Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.     

毛细管电泳法进行化妆品中砷的形态分析

从样品前处理、毛细管电泳修饰、进样方式、分离模式和检测条件等方面对毛细管电泳法研究化妆品中砷的形态进行讨论。在不同pH值的缓冲溶液中,用毛细管电泳法进行化妆品中砷的形态分析,测定波长为197nm。采用磷酸盐缓冲溶液,化妆品中As(Ⅲ)、二甲基胂(DMA)、对氨苯基胂酸(ANA)、一甲基胂(MMA)和As(V)等5种形态的砷可通过毛细管电泳法得到有效分离。     

Success and failure with phthalate buffers in capillary zone electrophoresis

Phthalate buffers are currently used in capillary electrophoresis as robust electrolyte systems for indirect detection. This contribution demonstrates that these buffers show regularly not only successful regions of mobilities of analytes (sample window) but also regions of failure where the migration of analytes is strongly deteriorated due to the presence of a system zone. System zones in phthalate buffers may be easily detected by UV detection and manifest themselves as peaks or dips. Peak shape diagrams are advantageously used for the prediction of the migration behavior of system zones in phthalate background electrolyte (BGE) systems at various pH. It is shown that the mobility of the system zone varies strongly with pH, is practically zero at pH values below 4 and above 7, and shows a maximum at pH 5. Thus, the system peak may coincide either with the peaks of various analytes or with the electroosmotic flow (EOF) peak. Experiments are given showing the effects of such coincidences as, e.g., zigzag detection patterns, double EOF peaks, and/or unusually broad peaks/dips. The message of this contribution is to show how to understand the electrophoretic properties of phthalate BGEs that, regardless of possible failure regions, may be successfully used in the analytical practice of capillary zone electrophoresis (CZE).     

Behaviour of inorganic and organic cations in the Debye–Hückel layer of DNA

Inorganic, monovalent cations (Li, Na, K, Rb, Cs), when present in the Debye–Hückel layer of DNA, are found to bind to the negatively charged groups of the helix solely on the basis of their charge/mass ratio. Thus, when an electric field is applied, the free mobility of the DNA is seen to increase from Li- to Cs-equilibrated DNAs, since the latter cation, having a weaker surface charge distribution and a larger physical size (in the non-hydrated state), is more loosely bound to the DNA helix, thus providing less screening of its negative charges. On the contrary, organic amines (Tris and a number of Good’s buffers) are found to bind not only via electrostatic interactions, but by additional bonds, notably H-bonds. In particular, Tris can form two H-bonds, with a purine and pyrimidine, respectively, and a third H-bond shared between the –OH groups of two adjacent Tris. Hence, these buffer components may be unwitting participants in reactions carried out in in vitro systems.     

General experimental aspects of the use of isoelectric buffers in capillary electrophoresis.

Four acidic, isoelectric buffers, for peptide and protein separations, have been recently described and adopted in capillary zone electrophoresis: cysteic acid [Cys-A, isoelectric point (pI) 1.85], iminodiacetic acid (IDA, pI 2.23), aspartic acid (Asp, pI 2.77) and glutamic acid (Glu, pI 3.22). These four buffers allow to explore an acidic portion of the titration curves of macroions, covering about 1.6 pH units (from pH 1.85 to ca. 3.45), thus permitting resolution of compounds having coincident titration curves at a given pH value. Given the rather acidic pI values of these buffers, their long-term stability has been investigated, by monitoring pH and conductivity changes upon increasing storage times. When dissolved in plain water, all four buffers appear to give constant pH and conductivity readings up to 15 days; after that, the conductivity keeps steadily increasing in a similar fashion. The same parameters, when the same buffers are dissolved in 6 M urea, appear to be stable for only one week, with the conductivity progressively augmenting after this period. A similar behaviour is exhibited by histidine (pI 7.70), a neutral, isoelectric buffer adopted for separation of DNA fragments. By mass spectrometry, Cys-A shows minute amounts (ca. 1%) of a degradation product after ageing for 3 weeks; in the same time period, Glu is extensively degraded (20%). No degradation species could be detected in IDA and Asp solutions. It is additionally shown that the acidic buffers are not quite stationary in the electric field, but can be transported at progressively higher rates (according to the pI value) from the cathodic to the anodic vessel. This is due to the fact that, at their respective pI values, a fraction of the amphotere has to be negatively charged in order to provide counterions to the excess of protons due to bulk water dissociation. Guidelines are given for the proper use and storage of such buffers.     

