Indirect UV Absorbance Detection Research Articles

Simultaneous separation of 17 anions by capillary electrophoresis with the addition of an organic solvent.

Seventeen inorganic and organic anions, that normally are insufficiently separated via ion chromatography, were completely separated by the addition of an organic solvent to a solution of BGE combined with an adjustment of the apparent pH via CE in combination with indirect UV absorbance detection. Methanol, ethanol, and acetonitrile were examined for their utility in manipulating the selective separation of anions. Methanol and acetonitrile were better modifiers than ethanol at enhancing the resolution of anions comigrating in an aqueous solution of BGE. Methanol was selected as the modifier that provided the largest separation window that could achieve a complete separation of the target analytes. Via the use of methanol, manipulation of the selectivity between inorganic anions and that between inorganic and organic anions was enhanced, but the separation between organic anions remained difficult when only methanol was used. By varying the apparent pH of the BGE in the presence of 10% v/v methanol, however, the separation selectivity between organic anions was substantially improved. Eventually, 7 inorganic and 10 organic anions were simultaneously separated using BGE at a pH of 6.3 in the presence of 10% v/v methanol.

Quantitation of underivatized branched-chain amino acids in sport nutritional supplements by capillary electrophoresis with direct or indirect UV absorbance detection.

The branched-chain amino acids (BCAAs) including leucine (Leu), isoleucine (Ile) and valine (Val) play a pivotal role in the human body. Herein, we developed capillary electrophoresis (CE) coupled with conventional UV detector to quantify underivatized BCAAs in two kinds of sport nutritional supplements. For direct UV detection at 195 nm, the BCAAs (Leu, two enantiomers of Ile and Val) were separated in a background electrolyte (BGE) consisting of 40.0 mmol/L sodium tetraborate, and 40.0 mmol/L β-cyclodextrin (β-CD) at pH 10.2. In addition, the indirect UV detection at 264 nm was achieved in a BGE of 2.0 mmol/L Na2HPO4, 10.0 mmol/L p-aminosalicylic acid (PAS) as UV absorbing probe, and 40.0 mmol/L β-CD at pH 12.2. The β-CD significantly benefited the isomeric separation of Leu, L- and D-Ile. The optimal conditions allowed the LODs (limit of detections) of direct and indirect UV absorption detection to be tens μmol/L level, which was comparable to the reported CE inline derivatization method. The RSDs (relative standard deviations) of migration time and peak area were less than 0.91% and 3.66% (n = 6). Finally, CE with indirect UV detection method was applied for the quantitation of BCAAs in two commercial sport nutritional supplements, and good recovery and precision were obtained. Such simple CE method without tedious derivatization process is feasible of quality control and efficacy evaluation of the supplemental proteins.

Determination of γ-hydroxybutyric acid in saliva by capillary electrophoresis coupled with contactless conductivity and indirect UV absorbance detectors.

The aim of the current study was to optimise and validate the methodology for determination of γ-hydroxybutyric acid (GHB) in saliva by CE combined with a contactless conductivity detector (C(4)D) and indirect UV absorbance detection (λ(ABS) = 210 nm). The optimized BGE, consisting of 8.5 mM maleic acid, 17 mM arginine, 255 μM cetyltrimethylammonium bromide (CTAB), and 15% acetonitrile, was evaluated for the separation of GHB in saliva within 6 min. The performance characteristics of the CE-C(4)D-indirect UV methodology was validated. The instrument detection and quantification limits were 0.49 and 1.6 mg/L for C(4)D, and 5.1 mg/L and 17.0 mg/L for indirect UV, respectively. The linearity was obtained over the range from 2.5 to 400 mg/L for C(4)D and from 12.5 to 400 mg/L for indirect UV. The interday precisions were within 2.3-5.7% and intraday precisions were within 1.6-9.0% for C(4)D as well as 2.1-9.3%, 5.6-10.1% for indirect UV in spiked saliva, respectively. The recoveries were within 87.2-104.4%. The matrix effects were +53.2% for small concentrations up to 25 mg/L for C(4)D and +23.6% for concentrations up to 75 for mg/L for indirect UV detection. No matrix effects were observed for higher concentration levels. In conclusion, CE-C4D-indirect UV can offer a rapid, accurate, sensitive, and definitive method for the determination of GHB abuse in saliva samples as a forensic screening tool.

