Sequential Elution Research Articles

Sequential Elution Technique in Moss Pseudoscleropodium purum: Comparison between the Commonly Used Extracellular Extractant NiCl2 and Other New Extractants

The sequential elution technique (SET) is used to determine the distribution of elements in the different cell fractions of mosses. The extracellular extractants most commonly used in this technique are NiCl2 and EDTA, although there are certain disadvantages associated with their use. In order to avoid such problems, we searched for new extractants that displace extracellularly bound metals, either because they are present at high concentrations (Ca) or because they have a high affinity for cation exchange sites (Hg and Au). The compounds HCl, NaCl, and CaCl2 were tested as extractants of the former type, as possible alternatives for the determination of extracellular metals in the moss Pseudoscleropodium purum. Calcium chloride was finally chosen as is it potentially the most successful in terms of binding to the cation exchange sites without altering the membrane permeability. The concentration chosen was 160 mM, as this yielded maximum displacement of Zn without membrane alterations. An experiment was then carried out to test the efficiency of Ca in extracting extracellular Zn, under laboratory and field conditions. In addition, Hg and Au were tested, at different concentrations, as extractants with high affinity for cation exchange sites, as neither of these elements has previously been used in the SET, and both display electronic characteristics that suggest their potential usefulness in displacing other cations from cation exchange sites. The results obtained show that extraction of extracellularly bound metal by high concentrations of Ca should be ruled out, as total extraction of Zn was not achieved. Both Hg and Au produced membrane alterations at low concentrations and moreover, neither was more efficient at extracting Zn than the reference extractant (20 mM NiCl2).

Obtaining of CD34+ cells from healthy blood donors: development of a rapid and efficient procedure using leukoreduction filters

Human CD34+ cells are mandatory to study many aspects of human hematopoiesis. Their low frequency in blood or marrow and ethical reasons limit their obtainment in large quantities. Leukoreduction filters (LRFs) are discarded after preparation of red blood cells. The CD34+ cell concentration in healthy donor blood is low (1×10(3) -4×10(3) /mL), but their number trapped in one LRF after filtration of 400 to 450mL of blood is high (0.4×10(6) -1.6×10(6) ). To develop a procedure allowing obtainment of purified CD34+ cells from LRFs with a good yield, white blood cell (WBC) recoveries after a 500-mL continuous or after sequential elution (50- or 20-mL fractions) were compared. Different WBC and mononuclear cell (MNC) centrifugation methods were tested to minimize their PLT contamination before the CD34+ cell immunomagnetic selection. Cell functionality was finally analyzed under various culture conditions. The 20-mL back-flushing of LRFs allowed the most efficient WBC recovery. The next steps (110×g centrifugation, MNC separation on Ficoll, and washes) resulted in a cell suspension in which the lymphocyte recovery was approximately 76±10% and the PLT contamination below 1.6%. After immunomagnetic selection, 4×10(5) to 6×10(5) cells containing approximately 85% of functional CD34+ cells were obtained. This procedure allows the easy, rapid (<5hr), and efficient preparation of large quantities of CD34+ cells having functional activities similar to those of CD34+ cells from other sources. Therefore, easily available and virally safe, LRFs represent an important and regular WBC source to work with human CD34+ cells, but also with other WBC types.

Excretion of bisphenol A into rat milk

Bisphenol A (BPA), an endocrine-disrupting chemical, is widely used in the production of polycarbonate plastic and epoxy resins. This study analyzed the BPA concentration in rat milk, in order to assess the risk of BPA transfer to the offspring via milk. The rats ingested BPA by oral administration or by drinking the water in a polycarbonate bottle, and the milk samples were collected using an automated experimental milker. The BPA concentration in the samples of milk, drinking water, and food was analyzed by LC/MS. In the case of milk samples obtained from rats injected with BPA at 2, 4, 8, and 24 h prior to milking, the BPA concentrations were 0.462 ± 0.182 ppm, 0.138 ± 0.0185 ppm, 0.080 ± 0.0197 ppm, and 0.0232 ± 0.0051 ppm, respectively. Also, in the cases of the water sample left in polycarbonate bottle and the milk sample obtained from rats provided it as drinking water, the concentrations of BPA were 0.000332 ± 0.00015 ppm and 0.0184 ± 0.0050 ppm, respectively. The results indicate that the BPA administered to the dams was transferred to their milk, and that BPA concentration in milk was higher at the early period after the single bolus dose. Additionally, these results reveal that sequential elution of BPA from polycarbonate containers in a much diluted form would undergo bioaccumulation in dams and likely be transferred to pups via milk in a much concentrated form.

