Figure5
Ultra IBD separates cytosine, cytidine, CTP, CDP, and CMP with simple isocratic reverse phase conditions.
1
LC_0130
RestekCorporation • (800)356-1688 • (814)353-1300 •
Figure6
Ultra IBD separates thymine, thymidine, TTP, TDP, TMP with simple isocratic reverse phase conditions.
LC_0132
side and mono-, di-, and triphosphate nucleotide. Guanosine,
uridine, cytidine, and thymidine are ribonucleosides derived
from guanine, uracil, cytosine, and thymine, respectively.
Note that each of these separations was achieved using the
same conditions and that, in each case, the order of elution is
the same: the triphosphate, the diphosphate, then the
monophosphate nucleotide, followed by the base, and lastly
the nucleoside. There are slight “shoulders” on the peaks for
GDP (Figure 4), TTP (Figure 6), and UMP (Figure 7). These
compounds were present in the standards and were presumed
to be impurities or degradation products.
Table 1 lists the typical retention times obtained for all 25 of
the compounds separated in Figures 3–7. While not all 25
compounds can be resolved in a single HPLC analysis, it is
possible to analyze all of them using these chromatographic
conditions and MS or MS/MS detection. Note that the
mobile phase is compatible with MS detection, as all of its
0 2 4 min.
Peak List:
Conc. (µg/mL):
1. CTP
248
2. CDP
322
3. CMP
248
4. cytosine
100
5. cytidine
340
Sample:
Inj.:
20µL
Solvent:
20mM ammonium
acetate, pH 5.8
Column:
Ultra IBD
Catalog #:
9175565
Dimensions:
150 x 4.6mm
Particle Size:
5µm
Pore Size:
100Å
Conditions:
Mobile Phase:
20mM ammonium
acetate, pH 5.8:
methanol (97.5:2.5)
Flow:
1.0mL/min
Temp.:
35°C
Det.:
UV @ 260nm
2
3
4
5
Peak List:
Conc. (µg/mL):
1. TTP
224
2. TDP
152
3. TMP
346
4. thymine
88
5. thymidine
318
Sample:
Inj.:
20µL
Solvent: 20mM ammonium
acetate, pH 5.8
Column:
Ultra IBD
Catalog #:
9175565
Dimensions:
150 x 4.6mm
Particle Size:
5µm
Pore Size:
100Å
Conditions:
Mobile Phase:
20mM ammonium
acetate, pH 5.8:
methanol (97.5:2.5)
Flow:
1.0mL/min
Temp.:
35°C
Det.:
UV @ 260nm
1
2
0 2 4 6 8 10 min.
3
4
5
