Simultaneous determination Simultaneous determination of inorganic anions and of inorganic anions and

cations by capillary cations by capillary electrophoresis with electrophoresis with indirect UV detectionindirect UV detectionI. Haumann, J. Boden, A. Mainka, U. I. Haumann, J. Boden, A. Mainka, U. Jegle Jegle

Journal of Chromatography (2000)Journal of Chromatography (2000)895 , 269-277 895 , 269-277

Purpose:Purpose:

Investigate the separation of low molecular Investigate the separation of low molecular mass anions and cations by capillary mass anions and cations by capillary electrophoresiselectrophoresis

Use of indirect UV detection to analyze Use of indirect UV detection to analyze anions or cations.anions or cations.

Background:Background:

What is capillary electrophoresis (CE) ?What is capillary electrophoresis (CE) ?

Advantages of CEAdvantages of CE High separation efficiencyHigh separation efficiency Small sample size requiredSmall sample size required Fast separationFast separation ReproducibilityReproducibility AutomationAutomation

Electroosmosis is the fundamental Electroosmosis is the fundamental process that drives CE.process that drives CE.

Fused silica capillaries are used for Fused silica capillaries are used for separationsseparations

pI of fussed silica is about 1.5pI of fussed silica is about 1.5

Degree of ionization is controlled by the Degree of ionization is controlled by the pH of the bufferpH of the buffer

Experiment/Instrumentation:Experiment/Instrumentation:

Chemicals used- Chemicals used- Dimethyldiphenylphonium (DIPP) and Dimethyldiphenylphonium (DIPP) and trimesic acidtrimesic acid All solutions, electrolytes and standards were All solutions, electrolytes and standards were

prepared using water purified with a Mill-Q prepared using water purified with a Mill-Q System. System.

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

Fused Silica CapillaryFused Silica Capillary

Results/Discussion:Results/Discussion:

The article compares three different The article compares three different principles of simultaneous determination of principles of simultaneous determination of anions and cations using CE.anions and cations using CE. Utilization of electroosmotic flowUtilization of electroosmotic flow Electrolyte flow generation by external Electrolyte flow generation by external

pressurepressure Sample infection on both sides of the capillarySample infection on both sides of the capillary

Utilization of the electroosmotic flowUtilization of the electroosmotic flow Requires a strong electroosmotic flow (EOF)Requires a strong electroosmotic flow (EOF) Sample injection is taken from the side of Sample injection is taken from the side of

capillary opposite of the detectorcapillary opposite of the detector Cations reach the detector firstCations reach the detector first EOF transports neutral species inside sample EOF transports neutral species inside sample

zone.zone. Its necessary to increase the velocity of the Its necessary to increase the velocity of the

EOFEOF

Electrolyte flow generation by external Electrolyte flow generation by external pressurepressure Separation is carried out under conditions (pH Separation is carried out under conditions (pH

of 6.0)of 6.0) EOF is not strong enough to transport small EOF is not strong enough to transport small

anions towards the detectoranions towards the detector Analysis of the cations is not possible Analysis of the cations is not possible Additional flow added to the systemAdditional flow added to the system Optimum conditions concerning velocities can Optimum conditions concerning velocities can

be adjusted be adjusted

Sample injection on both sides of the capillarySample injection on both sides of the capillary Electric field is appliedElectric field is applied Cations and anions migrate to the detector from Cations and anions migrate to the detector from

opposite sidesopposite sides Position of the detector is chosen in regards to the Position of the detector is chosen in regards to the

migration velocitymigration velocity Hydrodynamic injectionHydrodynamic injection Advantages- no strong EOF needed and no additional Advantages- no strong EOF needed and no additional

broadening caused by applying pressurebroadening caused by applying pressure

Selection and optimization of the Selection and optimization of the electrolyte systemselectrolyte systems Indirect UV detection is used if the analyte Indirect UV detection is used if the analyte

does not absorb in the UV-visible region of does not absorb in the UV-visible region of the spectrum. the spectrum.

In order to apply indirect UV detection the In order to apply indirect UV detection the electrolyte system must contain UV active electrolyte system must contain UV active cations as well as UV active anions. cations as well as UV active anions.

Imidazole-thiocyanate systemImidazole-thiocyanate system Imidazlole as cationic component Imidazlole as cationic component Thiocyanate as anionic componentThiocyanate as anionic component 18-crown-6 was added to the electrolyte to resolve the 18-crown-6 was added to the electrolyte to resolve the

ammoniumammonium Citric acid was used to separate the alkali earth ionsCitric acid was used to separate the alkali earth ions To avoid overlapping of signals, slowest cation must leave To avoid overlapping of signals, slowest cation must leave

point of detection before the first anion arrives at the point of detection before the first anion arrives at the detector. detector.

Signals of the inorganic cations appear first in the Signals of the inorganic cations appear first in the electropherogram then less mobile cations, inorganic anionselectropherogram then less mobile cations, inorganic anions

DIPP-TMA systemDIPP-TMA system Creates a strong EOF Creates a strong EOF Guarantees no overlapping of anion signals Guarantees no overlapping of anion signals

by cationic species if the EOF signal appears by cationic species if the EOF signal appears in front of the anion peaksin front of the anion peaks

DIPP as cationic UV absorbentDIPP as cationic UV absorbent TMA as anionic UV absorbent TMA as anionic UV absorbent HIBA as additive for optimization of the cation HIBA as additive for optimization of the cation

separtationsepartation

All analytes must be separated from each All analytes must be separated from each other and there must be enough space other and there must be enough space between the EOF signal and first anion between the EOF signal and first anion peak. peak.

Separation was developed by optimization Separation was developed by optimization of three parameters: pH, temperature and of three parameters: pH, temperature and capillary lengthcapillary length

pH:pH: Migration time of EOF, charge of the analytes Migration time of EOF, charge of the analytes

and the electrolyte components depend on pH and the electrolyte components depend on pH valuevalue

pH<4.5 (anionic analytes show peak pH<4.5 (anionic analytes show peak broadening and smaller peaks. broadening and smaller peaks.

Lower effective charge of the electrolyte anion Lower effective charge of the electrolyte anion TMA resulting in lower mobility.TMA resulting in lower mobility.

Fronting of the anionic analyte peaks Fronting of the anionic analyte peaks becomes stronger.becomes stronger.

pH>5.5pH>5.5 Velocity of EOF becomes higherVelocity of EOF becomes higher Resolution of the cations become lowerResolution of the cations become lower Longer migration timesLonger migration times

4.8 was the most advantageous pH4.8 was the most advantageous pH

Capillary Length:Capillary Length: Distance from EOF signal to anion peaks can Distance from EOF signal to anion peaks can

be influenced by changing the capillary length be influenced by changing the capillary length between the cathodic end of the capillary and between the cathodic end of the capillary and the detectorthe detector

Migration time of the EOF signal and the first Migration time of the EOF signal and the first anion should be at least 30 sec to guarantee anion should be at least 30 sec to guarantee quality of the separationquality of the separation

Temperature:Temperature: 20 degrees Celsius- anion peaks were too 20 degrees Celsius- anion peaks were too

small, which lead to overlappingsmall, which lead to overlapping 25 degrees Celsius- velocity of EOF increases 25 degrees Celsius- velocity of EOF increases

and distance between EOF and anions and distance between EOF and anions increasesincreases

Varying the temperature of the electrolyte Varying the temperature of the electrolyte system is a fast and easy method to choose system is a fast and easy method to choose the best conditions for different analytical the best conditions for different analytical tasks.tasks.

Conclusion:Conclusion:

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

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

QUESTIONS?????????QUESTIONS?????????