Name: A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants (Video) | JoVE Journal
Uploaded: 2016-01-01T14:00:00.000+00:00
Duration: 7 min 26 s
Description: Learn automated solid phase extraction for pre-concentrating organic pollutants from water samples using a cost-effective miniaturized system. This method enhances immunodetection analysis with low solvent content and high efficiency.

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5 minutes

This work was funded by the European Union Seventh Framework Program FP7/2007-2013 under grant agreement no. 265721. The authors thank the RIKILT Institute for Food Safety (NL) for their support in this project.

Note: The following protocol describes the SPE performed on 100 ml water sample with C18 sorbent and elution with 50% v/v methanol. The enriched sample is diluted to reach 10% v/v methanol before analysis with an enzyme linked immunosorbent assay (ELISA) kit.
1. Preparing the Reagents
<ol>
	<li>Prepare the water samples
	<ol>
		<li>Prior to any other step, filter each 100 ml water sample with 0.2 &#181;m pore size filters.</li>
		<li>Spike the sample with desired concentration by diluting appropriate volume of reference solution into a volume of water. For example, prepare 100 ml of water sample with 100 ng/L of E2 by diluting 3.3 &#181;l of E2 reference solution at a concentration of 300 &#181;g/L. Dilute this solution ten times to get a sample spiked with 10 ng/L E2. Dilute this latter another ten times to obtain a sample of 100 ml with 1 ng/L E2.</li>
		<li>Place the filtered sample (unmodified or spiked) in a glass bottle with GL45 thread. Use the sample on the same day. Take a small fraction to be analyzed with the ELISA to estimate initial concentration.</li>
	</ol>
	</li>
	<li>Prepare 300 &#181;l of eluent by diluting methanol in de-ionized (di-) water to 50% v/v in a tight tube.</li>
</ol>
2. Preparation of the SPE Column
<ol>
	<li>Prepare a 20 mg/ml suspension of octadecyl-silica sorbent particles by adding first 1,600 &#181;l of methanol and then 400 &#181;l of di-water to 40 mg of reverse phase sorbent. Tightly close the lid and agitate with a vortex.</li>
	<li>Installing the bottom membrane in column
	<ol>
		<li>Select one Nylon membrane with pore size 11 &#181;m and place it on a double layer of anti-dust tissue. With a 3 mm diameter punch, cut two small parts in the membrane.</li>
		<li>Grab one of the small membranes with a flat-end filter forceps and place it on one side of the column.</li>
		<li>Screw the flat-bottom connector with the tube and tighten. The membrane is now in place and the sorbent can be added securely. Draw an arrow on the body of the column pointing towards the end where the membrane was placed.</li>
	</ol>
	</li>
	<li>Preparing the packed sorbent column
	<ol>
		<li>Secure the column on the holder with the arrow pointing towards the bottom. The column must be set as vertically as possible.</li>
		<li>Attach an empty 10 ml disposable syringe to the end of the tube of the column, by using the Luer-Lock connectors.</li>
		<li>Prepare a micro-pipette with a 20-200 &#181;l tip, set the volume to 100 &#181;l. This pipette tip format is adapted to the size of the sorbent column to be prepared. The procedure would be more difficult with larger pipette tips such as the ones used with 1 ml pipettes.</li>
		<li>Agitate the sorbent suspension with a vortex, and rapidly pipet 100 &#181;l in the center on the column. While injecting, aspirate gently all the pipetted solution trough the membrane by using the syringe with the other hand. At this stage, the solution filling the syringe must be clear of particles, and a particle bed can eventually be observed in the column.</li>
		<li>Repeat this process 2 more times by agitating the stock suspension between all pipetting steps to ensure homogeneous suspension of the particles in the solution. The resulting column contains 6 mg of sorbent.</li>
		<li>When the 300 &#181;l of suspension have been loaded and dried by aspirating with the syringe, keep the syringe in position and place the second Nylon membrane on the top by using the forceps with the other hand.</li>
		<li>Screw down the second connector with tube and dispose the syringe. The SPE column is ready for use.</li>
