Aqueous SEC (GFC) Analysis

Calibration curves for PROTEIN KW-800 series (columns for aqueous SEC (GFC)) are shown.

Sample :
1. Thyroglobulin (MW: 669,000)
2. Aldolase (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. Adenylate kinase (MW: 32,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Columns      : Shodex PROTEIN KW-800 series (8.0mmID*300mm each)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Calibration curves for PROTEIN KW-802.5 (a column for Aqueous SEC (GFC)) are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)
Proteins

Column       : Shodex PROTEIN KW-802.5 (8.0mmID*300mm)
Eluent       : (Proteins); 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
               (Pullulan, PEG, PEO); H2O
Flow rate    : 1.0mL/min
Detector     : (Proteins); UV(220nm), (Pullulan, PEG, PEO); Shodex RI 
Column temp. : Room temp.

Calibration curves for PROTEIN KW-803 (a column for Aqueous SEC (GFC)) are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)
Proteins

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : (Proteins); 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
               (Pullulan, PEG, PEO); H2O
Flow rate    : 1.0mL/min
Detector     : (Proteins); UV(220nm), (Pullulan, PEG, PEO); Shodex RI 
Column temp. : Room temp.

Calibration curves for PROTEIN KW-804 (a column for Aqueous SEC (GFC)) are shown.

Sample :
Pullulan
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)
Proteins

Column       : Shodex PROTEIN KW-804 (8.0mmID*300mm)
Eluent       : (Proteins); 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
               (Pullulan, PEG, PEO); H2O
Flow rate    : 1.0mL/min
Detector     : (Proteins); UV(220nm), (Pullulan, PEG, PEO); Shodex RI 
Column temp. : Room temp.

KW400-4F series is aqueous SEC (GFC) columns and downsized KW-800 series. Figure is calibration curves of KW400-4F series columns with pullulan. KW405-4F is newly line-upped column, and it was designed having larger pore size than KW-804. KW405-4F can analyze high molecular weight substances more than 1,000,000 as protein like agglomerated protein. KW400-4F series can be applied wider range of molecular weight substances than KW-800 series.

Sample : Pullulan

Columns      : Shodex KW400-4F series (4.6mmID*300mm each)
Eluent       : H2O
Flow rate    : 0.33mL/min
Detector     : Shodex RI (small cell volume)
Column temp. : Room temp.

KW400-4F series is aqueous SEC (GFC) columns and downsized KW-800 series. Figure is calibration curves of KW400-4F series columns with pullulan. KW405-4F newly line-upped column, and it was designed having larger pore size than KW-804. KW405-4F can analyze high molecular weight substances more than 1,000,000 as protein like agglomerated protein. KW400-4F series can be applied wider range of molecular weight substances than KW-800 series.

Sample : Proteins

Columns      : Shodex KW400-4F series (4.6mmID*300mm each)
Eluent       : 50mM Sodium phosphate buffer (pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

KW400-4F series is aqueous SEC (GFC) columns and downsized KW-800 series. Figure is calibration curves of KW400-4F series columns with pullulan. KW405-4F newly line-upped column, and it was designed having larger pore size than KW-804. KW405-4F can analyze high molecular weight substances more than 1,000,000 as protein like agglomerated protein. KW400-4F series can be applied wider range of molecular weight substances than KW-800 series.

Sample :
PEG, Poly(ethylene glycol)
PEO, Poly(ethylene oxide)

Columns      : Shodex KW400-4F series (4.6mmID*300mm each)
Eluent       : H2O
Flow rate    : 0.33mL/min
Detector     : Shodex RI
Column temp. : 25deg-C

Shodex PROTEIN KW-803 (a column for aqueous SEC (GFC)) shows good efficiency until 0.5mg of BSA (Bovine serum albumin) injection. If the injection to KW-803 exceeds 1mg, chromatogram of BSA becomes gradually broad. Recommended sample loading is less than 0.5mg of BSA per column.

