Direct analysis of hydrogen peroxide by capillary electrophoresis

A method is described for analysis of hydrogen peroxide directly by CZE in borate buffer based on its absorption in UV light at 185 nm, without reaction with dyes. The absorption at 185 nm was about 3.5 times better than that at 214 nm. Hydrogen peroxide was generated enzymatically from glucose in aqueous solutions and in serum and was removed by the catalase enzyme. To improve the sensitivity of detection, samples were concentrated on the capillary based on stacking by ACN. The method is rapid (approximately 7 min) and specific.     

Analysis of Tamm-Horsfall protein by high-performance liquid chromatography with native fluorescence

Tamm-Horsfall (TH) is a large glycoprotein which originates in the kidney and is very abundant in the urine. This protein has been measured mainly by immunoassays. Here we describe a different approach for its measurement based on high-performance liquid chromatography (HPLC) using a molecular exclusion column with native fluorescence detection in the ultraviolet range. This method in addition to measuring the level of the protein has the advantage of detecting changes in size or aggregation. Urine, 1 ml was mixed with 100 microl of 30% NaCl and left at 37 degrees C for 30 min. The urine was centrifuged at 12000 rpm for 20 s. The precipitate was vortex-mixed and dissolved in a triethanolamine buffer. A 20 microl aliquot was injected on a Macrosphere GPC column which was eluted with phosphate buffer and the effluent was detected by a fluorometer set at 280 nm for excitation and 325 nm for emission. Since the protein has a very large molecular mass compared to other urinary and serum proteins we did not experience any interference. It elutes as the first peak (in approximately 2.5 min on a 500 A and 2.7 min on 1000 A). The protein precipitates rapidly < 60 min at 37 degrees C. The detection in the UV is sensitive for this protein down to 1 mg/l in absence of any concentration steps. The method was linear between 1 and 100 mg/l. The R.S.D. was 10.4% (mean 62, n = 10). The mean level in 42 normal individuals was 31 mg/g creatinine and in 30 patients with proteinuria (different renal disorders) was 23 mg/g creatinine.     

Effect of sample composition on electrophoretic migration application to hemoglobin analysis by capillary electrophoresis and agarose electrophoresis

The electrophoretic migration, in routine analysis, is crucial for compound identification especially when multiple components are present in the sample. In complex or crude samples, such as those obtained from biological fluids, electrophoretic migration often does not correspond well to that of a pure standard compound. Several factors, related to the sample itself, have been identified as modulating the electrophoretic migration in zone electrophoresis both in gel and capillary electrophoresis (CE): solute mobility and concentrations, salt content, and protein interaction in the sample. Peak shape asymmetry often signals changes in migration especially when comparing samples with wide differences in concentration or those containing high ionic strength. Also, the migration of a protein can be influenced by the presence of a high concentration of another slowly migrating protein in the sample. A weak interaction during the separation between the two proteins which lead to a decreased velocity has been postulated. This was confirmed by finding a curve-linear relationship between the ratio of the two hemoglobin (Hb) variants, hemoglobin F (Hb F) and hemoglobin S (Hb S), and the distance between the two in gel electrophoresis (GE); and also by the observation of formation of a new small peak based on the analysis of hemoglobin F by capillary electrophoresis upon the addition of Hb S to the separation buffer. These factors when present together have an additive effect on the migration. As an example, Hb F, present in low but variable concentration in patients with sickle cell disease (Hb S), migrates in gel electrophoresis slightly slower than it is expected; enough to be confused with other unknown variants. However, the small peaks with different migration distances between Hb S and the adult Hb (Hb A) correlated well (r = 0.98) with Hb F performed by an alkali-denaturing assay indicating that these peaks are indeed Hb F in spite of the difference in their migration.     

Organic solvent high-field amplified stacking for basic compounds in capillary electrophoresis

Many water-miscible organic solvents, especially acetonitrile and acetone, bring along significant degrees (approximately 30 times) of stacking by electroinjection through high-field amplified injection for the basic compounds compared to that for aqueous buffers or water. The relative stacking of different compounds in acetonitrile or acetone is different compared to that for water. Stacking by electroinjection in organic solvents is less stringent and easier to accomplish in practice. Acids and salts, in aqueous solutions, can ruin the stacking for both organic and aqueous solvents; however, this effect can be better tolerated by diluting the sample in acetonitrile. Thus, this stacking is termed "organic solvent high-field amplified injection". This stacking by electroinjection is enhanced by increasing the electrophoresis buffer concentration and can be better than that by pressure injection. From the practical aspects, some cationic drugs present in serum such as amiodarone can be detected at the therapeutic levels by electroinjection on the capillary after protein precipitation by acetonitrile.     