	</ol>
	</li>
</ol>
3. Preparing the System
<ol>
	<li>Tighten the SPE column on the device by using the Luer-Lock connectors. The arrow must be pointing to the same direction as the one shown on the device.</li>
	<li>Connect the bottle containing the sample to the device by screwing the provided GL45 safety cap on it.</li>
	<li>Load 200 &#181;l of eluent in the &#39;Eluent&#39; reservoir.</li>
	<li>Load 800 &#181;l of di-water in the &#39;Dilution&#39; reservoir.</li>
	<li>Place a bottle at the waste outlet to collect the processed water during extraction step. The format is not important but the volume needs to be sufficiently large with regard to the sample volume.</li>
	<li>Place a small vial at the sensor outlet with the minimum volume capacity of 1.5 ml. A maximum volume of 1 ml will be too small as bubbles will form at this outlet when collecting enriched eluent and dilution buffer.</li>
	<li>Verify the pressure regulator is in closed position by turning it manually, reverse-clockwise until further movement is not possible.</li>
</ol>
4. SPE with the Prototype
<ol>
	<li>Switch on the prototype by pressing the button on the back.</li>
	<li>Preparing the user interface and selecting the program
	<ol>
		<li>Open the user interface. Select the communication port of the computer to which the device is connected from the list, and click the button &#39;Next&#39;.</li>
		<li>Enter the values 580 for pset and 30 for dpset. The pump will adjust to maintain pressure in the pressurized system and reservoirs at 580 &#177; 30 mbar when running.</li>
		<li>Select the automated mode.</li>
		<li>In the automated mode corner, load the program file in the box &#39;configuration file path&#39;.</li>
	</ol>
	</li>
	<li>Adjusting the pressure regulator
	<ol>
		<li>Start the pump.</li>
		<li>Turn manually the pressure regulator until the value read for preg is inferior but close to 320 mbar.</li>
		<li>Stop the pump.</li>
	</ol>
	</li>
	<li>Starting the SPE procedure
	<ol>
		<li>Press &#39;start&#39; in the automated mode corner. The pump will switch on and the extraction, elution and dilution steps will automatically follow one another. The whole procedure for a 100 ml sample is performed in 50 min.</li>
		<li>Verify the value of preg. It must be in the range 320 - 350 mbar during the extraction step to ensure an optimal flow-rate.</li>
		<li>Close the small vial and store at 4-5 &#176;C in the dark until analysis. Perform analysis in the following 30 hr to prevent degradation of the analyte.</li>
		<li>Dispose of the processed water.</li>
	</ol>
	</li>
	<li>Cleaning the system 
	Note: After each extraction procedure the system needs to be cleaned to prevent cross-contamination.
	<ol>
		<li>Prepare a 10 ml solution of methanol 70% v/v in a GL 45 glass bottle.</li>
		<li>Unplug the SPE column and plug the tubing with connectors.</li>
		<li>In the automated mode, select the &#39;cleaning&#39; file and start it with same pressure settings.</li>
	</ol>
	</li>
</ol>
5. Detection of Estradiol Concentration with ELISA
<ol>
	<li>Prepare the calibration samples with concentrations as indicated in the protocol provided with the ELISA kit that is used. Prepare one calibration set with the same matrix as the sample, and one calibration set with methanol 10% v/v.</li>
	<li>Dispense the necessary amount of sample in the wells on the plate, according to manufacturer&#39;s instructions. Use calibration samples, unmodified and spiked water samples that were filtered but not processed with SPE, and enriched samples in methanol 10% v/v. Use 3 wells per sample to reduce the error associated with the assay.</li>
	<li>Follow the indications of the protocol that is provided with the kit for the addition of reactants, incubation time, washing and further reaction with the enzyme.</li>
	<li>Read the optical signal in each well according to the kit manufacturer&#39;s instructions with a plate reader instrument and collect the data.</li>
	<li>Use the software of the plate reader instrument to fit the calibration curves and determine the E2 concentration in the initial sample with same matrix, and in the enriched samples with methanol 10% v/v.</li>
</ol>