Evaluation of Methods for the Extraction and Characterization of Waxes from Crude Oils

Wax precipitation is one of the most important flow assurance problems. The adequate characterization of the content and composition of the waxes present in the oil is one of the major obstacles in dealing with this issue. In this work, two different methods for the extraction of waxes from different crude oils are compared. The methods used differ significantly with regard to the separation of waxes from asphalthenes. Further purification of parafffins for characterization was carried out using sequential elution chromatography. The n-paraffin distribution determined by gas chromatography along with wax melting temperature and total n-paraffin content determined by differential scanning calorimetry show remarkable differences in the waxes depending upon the precipitation method used. The separation carried out by method 1 allows for heavier n-alkanes to be retained but with a higher content of impurities, as revealed by their lower melting points and n-paraffin content. Method 2 provides a better purific...

Evaluation of cations and chelating agents as extracellular extractants for Cu, Pb, V and Zn in the sequential elution technique applied to the terrestrial moss Pseudoscleropodium purum

Three experiments were carried out to select the best extractant for use in the sequential elution technique, to enable extraction of Cu, Pb, V and Zn from the extracellular fraction of the terrestrial moss Pseudoscleropodium purum. The optimal concentrations of the extractants tested (CoCl 2, NiCl 2, Pb(NO 3) 2, SrCl 2, dimercaprol, EDTA, penicillamine) were determined on the basis of the maximum extraction of Zn achieved without any alteration of the plasma membrane. The capacity of these agents (at the optimal concentrations established) to extract the extracellular fractions of Cu, Pb, V and Zn was then evaluated. Extraction with 10 mM EDTA is recommended for all 4 elements considered. As a second option, the use of 50 mM penicillamine is recommended to extract Cu, 30 mM dimercaprol to extract Pb and V and 20 mM NiCl 2 to extract Zn. It was also concluded that these results cannot be extrapolated to other cryptogams, and that separate assays are required.

Site-Specific Profiles of Estrogenic Activity in Agricultural Areas of California’s Inland Waters

To evaluate the occurrence and sources of compounds capable of feminizing fish in agriculturally impacted waterways of the Central Valley of California, water samples were extracted and subjected to chemical analyses as well as in vitro and in vivo measurements of vitellogenin in juvenile rainbow trout (Oncorhynchus mykiss). Among the 16 sites sampled, 6 locations frequently exhibited elevated concentrations of estrogenic substances with 17beta-estradiol equivalents up to 242 ng/L in vitro and 12 microg/kg in vivo. The patterns of activity varied among sites, with two sites showing elevated activity only in vitro, two showing elevated activity only in vivo, and two showing elevated activity in both assays. Sequential elution of solid-phase extraction (SPE) disks followed by bioassay-guided fractionation was used to characterize water samples from the two locations where activity was observed in both bioassays. The highest estrogenic activity was observed in the most nonpolar fractions (80-100% methanol eluent) from the Napa River, while most of the activity in the Sacramento River Delta eluted in the 60% methanol eluent. Quantitative analyses of SPE extracts and additional HPLC fractionation of the SPE extracts by GC-MS/MS and LC-MS/MS indicated concentrations of steroid hormones, alkylphenol polyethoxylates, and herbicides that were at least 1-3 orders of magnitude below bioassay 17beta-estradiol equivalent calculations. Given the different patterns of activity and chemical properties of the estrogenic compounds, it appears that estrogenic activity in these agriculturally impacted surface waters is attributable to multiple compounds. Further investigation is needed to identify the compounds causing the estrogenic activity and to determine the potential impacts of these compounds on feral fish.

Co-purified gelatinases alter the stability and biological activities of human plasma fibronectin preparations.

