Polyacrylamide-based monolithic capillary column with coating of cellulose tris(3,5-dimethylphenyl-carbamate) for enantiomer separation in capillary electrochromatography

A hydrophilic chiral capillary monolithic column for enantiomer separation in CEC was prepared by coating cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) on porous hydrophilic poly(acrylamide-co-N,N'-methylene-bisacrylamide) (poly(AA-co-MBA)) monolithic matrix with confine of a fused-silica capillary. The coating conditions were optimized to obtain a stable and reproducible chiral stationary phase for CEC. The effect of organic modifier of ACN in aqueous mobile phase for the enantiomer separation by CEC was investigated, and the significant influence of ACN on the enantioresolution and electrochromatographic retention was observed. Twelve pairs of enantiomers including acidic, neutral, and basic analytes were tested and nine pairs of them were baseline-enantioresolved with acidic and basic aqueous mobile phases. A good within-column repeatability in retention time (RSD = 2.4%) and resolution (RSD = 3.2%) was obtained by consecutive injections of a neutral compound, benzoin, on a prepared chiral monolithic column, while the between-column repeatability in retention time (RSD = 6.4%) and resolution (RSD = 9.6%) was observed by column-to-column examination. The prepared monolithic stationary phase showed good stability in either acidic or basic mobile phase.     

In situ synthesis of monolithic stationary phases for electrochromatographic separations: study of polymerization conditions

Acrylate-based monolithic capillary columns were prepared from fused-silica capillaries using UV photopolymerization. The effect of the pretreatment of the capillary wall surface before polymerization was investigated and several procedures were compared. The columns were characterization by van Deemter curves and SEM imaging. The results indicated that a pre-silanization of the capillary wall in order to introduce methacrylate groups at the wall surface gave similar efficiencies but more homogeneous structures than when the silanization agent was introduced in the polymerization mixture. The conditioning of the capillary before silanization, especially the conditions of basic rinsing was also an important factor. The effect of the dose of UV light that was applied for the polymerization had also been investigated. The results demonstrated that the irradiation energy is a critical parameter. The minimum energy threshold required to obtain a suitable monolith was 3 J/cm(2) and the maximum was around 12 J/cm(2). A higher energy destroys the monolith. Within the convenient range of energy, the columns had the same efficiency and a good structure as seen by SEM imaging. Using the optimized procedure for the pretreatment and an adequate energy, the column-to-column repeatability was found good (n = 12). The repeatability was obtained for the plate height at two velocity values, the retention factor and the electroosmotic mobility with RSD values below 10.     

Life-time studies with capillary electrochromatography columns operated under different conditions

A test system has been established to permit the monitoring of the life-time performance of several reversed- phase capillary electrochromatography (CEC) columns. The retention factors, k(cec), peak symmetry coefficients, lambda(sym), and column efficiencies, N, of three neutral n-alkylbenzene analytes, namely ethyl-, n-butyl- and n-pentylbenzenes, were determined for Hypersil 3 microm n-octylsilica and n-octadecylsilica packed into CEC capillary columns of 100 microm I.D., with a packed length of 250 mm, and a total length of 335 mm. The performances of these CEC capillary columns were examined for a variety of eluents with pH values ranging between pH 2.0 - 8.0, similar to those employed to study the retention behaviour of peptides that we have previously reported. The relative standard deviation (RSD) of the retention factors (k(cec) values) of these n-alkylbenzenes, acquired with an eluent of (25 mM Tris-HCl, pH 8.0,)-acetonitrile (1:4, v/v), when the CEC capillary columns were used for the first time (virgin values), were 4% (based on data acquired with 4 CEC capillary columns) for the n-octyl bonded silica capillary columns, and 6% (based on 8 columns) for n-octadecyl bonded silica capillary columns. The RSD values of the k(cec) values of the n-alkylbenzenes for one set of replicates (n=6) with one CEC capillary column was < 0.5%. The theoretical plate numbers, N, for the virgin CEC capillary columns were ca. 60,000, whilst the observed N values for all new CEC capillary columns were > or = 40,000 for n-octyl bonded silica capillary columns and > or = 50,000 for n-octadecyl bonded silica capillary columns. The peak symmetry coefficients, lambda(sym), of the n-alkylbenzenes for virgin CEC capillary columns and for CEC capillary columns used for more than 1,000 injections were always in the range 0.95-1.05. The experimental results clearly document that the life-time performance of the CEC capillary columns depends on the eluent composition, as well as the nature of the analytes to which the CEC capillary columns are exposed.     

