Development of a continuous flow interface for stacking in capillary electrophoresis

The main purpose of the present work was to develop and optimize a continuous flow interface to improve the poor concentration detection limits of capillary electrophoresis which are one of its main limitations. The question was how the flow rate, applied voltage, interface and capillary dimensions and conductivities of background electrolyte and sample solution affect the electrokinetic sample injection in a continuous sample flow interface. Optimizing these parameters has the potential to perform near quantitative injection from large sample volumes in a short time. This can lead to the improvement of a variety of existing techniques that aim at lowering the concentration detection limits of CE. The injection voltage and flow rate have been optimized and their effect on the injected sample amount has been investigated using a tee connector in a commercial capillary electrophoresis instrument. The effect of sample injection from both flowing and static sample volumes was investigated. Using a tee connector interface with flowing sample injection, four times more analyte could be injected into the capillary than in a static system. Theoretical simulations along with experiments were performed to investigate the effect of flow rate and injection voltage on the injected sample. The results confirmed that more analyte could be injected into the capillary in a flowing sample interface due to depletion of the ions from the flowing stream indicating near quantitative injection of all of the ions. Significant enhancement in the proportion of sample ions that are injected when injecting from a flowing sample stream has been demonstrated and this work is the only to compare electrokinetic injection of the same sample volume, under the same conditions with the only difference being whether the sample stream was flowing or static. After having established the influence of the flow rate and injection voltage on the injected sample amount a mathematical model of the continuous sample flow interface was developed. The aim was to investigate the influence of the interface dimensions on the depletion flow rate, which is the maximum flow rate at a given voltage at which > 90% of all sample ions are being injected. Besides this the influence of the capillary dimensions and the conductivity ratio of the sample and backgroundelectrolyte on the depletion flow rate were investigated. The mathematical model proposed that the total applied voltage, the electrophoretic sample mobility and the conductivity ratio between the liquid in the interface and the capillary should be as high as practically possible to give high depletion flow rates. The conductivity ratio and the electrophoretic sample mobility are determined by the chosen stacking method and analyte of interest in an experimental setup. High currents pose a practical limitation to the total voltage that can be applied. The results proposed further that there is an optimum interface diameter and length at which the depletion flow rate reaches a maximum. It should be noted that the depletion flow rate changed only around 5% when changing the interface length within a range of 2 to 20 mm and the interface diameter within 450 to 2750 ˜í¬¿m. The mathematical model revealed that the depletion flow rate increases exponentially with the capillary inner diameter. Therefore the capillary inner diameter should be as big as practically possible. Out of all investigated variables a reduced capillary length showed the biggest improvements in depletion flow rate. The limitation when using a short separation capillary would be that the voltage needs to be reduced accordingly to avoid high currents. To study the predictions of the mathematical model experiments were performed. First the effect of the interface inner diameter on the depletion flow rate was investigated. The mathematical model predicted a less than 4 % change in depletion flow rate when increasing the interface diameter from 500 to 1500 ˜í¬¿m. A 500, 1000 and 1500 ˜í¬¿m inner diameter sample flow interface was constructed and integrated into a homemade CE system. A fluorescence microscope was used to observe the injection of a fluorescent dye in the transparent interface. During the injection process a plug of incoming sample replaced the BGE in the interface. It was found that the majority of BGE had to be replaced by the sample in order for the injection to reach stable conditions. In the 1000 ˜í¬¿m inner diameter interface the depletion flow rate was found to be 0.08 ˜í¬¿L/s. In the 1500 ˜í¬¿m interface the main challenge was the formation of electrolysis bubbles around the electrode which prevented the determination of a depletion flow rate. In the 500 ˜í¬¿m interface bubbles formed not only around the electrode but throughout the interface channel. This limited the number of injections that could be performed and no depletion flow rate could be determined. Bubble formation was attributed to overheating of the sample solution since the 500 ˜í¬¿m channel is four times smaller in volume compared to the 1000 ˜í¬¿m inner diameter interface. The formation of bubbles from electrolysis and overheating progressed with ongoing injection time in all interfaces used. Thus an injection approach was required that reached stable stacking conditions within a shorter timeframe. To achieve this sample was placed inside the interface from the start of the injection while the capillary was filled with BGE. This was expected to allow stable conditions from the start of the injection. Unexpectedly this approach caused the stacked sample zone to be move towards the capillary outlet within 10 sec of injection and did not allow the determination of a depletion flow rate either. A different BGE was chosen which was expected to stabilize the stacked sample zone at the capillary entrance during injection. This in contrast led to the formation of a stacked sample zone outside the capillary entrance and did not allow finding the depletion flow rate. For prospective future work fine tuning of the BGE parameters is therefore required. This would allow the formation of a stacked sample zone before bubble formation and to find the depletion flow rate in the 500 and 1500 ˜í¬¿m inner diameter interface. The mathematical model is a simplification that did not take into account the parabolic flow profile of the liquid flowing through the interface and the exact electric field line distribution in the interface. A simulation model was developed to get better guidelines on how to choose the interface inner diameter for maximizing the depletion flow rate. The simulation model proposed that the depletion flow rate increases with bigger interface inner diameters. This result stands in contrast to the mathematical model which predicts a decrease in depletion flow rate with bigger interface diameters. It was found that the concentrations in the simulation model are not regulated by the Kohlrausch function. In contrast the concentration changes in the mathematical model were assumed to follow Kohlrausch's regulating function. This difference was found to be the cause for the differences in the predictions of the two models. A closer look at the simulation model revealed that stacking of ions occurred without the presence of a conductivity difference between the liquid in the capillary and the liquid in the interface. A combination of hydrodynamic flow of liquid into the capillary that counteracts the electrophoretic movement of ions out of the capillary entrance was found to be the cause for stacking without a conductivity difference. These findings were experimentally confirmed when stacking of a fluorescent dye in the absence of a conductivity difference was achieved. Therefore it can be assumed that the simulation model predictions are correct. Thus it is anticipated that with bigger interface diameters higher depletion flow rates can be achieved which can enhance the sensitivity of CE equipment when used with a continuous flow interface. The increase of depletion flow rate with interface inner diameter will have to be investigated in future work. This is a promising new direction and presents great potential for the sensitivity enhancement by electrokinetic injection from a flowing sample stream.