ࡱ> q`  qbjbjqPqP .::fnnnn1111 2L5Jb2x2x2x2@2333IIIIIII$%LhNIn@46335$$5Inn22I88885n2n2I84I88H|nnLI2V2 e1(76IIJ05JI67O^8(7O$LILI7O\Z`IT303"834333II83335J4444j#j#nnnnnn ICP Protocol-written by Dusty Wood, December 2008. Updated by Corrie Blodgett, February 2, 2009 Updated by Corrie February 12, 2009 Updated by Corrie February 20, 2009 Updated by Corrie March 25, 2009 This is ICP protocol for Ruths students and technicians. It is most commonly used for analyzing diluted digestions of leaf, wood, bark, and root tissue. Much of this is also relevant to soil extractions or solutions (e.g. for nutrient uptake experiments). Helpful Contact Information ICP Lab - 470-6882 Deb - 470-4744, debdris@esf.edu Marlene - 470-6869, mabraun@esf.edu Preparation Scheduling an ICP Date: Generally ICP appointments can be made Deb 2 weeks in advance of the desired time. When making an appointment, they will want to know how many samples you have, the type of tissues, the digestion method (acid matrix, i.e. 1.2 N HNO3), and the expected concentrations. The first three pieces of information are probably known already, but you may have to dig some information up on what concentration ranges to expect in your tissues. Once you have your own ICP standards and dont need much help from Deb you can also schedule the ICP online at  HYPERLINK "http://faces.ccrc.uga.edu/" \t "_blank" http://faces.ccrc.uga.edu using the following information: Group: ESFICP Username: foresteco Password: forestecology Know Your Samples You need to know what concentrations to expect in your samples so that Deb or Marlene can make an ICP calibration standard, which is a solution with known concentrations of elements to use as a baseline. Deb and Marlene will expect these concentrations in mg/L, the units that the ICP works with. In order to give her the concentration in mg/L, you need to know the dilution of your samples and their weight before digestion. The formula looks like this: Solution concentration for ICP(mg/L)= Expected tissue concentration (mg/g)*sample weight (g) Dilution (L) Give safe estimates of your expected concentrations to Deb or Marlene, because the ICP cannot accurately measure concentrations higher than your calibration standard. This applies to your tissue samples AND your digested reference material standards (likely apple or citrus leaves). We have run into problems before when we gave Marlene expected concentrations based on only our tissues and not our reference material standards (which turned out to be higher than the ICP calibration standards she made). The calibration standards should closely bracket the sample concentrations. It is best to have at least a 3-point calibration standard (using one blank and at least 2 standards). Joel Blum recommended to us to use 4-5 up to as many as 8 standards to calibrate the ICP. This reduces the uncertainty in the calibration and thus the potential for error to propagate through the analysis. I have used Whittakers paper on Chemical Analysis of plant tissues at Hubbard Brook. The tables show the chemical composition of leaves, twigs, branches, bark, light wood, dark wood, and roots for six common northern hardwood species. He gives a mean concentration (plus or minus the standard error) for several important elements. To be safe, I have added the standard error to the means and used the values as my expected concentrations, rounding off to an even number (like 100 mg/L calcium). Here are some safe estimates for northern hardwood foliage, branches, wood, and roots (and apple leaves), based on A DIGESTION OF 1 GRAM SAMPLES: Ca: 300 mg/L K; 500 mg/L Mg: 100 mg/L P: 50 mg/L Mn: 30 mg/L Na: 10 mg/L Sr: 1 mg/L If the concentrations of the samples will be much less than the tissue standards... need to dilute the standards, or we won't have good sensitivity on the samples. We had wood samples so small the P couldn't be detected on ICP-OES (and Deb ran them on ICP-MS). Create a Sample Information File You need to create a sample information file (SIF). This is a list of the sample IDs that you are running. Note* it is best to label your procedural standards and blanks as such (Digestion Blank, Apple Standard etc.), so that they arent mistaken for the ICP blanks and standards (which is not easy to do, but still lessens the chance of confusion). To have the ICP automatically calculate the concentration of your sample your SIF file should contain the following information: Column A: A/S location (autsampler location) if using the small ICP test tube tray, this will start at location # after your blank, standards, and QC Column B: Sample ID a unique ID for each sample If you want the ICP to calculate your sample calculation automatically you need to have the following information in your SIF file: Column C: Initial Sample weight (for leaf tissue digestions our values are around 1 g but there is one specific weight for each sample) (Column D: Sample prep volume Column E: Aliquot volume - Im not sure which of these two we need, Im going to have to test it out on the ICP, by reprocessing some old data and seeing if I get the same results, Deb wasnt sure because she doesnt use it, but I believe it would be the 10 mL of nitric we use for the digesting) Column F: Diluted to volume (this will likely be the same for all of your samples if they are run from the same