Biomarker or geochemical fossils are the recent research methods for the identification of the paleo earth history where we did not find any bony fossil. Let’s discuss how we extract biomarker or Geochemical fossil like pristane, phytane, cadalene, dibenzofuran, etc.
At first, I took 120 gm (Approx.) of each sample in a biker for biomarker analysis of my study area. The total number of sample was 24 of different sedimentary rocks of paleoenvironment. The analysis is divided into 2 phases, In 1st Phase, I analyzed 12 samples and in the 2nd phase, I analyzed the rest of the 12 samples.
Each sample was washed with dichloromethane and methanol in the ratio of 6:4 solutions in a vacuum chamber. Then covering with foil, place it in an ultrasound chamber to agitate the molecules for approximately 3 minutes. Then pour this dichloromethane and methanol solution in a jar for washes the sample.
In this way, the total process is repeated once again.
Then, instead of dichloromethane and methanol of 6:4 ratio solutions, repeat the process again with dichloromethane 5000 and methanol 5000 of 6:4 ratio solution.
In this way, wash the samples of different rocks.
Now, wash the heavy weighted mortar 3 times with dichloromethane and methanol of 6:4 ratio solutions in the same way above. Then wait a while for drying the mortar.
Now, for the 12 samples of the following. carefully crush the samples to make grain size of less than 3 mm by the heavy weighted mortar. Keep the samples in the biker with a new foil cover which is burnt in 550° C which will be used after making powder by the powder making-machine.
Prepare the powder making machine by washing the dichloromethane and methanol in the ratio of 6:4 solutions 3 times.
Now, pour the sample into the power making tray and gradually push the key for closing the tray cover of the machine. Then, set 30 minutes for each sample to make fine grain powder. After completing each sample, I washed the tray by sand grain for 5 minutes rotation to remove any previous sample and other OM from the tray. Then each sample is covered by foil paper and writes the sample number. All the process is done in the vacuum chamber. Keep all the samples in an air-free glass storage tank/detector.
Now, prepare for the Soxhlet extraction process.
Cut from the upper part of thimble filter of 1.5 cm, cover by foil paper/Aaromaki Aluminum paper upper and lower part of thimble paper first, collect the thimble filter and cut in 1.5 cm from the open part of the filter. Then cover it with the foil and total no. of the thimble filter is 12. Place it in a high-temperature oven to be heated to 525° C for five hours. Then, I collect the thimble filter, burnt forceps, a syringe of 50-micron size washed with n-hexane solution, corpus cotton, a balance and standard solution of n C22D46 53.1 ppm – phenanthrene D 50.3 ppm mixer.
At first, weight 27 gm of power sample by a balance and put it within the thimble filter, cover the sample by corpus cotton then add another 27 gm of power sample and inject 50 microns of the standard solution of n C22 D46 53.1 ppm and phenanthrene D 50.3 ppm on the sample powder. Cover the sample by corpus cotton and again add 27 gm of sample powder and cover the sample by corpus cotton finally. Mark the sample number by marker pen.
Need small capillary glass tube divide and prepare for burn, 500 ml round shape flask, Pour 350 ml: 50 ml of dichloromethane 5000 and methanol 5000 solutions in burned biker, 27 no. flask,
Now, I took a burnt round 500 ml flask, keep 3 pieces of the capillary tube which is divided by flame tube in it, place a soxhlet funnel on the round 500 ml flask, carefully keep a thimble filter filled with the sample within the soxhlet funnel, and clip it. Then, Pour 350 ml: 50 ml of dichloromethane 5000 and methanol 5000 solutions in another 27 no. flask. Shake it well and pour it into the soxhlet slowly through the sample. Now, place it in a 60° C water temperature panel carefully for the Soxhlet extraction from the samples in which OM evaporates and cool down by cooled water and the evaporates return back to the round 500 ml flask. Keep the process to be continued for 48 hours continuously. In this way 1st 12 samples were kept under the soxhlet extraction process.
Then, after 48 hours, at first switched off the 60 degree heat panel, pour in it 6 piece/some ice bag to be cool the water temperature, when evaporation is completely stopped (10 Minutes) then to take off the soxhlet extraction panel carefully, loosen the screw and take off the flask filled with sample solution carefully then pour the sample solution to a new 29/42 size,500 ml dome shape flask, add approximately 60 gm (Approx.)(depends on water content within the sample solution) of NaHSO4, shale well, closely tied the flask. All the process is done in the vacuum chamber. In this way, 12 samples from the 1st phase were completed successfully.
