Use of electrophoretic techniques and MALDI–TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and “washed pellets”

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and “washed pellets”) were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of “washed pellets” and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI–TOF MS of “washed pellets” enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI–TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.     

Dynamic labeling of diagnostically significant microbial cells in cerebrospinal fluid by red chromophoric non-ionogenic surfactant for capillary electrophoresis separations

During bacterial infections of the central nervous system the number of microorganisms in the cerebrospinal fluid is often ranging from few up to hundreds of cells per milliliter. The electrophoretic techniques with the UV-detection reach a detection limit for whole cells of approximately 10⁷ cells per milliliter. The coupling of the filtration cartridge with capillary isoelectric focusing can improve the detection limit by four orders of magnitude. In order to improve the detection limit the red non-ionogenic surfactant 1-[[4-(phenylazo)phenyl]azo]-2-hydroxy-3-naphthoic acid polyethylene glycol ester, PAPAN 1000, has been prepared and used for the dynamic labeling of analytes before filtration of the sample with a concentration modulation in the analysis of proteins or microorganisms. Values of isoelectric points of labeled analytes have been calculated using pI markers detectable at 515 nm and have been found comparable with pI of the native compounds. Minimum detectable amounts of proteins and microorganisms were lower than nanograms and a hundred labeled cells, respectively. The introduced method, coupling of the filtration cerebrospinal fluid spiked with microorganisms and labeled by PAPAN, facilitates their rapid CIEF separation in the pH gradient pH range of 2–5 at their clinically important level 10¹ to 10² cells per milliliter.     

Capillary isoelectric focusing of microorganisms in the pH range 2–5 in a dynamically modified FS capillary with UV detection

The isoelectric points of many microbial cells lie within the pH range spanning from 1.5 to 4.5. In this work, we suggest a CIEF method for the separation of cells according to their isoelectric points in the pH range of 2–5. It includes the segmental injection of the sample pulse composed of the segment of the selected simple ampholytes, the segment of the bioanalytes and the segment of carrier ampholytes into fused silica capillaries dynamically modified by poly(ethylene glycole). This polymer dissolved in the catholyte, in the anolyte and in the injected sample pulse was used for a prevention of the bioanalyte adsorption on the capillary surface and for the reduction of the electroosmotic flow. Between each focusing run, the capillaries were washed with the mixture of acetone/ethanol to achieve the reproducible and efficient CIEF. In order to trace of pH gradients, low-molecular-mass pI markers were used. The mixed cultures of microorganisms, Escherichia coli CCM 3954, Candida albicans CCM 8180, Candida parapsilosis, Candida krusei, Candida glabrata, Candida tropicalis, CCM 8223, Proteus vulgaris, Klebsiela pneumoniae, Staphylococcus aureus CCM 3953, Streptococcus agalactiae CCM 6187, Enterococcus faecalis CCM 4224 and Staphylococcus epidermidis CCM 4418, were focused and separated by the CIEF method suggested here. This CIEF method enables the separation and detection of the microbes from the mixed cultures within several minutes. The minimum detectable number of microbial cells was less than 10³.     

On-target fraction collection for the off-line coupling of capillary isoelectric focusing with matrix-assisted laser desorption/ionization mass spectrometry.

In the present work, we describe a collection system for the off-line coupling of capillary isoelectric focusing (CIEF) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In this system, the capillary effluent is directly deposited in fractions onto the MALDI target via the use of a sheath liquid. The collected fractions are subsequently supplemented with matrix and further analysed by MALDI-TOF mass spectrometry for mass assignment. The experimental set-up includes a fiber optic based UV detector operating at 280 nm, which allows the study of the influence of the sheath liquid composition on the CIEF separation. The influence of the carrier ampholyte concentration on the protein MALDI spectra was also evaluated and the feasibility of the collection method was finally demonstrated with a mixture of four standard proteins.     

MPTS-silica coated capillary microextraction on line hyphenated with inductively coupled plasma atomic emission spectrometry for the determination of Cu, Hg and Pb in biological samples