<ol><li>Chang, H. S., Choo, K. H., Choi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+methods+of+identification%2C+analysis+and+removal+of+endocrine+disrupting+compounds+%28EDCs%29+in+water%5BTitle%5D)+AND+%22J.+Hazard.+Matter%22%5BJournal%5D)">The methods of identification, analysis and removal of endocrine disrupting compounds (EDCs) in water.</a> J. Hazard. Matter. 172, 1-12 (2009).</li>
<li>Azzouz, A., Souhail, B., Ballesteros, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Continuous+solid-phase+extraction+and+gas+chromatography-mass+spectrometry+determination+of+pharmaceuticals+and+hormones+in+water+samples%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Continuous solid-phase extraction and gas chromatography-mass spectrometry determination of pharmaceuticals and hormones in water samples.</a> J. Chromatogr. A. 1217, 2956-2963 (2010).</li>
<li>Tomsikova, H., Aufartova, J., Solich, P., Novakova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+sensitivity+analysis+of+female-steroid+hormones+in+environ-mental+samples%5BTitle%5D)+AND+%22Trends+Anal.+Chem%22%5BJournal%5D)">High sensitivity analysis of female-steroid hormones in environ-mental samples.</a> Trends Anal. Chem. 34, 35-58 (2012).</li>
<li>Ciofi, L., Fibbi, D., Chiuminatto, U., Coppini, E., Checchini, L., Del Bubba, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fully+automated+on-line+solid+phase+extraction+coupled+to+high-performance+liquid+chromatography-tandem+mass+spectrometric+analysis+at+sub-ng%2FL+levels+of+selected+estrogens+in+surface+water+and+wastewater%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Fully automated on-line solid phase extraction coupled to high-performance liquid chromatography-tandem mass spectrometric analysis at sub-ng/L levels of selected estrogens in surface water and wastewater.</a> J. Chromatogr. A. 1283, 53-61 (2013).</li>
<li>Robles-Molina, J., Lara-Ortega, F. J., Gilbert-Lopez, B., Garcia-Reyes, J. F., Molina-Diaz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-residue+method+for+the+determination+of+over+400+priority+and+emerging+pollutants+in+water+and+wastewater+by+solid-phase+extraction+and+liquid+chromatography-time-of-flight+mass+spectrometry%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Multi-residue method for the determination of over 400 priority and emerging pollutants in water and wastewater by solid-phase extraction and liquid chromatography-time-of-flight mass spectrometry.</a> J. Chromatogr. A. 1350, 30-43 (2014).</li>
<li>Huang, C. H., Sedlak, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+estrogenic+hormones+in+municipal+waste+water+effluent+and+surface+water+using+enzyme-linked+immunosorbent+assay+and+gas+chromatography%2Ftandem+mass+spectrometry%5BTitle%5D)+AND+%22Environ.+Toxicol.+Chem%22%5BJournal%5D)">Analysis of estrogenic hormones in municipal waste water effluent and surface water using enzyme-linked immunosorbent assay and gas chromatography/tandem mass spectrometry.</a> Environ. Toxicol. Chem. 20, 133-139 (2001).</li>
<li>Hintemann, T., Schneider, C., Schöler, H. F., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Field+study+using+two+immunoassays+for+the+determination+of+estradiol+and+ethinylestradiol+in+the+aquatic+environment%5BTitle%5D)+AND+%22Water+Res%22%5BJournal%5D)">Field study using two immunoassays for the determination of estradiol and ethinylestradiol in the aquatic environment.</a> Water Res. 40, 2287-2294 (2006).</li>
<li>Farré, M., Kuster, M., Brix, R., Rubio, F., Lopez de Alda, M. J., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+study+of+an+estradiol+enzyme-linked+immunosorbent+assay+kit%2C+liquid+chromatography-tandem+mass+spectrometry%2C+and+ultra-performance+liquid+chromatography-quadrupole+time+of+flight+mass+spectrometry+for+part-per-trillion+analysis+of+estrogens+in+water+samples%5BTitle%5D)+AND+%22J.+Chromatogr.+A%22%5BJournal%5D)">Comparative study of an estradiol enzyme-linked immunosorbent assay kit, liquid chromatography-tandem mass spectrometry, and ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry for part-per-trillion analysis of estrogens in water samples.</a> J. Chromatogr. A. 1160, 166-175 (2007).</li>
<li>Pu, C., Wu, Y. F., Yang, H., Deng, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Trace+analysis+of+contraceptive+drug+levonorgestrel+in+waste+water+samples+by+a+newly+developed+indirect+competitive+enzyme-linked+immunosorbent+assay+%28ELISA%29+coupled+with+solid+phase+extraction%5BTitle%5D)+AND+%22Anal.+Chim.+Acta%22%5BJournal%5D)">Trace analysis of contraceptive drug levonorgestrel in waste water samples by a newly developed indirect competitive enzyme-linked immunosorbent assay (ELISA) coupled with solid phase extraction.</a> Anal. Chim. Acta. 628, 73-79 (2008).</li>
<li>Van Emon, J. M., Gerlach, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+status+report+on+field-portable+immunoassay%5BTitle%5D)+AND+%22Environ.+Sci.+Technol%22%5BJournal%5D)">A status report on field-portable immunoassay.</a> Environ. Sci. Technol. 29 (7), 312A-317A (1995).</li>
<li>Farré, M., Brix, R., Barcelò, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Screening+water+for+pollutants+using+biological+techniques+under+European+Union+funding+during+the+last+10+years%5BTitle%5D)+AND+%22Trends+Anal.+Chem%22%5BJournal%5D)">Screening water for pollutants using biological techniques under European Union funding during the last 10 years.</a> Trends Anal. Chem. 24 (6), 532-545 (2005).</li>
<li>Schneider, C., Schöler, H. F., Schneider, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+enzyme-linked+immunosorbent+assay+for+ethinylestradiol+using+a+long-chain+biotinylated+EE2+derivative%5BTitle%5D)+AND+%22Steroids%22%5BJournal%5D)">A novel enzyme-linked immunosorbent assay for ethinylestradiol using a long-chain biotinylated EE2 derivative.</a> Steroids. 69, 245-253 (2004).</li>
<li>Heub, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+and+portable+solid+phase+extraction+platform+for+immuno-detection+of+17%CE%B2-estradiol+in+water%5BTitle%5D)+AND+%22J.+Chrom.+A%22%5BJournal%5D)">Automated and portable solid phase extraction platform for immuno-detection of 17β-estradiol in water.</a> J. Chrom. A. 1381, 22-28 (2015).</li>
<li>Petrovic, M., Eljarrat, E., Lopez de Alda, M. J., Barcelo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endocrine+disrupting+compounds+and+other+merging+contaminants+in+the+environment%3A+a+new+survey+on+new+monitoring+strategies+and+occurrence+data%5BTitle%5D)+AND+%22Anal.+Bioanal.+Chem%22%5BJournal%5D)">Endocrine disrupting compounds and other merging contaminants in the environment: a new survey on new monitoring strategies and occurrence data.</a> Anal. Bioanal. Chem. 378, 549-562 (2004).</li>
<li>Richardson, S. D., Ternes, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Water+analysis%3A+Emerging+contaminants+and+current+issues%5BTitle%5D)+AND+%22Anal.+Chem%22%5BJournal%5D)">Water analysis: Emerging contaminants and current issues.</a> Anal. Chem. 86, 2813-2848 (2014).</li>
</ol>