Step 10: Date; 14.10.2014
At first, prepare the rotation evaporation machine then keep an ice bottle in the condensation chamber of the rotation evaporation machine. Wash a two open mouth flask by dichloromethane 5000: methanol 5000 in a 7:1 ratio solution. Clip the conical flask with the rotation evaporation end now tied with clip in another mouth of the conical flask with a sample containing flask. Switch on the machine for rotation of sample containing flask in a 60-degree temperature water pan, RMP is 58. Pressure is 940 mbar of the machine but may vary from both if seen no evaporated condensed solution then lower the pressure to 500 mbar. When very little is present in the sample flask, stop the machine. The volume of sample and amount of NaHSO4 should be the same.
(as required per sample number) burnt 24/40 size 50 ml small flask of 12 piece, 1.0 ml size pipette pipette of 12 piece, 2 syringe, flask cap,7:1 ratio solution of dichloromethane can 5000 and methanol 5000 solutions for 80 ml in a flask, one syringe for dichloromethane 5000 and methanol 5000 solutions, and another for sample solution,
Then, Use different pipette for different sample, pour out all the sample from the conical flask to the small 50 ml flask by 1.0 pipette, pour 7:1 ratio solution of dichloromethane 5000 and methanol 5000 solution to the conical flask of 5 ml, shake the 500 ml NaHSO4 containing flask by ultrasonic wave for 30 sec, again pour out sample solution by pipette and add 7:1 ratio solution of dichloromethane 5000 and methanol 5000 solution of 5 ml (5 times by 1. ml pipette)to the 500 ml NaHSO4 containing flask shake by ultrasonic wave for 30 sec , pour out the sample solution, repeat the process one more time,
Now, shake the NaHSO4, containing conical flask by hand for further good washing, keep the 50 ml small sample solution in a dark place for again rotation evaporation process.
Again, do the same processes as we do above for evaporation. That means set all 50 ml sample containing flask in a evaporation machine again, When very little is present in the 50 ml sample flask, stop the machine , Now, Put the samples in a ampoule by the previous used 12 pipette, add 7:1 ratio solution of dichloromethane 5000 and methanol 5000 solution of 1.0 ml to the 50 ml sample flask, Again repeat for collecting all the samples by adding 7:1 ratio solution of dichloromethane 5000 and methanol 5000 solution of 1.0 ml to the 50 ml sample flask. Do one more time. Now, Name the samples of different ample for N2 gas evaporation.
wash the nitrogen gas pannel by Hexen-1 & Hexen-2 for 3 times. Nitrogen gas panel (Pressure is 0.1 Mpa) and dry the samples which is taken off from the rotation evaporation machine. Collect burned small ampoules of 12 piece, pipette, place small ampoule in a case.
Now, set all the sample containing ampoules in Nitrogen gas panel to be dry in this way 12 samples are done. After drying the sample inject out all the sample in a small ampoule. Add 0.5 ml of 7:1 ratio solution of dichloromethane 5000 and methanol 5000 solutions by pipette & syringe. Shake by ultrasonic wave and vibrator for 3 sec. repeat the process once again. In this way, all the ampoule solution is injected out to the small ampoule.
Again go to N2 pannel and evaporate from the small ampoule when small amount remains / After drying, add 0.5 ml of Hexane instantly by pipette & 0.5ml size syringe.
Step 14: Three fraction
- A) Silica gel-hexane solution
- b) Burned pipette according to sample number
- C) Small pipette filled with cotton
- d) Large ampoule 4 multiple times of a number of samples, Name the ampoules; 1st line blank, F1a with sample name, F1b+ 2 with sample name, F3-8 with sample name in 4 lines in an iron case.
- e) Solution preparation of; 1) Hexen-100 ml approx. 2) Hexen:Toluene -30 ml:10ml
3)Ethyle acetate- 10ml approx. 4) Methanol-60ml approx.
- f) 1.0 ml size syringe -2 piece
- g) 0.5 ml size syringe -1 piece
- h) Solution mixing chart according to Supervisor
For silica gel-hexane preparation; collect burned silica gel in a 50 ml conical flask, add 0.5 ml distilled water ( from distilled water ) drop-wise on a vibrator, now, pour hexane little bit more of silica gel in the flask).