digestions, for example, for our leaf tissue digestions it is 50 mL). Starting the ICP We use the finer of the two nebulizers. The tubing needs to be set on the nebulizer and clamped down. The tubing for the autosampler needs to be set (this is located on the side of the auto sampler tray). The ICP plasma has to be ignited, and the axial and radial viewing positions need to be aligned. This can be done by going to the Tools menu -> Spectrometer Control Here you can align the axial and radial views. Since we use both in our methods you need to do both Click on Axial and place the dip stick into 1 ppm Mn Click on Align View A spectrum display with pop up Click on the results window Click on Radial and place dip stick into 10 ppm Mn Click Align View Note your results The views align themselves for optimal measurement intensity, in intensity units. These units should be in the 10,000-100,000s. What you are looking for is Axial to give you a reading in the millions and radial to give you a reading in the tens to hundreds of thousands (ie. a good reading would be 2.4 million for axial, and 750 k for radial). The axial is more sensitive, and therefore will give much higher intensity readings, where the radial allows us to get readings of higher intensity samples without saturating. It is good to let the ICP warm up. It is good practice to run a couple blanks and see if they come out low. You can calibrate and run another blank to see if there are any high readings. Go to options: and turn autosampler on. Set your Results Data Set Name. If you do not create a file to for the Auto-Sampler to save to, you will not save any of your data. On this screen you can also check the box to print log during analysis. This is very useful because you should make notes right on the log during the analysis. *note: there is a difference between Sample Number, and Sample Location, when using the Auto-Sampler. Your sample number is the number the ICP assigns your sample where the location is the number that is assigned your sample on the auto sampler tray. It is best to create a page that is a summary of the information for your run. This page should include the following: 1) name; 2) date of analysis; 3) samples being run; 4) sample matrix; 5) QC concentrations; 6) number of standards and their concentrations; 7) ICP method being used; 8) location of your results file and file name; 9) if you used a linear control standard. This page should be stapled to the front of the log that is printed out during the analysis. I also include notes as the run goes along to help with sorting out the data afterwards. You can run your samples out of small test tubes, or larger centrifuge tubes. There are two trays that can work with the auto-sampler. You should only need around 5 mL of sample for analysis, but its safe to have more incase a sample needs to be rerun multiple times. Before the analysis begins, you should check that the auto-sampler is set up to send the dipstick to the right sample location according to your SIF file. You can do this by going to () menu Go to sample location Enter a random sample location number Watch the dip stick to make sure it goes to the correct number Send the dip stick back to wash (under the same menu) so it doesnt continually suck up that sample If you are running your samples out of centrifuge tubes make sure that you go to Tools -> autosampler -> and browse for the tray name that has 67 in it. You can also turn on the auto sampler pump in this dialogue box. Set it to -> Pump always on. *Note when creating your SIF, which influences where you place your samples in the auto-sampler rack, all samples should be randomized. In the event that you need to drift correct after the analysis this diminishes your chance of creating a false effect of one of your factors (such as site or soil depth) because a set of your samples was drift corrected (i.e. all samples from one site were drift corrected and another was not, increasing the potential to create a site effect). The Method Choose your method by clicking on the Method button on the top menu in WinLab. This stores the information on the elements being analyzed, the matrix, the calibration standard concentrations, the limits for acceptance for your QCs and blanks (for when using the auto-sampler), the viewing positions, gas flow, etc. Most of this information is supplied by Deb or Marlene. The method is saved on the computer for you to use in the future, provided you use the same sample prep procedures. For example, now we are using the method DustyHNO3HCl for leaf tissue digestions. The method also includes the routine for the run, meaning you designate how often you want to run QCs, how often to retry QCs if they fail, when to recalibrate and when to run blanks. This information is saved in the method as your QC, blank, and standard concentrations and limits. In the method editor: Start with a calibration (4-5 standards), follow with a QC, run periodic QCs and blanks, and end with a QC. EPA protocol suggestions running periodic QCs always followed by a blank. We run a QC every 10 samples followed by a blank check (just matrix - same blank as what is used in recalibration). I like to have the auto sampler set up to stop after a QC fails so I can check the numbers. Then you can decide if you want to retry the QC once (if it has barely failed), or you can recalibrate. If it still fails, then you have to problem solve. However, you can have the auto-sampler set up to automatically retry and recalibrate etc. This is all saved in the method, including your acceptable ranges for your QC (we are trying to have this set to 5 %). Also saved in your method are the acceptable ranges for your blanks. You can set the blank to range from -.4 to .4 if you have a problem you can stop, but this saves you from failing every time, however if it is failing largely you should note this and try rerunning a blank or check out what might be going on. You need to know which ICP lines (wavelengths) you want to use for your analysis. For instance, Ca reads at 315.887 and at 317.933. We usually use the 315 line, because this usually reads at a higher intensity (discussed below). Here are element lines we use: Ca- 315.887 K-766.490 Mg-285.213 P-214.914 Mn-257.610 Na-589.592 Sr-407.771 The lines we have chosen to use usually have the highest intensity. However, if your sample concentrations diverge between wavelengths (this is not a very common) you can check the spectrum to see if there is any interference. You can also check other lines to see if they agree with either of the lines, and then choose to use the line that appears to be more consistent with the next highest intensity line. This is not always possible, when there is only one line available, such as with K. It is best to keep all of the lines used in the analysis (we generally do 2 per element when available) to look at in the results afterward. If Ca 315 is better than 317 in the QCs keep the data from Ca 315. At times there can be interference and you can check for this on the ICP then eliminate or add a wavelength to the anlaysis if you need to (if you are going to eliminate one, you may decide not to do so during the analysis, just dont use the data after). Before beginning analysis you need to set up a file path to retrieve the results from your ICP run. This can be done in the auto-sampler tab. There should be a SIF file and a Results file designated. The SIF file and the Results Log are two separate things, the SIF is your input, and your results log is where your output gets stored. You can update the SIF file (and rebuild list in the Auto-Sampler) and this will append to the Results Log. Your results log does not change with changes to your SIF file unless you rebuild your list. If you reprocess your data, also save this to a new results file, so it does not overwrite your old data (unless you want it to). You can create a new SIF file and copy and paste your information from the excel sheet you previously created into the auto-sampler tab. Go to Save As, SIF file, its a good idea to save it the same as your results file (your name and date of analysis). Now you can click build list. This combines your SIF file, with your method. You should see your list to be analyzed in the auto-sampler tab. It should start with some blanks, a calibration, QC, blank, then 10 samples, QC, blank, 10 more samples etc. This is exactly as the machine will run it if you dont change anything, therefore if you dont see your QCs and calibrations in your list, you need to rebuild your list until it is exactly as you want it to run. Before you begin running samples make sure that there is tubing into DI water, and that the waste bottle is empty. Make sure the tubing is all attached properly, and also check with Deb that the fine nebulizer is in (this matters in the method). The Run Once you begin and let to machine warm up (15 min or so), check the bubbles for consistency in size, shape, and frequency. You can click calibrate, run all, or run samples in your auto-sampler. To start off its good to just calibrate, check your linearity and your wavelengths (You can program in a pause, but Id rather just calibrate, look at results, then begin the analysis). This assures you the machine is warmed up as well. If everything comes out linear, you can check your QC, and then click on run samples. There will be a little green circle on whichever button you click. If you reclick this button, it will give you a menu and the option to stop immediately. Choose accordingly to stop. If you want to restart in the same place, a previous or new place, click on the same button again, and it will prompt you again to begin. Shouldn't it be the same as the matrix for the calibration standard? If you do more than a 2-point calibration, you won't get a straight line! Mary Margaret Koppers (from SU) recommended that the calibration blank should always be matrix matched. There are three (probably more) ways to diagnose that the machine has given you the correct concentration for your samples: 1- Concentration. You would be able to ball park this, because you researched these when giving Deb or Marlene the information to make your ICP calibration standards. You should know it exactly for your tissue standard, or better yet, a previously confirmed tissue standard (some don't make it through the digestion process perfectly). 2- Mean Corrected Intensity- It is probably a good reading when these are in the hundred-thousands. Some elements have higher intensities than others. For example, Ca seems to have intensities of 500,000 or more, while Na rarely exceeds 50,000. Be alarmed when major elements like Ca and K dip below 100,000 and minor elements like Na dip below 5,000. 