Solid-phase extraction of polar pesticides from environmental water samples on graphitized carbon and Empore-activated carbon disks and on-line coupling to octadecyl-bonded silica analytical columns

The suitability of Empore-activated carbon disks (EACD), Envi-Carb graphitized carbon black (GCB) and CPP-50 graphitized carbon for the trace enrichment of polar pesticides from water samples was studied by means of off-line and on-line solid-phase extraction (SPE). In the off-line procedure, 0.5-2 1 samples spiked with a test mixture of oxamyl, methomyl and aldicarb sulfoxide were enriched on EnviCarb SPE cartridges or 47 mm diameter EACD and eluted with dichloromethane-methanol. After evaporation, a sample was injected onto a C₁₈-bonded silica column and analysed by liquid chromatography with ultraviolet (LC-UV) detection. EACD performed better than EnviCarb cartridges in terms of breakthrough volumes (>2 1 for all test analytes), reproducibility (R.S.D. of recoveries, 4–8%, n=3) and smapling speed (100 ml/min); detection limits in drinking water were 0.05–0.16 μg/l. In the on-line experiments, 4.6 mm diameter pieces cut from original EACD and stacked onto each other in a 9 mm long precolumn, and EnviCarb and CPP-50 packed in 10×2.0 mm I.D. precolumn, were tested, and 50–200 ml spiked water samples were preconcentrated. Because of the peak broadening caused by the strong sorption of the analytes on carbon, the carbon-packed precolumns were eluted by a separate stream of 0.1 ml/min acetonitrile which was mixed with the gradient LC eluent in front of the C₁₈ analytical column. The final on-line procedure was also applied for the less polar propoxur, carbaryl and methiocarb. EnviCarb could not be used due to its poor pressure resistance. CPP-50 provided less peak broadening than EACD: peak widths were 0.1–0.3 min and R.S.D. of peak heights 4–14% (n = 3). In terms of analyte trapping efficiency on-line SPE-LC-UV with a CPP-50 precolumn also showed better performance than when Bondesil C₁₈/OH or polymeric PLRP-S was used, but chromatographic resolution was similar. With the CPP-50-based system, detection limits of the test compounds were 0.05–1 μg/l in surface water.     

Trace enrichment of polar pollutants from water and the influence of humic substances

Summary A practical liquid chromatographic early-warning system for polar pollutants in water ought to have detection limits at a level below a microgram per litre. Using ultraviolet absorbance detection this normally necessitates trace enrichment of the samples. In this study ten different sorbents have been tested with respect to their enrichment capacity. The hydrophobic polymeric PLRP-S material, in combination with a C-18 modified analytical column, proved to be the best choice. The influence of humic substances, present in surface water, was also studied. Lowering of the sample pH, resulted in a significant increase in the interferences due to the humic substances.     

Determination of beta-19-nortestosterone and its metabolite alpha-19-nortestosterone in biological samples at the sub parts per billion level by high-performance liquid chromatography with on-line immunoaffinity sample pretreatment

An immunoaffinity precolumn (immuno precolumn) packed with Sepharose-immobilized polyclonal antibodies against the anabolic hormone 17 beta-19-nortestosterone (beta-19-NT) was used for the selective on-line pretreatment of raw extracts of urine, bile and tissue samples by high-performance liquid chromatography. Using UV detection (247 nm), beta-19-NT and its metabolite 17 alpha-19-nortestosterone (alpha-19-NT) can be determined in biological samples with a detection limit of 0.05 microgram/kg. Owing to the high clean-up efficiency of the immuno precolumn and the large sample volumes used, confirmation by gas chromatography-mass spectrometry is possible at this level. In urine samples from a calf treated with 19-nortestosterone 17 beta-laurate, the maximum concentrations of beta-19-NT (1.3 micrograms/l) and alpha-19-NT (3.1 micrograms/l) were found seven days after intramuscular administration. In a bile sample from this calf only alpha-19-NT (55 microgram/l) was detected. In meat samples from three treated calves, the concentration of beta-19-NT varied from 0.1 to 1.6 micrograms/kg and no alpha-19-NT could be detected. In liver samples from these calves, the concentrations of beta-19-NT and alpha-19-NT were less than 0.05-0.1 and 0.5-0.9 micrograms/kg, respectively. In the corresponding kidney samples, the concentrations of beta-19-NT and alpha-19-NT were 0.4-0.5 and 0.5-1.6 micrograms/kg, respectively. The application of the same immuno precolumn to the determination of 17 beta- and 17 alpha-trenbolone, two structurally related steroids, is also demonstrated.     