Sample : BSA, 100micro-L

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 150mM NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Effects of flow rate when analyzing proteins using PROTEIN KW-802.5 and KW-803 (columns for aqueous SEC (GFC)) were shown.

Sample : BSA, Myoglobin, 0.2% each, 50micro-L

Columns      : (Left); Shodex PROTEIN KW-802.5, (Right); KW-803 (8.0mmID*300mm each)
Eluent       : 50mM Phosphate buffer + 150mM Na2SO4
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : 25deg-C

Five kinds of protein standards were analyzed using PROTEIN KW-802.5 (a column for aqueous SEC (GFC)).

Sample : Gel filtration standard
1. Thyroglobulin
2. gamma-Globulin
3. Ovalbumin
4. Myoglobin
5. Cyanocobalamin

Column       : Shodex PROTEIN KW-802.5 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

This figure shows the separation of standard proteins using aqueous SEC (GFC) mode. Two columns for GFC separation, PROTEIN KW-802.5 and KW-803 were used. KW-802.5 is suitable for the analysis of proteins with molecular weights of less than 150,000, while KW-803, which has a larger pore size than KW-802.5, is suitable for the analysis of proteins with molecular weights of more than 150,000.

Sample : Molecular weight marker
1. Glutamate dehydrogenase
2. Lactate dehydrogenase
3. Enolase
4. Adenylate kinase
5. Cytochrome c

Columns      : Shodex PROTEIN KW-802.5, KW-803 (8.0mmID*300mm each)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Ten kinds of protein standards were analyzed using PROTEIN KW-802.5 (a column for aqueous SEC (GFC)).

Sample :
1. Thyroglobulin (MW: 669,000)
2. Aldolase (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. Adenylate kinase (MW: 32,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Column       : Shodex PROTEIN KW-802.5 (8.0mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Ten kinds of protein standards were analyzed using PROTEIN KW-803 (a column for aqueous SEC (GFC)).

Sample :
1. Thyroglobulin (MW: 669,000)
2. Aldolase (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. Adenylate kinase (MW: 32,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Ten kinds of protein standards were analyzed using PROTEIN KW-804 (a column for aqueous SEC (GFC)).

Sample :
1. Thyroglobulin (MW: 669,000)
2. Aldolase (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. Adenylate kinase (MW: 32,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Column       : Shodex PROTEIN KW-804 (8.0mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

When a sample contains high molecular weight portions, PROTEIN KW-803 (a column for aqueous SEC (GFC)) will give a better result than Company A column.

Sample : Gel Filtration Standard (Bio Rad)

Peak Protein PROTEIN KW-803 Company A column
Rt(min) Width(min) Rs Rt(min) Width(min) Rs
1 Thyroglobulin 6.5657 0.7550 6.5657 0.7550
(MW: 670,000) 2.59 2.27
2 gamma-Globulin 8.2957 0.5825 7.7203 0.8450
(MW: 158,000) 2.33 1.84
3 Ovalbumin 9.5547 0.5000 8.9840 0.5250
(MW: 44,000) 1.93 2.39
4 Myoglobin 10.4380 0.4175 10.1290 0.4350
(MW: 17,000) 4.69 3.57
5 Vitamin B12 12.3423 0.3950 11.5647 0.3700
(MW: 1,350) 
Columns      : (Left); Shodex PROTEIN KW-803 (8.0mmID*300mm), (Right); Company A column
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

PROTEIN KW-804 (a column for aqueous SEC (GFC)) is duitable for the analysis of sample containing high molecular weight portions because the pore size of KW-804 is larger than that of Company A column.

Sample : 1. Ferritin (MW: 440,000), 2. Aldolase (MW: 158,000), 3. Chymotrypsin (MW: 25,000)

Columns      : (left); Shodex PROTEIN KW-804 (8.0mmID*300mm), (right); Company A column
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Protein standards were analyzed with Asahipak GF-510 HQ.

