Aim of Experiment

Describe about the experiment of Glucose Molecule?

To carry out initial structural analysis of α-D-(+) -Glucose using NMR techniques (1-dimensional) such as ¹³C-DEPT 135, ¹H-NMR and ¹³C-NMR. In addition to perform, further structural analysis by 2-dimensional NMR such as HSQC, COSY, HMBC and TOCSY. Also, observe the influence of its structural characteristics on coupling constant and chemical shift on the NMR spectrum.

Glucose molecule

Glucose is a simple monosaccharide sugar, which is considered as one of the most important carbohydrates. Glucose is used as a resource of energy in plants and animals. It is found that glucose is one of the main products used in respiration and photosynthesis. Glucose is found in nature as D-glucose, which is commonly known as dextrose. Food industries manufactured glucose always in dextrose form. A glucose molecule is consisted of carbon, hydrogen and oxygen molecules. The chemical formula of glucose is C₆H₁₂O_6. Glucose is composed of 6 carbon atoms and contains an aldehyde group as the functional group, therefore glucose can be classified as an aldohexose. Glucose molecule can further be classified into either L or D series depending on the maximum number of its chiral center, C-5. At this C-5 position, if OH functional group is situated at the left of the plane, it can be classified as L-glucose. On the other hand, D-glucose is the mirror image of L-glucose. it is observed that glucose is more stable in its ring structure compared to open-chain structure. The reason behind the stability of ring structure is, the hydroxyl group on C-5 is prone to react with the carbonyl group on C-1, which results in the production of a closed pyranose ring (Figure 1). Therefore, glucose is further found in two isomeric forms such as α-D-glucose and β-D-glucose. In α-D-glucose, the OH functional group on C-1 is formally trans to the CH₂OH (on C-5). On the other hand, these two groups are found in cis position in β-D-glucose (Figure 1). In this experiment, the structural properties of α-D-glucose are focused, as glucose is the most abundance aldohexose found in living organisms. To find out the structural properties of this monosaccharide, the researchers have used many NMR spectroscopy techniques.

Figure 1: Anomeric forms of D-glucose

NMR (Nuclear magnetic resonance) induces transitions between different nuclear spin states of samples by using radiofrequency radiation in a magnetic field (Stothers, 2012). It is found that NMR spectroscopy is generally used in quantitative measurements; however, it is most useful to determine the structure of molecules (along with mass spectrometry and IR spectroscopy) (Günther, 2013). The utilization of NMR spectroscopy to determine the structural characterization arises because different atoms in a molecule face different magnetic fields. This phenomenon is responsible for the transitions of different resonance frequencies in an NMR spectrum (Graaf, 2013). It is found that the interactions between different nuclei are responsible for the deviation of the spectra lines. Therefore, the splitting lines of spectra can be used to gain information about the closeness of different atoms in a molecule. NMR spectroscopy is therefore can be applied in a number of areas of science (Keeler, 2011). It is observed that the chemists used NMR spectroscopy (simple one-dimensional techniques) regularly to observe the chemical structure (Jiang et al. 2011). On the other hand, the chemists used two-dimensional techniques to study the structures of complicated molecules (Morales et al. 2012).

Glucose molecule

Preparation of sample

At first pure α-D-glucose (89mg) was weighed and dissolved in of deuterated dimethyl sulfoxide (d₆-DMSO). Then the sample was pipette into a dry and clean NMR tube. After that, the the NMR tube was sealed with parafilm and labeled. At last the sample was subjected to the NMR spectrometer.

There are three steps involved in acquiring a spectrum from NMR spectrometer and these are:

• Sample loading: At first, the NMR tube was placed in a sample holder, known as “spinner”. Depth gauge was used to maintain the correct height of the NMR tube. After that, the sample was positioned in the NMR sample tray waiting to be analyzed.
• Instrument Locking and Shimming.
• Data acquisition and processing

It was found that NMR spectrum was modified using “Spinworks” software. SpinWorks is a software package for the dealing out of 1D and 2D data. Spinworks also included modules for the simulation, dynamic NMR spectra and analysis of second order spectra. It can perform spectral simulations to determine chemical shifts and coupling constant. All the information obtained below was obtained through Dr. Ramesh. Dr. Ramesh separated the class into many groups and each group visited the NMR device. The NMR devise is located in the basement of the chemistry department where the guide explained details about the NMR device working procedure. It is observed that many precautions steps need to be taken before running the NMR sequence. These steps are do not bring mobile phones, any metallic objects, coins, etc. near to the NMR sequence, since it may cause damage to the magnet. The guide also advised not to touch the NMR magnet as it can hamper the NMR’s homogeneity properties. In addition, personnel with metallic im[plants and heart pace makers should not come near to the NMR spectrometer.