Liquid chromatography of phenolic compounds in natural water using on-line trace enrichment

Summary Two packing materials, C18 and PLRP-S, are studied for on-line trace enrichment of phenolic compounds in water. Various precolumns of different internal diameter are also tested and the addition of an ion-pair reagent to increase retention and thus, breakthrough volumes of phenolic compounds, is studied. Best results are obtained when a PLRP-S precolumn is coupled on-line with a C18 analytical column and DAD detector. Addition of TBA considerably increases breakthrough volumes. In contrast, when a C18 precolumn is used, breakthrough volumes are lower and it is impossible to determine TCP and PCP, under the experimental conditions used, because of interference of other nonpolar compounds in the samples. The performance of the system is evaluated with river and tap water and the preconcentration of 10 ml of sample in a PLRP-S precolumn involves a linear range between 1 g 1^–1 and 20 l^–1 and limits of determination between 0.5 g l^–1 and 1 g l^–1 are obtained.     

Trace-level monitoring of chloroanilines in environmental waters using on-line trace-enrichment and liquid chromatography with UV and electrochemical detection

Summary An on-line procedure is described for the trace-level determination of mono-, di- and methyl-chloroanilines in aqueous samples using selective preconcentration with a cation-exchanger and liquid chromatography with UV and electrochemical detection. Because direct percolation through a cation-exchanger has to be avoided owing to the high content of inorganic anions present in natural waters, a two-step on-line preconcentration was carried out: chloroanilines were first trapped on a precolumn packed with an apolar polymeric sorbent (PRP-1) in their neutral form. Then the PRP-1 precolumn was coupled in series with a second precolumn containing cation exchange material. The chloroanilines were removed from the first precolumn with 3 mL of deionised water: acetonitrile (31) at pH 1 and retained by the cation exchange column. The contents of the cation exchange column were finally desorbed onto the analytical column and eluted with a water: acetonitrile gradient. The combination of selective trace enrichment and sensitive electrochemical detection allows the simultaneous determination of chloroanilines from 150 mL of river water samples with detection limits below 30 ng/l. Identification is confirmed by the selective preconcentration and the two detection modes.     

Determination of polar aniline derivatives in aqueous environmental samples using on-line liquid chromatographic preconcentration techniques

Summary On-line precolumn sample handling is used to enrich polar aniline derivatives in order to preconcentrate them prior to their separation. Liquid-solid extraction is possible with a cation-exchanger precolumn after acidification of water samples at pH 3 and a clean-up in order to remove the high amounts of inorganic cations present in natural samples. Since inorganic removal cannot be total, overloading of the ion exchanger occurs rapidly. The volume which can be directly percolated through the cation-exchanger precolumn cannot exceed 30 ml and the amount preconcentrated is not sufficient for a determination at the 100 ppt level. A two-step preconcentration procedure is carried out in order to increase the sample volume: the direct percolation of samples through the cation-exchanger precolumn is avoided and the clean-up step is no longer necessary. Aniline derivatives are preconcentrated in their neutral form at pH 7 by a 9-cm long column packed with the copolymer-based PRP-1 sorbent; then, a small volume of water-methanol at pH 2 allows the cationic compounds alone to be desorbed from the PRP-1 column in their protonated form and to be transferred to a 1-cm long cation-exchanger precolumn. This precolumn is then coupled to an analytical C18 column and its content on-line analysed by an acetonitrile gradient. The PRP-1 column acts as a powerful filter to many neutral interferents and aniline derivatives can be thus determined from 150-ml drinking water samples with 10–50 ppt UV detection limits.Dedicated to Roland W. Frei     

Deformations of overloaded bands under pH-stable conditions in reversed phase chromatography

It has recently been demonstrated, using mathematical models, how peculiar overloaded band profiles of basic compounds are due to the local pH in the column when using low capacity buffers. In this study, overloaded peak shapes resulting after injection of carefully pH matched samples close to the pK(a) of the chosen solute are investigated primarily on two columns; one hybrid silica C18 column (Kromasil Eternity) and one purely polymeric column (PLRP-S), the latter lacking C18 ligands. It was found that distorted peaks of the basic test compound appear even though there is no difference in pH between the injected sample solution and the eluent; the previous explanation to why these effects occur is based on a pH mismatch. Thus, the unusual band shapes are not due to an initial pH difference. Furthermore, it was observed that the effect does not appear on polymeric columns without C18 ligands, but only on columns with C18 ligands, independently of the base matrix (silica, hybrid silica, polymeric).     

