Optimized RP-HPLC Method for Hydroxybenzoic Acids

Balanced Retention for a Range of Polarities, Using an Ultra Aqueous C18 Column

• Useful retention of more polar and less polar analytes.
• Ultra Aqueous C18 column is compatible with 100% aqueous mobile phases.
• Ideal for samples that encompass a broad range of analyte polarity.

Hydroxybenzoic acids serve as active drug substances (aspirin, for example), as well as preservatives in drug products. In some cases, they represent impurities in drug products. Their analysis sometimes can be difficult, not only because they represent a wide range of applications, but primarily because they encompass a wide range of polarity. Chemically, benzoic acid, the basic structure for these analytes, consists of a benzene ring with a carboxyl group (Figure 1). Hydroxybenzoic acids share the same basic structure, but contain additional hydroxyl groups on the benzene ring (Figure 1). The additional hydroxyl groups’ varied positions and numbers create differences among the analytes’ overall polarity and solubility. Because these compounds represent such varying chemistry and polarity, finding an alkyl (C18) HPLC column that can effectively assay them all could be very difficult, but such a column could be of value for resolving these compounds from active drugs or from chemically similar impurities.

Figure 1  Hydroxybenzoic acids share the same basic structure, but have varying polarity.

Using identical conditions, we analyzed a group of hydroxybenzoic acids on a conventional C18 stationary phase column, on a C18 column with a polar group within (intrinsic to) the alkyl bonded phase (an IBD phase[2]), and on an Ultra Aqueous C18 column. Our objective was to find the optimum stationary phase for resolving analytes with a varying number of polar functional groups.

Overall, the Ultra Aqueous C18 column provided the best balance of retention for more polar and less polar analytes (Figure 2A), completely resolving our test mix when used with a simple gradient mobile phase. The conventional C18 column exhibited retention very similar to that of the Ultra Aqueous C18 column for the less polar analytes, benzoic acid and salicylic acid, but it showed less retention and resolution for the more polar compounds (Figure 2B). The intrinsically base deactivated column, on the other hand, exhibited opposite characteristics — retention similar to the Ultra Aqueous C18 column for the more polar compounds, but little retention of the less polar compounds (Figure 2C).

It is well documented that Ultra Aqueous C18 columns are compatible with 100% aqueous mobile phases, because the stationary phase has sufficient polar character to prevent dewetting or hydrophobic collapse.[1] Our current analyses reveal yet another advantage to the slight polar character of this column: by providing the best resolution of analytes exhibiting a wide range of polarity, the Ultra Aqueous C18 column demonstrates that it also can be used to retain, and separate, more polar or less polar compounds — or mixtures of both.

Figure 2  An Ultra Aqueous C18 column shows suitable retention for polar or nonpolar compounds, providing enhanced selectivity for a broad range of polarity.

Peaks 1. 3,5-Dihydroxybenzoic acid 2. 2,5-Dihydroxybenzoic acid 3. 4-Hydroxybenzoic acid Peaks 4. 3-Hydroxybenzoic acid 5. 2,4-Dihydroxybenzoic acid 6. Benzoic acid 7. Salicylic acid LC_PH0400 Column Ultra Aqueous C18 (cat.# 9178565) Dimensions: 150 mm x 4.6 mm ID Particle Size: 5 µm Pore Size: 100 Å Temp.: ambient Sample Diluent: mobile phase Conc.: 50 µg/mL each component Inj. Vol.: 5 µL Mobile Phase A: 20 mM potassium phosphate (pH 2.5) B: acetonitrile Time (min) %B 0.00 20 5.00 50 10.00 50 Flow: 1.0 mL/min Detector UV/Vis @ 210 nm

Options for Analyzing Polar Compounds

Retention by Reversed Phase (RP) HPLC

Many types of alkyl phases currently are available to the analyst, making column selection difficult. Although all alkyl phases possess the same basic structure — a specific length of alkyl chain bonded to a silica surface (typically C1-C30, with C18 being the most common) — various attached polar groups create selectivity and retention differences among columns. For example, a conventional C18 phase is comprised of a monomerically bonded straight 18 carbon alkyl chain, meaning every alkyl chain has a single, direct attachment to the silica surface. These phases are excellent for retaining nonpolar compounds, but they show very limited retention for polar compounds. One common bonding technique for increasing retention of polar compounds on an alkyl phase is to attach a polar group within, or intrinsic to, the alkyl phase. These phases, known as intrinsically base deactivated (IBD) phases, show increased retention for polar compounds because the embedded polar groups are capable of interaction with polar portions of analyte molecules. (These phases also have a deactivating effect on basic compounds, by creating an electrostatic barrier.) Polarity also can be added to an alkyl phase by adding polar end caps to active sites on the silica surface, or by adding polar side chains to the alkyl attachment. Interactions with polar compounds also can be increased through the use of a polymeric bonding chemistry.