LAAN-A-LC-E201
High Perform ance Liquid Chromatography
No.L423
High Speed, High Resolution Analysis (Part 40)
Analysis of Nucleobases, Nucleosides, and Nucleotides by the Nexera UHPLC System
Nucleic acids are biological macromolecules
consisting of linear chains of nucleotides, each of
which is made up of a base, a sugar, and a phosphate
group, and are important components that bear an
organism’s genetic code. In addition, nucleic acidrelated compounds, including nucleobases,
nucleosides, and nucleotides have a variety of
functions.
Here, using the Nexera UHPLC (Ultra High
Performance Liquid Chromatography) System, and
the Shim-pack XR-ODSⅢ and Phenomenex Kinetex
C18 high-speed, high-resolution columns, we
introduce examples of ultra-high-speed analysis and
ultra-high-resolution analysis of nucleic acid-related
compounds.
n Analysis of Nucleobases and Nucleosides
We prepared a sample solution consisting of a
standard mixture of 10 nucleic acid-related substances,
including 5 nucleobases (adenine, guanine, uracil,
thymine, cytosine) and 5 nucleosides (adenosine,
guanosine, uridine, thymidine, cytidine), each at a
concentration of 10 mg/L, and conducted analysis
using the Phenomenex Kinetex C18 column (particle
size 1.7 µm, 100 mm L. × 2.1 mm I.D.). The Phenomenex
Kinetex C18 is a Core-shell column consisting of a
1.25-µm solid core coated with a bonded 0.23 µm
multilayer of porous film.
Fig. 1 shows the chromatogram obtained using a 1 µL
injection of the prepared standard mixture, and Table 1
shows the analytical conditions used.
This analysis, which took 30 minutes to complete
using conventional conditions, took about 1/10 as long
(3 minutes) using these analytical conditions. The
system back pressure during this analysis was about
75 MPa.
Table 1 Analytical Conditions
Column
Mobile Phase
: Kinetex 1.7 μm C18 100 Å (100 mm L. × 2.1 mm I.D., 1.7 μm)
: 200 mmol/L Sodium perchlorate,
100 mmol/L (Sodium) phosphate buffer (pH=2.1) aq.
: 0.7 mL/min
Flow Rate
Column Temp. : 40 °C
Injection Volume : 1 μL
: SPD-20AV at 260 nm
Detection
: Semi-micro Cell
Flow Cell
40
mAU
4
35
2
30
1
25
3
5
20
6
15
7
8
9
10
10
5
0
0.0
1.0
2.0
min
■Peaks
1. Cytosine, 2. Uracil, 3. Guanine, 4. Adenine, 5. Cytidine,
6. Uridine, 7. Thymine, 8. Adenosine, 9. Guanosine, 10. Thymidine
Fig. 1 Chromatogram of a Standard Mixture of Nucleobases and
Nucleosides (10 mg/L each)
Note:When using a 100 % aqueous mobile phase or a composition close to that, as indicated in the analytical conditions in this document the
retention times may become smaller by temporarily stopping solvent delivery, and then restarting. To prevent the occurrence of this
phenomenon, after completion of the analysis, it is recommended to replace the mobile phase with one containing an organic solvent
(example: water/acetonitrile = 1/1) before stopping solvent delivery. In addition, if the retention times gradually become faster, perform a
rinse using the same mobile phase.
No.L423
n Analysis of ATP-related Compounds
We prepared a sample solution consisting of a
standard mixture of 6 ATP-related substances
(hypoxanthine, inosine, IMP, AMP, ADP, ATP)*, each at
a concentration of about 10 mg/L, and conducted
analysis using the Shim-pack XR-ODSⅢ column
(1.6 μm particle size, 50 mm L. × 2.0 mm I.D.).
Fig. 2 shows the chromatogram obtained using a 1 μL
injection of the prepared standard mixture, and Table 2
shows the analytical conditions used.