Intact-protein trapping columns for proteomic analysis in capillary high-performance liquid chromatography

A new type of monolithic trapping columns with high mechanical strength was prepared by thin-layer sol–gel coating method and applied to trapping intact proteins for on-line capillary liquid chromatography. Monolithic trapping columns were fabricated by entrapping C8 reversed-phase particles into the capillary columns through a sol–gel network, which was formed by hydrolysis and polycondensation of methyltriethoxysilane. Hundreds times of trapping/untrapping for intact proteins were carried out. The trapping columns showed long-term stability up to 300 bar. Recovery, loading capacity and reproducibility of trapping columns were evaluated using four proteins. The recovery of four protein mixtures for the C8 monolithic trapping columns was 99.3% on average. The loading capacity of 5 mm × 320 μm i.d. C8 trapping columns for the protein mixtures was 30 μg. Day-to-day relative standard deviation (RSD) values for recoveries of protein mixtures on the same C8 trapping column ranged from 2.34 to 5.87%, column-to-column RSD values were from 3.01 to 6.81%. The C8 trapping columns were used to trap normal mouse liver intact proteins in a capillary liquid chromatography system. Results demonstrated high efficiency of the monolithic trapping columns for trapping intact proteins for proteomic analysis in on-line capillary liquid chromatography system.     

毛细管电色谱柱重复性考察及实验条件的选择

成功地研制了７５μｍ内径偶联式和７５,１００μｍ内径非偶联式毛细管电色谱柱。在自制电色谱柱上获得了小于２．１２％的保留时间ＲＳＤ值,考察了ｐＨ值、有机溶剂浓度对保留行为的影响,并用以指导实验条件的选择。比较了两种方式电色谱柱的峰形。     

LC with a Teicoplanin Aglycone Chiral Sorbent for the Separation of the Enantiomers of Non-Steroidal Anti-Inflammatory Drugs: An Evaluation of Chiral Capillary Columns

A capillary column with a teicoplanin aglycone (TAG) stationary phase (CSP) was used for enantioselective separation of selected profen non-steroidal anti-inflammatory drugs in capillary liquid chromatography (cLC). The effect of variations in the mobile phase composition on the retention and enantioselective separation was examined. The best resolution was attained in the mobile phase composed of 40/60 (v/v) methanol/1.0% triethylamine acetate buffer, pH 4.0 or 4.5. Under the optimized separation conditions, five of the set of eight analytes were enantioresolved with resolution values better than 0.9. Only fenoprofen was not enantioseparated in any system tested. The optimized separation conditions were used for evaluation of three chiral capillary columns (all prepared in the same way in our laboratory) in terms of the repeatability and reproducibility of the results. The run-to-run repeatability was expressed in terms of the relative standard deviation (RSD) values, obtained from ten independent measurements, for the following parameters: the retention factor for the first eluted enantiomer (k ₁), the selectivity (α), the enantioresolution (R), the theoretical plate count per meter for the first eluted enantiomer (N ₁) and the elution curve asymmetry for the first eluted enantiomer (As ₁). None of the RSD values exceeded 8%. The column-to-column reproducibility of these parameters ranged between 1 and 9%. The results obtained with TAG based CSPs in cLC (a laboratory packed capillary column) were compared with those obtained by classical high-performance liquid chromatography (HPLC) with a commercially available column. The cLC procedure provided a better enantioresolution and the elution curves had a better symmetry.

     

Lauroyl peroxide as thermal initiator of lauryl methacrylate monolithic columns for CEC

The preparation of lauryl methacrylate (LMA)-based monolithic columns for CEC using lauroyl peroxide (LPO) as thermal initiator of polymerization has been investigated. The influence of initiator amount and composition of porogenic solvent on the physical and electrochromatographic properties of the resulting LMA-based monoliths was evaluated. A comparison with LMA-based columns thermally polymerized with AIBN was performed. At a given porogenic solvent composition, LMA stationary phases initiated with LPO showed higher permeabilities and better efficiency values than those prepared using AIBN as initiator. The optimum polymerization mixture found for LPO initiator provided a minimum plate height of 9.5 mum in a polycyclic aromatic hydrocarbon mixture. The produced monolithic beds also exhibited a good run-to-run repeatability and column-to-column and mixture-to-mixture reproducibility, with RSD values below 5.3% for the retention factors, areas and plate heights.     

