Introduction

In recent years there has been an increase in interest regarding ‘AQ’ columns due to their ability to retain polar

analytes reproducibly under high aqueous conditions and with no phase collapse as a traditional C18 column would exhibit.

There are two general approaches to the bonded phase chemistry of AQ columns. The first is to employ a polar or hydrophilic end-capping. The second method is to incorporate polar-embedded groups within the alkyl chains. These two methods provide a high degree of polar character to the final alkyl bonded phase, allowing full interaction with the alkyl chains upon wetting the silica surface with water. Additionally, the added polar functionality introduces a secondary separation mechanism (dipole-dipole interactions) to facilitate alternative selectivity for polar compounds.1

This application brief describes the use of Quasar™ C18, AQ and AQ Plus columns for the analysis of polar compounds, outlining the benefits of stationary phases offering alternative selectivity. Figure 1 shows the compounds used in this study (catechol, resorcinol and various derivatives of these compounds).

HPLC Analysis of Catechols and Resorcinols Using Quasar C18, AQ and AQ Plus Columns

A P P L I C A T I O N B R I E F

Author:

Kathryn Lawson-Wood

PerkinElmer, Inc. Seer Green, UK

Liquid Chromatography

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Experimental Conditions

Method ParametersHPLC method parameters are shown in Table 1.

Instrument PerkinElmer Flexar™ with PDA Plus™ Detector

Columns 150 mm 4.6 mm 5 µm

Quasar C18 (N9308802)

Quasar AQ (N9308841)

Quasar AQ Plus (N9304441)

Mobile PhaseA: 20 mM phosphoric acid

B: ACN 75 % A 25% B

Flow Rate 1.5 mL/min

Temp 30 °C

Wavelength 270 nm

Injection Volume 25 µL

Table 1. HPLC method parameters.

Solvents and Samples

All solvents were HPLC grade and samples were filtered using a 0.22 μm PTFE filter, P/N 02542924.

Individual stock standard solutions (1 mg/mL) were prepared in 20 mM phosphoric acid and sonicated. From these solutions, a combined working standard solution (0.05 mg/mL) was prepared using 20 mM phosphoric acid as the diluent.

Results and Discussion

The analysis of resorcinol, catechol and various derivatives is shown in Figure 2 using the Quasar C18, AQ and AQ Plus columns. Using the Quasar C18 column, compounds two and three co-elute and the remaining compounds are baseline resolved (Rs > 1.5). This C18 phase has C18 alkyl ligands bonded to the surface of the silica, with hydrophobic end-capping. Therefore, the only interaction between analyte and stationary phase is hydrophobic interaction.

Using the same experimental conditions on a Quasar AQ column allows the benefits of changes in selectivity to be realized. The Quasar AQ phase utilizes polar end-capping. The addition of a polar end-cap provides a higher degree of polar character to the final alkyl bonded phase, facilitating dipole-dipole interactions between the analyte and the stationary phase. The introduction of a secondary separation mechanism can lead to different retention behavior for polar analytes over a standard alkyl phase. In this case, compounds two and three begin to separate but are not baseline resolved (Rs = 1.03). The remaining peaks are baseline resolved and demonstrate the same elution order as the Quasar C18. To further increase resolution and improve retention, the aqueous content of the mobile phase can be increased (Figure 3). By simply adding 5 % more aqueous content, the resolution between compounds two and three is greatly enhanced, achieving more than baseline resolution (Rs). Additionally, the resolution between compounds four and five has increased (Table 2).

Using an AQ Plus column, under the same conditions as the C18 and AQ columns (Figure 2), shows a large change in selectivity and baseline resolution between all compounds. The elution order has altered slightly, and greater retention has been achieved. The AQ Plus has proprietary end-capping and bonding with polar functionality to facilitate this change in selectivity. It utilizes a mixture of C18 alkyl ligands and polar embedded alkyl chain ligands.

It is evident that switching from a C18 column to another column such as an AQ or an AQ Plus can provide alternative selectivity for more polar compounds. Although these phases offer the possibility of strong hydrophobic interaction, the added polar functionalities introduce additional modes of interaction. The AQ and AQ Plus columns also have the added benefit of being able to robustly retain polar analytes under 100 % aqueous conditions with no phase collapse. A traditional C18 column, on the other hand, would not withstand these conditions. The column would de-wet and undergo hydrophobic collapse.

Figure 1. Chemical structure of resorcinol, catechol and some of their various derivatives.

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Figure 2. Analysis of resorcinol, catechol and derivatives using a Quasar C18, AQ and AQ Plus column under the conditions in Table 1.

Figure 3. Analysis of resorcinol, catechol and derivatives using the Quasar AQ with 75 % and 80 % buffer conditions.

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Conclusion

• Initial screening of various column stationary phase chemistries is often the most powerful approach in method development to observe changes in selectivity.

• The Quasar AQ and AQ Plus columns have shown to provide alternative selectivity for polar compounds (catechols and resorcinols), in comparison with a Quasar C18 column, due to the added polar functionality of these phases.

• Excellent peak shape was obtained due to Quasar’s ultra-high purity silica and optimized ligand bonding technology.

Reference

1. https://www.hplc.eu/Downloads/AQ_Columns01.pdf, Date Accessed: July 2020.

Consumables

Mobile Phase Buffer (%) R1,2 R2,3 R3,4 R4,5 R5,6

75 4.52 1.03 10.79 1.50 2.95

80 4.52 2.13 13.73 2.31 2.48

Table 2. Resolution between peaks using the Quasar AQ column under 75 % and 80 % buffer conditions.

Consumables Used

Component DescriptionPart

Number

Columns

Quasar C18 (150 x 4.6 mm, 5 μm) N9308802

Quasar AQ (150 x 4.6 mm, 5 μm) N9308841

Quasar AQ Plus (150 x 4.6 mm, 5 μm) N9304441

HPLC Vials 2 mL Amber 9 mm Screw Top Vial with Write-on Patch and Fill Lines (100/Pack)

N9307802

HPLC Vial Caps 9 mm Screw Top Blue (polypropylene) Cap with PTFE/Silicone pre-slit Septa (100/Pack)

N9306203

PEEK Fittings Finger Tight for 1/16’’ OD PEEK Tubing 09920513

Syringes Syringe 1 mL BD Luer-Lok Disposable, Pack of 100

02542890

Syringe Filters 0.22 μm PTFE (Hydrophilic) Syringe Filter, 13 mm

02542924