Capillary isotachophoresis, detection

The hyphenation of CE and NMR combines a powerful separation technique with an information-rich detection method. Although compared with LC-NMR, CE-NMR is still in its infancy it has the potential to impact a variety of applications in pharmaceutical, food chemistry, forensics, environmental, and natural products analysis because of the high information content and low sample requirements of this method [82-84]. In addition to standard capillary electrophoresis separations, two CE variants have become increasingly important in CE-NMR, capillary electrochromatography and capillary isotachophoresis, both of which will be described later in this section. 

Fanali et al. have described a capillary isotachophoresis method for the determination of procaine in pharmaceuticals [ 150]. The drug was determined in a 6 pL sample of solution (Spofa product, obtained from Czechoslovakia, and diluted 180-fold) by cationic isotachophoresis in the single column mode. The system used a PTFE capillary column (20 cm x 0.3 mm) and a conductivity detector. The separation was carried out at room temperature, at 50 pA (but switched to 25 pA during detection). 

Finally, when RPC methods are used in preparative studies with peptides, the opportunity routinely exists for subsequent analysis of the recovered fractions by a variety of analytical methods including high-speed RP-HPLC, HP-IEX, HP-HILIC, or HP-IMAC, zonal or micellar electrokinetic high-performance capillary electrophoresis (HP-CZE and MECK-CZE), capillary electrochromatography (CEC), or capillary isotachophoresis. The combination of the RPC information, drawn from the In k versus i > plots, with the data derived from on-line spectroscopic detection thus readily provides a comprehensive opportunity to assess the purity of an isolated peptide, many of the physicochemical features of the interaction, as well as a means to optimize the resolution in the RPC separation. 

Other applications include the online coupling of capillary isotachophoresis and CZE for the quantitative determination of flavonoids in Hypericum perforatum (Guttiferae) leaves and flowers. This method involved the concentration and preseparation of the flavonoid fraction before introduction into the CZE capillary. The limit of detection for quercetin 3-0-glycosides was 100 ng/ml. ... 

Another CE separation method that has been adapted to on-line NMR detection for trace level separations is capillary isotachophoresis [22]. In this case, after the separation, the analyte bands are slowly moved through the capillary until they lie directly within the coil. Precise positioning of the analyte bands in the NMR detection coil can be difficult. A recent enhancement is the use of several NMR detection coils on a single separation capillary [23], In this way, the first coil acts as a scout coil and is optimized for sensitivity (not necessarily linewidth) to locate the analyte band as it moves through the coil. After an analyte band is detected, the flow is stopped after the appropriate time-interval so that the analyte bands are now located in the second coil, which is used to acquire high-resolution NMR spectra. 

Capillary electrophoresis (CE) coupled to MS has the advantage of high resolution and soft ionization for biomolecules, which may be used to differentiate post-translational modifications and variants of intact proteins and oligonucleotides. Different modes of CE (capillary zone electrophoresis, capillary isoelectric focusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as online preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) are used to compensate for the restricted detection sensitivity of the CE methodology [77, 78]. 

The separation unit of the capillary isotachophoresis instrument used is shown in Fig. 13.1. A 0.85mm id capillary tube made of fluorinated ethylene propylene copolymer was used in the pre-separation (first) stage and a capillary tube of 0.30mm id made of the same material served for the separation in the second stage. Both tubes were provided with conductivity detection cells [18] and an ac conductivity mode of detection [15] was used for making the separations visible. 

Capillary zone electrophoresis (CZE), with direct or indirect photometry and conductivity has become popular in wine analysis. Very little, or sometimes no sample preparation is needed and short analysis times are also apparent advantages of CE and CZE in the analysis of wine. Capillary isotachophoresis (ITP), with conductivity, thermometric, and UV absorption detection, is suitable for the separation of various anionic constituents (organic acids and inorganic anions), currently occurring in wines (Masar et al., 2001). 

Kvasnicka, F. and Voldrich, M. 2000. Determination of fumaric acid in apple juice by on-line coupled capillary isotachophoresis-capillary zone electrophoresis with UV detection. Journal of Chromatography A 891 175-181. 

Capillary isotachophoresis is a rapid, accurate, and potential detection technique for TTX. A small amount of TTX in contaminated extracts can be determined by this method (Shimada et al, 1983). It is performed using a cationic system, as TTX exists as cation under acidic and neutral conditions. Conditions for capillary isotachophoresis composed of 5 mmol/liter potassium acetate (pH 6.0) as an electrolyte, containing 0.2% Triton X-100 and 0.5 volume of dioxane, and 10 mmol p-alanine adjusted to pH 4.5 with acetic acid as a terminating electrolyte. When TTX is applied to isotachophoretic analyzer (Shimadzu IR-2A) equipped with a potential gradient 0.32, it is eventually monitored by the detector. PU is expressed as (PGs-PGl), where PGs, PGl, and PGt stand for potential gradient values for sample, leading ion, and... 

In the development of the method itself, the increase in the temperature in the isotachophoretic zones played an important role since temperature measurement was the first universal detection procedure with sufficient sensitivity in the capillary isotachophoresis . In connection with the detection by means of a thermocouple, more attention was devoted to the study of interrelations between the longitudinal distribution of the temperature on the outside wall of the isotachophoretic column and the width and the position of the zone boundary inside the column. At present, the thermocouple detector lost greater significance however, the temperature regime in the isotachophoretic column still requires adequate attention. 

Kaniansky, D., Rajec, R, Svec, A., Havasi, R, and Macasek, F., Online radiometric detection in capillary isotachophoresis. 1. Preliminary experiments, J. Chromatogr., 258, 238, 1983. 

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