Jian-Hua Wang

Multiwalled carbon nanotubes (MWCNTs) have been employed for the first time as sorbents for the isolation of basic proteins from other protein species in biological sample matrices by solid-phase extraction (SPE). A microcolumn packed with MWCNTs was incorporated after appropriate pretreatment into a sequential injection system, which facilitates online selective sorption of basic protein species (hemoglobin and cytochrome c in this particular case). The retained protein species were afterwards separated from each other by sequential elution from the microcolumn through the employment of appropriate eluents. A 0.025 mol L⁻¹ phosphate buffer solution of pH 8.0 facilitated the efficient collection of hemoglobin, while a 0.5 mol L⁻¹ NaCl solution ensured the quantitative recovery of the retained cytochrome c. With a sample loading volume of 2.0 mL, enrichment factors of 11 and 15 were derived for hemoglobin and cytochrome c, along with retention efficiencies of 100 % for both species and recovery rates of 98 and 90 %, respectively. A sampling frequency of 8 h⁻¹ was achieved, and the precisions were 3.0 % and 0.8 % (RSD) for hemoglobin and cytochrome c at a concentration of 5.0 μg mL⁻¹. The practical applicability of this system was demonstrated by processing of human whole blood for isolation of hemoglobin, and satisfactory results were obtained by assay with SDS-PAGE.

A spectrofluorometric method was developed for the determination of total serum protein by exploring toluidine blue (TB) as the fluorescence probe. The fluorescence intensity of TB at 648 nm was significantly quenched in the presence of sodium dodecylbenzene sulfonate (SDBS) by forming a dimer of the dye, which can afterwards reconvert to monomer when proteins were added accompanied by the recovery of the fluorescence. This might be attributed to the modulated transferring of the dimer‐monomer equilibrium of TB in the anionic surfactant caused by the addition of protein. A linear calibration graph was obtained in the range of 0.5–50 mg/l BSA, with a detection limit of 0.15 mg/l and a RSD of 1.3% (n=11, 5.0 mg/l BSA). Total proteins in human serums were analyzed by using the present procedure and the results agreed well with those obtained by the Biuret method.

Sodium tetrahydroborate (NaBH₄) was, for the first time, coated onto PTFE (polytetrafluoroethylene), FEP (fluorinated ethylene propylene), Excelon (microline) and PEEK (polytetrafluoroethylene) surfaces and used for mercury vapor generation-atomic fluorescence spectrometric determination. A lab-on-valve (LOV) incorporating a miniature membrane phase separation chamber was employed to facilitate the operation. A 600–1200 nm thin film of NaBH₄ solution was coated onto the interior surface of tubes by sweeping through 30 μl of NaBH₄ solution with an argon stream. Mercury cold vapor was generated as sample solution flowing through the pre-coated tubing carried by argon. The film coating was renewed for each operating run. The vapor was either immediately excited in the outlet of the separator in the LOV and detected by directing the fluorescence to a photomultiplier with optical fibers, or directed to a commercial AFS-920 atomic fluorescence spectrometer. NaBH₄ coating not only minimized fluorescence quenching by concomitant hydrogen encountered in conventional AFS, but the coating process also effectively eliminated mercury memory effects. In addition, the coating substantially simplified the operation of vapor generation using a single syringe pump for fluidic delivery. When using PTFE tubing as a coating medium, limits of detection of 0.06 and 0.005 μg l⁻¹ were derived by monitoring with a LOV-AFS system and an AFS-920 spectrometer, respectively, with a sample volume of 500 μl. A sampling frequency of 60 h⁻¹ along with RSDs of 2.9% and 2.0% at 2.0 μg l⁻¹ were achieved. Mercury contents in certified reference materials and a local coastal seawater were measured.

In the aqueous phase, ethidium bromide (EB) intercalates into the double helix structure of dsDNA (ds=double‐stranded) with a notable enhancement in fluorescence and resonance light scattering (RLS). However, when dsDNA was extracted into an ionic liquid (IL), 1‐butyl‐3‐methylimidazolium hexafluorophosphate (BmimPF₆), an abnormal RLS arising from the interactions of IL–DNA–EB was observed, with a substantial decrease of the recorded RLS. The cationic Bmim⁺ groups of BmimPF₆ intercalate into the DNA helix structure, in which they interact with the PO bonds of phosphate groups in DNA strands and result in a reduction of the base‐pair interstice along with transformation of DNA conformations that consequently prohibits the intercalation of EB with DNA. Thus, in the IL phase, the interactions between ethidium and DNA were dominated by electrostatic interactions and hydrogen bonding, leading to a congregation of EB entities around the DNA strands that results in an increase of absorption by ethidium, and consequently the inner filter effect leads to a reduction of the RLS. The present observation has been applied to the direct quantification of DNA in an ionic‐liquid phase after DNA from human whole blood was extracted into BmimPF₆.