Stacking and discontinuous buffers in capillary zone electrophoresis

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.     

Novel, trifunctional diamine for silica coating in capillary zone electrophoresis

A novel compound ?quaternarized piperazine [(N-methyl,N-4-iodobutyl)-N'-methylpiperazine] (QPzI)? for the coating of a silica capillary able to reduce or invert the electroosmotic flow (EOF) in capillary zone electrophoresis is reported. Unlike standard oligoamines (like spermine and tetraethylene pentamine) which are very efficient in quenching macromolecule interaction with the silica wall, but only in acidic pH ranges, QPzI acts all along the pH scale, including alkaline pH ranges. It is believed that QPzI behaves like a trifunctional derivative: it forms ionic bonds with dissociated silanols via its quaternary nitrogen, hydrogen bonds via its tertiary nitrogen and, most importantly, a covalent bond via alkylation of ionized silanols through the terminal iodine atom in the butyl chain. Excellent separations are obtained with a variety of organic compounds, such as aromatic carboxylic acids, tryptophan metabolites and arylalkanoic acids. Such separations could not be obtained in naked capillaries in the presence of oligoamines and on some occasions not even with capillaries coated with a covalent layer of neutral polymers. In separations taking place in alkaline media, QPzI is not added to the background electrolyte, but is used simply in the capillary pre-conditioning step, a unique feature strongly supporting the hypothesis of its covalent binding to the silica surface. In difficult separations, such as in the case of o-/p-OMe-phenylacetic acids or nicotinic/picolinic acid, which would not normally occur under standard conditions, it is believed that QPzI acts as a discriminator, thus playing an active role in the separation process, rather than simply modulating the EOF.     

Characterization of poly(4-vinylpyridine 1-oxide) by free-solution capillary electrophoresis and micellar electrokinetic chromatography

The migration characteristics of poly(4-vinylpyridine 1-oxide) (PVP-NO) in phosphate buffers of acidic pH (20 mM H3PO4 or NaH2PO4) have been studied using both free-solution capillary electrophoresis (FSCE) and MEKC. To inhibit adsorption, 250 mM o-phosphoethanolamine (2-aminoethyl dihydrogen phosphate) was used. In FSCE, PVP-NO showed a narrow peak and a broader band, both having anionic behavior. These peak and band were attributed to the free and aggregated or micellized PVP-NO forms, respectively. According to surface tension measurements, the CMC of SDS in the BGE was 1.8 and 0.48 mM in the absence and in the presence of 1000 microg/mL PVP-NO, respectively, and the association of the polymer with SDS was completed at 9.7 mM SDS. Using MEKC, a narrow peak and a broader band also appeared at SDS concentrations of ca. 1 mM, and their intensity increased with the SDS concentration. These peak and band were attributed to the formation of mixed micelles constituted by both free PVP-NO/SDS and aggregated PVP-NO/SDS, respectively. The determination of PVP-NO by FSCE in commercial additives for laundry was demonstrated.     

A new strategy for optimizing sensitivity, speed, and resolution in capillary electrophoretic separation of DNA

DNA separations were performed in poly(ethylene oxide) (PEO) solutions prepared in 100 mM Tris-boric acid (TB) buffers using a capillary filled with TB buffers with concentrations up to 2.5 M, pH 10.0. The electroosmotic flow (EOF) increased with increasing the concentration of TB buffers till 1.5 M as a result of decreasing PEO adsorption on the capillary wall. At high TB concentrations (> 1.5 M), the peaks corresponding to small DNA fragments (11 and 8 base pairs) became sharper and were detected. Relative standard deviations of the EOF coefficient and the migration times of the DNA fragments were all less than 1% using a capillary filled with TB buffers at concentrations higher than 1.5 M. When separations were performed at different pH values of PEO solutions and TB buffers, better results in terms of sensitivity, speed, and resolution were generally achieved. The fluorescence intensity of the 2176 bp fragment obtained at pH values of TB buffers/PEO solutions 10.0/8.2 was 27-fold of that at pH values 8.2/8.2. The enhancement was related to effects of pH and borate on fluorescence intensity, DNA conformation, stacking, and interactions with the capillary wall. Using a capillary filled with 400 mM TB buffers, pH 10.0, the separation of DNA (pBR 322/HaeIII digest, pBR 328/Bg/I digest and pBR 328/HinfI digest) in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 9.0, at 375 V/cm was accomplished in less than 18 min.     

