EnAlgae SOP: Biogas analysis
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- Jade Ward
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1 Table of content 1. PURPOSE PRINCIPLE REQUIREMENTS EQUIPMENT AND MATERIALS REAGENTS PREPARATION OF MEASUREMENT PROCEDURE SAMPLE MEASUREMENT PROCESSING DATA OF MEASUREMENTS CALCULATION OF RESULTS QUALITY CONTROL ERRORS, CALIBRATION AND INTERFERENCES WASTE STREAM AND PROPER DISPOSAL HAZARDS AND PRECAUTIONARY STATEMENTS REFERENCES CONTRIBUTIONS
2 1. PURPOSE This procedure is used to analyze the composition of a biogas sample, specifically the relative quantity of methane and carbon dioxide. 2. PRINCIPLE Gas from either direct samples or gas sampling vials is injected into a gas chromatograph with thermal conductivity detector (GC-TCD). This GC-TCD determines the relative quantity of CH 4, CO 2 and the sum of H 2 and N 2 in function of the different retention times it takes for the sample to migrate through a heated column. By integration of the resulting data, the relative quantity of the main different components (CH 4 and CO 2) is determined. 2
3 3. REQUIREMENTS 3.1. EQUIPMENT AND MATERIALS GC-TCD number 2 (on the right side of number 1) (in room A206) (Brand: Agilent; Type: 6890 Series (GC Systems) Plus +) 3.2. REAGENTS HP GC Autosampler Controller (connection unit to computer) Computer with connection drivers and software for GC controlling and data analysis Agilent J&W Capillary GC column CP7354 Hamilton Gastight Syringe (250 µl) (in the drawer right under the computer) (Brand: Hamilton; Model: GasTight #1725) A series of gas samples in sampling vials to be analyzed Acetone Calibration gas (5% N 2, 5% H 2, 80% CH 4 and 10% CO 2; stored in a small gas bottle next to the GC) 3.3. PREPARATION OF MEASUREMENT Check if the right column is installed in the GC oven (Agilent J&W Capillary GC column CP7354). Check if there is pressure of helium in the helium supply pipe (manometer behind GC number 1). Helium is the carrier gas in this GC-analysis. Boot the computer if it was shut down. This computer is located between the two GC s. The computer should never be shut down after use. Turn on GC2 with the power switch located on the front lower left. Turn on the HP GC Autosampler Controller unit with the power switch located on the front lower left. Run the Bootp shortcut on the desktop of the computer. This links the computer to the controller unit to control the GC and acquisition data 3
4 4. PROCEDURE Fig. 1. GC-TCD (Agilent 6890 Series GC Systems Plus +). Run the GCµTCD shortcut on the desktop to start the software that will acquire data and log it with a preconfigured method. Before measurement of samples the column and detector should be cleaned, running a preconfigured method: 1. Load CLEAN.M from C:\MSDChem\2\METHODS. 4
5 2. Click Sample name in the Instrument control panel to create a log file of the cleaning. This saves the chromatogram of any method to the computer. a. Locate the directory: C:\MSDChem\2\DATA\(in which 2 is the number of the GCunit) b. Create a new directory and name it with the day of the GC-analysis (not the same date!) in the format of YYMMDD in which Y is the number of the year, M for month and D for day. For example: 4 th July 2013 becomes Additionally your name can be added like George. c. Within this directory label the log file CLEAN#.D in which # is the number of the cleaning action on this day. For example CLEAN2.D. 3. Now click START RUN on the computer (do not just click OK ). 4. Press the PREP RUN button at the GC2 panel as soon as not ready LED is longer lit up. 5. Then press the START button on the GC2 panel as soon as the start and run for about 4 minutes. Do not abort this action. After cleaning the GC column will have to cool down before any measurement can be done. 5
6 When cleaning is done, the measurement of samples can begin by loading the method BIOGAS.M from C:\MSDChem\2\METHODS. After the last measurement of a series of samples that have to be compared, method CLEAN.M should be run again as previously described SAMPLE MEASUREMENT Preparing the computer The following steps should be followed before any other gas samples are taken: Make sure the BIOGAS.M method is loaded in the software. This method will run the analysis at a constant 60 C. 6
