The picoSpin-45 NMR spectrometer is a complete 45 MHz pulsed Fourier-transform liquid-phase proton NMR spectrometer in a shoebox-sized package. It includes a capillary cartridge, a permanent magnet with temperature controller and shim system, radio-frequency transmitter and receiver, digital data acquisition and signal processing, a programmable pulse sequencer, and a web browser user interface. The picoSpin software system is hosted on the spectrometer and is ready to be used once the spectrometer is turned on.
In most respects, the picoSpin-45 hardware components have functions similar to those of a conventional superconducting-magnet NMR spectrometer. Apart from drastically lower cost and smaller size, the main differences are that the magnet is permanent rather than superconducting and the sample is delivered by flow through a capillary rather than by inserting a glass NMR tube. Operation is greatly simplified because there is no need to handle cryogens or other utilities, tuning and shimming are not required when samples are changed and there is no software installation required to set up the system. Users can produce high-resolution spectra with a new unit a few hours after the shipping package is opened. In normal operation, samples can be injected into the system and spectroscopic data can be obtained in less than 10 minutes.
This Quick Start assumes that the user is setting up a newly-received instrument. It will also be helpful to a user who is becoming familiar with a unit that has already been set up for operation. In this case, it will be possible to skip many of the steps. If the unit is already set up to communicate with a web browser, temperature stabilized, shimmed adequately and the Larmor frequency is known, skip to Injecting a Sample and then read Your First Spectrum.
Inside the shipping box you will find the following items:
The cartridge sub-panel, mounted on the left side of the front panel with four thumb screws, is a part of the replaceable capillary cartridge. (See Figure 1.) Do not remove the cartridge at this time. When a cartridge is replaced the unit has to be shimmed. It is better to gain some experience with the unit and with shimming before replacing a cartridge. The unit is delivered with the inlet and outlet fittings covered with protective tape. You will notice that the inlet fitting has a double nut. A stainless steel frit filter is held in place by the smaller, outer nut.
The LCD display on the upper right of the front panel is used to monitor the status of the instrument.
On the rear panel (Figure 2) you will see an Ethernet connector, an auxiliary output connector, a power input connector and an on/off switch. The auxiliary output is intended for controlling external hardware such as valves.
The test report includes the spectrometer and cartridge serial numbers, the shipped software version, the shim settings that were used for factory tests, a screen shot of the free-induction-decay (FID) of water, and a screen shot showing a measurement of the signal-to-noise ratio.
The first step is to establish communication with the spectrometer using a computer and a standard web browser. We recommend Mozilla’s free Firefox web browser for best compatibility with our software. (See the Appendix if you must use Microsoft Internet Explorer.) Any type of computer with an Ethernet port can be used. There are two options for making the initial connection: through a LAN (local area network) with the IP address of the spectrometer assigned by the LAN, or by a direct connection between the spectrometer and your computer without using any network. Connection through a LAN is more convenient in most cases. However, if you do not have a LAN, do not have access to an open Ethernet port on your LAN, or if your LAN cannot assign IP addresses using DHCP (dynamic host configuration protocol), then you should use a direct connection.
Connect the power cord to the power module and connect the multi-pin connector from the power module to the power input connector on the rear panel. The multi-pin connector has a threaded collar that should be tightened by hand to secure the connector to the rear panel. Check that the rear panel power switch is in the off (down) position and plug the power cord into an AC power source.
Connecting through a LAN
Connect the unit to an available Ethernet port on your LAN using the Ethernet cable provided, or a longer cable if necessary. Switch on the unit while watching the front panel LCD display. After about one minute an IP address should appear on the front panel. This address has been assigned by your LAN using DHCP. (See below if the address displayed is 192.168.42.31.) Point a web browser at the address and you will see the welcome screen shown in Figure 3. (For example, if the address displayed is 192.168.2.12, type http://192.168.2.12 into the address field of the browser.)
