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CHM

580
SPECTROCHEMICAL METHODS OF ANALYSIS
EXPERIMENT 1
FOURIER TRANSFORM INFRARED (FTIR) SPECTROSCOPY
ANALYSIS OF ASPIRIN-PHENACITIN-CAFFEINE (APC)
TABLET
EXPERIMENT 7
NUCLEAR MAGNETIC RESONANCE (NMR) SPECTROSCOPY
ANALYSIS OF ASPIRIN-PHENACITIN-CAFFEINE (APC)
TABLET


NAME: NABILAH BINTI ABD RAHMAN
STUDENT ID: 2015484718
GROUP: AS2454D1
MEMBERS NAMES: 1. ANIS NAJIHAN BINTI AHMAD
2. ANIZA BINTI ABDULLAH
3. FATIN QURAISYAH BINTI SALIMON
DATE OF EXPERIMENT: 11TH OCTOBER 2016
DATE OF SUBMISSION: 3RD JANUARY 2017
TITLE

Experiment 1 Fourier Transform Infrared (FTIR) Spectroscopy Analysis Of
Aspirin-Phenacitin-Caffeine (APC) Tablet
Experiment 7 Nuclear Magnetic Resonance (NMR) Spectroscopy
Analysis Of Aspirin-Phenacitin-Caffeine (APC) Tablet

OBJECTIVES
1. To identify functional groups in IR spectra of standard compounds
Aspirin, Phenacitin, and Caffeine.
2. To identify functional groups present in an unknown.
3. To identify major peaks in NMR spectra of standard compound of Aspirin,
Phenacitin and Caffeine.
4. To predict the chemical structure of unknown sample by using both data
from FTIR and NMR technique.

ABSTRACT

The objectives of these experiment is to identify the major functional groups in
IR spectra (using fourier transform infrared (FTIR) spectroscopy) of standard
compounds aspirin, phenacitin and caffeine and an unknown, unknown A. The
objective is also to identify the major peaks in NMR (using nuclear magnetic
resonance (NMR) spectroscopy) of standard compounds aspirin, phenacitin and
caffeine and to predict the structure of unknown A using both data from FTIR
and NMR. Four standards, aspirin, phenacitin, caffeine and acetylsalicylic acid
(ASA) and unknown A were analyzed in FTIR by pelleting method, mixing the
standards and unknown with KBr powder in a ratio 1:99 using agate pestle and
mortar and making it into a pellet using handpress and die set. Once the samples
were pressed into a pellet (separately), the pellets were analyzed in FTIR
instrument to obtain the spectrums for identification of major functional groups
in the standards and unknown. The standards were also analyzed using NMR
instrument to identify major peaks. This method is done by mixing
approximately 30 mg of standards with deuterated chloroform and put into
clean NMR tubes separately. The tubes were then inserted into the NMR
instrument for identification of major peaks of the standards as well as the
unknown by observing the spectrums obtained. Both data from FTIR and NMR
were then used to identify unknown A. By observing both peaks, the major
functional groups and major peaks revealed unknown A to be benzoic acid.

INTRODUCTION

Infrared spectroscopy is nowadays one of the most important analytical
techniques available to scientists. One of the greatest advantages of the infrared
spectroscopy is that virtually any sample in any state may be analyzed. For
example, liquids, solutions, pastes, powders, films, fibres, gases and surfaces can
all be examined with a judicious choice of sampling technique. Fourier transform
infrared spectroscopy (FTIR) has facilitated many different IR sampling
techniques, including attenuated total reflection and diffuses reflectance infrared
Fourier transform (DRIFT) spectroscopy. It has dramatically improved the
quality of infrared spectra and minimized the time required to obtain data. The
increased speed and higher ratio of signal-to-noise of FTIR relative to dispersion
infrared has lead to a substantially greater number of applications of infrared in
natural fibres research. In addition, the constant advancing of computer and
computing science has made infrared spectroscopy techniques striding further.
The availability of a dedicated computer, which is required for the FTIR
instrumentation, has allowed the digitized spectra to be treated by sophisticated
data processing techniques and increased the utility of the infrared spectra for
qualitative and quantitative purposes. With interferometric techniques, the
infrared spectroscopy is being launched into a new era and interest in this
technique is at an all time high.

Nuclear Magnetic Resonance (NMR) spectroscopy is an analytical
chemistry technique used in quality control and reserach for determining the
content and purity of a sample as well as its molecular structure. For example,
NMR can quantitatively analyze mixtures containing known compounds. For
unknown compounds, NMR can either be used to match against spectral libraries
or to infer the basic structure directly. Once the basic structure is known, NMR
can be used to determine molecular conformation in solution as well as studying
physical properties at the molecular level such as conformational exchange,
phase changes, solubility, and diffusion. In order to achieve the desired results, a
variety of NMR techniques are available.

