Nucleotides and Isomers

Nucleotides were analyzed using AXpak WA-624 (a column for weak anion exchange chromatography).

Sample : 20micro-L
1. 5′CMP 20μg/mL
2. 5′UMP 20μg/mL
3. 5′TMP 20μg/mL
4. 3′CMP 20μg/mL
5. 5′ADP 100μg/mL
6. 3′UMP 20μg/mL
7. 5′GMP 20μg/mL
8. 2′AMP 20μg/mL
9. 3′AMP 20μg/mL
10. 3′GMP 20μg/mL

Column       : Shodex AXpak WA-624 (6.0mmID*150mm)
Eluent       : 0.35M CH3COOH aq./0.35M CH3COONH4 aq.=240/100
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : 60deg-C

Ten nucleotides were separated using AXpak WA-624 (a column for weak anion exchange chromatography) with the eluent of pH5.0 isocratically. Compare with the cases of pH4.3 and pH5.5.

Sample :
1. 5′CMP
2. 5′UMP
3. 5′AMP
4. 5′GMP
5. 5′CDP
6. 3′AMP
7. 5′UDP
8. 5′ADP
9. 5′CTP
10. 5′UTP

Column       : Shodex AXpak WA-624 (6.0mmID*150mm)
Eluent       : 0.7M CH3COONH4 buffer(pH5.0)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : 60deg-C

Nucleotides were analyzed using AXpak WA-624 (a column for weak anion exchange chromatography).

Sample :
1. 5′CMP , 2. 5′TMP
3. 5′IMP , 4. 5′AMP
5. 5′GMP , 6. 3′GMP
7. 3′CMP , 8. 5′UDP
9. 5′ADP , 10. 5′CTP
11. 5′UTP , 12. 5′ATP
13. 5′GTP

Column       : Shodex AXpak WA-624 (6.0mmID*150mm)
Eluent       : (A); 0.1M NaH2PO4 aq./CH3CN=90/10
               (B); 0.5M NaH2PO4 aq./CH3CN=80/20
               Linear gradient: 0min to30min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : 60deg-C

AFpak AAF-894 (an affinity column) which makes use of biochemical interaction, is suited for the analysis, fractionation and purification of such heterocyclic compounds like nucleic acids.

Sample :
1. Adenosine-5′-monophosphate, AMP
2. Adenosine-5′-diphosphate, ADP
3. Adenosine-5′-triphosphate, ATP

Column       : Shodex AFpak AAF-894 (8.0mmID*50mm)
Eluent       : 0.1M 4-Ethylmorpholine acetate buffer(pH7.0)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : Room temp.

Nucleotides were analyzed using AFpak AAF-894 (an affinity column). When (A) is used as the eluent, AMP isinulin was absorbed to the gel material. Then shortly after using (B) as the eluent, AMP elutes from the column.

Sample : Adenosine-5′-monophosphate, AMP

Column       : Shodex AFpak AAF-894 (8.0mmID*50mm)
Eluent       : (A); 0.01M 4-Ethylmorpholine acetate buffer(pH7.0)
               (B); 0.1M 4-Ethylmorpholine acetate buffer(pH7.0)
               Step gradient (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : Room temp.

Nucleotides were analyzed using IEC DEAE-825 (a column for weak anion exchange chromatography).

Sample :
1. CMP
2. AMP
3. UMP
4. GMP
5. CDP
6. ADP
7. UDP
8. CTP
9. GDP
10. ATP
11. UTP
12. GTP

Column       : Shodex IEC DEAE-825 (8.0mmID*75mm)
Eluent       : (A); 0.1M NaH2PO4 aq./CH3CN=80/20
               (B); 0.5M NaH2PO4 aq./CH3CN=80/20
               Linear gradient: 0min to 60min, (A) to (B)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : 25deg-C

Nucleotides were analyzed using AXpak WA-624 (a column for weak anion exchange chromatography). pH of the eluent is lowered to 4.3. Retention becomes stronger and separation becomes better. Please compare with the cases of pH5.0 and pH5.5

Sample :
1. 5′CMP
2. 5′UMP
3. 5′TMP
4. 5′AMP
5. 5′GMP
6. 3′AMP
7. 3′GMP
8. 5′CDP
9. 5′UTP

Column       : Shodex AXpak WA-624 (6.0mmID*150mm)
Eluent       : 0.7M CH3COONH4 buffer(pH4.3)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : 60deg-C

Nucleotides were analyzed using AXpak WA-624 (a column for weak anion exchange chromatography). pH of the eluent is heightened to 5.5.
Please compare with the cases of pH4.3 and pH5.0

Sample :
1. 5′CDP
2. 5′UDP
3. 5′ADP
4. 5′GDP

Column       : Shodex AXpak WA-624 (6.0mmID*150mm)
Eluent       : 0.7M CH3COONH4 buffer(pH5.5)
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : 60deg-C

As Asahipak GS-320 HQ is packed with a packing material bonded with trace amounts of ion exchange groups, the multimode, which combines size exclusion mode predominately with ion exchange mode and reversed phase mode, can be used for the separation of nucleotides.

Sample : 20micro-g/mL each, 20micro-L
1. 2′AMP
2. 3′AMP
3. cAMP

Column       : Shodex Asahipak GS-320 HQ (7.5mmID*300mm)
Eluent       : 200mM NaH2PO4 aq./ 200mM Na2HPO4 aq.=500/15
Flow rate    : 1.0mL/min
Detector     : UV(260nm)
Column temp. : 30deg-C