Download Online Photolytic Optical Gating of Caged Fluorophores in Capillary

Online Photolytic Optical Gating of Caged Fluorophores in Capillary Zone Electrophoresis
Utilizing an Ultraviolet-Light Emitting Diode
Elyssia S. Gallagher,1 Troy J. Comi,1 Kevin L. Braun,3 and Craig A. Aspinwall1,2*
1. Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona,
85721
2. Bio5 Institute, University of Arizona, Tucson, Arizona, 85721
3. Department of Chemistry, Beloit College, Beloit, WI, 53511
*Corresponding author:
1306 E. University Blvd, Tucson, AZ 85721
520-621-6338 (phone)
520-621-8407 (fax)
[email protected]
Abbreviations:
Arg
CFSE
Cit
HTCE
LED-IF
NO
POG
UV
arginine
caged fluorescein succinimidyl ester
citrulline
Hadamard transformation capillary electrophoresis
LED – induced fluorescence
nitric oxide
photolytic optical gating
ultraviolet
Key words: Capillary zone electrophoresis, Hadamard transformation, Photolytic optical gating,
Ultraviolet-light emitting diode
Supporting Information
Figure SI-1: Diagram of UV-LED POG instrument.
Figure SI-2: Images of UV-LED array and beam profile. (A) Image of the two-by-two matrix of
UV-LEDs mounted on an aluminum cooling plate. LEDs are circled in red. (B) Image of the
beam profile before the beam passes through the shutter. Images were taken at an angle. Images
were collected on an iPhone using 100 % magnification. Four distinct regions of light are visible
in the beam path due to the presence of four LEDs.
A.
B.
Table SI-1: Calculation of system variances
σ2Det (spot size)
σ2Det (amplifier)
σ2Det (collection rate)
σ2Det (Total)
σ2LD
σ2X
Variance (s2)
9.64 x 10-7
1.88 x 10-4
1.01 x 10-7
1.89 x 10-4
3.44 x 10-2
9.34 x 10-2
The total variance is defined by [1,2]:
σ2Tot = σ2Inj + σ2Det + σ2LD + σ2X
(SI-1)
Where σ2Tot represents the total separation variance, σ2Det is the variance associated with the
detector, σ2Inj is the variance from injection, σ2LD is the variance associated with linear diffusion,
and σ2X represents all other sources of variance, including that from Joule heating [1,3,4]. The
variance from the detector is a combination of the variance associated with the detection spot
size, the amplifier electronics, and the collection rate [1]. The variance from the detector
window width (spot size) can be calculated from Sternberg’s equation [1,5]:
σ2Det = (window width)2 / 12
(SI-2)
The spot size is approximated based on the Gaussian distribution of light within a laser beam.
Though the LED used here is non-Gaussian, we have utilized this approximation for simplicity.
Window width = 4λF / πD
where λ is the detection wavelength (496 nm), F is the focal length of the lens which focuses
light onto the capillary (25 m), and D is the diameter of the beam at the lens (2.5 mm) [1].
With these equations, as well as the velocity of CFSE during the separation, the variance due to
the detector spot size was calculated as 9.64 x 10-7 s2. The variance from the detection amplifier
was calculated to be 1.88 x 10-4 s2 based on the rise time (30 ms) of the current amplifier.
Finally, the variance from the data collection rate (500 Hz) was calculated to be 1.01 x 10-7 s2.
The total detector variance was 1.89 x 10-4 s2 with the majority of the detection variance coming
from the amplifier. Thus, the total variance from the detector represents less than 0.1 % of the
total separation variance. Using the diffusion constant for fluorescein [1], the variance
associated with linear diffusion was approximated as 3.44 x 10-2 s2, representing 25 % of the total
separation variance. Thus, a large percentage of variance is represented by σ2X, which is likely
due to Joule heating in the capillary since a larger capillary i.d. (25 µm) was used in this study
due to the large spot size of the UV-LED used for injection (1 mm) into the capillary.
References
1. Moore, A. W., Jorgenson, J. W., Anal. Chem. 1993, 65, 3550-3560.
2. Tao, L., Thompson, J. E., Kennedy, R. T., Anal. Chem. 1998, 70, 4015-4022.
3. Kennedy, R. T., Jorgenson, J. W., Anal. Chem. 1988, 60, 1521-1524.
4. Karger, B. L., Martin, M., Guiochon, G., Anal. Chem. 1974, 46, 1640-1647.
5. Sternberg, J. C., in: Advances in Chromatography, Giddings, J. C., Keller, R. A., Eds.,
Marcel Dekker, Inc., New York, 1966, 205 - 270.