Gas dispersion concentration of trace inorganic contaminants from fuel gas and analysis using head-column field-amplified sample stacking capillary electrophoresis

The presence of inorganic elements in fuel gas generally accelerates the corrosion and depletion of materials used in the fuel gas industry, and even leads to serious accidents. For identification of existing trace inorganic contaminants in fuel gas in a portable way, a highly efficient gas-liquid sampling collection system based on gas dispersion concentration is introduced in this work. Using the constructed dual path gas-liquid collection setup, inorganic cations and anions were simultaneously collected from real liquefied petroleum gas (LPG) and analyzed by capillary electrophoresis (CE) with indirect UV absorbance detection. The head-column field-amplified sample stacking technique was applied to improve the detection limits to 2-25 ng mL(-1). The developed collection and analytical methods have successfully determined existing inorganic contaminants in a real LPG sample in the range of 4.59-138.69 μg m(-3). The recoveries of cations and anions with spiked LPG samples were between 83.98 and 105.63%, and the relative standard deviations (RSDs) were less than 7.19%.

Capillary electrophoresis of small ions using complex formation and indirect detection

The journal Electrophoresis was launched in about the same time as modern CE. Today, Electrophoresis is one of the leading journals in the field of analytical separations. With over 23% of all papers on CE published in the past 5 years Electrophoresis is also the number one journal covering CE applications. During the past three decades CE has matured into one of the key separation techniques for the analysis of a large variety of ions. Analysis of small organic and inorganic ions used to be a high flyer during the development of CE, often under the name capillary ion analysis. In this mode it was mostly applied for analysis of inorganic ions in waters and/or determination of food ingredients and additives. Complex forming equilibria were commonly used for optimization of the separation and indirect UV absorbance detection was often the only choice for the detection of non-absorbing ions. This short critical overview reflects some of the aspects of CE based on indirect UV absorbance detection and complex forming equilibria related to our work on the topic published in Electrophoresis almost 20 years ago.

Rapid method for the determination of organic acids in wine by capillary electrophoresis with indirect UV detection

A capillary electrophoresis method for organic acids in wine was developed and validated. The optimal electrolyte consisted of 10 mmol/L 3,5-dinitrobenzoic acid (DNB) at pH 3.6 containing 0.2 mmol/L cetyltrimethylammonium bromide as flow reverser. DNB was chosen because it has an effective mobility similar to the organic acids under investigation, good buffering capacity at pH 3.6, and good chromophoric characteristics for indirect UV-absorbance detection at 254 nm. Sample preparation involved dilution and filtration. The method showed good performance characteristics: Linearity at 6 to 285 mg/L ( r > 0.99); detection and quantification limits of 0.64 to 1.55 and 2.12 to 5.15 mg/L, respectively; separation time of less than 5.5 min. Coefficients of variation for ten injections were less than 5% and recoveries varied from 95% to 102%. Application to 23 samples of Brazilian wine confirmed good repeatability and demonstrated wide variation in the organic acid concentrations.

Separation of common organic and inorganic anions in atmospheric aerosols using a piperazine buffer and capillary electrophoresis

The ability to monitor and quantify anionic components of aerosols is important for developing a better fundamental understanding of temporal and spatial variations in aerosol composition. Of the many methods that can be used to detect anions, capillary electrophoresis is among the most attractive ones because of its high separation efficiency, high resolving power for ionic compounds, and ability to be miniaturized for in-field monitoring. Here we present a method to baseline resolve common aerosol components nitrate, sulfate, chloride, and over two dozen organic acids in a single separation. A capillary electrophoresis separation utilizing a pH 5.78 piperazine buffer with 1,5-naphthalenedisulfonic acid as a probe for indirect UV absorbance detection was developed for this analysis. Previously, two different buffers were required to adequately separate all of these compounds. Electrophoretic mobilities, limits of detection, and migration time reproducibilities were measured for 38 organic and 8 inorganic anions. For solutions of low conductivity, detection limits for electrokinetic injections were found to be up to two orders of magnitude lower (0.2–0.4 μM) than those for pressure injection (1–45 μM). This separation was optimized and used for routine analysis of aqueous extracts of ambient atmospheric aerosols, but may be extended to other samples containing similar mixtures of anions.