Ion chromatographic determination of bromate in drinking water by post-column reaction with fuchsin.

Bromate deriving from ozonation treatment of bromide containing waters are analyzed by ion-exchange chromatography with spectrophotometric detection after post-column reaction with fuchsin in low pH medium. An anion-exchange column was used with 2.7 mM carbonate-0.3 mM hydrogencarbonate eluent. The eluent from the column was then allowed to react with a SO2-reduced fuchsin solution and then with a diluted HCl solution at 65 degrees C. The developed colour of the final product was measured spectrophotometrically at 530 nm. Linearity was checked up to 50 micrograms/l with a 200-microliter injection loop (r2 = 0.9997) and up to 100 micrograms/l of bromate with 100 microliters loop (r2 = 0.9939). Nitrate, sulfate, bromide, phosphate, fluoride did not interfere at 100 mg/l concentration level; only nitrite at concentration levels greater than 3 mg/l caused partial overlapping with bromate peak, but this value is not likely to occur in common drinking water. The detection limit (3 sigma) is 0.1 microgram/l (1 microgram/l propagation error approach).     

Ethyl acetate for the desorption of a liquid chromatographic precolumn on-line into a gas chromatograph

The feasibility of using ethyl acetate for the desorption of trace pollutants from a liquid chromatographic precolumn on-line into a diphenyltetramethyldisilazane-deactivated retention gap and, subsequently analysis by means of capillary gas chromatography has been demonstrated. First 5% of methanol are added to the water sample to prevent sorption of analytes onto parts of the preconcentration system. About 1 ml of this aqueous sample is injected onto a precolumn containing a polymeric stationary phase, using water–methanol (95:5, v/v) for transport and clean-up. The precolumn is desorbed with ethyl acetate and a fraction of 75 μl is injected on-line into the retention gap; separation is then achieved on a capillary CP Sil 19 column. No breakthrough of the test compounds was observed in the preconcentration step. The recovery was quantitative and the response obtained with flame ionization detection was linear in the range 0.1–100 ng/ml. The effect of varying the sorption flow rate on the recovery was studied. The system was applied to the analysis of river water.     

Automated column-switching high-performance liquid chromatographic determination of flumequine and oxolinic acid in extracts from fish

Two methods for automated analysis of extracts from edible muscle tissue of Atlantic salmon are described. Oxolinic acid and flumequine are extracted with phosphate buffer pH 9, and the extracts are analysed by high-performance liquid chromatography using a column-switching system. One method applies on-line concentration and clean-up of the extracts on a precolumn packed with polystyrene-divinylbenzene. This method was useful for the analysis of oxolinic acid and flumequine in the microgram/g range. The other method was based on on-line dialysis and concentration of the dialysate on the polymeric precolumn. This method was shown to be a reliable method for residue analysis, and the limit of detection was 2 ng/g for oxolinic acid and 3 ng/g for flumequine with fluorescence detection.     

Trace-level determination of benzidine and 3,3'-dichlorobenzidine in aqueous environmental samples by online solid-phase extraction and liquid chromatography with electrochemical detection

A simple, rapid, and reliable online methodology for the determination of benzidine and 3,3'-dichlorobenzidine (3,3'-DCB) in natural waters is proposed. The analytes are extracted and preconcentrated from aqueous samples in a small stainless steel precolumn packed with a polymeric PLRP-S phase. The precolumn is further online-analyzed by reversed-phase gradient-elution chromatography with a highly sensitive and selective coulometric detection at E = 700 mV. Recoveries greater than 90% and a relative standard deviation of approximately 5% are achieved with samples spiked at low micrograms-per-liter concentration levels. The detection limits of the method in fortified reagent water samples are 100 ng/L for benzidine and 50 ng/L for 3,3'-DCB.     