A novel sol-gel 3-mercaptopropyltrimethoxysilane (MPTS) modified silica coating was developed for capillary microextraction (CME) of trace Cu, Hg and Pb prior to their on line determination by inductively coupled plasma-atomic emission spectrometry (ICP-AES). This organic-inorganic hybrid coating was in situ created on the inner walls of fused silica capillary using a sol solution containing TMOS (tetramethoxysilane) as a precursor, MPTS as a co-precursor, ethanol as the solvent and hydrochloric acid as a catalyst. The structure of the capillary coating was characterized by FT-IR spectroscopy, Raman spectroscopy, SEM and TEM. The factors affecting on the capillary microextraction of analytes such as pH, sample flow rate and volume, elution solution and interfering ions had been investigated, and the optimized experimental parameters were obtained. Under the optimized conditions, the absorption capacity of MPTS-silica coated capillary was found to be 1.17, 1.96 and 1.19 μg m⁻¹ for Cu, Hg and Pb, and the limits of detection were as low as 0.17 0.22 and 0.52 ng mL⁻¹, respectively. With a sampling frequency of 12 h⁻¹, the relative standard deviations (R.S.D.s) were 4.2, 2.6 and 3.8% (C=4 ng mL⁻¹, n = 7, sample volume = 1 mL) for Cu, Hg and Pb, respectively. The proposed method had been successfully applied to the determination of Cu, Hg and Pb in human urine, human serum and preserved egg. To validate the proposed method, certified reference materials of BCR151 milk powder, GBW07601 (GSH-1) human hair, GSBZ 50016-90 and GSB 07-1183-2000 water samples were analyzed and the determined values were in a good agreement with the certified values.     

FIA-potentiometry in the sub-Nernstian response region for rapid and direct chloride assays in milk and in coconut water

A simple and reliable FIA-potentiometric system for rapid assays of chloride in certain food samples is described and evaluated. The system is constituted by an aquarium air pump to propel the carrier solution, a manually operated injector, a homemade dialysis flow cell, a solid-state chloride detector (Ag/AgCl), a reference electrode and a multimeter connected to a microcomputer for data acquisition. The dialysis unit enables direct analysis of liquid food samples without any other previous treatment. The principal novelties are the precision (R.S.D. of 1.2% for whole milk) and rapidity (90 determinations/h) of FIA measurements near and below the lower end of the linear (Nernstian) response region of the chloride ion-selective electrode (ISE), with an estimated detection limit (3 s) of 0.4 mg L⁻¹ Cl⁻ in the sample injected in donor stream. Data of peak potential versus sample chloride concentration (donor stream) was accurately fitted with a quadratic polynomial over the range between 4 and 1000 mg L⁻¹ (r² = 0.9999) and used as a calibration curve. The method was applied to the determination of chloride in milk and in coconut water samples. The validation of the results was done by comparison with a NIST reference material (milk) or by capillary electrophoresis (coconut water). For all analysis, no significant difference at a 95% confidence level was observed.     

Application of gas-sensor array technology for detection and monitoring of growth of spoilage bacteria in milk: A model study

The aim of this study was to develop a novel, rapid system for detection and monitoring of growth of undesirable bacteria in food using gas-sensor array technology. Three spoilage bacteria isolated from a cheese-processing hall were identified as Serratia marcescens, Serratia proteamacufans and Pseudomonas putida. The growth of these bacteria in milk was investigated using a commercial solid state based gas-sensor array system. On the basis of the temporal sensor readings of the pure cultures, bacterial growth could be monitored and the individual strains identified and followed throughout the complete growth cycle in both single and mixed culture. The gas-sensor signals could be used as early indicators of the onset of bacterial growth. Start detection of volatile bacterial metabolites coincided with the start of the exponential growth phase taking place around 7 h after inoculation and corresponding to bacterial numbers of 10⁴ (cfu/ml). The results were confirmed by comparing the gas profiles with the cell counts and by headspace gas chromatography mass spectrometry (GC/MS) of volatile microbial metabolites. High correlation (r > 0.90, p < 0.001) was found between the gas-sensor readings and major secondary volatile metabolites. Using the sensor readings, cell numbers of single strain cultures could be predicted with an error of less than 5%. The results show that it is possible to monitor growth of individual strains of spoilage bacteria in a mixed culture in milk on the basis of the type and amount of volatile compounds which they produce, using a gas-sensor array system. The system thus affords possibilities for further development for quick, more accurate and full scale determinations of shelf life, the design of spoilage indicators, rapid identification of undesired microorganisms and rapid measurements of spoilage.     