Sample : Molecular weight marker
1. Glutamate dehydrogenase
2. Lactate dehydrogenase
3. Enolase
4. Adenylate kinase
5. Cytochrome c

Column       : Shodex Asahipak GF-510 HQ (7.5mmID*300mm)
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.1M NaCl
Flow rate    : 0.5mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

Ten kinds of protein standards were analyzed using KW402.5-4F (a semi-micro column for aqueous SEC (GFC)).

Sample : 5micro-L
1. Thyroglobulin (MW: 669,000)
2. gamma-Globulin (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. beta-Lactoglobulin (MW: 35,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Column       : Shodex KW402.5-4F (4.6mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector     : UV(220nm) (small cell volume)
Column temp. : 30deg-C

Ten kinds of protein standards were analyzed using KW403-4F (a semi-micro column for aqueous SEC (GFC)).

Sample : 5micro-L
1. Thyroglobulin (MW: 669,000)
2. gamma-Globulin (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. beta-Lactoglobulin (MW: 35,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Column       : Shodex KW403-4F (4.6mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector     : UV(220nm) (small cell volume)
Column temp. : 30deg-C

Ten kinds of protein standards were analyzed using KW404-4F (a semi-micro column for aqueous SEC (GFC)).

Sample : 5micro-L
1. Thyroglobulin (MW: 669,000)
2. gamma-Globulin (MW: 158,000)
3. BSA (MW: 67,000)
4. Ovalbumin (MW: 43,000)
5. Peroxidase (MW: 40,200)
6. beta-Lactoglobulin (MW: 35,000)
7. Myoglobin (MW: 17,000)
8. Ribonuclease A (MW: 13,700)
9. Aprotinin (MW: 6,500)
10. Vitamin B12 (MW: 1,350)

Column       : Shodex KW404-4F (4.6mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector     : UV(220nm) (small cell volume)
Column temp. : 30deg-C

Ten kinds of protein standards were analyzed using KW405-4F (a semi-micro column for aqueous SEC (GFC)).

Sample : 5micro-L
1. Thyroglobulin (MW: 669,000)
2. gamma-Globulin (MW: 158,000)
3. Ovalbumin (MW: 43,000)
4. Ribonuclease A (MW: 13,700)
5. Vitamin B12 (MW: 1,350)

Column       : Shodex KW405-4F (4.6mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector     : UV(220nm) (small cell volume)
Column temp. : 30deg-C

Asahipak GF-310 HQ, GS-320 HQ permit GFC analysis with good recovery of proteins.

Proteins Recovery(%)
GF-310 HQ GS-320 HQ
Ovalbumin 95 93
Myoglobin 93 96
Lysozyme 95 86
Cytochrome c 96 93 
Columns      : Shodex Asahipak GF-310 HQ, GS-320 HQ (7.5mmID*300mm each)
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.1M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(210nm)
Column temp. : 30deg-C

Asahipak GF-510 HQ, GS-520 HQ permit GFC analysis with good recovery of proteins.

Proteins Recovery (%)
GF-510 HQ GS-520 HQ
gamma-Globulin
85
85
Transferrin
88
92
BSA
90
95
Lysozyme
95
96
 
Columns      : Shodex Asahipak GF-510 HQ, GS-520 HQ (7.5mmID*300mm each)
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.1M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(210nm)
Column temp. : 30deg-C

PROTEIN KW-800 series permit aqueous SEC (GFC) analysis with good recovery of proteins and peptides.

Protein Recovery(%)
KW-802.5
KW-803
gamma-Globulin 91 96
Bovine serum albumin 94 91
Ovalbumin 89 90
Myoglobin 92 95
Cytochrome c 100 98
Lysozyme 98 97
alpha-Chymotrypsinogen A 93 93

Both PROTEIN KW-803 and Company A column (a column for aqueous SEC (GFC)) show high mass recoveries of ribonuclrease A and thyroglobulin. Such high recoveries are attributed to minimum adsorption properties. Furthermore, the calibration curve obtained by sample loading and peak areas shows good linearity in a wide range between 1 micro-g to 100 micro-g.