Trace analysis of sulfonylurea herbicides in water by on-line continuous flow liquid membrane extraction--C18 precolumn liquid chromatography with ultraviolet absorbance detection

An on-line system that consists of continuous-flow liquid membrane extraction (CFLME), C18 precolumn, and liquid chromatography with UV detection was applied to trace analysis of sulfonylurea herbicides in water. During preconcentration by CFLME, five target compounds, including metsulfuron methyl, bensulfuron methyl, tribenuron methyl, sulfometuron methyl, and ethametsulfuron, were enriched in 960 microl of 0.5 mol l(-1) Na2CO3-NaHCO3 (pH 10.8) buffer used as acceptor. This acceptor was on-line neutralized and transported to the C18 precolumn where the analytes were absorbed and focused. Then the focused analytes were injected onto a C18 analytical column for separation and detection at 240 nm. The proposed method was applied to determine sulfonylurea herbicides in water, river, and reservoir water with detection limits of 10-50 ng l(-1) when enriching a 120-ml sample. Throughput is typically one sample per hour.     

Large-volume liquid chromatographic trace-enrichment system for environmental analysis

The fully automated on-line trace enrichment of 27 (polar) pollutants, using volumes of up to 1,000 mL, on a polymeric precolumn followed by liquid chromatography —diode-array detection has been studied. Various parameters influencing the reproducibility of these large volume injections like breakthrough volume, precolumn capacity, matrix effects, and enrichment flow rate are discussed. The relative standard deviation of the recoveries after enrichment of 25–500 mL of sample is 1–20%. Enrichment flow rates up to 15 mL min⁻¹ can be used with an optimum at 10 mL min⁻¹. A pore size of 300 A provides the best results using the polymeric PLRP-S material. The breakthrough volumes show significant dependence upon the concentration of the analytes in the sample.     

Determination of an aldosterone antagonist in urine by high-performance liquid chromatography using automated column switching

An automated high-performance liquid chromatographic assay for the determination of an aldosterone antagonist (I) is described using column switching for direct injection of urine samples. After dilution with buffered internal standard solution, the sample was injected onto a clean-up column (17 X 4.6 mm I.D.), dry-packed with C18 reversed-phase material (particle size 30 micron). Polar urine components were removed by flushing the clean-up column with water. Retained substances, including I and the internal standard, were desorbed by backflush elution onto a 5-micron ODS-silica analytical column (125 X 4 mm I.D.), separated with water-methanol-tetrahydrofuran, and detected at 295 nm. After backflushing the analytical column and re-equilibrating the clean-up column, the system was ready for the next injection. The limit of quantification was ca. 100 ng/ml, using a 100-microliter specimen of diluted urine. The mean inter-assay precision of the method up to 25.6 micrograms/ml was 2%. Practicability and accuracy of the new method were demonstrated by the application to excretion studies performed with human volunteers.     

On-line trace enrichment for the simultaneous determination of microcystins in aqueous samples using high-performance liquid chromatography with diode-array detection.

The need for a rapid, sensitive and reliable analytical method for cyanobacterial toxins, microcystins, has been emphasized by the awareness of toxic cyanobacteria as a human-health risk through drinking water. A new high-performance liquid chromatographic method with column switching was developed for the determination of microcystin-LR, -RR and -YR from water samples without pre-purification. The filtered water sample was passed through a Zorbax CN precolumn at a flow-rate of 3 ml/min for on-line trace enrichment. After valve switching, concentrated analytes were eluted in back-flush mode and separated on a Luna C18 column with a gradient of acetonitrile -20 mM phosphate buffer (pH 2.5). The method showed excellent precision, accuracy and speed with detection limits of 0.02 microgram/ml from 100 ml of surface water. The total analysis time per sample was about 90 min. This method improves reliability, sensitivity and sample throughput, and shortens the analysis time compared to analysis methods using off-line solid-phase extraction.     

Stability of Parathion-Methyl,Methiocarb, DDT and 2,4-D in Solid-Phase Extraction Precolumns Packed with a Polymeric Reversed Phase

Abstract

The stability of parathion-methyl, methiocarb, DDT and 2,4-D, adsorbed on the polymeric PLRP-S phase packed in small stainless steel precolumns was examined, with a view to propose the use of these precolumns as alternative means for the transport of water samples. First, water samples spiked with the studied pesticide at low μg/l concentration levels were extracted and preconcentrated in the precolumns, using appropriate conditions for a total recovery. Then, the precolumns were stored at room temperature (15—20°C) or at 35°C for different time periods. At the end of the respective period each precolumn was coupled to an HPLC column via a switching valve and was on-line analyzed by reversed phase chromatography with UV detection. The four pesticide recoveries after one week in the precolumn at room temperature were higher than 90%. The same was true at 35°C except for DDT, which suffered a 30% degradation in one week. Further studies showed that DDT and parathion-methyl were stable at least for five weeks in precolumns stored at room temperature. Methiocarb also was stable for this period but storing the precolumns at 4°C.     