A new type of migrating zone boundary in electrophoresis: 2. Transient sample zone shapes

A new type of migrating zone boundary in electrophoresis is presented theoretically and evidenced experimentally. This type of the boundary (called hybrid boundary) shows simultaneously a steady-state part with self-sharpening properties and an unsteady-state part with time-dependent electromigration dispersion. It is shown that a sample zone may possess a hybrid boundary both as its front and rear one simultaneously. In such a case, the evolution of a sample zone injected originally as a rectangular pulse exhibits very complex transient shapes before it reaches the well-known fronting or tailing triangular shape. Depending on the stage at which detection of such a sample zone occurs, variable and peculiar-shape peaks may appear in the electropherogram. Based on theoretical predictions, experimental examples of the above-mentioned phenomena are presented in this contribution for direct and indirect UV absorbance detection of sample zones. Excellent agreement of theoretical predictions with the experiments has been found. The knowledge of hybrid boundaries is of key significance for correct interpretation of records of CE analyses in practice.

Isoelectric buffers, part 3: Determination of pKa and pI values of diamino sulfate carrier ampholytes by indirect UV‐detection capillary electrophoresis

Ampholytes with close pK(a) values (i.e., good carrier ampholytes (CAs)) are needed as buffers in pH-biased isoelectric trapping (IET) separations. The syntheses of two families of such good CAs were reported recently. Members of the family of diamino sulfate ampholytes (first series) had pI values in the 5.7 < pI < 9.0 range. Members of the family of quaternary ammonium dicarboxylic acid ampholytes (second series) had pI values in the pI < 4.3 range. To further characterize the diamino sulfate ampholytes, their effective mobilities were measured by indirect UV-absorbance detection capillary electrophoresis in a series of background electrolytes (BGEs) with different pH values. The pK(a) and limiting ionic mobility values of the CAs were obtained by fitting these mobility values, as a function of the pH and the ionic strength of the BGEs, to the theoretical mobility expression. These diamino sulfates complete the list of CAs suitable for IET separations.

Determination of fosfomycin in pus by capillary zone electrophoresis

A method is described for the determination of fosfomycin in pus by capillary zone electrophoresis with reversed electroosmotic flow, and indirect UV absorbance detection. Sample pre-treatment is limited to removal of proteins and cell debris by adding the double volume of methanol, followed by vortexing for few seconds, and centrifugation at 15,000 × g for 2 min. The supernatant is directly injected into the instrument. Fosfomycin is separated from sample constituents with a background electrolyte at pH 7.25 (25 mM benzoate buffer with 0.5 mM hexadecyltrimethylammonium bromide added, adjusted to pH with tris(hydroxymethyl)-aminomethane (TRIS)). Separation is carried out in a capillary with 50 μm I.D., 64.5 cm total length, 56.0 cm to the detector, at 25 °C with −25 kV voltage applied. Due to the low absorbance of the analyte, indirect UV detection was performed at 254 nm using a bubble cell capillary. Sample was injected by pressure (450 mbar s). Repeatability for fosfomycin in spiked pus (from 8 or 10 consecutive injections of three different series at concentrations of 100 μg/mL of the antibiotic) was between 2.4 and 8.2% relative standard deviation (RSD). Accuracy (expressed as recovery of fosfomycin determined by three independent analysis at 10, 100 and 300 μg/mL fosfomycin added to plain pus) was between 75 and 102%. Intermediate reproducibility ( n = 9 at three different days) was between 2 and 12% RSD. Limit of detection and limit of quantitation were 4.5 and 15 μg/mL, respectively. The concentration of fosfomycin in pus of patients treated with the antibiotic ranged up to 240 μg/mL. The concentration of other anionic pus constituents identified beside chloride (acetate, succinate, lactate, phosphate) ranged between 20 and 7800 μg/mL.

Calibrationless quantitative analysis by indirect UV absorbance detection in capillary zone electrophoresis: The concept of the conversion factor

A new, fast and efficient procedure is described for the simultaneous quantitative analysis of various non-UV absorbing species in a sample, by capillary zone electrophoresis with indirect UV absorbance detection. The procedure is based on the concept of the conversion factor (CF). The CF of an analyte is defined as the ratio of the measured temporal peak area and the product of its migration time and transfer ratio (TR). Thus defined, the CF is of general validity for all analytes separated and detected in a given background electrolyte (BGE), since it has the same value for the same amounts of various analytes. If a sample is enriched with a known concentration of a standard component and analyzed by CZE, the CF of this standard component can be calculated and then the concentrations of all other analytes can be determined, without the use of any calibration graph. The individual TRs can be determined a priori from tabulated ionic mobilities and pK values of the analytes and of the constituents of the BGE or, for strong analytes, by using experimental data from the electropherogram of the analysis itself. The practical procedure of the analysis includes enrichment of the sample with a known quantity of a suitable standard and a single CZE run of the resulting mixture. The injected volume does not need to be known and thus the procedure also eliminates the injection error. The proposed procedure has been verified experimentally and reproducible and accurate values were obtained by using four different CZE apparatus for the analyses of standard mixtures of cations in three different BGEs.