The authors have nothing to disclose.

A new method for the preparation of water samples followed by analysis using immunoassay was proposed. The instrument enables to perform solid phase extraction in an automated and user-friendly way.
The filtration of the water sample prior to its injection into the system is critical. Any particulates still present in the solution would potentially cause clogging of the fluidic network and obstruct the SPE column. Another important step is the preparation of the SPE column. The amount of parti...

Sample preparation is an important step in any analytical process. In particular, removal of matrix effects, diminution of interferences, and enrichment of the analyte are necessary to obtain precise results and reach low limits of detection. Endocrine disrupting compounds (EDCs) are of particular concern due to their action on the living organisms even when present at very low levels in the environment. The natural hormone 17&#946;-estradiol is present on the EU water pollution Watch List and prone to be added to the list of priority substances regulated under the European Water Framework Directive. Solid phase extraction (SPE) is commonly applied for the analysis of small pollutants in water, with both chemical 1-5 (chromatography, mass spectrometry) and immunological 6-9 detection methods. The latter gained interest in the field of environmental monitoring, as immunoassays are available in large variety of formats, are specific to the target analyte, and reach low limits of detection.6, 7, 10, 11 Various enzyme linked immunosorbent assays (ELISA) are commercially available and enable to analyze multiple samples at once on a multi-well plate. The procedure consists in successive reaction steps that can take a few hours. The final product of reaction can be detected optically to determine the concentration of the target molecule based on a calibration curve.
Classical SPE procedures include sorbent pre-conditioning, sample extraction, washing, elution, and concentration by evaporation of the eluent. The solvent used for dilution of this extract is chosen depending on the detection method. For immunological methods, the amount of organic solvent influences strongly the sensitivity of the method.12
In addition to the recovery and the pre-concentration performances, the method also needs to be simple and cost efficient. Automation of the procedure helps to reduce human-related errors. In our previous work 13 we introduced our prototype for automated SPE, and our method was applied to the analysis of the natural hormone 17&#946;-estradiol in sea water samples. With the present video we would like to highlight the technical advantages of our method compared to traditional off-line and on-line SPE, and its particular compatibility with detection by immuno-reactions. We describe the protocol applied to water samples for the detection of 17&#946;-estradiol. SPE is performed with octadecyl-silica (C18) sorbent phase and elution is performed with diluted methanol.

