Physicochemical characterization of phosphinic pseudopeptides by capillary zone electrophoresis in highly acidic background electrolytes

Phosphinic pseudopeptides (i.e., peptide isosteres with one peptide bond replaced by a phosphinic acid moiety) were analyzed and physicochemically characterized by capillary zone electrophoresis in the pH range of 1.1-3.2, employing phosphoric, phosphinic, oxalic and dichloroacetic acids as background electrolyte (BGE) constituents. The acid dissociation constant (pK(a)) of phosphinate group in phosphinic pseudopeptides and ionic mobilities of these analytes were determined from the pH dependence of their effective electrophoretic mobilities corrected to standard temperature and constant ionic strength of the BGEs. It was shown that these corrections are necessary whenever precise mobility data at very low pH are to be determined. Additionally, it was found that the ionic mobilities of the phosphinic pseudopeptides and pK(a) of their phosphinate group are affected by the BGE constituent used. The variability of migration behavior of the pseudopeptides can be attributed to their ion-pairing formation with the BGE components.     

Monitoring folding transitions of synthetic,branched-chain polypeptides by capillary zone electrophoresis

The coil/helix transition of a synthetic, branched-chain polymeric polypeptide (poly (Lys(Glu(1)-DL-Ala(3))EAK), 50-Lys residues long in the backbone, as a function of increasing molarities of methanol in solution, is here studied by both, circular dichroism (CD) and capillary zone electrophoresis. CD spectra showed that, at 75% v/v methanol, the transition from random coil to fully helical structure was obtained, in a pH 1.1 HCI solution in the presence of 20 mM NaCI. CZE studies, run in parallel, exhibited the classical unfolding to folding sigmoidal transition, with mid-point at 60% v/v methanol concentration, plateauing at ca. 80% v/v organic solvent. Surprisingly, though, such unfolding to folding transition was accompanied by an expansion, rather than a contraction, of the resulting ordered polypeptide. As the charge of the polypeptide (a pure polycation at a pH of 2.1 in CZE) was kept rigorously constant, a plot of the radius of the polymer along the sigmoidal transition clearly showed that the radius of gyration of the helical, structured polypeptide was in fact larger than that of the random coil. Such results were confirmed by molecular dynamics simulations, which indicated that the dimensions of such polypeptide, in alpha-helix configuration, were 8.5 nm (in length) and 3.2 nm (in diameter), whereas those of the corresponding random coil were 7.2 nm (in length) and 5.1 nm (length of shorter axis). It would thus appear that the randomized structure assumes the shape of a more compact object, roughly resembling a "rugby ball".     

Splitless on-line coupling of capillary high-performance liquid chromatography,capillary electrochromatography and pressurized capillary electrochromatography with nuclear magnetic resonance spectroscopy

A mixture of unsaturated fatty acid methyl esters was separated with a new splitless capillary set-up. With the employed apparatus configuration different capillary separation techniques such as capillary high-performance liquid chromatography (cHPLC), capillary electrochromatography (CEC) and pressurized capillary electrochromatography (pCEC) could be applied. The detection and identification of the sample compounds were accomplished by hyphenating these capillary separation techniques with nuclear magnetic resonance (NMR) spectroscopy using a novel configuration of the detection capillary set-up. Using modified electrokinetically driven separation techniques, the electric field was applied solely across the separation column. With this improved interface for capillary liquid chromatography-NMR on-line coupling, the stereochemical assignment of the cis and trans configuration of unsaturated fatty acids could be easily accomplished. Finally, the results of cHPLC-NMR, CEC-NMR and pCEC-NMR coupling experiments were compared.Dedicated to Professor Günter Häfelinger on the occasion of his 65th birthday     

Field‐amplified sample injection combined with capillary electrophoresis for the simultaneous determination of five chlorophenols in water samples

A sensitive method of CZE‐ultraviolet (UV) detection based on the on‐line preconcentration strategy of field‐amplified sample injection (FASI) was developed for the simultaneous determination of five kinds of chlorophenols (CPs) namely 4‐chlorophenol (4‐CP), 2‐chlorophenol (2‐CP), 2,4‐dichlorophenol (2,4‐DCP), 2,4,6‐trichlorophenol (2,4,6‐TCP), and 2,6‐dichlorophenol (2,6‐DCP) in water samples. Several parameters affecting CZE and FASI conditions were systematically investigated. Under the optimal conditions, sensitivity enhancement factors for 4‐CP, 2‐CP, 2,4‐DCP, 2,4,6‐TCP, and 2,6‐DCP were 9, 27, 35, 43, and 43 folds, respectively, compared with the direct CZE, and the baseline separation was achieved within 5 min. Then, the developed FASI‐CZE‐UV method was applied to tap and lake water samples for the five CPs determination. The LODs (S/N = 3) were 0.0018–0.019 µg/mL and 0.0089–0.029 µg/mL in tap water and lake water, respectively. The values of LOQs in tap water (0.006–0.0074 µg/mL) were much lower than the maximum permissible concentrations of 2,4,6‐TCP, 2,4‐DCP, and 2‐CP in drinking water stipulated by World Health Organization (WHO) namely 0.3, 0.04, and 0.01 µg/mL, respectively, and thereby the method was suitable to detect the CPs according to WHO guidelines. Furthermore, the method attained high recoveries in the range of 83.0–119.0% at three spiking levels of five CPs in the two types of water samples, with relative standard deviations of 0.37–8.58%. The developed method was proved to be a simple, sensitive, highly automated, and efficient alternative to CPs determination in real water samples.     