Sample Column 1 micro-g 5 micro-g 10 micro-g 50 micro-g 100 micro-g
Ribonuclease A KW-803 108.6 96.3 98.2 98.8 99.0
Company A 110.5 100.6 97.7 98.3 98.9
Thyroglobulin KW-803 106.1 98.2 97.3 98.7 98.6
Company A 111.9 92.6 93.9 95.9 96.6 

Columns      : Shodex PROTEIN KW-803 (8.0mmID*300mm), Company A column
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(220nm)
Column temp. : Room temp.

Proteins in human serum were analyzed using PROTEIN KW-803 (a column for Aqueous SEC (GFC)).

Sample : Human serum, 20micro-L

Sample : Protein std. 0.1% each
1. Fibrinogen 50micro-L
2. α2 Macroglobulin 50micro-L
3. IgG 50micro-L
4. Transferrin 50micro-L
5. Plasminogen 50micro-L
6. Albumin 100micro-L
7. Antitrypsin 100micro-L
8. Hemoglobin 100micro-L

Columns      : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Dimer and trimer of BSA were analyzed using PROTEIN KW-803 (a column for Aqueous SEC (GFC)).

Sample : BSA(Sigma) Fraction V

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Crude albumin in chicken egg was analyzed using PROTEIN KW-803 (a column for Aqueous SEC (GFC)).

Sample ; Crude Albumin
1. Conalbumin
2. Ovalbumin
3. Lysozyme

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Crude lipoxidase was analyzed using PROTEIN KW-803 (a column for Aqueous SEC (GFC)).

Sample : Crude Lipoxidase

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Crude catalase was analyzed using PROTEIN KW-803 (a column for Aqueous SEC (GFC)).

Sample : Crude Catalase

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Crude hexokinase was analyzed using PROTEIN KW-803 (a column for Aqueous SEC (GFC)).

Sample : Crude Hexokinase

Column       : Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.

Calibration curves for Asahipak GF-510 HQ and GF-7M HQ (columns for GFC separation) are shown.

Sample:
1. IgM
2. IgA
3. IgG
4. Transferrin
5. Conalbumin
6. BSA
7. Ovalbumin
8. Myoglobin
9. Ribonuclease A
10. Cytochrome c
11. L-Phenylalanin
12. Creatinine

Columns      : Shodex Asahipak GF-510 HQ (7.5mmID*300mm)       : GF-7M HQ (7.5mmID*300mm) 
Eluent       : 0.1M Sodium phosphate buffer(pH7.0) + 0.1M NaCl : 0.2M Sodium phosphate buffer(pH7.0)
Flow rate    : 0.5mL/min                                       : 0.5mL/min
Detector     : UV(280nm)                                : UV(280nm)
Column temp. : 30deg-C                                         : 30deg-C

Human serum was analyzed using OHpak SB-802 HQ (a column for GFC separation)

Sample : Human serum
1. Proteins
2. Orotidine
3. unknown
4. Creatinine
5. Uric acid

Column       : Shodex OHpak SB-802 HQ (8.0mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 0.5mL/min
Detector     : UV(250nm)
Column temp. : Room temp.

Effect of injection volume when analyzing serum using MSpak GF-310 4B (a column for rapid analysis of drugs) was shown.

Sample : Control serum I Wako
(from Wako Pure Chemical Industries, Ltd.)

Column       : Shodex MSpak GF-310 4B (4.6mmID*50mm)
Eluent       : 50mM CH3COONH4 aq./CH3CN=70/30
Flow rate    : 0.25mL/min
Detector     : UV(220nm)
Column temp. : 30deg-C

When a tryptic digest of human lgG was analyzed using PROTEIN KW-803 and KW-802.5 (columns for Aqueous SEC (GFC)) with a UV detector (230nm) as well as with an optical rotation detector (ORD). The ORD shows a negative peak and it gives the information that human lgG is levorotatory.

Sample : Triptic digest of human IgG

Columns      : Shodex PROTEIN KW-803 + KW-802.5 (8.0mmID*300mm each)
Eluent       : 0.05M Tris-HCl buffer(pH7.6) + 0.15M NaCl + 0.3mM CaCl2
Flow rate    : 0.5mL/min
Detector     : UV(230nm), ORD
Column temp. : Room temp.