Fibronectin (FN) is an important cell adhesion molecule that is used widely to characterize cell behavior. Preparations of FN purified from human plasma by gelatin-Sepharose affinity chromatography typically also contain gelatin-binding gelatinases that may cleave FN, reduce its stability and alter its biological activities. Available methods for separating gelatinases from FN are resource demanding. Therefore, our objective was to devise a time- and cost-efficient protocol for purification of gelatinase-free FN. Experiments tested the elution profiles for FN and gelatinases from gelatin-Sepharose using a concentration range (1-7%) of dimethyl sulfoxide (DMSO) and 4 m urea as eluants. Subsequently, we explored the sequential application of those eluants for differential elution of gelatinases and FN using a single affinity column. Finally, experiments characterized the stability of purified FN with or without contaminating gelatinases, as well as the effects of FN degradation on cell attachment and migration. Assay optimization demonstrated that pre-elution with 3% DMSO efficiently eliminated gelatinases but not FN from gelatin-Sepharose, whereas subsequent elution with 4 m urea released FN. Sequential elutions with DMSO and urea produced gelatinase-free FN, which was more stable than FN eluted by urea only. Fibronectin degradation did not affect human gingival fibroblast attachment, but increased cell migration significantly. The present experiments devised a time- and cost-efficient protocol for eliminating gelatinases during purification of human plasma FN. Gelatinase-free FN preparations had greater stability, which may be essential for experiments because FN fragments have altered biological activities compared with intact FN.

Polarity based fractionation of fulvic acids

Fulvic acids from the soil of Peking University (PF) and a Nordic river (NF) were separated into well defined sub-fractions using sequential elution techniques based on eluent polarity. The chemical properties of the fractions including: PF1 and NF1 (eluted by 0.01 M HCl), PF2 and NF2 (eluted by 0.01 M HCl + 20% methanol), PF3 and NF3 (eluted by 0.01 M HCl + 40% methanol), and PF4 and NF4 (eluted by 100% methanol), were characterized using UV–Visible spectroscopy, elemental analysis and 13C NMR. The results showed that the UV absorptions of the elution peaks at 280 nm ( A280) increased from PF2 to PF4 and NF2 to NF4. No elution peaks were observed for PF1 and NF1. The carbon contents increased from 43.34% to 51.90% and 43.06% to 53.26% while the oxygen contents decreased from 46.39% to 36.76% and 49.76% to 40.03% for PF1–PF4 and NF1–NF4, respectively. As a polarity indicator, the (O + N)/C ratio for PF1–PF4 and NF1–NF4 decreased from 0.88 to 0.62 and 0.89 to 0.58, respectively. The aromatic carbon content increased from PF1 to PF4 and NF1 to NF4, suggesting an increase of the hydrophobicity of these fractions. The polarity was positively related to the ratio of UV absorption at 250 nm and 365 nm ( E2 /E3), and negatively related to the aromaticity. A high positive relationship between the aromaticity and E2 /E3 of fulvic acid fractions was also obtained. The use of an eluent with a decreasing polarity allowed to providing simpler fractions of soil and aquatic fulvic acids.

Intracellular and extracellular ammonium (NH4 +) uptake and its toxic effects on the aquatic biomonitor Fontinalis antipyretica

The objective of this work is to validate the use of the aquatic moss Fontinalis antipyretica as biomonitor of NH(4)(+) aquatic pollution. In order to achieve this objective we needed to understand the pattern of uptake of NH(4)(+) by the moss and evaluate the impact of high concentrations on its physiological performance. The cellular location of NH(4)(+) in the moss is crucial for understanding its monitoring capacity. We were able to show that a sequential elution technique, based on the use of NiCl(2) as an efficient displacing agent, allowed the quantification of the cellular location of NH(4)(+). This was done along a concentration gradient and time of exposure. The extracellular and intracellular NH(4)(+) concentrations that caused significant physiological impact in membrane permeability of F. antipyretica were the same that caused significant decreasing in the photosynthetic capacity of the same moss. The former NH(4)(+) concentration thresholds were shown to decrease with increasing exposure time. These results are important since under natural conditions lower concentration of NH(4)(+) are present in waters but for very long periods of time. The importance of applying this knowledge in biomonitoring studies to fulfil the requirements of the Water Framework Directive is discussed.

