Hydrophobic Interaction Analysis

Recovery of proteins and enzyme activity were measured using HIC PH-814 ( a hydrophobic interaction column ). With the exception of myoglobin, which had a recovery of 93%, all proteins show good recovery values. Enzyme activity for all proteins remained high, except for catalase which had a value of 88% and hexokinase which had a value of 94%.

Recovery of proteins
Protein Recovery(%)
Cytochrome c 104
Myoglobin 93
Ribonuclease A 100
Ovalbumin 103
Lysozyme 100
alpha Chymotrypsin 99
alpha Chymotrypsinogen A 96
Recovery of
enzyme activity
Enzyme Recovery(%)
Trypsin 96
Hexokinase 94
Catalase 88
Lysozyme 100
Lipoxidase 100


Sample load : 0.5mg each

Column : Shodex HIC PH-814

Effect of Sample load on peak width was measued using HIC PH-814 ( a hydrophobic interaction column ). A sample load of 0.4mg does not widen the peak. Even when injecting a higher load of 1mg sample, the peak only widens 1.3 times.

Sample :
1. alpha Chymotrypsinogen
2. Ribonuclease
3. Myoglobin

Column    : Shodex HIC PH-814 (8.0mmID*75mm)

Eluent    : (A); (B) + 1.8M Ammonium sulfate

            (B); 0.1M Phosphate buffer(pH7.0)

            Linear gradient: 0min to 60min, (A) to (B)

Flow rate : 1.0mL/min

Effect of salt concentration was measured using HIC PH-814 ( a hydrophobic interaction column ).

Sample :
1. alpha Chymotrypsinogen A
2. alpha Chymotrypsin
3. Lysozyme
4. Ovalbumin
5. Ribonuclease A
6. Myoglobin
7. Cytochrome c

Column    : Shodex HIC PH-814 (8.0mmID*75mm)

Eluent    : (A): (B) + Ammonium sulfate

            (B):  0.1M Phosphate buffer(pH7.0)

            Linear gradient: (A) to (B)

Flow rate : 1.0mL/min

Six kinds of protein standards were analyzed using HIC PH-814 ( a hydrophobic interaction column ).

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

Column       : Shodex HIC PH-814 (8.0mmID*75mm)
Eluent       : (A); (B) + 1.8M Ammonium sulfate
               (B); 0.1M Phosphate buffer(pH7.0)
               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 HIC PH-814 ( a hydrophobic interaction column ). Three large peaks were obtained. Enzyme activity was found at the sosition of arrow mark after 40 minutes.

Sample : Lipoxidase from soybean

Column       : Shodex HIC PH-814 (8.0mmID*75mm)
Eluent       : (A); (B) + 1.8M Ammonium sulfate
               (B); 0.1M Phosphate buffer(pH7.0)
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

Two trypsins, alpha and beta were separated using HIC PH-814 ( a hydrophobic interaction column ).

Sample :
1. alpha Trypsin
2. Trypsinogen
3. beta Trypsin

Column       : Shodex HIC PH-814 (8.0mmID*75mm)
Eluent       : (A); (B) + 1.8M Ammonium sulfate
               (B); 0.1M Phosphate buffer(pH3.0)
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C