What's the sugar in blueberries
Nuclear magnetic resonance (NMR) is a popular technique that measures intrinsic spin properties of nuclei which can not only measure the structure of compounds but also could quantify compounds concentrations with an internal standard. One of the great advantages is that NMR is a non-destructive method to measure samples which can save samples for other methods. Due to this advantage, in organic synthesis, NMR is almost the first method to use. In addition, NMR has a high tolerance to salt which makes it a great choice for many biological samples such as urine, stool, and blood samples. Compared to HPLC/MS which has a high requirement to low salt concentration, NMR also has a non-contact advantage which makes the cross-contamination negligible. However, NMR requires more sample volume than HPLC/MS and its detection limit is not as good as HPLC/MS which also limits its applications in many biological applications. In this experiment, we are using NMR to measure the glucose in the blueberry.
Objective:
1, Learn NMR data quality vs scan number (time)
2, Learn NMR study for natural compounds
3, Calculate the sugar percentage in one Blueberry
Graphical Abstract
Solution and supplies needed
D2O buffer (pH 7.4) with internal standard (5 mM) TSP, 1M phosphate and 0.5% sodium azide
Eppendorf tube 1.5 ml, NMR tube, 5mm and Transfer pipette (1 ml)
Sample preparation
1, Weight one blueberry and record the exact weight.
2, Use pestle and mortar to break the blueberries, and add 1 ml of water using a pipette.
3, Transfer most of the liquids to a Eppendorf tube and centrifuge it with a speed of about 12000 rpm.
4, Transfer 630 ml of the solution to a 1.5 ml Eppendorf tube and add 70 ml buffer, vortex it for about 1 min (630 ml solution and 70 ml buffer)
5, Centrifuge the solution with a speed of around 12000 in the biochemistry lab
6, Use the transfer pipette to take 600 ml solution and transfer it to an NMR tube, be sure to label the NMR tube with your sample ID
7, Use some force to spin the liquid to the bottom of the NMR tube, make sure no air bubbles are in your solution
8, Take the sample to the NMR room
NMR Procedure
The NMR tube must be positioned properly in a spinner before it can be introduced into the NMR instrument.
Make sure the blue spinner should be in the receiver like a gap.
NMR acquisition
1, Put your samples with a spinner to the NMR autosampler and record your sample position on the sampler.
2, We will use ICONNMR to run the sample automatically,
Click the Holder #where your sample is loaded. Key in the sample name, number and buffer, in this case, we use H2O + D2O with salt. We use the water suppression as the experiment and chose the parameters.
3, The Par has all the parameters needed, and we need to change the NS for the experiment, we used ns number of 2, 8 and 32 for our experiment with sample number 10, 11 and 12 or any other numbers you can record
4, The three experiments need to be added to the three different SN numbers on the same position for one sample. Different student groups need to be added to different positions, and submit all of them as shown in the figure
NMR data pre-treatment
1, the spectrum should be processed, but be sure to check the internal standard TSP, water and the baseline
2, Use a multiple displace window to overlay the three experiments and adjust the TSP to the same height.
2, The SN numbers should be related with the signal-noise ratio which can be observed by zooming in
2, Sugar level will be identified by the Chenomx software using the free evaluation and the concentration can be determined by the TSP concentration, but be careful about your dilution
4, Data analysis
1, What is the final concentration of phosphate, TSP, and sodium azide in your NMR tube?
2, Find a standard glucose or sucrose NMR spectrum on HMDB.ca and attach it to see if it matches any peak. Cycle at least 1 peak on your spectrum
3, Use the first-week data to get the concentration of sucrose (recorded in the lab). Remember, the sucrose concentration was diluted, be sure to convert it back to the original concentration.
4, Compare the first week's three experiments. Please describe the difference between the spectra under different scan numbers. Mention data quality and noise.
5, Compare the glucose peaks of the two weeks measurements (the 32 ns). Is glucose peak stable after 1 week?
6, Calculate the sucrose weight percentage in the blueberry.
Discussion
1, We re-ran the same sample for the second time after one week. Can you see the difference between the two spectra? Give a possible reason.
2, What may affect the NMR spectrum if no sample degradation? Check online sources for this question.
3, Why do we need to use a buffer for the NMR analysis? Please explain it considering the factors may affect NMR chemical shift.
Student examples in the Lab