A miniaturized atomic fluorescence spectrometric system was developed based on a novel lab-on-valve (LOV) configuration. The LOV integrates a micro-scale vapour generation chamber with a PTFE membrane (pore size 0.45 μm, thickness 60 μm) as the gas–liquid separation medium. The mercury cold vapour generated by reaction of sample with tetrahydroborate was separated and immediately excited in the outlet of the membrane gas–liquid separator by 253.7 nm incident light from a mercury hollow cathode lamp, while the fluorescence was monitored using a side-on photomultiplier. Both the incident light and the fluorescence were transmitted by fibre optics (600 or 1000 μm in diameter). A few important parameters governing the performance of the LOV atomic fluorescence spectrometric system were investigated, encompassing the positioning of the fibre optic tips in the excitation chamber, the concentration and volume of NaBH₄ solution, as well as the effect of the concomitant hydrogen from the reduction reaction of mercury. The limit of detection of the system for mercury was estimated to be 0.1 and 0.02 μg l⁻¹ by using fibre optics of 600 and 1000 μm diameter, respectively, with volume consumptions of 500 μl and 400 μl for sample and NaBH₄ solutions. A sampling frequency of 90 h⁻¹ was achieved along with RSD values of 2.7–4.4% at the concentration level of 1.0 μg l⁻¹ by using 600 μm diameter fibre optics. The present system was applied for the measurement of mercury in certified reference materials, i.e., CRM 176 (Trace Elements in a City Waste Incineration Ash), NASS-5 (Seawater) and SLRS-4 (Riverine Water).

Bead injection in a lab-on-valve (LOV) system was adopted for DNA purification via micro solid-phase extraction (SPE) with a renewable silica microcolumn packed in a channel of the LOV unit. The complex matrix components in human whole blood, including proteins, were well eliminated by choosing properly the sample loading and elution media. The DNA purification process was monitored on-line by using laser-induced fluorescence in a demountable side part of the LOV unit incorporating optical fibers. The practical applicability of the entire system was demonstrated by separation/purification of λ-DNA in a simulated matrix and human blood genetic DNA by performing SPE, in situ monitoring of the purified products, and postcolumn PCR amplification. When DNAs in a simulated matrix (10.0 ng μl⁻¹ λ-DNA, 50 ng μl⁻¹ bovine serum albumin, 1.0% Triton X-100) were processed in the present system and laser-induced fluorescence was monitored at 610 nm, an overall extraction/collection efficiency of 70% was achieved by employing identical sample loading and an elution flow rate of 0.5 μl s⁻¹, along with a precision of 3.8% relative standard deviation. DNA separation and purification from human whole-blood samples were performed under similar conditions. Lab-on-valve mesofluidic system employed for DNA separation and purification integrating a demountable fluorescence flow cell for in-situ laser induced fluorescence detection

During the last 30 years, flow injection analysis has gone through three generations, that is, the first generation in 1970s, supplemented by sequential injection in the 1990s as the second generation, and the recently emerged lab‐on‐valve system as the third generation, which holds clear advantages for instrumental miniaturization. The three generations have revolutionized the concept of sample pretreatment by facilitating on‐line operation and coupling with various detection techniques, among which its hyphenation with electrothermal atomic absorption spectrometry (ETAAS) has been one of the most attractive research field offering vast potentials and versatilities in the quantification of ultra‐trace metal species in complex matrices. In the present mini‐review, the state‐of‐the‐art developments for flow injection on‐line sample pretreatment coupled to ETAAS for trace metal analysis since 2003 were summarized, with special emphasis on the exploitations of the lab‐on‐valve system.

An ionic liquid/aqueous two‐phase system based on the hydrophilic ionic liquid 1‐butyl‐3‐methylimidazolium chloride (BmimCl) and K₂HPO₄ has been employed for direct extraction of proteins from human body fluids for the first time. Proteins present at low levels were quantitatively extracted into the BmimCl‐rich upper phase with a distribution ratio of about 10 between the upper and lower phase and an enrichment factor of 5. Addition of an appropriate amount of K₂HPO₄ to the separated upper phase results in a further phase separation, giving rise to an improved enrichment factor of 20. FTIR and UV spectroscopy demonstrated that no chemical (bonding) interactions between the ionic liquid and the protein functional groups were identifiable, while no alterations of the natural properties of the proteins were observed. The partitioning of proteins in the two‐phase system was assumed to have been facilitated by the electrostatic potential difference between the coexisting phases, as well as by salting out effects. The system could be applied successfully for the quantification of proteins in human urine after on‐line phase separation in a flow system. The use of an ionic liquid, as a green solvent, offers clear advantages over traditional liquid–liquid extractions, in which the use of toxic organic solvents is unavoidable.

The potential of biological cell-sorption for separation/pre-concentration of ultra-trace heavy metals was exploited by immobilizing Chlorella vulgaris and Saccharomyces cerevisiae cells onto silica beads and using them for cadmium sorption. FT-IR investigations showed that when C. vulgaris was used, the functional groups on the cell wall, i.e., hydroxyl and ether, were both involved in the sorption, while for S. cerevisiae, hydroxyl, amide and acetyl served as binding sites, but ether was not active. Because both cells contribute to the sorption, a significant improvement in retention efficiency was observed by immobilizing a mixture of C. vulgaris and S. cerevisiae cells onto silica used for sorption, with respect to those obtained by a single type of cell. A novel procedure for cadmium pre-concentration was developed based on this observation with detection by electrothermal atomic absorption spectrometry (ETAAS), employing cell immobilized silica beads for packing a micro-column in a sequential injection system. The cadmium retained on the column was eluted with a small amount of nitric acid and quantified with ETAAS. Within a range of 0.005–0.2 μg l⁻¹ and a sample volume of 1000 μl, the retention efficiencies achieved by using mixed cells, individual cells of C. vulgaris and individual cells of S. cerevisiae were 97%, 74% and 65%, respectively, with respect to 32% with pure silica. When a cell mixture was employed, an enrichment factor of 38.6, a limit of detection of 1.0 ng l⁻¹, along with a sampling frequency of 20 h⁻¹, was attained, leading to a precision of 2.3% RSD (0.05 μg l⁻¹). The procedure was validated by analyzing cadmium in a certified reference material of riverine water and spike recovery in a lake water sample.