Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips.
MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.
Roman
Gregory T
GT
Department of Chemistry, Kansas State University, Manhattan, KS 66506, USA.
Carroll
Susan
S
McDaniel
Kevin
K
Culbertson
Christopher T
CT
D000818:Animals; D002021:Buffers; D020374:Chromatography, Micellar Electrokinetic Capillary; D004129:Dimethylpolysiloxanes / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D005464:Fluorobenzenes / Q000737:chemistry; D006801:Humans; D046210:Microfluidic Analytical Techniques; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity
C513915:AlexaFluor 488 carboxylic acid tetrafluorophenyl ester; D002021:Buffers; D004129:Dimethylpolysiloxanes; D005456:Fluorescent Dyes; D005464:Fluorobenzenes; D011506:Proteins