Nonlinear analysis of dynamic binding in affinity capillary electrophoresis demonstrated for inclusion complexes of β-cyclodextrin

The stability of the molecular host–guest inclusion complexes of β-cyclodextrin with benzoate and four different hydroxybenzoates is investigated. For the measurement of the binding constants an experimental method is devised that is based on affinity capillary electrophoresis (ACE) with indirect UV absorbance detection. We derive an explicit equation for effective mobilities in ACE experiments without violation of rigorous mass balance. This equation is employed in the nonlinear least-squares analyses of the experimental data yielding binding constants of 48±2 M −1 for benzoate, 299±38 M −1 for 2-hydroxybenzoate, 37±1 M −1 for 3-hydroxybenzoate, 228±9 M −1 for 4-hydroxybenzoate, and 895±110 M −1 in the case of 2,4-dihydroxybenzoate.

High-sensitivity determination of the degradation products of chemical warfare agents by capillary electrophoresis–indirect UV absorbance detection

Capillary electrophoresis coupled with indirect UV absorbance detection was employed for the determination of the chemical warfare agent degradation products: methylphosphonic acid, ethyl methylphosphonate, isopropyl methylphosphonate, and pinacolyl methylphosphonate. Glutamic acid was used as a buffering agent at its isoelectric point (pH 3.22). In its zwitterionic form, glutamic acid does not act as a competing co-anion in the system, thus providing buffering capacity while maintaining high sensitivity. The indirect probe (phenylphosphonic acid) concentration was lowered to 1 m M from the 10 m M in previous literature studies, further enhancing sensitivity. Detection limits of 2 μ M were achieved with hydrodynamic injection and up to 100-fold lower using electrokinetic injection. The increased buffering capacity of this system over previous methods led to migration time reproducibility RSD values of 0.18 to 0.22%. This represents a 10-fold improvement in reproducibility over previous studies with comparable or improved sensitivity.

A comparison of capillary electrophoresis using indirect UV absorbance and ICP-MS detection with a self-aspirating nebulizer interface

A comparison of capillary electrophoresis (CE) migration times using standard on-column UV detection and inductively coupled plasma mass spectrometry (ICP-MS) is presented for two different CE separations. The first is a separation of five arsenic compounds, four anionic species and one neutral, using reverse polarity in a chromate buffer with an electroosmotic flow modifier. The second CE separation employs normal polarity for the separation of eleven lanthanide cations using a hydroxyisobutyric acid (HIBA) buffer with an indirect UV detection reagent. The CE-ICP-MS interface is based on a tee design which introduces a sheath flow around the CE capillary using a self-aspirating make-up electrolyte with a microconcentric nebulizer (MCN-100). Migration times observed for CE-ICP-MS electropherograms are comparable to migration times acquired with UV detection, and may be adjusted by changes in the make-up liquid level position. The precision of peak areas (<2%) and migration times (<4%) obtained using CE-ICP-MS is comparable to that obtained using online UV absorbance detection. CE-ICP-MS limits of detection are in the low ng ml−1 range for the analytes studied.

Capillary electrophoresis for the determination of bromide, chloride and sulfate as impurities in calcium acamprosate

A capillary zone electrophoresis method with indirect UV absorbance detection is reported for the determination of bromide, chloride and sulfate as impurities in calcium acamprosate. Separation was carried out on a 60.2 cm (50 cm to the detector) × 75 μm i.d. fused-silica capillary at a potential of −10.6 kV and 25°C. For each electrophoretic run, the capillary was first coated with polybrene to reverse the electrosmotic flow, then separation was carried out using an electrolyte containing 10 mM potassium chromate as visualization agent and 1 mM sodium borate. Standard and test solutions were hydrodynamically injected at a pressure of 0.3 psi for 6 s. The tests performed for validation demonstrated the reliability of the method for the determination of chloride, bromide and sulfate as inorganic impurities in the drug substance, under the conditions used. The limit of quantitation ranged between 8 and 20 mg kg −1 for the different anions.