Electrochemical Behavior of Cu Electrode and Its Application in CE Determination of Polyols

The electrochemical behavior of a copper (Cu) electrode and its application in capillary electrophoresis determination of polyols was investigated in order to understand the redox property of Cu and achieve better separation efficiency. Electrochemical measurements were performed using a Cu electrode (fresh or oxidized) in buffer solutions having different pH values (7.8–13.0) by cyclic voltammetry. The Cu electrode showed higher electroactivity under stronger alkaline conditions. Further, the Cu electrode was found suitable for detecting weak oxidizing or reducing polyhydroxy compounds because of the redox reactions among Cu, Cu(I), and Cu(II) species. Thus, the Cu electrode was used in capillary zone electrophoresis (CZE) for separation and determination of propanediol and glycerol, weak electroactive polyols, using different separation (pH 8.7) and detection (pH 11.04) buffers. Separation and detection buffers with different pH values in CZE technology could offer efficient separation efficiency and detection limits at the same time.     

Importance of the counterion in optimization of a borate electrolyte system for analyses of anions in samples with complex matrices performed by capillary zone electrophoresis

Borate buffers are common background electrolytes for analyses of anions in capillary zone electrophoresis. Usually, sodium borate at a given pH is used and this specification seems to be sufficient for a successful analysis. In this paper, we show that free migration of OH(-) may deteriorate the analysis of a typical anionic analysis of clinical samples due to uncontrolled migration of OH(-) throughout the systems of analyzed zones and may damage the stacking of anionic analytes of interest. We have proven that the use of ammonium borate may remedy the situation where the presence of ammonium may selectively stop the free migration of OH(-) ions, slow down their effective mobility and bring their safe behavior resulting in reproducible stacking of clinically important anions. Results of real analyses of human serum samples confirmed the proposed method and proved that substitution of sodium for ammonium in borate buffers offers reliable analyses of clinical samples having chloride as the bulk component. The experimental results given in this paper are supported also by computer simulation, which can not only support the positive results but also show the dynamics of the separation that is otherwise hidden to any detection possibilities.     

Maximization of injection volumes for DNA analysis in capillary electrophoresis

We report concentration and separation of DNA in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solution. DNA fragments migrating against EOF stacked between the sample zone and PEO solution. To maximize the injection volume, several factors, such as concentrations of Tris-borate (TB) buffer and PEO solution, capillary size, and matrix, were carefully evaluated. The use of 25 mM TB buffers, pH 10.0, containing suitable amounts (less than 10 mM) of salts, such as sodium chloride, sodium phosphate, and sodium acetate, to prepare DNA is essential for the concentration of large-volume samples. In the presence of salts, the peaks also became sharper and the fluorescence intensity of DNA complexes increased. Using 2.5% PEO and a 150 microm capillary filled with 400 mM TB buffer, pH 10.0, up to 5 microL DNA samples (phiX 174 RF DNA-HaeIII digest or the mixture of pBR 322/HaeIII, pBR 328/Bg/I, and pBR 328/HinfI digests) have been analyzed, resulting in more than 400-fold improvements in the sensitivity compared to that by conventional injections (ca. 36 nL). Moreover, this method allows the analysis of 3.5 microL PCR products amplified after 17 cycles without any sample pretreatment.     

Screening for the beta-39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers

A novel method is reported for screening for point mutations in genomic DNA: free-zone capillary electrophoresis in very acidic buffers. This method exploits the charge difference among the four different bases (C, T, A, G) in a pH window between 2.5 and 3.5, where the four titration curves fan out. The method is applied to the detection of the beta-39 missense mutation in the beta-globin gene in thalassemias. A 60-mer fragment straddling the mutation site has been amplified. In an isoelectric buffer (iminodiacetic acid) of pH 3.3, partial resolution between the wild type and mutated strands is obtained. In a pH 3.0 phosphate buffer, baseline resolution is achieved between the two strands in a heterozygous individual. Due to the short size of the amplified fragment, this method can only be applied to routine screening for known mutations because resolution was lost in a fragment 100 bases long.     

