The overall goal of this method is to determine the lipid profile of fish oil supplements using Nuclear Magnetic Resonance spectroscopy. This method can help answer key questions for lipid analysis such as the lipid contents of fish oil supplements and the positional distribution of various fatty acids on the glycerol skeleton. The main advantage of this technique is that it makes the rapid and robust analysis of several different molecules feasible in one snapshot without using separation and purification steps.
To prepare the samples for proton and carbon NMR analysis, first extract 120 microliters, which is around 110 milligrams of fish oil, from a dietary capsule using a syringe and place it in a 4-mL glass vial. Record the weight of the fish oil. Use 500 microliters of a BHT stock solution to dissolve 100 milligrams plus or minus 10 milligrams of fish oil.
After dissolving the oil, transfer all of the solution directly into a high quality, five millimeter NMR tube and attach a cap. Analyze the samples within 24 hours of preparing the samples. Insert the NMR tube into a spinner turbine.
Place the spinner and the tube on the top of a graded depth gauge and gently push the top of the tube until its bottom part touches the bottom of the gauge. Then place the NMR tube in an open spot of the sample case. Note the slot number the sample is placed in.
To load the sample in the NMR instrument, return to the control computer and type SX-number, where number is the slot in the sample case holding the sample. Wait for the deuterium signal of deuterated chloroform to appear on the screen. As soon as the deuterium signal is visible, type lock"on the command line and select deuterated chloroform from the solvents list to lock the sample using its deuterium resonance.
Type bsmsdisp"in the command line to ensure spinning is not active. If the spin button is green, click it to deactivate spinning. After typing the command new"to create a new data set, enter a name for the data set in the name tab and the experiment number in the experiment number tab titled EXPNO.
Use number one in the process number tab titled PROCNO. Write the title of the experiment in the TITLE tab. In the Experiment tab, hit Select and choose the C13IG parameter file.
Then hit Set selected item in editor"and click OK.Type getprosol"in the command line to obtain the standard parameters for the current NMR probe and solvent. Then type the command atma"to perform automatic tuning and to matching of the probe for both carbon and proton nuclei. Next, perform one-dimensional gradient shimming to achieve a highly homogeneous magnetic field and thus optimum line shape for the NMR signals.
To do so, use the standard automatic procedure for 1D shimming simply by sequently executing the commands qu topshim 1dfast ss;qu topshim tuneb ss;and qu topshim report"in the command line. After parameter optimization as described in the text protocol, acquire one dimensional carbon NMR spectra. To do so, go to the carbon data set and use an inverse gated decoupled pulse sequence by typing pulprog zgig"in the command line.
Type commands on the command line to set up the spectral with nppm, the center of the RF transmitter, the number of scans, the number of dummy scans, the number of data points, and the pulse duration for a ninety degree pulse angle. Type digmod baseopt"in the command line to acquire a spectrum with an improved base line. Then set a relaxation delay of 60 seconds for the 850 megahertz instrument by typing di 60s"in the command line.
Set the receiver gain to an appropriate value using the command rga"for automatic calculation of receiver gain. Finally, start the acquisition by typing the pulse acquire command zg"in the command line. To process the data, first type si 64K"in the command line to apply zero filling and set the size of the rail spectrum to 64K.
Then set the line broadening parameter to 1.0 hertz by typing lb 1.0"in the command line to apply a waiting function with a line broadening factor of 1.0 hertz prior to 4ea transform. Execute 4ea transformation by typing efp"in the command line. Next perform automatic phase correction by typing the command apk"in the command line.
If additional phase adjustments are required to further improve the spectrum, click on the Process tab. Then click on the Adjust Phase icon and the Phase Correction icons for zero order and first order phase correction. While clicking on the zero order and first order phase correction icons, drag the mouse until all of the signals are in positive absorption mode.
Apply and save the phase correction values by clicking the Return and Save button to exit the Phase Correction mode. Apply a polynomial forth order function for baseline correction upon integration by typing the command abs n"in the command line. To report chemical shifts in ppm from TMS, zoom in on the TMS signal, then click on the Calibration icon and place the cursor with the red line on top of the NMR signal to be referenced, TMS in this case.
To adjust the threshold intensity, use the middle mouse button if needed. Left click and type in 0. When using BHT as an internal standard, integrate the peak at delta 151.45 extending five hertz from each side of the peak.
Then set the integral equal to the micromoles of BHT per 0.5 milliliters of the stock solution. Integrate the peaks of interest extending fiver hertz from each side of the peak. If there is a need to focus on a region, click on the highlight icon to deactivate it.
Then left click and drag to zoom in on the region. Click on the highlight icon again to make the integration function active and move to the next peak. Left click and drag the cursor through the integral.
Click the Return Save Regions button. Shown here is a representative spectrum of the Carbon NMR analysis in the carbonyl carbon region. The NMR signals of EPA and DHA on the sn-1, 3 and sn-2 positions are shown.
These signals can be used for the quantitative determination of EPA and DHA. Shown here is a representative spectrum of the proton NMR analysis. The NMR signals of EPA and DHA that can be used for their determination are shown.
The proton NMR spectrum is characterized by a narrower spectral width as compared to the carbon NMR spectrum and thus a lower spectral resolution. Once mastered, this technique can be done in less than 30 minutes if it is performed properly. After its development, this technique paved the way for researchers in the field of lipid analysis to rapidly analyze various lipid components that appear in multi-component matrices, such as dietary supplements, vegetable oil, and biofuels.
After watching this video, you should have a good understanding of how to apply proton and carbon, NMR spectroscopy for the analysis of the lipid profile of fish oil supplements. Don't forget that working with strong magnetic fields produced by NMR spectrometers can be extremely hazardous to pacemakers and surgical protheses as well as electronic items such as credit cards and watches.
