Capillary Zone Electrophoresis

Weinberger, Robert

doi:10.1016/b978-012742356-2/50004-3

Cited by 56 publications

(67 citation statements)

References 59 publications

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“…Under the typical conditions optimized for limited power dissipation, effective mobilities are generally considered to be independent of applied voltage. 1,15 However, Figure 2 shows for dendrimers a strong dependence on applied electric field, with a significant role of ionic strength. The effect of ionic strength is continuous, and at an ionic strength of 0.05 M, i.e., intermediate between the cases of low and high salt shown in Figure 2, the mobility becomes field independent, as shown in Figure 3 for G5.…”

Section: Resultsmentioning

confidence: 99%

Effect of Electric Field on the Mobility of Carboxyl-Terminated Dendrimers

2004

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The effect of an applied electric field on the mobilities of carboxyl-terminated dendrimers has been measured by capillary electrophoresis at applied voltages V ranging from 5 to 30 kV. The mobility increased with the field strength at moderate ionic strength, but varied inversely with V at low ionic strength. These results did not arise from Joule heating or from electroosmotic flow effects. It is suggested that the remarkably high velocities of these dendrimers enables them to outstrip their ion atmospheres at high V, in a manner related to the increase in conductance at high field strength first described by Wien in 1927. At low ionic strength, the increase in the size of the ion atmosphere leads to increased frictional drag with increasing field strength.

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Section: Resultsmentioning

confidence: 99%

Effect of Electric Field on the Mobility of Carboxyl-Terminated Dendrimers

2004

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“…The SphFl concentrations used were 2. 5,5,10,15,20,30,40, and 60 μM, using 2% fatty-acid free BSA as a carrier for SphFl. The K M and V max were determined assuming Michaelis-Menten kinetics and fitting the data points in Origin (OriginLab Corporation, Northampton, MA).…”

Section: Measurement Of Michaelis-menten Kineticsmentioning

confidence: 99%

“…The value was calculated using the equation N = 5.54(t m /W 1/2 ) 2 where t m is the migration time and W is the peak width at half-height. 40 The resolution was calculated as described by Baker and Skoog. 41,42 The SphFl and SphFl-1-P peaks were normalized against the Bodipy-Fl internal standard.…”

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confidence: 99%

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

et al. 2008

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Regulation of sphingosine and sphingosine-1-phosphate concentrations is of growing interest due to their importance in cellular signal transduction. Furthermore, new pharmaceutical agents moderating the intracellular and extracellular levels of sphingosine metabolites are showing promise in preclinical and clinical trials. In the present work, a quantitative assay relying on capillary electrophoresis with laser-induced fluorescence detection was developed to measure the interconversion of sphingosine and sphingosine-1-phosphate. The assay was demonstrated to be capable of determining the in vitro activity of both kinase and phosphatase using purified enzymes. The K M of sphingosine kinase for its fluorescently labeled substrate was 38 ± 18 μM with a V max of 0.4 ± 0.2 μM/min and a k cat of 3900 s −1 . Pharmacologic inhibition of sphingosine kinase in a concentration-dependent manner was also demonstrated. Moreover, the fluorescent substrate was shown to be readily taken up by mammalian cells making it possible to study the endogenous activity of sphingosine kinase activity in living cells. The method was readily adaptable to the use of either bulk cell lysates or very small numbers of intact cells. This new methodology provides enhancements over standard methods in sensitivity, quantification, and manpower for both in vitro and cell-based assays.The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration. [1][2][3][4][5][6] These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die. 7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis. 4,[7][8][9] S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2. 6,[9][10][11] In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine. 6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies. 7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases. 10,[15][16][17][18] Thus, sphingosine signaling is proving to be extremely important in clinical medicine. [17][18][19][20][21] *Corresponding authors. Phone: 919-966-2291 (C.E.S. and N.L.A.). Fax: 919-962-2388 (C.E.S. and N.L.A.). cesims@unc.edu (C.E.S.); nlallbri@unc.edu (N.L.A. Although S1P plays a major role as an extracellular signaling molecule, it is predominately syn...