Rapid determination of globin chains in red blood cells by capillary electrophoresis using INSTCoated fused‐silica capillary

A laboratory‐made INSTCoated fused‐silica capillary has been newly used for CE separation of four mixtures of proteins in sodium phosphate BGEs at pH 3.0 and 2.5, respectively. The obtained separation efficiencies range from 145 000 theoretical plates per meter for myoglobin to 1 216 000 m^?1 for lysozyme. A total of 49–89% of the number of theoretical plates was obtained in a commercial polyvinyl alcohol coated capillary compared to the INSTCoated capillary under the same experimental conditions, 0–86% was obtained in a laboratory polyacrylamide‐coated capillary, and only 0–6% was obtained in an uncoated fused‐silica capillary. The RSD values for the intraday repeatability for an INSTCoated capillary were 0.1–1.0% (migration time) and 0.3–2.4% (peak area); RSD values for the interday repeatability in the same capillary are 0.6–1.4% (migration time) and 2.4–5.5% (peak area); RSD values for interday repeatability between different capillaries equaled 1.7–2.1% (migration time) and 2.8–10.9% (peak area). The INSTCoated capillary has been further used for rapid determination of globin chains isolated from red blood cells. A separation of α and β chains prepared from adult blood has been completed in 3 min with a peak resolution of 1.3, and the separation of α and ^Gγ chains prepared from newborn blood took 3 min with a peak resolution of 3.6.     

Gas chromatographic study of acetyl derivates of chlorinated guaiacols

Summary The gas chromatography of acetyl derivatives of guaiacol and all chlorinated guaiacols has been studied on SE-30, OV-351 and Carbowax 20M capillary columns. The retention order and relative retention times under isotermal and temperature-programmed conditions are discussed. Different retention times for all mono- and dichloro compounds have been observed on the SE-30 column. However, all trichloroguaiacol isomers were separated only on polar columns. The best overall temperature-programmed separations have been achieved on an SE-30 capillary column.     

Chromatographic selectivity of poly(alkyl methacrylate-co-divinylbenzene) monolithic columns for polar aromatic compounds by pressure-driven capillary liquid chromatography

In this study, divinylbenzene (DVB) was used as the cross-linker to prepare alkyl methacrylate (AlMA) monoliths for incorporating π-π interactions between the aromatic analytes and AlMA-DVB monolithic stationary phases in capillary LC analysis. Various AlMA/DVB ratios were investigated to prepare a series of 30% AlMA-DVB monolithic stationary phases in fused-silica capillaries (250-μm i.d.). The physical properties (such as porosity, permeability, and column efficiency) of the synthesized AlMA-DVB monolithic columns were investigated for characterization. Isocratic elution of phenol derivatives was first employed to evaluate the suitability of the prepared AlMA-DVB columns for small molecule separation. The run-to-run (0.16–1.20%, RSD; n = 3) and column-to-column (0.26–2.95%, RSD; n = 3) repeatabilities on retention times were also examined using the selected AlMA-DVB monolithic columns. The π-π interactions between the aromatic ring and the DVB-based stationary phase offered better recognition on polar analytes with aromatic moieties, which resulted in better separation resolution of aromatic analytes on the AlMA-DVB monolithic columns. In order to demonstrate the capability of potential environmental and/or food safety applications, eight phenylurea herbicides with single benzene ring and seven sulfonamide antibiotics with polyaromatic moieties were analyzed using the selected AlMA-DVB monolithic columns.     

LC with a Teicoplanin Aglycone Chiral Sorbent for the Separation of the Enantiomers of Non-Steroidal Anti-Inflammatory Drugs: An Evaluation of Chiral Capillary Columns

