Аннотация:Modern chromatographic methods using bonded phases based
on silica are restricted in the choice of the eluent due to the limited
substrate operating stability range (pH 2-8). The use of metal oxides or
polymers that are stable in a wider pH range as alternative substrates
can contribute not only an efficiency increase, but also an alternative
separation selectivity due to the different electrostatic charge of their
surface and the possibility of converting the analytes into appropriate
ionic form, which is important for the separation of polar compounds
in HILIC mode.
In the present work alternative substrates for HILIC were explored,
such as titanium dioxide (particle diameter of 5 μm) and polystyrenedivinylbenzene
(particle diameter of 5.5 μm) with a crosslinking degree
of 50% and a hydrophilic surface layer.
Titanium dioxide has a wider spectrum of possible interactions with
sorbates as compared with silica, namely, cationic, anionic, and also
ligand-exchange ones. The retention characteristics for water-soluble
vitamins (thiamine, nicotinic acid and amide, riboflavin, pyridoxine,
cyanocobalamin) on the surface of titanium dioxide particles in HILIC
mode were studied in the present work. Mobile phase parameters such
as water/acetonitrile ratio, pH, type, and concentration of the buffer
solution were varied to establish the optimal separation conditions. It
was demonstrated that separation efficiency up to 20000 N/m could
be achieved using 100mM ammonium acetate and pH 5.8, while
selectivity increases with decreasing buffer concentration in the mobile
phase down to 10-50 mM.
Chemically and mechanically stable, but hydrophobic polystyrenedivinylbenzene
(PS-DVB) was another tested substrate. The
proposed novel method of its surface hydrophilization by covalent
attachment of polyethyleneimine made it suitable for the separation
of polar compounds in HILIC. The mixture of thiamine, riboflavin,
cyanocobalamin, nicotinic, and ascorbic acids were separated in 20 min
with efficiency of up to 10000 N/m in gradient elution mode using 100
mM ammonium acetate, pH 5.8 and 8-30% acetonitrile.
The proposed substrates indicated new prospects in the development
of HILIC method due to the increased chemical stability, expansion of
the operating pH range, and also the alternative selectivity of separating
polar compounds.
