High performance capillary electrophoresis - T.E.A.M.

detectors the absorbance of a solute is dependent on path
length, b, concentration, C, and molar absorptivity, e, as
defined by Beer’s law
A = bCe (31)
The short pathlength is the factor that mainly limits sensitivity
in CE. Due to the curvature of the capillary, the actual
pathlength in the capillary is less than the inner diameter
since only a fraction of the light passes directly through the
center. The actual pathlength can be determined by filling
the capillary with a solute of known concentration and
molar absorptivity.
High sensitivity can often be realized by use of low-UV
detection wavelengths. Peptides and carbohydrates, for
example, have no strong chromophores but can be adequately
detected at 200 nm or below (figure 56). Detection
at these low wavelengths necessitates the use of minimallyabsorbing
running buffers since high background absorbance
increases baseline noise and decreases signal.
Phosphate and borate are useful in this respect. Many biological
buffers such as HEPES, CAPS, and Tris are inappropriate
for use below about 215 nm.
Instrumentation/Operation
mAU
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1
0
Lysozyme peptide map
200 nm
214 nm
Figure 56
Use of low detection wavelengths to
increase signal-to-noise ratio
14.00 18.00 22.00 26.00
Time [min]
99