In figure2, hydrogen and carbons nuclei are numbered for α-D-glucose molecule to interpret NMR result. In the following structure of glucose, 6 carbon atoms are shown, which are referring to 6 different environments. There are also 11 hydrogen atoms, which are referred to 10 different environments. 

Figure 2: Carbon and hydrogen nuclei numbering for α-D-glucose molecule

Table 1: ¹H-NMR showing the chemical shift of assigned proton nuclei (Figure 2), its multiplicity patterns and the coupling constant J (Hz)

 
Table 2: ¹³CNMR analysis showing the chemical shift of assigned carbon nucleiand its phasing behavior in ¹³C DEPT-135 NMR spectrum in (Figure 2)

Table 3: shows the observed correlation of ¹H nuclei (Figure 2) in 2D Correlation Spectroscopy (COSY) NMR spectrum and 2D Total Correlation Spectroscopy (TOCSY) NMR spectrum

*C indicate ¹H-¹H nuclei in COSY spectrum, D indicate ¹H-¹H nuclei in TOCSY spectrum

Table 4: shows the observed correlation of ¹H-¹³C nuclei (Figure 2) in 2D Heteronuclear Single Quantum Correlation (HSQC) NMR spectrum and 2D Heteronuclear Multiple Bond Correlation (HMBC) spectrum

*X symbol indicates¹H-¹³C nuclei in HSQC, Y symbol indicate ¹H-¹³C nuclei in HMBC

