FTIR-ATR Identification of a Food Oil Using Chemometrics

Transcription

1 FTIR-ATR Identification of a Food Oil Using Chemometrics *NOTE: BRING A FLASHDRIVE TO LAB ON THE DAY YOU INTEND TO DO THE EXPERIMENT AS YOU WILL NEED TO TRANSFER THE DATA FILES FOR FURTHER PROCESSING IN EXCEL. 1. Purpose This procedure is designed to determine the identity of a food oil using Fourier-transform infrared (FTIR) spectroscopic data obtained with an attenuated total reflectance (ATR) cell. 2. Background While scientists regularly do quantitative analysis to determine the concentration(s) of some unknown compound(s) in a particular sample, it is also common to do qualitative analysis to ascertain the identity of a sample and perhaps its chemical composition and overall purity. There are two experiments in Chm 466 that are qualitative in nature, both using a food oil as the unknown. Food oils are mixtures of triglycerides that consist of three long fatty acids attached to a glycerol backbone. While there are many different fatty acids, they differ only in the number of carbons and double bonds present in the long fatty acid tails. The figure below shows a triglyceride with three common fatty acids, the top one being the saturated palmitic acid, the middle being the monounsaturated oleic acid, and the bottom being the polyunsaturated α- linolenic acid. Note that the polyunsaturated linolenic acid has three cis- double bonds and so is chemically named all-cis-9,12,15-octadecatrienoic acid. Different food oils have different percentages of the various fatty acids. In the GC-MS experiment this semester, an unknown oil is identified based upon the relative amounts of its constituent fatty acids. Doing so requires chemical derivatization of the oil prior to analysis so that the triglyceride fatty acids are converted to their corresponding fatty acid methyl esters (FAMEs) with boiling points low enough to be done by gas chromatography. The unknown oil is then identified based upon the variable percentages of individual fatty acids as measured by the GC-MS. While this method is very accurate and can provide positive identification of the different fatty acids, it is somewhat time-consuming due to the derivatization and extraction process and subsequent chromatographic analysis. A simpler, though possibly less precise technique for determining the identity of an oil relies upon infrared (IR) spectroscopy. As you learned in organic chemistry, IR wavelengths interact with the vibrational modes of a molecule s bonds, with different bonding pairs absorbing IR radiation at different wavelengths (or wavenumbers, as is traditionally used in IR). In this procedure, you will obtain FTIR spectra of neat oil samples individually analyzed on an attenuated total reflectance (ATR) crystal. Using ATR-FTIR is much simpler than traditional spectroscopic methodologies as there is no sample cell. The minimal sample preparation results in a very simple procedure.

2 Schematic of ATR-FTIR instrument The ATR-FTIR data allow you to quickly fingerprint an unknown oil and then identify it by comparison to a set of standard spectra for various oils. However, since all food oils are very similar in their composition (all consist of the same C-14 to C-22 fatty acids attached to a glycerol backbone, the only difference being the relative amounts of the various fatty acids), the spectral differences are small and often almost indiscernible at least to the naked eye. As such, the data needs to be processed using a basic form of chemometrics you will do using an Excel spreadsheet. 3. Materials and Equipment Oil standards and unknowns obtained from instructor. Plastic or Pasteur transfer pipettes, kimwipes, cotton swabs, and acetone should be available by the instrument. 4. Safety Safety glasses should be worn at all times. Food oils are for analysis only and, like all other laboratory chemicals, are not intended for human consumption. For more general safety in the laboratory, please refer the appendix. 5. Procedure Obtain the set of five oil standards from your instructor along with your unknown and proceed to the instrumental room. You will possibly need to bring along several plastic transfer pipettes and your notebook. A bottle of acetone (wash bottle with the red top) should be near the Nicolet Avatar 370 FTIR instrument located near the north double doors of the laboratory. If there is no acetone, consult your instructor.

3 Picture of Thermo-Nicollet Avatar 370 with the cell assembly hood open Confirm that the computer and instrument are both on (the instrument on light is located at the top left of the instrument). If the instrument is not on, you will eventually get a computer message: Computer cannot communicate with spectrometer, when you open the OMNIC software. The switch for the instrument is located in the back left corner. The sample well and ATR crystal must be cleaned prior to each spectral measurement and before beginning the experiment. On the front of the Avatar FTIR is a latch. Turn the latch so that it is pointing upwards and raise the dark brown Avatar cell shield (hood). Gently unscrew the black ring counterclockwise until you can remove the ring and the cell cover with the white Teflon well (hole) in the center. Picture of ATR Assembly with the ATR crystal in the center of the white well Remove the black plastic ring and set it aside. Lift the stainless steel cell cover. Holding the cell cover in a Kimwipe, clean both sides of the cell cover with 2-3 drops of acetone, wiping