A capillary column with a teicoplanin aglycone (TAG) stationary phase (CSP) was used for enantioselective separation of selected profen non-steroidal anti-inflammatory drugs in capillary liquid chromatography (cLC). The effect of variations in the mobile phase composition on the retention and enantioselective separation was examined. The best resolution was attained in the mobile phase composed of 40/60 (v/v) methanol/1.0% triethylamine acetate buffer, pH 4.0 or 4.5. Under the optimized separation conditions, five of the set of eight analytes were enantioresolved with resolution values better than 0.9. Only fenoprofen was not enantioseparated in any system tested. The optimized separation conditions were used for evaluation of three chiral capillary columns (all prepared in the same way in our laboratory) in terms of the repeatability and reproducibility of the results. The run-to-run repeatability was expressed in terms of the relative standard deviation (RSD) values, obtained from ten independent measurements, for the following parameters: the retention factor for the first eluted enantiomer (k ₁), the selectivity (α), the enantioresolution (R), the theoretical plate count per meter for the first eluted enantiomer (N ₁) and the elution curve asymmetry for the first eluted enantiomer (As ₁). None of the RSD values exceeded 8%. The column-to-column reproducibility of these parameters ranged between 1 and 9%. The results obtained with TAG based CSPs in cLC (a laboratory packed capillary column) were compared with those obtained by classical high-performance liquid chromatography (HPLC) with a commercially available column. The cLC procedure provided a better enantioresolution and the elution curves had a better symmetry.     

Retention in coupled capillary column supercritical fluid chromatography with lipids as model compounds

Summary We have investigated to which extent retention data, acquired on single capillary columns, can be used for predicting retention factors in a coupled column system. For this purpose we utilized a model mixture of 18 lipid components with widely different vapor pressures and polarities. The sample was chromatographed on two columns, SB-biphenyl-30 (70% methyl-30% biphenylpolysiloxane) and SB-cyanopropyl-50 (50% methyl-50% cyanopropylsiloxane). Experimental retention factors, acquired in coupled column systems with two columns connected in different order, were thus compared with values calculated from runs on each single column. The agreement between calculated and experimental values generally was better than 5% without any pressure drop correction.To study the possibility of predicting retention behavior in a wide pressure range from a limited number of experiments, we also investigated the relation between solute retention and mobile phase density. We found that all data could be fitted to second order equations, which gives the possibility to optimize the resolution with respect to pressure from a limited number of runs at different pressures.     

Preparation and evaluation of a vancomycin-immobilized silica monolith as chiral stationary phase for CEC

Enantiomeric separations in CEC with the macrocyclic antibiotic vancomycin immobilized silica monolith as a chiral stationary phase are presented. The monolithic silica capillary columns were prepared by a sol-gel process in fused-silica capillaries with an inner diameter of 50 mum and subsequently in situ immobilization of vancomycin as a chiral selector by reductive amination. Enantioselectivity was obtained for eight pairs of enantiomers in nonaqueous polar organic or aqueous mobile phases and most of them were baseline-separated with high column efficiencies. It was observed that the organic modifier ratio (MeOH/ACN) in the polar organic mobile phase played a significant role in controlling the resolution and efficiency of the enantiomers. In enantiomeric separation of propranolol, repeatability for column efficiency and resolution in the nonaqueous mobile phase was given in terms of RSD values at 1.1 and 2.3% (n = 5) for run-to-run injections and 7.2 and 9.6% (n = 5) for column-to-column testing while repeatability for the separation of thalidomide in the aqueous mobile phase was given in terms of RSD values at 1.5, 2.8% and 6.1, 10.5%, respectively.     

Optimization of the porous structure and polarity of polymethacrylate-based monolithic capillary columns for the LC-MS separation of enzymatic digests

The porous structure as well as the polarity of methacrylate ester-based monolithic stationary phases has been optimized to achieve the separation of various peptides originating from enzymatic digestion. The porous structure, determined by the size of both pores and microglobules, was varied through changes in the composition of porogenic solvents in the polymerization mixture, while the polarity was controlled through the incorporation of butyl, lauryl, or octadecyl methacrylate in the polymer backbone. Both the morphology and the chemistry of the monoliths had a significant effect on the retention and efficiency of the capillary columns. The best resolution of peptidic fragments obtained by digestion of Cytochrome c with trypsin in solution was obtained in a gradient LC-MS mode using a monolithic capillary column of poly(lauryl methacrylate-co-ethylene dimethacrylate) featuring small pores and small microglobules. Raising the temperature from 25 to 60 degrees C enabled separations to be carried out at 40% higher flow rates. Separations carried out at 60 degrees C with a steeper gradient proceeded without loss of performance in half the time required for a comparable separation at room temperature. Our preparation technique affords monolithic columns with excellent column-to-column and run-to-run repeatability of retention times and pressure drops.     

