50 May / June 2016 Diffusion coefficient of molecule
Another factor to consider is the diffusion of the molecule through the mobile phase. In this case the larger the molecule, in general, the greater will be the resistance to movement and so the diffusion coefficient will be lower.
In terms of quantifying the amount of variance, Figure 1 shows the effect of varying the flow rate for a range of capillary connectors and the effect that this has on the overall system variance. It can be clearly seen that care has to be taken when choosing an appropriate tubing diameter
Other factors
The dispersion within the chromatographic system does not just come from the volume attributable to the volume of the tubing, but can also arise from the injection volume, the void volume of the column, the volume associated with an ill-fitting connector as well as the detector volume and can be summarised as;
V = Vinj + Vcol + Vtubing + Vconnectors + Vdetector cell
The volumes that each of these components contributes to the dispersion process within the chromatographic system can also be expressed in terms of a system variance with the individual variances having the same form as the above equation.
σ2 total = σ2 inj + σ2 col + σ2 tubing + σ2 connectors + σ2 detector cell
Thus when running with very small i.d. columns it is necessary to minimise injection volume which will clearly have an effect on potential detection limits. With the ids of capillary columns being so small, often less than 1 mm, the issues associated with the logistics of nanolitre manipulation. It is also important to consider the detector cell volume and ensure that the volume is reduced there as well. The injector volume and detector volume are variables that will normally be addressed by the manufacturer who will optimise these components for the user and consequently although awareness that these components can cause dispersion to occur, it is not normally a substantive issue for commercial derived systems.
Figure 1 shows the variance that is obtained from a variety of different diameter pieces of tubing and how the flow rate affects the amount of dispersion. It can be clearly seen that increasing the diameter of the tubing, the length of the tubing or increasing the flow rate will have a detrimental effect on the chromatographic performance of the system.
various components of the system. There are a range of different type of fittings that are available for the chromatographer. It is important that the correct fittings are used, otherwise extra dispersion will be introduced into the system. It is also important that the fitting is correctly installed as failure to do so can create a dead volume within the system, which on the larger scale columns is not such an issue but with a smaller i.d. column does become a significant issue.
Figure 2. Some of the common connectors used within HPLC and examples of correct and incorrectly installed fitting (Picture courtesy of Agilent Technologies).
Figure 2 shows some common examples of fittings that are routinely used within the chromatographic field. It is important to ensure that the user has an awareness of the different types of fittings and that the fittings are correctly used, thus minimising system dispersion. Figure 3 demonstrates the effect of a bad connector.
Figure 3. The consequences of an incorrectly installed fittings A and correctly installed fitting B (Picture courtesy of Agilent Technologies). 40% ACN isocratic
separation on 150 x 1 mm C18 column. Conclusion
The growth in mass spectrometry and the drive for ever more efficient chromatography columns has resulted in decreasing column i.d.’s, with fields such as proteomics leading the way in what is possible for very narrow i.d. columns. With more users venturing into this growing field it becomes increasingly important that users are aware of the potential challenges that micro and nano flow chromatography pose. Ensuring the use of the correct connectors, and also minimising the flow path is critical to ensuring that the performance of the column is not affected. Manufacturers are looking to aid the chromatographer with the introduction of technology that could remove the need for tubing and connectors, however having an understanding of what the dispersion processes are will ensure that chromatographers will get the best out of very low flow chromatography systems.
Figure 1. Effect of varying the tubing diameter and length of the tubing on the system variance (Picture courtesy of Agilent Technologies).
Connectors
One area that is of particular importance for chromatographers utilising nano and capillary systems is the use of the connectors that link the
References 1. Fick, A. Ann. der. Physik (in German) 94 (1855) 59 2. Aris, R., Proc. Roy. Soc. A., 235 (1956) 67–77
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