Then orderly arrange the large ampoule in an iron case, mark the sample name of small conical tube with 12 pieces.
1st line 12 pieces; without a name
2nd line 12 pieces; a name with F1a
3rd line 12 pieces; a name with F1b+ 2
4th line 12 pieces; a name with F3-8
Step 15: Three fraction (cont….)
Now, place pipettes on the 1st line then pour silica gel-hexane solution, up to 50.1-50.8 mm in height which is injected with a syringe containing Hexane. Be careful don’t keep dry of compacted silica gel solution, pour hexane if needed.
Shift the silica gel-hexane filled pipette to the 2nd line.
Now inject out sample solution from the small ampoule by new pipette then pour into 2nd line the silica gel filled pipette. Use different pipette for a different sample. Then add 0.5 ml hexane into the sample containing ampoule, shake by ultrasonic wave and vibrator for 3 sec, pour it into the Fla sample, then again add 0.50 ml and repeat the process then add directly 1.0ml of Hexen to the F1a ampoule (according to the supplied chart by Sensei (Dr. Kaiho).
Shift the silica gel-Hexen pipette from the F1a ampoule to the F1b+2 ampoule, then add 1.0 ml hexane to the sample ampoule mixed by ultra and vibrator for 3 sec, pour it to the F1b+2 ampoule by the same sample pipette and syringe.
Then add 1.00+ 1.00+ 1.00 ml hexen directly to F1b+2 ampoule each time.
Now add 1.00 ml of hexane +Toluene solution to the sample ampoule the mixed by ultrasonic and vibrator for 3 sec, pour it to the F1a+2 ampoule.
Shift the silica gel-Hexen pipette from the F1a+2 ampoule to the F3-8 ampoule, add 1.00 ml ethyl acetate to the F3-8 ampoule. Add 1.00 ml of methanol to sample ampoule, shake well, then inject to the F3-8 ampoule, repeat again, then add 1.00 ml methanol directly to the F3-8 for 7 times separately. Close the cap of each ampoule. Steps are closed. Amount of every solution (F1a, F1b+ 2, and F3-8) should be the same.
Burn clear vial 12 pieces, Pipette, and small tube-like glass 12 according to sample number.
Keep all the F1a, F1b+ 2, and F3-8 ampoules in a safe place on the molded-wood-made keeper for further nitrogen gas panel. From the F1a ampoule inject out all the sample to a green cap clear vial, add 0.5 ml of hexane to the F1a ampoule, shake well for extracting all the sample, pour it to a green cap clear vial, Name the sample name of the vial.
Go to an N2 gas chamber, set all the green cap vial F1a named sample when the sample solution is to get a small amount with a solution, remove from nitrogen gas chamber, add a standard solution of C24 D50 50.2 ppm of 30 microns and add 70 microns of Hexane.
For F1b+2, In this way after dying from N 2 chamber add a standard solution of C24 D50 50.2 ppm of 30 microns and add 70 microns of Hexane. then according to the color of the F1a, F1b+ 2, and F3-8 samples in the green cap clear vial dilute the samples of ½, 1/5, 1/10, 1/30, 1/50 etc. that is 1/50 is needed for more deeper color sample and ½ is for a more lighter color sample to be the same dilution. So for a 100 ml (30 stand. +70 micron Hexen) sample to be the same dilution add more 100 Micron of Hexen (for ½ ) and add more 400 micron (for 1/5)of hexane and add more 900 micron of hexane (for 1/10) to be the same dilution of different color sample. Keep sample in a safe place, now, ready for biomarker analysis in Gas Chromatography-mass spectrometry-mass spectrometry (GC MSMS) machine.
Preparation of standard solution for the GCMSMS machine (C24 D50 50.2 ppm + n C22 D46 53.1 ppm and phenanthrene D 50.3 ppm );
Take out from refrigerator of C24 D50 50.2 ppm and n C22 D46 53.1 ppm & phenanthrene D 50.3 ppm standard vial then wait for being liquid now, wash 100 micron syringe by n hexene 1 and 2 times then for making 2 vials of GCMSMS standard solution, take 60 micron of C24 D50 50.2 ppm and add 100 micron of n C22 D46 53.1 ppm and phenanthrene D 50.3 ppm in a new vial (green cap), pour 40 micron of n haxen total 200 microns then divide into 2 vials (100 +100 stand solution.) by micron syringe.