A method for quantitative analysis of an anticancer drug in human plasma with CE-ESI-TOF-MS

In this study, the extraction recoveries of an anticancer drug (Imatinib) from human plasma using a common liquid-liquid extraction (LLE) method and a new strong cation exchange (SCX) solid-phase extraction (SPE) column was investigated. The extracts were analyzed with CE coupled on-line to electrospray ionization (ESI) time-of-flight mass spectrometry (TOF-MS) using a monoquaternarized piperazine compound (M7C4I) for capillary coatings. Clean extracts with high and reproducible extraction recoveries ranging between 85 and 91% with % RSD values of 2.5% (n = 3) were obtained using the SCX-SPE columns. This can be compared with the recoveries obtained with the LLE method ranging between 30 and 35%. The CE-ESI-TOF-MS analysis was performed in <10 min with peak efficiencies up to 4.7 × 10? plates/m and an average % RSD for the repeatability of the migration times of the analytes of 1.9% (n = 3) using acidic buffer and adding 5% ACN to the sample. The calibration curves were linear over the range of 0-1500 ng/mL with correlation coefficient (R2 ≥ 0.997 and % RSD values of 0.5% (n=3). The intra-day and inter-day assay variations were lower than 8%. The presented CE-ESI-TOF-MS method with the use of SCX-SPE columns yielded rapid, efficient and high extraction recoveries together with high sensitivity (LOD 5 ng/mL), selectivity and good linearity. Accordingly, the method can readily be used for accurate determination and therapeutic monitoring of the Imatinib blood levels for more effective patient treatment. In addition, it can be applied for the extraction, quantification and clinical assessments of metabolites of Imatinib and other basic pharmaceutical drug molecules in biological fluids or pharmaceutical dosage forms.     

Practical aspects of using methacrylate-ester-based monolithic columns in capillary electrochromatography

Methacrylate-ester-based monoliths containing quaternary ammonium groups were prepared in situ in capillary columns and in simultaneous experiments in vials, employing thermal initiation. The chromatographic properties of the monoliths were determined with capillary electrochromatography (CEC), and their morphology was studied with mercury-intrusion porosimetry on the bulk materials. Materials with different, well repeatable pore-size distributions could be prepared. A satisfactory column-to-column and run-to-run repeatability was obtained for the electro-osmotic mobility, the retention characteristics (k-values) and the efficiency on the columns prepared and tested in the CEC mode. A relatively high electro-osmotic flow was observed in the direction of the positive electrode. The electro-osmotic mobility was found to be influenced only marginally by mobile-phase parameters such as the pH, ionic strength, and acetonitrile content. The retention behavior of the monolithic columns was similar to that of columns packed with C18-modified silica particles. Columns could be prepared with optimum plate heights ranging from 6 microm for unretained compounds to 20 microm for well retained (k=2.5) polyaromatic hydrocarbons. However, for specific analytes a - still unexplained - lower chromatographic column efficiency was observed.     

Poly(cyclooctene)-based monolithic columns for capillary high performance liquid chromatography prepared via ring-opening metathesis polymerization

Monolithic columns for capillary HPLC were prepared via ring-opening metathesis polymerization (ROMP) from cis-cyclooctene (COE), tris(cyclooct-4-enyl-1-oxy)methylsilane (CL) as monomers, 2-propanol and toluene as porogens and RuCl(2)(Py)(2)(IMesH(2))(CHC(6)H(5)) (Py=pyridine, IMesH(2)=1,3-dimesityl-4,5-dihydroimidazolin-2-ylidene) as initiator within the confines of 200 microm i.d. fused silica columns. For evaluation of the novel monolithic capillary HPLC columns, a protein standard consisting of six proteins in the molecular weight range of 5800-66000 g/mol, i.e. ribonuclease A, insulin, albumin, lysozyme, myoglobin and beta-lactoglobulin, was used. Reproducibility of synthesis was checked by determining the relative standard deviation (RSD) in retention times (t(R)), which was found to be in the range of 2.9-3.9% for all analytes. Variations in polymer kinetics were realized by adding different amounts of free pyridine and had a significant influence on the monolith's morphology, the backpressure and retention times. On the contrary, variations in monomer content and COE to CL ratio showed only minor changes on these parameters. Long-term stability of 1000 runs at 50 degrees C showed excellent stability of the columns and no significant alteration in separation performance was observed in combination with slightly decreased retention times (approx. 1.6-7.2% for all analytes).     