Nuclear fission product analysis using capillary separation techniques

Capillary electrophoresis has been used to separate metal ions characteristically associated with nuclear fission. Electrokinetic injections and transient isotachophoretic techniques were employed to increase sample loading and provide on-column concentration of the analyte. On-line concentration factors of approximately 700-fold have been achieved. Indirect-UV absorbance, on-line radioactivity, and indirect laser-induced fluorescence detection were used to monitor analytes of interest. The radioactivity detector consists of a plastic scintillator and photomultiplier tube with a 4π detection geometry. The efficiency was determined to be approximately 80%, enabling samples resident in the detector window for 0.1 minutes to be reliably assayed. Detection of152Eu and137Cs was achieved at the low nCi level. Indirect fluorescence was performed with quinine sulfate as the background fluorophor with α-hydroxysobutyric acid added as a complexing agent. An argon ion laser was used as the excitation source with a diode array detector. Limits of detection for La3+, Ce3+, Pr3+, Nd3+, Sm3+, and Eu3+ were determined to be in the sub — 10 ppb range (6–11 nM) with indirect laser-induced fluorescence detection.

Capillary electrophoretic separation of mono- and dimaltosyl-β-cyclodextrins and determination of the stability constants of their benzoate complexes

Capillary zone electrophoresis method has been developed for the separation (and determination) of monoand dimaltosyl-β-cyclodextrins using indirect UV absorbance detection. The separation is based on inclusion complex formation with benzoate ion which is used as background electrolyte and absorbance provider simultaneously. Good resolution of cyclodextrin mixtures can be achieved optimizing the pH and the benzoic acid concentration in the background electrolyte. Capillary zone electrophoresis method has also been developed for the determination of formation constants of inclusion complexes.

Improved Separation and Determination of Rare Earth Metal Ions in Real Samples by Capillary Electrophoresis with Indirect UV Detection

The separation of rare earth metal ions by capillary electrophoresis in the presence of imidazole as the UV-absorbing background electrolyte, and lactic acid or α-hydroxyisobutyric acid (α-HIBA) as the complexing agent, with indirect UV absorbance detection was investigated. The effects of various acids in the running buffer, pH of buffer and the concentration of complexing agent on the separation were examined. It was observed that the acid used for adjusting the pH of the buffer was another important factor, besides the concentration of the complexing agent that affects the separation of rare earth metal ions. The results showed that under a positive voltage of 10.0 kV, fourteen rare earth metal ions could be baseline separated within 9 minutes using 0.02 mol/1 imidazole containing 8x10−3 mol/1 α-HIBA at pH 3.4 adjusted with hydrochloric acid. The detection limits of La, Ce, Pr, Nd, Sm and Tb were 5x10−6 mol/1. The contents of La, Ce, Pr, Nd and Sm in real samples from Gansu Rare Earth Company were determined quantitatively using Tb (III) as an internal standard. The results were consistent with those of ICP.

Analysis of Fission Products Using Capillary Electrophoresis with On-Line Radioactivity Detection

Capillary electrophoresis has been used to separate metal ions characteristically associated with nuclear fission. Indirect UV absorbance and on-line radioactivity detection were used simultaneously to monitor the analytes. The radioactivity detector consists of conical plastic scintillating material with the capillary passing through the center to provide a 4π detection geometry. The wide end of the cone is optically coupled to a photomultiplier tube. Transient isotachophoretic techniques were employed to stack large volumes of samples which had low specific activities. Radioactivity detection of (152)Eu and (137)Cs was achieved at the nanocurie level for 80-100 nL injections. The detector is approximately 80% efficient, enabling samples resident in the detector window for 0.1 min to be reliably assayed. The separation of (137)Cs and (137m)Ba isotopes, which are in secular equilibrium, was modeled to demonstrate the effects of the rapid decay of (137m)Ba.

Determination of oxalate in parenteral nutrition solutions by capillary electrophoresis

A rapid, sensitive and reliable capillary electrophoretic (CE) method based on indirect UV absorbance detection has been developed, optimized and applied to the separation and determination of low (mg/1) levels of oxalate in commercial parenteral nutrition preparations. The determination of oxalate requires no extraction, derivatization or other complex pre-analysis steps. The method used chromate as the UV-absorbing background electrolyte and oxalate was detected indirectly at 254 nm. The analysis time for oxalate is just under 7 min per sample with oxalate migrating at approximately 4 min. The method calibration curves were shown to be linear over a minimum of 1.5 orders of magnitude with a limit of detection for oxalate of approximately 240 μg/1. The methods linear dynamic range for oxalate was shown to extend over more than 2.5 orders of magnitude. The final optimized method was used to separate and quantify oxalate in parenteral nutrition solutions that had been submitted to 24-h neonatal phototherapy procedures.