On-line amino acid-based capillary isoelectric focusing-ESI-MS/MS for protein digests analysis

Six amino acids with pIs that ranged from 3.2 to 9.7 were used as ampholytes to establish a pH gradient in capillary isoelectric focusing. This amino acid-based capillary isoelectric focusing (cIEF) was coupled with ESI-MS/MS using an electrokinetically pumped sheath-flow interface for peptide analysis. Amino acid-based isoelectric focusing generates a two-order of magnitude lower background signal than commercial ampholytes in the important m/z range of 300–1800. Good focusing was achieved for insulin receptor, which produced ∼10 s peak width. For 0.1 mg mL⁻¹ bovine serum albumin (BSA) digests, 24 ± 1 peptides (sequence coverage 47 ± 4%) were identified in triplicate analysis. As expected, the BSA peptides were separated according to their pI. The concentration detection limit for the BSA digests is 7 nM and the mass detection limit is 7 fmole. A solution of six bovine protein tryptic digests spanning 5 orders of magnitude in concentration was analyzed by amino acid based cIEF-ESI-MS/MS. Five proteins with a concentration range spanning 4 orders of magnitude were identified in triplicate runs. Using amino acid based cIEF-ESI-MS/MS, 112 protein groups and 303 unique peptides were identified in triplicate runs of a RAW 264.7 cell homogenate protein digest. In comparison with ampholyte based cIEF-ESI-MS/MS, amino acid based cIEF-ESI-MS/MS produces higher resolution of five acidic peptides, much cleaner mass spectra, and higher protein spectral counts.     

Conjugated linoleic acid determination in human milk by fast-gas chromatography

An efficient direct method for measuring c9,t11- and t10,c12-conjugated linoleic acid (CLA) isomer content in human and rat milk was developed and validated using an RTX-2330 capillary column (40 m × 0.18 mm × 0.1 μm). In comparison with the commonly used 100 m × 0.25 mm × 0.20 μm columns, this new type of fast column allowed the separation of FAMEs with the same resolution but in much less time. An additional advantage for biological samples was that only a small volume of sample was needed. Two different procedures were tested in order to select the best methylation of CLA isomers, and the alkali plus acid-catalyzed procedure was selected. The precision results showed relative standard deviations (R.S.D.) of repeatability and reproducibility ranging between 0.10 and 8.71%. The application of this method to human and rat milk samples showed that it was a rapid, simple and reliable method for the analysis of biological samples.     

Determination of aromatic amines in food products and composite food packaging bags by capillary electrophoresis coupled with transient isotachophoretic stacking

A capillary electrophoretic method was explored to assay aromatic amines in food samples. With an inline-coupled transient isotachophoretic stacking approach, the method has yielded about 200-fold improvement of sensitivity in UV detection of three primary aromatic amines and melamine. By using K⁺ as a leading ion and Tris⁺ as a terminating ion, a plug of 10 cm (equivalent to 0.44 μL) sample solution was allowed to introduce into a 60 cm (50 cm effective) capillary for separation, giving limits of detection down to 2.0 × 10⁻⁸ M. Baseline separation was achieved within 10 min, with relative standard deviation of 0.41–0.75% (intra-day) or 1.2–1.5% (inter-day) for migration time and 3.8–4.3% (intra-day) or 5.2–6.7% (inter-day) for peak area. The method was directly applicable to assaying the melamine in powder milk samples, with recovery in between 92.0% and 107.1%. The method could also be applied to the analysis of trace primary aromatic amines migrating from composite food packaging bags after combination of a 10-fold off-line concentration step, with limit of detection down to less than 1 μg/L. By this method, 4,4′-diaminophenylmethane and 2,4-diaminotoluene were thus found in three types of composite food packaging bags.     

Development and application of a capillary electrophoresis based method for the simultaneous screening of six antibiotics in spiked milk samples

An easy to use and low time consuming capillary electrophoresis (CE) method was developed and applied to the simultaneous determination of six antibiotics (ampicillin, amoxicillin, cloxacillin, penicillin, tetracycline and chloramphenicol) in spiked milk samples. Samples of milk were cleaned up by solid-phase extraction (with a C₁₈ cartridge) after protein precipitation. Analysis was performed by CE and results compared with the obtained via HPLC, both coupled to a UV-vis detector (210 nm). CE employed a 58.5 cm long fused-silica capillary (50 cm to detector), 75 μm i.d., a 2.7 × 10⁻² M KH₂PO₄, 4.3 × 10⁻² M Na₂B₄O₇ separation buffer, pH 8; an applied voltage of 18 kV; a hydrostatic injection of 0.5 psi during 3 s; and a run temperature of 25 °C. Under the described conditions, amoxicillin was not separated by HPLC, while CE was able to separate, and, therefore, allow detection. Regardless of amoxicillin, comparable results were obtained by HPLC and CE. The average recoveries of antibiotics, from milk fortified at 2.5 and 5 μg/mL, was over 72% with R.S.D.s within 5%. Recovery levels were essentially dictated by the used SPE cartridge.     