Preparation and characterisation of anion-exchange latex-coated silica monoliths for capillary electrochromatography

Silica monoliths coated with functionalised latex particles have been prepared for use in monolithic ion-exchange capillary electrochromatography (IE-CEC) for the separation of inorganic anions. The ion-exchange monoliths were prepared using 70 nm quaternary ammonium, anion-exchange latex particles, which were bound electrostatically to a monolithic silica skeleton synthesised in a fused silica capillary. The resulting stationary phases were characterised in terms of their chromatographic performance and capacity. The capacity of a 50 microm diameter 25 cm latex-coated silica monolith was found to be 0.342 nanoequivalents and 80,000 theoretical plates per column were typically achieved for weakly retained anions, with lower efficiency being observed for analytes exhibiting strong ion-exchange interaction with the stationary phase. The electroosmotic flow (EOF) was reversed after the latex-coating was applied (-25.96 m2 V(-1) s(-1), relative standard deviation (RSD) 2.8%) and resulted in anions being separated in the co-EOF mode. Ion-exchange interactions between the analytes and the stationary phase were manipulated by varying the ion-exchange selectivity coefficient and the concentration of a competing ion (phosphate or perchlorate) present in the electrolyte. Large concentrations of competing ion (greater than 1M phosphate or 200 mM perchlorate) were required to completely suppress ion-exchange interactions, which highlighted the significant retention effects that could be achieved using monolithic columns compared to open tubular columns, without the problems associated with particle-packed columns. The latex-coated silica monoliths were easily produced in bulk quantities and performed reproducibly in acidic electrolytes. The high permeability and beneficial phase ratio makes these columns ideal for micro-LC and preconcentration applications.     

Ring-opening metathesis polymerization-derived monolithic capillary columns for high-performance liquid chromatography. Downscaling and application in medical research

Monolithic capillary columns were prepared via ring-opening metathesis polymerization (ROMP) using norborn-2-ene (NBE) and 1, 4, 4a, 5, 8, 8a-hexahydro-1, 4, 5, 8-exo,endo-dimethanonaphthalene (DMN-H6) as monomers. The monolithic polymer was copolymerized with Grubbs-type initiator RuCl(2)(PCy(3))(2)(CHPh) and a suitable porogenic system within the confines of fused silica capillaries of different inner diameter (I.D.). The first part of the study focused on batch-to-batch reproducibility of ROMP-derived capillary monoliths. Capillary monoliths of 200 microm I.D. showed good reproducibility in terms of retention times, with relative standard deviations (RSD) of 1.9% for proteins and 2.2% for peptides. However, the separately synthesized capillary monoliths revealed pronounced variation in back pressure with RSD values of up to 31%. These variations were considerably reduced by cooling of the capillaries during polymerization. Using this optimized preparation procedure capillary monoliths of 100 and 50 microm I.D. were synthesized and the effects of scaling down the column I.D. on the morphology and on the reproducibility of the polymerization process were investigated. In the second part, the applicability of ROMP-derived capillary monoliths to a separation problem common in medical research was assessed. A 200 microm I.D. monolithic column demonstrated excellent separation behavior for insulin and various insulin analogs, showing equivalent separation performance to Vydac C4 and Zorbax C3-based stationary phases. Moreover, the high permeability of monoliths enabled chromatographic separations at higher flow rates, which shortened analysis time to about one third. For the analysis of insulin in human biofluid samples, enhanced sensitivity was achieved by using a 50 microm I.D. ROMP-derived monolith.     

Parameters affecting reproducibility in capillary electrophoresis

It is well known that poor quantitative reproducibility substantially limits the practical implementation of capillary electrophoresis (CE) separations in chemical analysis. The principal sources of variance in observed peak areas are irreproducible flow rate, which influences on-column detector response, and inconsistent injection volume or amount. An overview of studies by researchers to address the reproducibility issue will be presented. In addition, current efforts in our laboratory to assess sources of quantitative variance for separations of dansylated amino acids using an automated CE system are presented and related when appropriate to the body of existing knowledge on this important topic. A comparison of different injection methods (hydrostatic vs. electrokinetic) and approaches (e.g., high vs. low pressure), the effect of random changes in electroosmotic flow (EOF) due to air bubbles in the CE capillary, and choice of certain peak integration parameters in terms of peak area reproducibility are presented. Under optimum conditions relative standard deviation (RSD) values in raw peak area are typically 2.0%. With nonoptimum conditions (e.g., with air bubbles in capillary), RSD values can substantially degrade. However, normalizing with retention times, internal standards, or observed electrophoretic current produces RSD values in a range of 1.4-2.3%.