3-Relative Standard Deviation (RSD)- Similar to describing variability in data, like a coefficient of variation, this is a measure of precision of the reading. RSDs have different ranges for ICP calibration blanks, calibration standards, and actual samples. The RSD for a calibration blank is usually high (up to 20%). The RSD for a calibration standard should not be above 2%. Similarly, the RSD for a Quality Control (QC; DISCUSSED BELOW) should not be above 2%. This sounds like we're doing a 2-point calibration. We're doing more now, right? If some of the sample concentrations also turn out to be much lower than the highest standard in the calibration curve, a note should be made on the sample. These can then be rerun at the end of the analysis using a calibration curve without the higher standards to more closely target the range of the samples you are analyzing. This is good to keep in mind if you have a wide spectrum of concentrations in your samples, because if you run the calibration curve to a very high concentration it could bias the results of very low concentration samples. For the calibration curve, you should set up the Auto-Sampler to automatically subtract out your ICP blank to account for the matrix. This will be set up by: Line through 0. This then adjusts your standards and your results. You can also have the auto-sampler set up to subtract your procedural blanks from your results, however this may not always be a good idea unless you know there is consistent contamination in all of the samples. A number of blanks can be run at the beginning of a sequence. This can be used to determine the detection limit of the ICP. After we have done this successfully, Mary Margaret believes that if you run your blank, calibration curve and QC, if this passes, this is good enough. If it doesnt pass then recalibrating would serve the same purpose as running 5 blanks at the beginning of a run. There are many different ways to define the detection limit of the ICP. One way is to run 4-5 blanks. What is 6 standard deviations away from the blank is considered to be the detection limit. 3 stddevs away is considered detectable but unquantifiable. This an IDL as defined by the ASA (need to look up to confirm and check # of blanks to run). After running the standard, run a QC, which is a quality control to help ensure the accuracy of your readings. The QC is a solution of known concentration made up by Marlene or Deb. The concentration is the most important diagnostic for a QC.The concentration of the QC should be within 5 % of the expected concentration. The is an industry standard is 10 %. If just one of your elements fails, you may deem this QC inaccurateHowever, often you may have to use your best judgment. For example, if you are monitoring an element but not using it in your results, you may not care if it does not pass in the QC. We often have very low Na values, but monitor it otherwise (for potential contamination etc.), therefore if it doesnt pass in every QC we may not always re-run those samples. Wouldn't you stop if you were even close to 10%? Because if you drift past 10% by the next QC, you won't have confidence in the samples run between them. Ruth Im not sure if you would stop if you were close to the limit (which will hopefully now be 5 %) because if the other end of the QC bracket fails, you dont have any confidence in those, however if it doesnt fail, then both the QCs pass and those samples should be fine. If you are seeing clear and consistent drifting then maybe, but I havent seen anything like that yet If you diagnose a problem with the calibration blank, calibration standard, or QC, you need to RE-CALIBRATE (start the process over again) until everything looks good. Whether you are using the auto-sampler or running the ICP manually, you need to run a QC at least every 10 samples, to assure that the ICP is giving you accurate results. If your QC fails (and your RSD and intensities are poor), you need to RE-RUN your last bracket of samples, because you cannot be sure that the ICP was giving accurate readings before this. This can be a frustrating headache, because RE-CALIBRATION is needed. I would run them in reverse order, and stop when you get confirmation of the first analysis (in the case of drift). As discussed earlier, you can set up the Auto-Sampler to retry the QC once after failing. If it fails a second time, you can set up the Auto-Sampler to stop and recalibrate. Use your judgment, if it failed the first time and passed the second time, you may or may not feel comfortable accepting the previous bracket of samples, but should consider recalibrating anyway. You should run blanks intermittently between samples. This will give us an indication of any carry-over between samples. I am also in favor of running at least one of what I call an ICP duplicate. This is not a duplicate created in the digestion process, but simply running the same sample twice in different brackets of samples. This is something that doesnt hurt to do for now, while we are figuring out procedures, but once we start getting consistent results shouldnt need to do. We should only do this however, if there is enough of the extractant. To me, it just lets us know how similar results