Chromatography
Biphenyl Modifi ed Silica Stabilised by Bulky Substituents - A New Stationary Phase for HPLC
Authors: Helmut Riering*, Natalie Bilmann, MACHEREY-NAGEL GmbH & Co. KG * Corresponding author
Biphenyl modifi ed HPLC phases are, due to their orthogonal selectivity, an interesting alternative to conventional reversed phase silica. However, the use of UV detection in combination with aromatic phases can result in spurious peaks which make interpretation of the chromatogram considerably more diffi cult. The reason is the hydrolysis of biphenyl ligands. The introduction of bulky isobutyl substituents at the silicon atom of the ligand improves the stability. Therefore, the noise due to phase bleed can be almost completely avoided. In addition, these bulky substituents also infl uence the selectivity of this new biphenylpropyldiisobutylsilyl modifi ed silica. The chromatography of forty-fi ve organic substances is studied comparatively on octadecyl, octyl, and biphenyl sorbents.
Introduction
Octadecyl and octyl phases are probably the most widely used sorbents in reversed phase (RP) chromatography. Their ability to separate substance mixtures is enormous [1]. In addition, the use of mass spectrometry or diode array detectors is increasing. These detectors also provide information for substance identifi cation and can detect coelutions. Nevertheless, the quantifi cation of coeluting substances can still be challenging. There are numerous compounds that cannot be distinguished by MS or whose UV spectra are very similar. Therefore, stationary phases with orthogonal selectivity are still of major interest. Ligands with aromatic groups are alternatives in RP chromatography [2]. They are chemically inert and the selectivity of the stationary phase is additionally infl uenced by the possibility of p-p interactions [3, 4]. However, the frequently used phenylpropyl or phenylhexyl groups show less retention compared to octadecyl ligands [5]. As an alternative, biphenyl groups can be utilised, which exhibit stronger interactions through a larger conjugated p-system and a higher carbon number. Biphenyl modifi ed silica shows comparable retention times to octadecyl phases [5].
Experimental
Stainless steel columns (100 mm length x 3 mm ID and 50 mm length x 4 mm ID) with the stationary phases NUCLEOSHELL®
Biphenyl, 2.7 µm and NUCLEOSHELL® p² and NUCLEOSHELL®
2.7 µm both based on core shell silica are commercially available (MACHEREY-NAGEL, Germany). NUCLEOSHELL®
RP 8plus are not commercially
available. For comparison, they were produced according to the confi dential procedures of NUCLEODUR®
p² and NUCLEOSHELL®
steel columns by a slurry packing method. The structures of the ligands are shown in Table 1. The HPLC columns Kinetex® Raptor®
length x 3 mm ID).
HPLC grade solvents were used for the preparation of the eluents. Water was prepared by an ultra clear GP UV UF purifi er (Evoqua, Germany). The various test compounds were of reagent grade or higher purity and were purchased from several sources.
The HPLC equipment used in this work were a Nexera XR (Shimadzu, Germany) and a Vanquish UHPLC system (Thermo Scientifi c, Germany). Solutions (c = 1 mg/mL) were prepared in methanol. The void volume was determined by injection of uracil solution.
RP 18plus. These sorbents were fi lled in stainless Biphenyl, 2.6 µm (Phenomenex, Germany) and
Biphenyl, 2.7 µm (Restek, USA) were purchased as comparison columns (100 mm Results and Discussion
Si R
R R=Me, i-Bu Figure 1. Hydrolysis of biphenyl ligands. H+ (CH2 )3 /H2 O
Si R
R (CH2 )3
Ligands are usually attached to silica via a siloxane bond. Partial hydrolysis of this siloxane bond can occur under acidic conditions [6]. In the case of a biphenyl containing modifi cation, the hydrolysis leads to compounds with biphenyl containing groups (Figure 1). These compounds show a strong absorption of UV light in the wavelength range of 220 to 300 nm and can cause rising baselines in the chromatogram (Figure 2). This can complicate the quantifi cation of the analytes. The similarity of the UV spectra of eluate and the pure substance allylbiphenyl demonstrates the biphenyl containing structure of the molecules that cause this disturbance. This malfunction is only visible in gradient mode. At high water contents of the eluent hydrolysis of the siloxane bond occurs. Due to their hydrophobic character, the compounds accumulate on the stationary phase. The enriched impurities elute at higher acetonitrile levels. This gradient method was used to compare the bleed behaviour of four biphenyl modifi ed core shell silica (Figure 2). Biphenylpropyldimethylsilyl modifi ed NUCLEOSHELL® peak followed by Kinetex®
Biphenyl and Raptor®
p² shows the most intense spurious Biphenyl (both biphenyldimethylsilyl-
modifi ed). Obviously, the direct attachment of the biphenyl group to the silicon has a stabilising effect, most likely due to steric shielding of the siloxane bond. In addition, the attachment of the biphenyl group via a propyl spacer leads to stronger hydrophobic interactions [5], which indicate a higher occupancy density. This would also explain the
RP 18plus, NUCLEOSHELL® RP 8plus * i-Bu Si i-Bu * This stationary phase is not available commercially Table 1. Surface modifi cations of the investigated core-shell silicas.
Stationary Phase NUCLEOSHELL®
Biphenyl
Ligand structure i-Bu
Si i-Bu NUCLEOSHELL® p² * CH3 Si (CH2 CH3 NUCLEOSHELL® RP 18plus i-Bu Si i-Bu )3 (CH2 )3
LABMATE UK & IRELAND - SEPTEMBER 2020
silica
silica
silica
silica
silica
silica
OH +
HO
O
O
O
O
O
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