<table><tbody><tr><td>Filter membrane 0.2 &amp;mu;m pore size</td><td>Merck Millipore</td><td>GNWP04700</td><td>For sample filtration</td></tr><tr><td>Nylon membrane 11 &amp;mu;m pore size</td><td>Merck Millipore</td><td>NY1104700</td><td>For SPE column</td></tr><tr><td>Disposable biopsy punch 5 mm</td><td>Medical Budget</td><td>39302439</td><td></td></tr><tr><td>Nucleodur C18 ec&amp;nbsp;</td><td>Macherey Nagel</td><td>713550.01</td><td>50 &amp;mu;m particle diameter</td></tr><tr><td>Synthetic sea water</td><td>Sigma Aldrich</td><td>SSWS500-500ML</td><td></td></tr><tr><td>Methanol</td><td>VWR</td><td></td><td></td></tr><tr><td>17beta-estradiol standard</td><td>Enzo Life Science</td><td></td><td>300 ng/ml</td></tr><tr><td>17beta-estradiol ELISA kit</td><td>Enzo Life Science</td><td>ADI-900-008</td><td>96 wells, range 30 - 3,000 ng/L</td></tr></tbody></table>

a simple method for automated solid phase extraction of water samples for immunological analysis of small pollutants

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and enables to perform rapid, automated and simple SPE. The pre-concentrated solution is compatible with analysis by immunoassay, with a low organic solvent content. A method is described for the extraction and pre-concentration of natural hormone 17&#946;-estradiol in 100 ml water samples. Reverse phase SPE is performed with octadecyl-silica sorbent and elution is done with 200 &#181;l of methanol 50% v/v. Eluent is diluted by adding di-water to lower the amount of methanol. After preparing manually the SPE column, the overall procedure is performed automatically within 1 hr. At the end of the process, estradiol concentration is measured by using a commercial enzyme-linked immune-sorbent assay (ELISA). 100-fold pre-concentration is achieved and the methanol content in only 10% v/v. Full recoveries of the molecule are achieved with 1 ng/L spiked de-ionized and synthetic sea water samples.

Reproducibility of sorbent packing was evaluated by drying and weighting the pipetted sorbent in glass vials and the result is shown in Figure 1. Reproducibility of the time of injection was tested for 100 ml samples, as shown in Figure 2. Concentration in initial and pre-concentrated spiked samples were determined by using a commercial ELISA kit for 17β-estradiol and are shown in Figure 3.
<p class="jove_content" fo:keep-together.wit...

A protocol for the extraction and pre-concentration of estradiol from water samples by using an automated and miniaturized system is presented.

A new method for solid phase extraction (SPE) of environmental water samples is proposed. The developed prototype is cost-efficient and user friendly, and ...