Protocol of capillary isoelectric focusing to separate extremely acidic and basic proteins

A new set-up was constructed for capillary isoelectric focusing (CIEF) involving a sampling capillary as a bypass fixed to the separation capillary. Sample solutions were subjected to a previously established pH gradient from the sample capillary. Besides performing conventional CIEF, the separation of ampholytic compounds with isoelectric points (p/s) beyond the pH gradient was carried out on this system. This method was termed as pH gradient driven electrophoresis (PGDE) and the basic mathematical expressions were derived to express the dynamic fundamentals. Proteins such as lysozyme, cytochrome C, and pepsin with p/s higher than 10 or below 3 were separated in a pH gradient provided by Pharmalyte (pH 3-10). Finally, this protocol convincingly exhibited its potential in the separation of a solution of chicken egg white.     

Pretreatment-free fast ultraviolet detection of melamine in milk products with a disposable microfluidic device

A new method for sensitive and fast screening of melamine (MEL) in milk products was developed with a low-cost disposable microfluidic device coupled with ultraviolet (UV) detection. This method avoided the need of sample pretreatment prior to the separation process, thus was simple and green. Due to the advantages of the device and fracture sampling technique, milk sample could be directly sampled through the fracture to achieve baseline separation from amino acids, and proteins in the sample did not interfere with the detection. Using 20 mM phosphate running buffer (pH 9.0), a sampling time of 3 s at +180 V and a separation voltage of +1800 V (240 V/cm), this method could detect MEL in milk within 75 s. At the detection wavelength of 202 nm, the linear range for MEL was from 1.0 to 100 μg mL⁻¹ with a detection limit of 0.23 μg mL⁻¹ (S/N = 3). The novel protocol had been successfully used to screen MEL in milk samples with recovery more than 82%. The environmentally friendly methodology for pretreatment-free sensitive screening of MEL provided promising applications in monitoring the quality of different foods.     

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 × 10⁻¹³ m²). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.     

Effect of Collagen Types,Bacterial Strains and Storage Duration on the Quality of Probiotic Fermented Sheep’s Milk

Collagen has become popular in dietary supplements, beverages and sports nutrition products. Therefore, the aim of this study was to evaluate the possibility of using various doses of collagen and collagen hydrolysate to produce probiotic sheep’s milk fermented with Lactobacillus acidophilus, Lacticaseibacillus casei, Lacticaseibacillus paracasei and Lacticaseibacillus rhamnosus. The effects of storage time, type and dose of collagen, and different probiotic bacteria on the physicochemical, organoleptic and microbiological properties of fermented sheep’s milk at 1 and 21 days of refrigerated storage were investigated. The addition of collagen to sheep’s milk increased the pH value after fermentation and reduced the lactic acid contents of fermented milk compared to control samples. After fermentation, the number of probiotic bacteria cells was higher than 8 log cfu g⁻¹. In sheep’s milk fermented by L. acidophilus and L. casei, good survival of bacteria during storage was observed, and there was no effect of collagen dose on the growth and survival of both strains. The addition of collagen, both in the form of hydrolysate and bovine collagen, resulted in darkening of the color of the milk and increased the sweet taste intensity of the fermented sheep’s milk. However, the addition of hydrolysate was effective in reducing syneresis in each milk sample compared to its control counterpart.     

Integration of microfiltration and anion-exchange nanoparticles-based magnetic separation with MALDI mass spectrometry for bacterial analysis

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is powerful in characterizing and identifying bacterial isolates. However, sufficient quantities of bacterial cells are required for generating MALDI mass spectra and a procedure to isolate and enrich target bacteria from sample matrix prior to MALDI-MS analysis is often necessary. In this paper, anion-exchange superparamagnetic nanoparticles (NPs), i.e., fluidMAG-DEAE and fluidMAG-Q, were employed to capture Aeromonas, Salmonella, Pseudomonas, Enterococcus, Bacillus, Staphylococcus and Escherichia coli from aqueous solutions and fresh water. The magnetically isolated bacteria were then characterized by whole cell MALDI-MS. The capture efficiency was found to be dependent on bacterial species, medium pH, the functional group and concentration of the NPs. The experimental results demonstrated that fluidMAG-DEAE and fluidMAG-Q were broad spectrum probes for bacteria. Furthermore, both dead and live bacteria could be captured by the NPs, and the live bacteria captured remained viable. Membrane filtration prior to the magnetic isolation could increase enrichment factor and eliminate potential matrix interference. A detection limit of 1 × 10³ cfu/ml was achieved for the bacteria spiked in tap water and reservoir water, and the analytical time was around 2 h.     

Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing

Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05 ± 0.83 × 10⁻³ s⁻¹. The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.     

Determination of oxytocin in milk of cows administered oxytocin

To address people's concerns of exogenous oxytocin (OT) administration to lactating bovines, a study was undertaken to (a) establish an enzyme immunoassay (EIA) for OT determination in milk, (b) quantify OT in milk of cows administered OT, and (c) study influence of pasteurization on OT stability in milk. A sensitive EIA validated according to the criteria of European Union—Decision 2002/657/EC was developed for OT in skim milk in an analytical range of 10-250 pg mL⁻¹ with a decision limit (CCα) of 30 pg mL⁻¹ and detection capability (CCβ) of 41.5 pg mL⁻¹. Milk samples collected from cows (n = 38) administered either 25 or 50 IU OT prior to milking were investigated for the presence of OT. There was no significant difference among both groups with the mean concentrations of OT being 15.8 and 14.9 pg mL⁻¹ for cows subjected to 25 and 50 IU OT administration, respectively. The OT levels in skim milk of control cows (n = 30; untreated) were basal (around 10 pg mL⁻¹). All the analyzed milk samples were below the CCα value of 30 pg mL⁻¹. Pasteurization of OT spiked milk samples at different temperature and sample holding conditions reduced the immunological activity of OT to 43% at 110 °C. However, no further decline occurred in the immunological activity with increased pasteurization temperature and time. It was concluded that the milk OT concentrations after OT administrations were minimal and below the assay decision limit. However, OT was quite stable to pasteurization in OT spiked milk.     

Diffusivity enhancement of water vapor in poly(vinyl alcohol)–fumed silica nano-composite membranes: Correlation with polymer crystallinity and free-volume properties

The diffusion coefficients of water vapor in poly(vinyl alcohol)–fumed silica (PVA–FS) nano-composite membranes were determined using the gravimetric method. Water vapor was observed to diffuse more rapidly in membranes with increased FS content. The vapor diffusion coefficient was determined as 1.2 × 10⁻¹³ m²/s in pure PVA and was observed to increase to 3.0 × 10⁻¹³ m²/s in PVA composites containing 30% FS nano-particles. The free-volumes of PVA–FS membranes were characterized using positron annihilation lifetime (PAL) spectroscopy. PAL results showed that both the ortho-positronium (o-Ps) lifetime and intensity increased with the addition of FS. The intensity (I₃) was found to be higher than the estimated value determined from the linear combination of the data from pristine PVA and FS, and correlated excellently with the polymer amorphous content. The PAL results indicate that a higher FS content in PVA increases the free-volume hole size (a volume increase from 40 to 55 Å³) and free-volume hole density (an I₃ increase from 23 to 28%), resulting in a higher fractional free-volume in the nano-composites. The increase in the relative polymer free-volume with higher FS content was associated with a decrease in the PVA crystallinity, as determined from differential scanning calorimetry measurements. It is postulated that the incorporated FS nano-particles interrupt polymeric chain packing and retard crystallization during membrane formation. More crystalline segments were transformed into amorphous regimes in the nano-composites containing more FS. A correlation between water diffusivity and the fractional free-volume was obtained, and the water diffusivity was successfully expressed by the free-volume theory.     

Miniaturized capillary electrophoresis system with ultraviolet photometric detection combined with flow injection sample introduction using a modified falling-drop interface

A miniaturized capillary electrophoresis (CE) system with UV-Vis detection was coupled to a flow injection (FI) system for achieving high throughput continuous sample introduction. The cassette of a commercial CE instrument was modified to hold a 6.5 cm long silica capillary and a flow-through waste reservoir. The cassette was inserted into the flow-cell chamber of a commercial UV detector, with the light beam focused on the capillary and collected by two ball lenses on the cassette. The capillary inlet, left outside the cassette and detector, was positioned on the top of a vertical 3.5 mm diameter glass rod, in close contact with an electrode. Samples injected through the FI system dropped freely on top of the pillar, covering the capillary inlet and electrode. Continuous sample introduction was achieved for CE separations under non-interrupted separation voltage, which was isolated from the FI system through the discontinuity of droplets. The newly developed interface and UV detection system was used for fast separation of sulphamethoxazole (SMZ) and trimethoprim (TMP) in sulphatrim tablets, achieving a high throughput of over 48 h⁻¹, and a low carryover of 2%. Separation efficiencies of 8 μm plate height and detection limits of 1.0 mg l⁻¹ for SMZ and 0.5 mg l⁻¹ (3σ) for TMP were obtained.