7 Prepare saving of the log file to C:\MSDChem\2\DATA\ YYMMDD \ with the correct sample name that is to be analyzed next. For example 0703BIOREACTORSOFIE7.D. Do this by clicking Sample name in the Instrument control panel to create a data log file as done before during cleaning Taking the gas sample Prepare the Hamilton gas syringe (250 µl) by reassembling it in case it was taken apart. Before taking any sample, align the valve of the glass Hamilton syringe with the cylinder axis so it is opened. Then rinse the syringe by filling and completely ejecting it 2 or 3 times with air from the room you are in making sure there is nothing but gas inside (no liquids). Finally eject all air and close the valve again. There are two options on how to take gas samples: gas samples can be either taken from sampling gas vials or directly from the reactor. 1. Taking gas sample from a sampling vial a. Turn the second sampling vial (the one with the most gas (± 2 ml)) upside down and pierce the inner ring of the butyl stopper with the gas syringe and aim for the gas headspace. The first sampling vial with the small gas bubbles (± 0.5 ml) are for backup in case something goes wrong with the second one. b. Open the valve of the syringe and rinse the volume of the syringe a couple of times with the content of the gas headspace in the vial. Finally take the gas sample of 250 µl and wait for a few seconds for gas pressure to equalize. c. Close the valve at the top of the syringe and pull out the syringe again in a smooth movement. Place the sampling vial in the correct container again. 7
8 2. Taking direct gas samples from a reactor a. Shake the reactor so that the gas composition is representative for a certain time. b. Pierce the gas syringe all the way down through the septum of the bioreactor (it shouldn t hit the sludge level inside the reactor). c. Open the valve and rinse a few times with gas in the reactor. Finally take the gas sample of 250 µl and wait for a few seconds for pressure to equalize. d. Close the valve at the top of the syringe and take out the syringe again. e. Without opening the valve, pressurize the gas volume from 250 µl to 200 µl while carrying the syringe with the gas to the GC inlet. This makes sure no air tries to enter the syringe, as only gas could escape in case of malfunction Injecting gas sample into GC a. When the correct data saving file name has been entered in the computer and you have the sample ready in the syringe, click START RUN. b. Press the PREP RUN button on the panel of GC2. Wait until the not ready LED is no longer lit up. Carefully bring the gas syringe towards the inlet of the liner of GC2. Hold the syringe downwards near the opening. Do mind the heat of the metal ring on the outside of the liner as you can burn your fingers here. c. Now, without opening the valve of the syringe yet, pressurize the gas in the syringe by reducing the volume from 200 µl to exact 100 µl outside the GC. d. Now quickly but carefully open the valve outside the inlet and then immediately insert the syringe all the way down into the inlet of the liner of GC2 and empty it with a fluent and short movement of the piston from 100 µl to 0 µl. Immediately and simultaneously, press the START button on the GC2 s panel and retract the needle from the inlet. This series of actions should be fluent and identical in timing to the other times when performed and as short as possible. The computer should now have started logging and visualizing the chromatogram until the programmed time of 6 minutes runs out. Do not abort this action. After this time the data is no longer logged, however the GC keeps drawing a chromatogram on the PC screen until the next action is started (for example the run on another sample). e. Wait at least one minute before injecting the next sample as remains of other compounds can still be migrating through the column which is better not included in the next chromatogram as they would be part of the area when integrating. f. Retake all steps from part (Preparing the computer) through and for the amount of samples to be analyzed. 8
9 Finalizing the measurement When all sample measurements are done, check if you see dirt or condensate in the gas syringe. If so, it should be thoroughly cleaned with acetone, disassembled and stored to dry disassembled so acetone can vaporize and escape well. Do not shut down the computer after use; only close the programs you started PROCESSING DATA OF MEASUREMENTS To gain time, this action can be performed on previously acquired data while the next measurement is running, since it is done through a different program. Alternatively, this can be done after all measurements at a later time. Run the GCµTCD Enhanced Data Analysis shortcut on the desktop of the computer to start the software that will enable you to interpret the data. Select the data file (.D ) to be analyzed. Choose Chromatogram in the menu and choose Integrate. Check if the program is able to distinguish 3 peaks at the expected and known succession of retention timings: a) the sum of N 2 and H 2 around 4.23 minutes; b) CH 4 around 4.43 minutes; c) CO 2 around 4.84 minutes. They are cut off by blue lines in the graph. Possibly a fourth peak can be found in front of or at the back of the other peaks, but this can be ignored, as only the ratio of CH 4 and CO 2 is of importance. However if the size of this unknown peak is very large compared to 3 being distinguished, the integration detection of the smallest peak may fail to compute and produce an area of significance. In this case the initial threshold setting should be lowered in steps of 1 unit, until after reintegration a representative area can be computed. This setting can be found in the menu Chromatogram > Signal Integration Parameters. Once changed, choose Integrate again to recompute using the changed parameters. Restore the initial threshold setting to the original setting of 16.5 before loading the next sample data for integration. 9