are we getting between different sets of QCs - one more piece of information to help us get consistent results. Mary Margaret has suggested subtracting from the results of anything significant in the procedural blanks (blanks that have gone through the digestion process) from our results. What this means is that if the sample concentration is greater than 10x the blank you shouldnt subtract anything. If the sample concentration is 5-10x the concentration of the blank, then you should subtract out the blank from the sample concentration. If your sample concentration is less than 10x the concentration of the blank, then you have some kind of a problem and should figure that out. (*note: She said that she doesnt think you need to do this for all or none of the elements, but can pick and choose specific ones as long as it is designated and noted, but this is something we should discuss). End your run with a QC, for the same reason mentioned above (without it, we can't be confident of any results after the last QC). You should set the Auto-Sampler to run a QC at the end of your run in the same fashion as the rest of your QCs. Other notes: Never allow the probe to suck air. Always keep the probe in a solution, but remember, it will continue to deplete your solutions if you leave it in them. So, between samples, stick the probe back in your blank. Detection limits: Some elements may have true concentrations below the detection limit of the ICP. Simply put, the ICP may not be able to see all of whats there. This isnt common for most elements. We should add instructions for determining the detection limit. Run repeated blanks? The more standards you use in your calibration curve the more confidence we can have in our calibration. Use your judgement to how many you think are necessary. To be conservative we are running a 6 point calibration curve for leaf tissue digestions (one blank and 5 standards). Your QC should have concentrations that are about in the middle of your calibration curve, however it should not have the same concentrations as one of your standards. If you run into problems with a QC and arent sure if its the QC or something else, you can always use one of your standards as a QC to test for accuracy. Keep the ICP calibration standard(s) and QC with you-Deb and Marlene lose (or dump) their standards. It may also benefit you to bring a set of procedural standards that you already know are good standards. If you run these it may help you with problem solving later since you already have previous results and can compare. It may increase confusion, but hey you never know what might help. Finishing your run Collect your printed results from the printer. Print your ICP log and attach these sheets together. Before you exit WinLab, go to the Data Manager to retrieve your results. Select locate result name. Then go to task and export data. There are certain formats for exporting your data. I have saved a method as Corrie. This exports your results file to a thumb drive. Check your thumb drive to make sure that the file has exported there and there is data in the file. If no one will be using the ICP after you follow these instructions to shut it down: Flush ICP for 5 minutes in H20 or HNO3 with the plasma and pump both on Turn off the plasma Pump should automatically go off If the pump does not shut off, turn it off through the menu option pump always off Release the tension on the tubes that go around the pump, and also the pump for the auto sampler (one is horizontal, one is vertical, they are close to one another). Close the WinLab program Select Yes when prompted if you are sure you want to close Choose to save or not save any changes in your SIF file Open the door on the ICP to the nebulizer and check for any leaks If there is any liquid in there soak it up with a paper towel Gather samples, standards, results and USB as anything left is easily thrown out. Back in the lab: Check through your results, looking at the diagnostics, to identify any problems or concerns. Sometimes the ICP will drift, which involves a whole headache-invoking procedure. Explain. You now have a spreadsheet of your ICP results, and now you need to determine what the concentrations of the actual plant tissues are. To do this, use this formula: Tissue concentration (mg/g) = ICP solution concentration (mg/L) * solution volume (L) Sample weight (g) To convert from mg/g to %, divide by 10. Dusty or Deb said that there's a way to provide the sample mass and dilution to the ICP computer, so the results come out in the units of tissue concentration and not just the solution concentration. PAGE \# "'Page: '#' '" Likens and Bormann (1970)? We have some of the data in excel and I have a pdf of the paper, which is a pin to get. Some of the data are also in a Whittaker paper (of a different name). PAGE \# "'Page: '#' '" Is this list relevant? If apple leaves are the highest, then the rest aren't relevant. PAGE \# "'Page: '#' '" I think he means accurate. PAGE \# "'Page: '#' '"  When do you accept that the first attempt and the second attempt of the sample are close enough so that the others do not have to be rerun? Within 5 %? PAGE \# "'Page: '#' '" Why not? This is bizarre. It introduces a bias between low and high concentration samples. 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