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.