Combinatorial peptide ligand libraries for urine proteome analysis: Investigation of different elution systems

Proteome treatments with peptide libraries in view of reducing high-abundance proteins and increasing the concentration of rare species involve the adsorption on solid-phase material. Subsequent elution of captured proteins may not be fully effective except when sequences of eluting agents are used. The standard way utilized up to the present has been a three- to four-step, sequential elution system consisting of various agents mixed together such as urea, thiourea, CHAPS, sodium chloride, citric or acetic acid and some polar solvents such as ACN and isopropanol. Elution sequences produce distinct fractions adding to the burden of having to analyze all of them. An alternative, highly effective, single elution to reduce the workload is here reported for the first time, namely elution in boiling 10% SDS added with 3% DTE. This single step elutes almost quantitatively the adsorbed proteins, thus ensuring, for all practical purposes, a full recovery. This high efficiency is believed to be due to the fact that the SDS micelles bury the polypeptide chains within their hydrophobic core, thus shielding them from the surroundings and impeding accidental adsorption to surfaces. Suggestions for selecting the best method to eliminate the excess of SDS for further protein analysis are also evaluated. The merits and limits of this novel system are assessed and discussed.

Mercury(II) and methyl mercury determinations in water and fish samples by using solid phase extraction and cold vapour atomic absorption spectrometry combination

A method has been developed for mercury(II) and methyl mercury speciation on Staphylococcus aureus loaded Dowex Optipore V-493 micro-column in the presented work, by using cold vapour atomic absorption spectrometry. Selective and sequential elution with 0.1 mol L −1 HCl for methyl mercury and 2 mol L −1 HCl for mercury(II) were performed at the pH range of 2–6. Optimal analytical conditions including pH, amounts of biosorbent, sample volumes were investigated. The detection limits of the analytes were 2.5 ng L −1 for Hg(II) and 1.7 ng L −1 for methyl mercury. The capacity of biosorbent for mercury(II) and methyl mercury was 6.5 and 5.4 mg g −1, respectively. The validation of the presented procedure is performed by the analysis of standard reference material. The speciation procedure established was successfully applied to the speciation of mercury(II) and methyl mercury in natural water and microwave digested fish samples.

Testing differences in methods of preparing moss samples. Effect of washing on Pseudoscleropodium purum

When terrestrial mosses are used to study the risks associated with the accumulation of airborne pollutants, samples must be washed to remove deposits adhering to the surface. Here, we studied the effects of washing the samples as well as of freezing, drying, and acclimatization treatments. The differences between the concentrations of Al, As, Cd, Cr, Cu, Fe, Hg, K, Ni, Pb, Se, V, and Zn in washed and unwashed samples were significant, and therefore, it would not be valid to combine or compare the data corresponding to samples treated thus. We also investigated whether there were significant differences in the concentrations of various metals and metalloids yielded from samples subjected to different treatments prior to washing (freezing, drying, acclimatization after drying, and acclimatization only), while also considering possible effects of the level and type of contamination and the weather conditions. For many of the elements, we found significant differences that depended on both the levels of contamination at each sampling site and the weather. As these differences may be due to changes in the contents of the different cellular fractions, the sequential elution technique was applied to determine the locations of Cu, K, and Zn. The freezing and drying procedures were rejected because they alter the permeability of the membranes and lead to underestimation of the concentrations of elements. With the acclimatization procedure, particles of some elements adhere to the surface, making this option unsuitable when a description of bioconcentration is required. Taking into account these limitations and the lack of an optimal procedure, acclimatization after drying is proposed as the best option for harmonizing the method.

Mercury(II) and methyl mercury speciation on Streptococcus pyogenes loaded Dowex Optipore SD-2

A solid phase extraction procedure based on speciation of mercury(II) and methyl mercury on Streptococcus pyogenes immobilized on Dowex Optipore SD-2 has been established. Selective and sequential elution with 0.1 mol L −1 HCl for methyl mercury and 2 mol L −1 HCl for mercury(II) were performed at pH 8. The determination of mercury levels was performed by cold vapour atomic absorption spectrometry (CVAAS). Optimal analytical conditions including pH, amounts of biosorbent, sample volumes, etc., were investigated. The influences of the some alkaline and earth alkaline ions and some transition metals on the recoveries were also investigated. The capacity of biosorbent for mercury(II) and methyl mercury was 4.8 and 3.4 mg g −1. The detection limit (3 sigma) of the reagent blank for mercury(II) and methyl mercury was 2.1 and 1.5 ng L −1. Preconcentration factor was calculated as 25. The relative standard deviations of the procedure were below 7%. The validation of the presented procedure is performed by the analysis of standard reference material (NRCC-DORM 2 Dogfish Muscle). The procedure was successfully applied to the speciation of mercury(II) and methyl mercury in natural water and environmental samples.

