Cruces-Blanco, C., Gamiz-Gracia, L., Garcia-Campana A.M., Applications of Capillary Electrophoresis...
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Transcript of Cruces-Blanco, C., Gamiz-Gracia, L., Garcia-Campana A.M., Applications 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
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