4 off the excess acetone until the cell cover is dry. Place the cell cover on the bench next to the black ring. Next, you need to clean the cell itself. Clean around the outside of the crystal by gently rubbing with a Kimwipe and acetone. DO NOT TOUCH THE SMALL CENTER CRYSTAL WITH A KIMWIPE AS THAT CAN SCRATCH AND DAMAGE THE DELICATE CRYSTAL. Put 2-3 drops of acetone on the small shiny center cell and gently wipe in circular fashion with a Q-tip. Once you have wiped off the acetone, you have fully cleaned the apparatus and are ready to begin analysis of the first standard. Reassemble the apparatus by first replacing the metallic cell cover with the white Teflon well and then securing it with the plastic black ring. Gentle screw the black ring until it is fingertight. DO NOT OVERTIGHTEN. Close the brown Avatar hood assembly and tighten the front latch. You will repeat this cleaning procedure for each trial of your standard and unknown. Before starting the software to acquire the data on FT-IR, plug your USB flash drive into a USB extension cable labeled USB plug. Once you have your USB flash drive plugged into the computer, open the OMNIC software program (desktop icon with the multicolored triangle), again assuring that the spectrometer is on by noting the green light at the top lefthand corner. Wait for a few seconds until you see a message about an accessory change, simply press the OK button to continue. Expand the window to full-size. Windows desktop showing OMNIC icon Upon starting OMNIC, you may see this window. Simply click OK to continue. Go to the Collect menu, select Experiment Setup. The Experimental Setup window should open. At the bottom of this window, click on the Open command and load/open 466 Lab Food Oil Analysis.exp that has the correct instrument settings for running the oils. Click OK (bottom right of window) to accept the experimental parameters.

5 Open Experiment window. Select 466 Lab Food Oil Analysis.exp. Once you select the correct experiment file, you should see Experiment Setup shown right above. You are now ready to begin your first trial. On the icons directly above the blank spectrum, click the Col Smp (Collect Sample). OMNIC tool buttons. You will be prompted with a window that says, Enter the spectrum title where you will enter your initials, the date of the analysis, and some identifier of the sample type (e.g., AZ0904Walnut1 or DD1021Unk7121 ). You MUST NOT have any spaces in your file names. Enter the sample name here. DO NOT have space in the sample name here. As you will run your standards and unknown in triplicate, make the last letter of your title 1, 2 or 3 to denote the different trials. Be sure to record the file name(s) in your notebook. After you click on the OK button, you will possibly get a prompt stating, A background spectrum must exist to produce the specified final format. Click OK. You will subsequently see a prompt that says, Background. Please prepare to collect the background spectrum. As the ATR crystal is clean, press OK, at which point the instrument will make 16 scans of the background spectrum.

6 OMNIC will ask to perform the background scan. Click OK buttons to continue with no samples loaded in ATR. A typical background scan should look like shown above. Notice the data is still being acquired (shown as 7/16 on the lower left corner of the window here) You can monitor the progress of the background collection by the black bar icon in the lower left-hand corner. This will be the only blank background run during the entire experiment. After completion of background, the computer will prompt Sample. Please prepare to collect the sample spectrum. Once the background is done, you will be prompted for your sample. Load your sample BEFORE clicking OK button. It is now time to run the first trial of your first standard. The order in which you run the standards is unimportant, but they should be run before your unknown. Open the Avatar FTIR and use a Pasteur pipette to slowly add 1-2 drops of the oil into the well (the exact amount is

7 not important; add just enough so that a film of oil covered the shiny metal crystal but not so much that the well is overfilled) above the FTIR crystal. After adding the oil, close the instrument and run the sample by pressing OK on the computer. The computer will acquire and average 16 scans over the next seconds with the progress again shown by the black bars in the lower left corner. Once the 16 scans have been averaged, you will be prompted to Add to Window 1? Enter Yes. Once the sample spectrum is collected, OMNIC will ask if you would like to add the spectrum to the active window. Answer Yes to continue. At this point, you should have only the corrected %Transmittance showing on your screen (usually in red color) with %Transmittance plotted versus Wavenumber with negative peaks. Note that as transmittance and absorbance are inverse functions, the peaks with the lowest %T are at wavelengths where the oil exhibits maximum absorbance. You do not need to print off the spectra, but you MUST save your data. Typical spectrum of an oil sample To save the data, you need to first change the file type to CSV text, a format that is compatible with Excel. To do so, go to File menu, select Save As dialogue box (also can be done by clicking the F12 hot key). DO NOT click on Save button (nor select Save from File menu, as that

