A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. It was found that a dynamic pH junction between sample and background electrolyte zones can be used to focus zwitterionic catecholamines and weakly acidic compounds without the use of special ampholytes. Differences in pH and concentration of complexing agents, such as borate, in the sample and background electrolyte zones were determined to cause focusing through changes in the local velocity of the analyte in two different segments of the capillary. Velocity-difference induced focusing (V-DIF) of analytes using a dynamic pH junction allowed the injection of large sample volumes and significantly improved the concentration sensitivity of CE. Under optimized conditions, the limit of detection for epinephrine was determined to be about 4 x 10(-8) M (the original sample) with conventional UV absorbance detection. Moreover, separation efficiencies greater than a million theoretical plates can be achieved by focusing such large sample volumes into narrow zones. Multisection electrolyte systems, which lead to the formation of a dynamic pH junction, can be tuned toward improving the concentration sensitivity of specific analytes if their chemical properties are known.
Velocity-difference induced focusing (V-DIF) of nucleotides was achieved by using a dynamic pH junction in capillary electrophoresis (CE) with UV detection. The influence of specific analyte properties, such as nucleotide base structure, sugar structure, and degree of phosphorylation, is examined. The pKa values and borate complexation with vicinal diols are important factors that caused the focusing. Therefore, the pH and borate content in the sample and background electrolyte can be adjusted to optimize the focusing effect. This method allows the injection of large volumes of sample (approximately 300 nL), resulting in at least 50-fold improvement in concentration sensitivity. The detection limit of 4.0 x 10(-8) M for nucleotides can be achieved in favorable conditions. V-DIF can be also applied to nucleotide pool analysis from cell extracts to improve the concentration sensitivity of CE and to reduce the time-consuming steps of desalting and off-line preconcentration that are often required for assays of nucleotides from biological samples.
Capillary electrophoresis is coupled with a single molecule
detector based on laser-induced fluorescence.
Individual
molecules migrating from the capillary are detected and
counted with 50% efficiency. Injection of 30 000
analyte
molecules generates a reproducible peak consisting of at
least five components. However, injection of 3000 or
fewer molecules leads to a noisy and irreproducible peak.
Monte Carlo simulation demonstrates that this irreproducibility results from molecular shot noise or stochastic
fluctuations in the number of injected molecules. The
model predicts that the relative standard deviations of
peak area, peak center, and peak width are inversely
proportional to the square root of the number of injected
molecules. At least 104 analyte molecules (17 zmol)
are
required to define peak area and width with 1% relative
precision. Fluctuation in the number of molecules
taken
for chemical analysis is a fundamental and irreducible
source of uncertainty.
An interface for CE-ESI-MS that decouples both the electrical and the solution flow rate requirements of the separation and ionization processes is presented. The interface uses a tapered and beveled stainless steel hollow needle surrounding the separation capillary terminus so that the inside of the electrode acts as the CE outlet vial and the outside tip acts as the electrospray emitter. No capillary pre-treatment is required, enabling the use of capillaries with any type of surface modification. A chemical modifier solution is introduced through a second capillary connected to the needle via a tee junction and can be used to improve the compatibility of the CE BGE with electrospray. The flow rate of modifier solution can be as low as 0.1 microL/min, much less than that in a typical sheath-flow interface, thus minimizing dilution of the CE effluent in order to maximize sensitivity. The presence of the modifier solution also allows the use of neutral-coated capillaries for protein analysis by CE-MS without using an assisting pressure, despite the absence of EOF under these conditions. The interface is easily integrated into a commercial CE instrument, such that all operations can be carried out by the automated controls. Compared with a commercial sheath-flow CE-MS interface operating under optimized conditions, LODs for amino acids were, on average, improved fivefold.
Capillary gel electrophoresis is demonstrated for the four-spectral-channel sequencing technique of Smith, the two-spectral-channel sequencing technique of Prober, and the one-spectral-channel sequencing technique of Richardson and Tabor. Sequencing rates up to 1000 bases/h are obtained at electric field strengths of 465 V/cm. At lower electric field strengths, capillary electrophoresis produces useful data for fragments greater than 550 nucleotides in length with 2 times better resolution than slab gel electrophoresis. An on-column detector produces detection limits of 200 zmol (1 zmol = 10(-21) mol = 600 molecules) for the four-spectral-channel technique. A postcolumn detector, based on the sheath flow cuvette, produces detection limits of 20 and 2 zmol for the two- and one-spectral-channel techniques, respectively.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.