Methacrylate monolithic capillary columns for gradient peptide separations

 

 

 

 

 

For the separation of peptides with gradient-elution liquid chromatography a poly(butylmethacrylate-

coethylene

dimethacrylate) (BMA) monolithic capillary column was prepared and tested. The conditional

peak capacity was used as a metric for the performance of this column, which was compared with a

capillary column packed with C18-modified silica particles. The retention of the peptides was found to

be smaller on the BMA column than on the particulate C18 column. To obtain the same retention in

isocratic elution an approximately 15% (v/v) lower acetonitrile concentration had to be used in the mobile

phase. The retention window in gradient elution was correspondingly smaller with the BMA column.

The relation between peak width and retention under gradient conditions was studied in detail. It was

found that in shallow gradients, with gradient times of 30 min and more, the peak widths of the least

retained compounds are strongly increased with the BMA column. This was attributed to the fact that

these compounds migrate and elute with an unfavorable high retention factor. More retained compounds

are eluted later in the gradient, but with a lower effective retention factor. With shallow gradients the

peak capacity of the BMA column (

≈90) was clearly lower than that of a conventional packed column

(

≈150). On the other hand, with steep gradients, when components elute with a low effective retention

factor, the performance of the BMA column is relatively good. With a gradient time of 15 min similar peak

widths and thus similar peak capacities (

≈75)were found for the packed and the monolithic column. Two

strategies were investigated to obtain higher peak capacities with methacrylate monolithic columns. The

use of lauryl methacrylate (LMA) instead of butyl methacrylate (BMA) gave an increase in retention and

narrower peaks for early eluting peptides. The peak capacity of the LMA column was

≈125 in a 60min

gradient. Another approach was to use a longer BMA column which resulted in a peak capacity of

≈135

could be obtained in 60 min.

Journal of Chromatography A, 1208 (2008) 109–115

 

Capillary gel electrophoresis analysis of G-quartet forming oligonucleotides used in DNA–protein interaction studies

DNA–protein binding is among the most frequently studied biomolecular interactions with high importance in modern systems biology research.

One interesting aspect of this rapidly developing field is the affinity capture of proteins by G-quartet forming oligonucleotides also referred to

as aptamers. G-quartets are structural motifs formed by guanine-rich sequences commonly occurring in the human genome. In this paper, we

describe a capillary gel electrophoresis based method to validate G-quartet formation of in-house designed oligonucleotides and discuss the effect

of monovalent cation concentration on the development of this structure. The relevant aptamer was then bound to magnetic beads to form an affinity

capture surface for target proteins, which were then analyzed by matrix-assisted laser desorption/ionization mass spectrometry.

Journal of Chromatography A
Volume 1161, Issues 1-2, 17 August 2007, Pages 15-21

Single cell determination of nitric oxide release using capillary electrophoresis

Measurements of nitric oxide (NO) release at single cell level are fundamental to understand the

diverse physiological functions of this remarkable molecule. To achieve this purpose, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was originally described for the sensitive

determination of NO release in individual neuron and mammalian cell after 8-(3,4-diaminophenyl)-2,6-

bis(2-carboxyethyl)-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-

s-indacene (DAMBO-PH) was

chosen as the fluorescent probe. Various parameters affecting NO trapping

in vivo and CE separation

were systematically studied. Under the optimal conditions, complete and fast separation of the resulted

targeted high-fluorescent triazole (DAMBO-P

H-T) was achieved in about 3 min (2.89 min), and the relative

standard deviations (RSDs) values of migration time and peak area were less than 5% and 9% for

intra-day and inter-day assays, respectively. The detection limit was 42 amol (at a signal-to-noise ratio

of 3). The feasibility of application of the developed method was validated by successfully applied to the

measurements of NO release from four single cell study models. This original application of this method

in diverse samples represents a powerful tool to study the kinetics of NO release by neuronal cells during

neurotransmission, as well as for the understanding of the pathobiological and therapeutic basis of this

molecule for cardiovascular diseases and under oxidative stress.

http://www.sciencedirect.com/science/journal/00219673