Automatic assignment of the IP address by the LAN will only be successful if the unit is switched on after it has been connected to an active Ethernet port on your LAN, and then only if your LAN has DHCP capability. If the unit is not assigned an IP address by DHCP it will display a factory default IP address of 192.168.42.31. In most cases, you will not be able to communicate through your LAN to this address because LANs are configured so they can only communicate with a sub-set of all IP addresses. For example, your LAN might only allow communication between IP addresses of the form 192.168.2.x, where x is a number between 0 and 255. If you see the default IP address and you are sure that your LAN does support DHCP, try using the same cable to connect another device to the LAN Ethernet port you have chosen to make sure it is active. If you are unable to connect to the spectrometer through your LAN, make a direct connection as described below.
The computer must have an available Ethernet port with a working network adapter. If you are unsure if the Ethernet port is working, try using it to connect to a network. Turn the spectrometer power switch on before connecting it to the computer with the Ethernet cable. After about a minute, the front panel LCD display will show the factory default IP address of 192.168.42.31. Now connect the computer to the spectrometer using the provided Ethernet cable. Configure the Ethernet port on the computer so that it can communicate with the default IP address. (To do this, you must set the IP address of the computer to another address on the same sub-net. For example, you could set the sub-net mask to 255.255.255.0 and the IP address to 192.168.42.10.) The details of setting up Ethernet ports and network adapters on different computers vary widely. For help, consult your computer’s documentation, your IT support staff, or see the Appendix. Once the port has been set up, type http://192.168.42.31 into the address field of your browser to see the welcome screen shown above.
After the welcome screen has been displayed for a moment, it will be replaced by the Run page, which is used to enter experiment parameters, start experiments and monitor their progress. The orange text links at the upper right of the page are used to navigate between pages. Go to the System page and click on Version to find the current installed software version. If the installed software version is not the same as the documentation you are reading, go to the picoSpin web site and get the documentation for the software version you are using. Do not click on the System Update link at this time. We recommend that you do not update the software on a new unit until after you have verified correct operation by following the steps in this Quick Start guide. This will reduce confusion if it becomes necessary to communicate with picoSpin support staff, because you will be using the same software version that was used to generate the factory test report.
The permanent magnet in the picoSpin-45 has a magnetization temperature coefficient of about -785 ppm/°C. As a consequence, the stability of the instrument depends upon high-resolution control of the magnet temperature. The controller can stabilize the magnet temperature to better than one millidegree over a period of several hours and to even higher precision over shorter times.
Click on the Temperature link at the upper right to go to the temperature controller page. Also click on the Controller button near the top of the page to display the temperature controller settings. The screen shot in Figure 4 shows the temperature page after the temperature is fully stabilized.
If the settings are not as shown, change them to P=10.2, I=0.02, heater=on, closed loop=on, set-point=42.0 °C and click the Submit button. A set-point of 42.0 °C was used when the unit was tested at the factory. Both the magnet Larmor frequency and the optimal shim settings are a function of the magnet set-point temperature, so it is important to use the factory set-point temperature for initial set-up.
The three plots on the temperature page display the magnet temperature, the ambient or case temperature, and a number proportional to the magnet heater current. These plots help the user monitor the magnet temperature and check that the set-point and other controller settings are suitable. After start up, it may take from 20 minutes to 2 hours for the magnet to stabilize at the set-point, depending on the starting temperature of the magnet. When the magnet temperature reaches the set-point and stabilizes, the heater current will be between 0 and its maximum value of 65535. If the ambient temperature is too low, the current will saturate at 65535 and a lower set-point has to be used. If the ambient temperature is too high, the heater current will be at 0 and a higher set point is needed. It will be possible to operate with a 42.0 C set-point in almost all situations.
While the magnet is stabilizing you can inject a sample into the spectrometer. For initial shimming we need a sample that will generate a strong singlet. Water is the best choice. If you must avoid water, you can use acetone instead but note that all signal amplitudes will be reduced to 37% of the values in your test report. The sample fluid should be free of particulates that could clog the 2 µm inlet filter. Otherwise there are no critical purity requirements.
The contents of the accessory packet are shown in Figure 5. From top to bottom, the items included are a 1 mL polypropylene syringe, a fill tube assembly, a drain tube assembly and several spare nuts and ferrules. Two PEEK plugs for the inlet and outlet ports are also provided. The fill and drain tube assemblies have PEEK ferrules and nuts for attachment to the front panel inlet and outlet fittings.
