Simultaneous determination of inorganic anions and cations by capillary electrophoresis with indirect UV detection I. Haumann, J. Boden, A. Mainka, U. Jegle Journal of Chromatography (2000) 895,

Purpose: Investigate the separation of low molecular mass anions and cations by capillary electrophoresis Use of indirect UV detection to analyze anions or cations.

Background: What is capillary electrophoresis (CE) ? Advantages of CE High separation efficiency High separation efficiency Small sample size required Small sample size required Fast separation Fast separation Reproducibility Reproducibility Automation Automation

Electroosmosis is the fundamental process that drives CE. Fused silica capillaries are used for separations pI of fussed silica is about 1.5 Degree of ionization is controlled by the pH of the buffer

Experiment/Instrumentation: Chemicals used- Dimethyldiphenylphonium (DIPP) and trimesic acid All solutions, electrolytes and standards were prepared using water purified with a Mill-Q System. All solutions, electrolytes and standards were prepared using water purified with a Mill-Q System.

Instruments used- modular composed device combining a high-voltage power supply (P/ACE Capillary Electrophoresis System) Fused Silica Capillary

Results/Discussion: The article compares three different principles of simultaneous determination of anions and cations using CE. Utilization of electroosmotic flow Utilization of electroosmotic flow Electrolyte flow generation by external pressure Electrolyte flow generation by external pressure Sample infection on both sides of the capillary Sample infection on both sides of the capillary

Utilization of the electroosmotic flow Requires a strong electroosmotic flow (EOF) Requires a strong electroosmotic flow (EOF) Sample injection is taken from the side of capillary opposite of the detector Sample injection is taken from the side of capillary opposite of the detector Cations reach the detector first Cations reach the detector first EOF transports neutral species inside sample zone. EOF transports neutral species inside sample zone. Its necessary to increase the velocity of the EOF Its necessary to increase the velocity of the EOF

Electrolyte flow generation by external pressure Separation is carried out under conditions (pH of 6.0) Separation is carried out under conditions (pH of 6.0) EOF is not strong enough to transport small anions towards the detector EOF is not strong enough to transport small anions towards the detector Analysis of the cations is not possible Analysis of the cations is not possible Additional flow added to the system Additional flow added to the system Optimum conditions concerning velocities can be adjusted Optimum conditions concerning velocities can be adjusted

Sample injection on both sides of the capillary Electric field is applied Electric field is applied Cations and anions migrate to the detector from opposite sides Cations and anions migrate to the detector from opposite sides Position of the detector is chosen in regards to the migration velocity Position of the detector is chosen in regards to the migration velocity Hydrodynamic injection Hydrodynamic injection Advantages- no strong EOF needed and no additional broadening caused by applying pressure Advantages- no strong EOF needed and no additional broadening caused by applying pressure

Selection and optimization of the electrolyte systems Indirect UV detection is used if the analyte does not absorb in the UV-visible region of the spectrum. Indirect UV detection is used if the analyte does not absorb in the UV-visible region of the spectrum. In order to apply indirect UV detection the electrolyte system must contain UV active cations as well as UV active anions. In order to apply indirect UV detection the electrolyte system must contain UV active cations as well as UV active anions.

Imidazole-thiocyanate system Imidazlole as cationic component Imidazlole as cationic component Thiocyanate as anionic component Thiocyanate as anionic component 18-crown-6 was added to the electrolyte to resolve the ammonium 18-crown-6 was added to the electrolyte to resolve the ammonium Citric acid was used to separate the alkali earth ions Citric acid was used to separate the alkali earth ions To avoid overlapping of signals, slowest cation must leave point of detection before the first anion arrives at the detector. To avoid overlapping of signals, slowest cation must leave point of detection before the first anion arrives at the detector. Signals of the inorganic cations appear first in the electropherogram then less mobile cations, inorganic anions Signals of the inorganic cations appear first in the electropherogram then less mobile cations, inorganic anions

DIPP-TMA system Creates a strong EOF Creates a strong EOF Guarantees no overlapping of anion signals by cationic species if the EOF signal appears in front of the anion peaks Guarantees no overlapping of anion signals by cationic species if the EOF signal appears in front of the anion peaks DIPP as cationic UV absorbent DIPP as cationic UV absorbent TMA as anionic UV absorbent TMA as anionic UV absorbent HIBA as additive for optimization of the cation separtation HIBA as additive for optimization of the cation separtation

All analytes must be separated from each other and there must be enough space between the EOF signal and first anion peak. Separation was developed by optimization of three parameters: pH, temperature and capillary length

pH: Migration time of EOF, charge of the analytes and the electrolyte components depend on pH value Migration time of EOF, charge of the analytes and the electrolyte components depend on pH value pH<4.5 (anionic analytes show peak broadening and smaller peaks. pH<4.5 (anionic analytes show peak broadening and smaller peaks. Lower effective charge of the electrolyte anion TMA resulting in lower mobility. Lower effective charge of the electrolyte anion TMA resulting in lower mobility. Fronting of the anionic analyte peaks becomes stronger. Fronting of the anionic analyte peaks becomes stronger.

pH>5.5 Velocity of EOF becomes higher Velocity of EOF becomes higher Resolution of the cations become lower Resolution of the cations become lower Longer migration times Longer migration times 4.8 was the most advantageous pH

Capillary Length: Distance from EOF signal to anion peaks can be influenced by changing the capillary length between the cathodic end of the capillary and the detector Distance from EOF signal to anion peaks can be influenced by changing the capillary length between the cathodic end of the capillary and the detector Migration time of the EOF signal and the first anion should be at least 30 sec to guarantee quality of the separation Migration time of the EOF signal and the first anion should be at least 30 sec to guarantee quality of the separation

Temperature: 20 degrees Celsius- anion peaks were too small, which lead to overlapping 20 degrees Celsius- anion peaks were too small, which lead to overlapping 25 degrees Celsius- velocity of EOF increases and distance between EOF and anions increases 25 degrees Celsius- velocity of EOF increases and distance between EOF and anions increases Varying the temperature of the electrolyte system is a fast and easy method to choose the best conditions for different analytical tasks. Varying the temperature of the electrolyte system is a fast and easy method to choose the best conditions for different analytical tasks.

Conclusion: Simultaneous determination of low molecular mass cation and anions can be carried out with different separation techniques Most advantageous method utilized the sample injection at both sides of the capillary and opposite migration directions of anions and cations. The electrolyte system has to be selected with respect to special requirements for separation and the indirect UV detection of both cationic and anionic analytes

Effective separation was developed by optimization of pH, capillary length and temperature

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