24 February / March 2019
The Role of Methanol and Acetonitrile as Organic Modifiers in Reversed-phase Liquid Chromatography
Acetonitrile (MeCN) and methanol (MeOH) are the most commonly used organic modifiers in reversed-phase chromatography. Although both solvents offer certain advantages and disadvantages, one of their key strengths, from a chromatographic perspective, is that they offer substantially different selectivity, and as such, are valuable for method development purposes. This article will compare various characteristics of the two different solvents, such as UV cut-off, pressure and elution strength and discuss how organic eluent selection can be used as a method development tool.
Introduction
Reversed-phase mobile phases typically contain water and a less polar organic solvent (the organic modifi er), together with additives such as buffers, acids or bases. In reversed-phase, the aqueous component of the mobile phase has weak analyte elution strength, whilst the organic component has a higher elution strength. The relative proportions of aqueous and organic can therefore be adjusted to control analyte retention.
A range of organic solvents are suitable for use as the organic modifi er in reversed- phase liquid chromatography, although in practice, only a few have been used routinely. When selecting an organic solvent, properties such as miscibility with water, polarity, UV cut-off, viscosity and safety are important to consider. Each organic modifi er has advantages and disadvantages that should be considered before their use. For example, Tetrahydrofuran (THF) and isopropanol (IPA) can be useful as both provide high elution strength. However, IPA use is limited due to its high viscosity (leading to low performance and high back pressures), whilst THF can degrade pump seals, along with PEEK tubing and fi ttings and requires the use of stabilising agents such as BHT to prevent peroxide formation. Acetone is an inexpensive solvent, with similar elution properties to acetonitrile, although its high UV absorbance limits its applicability for some applications.
Over the years, acetonitrile and methanol have become the two organic modifi ers of choice for many reversed-phase applications. Both solvents are fully miscible with water and are compatible with common mobile phase additives and buffers (although care should be taken when using buffer salts at high organic compositions). Both solvents have advantages and disadvantages that should be considered, with fi nal modifi er choice often application dependant. For example, the cost of acetonitrile is signifi cantly higher than that of methanol and methanol is also less toxic. However, acetonitrile
has a lower UV cut-off than methanol (190 nm vs 205 nm), making it more suitable for use in applications requiring low UV detection wavelengths (note however, that it is important to use HPLC grade or better solvents for LC analyses). In addition, acetonitrile/water mixes have lower viscosity than methanol/water mixes and therefore generate substantially lower back pressures across the LC column (Figure 1). This lower backpressure is often seen as advantageous as it puts less strain on the LC system components and column, and provides scope to increase fl ow rate and reduce run times.
Figure 1: Experimentally determined backpressures for different compositions of methanol and acetonitrile with water on a C18 column, 100 x 3.0 mm (fl ow rate: 0.43 mL/min, temperature: 30°C).
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