Determination of Epinephrine Molecular Structure through IR and

NMR (1H & 13C) Spectrum

Group members : Tan Pei Ni Goh Mei Ying

Mishalni a/p Kanakarajah

Introduction to Epinephrine183.20442 g/mol

(Pubchem.ncbi.nlm.nih.gov, 2016)

Introduction to Epinephrine

A.k.a Adrenaline First hormone to be discovered. Secreted by the medulla of the adrenal glands Stimulates production of cyclic AMP, a second

messenger that regulates cell activity. Prepares the body for “fight or flight” It is a powerful

Vasopressor (rise in blood pressure) Heart stimulant (restores heart rhythm) Bronchodilator (relieves breathing distress)

(Encyclopedia.com, 2016)

Biosynthesis of Epinephrine

Step 1: Hydroxylation; Phenylalanine-hydroxylaseStep 2: Hydroxylation; Tyrosine-hydroxylaseStep 3: Decarboxylation; Aromatic amino-acid decarboxylaseStep 4: Hydroxylation; Dopamine-β-hydroxylase Step 5: Methylation; Noradrenaline-N-methyltransferase

(Oellien, 2016)

Introduction to Infrared Spectroscopy (IR) & Nuclear

Magnetic Resonance Spectroscopy (NMR)

A form of electromagnetic radiation of slightly lower energy (longer wavelength) than visible light (Lohey, 2016).

Most common and widely used spectroscopic technique for: Determination of molecular structure. Detection of functional groups in organic

compounds.

An instrument that passes infrared light through an organic

molecule and produces a spectrum – where amount of light transmitted is plot against the wavelength of infrared radiation (Infrared Spectroscopy, 2014).

Infrared portion of the electromagnetic spectrum is divided into three regions; the near-, mid-, and far-infrared (Lohey, 2016).

Introduction to Infrared Spectroscopy (IR)

Introduction to Nuclear Magnetic Resonance Spectroscopy (NMR)

The study of absorption of radio frequency radiation by nuclei in a magnetic field is called Nuclear Magnetic Resonance (Chavan, 2015).

It involve change of the spin state of a nucleus when the nucleus absorb electromagnetic radiation in a strong magnetic field (Chavan, 2015).

Source of energy – Radio waves, which have long wavelengths, resulting in low energy and frequency (Najwa, 2012).

When low-energy radio waves interact with a molecule, they can change the nuclear spins of some elements, such as 1H and 13C (Najwa, 2012).

Principles of Infrared Spectroscopy (IR) & Nuclear

Magnetic Resonance Spectroscopy (NMR)

Principle of Infrared Spectroscopy (IR)

Principles:-

Study of absorption of infrared radiation, which causes vibrational/rotational transition in the molecule (Kshetri, 2013).

Results in a net change in dipole moment of the molecule (Perkins, 2015).

Alternating electrical field of radiation interacts with fluctuations in the dipole moment of the molecule (Perkins, 2015).

Freq of radiation = Vibrational/Rotational freq of molecule, absorption of radiation occurs, resulting a change in amplitude of molecular vibration/rotation (Perkins, 2015).

Principle of Nuclear Magnetic Resonance Spectroscopy (NMR)

Principles:-Theory behind NMR comes from the spin of a nucleus

and it generates a magnetic field (Solairajan A., 2012).

Without an external applied magnetic field, the nuclear spins are random in directions and they cancel each other’s fields (Solairajan A., 2012).

With strong external applied magnetic field, the nuclei align themselves either parallel (low-energy) or against (high-energy) the field of the external magnet (Solairajan A., 2012).

Nucleus precesses under applied magnetic field.

Frequency of precession (Larmor frequency) increases with the strength of applied magnetic field.

Nuclei exposed with radio frequency radiation where its frequency equals to the frequency of precession.

Absorption of radiation occurs, resulting in resonance, where nuclei in the lower energy spin state transit to the higher energy spin state by absorbing radiation.

The amount of radiation absorbed is detected by the detector.

Spectrums of Epinephrine

Infrared spectrum of Epinephrine

(Rawas-Qalaji et al., 2016)

Figure 1. Molecular structure of epinephrine (Bmrb.wisc.edu, 2016)

Interpretation of infrared spectrum of Epinephrine

Table 1. Assignation of peaks based on infrared spectrum of Epinephrine.

Wavenumber (cm-1) %T Deduction

3325.96 Weak Secondary amines

3019.05 Weak C-H bond in aromatic ring

2694.26 Weak O-H stretch

1221.21 Strong C-O stretch

1417.17 Strong C-C stretch and bend

1464.78 Medium C-H stretch

(Bmrb.wisc.edu, 2016)

1H-Nuclear magnetic resonance spectrum of Epinephrine

Figure 2. Molecular structure of epinephrine (Bmrb.wisc.edu, 2016)

Interpretation of 1H-NMR spectrum of Epinephrine

Table 2. Assignation of peaks based on 1H-nuclear magnetic resonance spectrum of Epinephrine.

Chemical shift (ppm) Integration Spin-spin coupling Atom

6.953 1 Singlet H17

6.938 1 Doublet H20

6.856 1 Doublet H18

4.931 1 Triplet H14

3.271 2 Doublet H15

3.271 2 Doublet H16

2.755 3 Singlet H22

2.755 3 Singlet H23

2.755 3 Singlet H24

13C-Nuclear magnetic resonance spectrum of Epinephrine

(Bmrb.wisc.edu, 2016)

Figure 3. Molecular structure of epinephrine (Bmrb.wisc.edu, 2016)

Interpretation of 13C-NMR spectrum of Epinephrine

Table 3. Assignation of peaks based on 13C-nuclear magnetic resonance spectrum of Epinephrine.

Chemical Shift (ppm) Atom147.086 C10147.086 C12

134.7631 C6121.2007 C9119.0664 C11116.5832 C871.0611 C557.2394 C735.662 C13

Conclusion 1H-nuclear magnetic resonance spectroscopy is

found to be the best spectroscopy technique to characterize epinephrine in better precision.

Able to detect very fine structural components.

Works on organic & inorganic compounds.

Provides qualitative and quantitative data.

Only requires few milligrams of sample.

High isotopic abundance of 1H (>99%) – occurrence of spin-spin coupling.

References Chavan, B. (2015). PROTON NMR SPECTROSCOPY. Encyclopedia.com. (2016). epinephrine Facts, information, pictures |

Encyclopedia.com articles about epinephrine. [online] Available at: http://www.encyclopedia.com/topic/epinephrine.aspx [Accessed 21 June 2016].

Infrared Spectroscopy. (2014). Kshetri, R. (2013). Introduction and Principle of IR spectroscopy. Lohey, R. (2016). INFRARED SPECTROSCOPY. Najwa, L. (2012). Introduction to NMR Spectroscopy. Oellien, F. (2016). Molecule of the Month - Adrenaline. [online] Www2.ccc.uni-

erlangen.de. Available at: http://www2.ccc.uni-erlangen.de/projects/ChemVis/motm/index.html [Accessed 16 June 2016].

Perkins, D. (2015). Fourier Transform Infrared (FT-IR) Spectroscopy: Theory and Applications.

Pubchem.ncbi.nlm.nih.gov. (2016). epinephrine | C9H13NO3 - PubChem. [online] Available at: https://pubchem.ncbi.nlm.nih.gov/compound/epinephrine#section=Top [Accessed 19 June 2016].

Solairajan A. (2012). 1H-NMR SPECTROSCOPY.

Teaching.shu.ac.uk. (2016). NMR Spectroscopy - Theory. [online] Available at: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm [Accessed 26 June 2016].

Thank you! Q & A Session