Chromatography focus on


Turn Tony Edge*1 , Monica Dolci1 , Luisa t And Go Green horsten Teutenberg2


1. Thermo Fisher Scientific, Tudor Road, Runcorn, WA7 1TA 2. Institut für Energie- und Umwelttechnik e. V., Bliersheimer Straße 58-60, 47229 Duisburg, Germany *Corresponding author


The use of organic solvents in liquid chromatography is common; indeed trying to perform a separation with just an aqueous mobile phase in most laboratories is one that is not readily considered. Here we will discuss how a greener approach to separation science can be employed by raising the mobile phase temperature and decreasing or even eliminating the need for an organic solvent. Other advantages associated with the use of temperature will also be discussed, in particular the selectivity changes that can occur as the temperature is varied.


The exploitation of temperature as a key experimental parameter does require an understanding of what the appropriate operating conditions should be and data will be presented to look at the effect that temperature has on the operating pressure as well as the solubility of the mobile phase.


There are, however, perceived disadvantages of this approach, specifically the thermal stability of the analyte and column will be investigated and an approach to minimise these effects will be discussed.


Experimental Parameters to be Considered


One of the first considerations in going to green HPLC, that is chromatography that does not use organic solvents, is to determine what experimental conditions are required to allow the use of a purely aqueous mobile phase. Clearly this will be dependent on the analyte being investigated, but it will also depend on the physical properties of the mobile phase, in this case water. To fully understand the advantage that using an aqueous mobile phase at elevated temperatures has, it is important to be aware of the physical changes that occur with water at elevated temperatures, both in terms of the viscosity and hence operating pressure, but also in terms of the solubility of the mobile phase, and also the chemical changes with an increase in the static permittivity resulting in organic compounds becoming soluble in water at elevated temperatures, thus behaving more like an organic solvent.


Viscosity – Pressure – Temperature Variation


The variation of temperature and viscosity for water has been well documented, and there are many models [1-5] which have been developed to account for the variation in the viscosity over a specified temperature range. One of the simplest models was developed by Andrade [2], and is given in Equation 1.


Where;


λ – constant relating to particle packing, dp – particle diameter,


γ – mobile phase visocosty, Dm, Ds – diffusion coefficients in mobile phase and stationary phase for analyte, Φ – phase ratio


ω - constant, df – film thickness, k - retention factor for analyte, c’,c’’ – constants


u – Linear velocity of mobile phase


The key temperature dependent parameters in this equation are the Dm and Ds terms which relate to the diffusion of the solute in the mobile and stationary phases, respectively.


Increasing the temperature will not only affect the viscosity, but it will also affect the diffusion of the analytes within the mobile phase, as given by the van Deemter equation in Table 1, which will in turn affect the band dispersion process occurring within the column. The rate of diffusion of a molecule can be explained by an Arrhenius type expression as given in Equation 2 [8].


Equation 1. Variation of viscosity with temperature where:


ηT – is the viscosity at a temperature T in Kelvin. b – is a constant which is dependent on the fluid. T – is the thermodynamic temperature in Kelvin.


η0 – is the viscosity at 0 Kelvin, this is clearly a theoretical value. It can be seen that as the temperature is increased the viscosity of the mobile phase drops, which means that the flow rate can be increased, since this results in a reduction in the pressure drop across the column, clearly this is an advantage to most separation scientists as it allows for faster analysis [6].


Variation of optimal flow rate with temperature


An examination of the van Deemeter [7] equation reveals that increasing the rate of diffusion will affect both the ‘B’ and ‘C’ terms. The ‘B’ term, the rate of longitudinal diffusion, will increase as the temperature is increased. As a consequence the optimal flow rate will also increase.


Increasing the diffusion rate will also have an effect on the ‘C’ term, which relates to the radial dispersion of the analyte, which will improve the mass transfer, thus lowering the effect that the ‘C’ term has on the dispersion process. See Table 1.


Table 1. Van Deemter equation broken into three terms. A term B term


C term


Equation 2. Variation of diffusion with temperature


Where Dm, Ds – the diffusion rates in the mobile and stationary phases, α, β - constants, Ea1, Ea2 - activation energies, R – universal gas constant, and T - temperature in Kelvin.


It can be seen from this that the diffusion rate will increase with the temperature for both of these parameters. It is a reasonable assumption that the rate of diffusion on the surface is related to the rate of diffusion of the molecule in the bulk mobile phase [9]. If this is the case then it becomes possible to determine what the optimal flow rate is to obtain the minimum peak dispersion.


INTERNATIONAL LABMATE - JANUARY/FEBRUARY 2013


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