0 min to 20min, (A) to (B)
Detector     : UV(280nm)
Column temp. : Room temp.

When IEC CM-825 (a column forweak cation exchange chromatography) is used, the effect of sample load on the resolution is shown. The resolution (Rs) of a mixture of ribonuclease A and (alpha) chmotrypsinogen A remains at approximately 3 until the sample load reaches 1.0mg. Even when the sample load is 5.0mg, the Rs is approximately 1.1, which means the substances can still be separated.

Sample :
Ribonuclease A
alpha Chymotrypsinogen A

Column       : Shodex IEC CM-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 20min, (A) to (B)
Detector     : UV(280nm)
Column temp. : Room temp.

When the buffer eluent is acidic, four types of proteins are separated using IEC DEAE-825 (a column for weak anion exchange chromatography) well. In opposition to this, when the eluent is alkaline, the separation of the proteins is poor. By increasing the pH value, the four peaks tend to converge into one peak. The reason for that is the ion exchange groups of the packing material cannot dissociate adequately when the pH value is high.

Sample:
1. Conalbumin
2. Transferrin
3. Ovalbumin
4. Trypsin inhibitor

Column    : Shodex IEC DEAE-825 (8.0mmID*75mm)
Eluent    : (A); 20mM Piperazine-HCl buffer(pH6.0)
                 20mM Tris-Trispropanol-HCl buffer(pH7.0)
                 20mM Tris-HCl buffer(pH8.0)
                 20mM Ethanolamine-HCl(pH9.0)
                 20mM 1,3-Diaminopropane-HCl buffer(pH10.0)
            (B); (A) + 0.5M NaCl
             Linear gradient: 0min to 20min, (A) to (B)
Flow rate : 1.0mL/min

Using an eluent at pH of 7.0 or 8.0, five types of proteins are well separated using IEC SP-825 (a column for strong cation exchange chromatography). The retention time of ribonuclease A increases with decreasing pH value and, at pH of less than 6.0, ribonuclease A elutes after alpha-chymotrypsinogen A. At pH 6.0, the separation of ribonuclease A from alpha-chymotrypsinogen A is insufficient. At pH 4.0 or 5.0, myoglobin does not elute sharply.

Sample :
1. Myoglobin, 2. Ribonuclease A, 3. alpha Chymotrypsinogen A, 4. Cytochrome c, 5. Lysozyme

Column    : Shodex IEC SP-825 (8.0mmID*75mm)
Eluent    : (A); 20mM Sodium formate buffer(pH4.0)
                 20mM Sodium malonate buffer(pH5.0 and 6.0)
                 20mM Sodium phosphate buffer(pH7.0)
                 20mM HEPES(pH8.0)
            (B); (A) + 0.5M NaCl
            Linear gradient: 0min to 20min, (A) to (B)
Flow rate : 1.0mL/min

Four kinds of protein standards were analyzed using IEC QA-825 (a column for strong anion exchange chromatography). Since the pH value of the eluent is 6.0 and the pI value of conalbumin is in the range of 6.0 to 6.8, the conalbumin cannot be retained by the packing material and therefore it elutes rapidly. The other proteins like transferrin, ovalbumin and trypsin are negatively charged because their pIs are lower than 6.0 and therefore they are retained by the packing material. These proteins are released from the surface of the packing material and elute from the column by gradient elution with increasing salt concentration.

Sample :
1. Conalbumin
2. Transferrin
3. Ovalbumin
4. Trypsin inhibitor

Column       : Shodex IEC QA-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Piperazine-HCl buffer(pH6.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 30min, 100% (A) to 50% (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

The base packing material of the IEC DEAE-825 (a column for weak anion exchange chromatography) is poly(hydroxy methacrylate) and that of the Asahipak ES-502N 7C (a column for weak anion exchange chromatography) is poly(vinyl alcohol). In case of poly(vinyl alcohol) material, not only the ion exchange mode affect the separation, but also the reversed phase mode contributes to some degree to the separation of high molecular weight compounds such as proteins. There are some cases in which the difference in the packing material base may affect the separation.