10 Now choose Chromatogram again in the menu and choose Generate Percent Report. 10
11 Scroll down to the bottom and note this data (or copy it to your personal data file). Data is usually noted up to 4 significant numbers. For example % becomes %. 11
12 o o o Note retention times of the peaks for CH 4 and CO 2 (R.T min column) (this is the top of the retention peak, not the start or end of steep slopes). Time is displayed in digits, so 5 minutes and 30 seconds is written as 5.5. Note the relative percentage in the last column in this report (% of total): - 2 nd peak: CH 4 (around 4.43 minutes) - 3 rd peak: CO 2 (around 4.84 minutes) There is no need to note the peak for N 2 and H 2. In case there is a fourth peak, make sure you read or copy the 2 correct peaks using the retention timing as reference, as data from the fourth peak is irrelevant. Important remark: DO NOT try to open a sample data file with the GCµTCD Enhanced Data Analysis tool or any other program when this file is still being written to by the GCµTCD logging program that controls the GC. Doing so will result in a corrupted.d data file and the sample will have to be measured all over again! Do not shut down the computer after use, only close the programs you started. 5. CALCULATION OF RESULTS It is advised to use a spreadsheet for calculations. Use the calibration factors mentioned earlier (or determine them again yourself using calibration gas of known contents) to multiply the measured area percentage to the correct area percentage. Then calculate the relative quantity of the two peaks (CH 4 and CO 2) with the following formulas by dividing each one through the sum of the two to obtain the relative quantity in the gas sample that is only supposed to consist of these gasses. The final percentages acquired for CH 4 or CO 2 can be divided by the sum of percentages of CH 4 and CO 2 to find the relative quantity of produced gas in this biogas sample. Formulas: ([CH 4 ] area CF CH4 ) 100 [CH 4 ] sample = ([CH 4 ] area CF CH4 ) + ([CO 2 ] area CF CO2 ) [CO 2 ] sample = ([CO 2 ] area CF CO2 ) 100 ([CH 4 ] area CF CH4 ) + ([CO 2 ] area CF CO2 ) With [CH 4 ] sample : concentration of CH 4 in the biogas sample (%v) [CO 2 ] samle : concentration of CO 2 in the biogas sample (%v) 12
13 [CH 4 ] area : area peak percentage of CH 4 (%) [CO 2 ] area : area peak percentage of CO 2 (%) CF CH4 : calibration factor for CH 4 (no unit) CF CO2 : calibration factor for CO 2 (no unit) 6. QUALITY CONTROL A sample of calibration gas should be measured during each sampling run as a quality control. Biogas samples originating from anaerobic digestion batch test should contain 50-90% CH 4 and 10-50% CO ERRORS, CALIBRATION AND INTERFERENCES This GC unit always measures a difference in composition compared to the actual composition as was determined with a calibration gas (consisting of 5 % N 2, 5% H 2, 80% CH 4 and 10% CO 2). The ratio of this measurement to the actual composition was turned into a calibration factor which is applied to all samples to determine the actual composition of gas samples in analysis. Multiply the obtained area percentage with the calibration factors in order to read the correct area percentage of the samples. Table 1 shows an example of calibration factors which are to be determined calibration gas. Table 1: Example of calibration factors for methane and carbon dioxide CH 4 CO 2 Calibration factor WASTE STREAM AND PROPER DISPOSAL Dispose any captured spills or contents of vials in the correct liquid waste stream disposal barrel in the lab (acids). Clean all gas vials and remove their labeling with acetone. 13
14 9. HAZARDS AND PRECAUTIONARY STATEMENTS Mind the heat of the liner opening in which the gas sample is inserted into the GC. 10. REFERENCES APHA, Awwa, WEF, Standard Methods for the Examination of Water and Wastewater, 21st ed. American Public Health Association, Washington DC. Laurent C., Biogaspotentieel van microalgen bacteriën vlokken bij verschillende groeicondities en voorbehandelingen. Annex: Biogas sampling. Master thesis, Ghent University, Campus Kortrijk, 123p. 11. CONTRIBUTIONS SOP written by: Cedric Laurent (master thesis student EnAlgae project). SOP restructured by: Alexandra Lefoulon (internship student EnAlgae project). SOP approved by: Sofie Van Den Hende (staff EnAlgae project). 14
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