En bloc elution of proteomes from combinatorial peptide ligand libraries

In contrast to the three to four sequential elution steps routinely adopted for recovering proteomes adsorbed onto combinatorial peptide ligand libraries, we report here two en bloc elution systems, which are able to achieve recoveries in the order of 95% in a single step. One consists of TUC (7 M urea, 2 M thiourea, 3% CHAPS) added with 40 mM formic acid, the other of TUC added with 25 mM cysteic acid (Cys-A). Although both systems are almost equally performing, the formic acid eluant has as a drawback, namely the potential to modify proteins by formylation of Ser and Thr residues. On the contrary, the Cys-A system is unreactive towards proteins. Additionally, Cys-A, due to its very low pI value (1.80) does not interfere with subsequent 2D map analyses since, during the first isoelectric focusing step (in general performed in immobilized pH gradients), it migrates to the anodic compartment and thus vacates the gel. Conversely, formic acid would mostly collect around pH 3 and acetic or citric acid, formerly used in the UCA (9 M urea, 50 mM citric acid) eluant, would condense around pH 4 in the focusing step, interfering thus with 2D map analyses. Elution by boiling SDS of the small amount of protein left over after three sequential elution steps in TUC and 25 mM Cys-A and analysis by nanoLC-MS/MS has demonstrated that these residual proteins are indeed a residue left over from proteins already eluted in TUC-Cys-A and not new species absent in the latter eluate.

A search for an extracellular extractant of Hg for use in the sequential elution technique with Pseudoscleropodium purum

The extraction efficiency of several metal cations (50 mM CoCl2, 20 mM NiCl2, 30 mM Pb(NO3)2 and 50 mM SrCl2) and chelating agents (30 mM dimercaprol, 10 mM EDTA and 50 mM D-penicillamine) was evaluated in order to determine the optimal extracellular extractant for extracting Hg from the moss Pseudoscleropodium purum, by the sequential elution technique (SET). Moss samples were incubated with a high concentration of Hg (40 μg L–1) for one hour, and after the intercellular fraction was removed, the samples were subjected to the SET with the above-mentioned extractants. The chelating agents dimercaprol and D-penicillamine displaced most extracellular Hg, probably because both contain -SH groups, which have a high affinity for Hg. Dimercaprol was chosen as the best extractant because it displaced up to 93% of the extracellular metal. This compound was then used to extract Hg in an industrial gradient of Hg contamination, and the concentration of extracellular Hg was calculated relative to the total amount (80%). A new modification to the SET was also proposed. In the new version of the technique, the metals were determined in solid moss samples rather than in extracts. One advantage of the modification is that all of the determinations are carried out with the same analytical technique (direct mercury analysis), thereby minimizing the errors associated with the use of different techniques as in the previous version of the method. It was found that the cell compartment where the metals are located is not always accurately reflected by the SET, and therefore the use of other complementary techniques that show the correct location of the element under study is recommended.

Rapid solid-phase extraction method to quantify [11C]-verapamil, and its [11C]-metabolites, in human and macaque plasma

P-glycoprotein (P-gp), an efflux transporter, is a significant barrier to drug entry into the brain and the fetus. The positron emission tomography (PET) ligand, [(11)C]-verapamil, has been used to measure in vivo P-gp activity at various tissue-blood barriers of humans and animals. Since verapamil is extensively metabolized in vivo, it is important to quantify the extent of verapamil metabolism in order to interpret such P-gp activity. Therefore, we developed a rapid solid-phase extraction (SPE) method to separate, and then quantify, verapamil and its radiolabeled metabolites in plasma. Using high-performance liquid chromatography (HPLC), we established that the major identifiable circulating radioactive metabolite of [(11)C]-verapamil in plasma of humans and the nonhuman primate, Macaca nemestrina, was [(11)C]-D-617/717. Using sequential and differential pH elution on C(8) SPE cartridges, we developed a rapid method to separate [(11)C]-verapamil and [(11)C]-D-617/717. Recovery was measured by spiking the samples with the corresponding nonradioactive compounds and assaying these compounds by HPLC. Verapamil and D-617/717 recovery with the SPE method was >85%. When the method was applied to PET studies in humans and nonhuman primates, significant plasma concentration of D-617/717 and unknown polar metabolite(s) were observed. The SPE and the HPLC methods were not significantly different in the quantification of verapamil and D-617/717. The SPE method simultaneously processes multiple samples in less than 5 min. Given the short half-life of [(11)C], this method provides a valuable tool to rapidly determine the concentration of [(11)C]-verapamil and its [(11)C]-metabolites in human and nonhuman primate plasma.