This figure shows the chromatogram of protein mixtures analyzed with OHpak SB-803 HQ, Asahipak GF-510 HQ or PROTEIN KW-803. KW-803 is the best column for the separetion of these proteins.

Sample :
A: Thyroglobulin (bovine)
B: gamma-Globulin (bovine)
C: Ovalbumin (chiken)
D: Myoglobin (horse)
E: Cyanocobalamin

Columns      : Shodex OHpak SB-803 HQ (8.0mmID*300mm) 
               Shodex Asahipak GF-510 HQ (7.5mmID*300mm)
               Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 0.2M Phosphate buffer(pH6.9)
Flow rate    : 0.5mL/min
Detector     : UV(280nm)
Column temp. : 30deg-C

High sensitive analysis of metalloproteins in lat liver cell was performed using GS320A-M5D (0.5mmID x 150mm) and ICP-MS. GS320A-M5D is a micro-column of Asahipak GS-320 HQ.

Sample : 100nL
Cu,Zn-SOD (Supe roxide dismutase)
Cu-MT (Metallothionein)

Column    : Shodex GS320A-M5D (0.5mmID*150mm)
Eluent    : 50mM Ammonium acetate buffer(pH6.5)
Flow rate : 2.0micro-L/min
Detector  : ICP-MS(Cu and Zn at m/z=65 and 66,respectively)

Courtesy of Dr. Yasumitsu Ogra, Graduate School of Pharmaceutical Sciences, Chiba University

Protein analysis data using KW402.5-4F, high performance semi-micro aqueous SEC (GFC) column, and current PROTEIN KW-802.5. Theoretical plate number of KW400-4F series has improved 1.5 times than KW-800 series. Sensitivity becomes 3 or 4 times better than current column. KW400-4F series is suitable for high resolution and high sensitivity analysis.

Sample : 10micro-L
1. Blue dextran 2000, 0.2mg/mL
2. gamma-Globulin, 0.8mg/mL
3. Ovalbumin, 0.8mg/mL
4. Myoglobin, 0.56mg/mL
5. Uridine, 0.04mg/mL

Columns      : (A); Shodex KW402.5-4F (4.6mmID*300mm)
               (B); Shodex PROTEIN KW-802.5 (8.0mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer (pH7.0) + 0.3M NaCl
Flow rate    : (A); 0.33mL/min, (B); 1.0mL/min
Detector     : UV(280nm)
Column temp. : Room temp.(25deg-C)

Recovery data of seven kinds of standard protein using KW402.5-4F and KW403-4F was shown. Both columns showed good recovery data.

Protein Recovery(%)
KW402.5-4F
KW403-4F
gamma- Globulin 98 96
Bovine serum albumin 89 96
Ovalbumin 89 97
Myoglobin 90 89
Cytochrome c 92 92
Lysozyme 87 98
alpha-Chymotrypsinogen A 95 94

Oligopeptides were analyzed using SB401-4E (a column for high perfonmance semi-micro aqueous SEC (GFC)).

Sample : 10micro-L
1. Gly-Gly-Gly
2. Gly-Gly
3. Glycine

Column       : Shodex SB401-4E (4.6mmID*250mm)
Eluent       : 50mM CH3COONH4 aq./CH3CN=90/10
Flow rate    : 0.2mL/min
Detector     : UV(210nm) (small cell volume) 
Column temp. : 25deg-C

Modification of protein is one of research methods of protein function. Functional performance or stability is changed by the modification. PEG modified chymotrypsin is analyzed using PROTEIN KW-802.5.