Sample :
1. Conalbumin
2. Ovalbumin
3. Trypsin inhibitor
4. beta-Lactoglobulin

Column       : Shodex Asahipak ES-502N 7C (7.5mmID*100mm)
Eluent       : (A); 50mM Bis-Tris propane HCl(pH7.0)
               (B); 50mM Bis-Tris propane HCl(pH7.0) + 500mM NaCl 
               Linear gradient: 0min to 20min, 100% (A) to 100% (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

Six kinds of protien standard were analyzed using IEC CM-825 ( a column for weak cation exchange chromatography ) and IEC SP-825 ( a column for strong cation exchange chromatography ).

Sample :
1. Myoglobin, 2. Trypsinogen, 3. Ribonuclease A, 4. alpha Chymotrypsinogen A, 5. Cytochrome c, 6. Lysozyme

Columns      : (Left) Shodex IEC CM-825 (8.0mmID*75mm)
               (Right) Shodex IEC SP-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Four kinds of protien standard were analyzed using Asahipak ES-502C 7C (a column for weak cation exchange chromatography).

Sample :
1. Myoglobin
2. Ribonuclease A
3. Chymotrypsinogen A
4. Lysozyme

Column    : Shodex Asahipak ES-502C 7C (7.5mmID*100mm)
Eluent    : (A); 50mM Sodium phosphate buffer(pH7.0)
            (B); (A) + 0.5M NaCl
            Linear gradient: 0min to 20min, (A) to (B)
Flow rate : 1.0mL/min
Detector  : UV(280nm)

Chicken egg albumin was analyzed using IEC QA-825 ( a column for strong cation exchange chromatography).

Sample: Crude albumin (chicken egg)

Column       : Shodex IEC QA-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Tris-HCl buffer(pH8.5)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

Human serum was analyzed using IEC DEAE-825 (a column for waek anion exchange chromatography).

Sample : Human serum

Column       : Shodex IEC DEAE-825 (8.0mmID*75mm)
Eluent       : (A); 0mM Tris-HCl buffer(pH8.0)
               (B); (A) + 0.5M NaCl 
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Proteins in Human serum were analyzed using IEC QA-825 ( a column for strong anion exchange chromatography ).
In this analysis, the sample was separated into four peaks including the transferrin peak; the largest one is serum albumin.

Sample : Human serum
1. IgG
2. Transferrin
3. (Unknown)
4. Serum albumin

Column       : Shodex IEC QA-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Tris-HCl buffer(pH8.6)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, 100% (A) to 50% (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Proteins in human serum were analyzed using IEC DEAE-825 (a column for weak anion exchange chromatography).

Sample : Human Serum
1. Albumin
2. alpha1 Acid glycoprotein
3. Transferrin
4. IgA
5. IgM
6. IgG

Column       : Shodex IEC DEAE-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Tris-CH3COOH buffer(pH8.0)
               (B); (A) + 0.5M CH3COONa
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Lipoxidase from soybean was analyzed using IEC QA-825 (a column for strong anion exchange chromatography) and IEC DEAE-825 (a column for weak anion exchange chromatography). The enzyme activity of this sample was measured by using hydrogen peroxide as a substrate. Enzyme activity was found at the arrowhead position.

Sample : Lipoxidase from soybean

Columns      : Shodex IEC QA-825 (8.0mmID*75mm)           : Shodex IEC DEAE-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Ethanolamine-HCl buffer(pH9.0)   : (A); 20mM Tris-HCl(pH8.0)
               (B); (A) + 0.5M NaCl                         (B); (A) + 0.5M NaCl
               Linear gradient:0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Lipoxidase, an oxidoreductase was analyzed using IEC SP-825 (a column for strong anion exchange chromatography). It is a glycoprotein of MW 80,000. Lipoxidase from soybean consists of three fractions. Enzyme activity was found at two peaks around 25 minutes.

Sample: 0.5% Lipoxidase, 150micro-L

Column       : Shodex IEC SP-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Citric acid-NaOH buffer(pH4.3)
               (B); (A) + 0.5M Na2SO4
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

Collagen and BSA were analyzed using Asahipak ES-502N 7C (a column for weak anion exchange chromatography).