8 will NOT save in CSV format). In the Save as type dialogue box and scroll down until you find the CSV Text file type. After choosing the CSV option, click on the Set Filename to Title button and save with the CSV file type. Once again, you MUST NOT have any spaces in your file names. Check the file name before you save. If you have a space in your file name, please remove the space. This will save your data with the same name as before but with a CSV file type extension. Make sure you save each trial with the CSV extension. To save your data, you should go to your USB flash drive. You should also make sure that you are saving the data as a CSV file as shown on right. Once you have saved your data, use a Q-tip to soak up the oil in the well and then clean the crystal as described above. As before, the yellow cell crystal should only be cleaned with a Q-tip and acetone. You are then ready to run the second and third trials of your first standard. These and all successive runs will be made with the original blank. Trials should be treated identically as the first trial (save with csv file extension using a slightly different filename, etc.). As such, you will have three data files for each standard and unknown. Again, you do not need to run the blank background other than for the first trial of the first standard. After running each standard and unknown in triplicate, you can exit the program, closing all open windows. Leave the instrument and computer both on. Make sure that no oil remains on the crystal and that the station is clean; if the acetone bottle is low, inform the instructor. At this point, you should have 18 CSV files with unique name on your flash drive. If you do not have 18 files, check your work. You should also tabulate all the 18 file names in your lab notebook for a reference. Before leaving, transfer your data files to your flashdrive and proceed to the data analysis section.

9 6. Data Analysis Data analysis will be done using Excel and the computers in the balance room. There are four spectral regions that have been deemed particularly useful for determining the identity of the unknown. Those FTIR wavenumber regions are: (Region A identified as being a cis-c=c stretching mode)* (Region B identified as being an asymmetric C-H (-CH 2 -) n stretching mode)* (Region C identified as being a cis-c=c bending mode)* (Region D identified as being a trans-c=c bending mode)* *Lerma-Garcίa, M.J., et al., Food Chem 118 (2010) FTIR-ATR Spectrum of Corn Oil Region A Region D Region C %T Region B wavenumber (cm -1 ) To process your data, you will need to use one of the computers in the balance room. Begin by inserting your flash drive into a USB port on the back of the desktop tower. You should get a message on the desktop showing that the computer has recognized the flash drive. If you wish, open the My Computer icon on the upper-left corner of the desktop and then open your Removable Disk flash drive. Once the flash drive files are seen, open the Excel file FT-IR on the desktop. This program will combine all the 18 CSV files you have created into one file that can be analyzed in Excel easily. If you do not have 18 files, you should check your work. It will first ask for the files for the standard 1 trials.

10 Upon starting the FT-IR program, it will ask you a series of questions. You will be asked to select your CSV files. Notice the title of the window indicates the file it is asking for. In this example, it is asking for the Standard 1 Trial 1 data. Select the file, then click OK to continue. It will ask more files once you do so. You will have to pick all the 15 files for the standard trials as well as the unknown trials. Once you are done selecting the files, it should ask you for the confirmation. Check your notebook for the accuracy, make sure the list in your notebook matches with the list shown on the computer screen. If the dialog box does not look like shown below, ask your laboratory instructor for help.

11 Once you have selected all the 18 files, it will show you which ones you have selected. Check what you have in your lab notebook against what is being shown BEFORE you click OK. If it does not match, click Cancel and start the data analysis part over from the beginning. If the list does not look shown above (if the Trial column does not look shown above), consult your lab instructor immediately. Any time during this part, if you have made any mistakes, click Cancel button. The program will stop, then, you will have to start over again for the data analysis (starting from selecting the standard 1 trial 1 file). One you have all the 18 files selected correctly, the program will combine all the 18 CSV files into one singular file called FT-IRdata.CSV in the same directory as where you have all of your 18 CSV files. Click OK to continue. Your data should be saved in the same directory as where your 18 CSV files are saved (should be on your flash drive). After that, Microsoft Excel should automatically start. Note the worksheet tabs at the bottom of the spreadsheet entitled Graph, Trials, and Averages.

12 Notice the different worksheet listed in this spreadsheet. There should be a button called Import CSV on the Graph worksheet, on A2-A3 cells. Click that button. Click Import CSV button shown above. Once you do, it will ask for your name. Enter your name (this is NOT an optional. You have to enter your name here to continue). You will be asked to enter your name. Enter your name. This is NOT optional, and the only way to get through is to enter your name. Once you enter your name, your CSV file should get imported automatically in Trials worksheet (and the Excel should switch the active worksheet to Trials worksheet). If this does not work, consult your laboratory instructor for help. You are now ready to extract and average (necessary because the data is noisy, making discrimination between the different oils impossible with merely the peak minimum) the data for the four spectral regions of interest. Go to the Averages worksheet tab at the bottom of the FTIR Template spreadsheet. If everything has been entered correctly, the headings in the first row will match the names of your standards and unknown. Go to Cell B3 to begin averaging the data for the first region by typing: =average(trials!b4970:b5012). You should now see a single value in Cell B3 that is the average of the %T for the wavenumber region from cm -1. Enter the equation as shown above. You need to average the three other regions for your first standard trial in the same way. Cell B4 of the Averages folder will contain the average %T for the wavenumber region between cm -1. To do this, go to Cell B4 and type: =average(trials!b4804:b4846). Once entered, you should see a single value representing the average %T.