The items provided in the accessory packet are intended for initial set-up of the picoSpin-45. Recommended sample handling supplies for routine laboratory and teaching applications are described in Sample Handling. Refer to the product page of the picoSpin web site for convenient kits containing sample handling supplies.
Remove the protective tape on the inlet and outlet fittings and insert the drain tube into the outlet fitting on the front panel. Gently finger-tighten the PEEK nut. You can direct the drain tube into a small bottle if you wish. Draw a few hundred microliters of fluid into the polypropylene syringe and fit it to the fill tube. Hold the syringe with the fill tube upwards, tap it with a finger and eject any bubbles. Next, insert the fill tube into the inlet fitting, gently finger-tighten the PEEK nut and inject fluid into the cartridge until you see it flowing out of the outlet. The only concern with this process is that you might inject a bubble into the cartridge. If a bubble is left at or near the center of the cartridge where the NMR RF coil is located, the signal may be weak or it may be completely absent. Any time you fail to find an expected signal keep in mind that it could be because of a bubble.
Remove the fill and drain tubes and plug the inlet and outlet fittings with the two PEEK plugs from the accessory package. These should be gently finger-tightened to seal the ports. When the spectrometer is in continuous use it is sufficient to plug just the inlet fitting and leave the drain tube in the outlet fitting. However, it is bad practice to leave the instrument like this for long periods because the sample will evaporate and may leave solid residue in the cartridge. Cartridges will last longest if they are left filled with clean solvent and with both ports plugged.
The first step towards making an NMR spectrum is to locate the proton Larmor frequency. According to specifications, the picoSpin-45 proton Larmor frequency is 45±1 MHz. Proton NMR is normally done in bandwidths of only a few kilohertz, so the Larmor frequency has to be known to much higher accuracy than this specification. The exact Larmor frequency varies from magnet to magnet, as a function of magnet set-point temperature, and it depends on shim settings and external fields. The magnet’s Larmor frequency will also change slowly with age (a few kilohertz per year) and it may change by several kilohertz when the magnet is temperature cycled, as during shipment or storage. When setting up a new spectrometer, the Larmor frequency will be found within 20 kHz of the frequency that was found at the factory, assuming that the magnet set-point temperature has not been changed. In normal laboratory use the unit should be left on at a fixed magnet set-point temperature for best stability.
Before we look for the signal, go to the Temperature page and check that the magnet temperature has stabilized to within a few millidegrees of the set-point. (The current temperature can be found in the upper left corner of the temperature plot.)
Next, go to the Files page (Figure 6). This page provides access to previously saved experiment runs, together with their settings and data. It also allows you to select shim settings files and experiment scripts.
The screen shot above shows the Files page as it appears in a new unit. The three groups of orange links down the left column are labeled Run Data, Shims and Scripts. In the screen shot, the link autoShim QuickStart has been selected. This is the name of a run that was saved in the unit at the factory. When this link is selected the settings for this run appear at the center of the screen. The Last Run link stores the settings of the most recent run.
The Current shims link stores the shims currently set on the Run page. The QuickStart shims were saved for this unit at the factory. (The Default shims will be used in future versions of the software.)
The three scripts available in the Scripts group are autoShim for finding optimal magnet shim settings, onePulse for 1D spectroscopy and search for finding the Larmor frequency when the approximate frequency is not known. (We will not need to use the search script in this Quick Start guide.) Scripts with default settings can be accessed through these links. More often, scripts are selected by choosing a previous run in the Run Data group.
To make sure you are using the shims saved at the factory, click on the QuickStart shims and then click on use these shims at Run. You will be transferred to the Run page.
Go back to the Files page and click on the autoShim QuickStart saved run. Although this script is normally used for shimming we will use it first to find the Larmor frequency. Next, click on use these values at Run. You will return to the Run page with the settings shown in Figure 7 below, except that tx frequency (transmitter frequency) will be set close to the value of the Larmor frequency that was found for your unit at the factory. (You can find this frequency in the shimmed FID plot in your factory test report.) The setting of pulse width (transmitter 90-degree pulse width) may also be set to a better value that was found at the factory. The data plots will be empty until the script is started. All of the other settings should be adjusted to agree with Figure 7 if they are not already the same.