Sample :
1. Collagen
Pepsin-digested product of collagen
soluble in acid extracted from pig skin
2. BSA

Column       : Shodex Asahipak ES-502N 7C (7.5mmID*100mm)
Eluent       : 20mM Monoethanolamine buffer(pH9.5) + 500mM NaCl 
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

Courtesy of Dr. Shirai, Tokyo University of Agriculture and Technology

beta Lactoglobulin subfractions were analyzed using Asahipak ES-502N 7C (a column for weak anion exchange chromatography).

Sample : beta-Lactoglobulin

Column       : Shodex Asahipak ES-502N 7C (7.5mmID*100mm)
Eluent       : (A); 20mM Piperazine(pH5.0)
               (B); (A) + 300mM NaCl
               Linear gradient: 0min to 12min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

Angiotensins were analyzed using IEC SP-825 (a column for strong cation exchange chromatography). Angiotensins are physically active peptides and cause blood pressure to rise. Angiotensin I is a decapeptide, Angiotensin II an octapeptide and Angiotensis III a hexapeptide. These three components separated very well by gradient elution with increasing salt concentration.

Sample : 250micro-L
1. Angiotensin II
2. Angiotensin I
3. Angiotensin III

Column       : Shodex IEC SP-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Weak cation exchange columns bonded with carboxy methyl groups such as Asahipak ES-502C 7C are suitable for the analysis of basic proteins.

Sample : Lysozyme chloride

Column       : Shodex Asahipak ES-502C 7C (7.5mmID*100mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 300mM NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

The pIs of trypsin and alpha-chymotrypsin are approximately 8.1-8.6 and 10.1-10.6 respectively. These basic proteins can be separated well with the IEC CM-825 (a column for weak cation exchange chromatography).

Sample : 30micro-L
1. Trypsin 0.5%
2. alpha-Chymotrypsin 0.25%

Column       : Shodex IEC CM-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Chytochrome P450 in rat liver microsome was analyzed using Asahipak ES-502C 7C (a column for weak cation exchange chromatography).

Sample : Cytochrome P450 in rat liver microsome

Column       : Shodex Asahipak ES-502C 7C (7.5mmID*100mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH6.5) + 20% Glycerol + 0.4% Emulgen 911
               (B); (A) + 1.0M Sodium Acetate
               Linear gradient: 0min to 50min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : VIS(417nm)
Column temp. : Room temp.

Courtesy of Dr. Funae, Osaka City University

Myosin subfragments were analyzed using Asahipak ES-502C 7C ( a column for weak cation exchange chromatography ).

Sample : Myosin subfragment

Column       : Shodex Asahipak ES-502C 7C (7.5mmID*100mm)
Eluent       : (A); 5mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.1M NaCl
               Linear gradient: 0min to 25min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : 10deg-C

Courtesy of Dr. Muno, Juntendo University

Commercially available catalase was analyzed using IEC QA-825 ( a column for strong anion exchange chromatography ). Enzyme activity was found at about 18 min at the position of arrow mark.

Sample : 0.5% Catalase, 250micro-L

Column       : Shodex IEC QA-825 (8.0mmID*50mm)
Eluent       : (A); 20mM Ethanolamine-HCl buffer(pH9.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

Purified Kallikrein from Pig Pancreas was analyzed using IEC QA-825 ( a column for strong anion exchange chromatography ). Enzyme activity was found at the large peak which has two peak tops after 30 minutes. The recovery of enzyme activity was 95%.

Sample : 0.5% Kallikrein, 200micro-L

Column       : Shodex IEC QA-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Tris-HCl buffer(pH7.5)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

Ribonuclease A from equine pancreas was analyzed using IEC CM-825 ( a column for weak cation exchange chromatography ). This enzyme contains rather small impurities.

Sample: 0.5% Ribonuclease A (pI=9.3), 30micro-L

Column       : Shodex IEC CM-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

Papain was analyzed using IEC SP-825 (a column for strong cation exchange chromatography). Papain is a polypeptide chain of MW 23,406 and pI 8.75. It is very stable protelytic enzyme. It is used as the reagent for the study of structure of proteins and also for industrial use.

Sample : Papain

Column       : Shodex IEC SP-825 (8.0mmID*75mm)
Eluent       : (A); 20mM Sodium phosphate buffer(pH7.0)
               (B); (A) + 0.5M NaCl
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C
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