Soil and Water Conservation Laboratory
Standard Operating Procedure
Sherman 230, Soil and Water Conservation Laboratory, UH Mānoa
Mid Infra Red (MIR) Diffuse Reflectance Spectroscopy (DRS) of Ball Milled Soil Samples with the Varian 2000 FT-IR
Purpose:
To scan soil samples in the mid infrared wavelengths to create spectral libraries and for chemometric analyses.
Soil Sample Preparation
1. Soil samples need to be oven dried for at least 24 hours at 105oC and then passed through a 2 mm sieve to remove rocks and coarse organic debris.
2. The dried and sieved soil samples then need to be ball milled to a fine, homogeneous powder. See SOP on Ball Milling of Soil and Plant Tissue Samples for more details on this process.
Reference Material Preparation
1. The reference spectra for the MIR is Potassium Bromide (KBr). Unlike the VNIR which uses Spectralon, the MIR uses KBr. The KBr needs to be oven dried for 24 hours at 105oC and then ball milled to a fine, homogeneous powder. KBr is a respiratory, skin, and eye irritant so always use gloves while dealing with this chemical.
2. KBr is stored in the desiccator cabinet next to the new drying oven.
Sample Holder Preparation
1. In the drawer below the FTIR labeled FTIR Parts, there are 2 sample holders. These are thin black metal bars, with 4 removable sampling cups. The sampling cups are considerably smaller than those for the VNIR so care must be taken to prepare a homogeneous sample. Get the sample holders set them up on the lab bench.
2. In position 1, carefully add the dried and ball milled KBr. Fill the cups completely and then gently smooth the surface with a spatula. In positions 2-4 carefully add soil samples to be scanned and smooth the surface with a spatula. Try not to tap the cups on the bench surface as this can lead to particle sorting and artifacts in the scans. The second sample holder can also be filled with additional soil samples. Keep track of which samples are added to which cup holder position. Samples should ALWAYS be scanned in a random order, even for a small set of samples.
Instrument Operation and Scanning
1. The Instrument itself is left running all the time to avoid problems associated with heating and cooling.
2. Open the Varian Resolutions Pro software package.
Start→All Programs→Varian Resolutions→Varian Resolutions Pro
3. Open a file where you will save the new spectra.
File→Open (Use the Soil Test Samples file to start)
Or
File → New → Multi-Spectral Document→ OK
4. Move sample cup 1 (with the KBr) into position. You may hear a click when it is position. If not, look down from the top of the chamber to see if cup is in position.
5. Collect a background scan with KBr
-Hit ‘Collect Background’ button
-Observe background spectrum
-Repeat and compare this scan to previous background scans.
-If this looks good (i.e. comparable to previous scans), then proceed to step 6.
6. Collect a sample scan with the KBr.
-Hit ‘Collect Sample’ button
-Wait- it averages 32 scans to get final scan
-Check the spectrum- The spectrum should read about 100% transmittance/reflectance across all the wavelengths and will be slightly noisy.
*If there is a lot of noise, or there are major dips in reflectance, it probably means your KBr has taken on moisture, and it needs to be redried, re ball milled, and repacked. Do not proceed with scanning soil samples until you have a good KBr white references. Remember that there will be more noise with the KBr white reference on the MIR than there will be with the Spectralon on the AgriSpec.
**Even though this plot says % Transmittance on the Y-axis, % relfectance is actually what you are measuring- they’re not the same, it’s just an artifact of the program.
-Change sample name to your naming convention in the spreadsheet.
-Save
7. Move sample cup 2 into position and collect a sample scan.
-Hit ‘Collect Sample’ button
-Wait- it averages 32 scans to get final scan
-Change sample name to your naming convention in the spreadsheet.
-Save
8. Repeat this process with the rest of the samples on the rack, then insert second rack and repeat for those samples.
9. Repeat KBr background after every two racks measured,if you are measuring more samples.
- prepare and load samples in the rack with KBr and repeat the background collection and scanning of KBr as you did before in steps 5 & 6.
10. When done measuring, make sure file is saved.
11. To export spectra:
File → Export Spectrum
-pick what/where you want to export and save
-for now each spectrum must be exported separately
12. Exit program
13. Put KBr back in dessicator
14. Make sure all sample cups and racks are clean, along with the counters.
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