13 Subsequently, go to Cell B5 and type: =average(trials!b635:b677), which will give the average %T for the wavenumber region from cm -1. Finally, go to Cell B6 and type: =average(trials!b738:b781), which will give the average %T for the wavenumber region from cm -1 You should be entering formulas shown below in each cells: Cell B3 B4 B5 B6 Formula =average(trials!b4970:b5012) =average(trials!b4804:b4846) =average(trials!b635:b677) =average(trials!b738:b781) To complete this part of the data analysis for the other trials, you can simply copy the formulas from Cells B3-B6 to all the adjacent cells to the right (C3-C6, D3-D6, etc.). To do so, use the mouse to highlight Cells B3-B6, move the mouse pointer to the right bottom corner of the selected cells until the pointer changes from a white cross to a black cross (see the figures below). You should see the mouse pointer changes to a black cross if you move the pointer over the small black square in the right bottom corner of the selected cells. Once you see the pointer changes to the black cross, while holding the left mouse button, scroll the mouse to the right so that all the remaining cells are highlighted. Once you release the left button, all averaged values should be shown on the Averages folder. OnceB3-B6 have formula entered, you can populate the rest of the worksheet as shown above. Notice the mouse pointer changes to a black cross from white cross on the right. Click the mouse button and drag to right as shown below.

14 If you drag it correctly, all the cells should be populated with the same formula. Alternatively, select cells B3-B6, go to Edit, select Copy from the menu, then, select cells C3-S6, and go to Edit select Paste from the menu. This should populate the range of the cells with the correct formula. At this point, click the Graph worksheet tab at the bottom of the FTIR Template worksheet. You should see your averaged data for all standards and unknowns. There should also be a graph of the region ratios (A/B on the x-axis and C/D on the y-axis). If you did the experiment correctly, you should see relatively close groupings of each standard and your unknown. Print the worksheet (be sure your printout contains both the table and the graph). Based on the A/B vs. C/D graph you obtain, determine the identity of the oil unknown. Points will be based upon not only making the correct choice, but also upon thoroughly explaining your choice and also on the overall quality of your data (graphical precision and accuracy). You should probably save your work. Your data will be saved automatically on your flash drive along side of the CSV files as FTIR Template.xls.

15 Instrumental Analysis Laboratory Safety Rules A. Instructions: Carry out all manipulations in accordance with instructions and the safety rules and procedures given herein. B. Eye Protection: All students and staff working in the laboratory must wear safety glasses at all times. If a student needs to be reminded more than three times to wear goggles, she/he will be dismissed from lab for the remainder of the day, and will not be given an opportunity to make up the work. C. Apparel: The clothes you wear in lab are an important part of your safety equipment, and should offer protection from splashes/spills. Closed toed shoes (sneakers are fine), Full-length pants or a full-length skirt, and A shirt that completely covers your torso (i.e. at minimum, a t-shirt). In other words, you must NOT wear shorts to lab. You must NOT wear flip-flops, sandals, or crocs. You must NOT wear tank tops, halter tops, spaghetti-strap tops, or low cut jeans to lab. Exposed abdomens, hips, and backs are not safe in the lab. D. Gloves: Gloves are an important part of personal protection. Gloves will be available at all times in the laboratory. Your instructor will require their use when appropriate. E. Food: Food, drinks, and gum are not allowed in lab. None at all, not even water bottles. F. Sanitation Issues: Be sure to wash your hands before leaving lab, before you eat anything outside of lab, and before you answer your cell phone. G. Music: Individual headphones are not allowed. Your may choose to play music for the entire class. H. Cell Phones and Other Electronic Devices: Cellular phones and other electronic devices that you do not need to perform your laboratory work should be put away. I. Other: All students are explicitly prohibited from: 1. conducting any unauthorized experiments. 2. removing chemicals or apparatus from the laboratory for any reason. 3. working in the lab alone, or at other than regularly scheduled lab periods. 4. smoking in the laboratory or within 20 feet of any doorway. 5. impeding movement in aisles or through doorways with bags, skateboards, etc.