Use of the hyper-crosslinked polystyrene sorbents “Styrosorb” for solid phase extraction of phenols from water

The analytical HPLC procedure for the quantitation of trace amounts of phenol and chlorophenols in aqueous media has been supplemented with the on-line preconcentration of phenols in a sorption cartridge packed with microporous hyper-crosslinked polystyrene. The cartridge is coupled in succession with a reversed-phase analytical column, which is operated isocratically in an aqueous acetonitrile eluent (1:1, v/v). Phenol concentrations down to 0.5 μg/l can be detected using a simple 254 nm UV detector. Biporous hyper-crosslinked polystyrene-type sorbents were shown to effectively purify about 100 bed volumes of water from phenol under neutral or acidic conditions and linear flow rates up to 4 cm/min, which could be of great practical importance. Rapid and quantitative elution of sorbates from the hyper-crosslinked polystyrene materials is facilitated by the unique ability of the latter to additionally swell with organic or aqueous-organic eluents applied.     

Separation and sensitive determination of i-urobilin and 1-stercobilin by high-performance liquid chromatography with fluorimetric detection.

i-Urobilin and 1-stercobilin were separated by high-performance liquid chromatography on a reversed-phase octadecylsilane-bonded column and detected fluorimetrically through formation of phosphor with zinc ions in the eluent. The separation and the intensity of the fluorescence response were affected by concentrations of zinc acetate and sodium borate buffer, pH and methanol content in the eluent. The optimal eluent used consisted of 0.1% zinc acetate in 75 mM boric acid buffer (pH 6.0)-methanol (25:75). The detection limit was 0.2 microgram/l for both i-urobilin and 1-stercobilin (signal-to-noise ratio 2), which makes the method 250-2500 times more sensitive than conventional methods.     

Rapid determination of clenbuterol residues in urine by high-performance liquid chromatography with on-line automated sample processing using immunoaffinity chromatography

A liquid chromatographic column-switching system for automated sample pretreatment and determination of clenbuterol in calf urine, using an immunoaffinity precolumn with Sepharose-immobilized polyclonal antibodies against clenbuterol, is described. A second precolumn packed with C18-bonded silica was used for the reconcentration of desorbed clenbuterol prior to the analytical separation. Urine, after 2-fold dilution with buffer (pH 7.4), was loaded directly onto the immuno precolumn, where clenbuterol was trapped by the immobilized antibodies. This immuno precolumn has been used for more than 200 runs with standard solutions and samples. Bound analyte was desorbed with 0.01 M acetic acid and transferred, via the second precolumn, to the analytical column. The total runtime per sample was 35 min. Using a sample load of 27 ml of dilute urine and UV detection at 244 nm, the detection limit was 0.5 ng/ml. The mean recovery of clenbuterol added to a blank urine sample at the 5 ng/ml level was 82 +/- 2% (n = 5) as determined with standard solutions loaded onto the same system. Urine samples from treated animals were analysed and the clenbuterol concentrations were comparable to those obtained by high-performance liquid chromatography using solid-phase extraction for sample clean-up.     

Determination of plasma homovanillic acid by liquid chromatography with electrochemical detection.

We describe a simple method for extracting homovanillic acid (HVA) from plasma. An aliquot of 0.5 ml of the internal standard solution (3-hydroxy-4-methoxycinnamic acid in 0.2 mol/l phosphoric acid) and 0.5 ml of the sample are applied to a 1-ml Bond Elut C18 column prewashed with methanol and 0.2 mol/l phosphoric acid. The sample is drawn through the column at low speed. The column is washed with water and eluted with dichloromethane. The eluate is evaporated under vacuum at ambient temperature and the residue reconstituted with 250 microliters of the mobile phase. A 10-microliters aliquot of the resulting solution is injected onto a 150 mm x 4.6 mm I.D. column packed with 5-microns octadecylsilyl silica particles (Beckman). Peaks are detected coulometrically in the screening-oxidation mode with E1 = +0.25 V and E2 = +0.38 V. In the resulting chromatogram, HVA and the internal standard give sharp peaks and are well separated from solvent and other endogenous electroactive acids. The extraction recovery is 90-95% which allows the determination of 0.5 microgram/l analyte.