Direct injection of organic solvent extracts for capillary electrophoresis

It is demonstrated here that organic solvents immiscible in water used in sample extraction, such as chloroform, can be injected directly and successfully on the capillary without the need for evaporation and reconstitution. Current continuity was maintained all the time during the run. In order to avoid the rapid evaporation of the organic solvent during the analysis, the aqueous layer was left over the chloroform. This simplified the extraction step, and enabled the injection from the same vial over several hours without dealing with problem of evaporation. The relative peak heights in the electropherograms can be modified by the inclusion in the chloroform of a more polar solvent, by adjusting the pH, or adjusting the salt content of the sample. Addition of a polar solvent to the chloroform improved greatly the precision of the analysis for both the peak height and migration time.     

Analysis of angiotension-converting enzyme by capillary electrophoresis.

A method is described for determination of serum angiotension-converting enzyme by capillary electrophoresis (CE) based on incubation of the substrate, a synthetic peptide, with the serum outside the capillary and cleaving hippuric acid and a dipeptide. The reaction is stopped by the addition of acetonitrile, followed by injection of the supernatant on the capillary. The acetonitrile allows injection of a large volume of sample on the capillary. Both the substrate and the reaction product (hippuric acid) can be monitored at the same time. The CE step is rapid and can be performed in about 6 min. The CE method compared well to a kinetic assay method (= 0.98).     

Transient pseudo-isotachophoresis for sample concentration in capillary electrophoresis

We show that many water miscible organic solvents such as acetonitrile, acetone and small alcohols can function as a terminating ion in transient isotachophoresis, which leads to sample concentration on the capillary. It is suggested that this method could be termed transient "pseudo-isotachophoresis" (pseudo-ITP). Because of their low conductivity, these water miscible organic solvents provide the high field strength necessary for band sharpening similar to that provided by the terminating ion. Salts, when present in such samples act briefly as leading ions, migrating rapidly in the organic solvent until they are slowed at the interface of the separation buffer. When the organic solvents are added to the sample, both the migrations as well as the stacking of the analytes are affected by the concentration of salts (leading ions) in the sample, similar to that observed in isotachophoresis. Our results show that this type of stacking offers good reproducibility and reliability for practical analysis. In practice, pseudo-ITP stacking is much easier to perform compared to that of true ITP with several added practical advantages as discussed.     

Stacking in capillary zone electrophoresis

Due to the short light path of the capillaries, the CE detection limit based on concentration, is far less than that of HPLC and not sufficient for many practical applications. Several methods, based on different electrophoretic maneuvers, can concentrate the sample (stack) easily on the capillary before the separation step of capillary zone electrophoresis (CZE). These methods incorporate different types of discontinuous buffers as the means for invoking different velocities to the same analyte molecules to produce a sharpening of the band (stacking). In CZE, these buffers can be often very simple such as sample dilution or adding to the sample a high concentration of a fast mobility ion. However, in other applications these buffers can be as complicated as those required for isotachophoresis. Stacking can often yield a concentration factor of 5-30-fold, which can improve greatly in CZE the detection limits bringing them very close to those of HPLC. Different methods of stacking, the importance of discontinuous buffers and the different mechanism for concentration on the capillary are reviewed here. As there is a need for more practical applications, there will be more methods devised for stacking in CZE.     

Stacking for nonaqueous capillary electrophoresis

Nonaqueous capillary electrophoresis (NACE) is a useful mode in CE for separation and quantification of hydrophobic compounds. However, because of the low conductivity of most of the organic solutions, stacking is not used often in this technique and the sample volume is very limited. As a result of the small sample volume, the detection limits are poor. Furthermore, NACE is affected greatly by the presence of salts in the sample. Here, we show that transient isotachophoresis (t-ITP) can be used easily in this type of electrophoresis to enhance the detection limits and also to reverse the deleterious effects of salts in the sample. Several factors, which affect the stacking in this type of electrophoresis, are described. For example, the presence of salts in the organic solvent, type of sample introduction, and the solvent for the terminating ion were all found to have profound effects on the degree of concentration. Furthermore, the separation time can be shortened by t-ITP.     

Stacking by electroinjection with discontinuous buffers in capillary zone electrophoresis

The work presented here demonstrates that electroinjection can be performed using discontinuous buffers, which can result in better stacking than that obtained by hydrodynamic injection. The sample can be concentrated at the tip of the capillary leaving practically the whole capillary for sample separation. This results in several advantages, such as better sample concentration, higher plate number and shorter time of stacking. However, sample introduction by electromigration is suited for samples free or low in salt content. Samples, which are high in salt content, are better introduced by the hydrodynamic injection for stacking by the discontinuous buffers. Different simple methods to introduce the discontinuity in the buffer for electroinjection are discussed.