Detection of neutral and charged mutations in alpha- and beta-human globin chains by capillary zone electrophoresis in isoelectric, acidic buffers

A simple and reliable method, for screening for point mutations in alpha- and beta-human globin chains, is reported here, utilizing capillary zone electrophoresis in isoelectric, acidic buffers. A solution of 50 mM iminodiacetic acid (pI 2.23) containing 7 M urea and 0.5% hydroxyethylcellulose (apparent pH 3.2) is used as background electrolyte for fast separation of heme-free, denatured globin (alpha and beta) chains. Due to the low conductivity of such buffers, high voltage gradients (600 V/cm) can be applied, thus reducing the separation time to only a few minutes. In presence of neutral to neutral amino acid substitutions, it is additionally shown that the inclusion of 3% surfactant (Tween 20) in the sample and background electrolyte induces the separation of the wild-type and mutant chains, probably by a mechanism of hydrophobic interaction of the more hydrophobic mutant with the detergent micelle, via a mechanism similar to "micellar electrokinetic chromatography". At this low operative pH, however, charged mutants, involving substitutions of acidic amino acids (Glu and Asp) are not detected, since these residues are extensively protonated. Curiously, however, they are still separated in presence of detergent, due to the large variation in hydrophobicity involved in such mutations. Of the 19 mutants analyzed, all but one were resolved: Hb St Nazaire (beta 103 Phe-->Ile). This is due to the fact that the delta G (in kcal/mol) in the substitution Phe-->Ile is zero, thus no separation can possibly take place between two chains exhibiting the same hydrophobicity parameter.     

Quenched phosphorescence, a new detection method in capillary electrophoresis

The applicability of quenched phosphorescence as a detection mode in capillary electrophoresis (CE) was explored for a number of analyte classes and buffer systems. The detection method is based on the quenching of biacetyl phosphorescence (biacetyl is a constituent of the CE buffer) by the analytes via various mechanisms (energy transfer, electron transfer and, possibly, hydrogen donation) and gives rise to negative peaks in the electropherograms. A number of buffers in the pH range 4.7-11.5, frequently used in CE, were tested for their compatibility with this detection mode. Borate, succinate, malonate, acetate, and phosphate buffers (pH 4.7-8.5) could be used without any problems. With a pH of ca. 8.5 or higher the baseline declined with time, while at a pH higher than 9.5 no signal at all was obtained. Obviously, the noise on the phosphorescence signal (i.e., the baseline) determines the ultimate analyte detection limits (LODs). The baseline signal-to-noise ratio, usually denoted as the dynamic reserve (DR), was enhanced ca. 25-fold compared to direct biacetyl excitation by sensitization of the biacetyl phosphorescence by 1,5-naphthalenedisulfonic acid, and by application of a total emission mirror (TEM). A concentration of 1 x 10(-3) M 1,5-naphthalenedisulfonic acid was found to be optimal. For the buffer systems considered, the DR was typically ca. 300-600 under optimized conditions (noise defined as 1 x sigma). Investigated analytes include naphthalenesulfonic acids (NS), nitrophenols, hydroxybenzoic acids, amino acids, and dithiocarbamates (DTCs.). For most of these, the LODs were in the 10(-7)-10(-8) M range, which is significantly lower than with direct or indirect absorption detection.     

A semipermanent coating for preventing protein adsorption at physiological pH in kinetic capillary electrophoresis

Protein adsorption to the inner capillary wall hinders the use of kinetic capillary electrophoresis (KCE) when studying noncovalent protein-ligand interactions. Permanent and dynamic capillary coatings have been previously reported to alleviate much of the problems associated with protein adsorption. The characteristic limitations associated with permanent and dynamic coatings motivated us to look at a third type of coating - semipermanent. Here, we demonstrate that a semipermanent capillary coating, designed by Lucy and co-workers, comprised of dioctadecyldimethylammonium bromide (DODAB) and polyoxyethylene (POE) stearate, greatly reduces protein adsorption at physiological pH - a necessary requirement for KCE. The coating (i) does not inhibit protein-DNA complex formation, (ii) prevents the adsorption of the analytes, and (iii) supports an electoosmotic flow required for many applications of KCE. The coating was tested in three physiological buffers using a well-known DNA aptamer and four proteins that severely bind to bare silica capillaries as standards. For every protein, a condition was found under which the semipermanent coating effectively suppresses protein adhesion. While no coating can completely prevent the adsorption of all proteins, our findings suggest that the DODAB/POE stearate coating can have a broad impact on CE at large, as it prevents the absorption of several well studied, highly adhesive proteins at physiological pH.     