An air‐assisted flow‐gating interface for capillary electrophoresis

A new kind of flow gating interface (FGI) has been designed for online connection of CE with flow‐through analytical techniques. The sample is injected into the separation capillary from a space from which the BGE was forced out by compressed air. A drop of sample solution with a volume of 75 nL is formed between the outlet of the delivery capillary supplying the solution from the flow‐through apparatus and the entrance to the CE capillary; the sample is hydrodynamically injected into the CE capillary from this drop. The sample is not mixed with the surrounding BGE solution during injection. The functioning of the proposed FGI is fully automated and the individual steps of the injection process are controlled by a computer. The injection sequence lasts several seconds and thus permits performance of rapid sequential analyses of the collected sample. FGI was tested for the separation of equimolar 50 μM mixture of the inorganic cations K⁺, Ba²⁺, Na⁺, Mg²⁺, and Li⁺ in 50 mM acetic acid/20 mM Tris (pH 4.5) as BGE. The obtained RSD values for the migration times varied in the range 0.7–1.0% and the values for the peak area were 0.7–1.4%; RSD were determined for ten repeated measurements.     

Resolving quantum dots and peptide assembly and disassembly using bending capillary electrophoresis

Despite the numerous techniques developed for the studying nanoparticle and peptide interaction nowadays, sensitive and convenient assay in the process of flow, especially to simulate the self‐assembly of quantum dots (QDs) and peptide inflow in blood vessels, still remains big challenges. Here, we report a novel assay for studying the self‐assembly of QDs and peptide, based on CE using a bending capillary. We demonstrate that the semicircles numbers of the bending capillary affect the self‐assembly kinetics of CdSe/ZnS QDs and ATTO‐D₃LVPRGSGP₉G₂H₆ peptide. Moreover, benefitting from this novel assay, the effect of the position on the self‐assembly has also been realized. More importantly, we also demonstrate that this novel assay can be used for studying the stability of the QDs–peptide complex inflow. We believe that our novel assay proposed in this work could be further used as a general strategy for the studying nanoparticle–biomolecule interaction or biomolecule–biomolecule interaction.     

PMOXA/PAA Brushes toward on-Line Preconcentration for BSA in Capillary Electrophoresis

In this work, a binary-mixed-brushes-coated (BBC) capillary with switchable protein adsorption/desorption properties was developed and applied for on-line preconcentration of proteins. Firstly, amine-terminated poly(2-methyl-2-oxazoline) (PMOXA-NH₂) and thiolterminated poly(acrylic acid) (PAA-SH) were synthesized by using cationic ring-opening polymerization (CROP) and reversible addition fragmentation chain transfer (RAFT) polymerization, respectively. Then, the BBC capillary based on poly(2-methyl-2-oxazoline) (PMOXA) and poly(acrylic acid) (PAA) was prepared by sequentially grafting of PMOXA-NH₂ and PAA-SH onto fused-silica capillary inner surface through poly(dopamine) (PDA) as an anchor. The obtained PMOXA/PAA coating formed on the capillary or capillary's raw material was characterized in terms of the thickness, surface chemical composition by using scanning electron microscope (SEM) and X-ray photoelectron spectrum (XPS). The switchable protein adsorption/desorption performance of the BBC capillary was investigated by using fluorescence microscope under di erent solutions with certain pH and ionic strength(I). The results showed that bovine serum albumin (BSA) could be adsorbed on BBC capillary at pH=5.0 (I=10^-5 mol/L), and then the adsorbed BSA could be released at pH=9.0 (I=0.1 mol/L). This switchable protein adsorption/desorption property of coated capillary was then used to preconcentrate proteins on-line for increasing the detection sensitivity of BSA in capillary electrophoresis (CE). With this method, a sensitivity enhancement factor (SEF) more than 5000 for BSA detection was obtained.     

Capillary electrophoresis with direct chemiluminescence detection for the analysis of catecholamines in human urine

A rapid and sensitive method for the analysis of three catecholamines by capillary electrophoresis(CE)with directchemiluminescence(CL)detection is described.The detection limits(S/N=3)were 1.3*10-8g/mL for isoprenaline,1.0*10-8g/mL for epinephrine and 2.8*10-8g/mL for dopamine.The proposed method was successfully applied to theanalysis of catecholamines in urine samples of cigarette smokers and nonsmokers.The results showed that there is a close relationbetween the release of dopamine in human body fluids and cigarette smoking/nonsmoking.     

Transient pseudo-isotachophoretic stacking in analysis of plasma for homocysteine by capillary zone electrophoresis

An analytical procedure enabling routine analysis of human plasma for total homocysteine has been developed and validated. The method includes reduction of homocysteine disulfides to thiol with tris 2-carboxyethylphosphine, derivatization of the thiol with 2-chloro-1-methylquinolinium tetrafluoroborate, separation of homocysteine 2-S-quinolinium derivative from those of plasma endogenous and exogenous thiol derivatives by capillary zone electrophoresis, and quantitation with the use of ultraviolet detection based on acetonitrile stacking. Method performance characteristics, for example recovery, calibration, precision, limit of detection, and limit of quantitation, are presented. The procedure was applied to analysis of plasma samples donated by apparently healthy volunteers.     

Temperature program optimization by computer simulation for the capillary GC analysis of fatty acid methyl esters on biscyanopropyl siloxane phases

The selectivity of capillary columns coated with biscyanopropyl siloxane stationary phases for the separation of fatty acid methyl esters has been optimized by means of computer-assisted column temperature optimization software. Temperature programming rates yielding the highest resolution in the shortest analysis time were selected for split, splitless, and on-column injection operated in the constant pressure and pressure programmed modes.