THE DRAWBACKS OF CAPILLARY ELECTROPHORESIS CrucesBlanco C GamizGracia

THE DRAWBACKS OF CAPILLARY ELECTROPHORESIS Cruces-Blanco, C. , Gamiz-Gracia, L. , Garcia-Campana A. M. , Applications of Capillary Electrophoresis in Forensic Analytical Chemistry Trends in Analytical Chemistry 2007 (26) 3

OVERVIEW Review of capillary electrophoresis Sensitivity issues Stacking issues Some specific flaws

THE CHEMISTRY OF CAPILLARY ELECTROPHORESIS Anode Cathode Electrophoresis is the differential movement of ions in an electric field Detection occurs as resolved components move past a detector, typically UV, with output shown as peaks on a baseline 2

Separation suffers if injection volume exceeds 1% of the column volume Sample stacking can be done to increase the concentration of the sample within the column CE flows through the column electro-osmotically rather than laminar

SENSITIVITY ISSUES Sensitivity: the smallest signal an instrument can measure The greater the sensitivity of the instrument, the better it can differentiate compounds Sensitivity defined as the slope of the signal vs. concentration line

FACTORS CONTRIBUTING TO LOW SENSITIVITY Small amount of analyte injected 1 Tiny peak volumes 1 UV-Vis detection is the most common detector 1 Beers Law A = ε*L*C A: absorbance ε: epsilon (L/mol*cm) L: path length (cm) C: concentration (mol/L)

LIMIT OF DETECTION IN CAPILLARY ELECTROPHORESIS CE typically uses “on tube” analysis as the cell The path length of the cell is the internal diameter of the tube ~ 50 µm Leads to LOD of ~ ppm Detector portion of the tube must be bare Could lead to breakage of the tube

LIMIT OF DETECTION IN HPLC uses 1 cm cells for UV analysis Increased path length leads to increased absorbance Leads to LOD of ~ ppb 3

LENGTHENING OF THE CELL Z shaped cells lengthen the path length of the cell Path must not be so long as to allow more than one analyte Determination of flow rates for this is time consuming

SAMPLE STACKING the concentration of the samples must be dramatically increased to obtain the same signal -to-noise ratio as would result from a typical LC experiment Field stacking uses two buffers of differing resistance to concentrate the sample If the sample matrix contains salts this will cause band broadening and a decreased signal-to-noise ratio Efficiency is limited by laminar flow Flow profile can become convex or concave causing band broadening Resolution can be decreased due to large injection volumes used in stacking

p. H in the capillary can be affected countering the stacking effect Ionic strength of the analyte must be significantly lower than that of the background analyte Large volume sample stacking involves using reverse polarity, but the electrophoretic current must be monitored carefully or analyte will be lost In p. H stacking if too much analyte is loaded the separation efficiency is reduced

OTHER CURRENT PROBLEMS Laser-induced fluorescence More complex More expensive Limited excitation wavelengths Lack of data regarding standard retention times and peak areas Inability to quantify analyte Reproducibility comes into question Irreproducible flow rates Inconsistent injection volumes Lack of data regarding the reliability of each method used Pre-treatment reduces time effectiveness and involves the dilution of the analyte

SEPARATION METHODS Substance being analyzed are of complex composition

Identification is difficult using one method, but multiple methodologies produce problems Limits development of a generally applicable method Makes Variations in SDS concentration, p. H, addition of tetraalkylammonium salts, capillary diameter, and injection times several runs necessary Inappropriate Sample conditions can cause sticking to capillary walls Lack of separation or focus in peaks Decreased species stability leading to new species peaks Inconsistent retention times

CONCLUSION Capillary electrophoresis is a technique with potential but currently has several problems Sensitivity issues Sample stacking problems Lack data regarding reliablility and reproducibility of methods No standardized method, determining appropriate test conditions for unknown sample Capillary electrophoresis is not suitable for producing independently conclusive results

REFERENCES 1. Cruces-Blanco, C. , Gamiz-Gracia, L. , Garcia-Campana A. M. , Applications of Capillary Electrophoresis in Forensic Analytical Chemistry Trends in Analytical Chemistry 2007 (26) 3 2. Cunico, R. L. , Gooding, K. M. , Wehr, T. , Basic HPLC and CE of Biomolecules 1998 Bay Bioanalytical Laboratories, Inc 3. Harris, D. Quantitative Chemical Analysis 2003 W. H. Freeman and Company 4. Michalke, B. Potential and limitations of capillary electrophoresis inductively coupled plasma mass spectrometry. J. Anal. At. Spectrom. , 1999, 14, 1297 -1302 5. Osbourn, D. M. , Weiss, D. J. , Lunte, C. E. On-line preconcentration methods for capillary electrophoresis. Electrophoresis 2000 August 21(14), 2768 -2779