Note that the test run check box is checked. Because of this, the autoShim script will only generate a single RF pulse and plot the FID once. Later, when we are ready to find the optimal shim settings, we will uncheck this box and the script will run the pulse sequence repeatedly, searching for optimal shim settings. Also note that the bandwidth is set to 64 kHz. This is a very large value that is only used for finding the Larmor frequency during set-up.
Press the Start Run button to start the script. When the script finishes, the plots will contain data as in Figure 7. The upper plot is the time-domain FID and the lower plot is the spectrum. Frequencies in the spectrum are plotted relative to the transmitter frequency, and with negative frequency to the right, as is conventional in NMR spectroscopy. The appearance of the plots will vary according to how far the signal frequency is from the transmitter frequency.
Now adjust the transmitter frequency so that the peak in the spectrum will appear between +1 kHz and - 1 kHz. To do this, add the peak frequency to the transmitter frequency. For the example shown, the peak is at -5 kHz so the transmitter frequency should be changed to 44.6255 MHz. (Note that the transmitter frequency is entered in MHz.) Press the Start Run button again. The peak should now appear in the range ±1 kHz. If not, try adjusting the transmitter frequency again. If you feel that you have become lost, try again starting with the transmitter frequency shown in the shimmed FID plot of your factory test report. If a signal with an amplitude similar to that shown in Figure 7 cannot be found, you may have injected a bubble into the cartridge. Try injecting the sample again, following the instructions above.
The resolution of an NMR spectrometer depends upon how uniform the magnetic field is over the sample volume. Since the proton Larmor frequency is directly proportional to the field strength, a resolution or line-width of 50 ppb (parts-per-billion) requires that the field strength be uniform to 50 ppb across the sample. This is a very demanding requirement. In all modern NMR spectrometers, the magnetic field is adjusted for uniformity by adjusting currents in shim coils within the magnet. The picoSpin-45 has 8 shim coils; 3 that create linear gradients and 5 that create quadratically varying fields. There is also a 9th coil for generating a uniform field. The shim settings can be examined on the Run page by clicking on the shims button just to the left of the orange navigation links in the upper-right corner of the page. Saved shim settings files can be examined by clicking on their names on the Files page.
New picoSpin-45 spectrometers are shimmed at the factory before shipment. However, during shipment and storage, inevitable temperature cycling of the magnet will degrade the shim somewhat. To obtain the best resolution it is necessary to readjust the shim currents after the unit has been switched on and the magnet temperature has stabilized. If the unit is then left on and the magnet temperature remains constant, only occasional and minor reshimming will be necessary. There is no need to reshim when samples are changed, and the magnet will tend to become more stable over time. More substantial reshimming will be needed if the unit is switched off for a time or if the magnet temperature set-point is changed. The most complete reshimming is necessary when a cartridge is changed. Consult Shimming and the autoShim script documentation for more information.
Go to the Run page, choose the autoShim QuickStart saved run, and click on use these values at Run. You will see a page with settings similar to those shown in Figure 8 below. The data plots will be blank.
In the first field, tx frequency (transmitter frequency), enter a value that you know to be within 1 kHz of the correct Larmor frequency. Do not change the value of pulse width. All of the other settings must be adjusted to agree with Figure 8. The test run box should still be checked. Note that we are now using a much smaller bandwidth of 4 kHz, which is appropriate for most shimming and spectroscopy situations.
Press the Start Run button. After a few moments you should see plots similar to those in the screen shot. Note the position of the peak in the spectrum and make another adjustment to the transmitter frequency to place the peak between 200 Hz and 500 Hz. Press the Start Run button again to verify that the peak is now between 200 Hz and 500 Hz.
