Figure 3
In gradient elution regime ion-paired reverse phase chromatography outperforms strong anion exchange HPLC both in sensitivity and resolution. (a) 2 nmol of nucleotide standard (ADP, GDP, ATP, GTP and ppGpp) were resolved in a SAX-HPLC run using gradient elution followed by tracking absorbance at 252 nm. Degradation of di-, tri- and tetraphosphates leads to the appearance of AMP and GMP in the standard. (b) Nucleotides extracted from an E. coli sample were resolved in a SAX-HPLC run using gradient elution both without (black trace) and with (red trace) a spiked-in 2 nmol nucleotide standard used to validate identity of the peaks. (c) 0.5 nmol of nucleotide standard (GMP, cAMP, GDP, ADP, CTP, GTP, UTP, ATP and ppGpp) were resolved in an IPRP-HPLC run using gradient elution. (d) Nucleotides extracted from an E. coli sample were resolved using IPRP with the aid of a spiked-in 0.25 nmol standard (red trace) used to validate the identity of the peaks. IMP and GMP were not resolved and co-migrate as one peak. Sample preparation is described in the Methods section. SAX-HPLC: A 5 µm Spherisorb 4.6 \(\times \) 150 mm column was run at 1 ml/min and 26°C. Buffer A: 0.05 M NH4H2PO4, pH 3.4. Buffer B: 0.5 NH4H2PO4, pH 3.4. IPRP: Kinetix C18 2.6 µm 4.6 \(\times \) 150 mm, 0.8 ml/min, 26°C. Buffer A: 5 mM Bu4NOH, 30 mM KH2PO4 pH 6.0. Buffer B: 100% acetonitrile.
