Some theoretical and practical aspects in the separation of humic substances by combined liquid chromatography methods.

Permanent need to understand nature, structure and properties of humic substances influences also separation methods that are in a wide scope used for fractionation, characterization and analysis of humic substances (HS). At the first glance techniques based on size-exclusion phenomena are the most useful and utilized for relating elution data to the molecular mass distribution of HS, however, with some limitations and exceptions, respectively, in the structural investigation of HS. The second most abundant separation mechanism is reversed-phase based on weak hydrophobic interactions beneficially combined with the step gradients inducing distinct features in rather featureless analytical signal of HS. Relatively great effort is invested to the developments of immobilized-metal affinity chromatography mimicking chelate-forming properties of HS as ligands in the environment. Surprisingly, relatively less attention is given to the ion-ion interactions based ion-exchange chromatography of HS. Chromatographic separation methods play also an important role in the examination of interactions of HS with pesticides. They allow us to determine binding constants and the other data necessary to predict the mobility of chemical pollutants in the environment. HS is frequently adversely acting in analytical procedures as interfering substance, so more detailed information is desired on manifestation of its numerous properties in analytical procedures. The article topic is covered by the review emphasizing advances in the field done in the period of last 10 years from 2000 till 2010.

On-line flow-through extraction-preconcentration-large volume injection-RP LC for trace determination of pyrethroids in Slovak soil.

A rapid micro-analytical multiresidue method was developed for analysis of pyrethroids (kadethrin K, cypermethrin C and permethrin P) in soil micro-sample (200 mg). It uses on-line flow-through extraction of soil micro-samples (packed into a short glass column) with a methanol-aqueous citric acid buffer mixture, successive on-line SPE preconcentration of analytes from the extract and on-line RP-HPLC analysis with UV photometric detection. The separation of pyrethroids is performed on a Purospher RP-18e column with methanol/water as mobile phase. Effects of sorbent placed at the bottom of a short column holding the soil sample and different kinds of on-line SPE columns were tested. Besides, the influence of volume of the effluent on the pyrethroids recovery was also studied. Calibration curves were linear over the range assayed from 0.01 to 0.2 microg/mL with correlation coefficients of linear regression (least-squares method) in the range 0.998-0.999. Recovery studies were carried out at 0.25-1.00 microg/g dry soil fortification level and obtained recoveries were for K 81-84%, C 56-59% and for P 58-63%. Achieved LOD (confidence band) of studied pyrethroids were for large-volume injection (1 mL) 4.5 ng K, 3.7 ng C, 3.6 ng P or 27 ng/g K, 32 ng/g C and 29 ng/g P in dry soil "solid sampling HPLC".

Reversed phase liquid chromatography trace analysis of pesticides in soil by on-column sample pumping large volume injection and UV detection.

The idea of utilization of one hydraulic line of a common commercial HPLC pump for direct on-column sample pumping injection of large sample volumes, 20 mL, was further investigated with the aim to develop multicomponent pesticides trace residues HPLC method in gram soil samples. Target pesticides group involve asulam, atrazine, 2,4-D, PCA, propazine, simazine, 4-chloro-2-methylphenoxyacetic acid, 2-(4-chloro-2-tolyloxy) propionic acid, chlortoluron, metoxuron, epoxiconazole. The results proved the applicability of this approach in experiments with mixtures of analytes at low ng/mL levels. Analysis of 20 mL of soil leachates and extracts of fortified soil samples containing these pesticides at the 10-50 ng/g level (in dry soil) revealed good figures of merit, also in the presence of large excess of humics. LODs achieved by detection at 220 nm evaluated from calibration runs of spiked soil extracts by Hubaux et al. method ranged from 5-12 ng per injected volume. For 20 mL large volume injection it represents 0.25-0.6 ng/mL of diluted soil extract, or 2.5-6 ng/mL of crude extract, or 6-5 ng/g dry soil. Recoveries of pesticides at concentration levels approaching half of maximum allowable concentration of pesticides in soil (100 ng/g) ranged from 85 to 98% with acceptable reproducibility, except asulam and metoxuron.

Method of analysis of a selected group of pyrethroids in soil samples using off-line flow-through extraction and on-column direct large-volume injection in reversed phase high performance liquid chromatography.

An analytical method using flow-through extraction of a soil sample filled in a short HPLC column with methanol or methanol-water mixtures and large-volume injection in RP-HPLC has been developed for the simultaneous determination of residues of three pyrethroids--kadethrin, cypermethrin, and permethrin--from soil samples. The developed RP-HPLC method enables separation of four diastereoisomers of cypermethrin into three peaks and resolution of two diastereoisomers of permethrin. The UV photometric detection limits of direct on-column large-volume injection of 1.00 mL of extract were 30 ng/mL of kadethrin, 37 ng/mL of total content of cypermethrin, and 65 ng/mL of trans-permethrin, which corresponds to a pyrethroid soil content of around 0.3 mg/kg. Effects of extractant flow rate and optimal extractant volume on the percentage recovery of pyrethroids from Slovak soil samples were studied. Recovery studies were performed at 0.5- 5.0 microg/g fortification level of kadethrin and 1.0-2.5 microg/g fortification level of cypermethrin and permethrin in a soil sample. Recoveries ranged from 83 to 90% for kadethrin, from 87 to 94% for total cypermethrin, and from 85 to 98% for trans-permethrin. This work comprises a basic study aimed at elaboration of an RP-HPLC method of direct analysis of pyrethroids in a soil matrix at low concentration levels achieved by a "solid sample injection" in HPLC--on-line interfacing of analyte extraction, extract clean-up, and analysis.

New approach to large-volume injection in reversed-phase high performance liquid chromatography: determination of atrazine and hydroxyatrazine in soil samples.

A well established method of direct injection of larger than conventional sample volumes ranging from 0.1 mL to 10 mL in HPLC is the injection valve method in which a loop of tubing is totally or partially filled with sample. Recent HPLC pumps have a flow-rate setting accuracy of +/- 1-2% over a flow-rate range from 0.1 mL/min to 10 mL/min and the flow stability is 0.2% or less. Quarternary low pressure gradient pumps are widely available and used, but all their hydraulic lines are seldom utilised. The idea of using one line of a common commercial HPLC quaternary low-pressure pump for direct on-column injection (pumping) of large sample volumes ranging from 1 mL to 100 mL was tested. This approach was evaluated during practical work on the development of an RP-HPLC method of determination of residual atrazine and hydroxyatrazine. In lysimetric environmental experiments hydroxyatrazine was formed in situ in a soil column by hydrolysis of atrazine. The results proved the applicability of this approach not only in experiments with model mixtures of analytes at microg/L levels in solutions. Analysis of 20 mL of soil leachates and extracts of soil samples containing atrazine and hydroxyatrazine at the 10 microg/kg level (in dry soil) revealed that good figures-of-merit were preserved, even in the presence of a large excess of humic substances.