Using Selectivity to Enhance Separation of Analgesics
Selectivity is the most powerful tool to optimize separations in HPLC. This parameter is changed by using different bonded phases, including C18, polar embedded, phenyl bonded phases and perflorophenyl, or by changing the mobile phase. In this work, 4.6 × 50 mm Poroshell 120 columns are used to quickly evaluate method development choices for the analysis of non-steroidal anti-inflammatory drugs (NSAIDS). The short column length and high efficiency provide short analysis times and rapid equilibration, leading to fast investigations of selectivity.
Experimental ConditionsInstrument: Agilent 1260 Infinity Binary LC System
Columns: Noted below
Flow rate: 2 mL/min
Mobile phase: A: 20 mM NH4HCO2 pH 3.0 B: Acetonitrile
Temperature: 40 °C
Detection: UV, 254 nm
The Agilent 1260 Infinity Binary LC System was configured as follows:
The following columns were used in this study.
Column Choice to Enhance Selectivity
The columns were chosen to improve selectivity in the separation. They included a highly end capped C18 column recommended as a first choice in method development (Poroshell120 EC-C18).
Poroshell 120 Bonus-RP can be used for many of the same separations as a C18 column while avoiding some of the disadvantages of C18, such as poor wettability in high aqueous mobile phases. In addition, it is much more retentive for those molecules that can interact by hydrophobic interactions and also by H-bonding with the amide group. Compared to alkyl only phases, Bonus-RP has enhanced retention and selectivity for phenols, organic acids, and other polar solutes due to strong H-bonding between polar group (H-bond acceptor) and H-bond donors, like phenols and acids. Bonus-RP gives retention slightly less than a C18 allows, for easy column comparison without the need to change mobile phase conditions. The Bonus-RP phase gives different selectivity than C18 for polar compounds. It is also compatible with 100% water.
Poroshell 120 Phenyl-Hexyl columns deliver unique selectivity for compounds with aromatic groups, providing superior resolution for these samples. Poroshell 120 Phenyl-Hexyl can also provide optimum separations of moderately polar compounds where typical alkyl phases (C18 and C8) do not provide adequate resolution. Acetonitrile tends to decrease the π–π interactions between aromatic and polarizable analytes and the phenyl-hexyl stationary phases, but methanol enhances those same interactions, giving both increased retention and changes in selectivity. This does not mean that acetonitrile should not be used with a phenyl bonded phase or that it might not provide an acceptable separation, but methanol is more likely to deliver the different selectivity that is desired from a phenyl phase.
Poroshell 120 PFP columns possess a pentafluorophenyl ligand. This can provide an orthogonal separation mechanism to traditional reverse phase columns. By specifically targeting many polar retention mechanisms, PFP phases can separate analytes based on small differences in structure, substitution, and steric access to polar moieties. The resulting selectivity for positional isomers, halogenated compounds, and polar analytes is particularly useful in the analysis of complex mixtures, and small molecule pharmaceuticals
Results and Discussion
Analysis problems can be quickly resolved by including survey methods with generic gradients as part of the method development scheme. This work demonstrates how different chemistries and organic modifiers such as acetonitrile and methanol can develop different selectivity and can be used to optimize the separation. In this case, using an alternative selectivity column such as Poroshell 120 PFP yielded better results than a more commonly used C18 chemistry. Fluorinated stationary phases are useful because of their enhanced interaction with halogens, and conjugated compounds.
(1) A. Streitweiser Jr. and C.H. Heathcock, Introduction to Organic Chemistry (MacMillan, New York, USA, 1981).
(3) J. Sangster, "Logkow: A Database of Evaluated Octanol/Water Partition Coefficents," (Log P), http://logkow.cisti.nrc.ca/logkow/index.jsp.
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