Multimode Analysis
Although Asahipak GS-HQ series is basically SEC columns, they permit different types of separation and analysis from conventional ones by use of a multimode which combines the hydrophobic interaction (RPC) and ionic interaction (IEX: ion exclusion). The GS-320 HQ column performs well in separating hydrophillic peptides, particularly acidic and basic peptides. As the columns can be used over a wide range of eluent pH because of being polymer-based column, they can be washed with alkaline solution leading to a longer column life, which is another feature of the column.
Sample : 20micro-L
1. Glu-Ala-Glu 0.025%
2. Arg-Asp 0.05%
3. Gly-His-Lys 0.025%
4. Arg-Pro-Lys-Pro 0.025%
| MW | pI | Σf | |
|---|---|---|---|
| Glu-Ala-Glu |
347
|
3.12
|
0.39
|
| Arg-Asp |
289
|
6.75
|
0.68
|
| Gly-His-Lys |
340
|
9.95
|
0.29
|
| Arg-Pro-Lys-Pro |
497
|
11.44
|
3.24
|
pI : Isoelectric point
Σf : Hydrophobic parameter
Column : Shodex Asahipak GS-320 HQ (7.5mmID*300mm) Eluent : 50mM Ammonium acetate buffer(pH6.7) Flow rate : 0.5mL/min Detector : UV(220nm) Column temp. : 30deg-C
A calibration curve for Asahipak GS-520 7G (a multimode column) is shown. When an eluent with a relatively high salt concentration is used for Asahipak GS series, substances are eluted in decreasing order of molecular weight as it is the GFC mode which function mainly. GS-520, which has a large pore size, can analyze proteins with molecular weight up to 1,000,000. Samples such as human and bovine serum albumins which exhibit strong affinity with the molecular structure of the packing material are eluted belatedly. Making use of this characteristics, GS-520 is used exclusively for the analysis of serum albumin.
GS-520 7G is an old type of GS-520 HQ.
Sample :
1. IgM
2. IgA
3. IgG
4. Transferrin
5. BSA
6. Ovalbumin
7. Chymotrypsinogen A
8. Myoglobin
9. Ribonuclease
10. Cytochrome c
Column : Shodex Asahipak GS-520 7G (7.5mmID*500mm) Eluent : 0.1M Sodium phosphate buffer(pH7.0) + 0.3M NaCl Flow rate : 1.0mL/min Detector : UV(210nm) Column temp. : Room temp.
When an eluent with a relatively high salt concentration is used for Asahipak GS series (multimode columns), substances are eluted in decreasing order of molecular weight as it is the GFC mode which function mainly. Asahipak GS-320 is suitable for the analysis of proteins and peptides with molecular weight of less than 100,000.
GS-320 7G is an old type of GS-320 HQ.
Sample :
1. gamma-Globulin
2. Transferrin
3. BSA
4. Ovalbumin
5. Chymotrypsinogen A
6. Myoglobin
7. Ribonuclease
8. Cytochrome c
9. Oxytocin
Column : Shodex Asahipak GS-320 7G (7.5mmID*500mm) Eluent : 0.1M Sodium phosphate buffer(pH7.0) + 0.3M NaCl Flow rate : 1.0mL/min Detector : UV(210nm) Column temp. : Room temp.
H.Wada et al., J. Chromatogr., 320 (1985) 369.
Metal-bonded proteins in plasma were analyzed using Asahipak GS-520 7G (a multimode column) without the interference by albmins because albumins eluted belatedly.
GS-520 7G is an old type of GS-520 HQ.
Sample : Rat Plasma
Column : Shodex Asahipak GS-520 7G (7.5mmID*500mm) Eluent : 10mM Tris-HCl buffer(pH7.4) + 0.9% NaCl + 0.05% NaN3 Flow rate : 1.0mL/min Detector : UV(254nm, 280nm) and ICP
Courtesy of Dr. Suzuki, National Institute for Environmental Studies
Peptides in extract from waste water sludge were analyzed using Asahipak GS-320 7G ( a multimode column ).
GS-320 7G is an old type of GS-320 HQ.
Sample : Peptides in extract from waste water sludge
Acetone
Angiotensin I
Angiotensin III
Dextran
Insulin
Myoglobin
Ovalbumin
Column : Shodex Asahipak GS-320 7G (7.5mmID*500mm) Eluent : [50mM NH3-NH4Cl(pH10.3) + 300mM NaCl]/CH3CN=80/20 Flow rate : 1.0mL/min Detector : UV(280nm) Column temp. : 30deg-C
Courtesy of Dr. Hirai, Tokyo Institute of Technology
Crude pronase E was analyzed using Asahipak GS-320 7G ( a multimode column ).
GS-320 7G is an old type of GS-320 HQ.
Sample : Crude pronase E
Column : Shodex Asahipak GS-320 7G (7.5mmID*500mm) Eluent : 1/15M KH2PO4 + 1/15M Na2HPO4(pH7.4) Flow rate : 1.0mL/min Detector : UV(210nm) Column temp. : Room temp.
H. Wada et al., J. Chromatogr. , 320 (1985) 369
Endorphins were analyzed using Asahipak GS-320 HQ ( a multimode column ).
Sample :
1. beta-Endorphin
2. alpha-Endorphin
3. gamma-Endorphin
Column : Shodex Asahipak GS-320 HQ (7.5mmID*300mm) Eluent : 0.1% TFA/CH3CN=95/5 Flow rate : 0.8mL/min Detector : UV(210nm) Column temp. : 30deg-C
Chymotrypsin-hydrolysated peptides of ascidian 29K protein were analyzed using Asahipak GS-320 7G ( a multimode column ).
GS-320 7G is an old type of GS-320 HQ.
Sample : A. Ser-CMC-Ser-Glu-Phe, B. Asp-Ser-Phe, C. Ala-Tyr, D. Thr-Ser-Ser-Leu, E. Ser-Gln-Asp-Trp
(1) Preliminary separation of Reverse phased chromatography
Column : ODS column from other manufacturer (4.6mmID*250mm)
Eluent : (A); 50mM Ammonium acetate bufer(pH6.9)
(B); 50mM Ammonium acetate buffer(pH6.9)/CH3CN=10/90
Linear gradient: (A) to (B)
Flow rate : 1.0mL/min
Detector : UV(215nm)
(2) Refined separation on GS-320 7G
Column : Shodex Asahipak GS-320 7G (7.5mmID*500mm)
Eluent : 50mM Ammonium acetate buffer(pH6.9)
Flow rate : 1.0mL/min
Detector : UV(215nm)
Courtesy of Dr. Takagi, Tohoku University
With Asahipak GS-320 7G, a multimode column, it is possible to make the direct analysis of drug components in Serum and urine without sample pretreatment. Proteins, nucleic acids and polysaccharides are eluted first by GFC mode, and then, drugs and metabolites, whose hydrophobic interaction is larger, are eluted by reversed phase mode. This method can be used for the analysis of extracted fluid from serum, urine and internal organs.
GS-320 7G is an old type of GS-320 HQ.
Sample :
1. Protein
2. Glutathione
Column : Shodex Asahipak GS-320 7G (7.5mmID*500mm) Eluent : 40mM EDTA-3Na(pH8.0) Reagent : 0.8mg/mL OPA in 20% CH3OH (for Post-column reaction) Flow rate : 1.0mL/min Detector : Fluorescence(Ex. 350nm, Em. 420nm)
Courtesy of Dr. Nimura, Kitazato Unversity