This analysis, which took 25 minutes to complete
using conventional conditions, took about 1/10 as long
(2.5 minutes) using these analytical conditions. The
system back pressure during this analysis was about
83 MPa.
Table 2 Analytical Conditions
Column
Mobile Phase
: Shim-pack XR-ODSIII (50 mm L. × 2.0 mm I.D., 1.6 μm)
: 100 mmol/L Phosphoric acid, 150 mmol/L Triethylamine aq.
/ Acetonitrile =100/1 (v/v)
Flow Rate
: 0.9 mL/min
Column Temp.
: 40 °C
Injection Volume : 1 μL
Detection
: SPD-20AV at 260 nm
Flow Cell
: Semi-micro Cell
30
mAU
1
25
20
15
10
3
4
2
5
5
6
0
0.0
0.5
1.0
1.5
2.0
min
■Peaks
1. Hypoxanthine, 2. IMP, 3. Inosine, 4. AMP, 5. ADP, 6. ATP
Fig. 2 Chromatogram of a Standard Mixture of ATP-Related Compounds
n Analysis of Nucleotides
We prepared a sample solution consisting of a
standard mixture of 18 nucleotides (AMP, ADP, ATP,
GMP, GDP, GTP, UMP, UDP, UTP, TMP, TDP, TTP, CMP,
CDP, CTP, IMP, IDP, ITP)*, each at a concentration of
about 50 mg/L, and conducted analysis using the
high-resolution Shim-pack XR-ODSⅢ column (2.2 μm
particle size, 200 mm L. × 2.0 mm I.D.).
Fig. 3 shows the chromatogram obtained using a 1 μL
injection of the prepared standard mixture, and Table 3
shows the analytical conditions used.
Analysis of these 18 substances was achieved at high
speed and with high resolution using these conditions,
and the system back pressure during the analysis was
about 78 MPa.
Table 3 Analytical Conditions
Column
Mobile Phase
: Shim-pack XR-ODSIII (200 mm L. × 2.0 mm I.D., 2.2 μm)
: A:100 mmol/L Phosphoric acid, 150 mmol/L Triethylamine aq.
B: Mobile Phase A / Acetonitrile =90/10 (v/v)
A/B=98/2 (v/v)
: 0.6 mL/min
Flow Rate
: 50 °C
Column Temp.
Injection Volume : 1 μL
: SPD-20AV at 260 nm
Detection
: Semi-micro Cell
Flow Cell
35
mAU
30
1
25
2
20
15
4
3
10
10
56 8
11
7 9 12 13
5
14
16
15
0
0.0
5.0
10.0
17
15.0
18
min
■Peaks
1. CMP, 2. UMP, 3. CDP, 4. GMP, 5. IMP, 6. UDP, 7. CTP, 8. GDP,
9. IDP, 10. AMP, 11.TMP, 12. UTP, 13. GTP, 14. ITP, 15. TDP,
16. ADP, 17. TTP, 18. ATP
Fig. 3 Chromatogram of a Standard Mixture of Nucleotides
* AMP: Adenosine 5'-monophosphate, ADP: Adenosine 5'-diphosphate, ATP: Adenosine 5'-triphosphate, GMP: Guanosine 5'-monophosphate,
GDP: Guanosine 5'-diphosphate, GTP: Guanosine 5'-triphosphate, UMP: Uridine 5'-monophosphate, UDP: Uridine 5'-diphosphate,
UTP: Uridine 5'-triphosphate, TMP: Thymidine 5'-monophosphate, TDP: Thymidine 5'-diphosphate, TTP: Thymidine 5'-triphosphate,
CMP: Cytidine 5'-monophosphate,CDP: Cytidine 5’-diphosphate, CTP : Cytidine 5’-triphosphate, IMP : Inosine 5’-monophosphate, IDP : Inosine
5’-diphosphate, ITP : Inosine 5’-triphosphate
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