Study on the extraction, purification and quantification of jasmonic acid, abscisic acid and indole‐3‐acetic acid in plants

Jasmonic acid (JA), abscisic acid (ABA) and indole-3-acetic acid (IAA) are important plant hormones. Plant hormones are difficult to analyse because they occur in small concentrations and other substances in the plant interfere with their detection. To develop a new, inexpensive procedure for the rapid extraction and purification of IAA, ABA and JA from various plant species. Samples were prepared by extraction of plant tissues with methanol and ethyl acetate. Then the extracts were further purified and enriched with C(18) cartridges. The final extracts were derivatised with diazomethane and then measured by GC-MS. The results of the new methodology were compared with those of the Creelman and Mullet procedure. Sequential elution of the assimilates from the C(18 )cartridges revealed that IAA and ABA eluted in 40% methanol, while JA subsequently eluted in 60% methanol. The new plant hormone extraction and purification procedure produced results that were comparable to those obtained with the Creelman and Mullet's procedure. This new procedure requires only 0.5 g leaf samples to quantify these compounds with high reliability and can simultaneously determine the concentrations of the three plant hormones. A simple, inexpensive method was developed for determining endogenous IAA, ABA and JA concentrations in plant tissue.

New practical algorithm for modelling analyte recovery in bioanalytical reversed phase and mixed-mode solid phase extraction

Solid phase extraction (SPE) is a widely used method for sample cleanup and sample concentration in bioanalytical sample preparation. A few methods to model the retention behaviour on SPE cartridges have been described previously but they are either not applicable to ionised species or are not suitable when using multiple wash and elution steps with solvents differing in volume, modifier concentration and acidity. Furthermore, these models were not applied to mixed mode SPE sorbents. In order to overcome these limitations a new SPE modelling algorithm was proposed. The retention behaviour was determined directly on the SPE cartridge by connecting the cartridge online with an HPLC system using a simple but suitable device that was developed and described. The results from these online experiments were used to model the elution behaviour using a quadratic retention function combined with an exponentially modified Gaussian peak shape model to predict analyte recovery under different wash and elution conditions. The validity of the proposed algorithm was tested using practical SPE experiments with an aqueous test mixture as well as with spiked human plasma. Different sequential wash and elution steps were performed using solvents differing in volume and composition. The predicted band shape and recoveries in each collected step were in good agreement with the results obtained from practical experiments. The proposed algorithm is very useful for the description of the SPE behaviour of the analytes on the actual used SPE cartridge and can be used in structural and automated SPE method development.

SIMAC (Sequential Elution from IMAC), a Phosphoproteomics Strategy for the Rapid Separation of Monophosphorylated from Multiply Phosphorylated Peptides

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 mug of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides.

Speciation of inorganic platinum–chloride complexes in spiked environmental samples by SPE and ICP–AES

A method for the separation of tetrachloroplatinate PtCl 4 2−, and hexachloroplatinate PtCl 6 2−, by solid-phase extraction, using a Dowex 1 × 10 anion exchange resin is proposed. The sequential elution and separation of PtCl 4 2−, and PtCl 6 2− is achieved using selective complexing agents. The eluates, containing Pt(II) and Pt(IV) were analyzed by inductively coupled plasma–atomic emission spectroscopy (ICP–AES). Recoveries of 102% for PtCl 4 2− and 94% for PtCl 6 2− and detection limit of 15 ng g −1 were achieved. Using this method determination of Pt(II) and Pt(IV) in soil samples, spiked with different platinum species was performed. The comparison with GFAAS determination showed a very good agreement.