The overall goal for this sample preparation method is to pre-concentrate small, organic pollutants from water samples, in a user friendly, cost effective and automated way. This method can replace standard SPE processes when studying the contamination of water samples using immunodetection methods, such as well plate immunoassays or biosensors. The main advantages of this technique is that SPE procedure is automated while the footprint and cost of the instruments are low compared to traditional approaches.In addition, the performances of the enrichment process match the requirement of analysis of immunoassays both in terms of low solvent content and pre-concentration efficiency. First, filter out 100 millimeter water sample with a zero point two micrometer pour size filter. Place the filtered sample in a glass bottle with GL 45 thread.Prepare 300 microliters of eluent by diluting methanol in deionized water, to 50 percent volume in a tight tube. Next, prepare a 20 milligram per milliliter suspension of octadecyl silica sorbent particles by adding 1, 600 microliters of methanol followed by 400 microliters of deionized water to 40 milligrams of reversed phase sorbent in a glass vile. Tightly close the lid, and agitate with a vortex mixer.Place a nylon membrane with pore size 11 micrometers on a double layer of anti-dust tissue. Cut two small parts in the membrane with a three millimeter diameter punch. Then, grab on of the small membranes with flat end filter forceps, and place it on one side of the column.Following this, screw the flat bottom connector with the tube and tighten. Then, draw an arrow on the body of the column, pointing towards the end where the membrane was placed. At this point, secure the column on the holder with the arrow pointing towards the bottom.Attach an empty 10 milliliter disposable syringe to the end of the tube of the column by using the lower lock connectors. Agitate the sorbent suspension with the vortex mixer, and rapidly pipette 100 microliters in the center of the column. While injecting, gently aspirate the pipetted solution through the membrane by using the syringe with the other hand.The SPE column preparation is very important. You have to make sure that you properly aspirate the solution through the membrane using the syringe while injecting the particle suspension in the column. Otherwise, the particles will not be packed densely.Repeat the previous process two more times, by agitating the stock suspension between all pipetting steps, to ensure homogeneous suspension of the particles in the solution. After the entire suspension has been loaded and dried by aspirating with a syringe, keep the syringe in position and place the second nylon membrane on the top by using the forceps with the other hand. Then, screw down the second connector with a tube and dispose of the syringe.At this point, tighten the column on the SPE device by using the lower lock connectors. Connect the bottle containing the sample to the device by screwing the provided GL 45 safety cap on it. Load 200 microliters of eluent in the eluent reservoir.Then, load 800 microliters of deionized water in the dilution reservoir. Verify the pressure regulator is in the closed position by turning it reverse clockwise until further movement is not possible. After switching on the prototype, enter the values 580 for pset and 30 for delta pset.To adjust the pressure regulator, start the pump. Then, manually turn the pressure regulator until the value read for preg is inferior but close to 320 millibar. After stopping the pump, start the SPE procedure by pressing start in the automated mode corner.Once the SPE procedure is complete, close the small vile of sample and store at four to five degrees Celsius in the dark, until ELISA analysis. To clean the system, prepare a 10 milliliter solution of 70 percent methanol by volume in a glass bottle with GL 45 thread. Unplug the SPE column, and plug the tubing with connectors.In the automated mode, select the Cleaning file and start it with the same pressure settings previously used for pset, delta pset, and preg. The reproducibility of sorbent packing was evaluated by drying and weighing the pipetted sorbent in glass viles. The reproducibility of time of injection was tested for the 100 milliliter samples.The concentrations of the initial and pre-concentrated spiked samples were determined using a commercial ELISA kit of 17 beta-estradiol. It is clear from the calibration curves that the methanol ratio from the enriched sample does not effect the sensitivity of the immunoassay. Recoveries of 128, plus or minus 22 percent, and 107 plus or minus six percent, were calculated for deionized water and artificial sea water respectively.Once mastered, the preparation of an SPE column can be done in less than five minutes, if it is performed properly. The entire SPE procedure can be completed in less than one hour. After its development, this technique paved the way for researchers in the field of environmental science to explore the development of immunodetection methods for monitoring of small organic pollutants.After watching this video, you should have a good understanding of how to prepare water samples using our cost effective, automated instrument for the analysis of pollutants by immunoassays. Don't forget that working with organic pollutants and solvents can be hazardous, and precautions such as wearing appropriate gloves and protection goggles should always be taken when performing this process.

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A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Here, we present a protocol for the sequential targeted quantification and non-targeted analysis of fluorinated compounds in water by mass spectrometry. This methodology provides quantitative levels of known fluorochemical compounds and identifies unknown chemicals in related samples with semi-quantitative estimates of their abundance.

Identifying Per- and Polyfluorinated Chemical Species with a Combined Targeted and Non-Targeted-Screening High-Resolution Mass Spectrometry Workflow

This protocol presents a fully automated workflow for the extraction of geosmin and 2-methylisoborneol from water and lipid-rich fish tissues. The method allows for the early detection of these molecules before they reach odor thresholds. Representative data from an aquaculture setting are provided.

Extraction and Detection of Geosmin and 2-Methylisoborneol in Water and Fish using High-Capacity Sorptive Extraction Probes and GC-MS

Learn automated solid phase extraction for pre-concentrating organic pollutants from water samples using a cost-effective miniaturized system. This method enhances immunodetection analysis with low solvent content and high efficiency.

A Simple Method for Automated Solid Phase Extraction of Water Samples for Immunological Analysis of Small Pollutants

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