Separation and migration behavior of positional and structural naphthalenesulfonate isomers by cyclodextrin-mediated capillary electrophoresis

The effects of the type of buffer system, buffer pH, the polarity of electrode, and both the type and the concentration of cyclodextrins (CDs) on the separation and migration behavior of seven positional and structural naphthalenesulfonate isomers in CD-mediated capillary electrophoresis were systematically investigated. The most effective separation conditions were to use 20 mM phosphate buffer with beta-CD at pH 3.0, while the polarity of the electrodes were reversed across the capillary. Under such conditions, these isomers can be separated in 10 min. The results also indicate that the interactions of naphthalenesulfonate derivatives with CDs are strongly affected by the position of the substituent(s) on the aromatic ring. The inclusion complex formation constants of these compounds were evaluated to improve our understanding of the interaction between the naphthalenesulfonate derivatives and CDs. Moreover, the formation constants of naphthalene-2-sulfonate to beta-CD agreed closely with the data in the literature obtained by a spectrophotometric method and by CE methods in various pH buffers.     

Use of quasi-isoelectric buffers to limit protein adsorption in capillary zone electrophoresis

The use of quasi-isoelectric buffers consisting of narrow pH cuts of carrier ampholytes (NC) has been investigated to limit protein adsorption on capillary walls during capillary zone electrophoresis experiments. To quantify protein adsorption on the silica surface, a method derived from that of Towns and Regnier has been developed. alpha-Lactalbumin (14 kDa, pI 4.8) and alpha-chymotrypsinogen A (25 kDa, pI 9.2) have been used as model proteins. Acidic narrow pH cuts of carrier ampholytes (NC, pH 3.0) obtained from fractionation of Serva 4-9 carrier ampholytes were used as BGE in bare-silica capillaries, and allowed to decrease significantly protein adsorption, as compared to experiments performed with classical formate buffer. The use of NC as BGE appeared to be as efficient as the use of polydimethylacrylamide coating to prevent protein adsorption. This increase of protein recovery when using NC was attributed to the interaction of carrier ampholytes with the silica surface, leading to a shielding of the capillary wall.     

Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis

Denaturing CE (DCE) is a powerful tool for analysis of DNA variation. The development of commercial multi-CE instruments allows large-scale studies of DNA variation (many samples and many fragments). However, the cost of consumables like capillary arrays and sieving matrix might limit the use of DCE in such studies. Thus, we have tested 72 different in-house formulated sieving matrices' ability to suppress EOF and separate PCR-amplified alleles with the DCE variant, cycling temperature CE (CTCE). The data herein demonstrate that alleles can be baseline-separated by use of PVP and poly(N,N-dimethyl acrylamide) polymers at various percentages and pH. Allele separation by CTCE is matrix-independent and consequently applicable to any capillary instrument used for DNA separation. Formulation of sieving matrix for CTCE was done by dissolving appropriate amount of polymer powder into the running buffers. Allele separation was observed at different pH (7.5-8.5), concentrations and molecular size of the polymer, without compromising the separation and reproducibility. Finally, the cost reduction of homemade matrices is more than 1000-fold as compared to commercial sieving matrices.     

The use of metal ion-supplemented buffers to enhance the resolution of peptides in capillary zone electrophoresis

The potential of metal ion-containing buffers to enhance the resolution of peptides in capillary zone electrophoresis was evaluated. The impact of adding Cu(II) and Zn(II) salts to electrophoresis buffers is shown to affect the migrational behavior of several dipeptides containing histidine. Interaction with a metal ion differentially decreases the electrophoretic mobilities of peptides which comigrate in the absence of metal ions, thus causing their separation. This effect is obtained at low pH where the large net charge on the samples yields short analysis times. The dependence of the resolution on Zn(II) concentration is presented for two different samples. The influence of the background buffer is discussed.