42 February / March 2021
Figure 3. Comparison of chromatograms for the separation of a mixture of AMP and ATP using a standard UPLC system and column, a standard system with a MaxPeak HPS column and a MaxPeak HPS system and column. ACQUITY UPLCTM
BEH C18 1.7 µm 2.1 x 50 mm columns were used with an aqueous 10 mM ammo-
nium acetate (pH 6.8) mobile phase, 30°C column temperature and a 0.5 mL/min flow rate. The peaks were detected by absorbance at 260 nm. A series of fifteen injec- tions was made (20 ng of each nucleotide) , and the results for injections 1, 6, 11 and 15 are shown
Results and Discussion
The benefits of MaxPeak HPS technology may be demonstrated for the separation of nucleotides, which have been shown to adsorb onto stainless steel surfaces [5 - 8]. Adenosine triphosphate (ATP) (see Figure 2 for chemical structure) is a nucleotide that, in addition to being a precursor of DNA and
RNA, is an important metabolite, providing energy to drive many cellular processes [18]. Consequently, the quantification of ATP is important in a number of different application areas. Shown in Figure 3 are a series of chromatograms demonstrating the separation of adenosine monophosphate (AMP) and ATP using standard vs MaxPeak
HPS systems and columns. In each case, a series of fifteen injections was made. In the chromatograms obtained using the standard system and column, the peak for AMP shows significant tailing, which gradually decreases as more injections are made. ATP, which adsorbs more strongly due to its three phosphate groups, shows severe tailing and very low intensity, and is barely detectable in the fifteenth injection. When using the same standard system with a column that incorporates MaxPeak HPS technology, the AMP peak shows improved symmetry. However, the ATP peak is severely tailed, improving over the series of injections, but still showing severe tailing and low intensity in the fifteenth injection. In contrast, when using both a system and column incorporating MaxPeak HPS technology, the chromatograms show symmetrical peaks from the first to the last injection.
Figure 4. Comparison of peak areas of AMP and ATP vs injection number using a standard UPLC system and column, a standard system with a MaxPeak HPS column and a MaxPeak HPS system and column. The test conditions are described in the caption for Figure 3.
In Figure 4 we show the areas of the AMP and ATP peaks vs injection number for the three combinations of system and column. It is evident that besides the peak tailing observed when using a standard system and/or column, the peak areas are also lower relative to those observed when using both a system and column that incorporate MaxPeak HPS technology. This is particularly true for ATP, due to its strong adsorption on stainless steel surfaces. Thus, without substantially more conditioning than used in this experiment, the quantitative results
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