Many NMR techniques are used to analyze glucose molecule. In the H-NMR spectrum, it is found that C-H peaks are clustered and overlapped comparing to the O-H peaks, which are well separated among each other. The hydroxyl protons (O-H) are mostly located in the middle of the spectrum ranging from 4.34ppm to 4.82 ppm (except O-H2 that appeared more deshielded at approximately 6.2ppm). On the other hand, the C-H protons are mostly located at around 2.99ppm to 3.61ppm (except C-H1, which appeared at 4.9ppm). The proof of this pattern can be found in the 2D-HSQC spectrum in which the O-H region stated above was blank (showing no interaction) while the C-H regions showed correlation signal between the positioned carbon with its bonded hydrogen ( for example: C5-H5). HSQC helped to determine the correlation of hydrogen and carbon in a single bonded position. Hydroxyl protons are more deshielded compared to C-H protons due to the presence of electronegative atom, oxygen; which is the reason behind the shifting of hydroxyl-protons more to the left compared to C-H protons. The presence of electronegative atom pulls the electron density away from proton nucleus, which in turn makes it more vulnerable towards the applied magnetic field, thus appeared downfield of NMR spectrum. However, an exception of for this pattern is also observed. Further analysis of C-H nuclei at C-1 showed that it is highly deshielded compared to the other C-H nuclei. This is because of the same reasons as hydroxyl groups (the presence of an electron withdrawing atom, oxygen). In fact it is observed that the O-H proton at this carbon is the most deshielded one among its species located in the other carbon skeletons. Therefore, positioning the C-H protons and O-H protons on the skeleton of glucose is hard while only depending on the ¹H-NMR spectrum. The reason is, the O-H groups of protons are rather quite the same chemical environment between one each other except O-H2, which are difficult to determine the sequence and it is also found that the C-H are even worse as it exist as multiples except for C-H1. Therefore, to locate the proton peak, 2D-COSY NMR analysis of this molecule is very helpful. 2D-COSY NMR is a technique, which is helpful to track the correlation of ¹H-¹H up to 3 bond lengths. By studying the correlation peaks of this spectrum one can resolve the complex multiplets of C-H protons in which for the first multiplet (δ=3.37ppm – 3.61ppm), there are four C-H protons resonate at this frequency: C-H5,C-H9 and C-H10,10’and for the second multiplet (δ=3.37ppm – 3.61ppm) there are only two protons resonate: C-H3 and C-H7. It is found that the integral values in ¹H-NMR is equivalent to the number of protons in each of this multiplet (which is 4.4 and 2.1 respectively). However, it is observed that the multiplicities of C-H protons are rather difficult to study except for C1-H1. On the other hand, The O-H proton splitting patterns are easy and simple to interpret. All of the C1-H1 and O-H protons followed the multiplicity rules, (2nI+1). Such as, the reason behind the triplet appearance of C1-H1 peaks is that it is split by two hydrogen protons O-H2 and C-H3 and for the O-H11. In addition, it is also appeared as triplet because it is split by two hydrogens at C-6. In ¹H NMR spectrum, the coupling constant can be observed. It is observed that J value for the splitting of O-H protons are larger compared to C-H protons. the analysis ¹³C NMR revealed there are 6 different environments of carbon atoms which one of it is highly deshielded and this is certainly C-1 due to the presence of oxygen atom bonded to it. C-6 is surrounded by more electrons due to the presence of two hydrogen atoms attached to it, therefore it is The most shielded carbon in this molecule. Evidence of this peak is C-6 can be observed, firstly in ¹³C-DEPT 135, but in the chemical shift the peak is negative phasing (indicate CH₂), however, the other CH carbons are in the positive phasing. In HSQC spectrum, it is found that there are two spots plotted at F1 axis of around 61.0 ppm (the carbon is directly bonded to two hydrogen atoms). The other carbon peaks appeared in the ¹³C NMR spectrum was also being determined by studying the correlation patterns in HSQC NMR. The HMBC NMR spectrum and TOCSY NMR spectrum were used to confirm the structure once again. In TOCSY NMR, the interaction of ¹H-¹H nuclei up to five chemical bonding is observed. As an example, an interaction observed between C-H1 and O-H6, which is in this molecule, is 5 bonding apart. Examples of interaction of 4 bonding includes O-H8 with C-H5 and C-H5 with C-H1. On the other hand, HMBC is helpful to observe the heteronuclear interaction of ¹H-¹³C nuclei typically 2-4 bonds away. Due to peaks overlapping, In this experiment it is difficult to observe a single interaction between carbons in the position of C2 up to C5. The interaction presented in Table 4 for C2, C3, C4 and C5 is just a rough plotting based on the theoretical concept of these techniques. However, a clear isolation of peaks spotting for C1 and C6 are found in this glucose spectrum. The interaction of C1 with H4 and H2 were clearly observed as well as the interaction of C6 with H7 and H11.

6.0 Conclusion

It is found that new beginning in carbohydrate researches being fostered by a blend of new synthetic methods and developments in analytic techniques. NMR spectroscopy is sure to play an important role in the carbohydrate study since, it has a capability to provide close structural details of Biomolecular interactions. To analyze the structural properties of α-D-(+) -glucose molecule application of NMR spectroscopy offers the unique capacity. With ¹H-NMR and ¹³C NMR coupling constant, the chemical shift, integral values of each nuclei peak and multiplicity patterns can be observed. On the other hand, 2D HMQC NMR and 2DCOSY NMR is helpful to locate each of these nuclei into its position in the cyclic form of the glucose molecule. In addition, 2D HMBC and 2D TOCSY are strong tools used in evidencing of each of these nuclei located in the molecule.

7.0 References:

De Graaf, R. A. (2013). In vivo NMR spectroscopy: principles and techniques. John Wiley & Sons.

Flores-Morales, A., Jiménez-Estrada, M., & Mora-Escobedo, R. (2012). Determination of the structural changes by FT-IR, Raman, and CP/MAS 13 C NMR spectroscopy on retrograded starch of maize tortillas. Carbohydrate Polymers, 87(1), 61-68.

Günther, H. (2013). NMR spectroscopy: basic principles, concepts and applications in chemistry. John Wiley & Sons.

Jiang, F., Zhu, Q., Ma, D., Liu, X., & Han, X. (2011). Direct conversion and NMR observation of cellulose to glucose and 5-hydroxymethylfurfural (HMF) catalyzed by the acidic ionic liquids. Journal of Molecular Catalysis A: Chemical, 334(1), 8-12.

Keeler, J. (2011). Understanding NMR spectroscopy. John Wiley & Sons.

Stothers, J. (2012). Carbon-13 NMR spectroscopy (Vol. 24). Elsevier.