NMR Questions

1. How should I prepare my NMR sample?
2. My NMR tube is loose in the spinner. Is this OK?
3. What should I do if an NMR tube breaks?
4. The NMR won't lock onto my deuterated solvent. How do I fix this?
5. Topshim is taking a long time or not working. How do I fix this?
6. I'm having issues with probe tuning. What should I do?
7. I'm having another hardware problem not listed here. What can I do?
8. How do I get my data from the NMR computers?
9. What are the current user fees?

MALDI Questions

1. Which matrix should I use?
2. How are the stock matrix solutions prepared?
3. My spots are taking a long time to dry. How do I fix this?
4. Should I use the "pep CCA" or "prot SA" calibrant spot?
5. How do I make a PDF of my spectrum?
6. How do I get the PDF spectrum off the MALDI computer?
7. What are the current user fees?

NMR Answers

1. How should I prepare my NMR sample?

   Dissolve your sample in at least 0.6 mL of a deuterated solvent. If your sample does not dissolve in the solvent, you will not see an NMR signal from it. Next, transfer the sample to a clean 5 mm NMR tube (you cannot use an EPR tube). Be sure to cap the tube. ▲
   

2. My NMR tube is loose in the spinner. Is this OK?

   No! If your tube slips in the spinner, even by a few millimeters, it will hit the thermocouple at the bottom of the probe and possibly shatter. ▲

3. What should I do if an NMR tube breaks?

   Outside the magnet: please use the broom and dustpan to sweep up any broken glass and chemicals. Leave the glass in the dustpan and notify the facility manager.

   Inside the magnet: if you were spinning the sample, stop the spinning to avoid further damage. Do not try to eject the tube or do anything else - that runs the risk of causing (more) damage. Leave a note on the spectrometer saying "BROKEN TUBE INSIDE PROBE, DO NOT USE THE NMR". Notify the facility manager. ▲

4. The NMR won't lock onto my deuterated solvent. How do I fix this?

   If you did not do so already, load a standard shimset (rsh), then try locking once again. Be sure to select the correct solvent from the list!
   If your sample is cloudy, or has a volume less than 0.6 mL, you may have no choice but to correct those issues prior to being able to lock. ▲

5. Topshim is taking a long time or not working. How do I fix this?

   Topshim often takes a long time with CDCl₃ - this is normal. If Topshim fails, the usual cause is a sample volume of less than 0.6 mL. Add more solvent! ▲

6. I'm having issues with probe tuning. What should I do?

   If you have not been trained, or feel uncomfortable, do not tune the probe. Broken tuning rods cost thousands to repair.

   If wobb shows a flat noisy line at the bottom instead of a tuning curve, type stop to stop tuning, then ii to re-initialize the spectrometer hardware. This command can take a minute and may produce some error messages. If it does, run ii again until no errors pop up.

   If wobb shows a featureless (but not noisy) line near the top of the display, try changing the tuning width (wbsw) to 60 and re-tuning. This is often necessary when tuning to different nuclei such as ¹⁹F or ³¹P. ▲

7. I'm having another hardware problem not listed here. What can I do?

   First, call 9-3163 and leave a message. This 'pages' the instrumentation specialist, and you will hopefully get a response in five minutes.

   If the above does not work, you can try to re-initialize the spectrometer hardware by typing ii in the Topspin commandline. This command can take a minute and may produce some error messages. If it does, run ii a second (or third) time, until no errors pop up. If, after the third try, ii still produces errors, please eject your sample, leave a note on the spectrometer, and send an e-mail describing what happened. ▲

8. How do I get my data from the NMR computers?

   The data is available on the facility's SFTP file server (the server name, username, and password can be found in any of the NMR labs). Please note, this is different from a regular FTP file server, and you will need to use an SFTP-capable client to download your data. One such client is Cyberduck, a free program available for Windows, and Mac. Here's how you use it:

   1. Click the Open Connection button in the top left.
   2. From the dropdown at the top, select SFTP (SSH File Transfer Protocol)
   3. Enter the Server, Username, and Password, then click Connect.
   4. If an "Unknown Fingerprint" dialog appears, select Always then click Allow.
   5. Open the folder for the NMR you want data from (e.g. nmr_200, nmr_300, nmr_500), find your datasets, and copy the desired folders (you can drag and drop onto your desktop).
   6. The transfer will appear in a new window. You can quit Cyberduck when the transfer has finished.▲
   
   
9. What are the current user fees?

   For internal users, the current rates are:
   DPX200: $3.00 / hour
   DPX300: $4.00 / hour (M-F 8a-8p), $2.50 / hour (evenings/weekends)
   AV500: $5.00 / hour (M-F 8a-8p), $2.50 / hour (evenings/weekends)

   Users are billed for the time that they are logged into the spectrometer. There is a no-charge data-processing workstation located in HUG065.

   Non-Miami users are asked to please contact the instrumentation specialist for rate information. ▲

   MALDI Answers

   
   

1. Which matrix should I use?

   Of course, this question has no one correct answer! Some starting guidelines:
   For peptides and small proteins, try HCCA.
   For larger proteins (> 8 kDa), try SA.
   For other molecules, try HCCA or DHB if the literature has no suggestions. ▲

2. How are the stock matrix solutions prepared?▲

   Our stock HCCA and SA solutions are made by dissolving 10 mg of matrix in 0.25 mL of acetonitrile, then adding 0.25 mL of 0.1 % TFA in milliQ water. Stock DHB is made by dissolving > 50 mg of matrix in the same 0.25 mL ACN and TFA solutions. For both SA and DHB, this often results in a less-than-saturated solution - not optimal, but still usable.

3. My spots are taking a long time to dry. How do I fix this?

   Long drying times can lead to poor crystal formation. Try increasing the acetonitrile (or other volatile solvent) content of your sample. Also, try increasing proportion of matrix solution used, as it tends to dry more quickly ▲

4. Should I use the "pep CCA" or "prot SA" calibrant spot?

   This depends on the method you select in FlexControl. For any of the "pepmix" methods, use the "pep CCA" spot; for any "protmix" method, use the "prot SA" spot. When calibrating, it does not matter which matrix you used with your sample. ▲

5. How do I make a PDF of my spectrum?

   You will need to save your spectrum in FlexControl, then open the spectrum in FlexAnalysis. When you are ready to print, choose the following options: Printer: "Adobe PDF" printer, Layout: "Spectrum(landscape)+MASSLIST_IRP". Click Print, and the PDF will appear on the desktop. ▲

6. How do I get the PDF spectrum off the MALDI computer?

   Move the PDF into the "SFTP Shared" folder on the desktop to make it avaiable on the facility's SFTP server (the server name, username, and password can be found in the MALDI lab). Please note, this is different from a regular FTP file server, and you will need to use an SFTP-capable client to download your PDF onto your computer. One such client is Cyberduck, a free program available for Windows, and Mac. Here's how you use it:

   1. Click the Open Connection button in the top left.
   2. From the dropdown at the top, select SFTP (SSH File Transfer Protocol)
   3. Enter the Server, Username, and Password, then click Connect.
   4. If an "Unknown Fingerprint" dialog appears, select Always then click Allow.
   5. Open the folder for the NMR you want data from (e.g. nmr_200, nmr_300, nmr_500), find your datasets, and copy the desired folders (you can drag and drop onto your desktop).
   6. The transfer will appear in a new window. You can quit Cyberduck when the transfer has finished.▲
   
   
7. What are the current user fees?

   For internal users, the current rates are $6 per spot for user-run samples and $12 per spot for staff-run samples.

   Non-Miami users are asked to please contact the instrumentation specialist for rate information. ▲