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“…Desde então disseminou-se o uso de capilares com d.i. cada vez menor, o que permitiu aumentar a intensidade do campo elétrico sem necessidade de rotação do capilar 8 . Em 1981, Jorgenson e Lukacs 9 utilizaram capilares com 75 μm de d.i.…”

Section: Introductionunclassified

Um equipamento de eletroforese capilar com detecção condutométrica construído em laboratório

et al. 2005

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Recebido em 11/8/04; aceito em 30/3/05; publicado na web em 24/8/05A LABORATORY-MADE CAPILLARY ELECTROPHORESIS EQUIPMENT WITH CONDUCTOMETRIC DETECTION. A new construction of a capillary electrophoresis instrument with a universal conductivity detector (oscillometric detector) is described. The performance of the electrophoresis equipment was evaluated through the separation and detection of inorganic anions and cations in aqueous solutions. The results reproduced those found in the literature for the investigated probe ions, showing an efficient separation and good repeatability.Keywords: capillary electrophoresis instrument; oscillometric detector; inorganic ions. INTRODUÇÃOA importância da eletroforese capilar (CE) na análise de misturas complexas decorre da alta eficiência de separação e da simplicidade do equipamento utilizado 1 . O processo de separação dos componentes da mistura, injetada em um eletrólito de corrida que flui através do capilar, consiste no deslocamento do soluto sob a ação de um campo elétrico. As partículas ou íons migram na direção do cátodo ou do ânodo em função de sua carga específica 2 e a separação decorre das diferenças de mobilidade dos compostos iônicos ou ionizáveis presentes, da razão carga/volume das espéci-es e de fatores estruturais 3,4 . O eletroferograma, isto é, o registro gráfico do sinal do detector em função do tempo decorrido a partir da injeção da amostra, permite identificar e quantificar os componentes da mistura.A análise por eletroforese tem sua origem no método da fronteira móvel 5,6 . Posteriormente, para evitar o processo de convecção devido à força gravitacional, o tubo de vidro foi substituído por uma tira de papel ou empacotado com um meio quimicamente estável como pó de vidro, celulose ou gel 7 . Por sua vez, o efeito de convecção causado pela geração de calor devido à passagem de corrente elétrica através da solução foi minimizado por Hjertén pela rotação de um capilar com 3 mm de diâmetro interno (d.i. A escolha do método de detecção na eletroforese capilar depende, entre outros fatores, da compatibilidade do detector com as dimensões do capilar. Tradicionalmente, tem sido empregada a espectrometria de absorção molecular no UV-visível e, mais recentemente, a espectrometria de massas 1,11 . Por outro lado, as téc-nicas de detecção eletroquímica são especialmente adequadas à determinação de analitos que não são detectáveis por métodos ópticos 1,12-14 e quando são empregados tampões biológicos, que absorvem fortemente na região do ultra-violeta 15 . Para minimizar a interferência do elevado campo elétrico empregado na separação dos analitos no potencial do eletrodo de trabalho, emprega-se um desacoplador. Este consiste basicamente de uma fissura feita pró-xima da extremidade do capilar e coberta com um material poroso 1 . Este tipo de detecção apresenta como inconvenientes 13,14,16,17 a geração de gás no eletrólito de corrida, o maior tempo necessário para limpeza do eletrodo, seu alinhamento em relação à extremidade do capilar e sua calibração. O método ele...

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Capillary Zone Electrophoresis

Cited by 56 publications

References 59 publications

Effect of Electric Field on the Mobility of Carboxyl-Terminated Dendrimers

Effect of Electric Field on the Mobility of Carboxyl-Terminated Dendrimers

Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

Um equipamento de eletroforese capilar com detecção condutométrica construído em laboratório

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