Your starting shim may be better or worse than is shown here. Better shim corresponds to a slower decay of the FID and a stronger peak in the spectrum, while worse shim corresponds to a FID that decays more quickly and a spectrum with a broader and weaker peak. The shape of the FID envelope may vary widely. Note also when max plot points is set to a value less than acq. points, not all of the acquired points will be displayed in the plot. In this case the appearance of the FID may not be as expected because of aliasing.
Once your transmitter frequency is adjusted correctly, uncheck the test run box. Then set max iterations to 100, Max plot points to 200, Min freq to plot to -1000, Max freq to plot to +1000 (click on show all to access these settings) and click on Start Run to start the autoShim script. The changed plot settings will make the display less smooth but they also reduce load on the server because fewer points have to be sent to the web browser. This makes it possible to view live plots while the shimming algorithm is operating.
The autoShim script uses the Nelder-Mead simplex algorithm to find shim settings that optimize the peak height of the magnitude spectrum. With the settings shown, the script will reach 100 iterations and stop after about 30 minutes. (The execution time is not deterministic because the simplex algorithm uses a variable number of evaluations of the spectrum peak height for each iteration.) When the script stops it will enter the best shim settings found into the shim fields on the Run page. These can be examined by clicking on the shims button near the top of the page, or by viewing the Current shims file on the Files page. The autoShim script does not save the shims to a named shim file when it completes unless a name has been entered in the Shims Name field. When autoShim completes, save the shims by typing a name into the Shims Name field and clicking on the Save button. The new shim file will then appear on the Files page.
Once the shims have been saved, go to the Run page and click on the test run check box, set max plot points back to 1000 and click on Start Run again. An example of the resulting FID is shown in Figure 9. The shim you achieve may be better or worse than is shown. The spectrum peak height is reported in the message screen at the bottom of the run page. You can compare your shimmed peak height and the shape of your FID envelope to the screen shot included in the factory test report. If the unit has been stored for many months since it was tested at the factory, or if it has been exposed to temperature extremes, it may be necessary to run the autoShim script several times (as described above) before the performance found at the factory is re-established.
For your first attempt at spectroscopy, pick a simple molecule without labile protons. Ethyl acetate is a good choice. Following the instructions on Injecting a Sample, fill the cartridge capillary with the sample fluid. In most cases the new sample can be used to flush out a previous one, although one should keep in mind the possibility of reactions or precipitation where the two fluids mix. In cases of concern, a compatible solvent can be used to flush the previous sample.
Go to the Files page, choose the onePulse QuickStart saved run, and click on use these values at Run. You will see a page with settings similar to those shown in the screen shot below. As before, the plots will remain blank until the script is started.
Set the transmitter frequency to the known Larmor frequency and scans to 1. Also set min freq. to plot to -2000 and max freq. to plot to +2000. Click the Start Run button and examine the plots. Make adjustments to the transmitter frequency, the plot frequency limits and phase correction until the spectrum looks similar to Figure 10. With a neat sample you will be able to see the main spectral features from a single FID. In this case, we see a CH3 singlet, a CH3 triplet, and a CH2 quadruplet.
Once you are happy with the settings, set Scans to 25, max plot points to 300 and click on the Start Run button. Plotting fewer points reduces the load on the server so that live plots can be viewed during the run without extending the desired T1 recovery time. The plot settings have no effect on the data that is recorded. After the run finishes (about 3 minutes), go to the Files page and click on Last Run. The list of files written will appear near the bottom of the page. Click on the file fid-avg.jdx which contains the averaged FID data from the 25 scans in JCAMP-DX format. Download the file to your computer.
Every picoSpin-45 comes with a one-year free license to the MNova NMR data analysis package from Mestrelab Research. MNova is a powerful package that makes all common NMR analysis tasks fast and easy. To learn the basics of MNova, download the manual from the Mestrelab site and read the first 5 sections of Chapter VIII, Processing Basics.
Figure 11 shows the result of processing the fid-avg.jdx file in MNova as follows:
These steps were done manually but MNova can do them all automatically and perform many other useful processing steps, including multiplet analysis, peak integration and global spectral decomposition.
We hope this Quick Start guide has been useful. If you have encountered any difficulties, please follow the instructions on the support page of the picoSpin web site or consult the full documentation Contents.