Sample : 50micro-L, 0.1% PEG-Chymotrypsin

Column     　: Shodex PROTEIN KW-802.5 (8.0mmID*300mm)
Eluent       : 10mM Sodium Phosphate Buffer(pH7.0) 
Flow rate    : 1.0mL/min
Detector　   : UV(210nm) 
Column temp. : 40deg-C

Human control serum was analyzed using aqueous solvent SEC (GFC) high performance semi-micro columns, KW403-4F and KW404-4F. The bigger pore size (1,500A compared to 800A) of packed gel in KW404-4F is suitable for the analysis of large molecules that were excluded as void when using a KW403-4F column.

Sample : 1micro-L
Control serum I wako/B

Columns      : Shodex KW403-4F, KW404-4F (4.6mmID*300mm each)
Eluent       : 50mM Sodium phosphate buffer (pH7.0) + 0.3M NaCl 
Flow rate    : 0.20mL/min
Detector　   : UV(280nm) 
Column temp. : 25deg-C

Whey protein in yogurt was analyzed using aqueous solvent SEC (GFC) high performance semi-micro columns, KW402.5-4F and KW403-4F.

Sample : 5micro-L
Whey protein in Yogurt

Columns    　: Shodex KW402.5-4F + KW403-4F (4.6mmID*300mm each)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl 
Flow rate    : 0.20mL/min
Detector　   : UV(280nm) 
Column temp. : 30deg-C

Lectin from various sources was analyzed using aqueous solvent SEC (GFC) high performance semi-micro column, KW402.5-4F. Lectin is a protein that has specific binding characteristic to saccharide and can exist as compounds of saccaride-binding protein lectin or metal-binding lectin.

Sample : 5micro-L
1. Lectin from Soybean
2. Lectin from Arachis hypogaea
3. Lectin from Canavalia ensiformis (Con A)
4. Lectin from Lens culinaris
5. Lectin from Triticum vulgaris

Column     　: Shodex KW402.5-4F (4.6mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector     : UV(220nm) 
Column temp. : 30deg-C

Various peptides with MW ranged from 269 to 1734 was analyzed using aqueous solvent SEC (GFC) high performance semi-micro column, KW402.5-4F. Since each peptide may contain different amino-functional groups, the separation mode occur may not be as a “pure” size exclusion mode like for the analysis of proteins. This is more significant when peptides contain more hydrophobic-amino acids and/or basic-amino acids.

Sample : 5micro-L
1. Gly-Gly-His
2. Thrombin Receptor Activator Peptide (Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe)
3. Angiotensin Ⅱ (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe)
4. Angiotensin Ⅰ (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu)
5. Leu-enkephalin (Tyr-Gly-Gly-Phe-Leu)

Column     　: Shodex KW402.5-4F (4.6mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 0.33mL/min
Detector　   : UV(220nm) 
Column temp. : 30deg-C

IgM from human serum was analyzed using aqueous solvent SEC (GFC) columns, OHpak SB-805 HQ and SB-806 HQ.

Sample : 50micro-L
IgM, Immunoglobulin M from Human Serum
(Wako Pure Chemical Industries, Ltd.) 0.1%

Column     　: Shodex OHpak SB-806 HQ + SB-805 HQ (8.0mmID*300mm each)
Eluent       : 100mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector　   : UV(254nm) 
Column temp. : 30deg-C

Basic polypeptide Poly-Lysine was analyzed using PROTEIN KW-803 (a column for GFC separation).

Sample : 100micro-L
Poly-L-lysine hydrobromide (Sigma-Aldrich) 0.1%

Column     　: Shodex PROTEIN KW-803 (8.0mmID*300mm)
Eluent       : 50mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector　   : UV(220nm) 
Column temp. : 30deg-C

Avidin, one of the saccaride-binding proteins, was analyzed using aqueous solvent SEC (GFC) column, PROTEIN KW-804.

Sample : 50micro-L
1. Avidin (Wako Pure Chemical Industries, Ltd.) 0.1%

Column     　: Shodex PROTEIN KW-804 (8.0mmID*300mm)
Eluent       : 100mM Sodium phosphate buffer(pH7.0) + 0.3M NaCl
Flow rate    : 1.0mL/min
Detector　   : UV(254nm) 
Column temp. : 30deg-C