Cruces-Blanco, C., Gamiz-Gracia, L., Garcia-Campana A.M., Applications of Capillary Electrophoresis in Forensic Analytical Chemistry Trends in Analytical.

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

Review of capillary electrophoresis Sensitivity issues Stacking issues Some specific flaws

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 baseline2

Anode

Cathode

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: 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

Small amount of analyte injected1

Tiny peak volumes1

UV-Vis detection is the most common detector1

Beers Law A = ε*L*C A: absorbance ε: epsilon (L/mol*cm) L: path length (cm) C: concentration (mol/L)

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

HPLC uses 1 cm cells for UV analysis

Increased path length leads to increased absorbance

Leads to LOD of ~ ppb3

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

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

pH 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 pH stacking if too much analyte is loaded the separation efficiency is reduced

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

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 Variations in SDS concentration, pH, addition of tetra-

alkylammonium salts, capillary diameter, and injection times

Makes several runs necessary

Inappropriate conditions can cause Sample sticking to capillary walls Lack of separation or focus in peaks Decreased species stability leading to new species

peaks Inconsistent retention times

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

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 Company4. Michalke, B. Potential and limitations of capillary electrophoresis inductively coupled plasma mass spectrometry. J. Anal. At. Spectrom., 1999, 14, 1297-13025. Osbourn, D.M., Weiss, D.J., Lunte, C.E. On-line preconcentration methods for capillary electrophoresis. Electrophoresis 2000 August 21(14), 2768-2779