DISCUSSION
Separation of Organic Phosphorus Compounds
Separation of four organic P compounds was successful using RP-IPC.
This method required minimal sample preparation, which decreased the time
and cost of analyzing large sample sets. The separation speed, efficiency, low
buffer salt concentrations and back-pressures made this an attractive
alternative to anion-exchange HPLC methods. In addition, there was no
observable effect of the extracting solution matrix on compound separation
and detection.
The separation of the nucleotides (ATP, ADP, and AMP) was
accomplished with a mobile phase composition containing 7% acetonitrile.
While this did allow separation of the compounds, the separation of ADP and
AMP could possibly be improved by further decreasing the concentration of
acetonitrile in the mobile phase. The use of a gradient pump may also improve
the separation, by altering the acetonitrile concentration over time. 171
Analysis of IHP was possible with the RP-ICP method investigated.
Detection of the lower Inositol phosphate esters (such as tetra-, tri-, di- or
mono phosphates) would not be possible with this method unless the pH of the
mobile phase was increased. However, this pH change in the mobile phase
would create problems with the peak shape of IHP. Sandberg and Ahderinne[61
successfully used this separation technique for the determination of tri-, tetra-,
penta-, and hexaphosphates in foods and intestinal contents. The Sandberg and
Ahderinne[61 technique required extensive sample preparation (pre-separation
with ion-exchange) prior to analysis, which was not performed with the
soil samples used in this study. Rounds and Nielsen [91 used a gradient
anion-exchange HPLC technique to successfully separate 1HP and its lower
esters from plant materials with minimal sample preparation, which could
provide an alternative to the RP-ICP technique. Clarkin et a1. E233 also used a
gradient anion-exchange technique to separate Inositol phosphate congeners
in aqueous samples. While they obtained good compound separation with this
technique, the time requirement was very long (160 min/sample) which is not
practical for analyzing large numbers of samples.
CONCLUSIONS
Methods for using RP-IPC for the separation of soil organic P compounds
in solutions and extracts were developed. These methods will allow more
accurate determination of important P-containing compounds in soils, leading
to improved characterization of their reactivity and mobility in soil. The
ability to directly measure these compounds is important, due to increasing
concern over the addition of organic P compounds to soils and how their
behavior will affect overall P losses to the environment.
Extraction methods were developed that gave up to 95% recovery of
nucleotides from soil while minimizing their degradation. These extraction
procedures were developed to be compatible with the RP-ICP system
developed for their separation. In contrast, extraction of IHP using strong acid
or alkaline extracting solutions caused significant decrease in peak areas and
was unsuitable for studies where accurate measurement of IHP was needed.