Aspirin, or acetylsalicylic acid (ASA), is a common drug that is generally
used as a pain reliever for minor aches and pains, to reduce fever, and also as an
anti-inflammatory drug. Aspirin has also become increasingly popular as a drug
to prevent clot-forming; it is used long-term in low doses to prevent heart
attacks and strokes in high-risk patients. Nowadays, aspirin is often given to
patients immediately after a heart attack to prevent recurrence or cardiac tissue
death. Aspirin is a non-steroidal anti-inflammatory drug (NSAID). As analgesics,
NSAIDs are generally non-narcotic (do not cause insensibility or stupor). Aspirin
was the first NSAID to be discovered.

Caffeine an alkaloid of the methylxanthine family is a naturally occurring
substance found in the leaves, seeds or fruits of over 63 plants species
worldwide. The most commonly known sources of caffeine are coffee, cocoa
beans, cola nuts and tea-leaves. In its pure state, it is an intensely bitter white
powder. Its chemical formula is C8H10N4O2, its systematic name is 1, 3, 5-
trimethylxanthine (Aurnaud, 1987). Caffeine is a pharmacologically active
substance and depending on the dose, can be a mild central nervous system
stimulant. Caffeine does not accumulate in the body over the course of time and
is normally excreted within several hours of consumption (Barone and Roberts,
1996). Caffeine belongs to a family of naturally occurring components known as
xanthines. The xanthines, which come from plants, are possibly the oldest known
stimulants. Caffeine is the most powerful xanthine, in its ability to increase
alertness, put off sleep and to improve attention in study (Boltonad, 1981),
caffeine is a vasodilator (relaxes the blood vessels) as well as a diuretic (increase
urination). On the other hand, sever restlessness and excitement, leading to mild
delirium, muscular tension and twisting and cardiovascular disturbances such as
tachycardia, are negative effects of caffeine at large doses (Boltonad, 1981). The
spinal cord is stimulated at higher doses, convulsions and death may result
(Bolton and Null, 1981).

Hypothesis

The basis of this experiment is that FTIR and NMR techniques were used
to identify the major functional groups and major peaks of standard compounds
aspirin, phenacitin, caffeine and acetylsalicylic acid (ASA) and unknown A. Both
datas from FTIR and NMR revealed unknown A to be benzoic acid. All these are
known to be drugs used for medical purposes. An overdose of these drugs can
lead to severe health consequences.

EXPERIMENTAL PROCEDURE

Experiment 1: Fourier Transform Infrared (FTIR) Spectroscopy Analysis Of
Aspirin-Phenacitin-Caffeine (APC) Tablet

A. Mixing of Sample and KBr:

The agate mortar and pestle was removed from the desiccator. 0.001 g of
sample was grinded in agate mortar into powder for 1 minute. 0.080 g of KBr
powder was added into the sample powder and was grinded for about 30
seconds with pestle. The mixture in the center of the mortar was scrappe and
mixture was grinded again for 15 seconds. The mixture in the center of the
mortar was heaped using a spatula. The remaining KBr was returned into the
desiccators after use.

B. Preparation of KBr Pellets

One fourth of the KBr mixture was transferred into the collar of the
handpress. The anvil was placed along with the die pin, allowing it to come into
contact with the sample. The die set was lifted carefully by holding the lower
anvil. The collar was ensured to stay in place. The handle of the handpress was
opened slowly and the die set was inserted into the handpress. The handle was
closed. The dial pressure was rotated until the upper ram of the handpress
slightly touches the upper anvil on the die assembly. The unit was tilted back in
order to hold the die set from falling off. The handle was opened. The pressure
dial was rotated clockwise in one half turn. The mixture was slowly compressed
while closing the handle in two minutes. The unit was tilted back, and the handle
was opened and the die set was removed from the unit carefully. The pellet was
weighed and inspected. The collar containing the KBr pellet was placed onto the
sample holder.

C. Operations of Instrument

1. Click on spectrum software.
2. To scan background > click on instrument.
3. Click on scan and on column sample name, type background.
4. Click on scan parameters > click on scan type to choose background
at the dropdown menu and click on button scan > highlight background.
5. To calibrate instrument > use polystyrene film.
6. To scan polystyrene film > click on instrument.
7. Click on scan and on column sample name, type polystyrene film.
8. Click on scan parameter > click on scan type to choose sample at the
drop down menu.
9. To scan sample for example: phenacitin
10. Click on instrument > click on scan > change sample name to
phenacitin.
11. Click on scan > highlight the sample at the bottom of software.
12. Click processes > click on baseline correction > choose automatic.
13. Highlight the sample at the bottom of the old spectrum, then delete.
Highlight the new spectrum > click processes > click smooth baseline
> click on automatic>
14. To adjust peak > click on axis to adjust bottom or top and left or right.
15. To number peak > click on peak
16. Click on text to type sample name of spectrum > type: phenacitin

Experiment 7: Nuclear Magnetic Resonance (NMR) Spectroscopy Analysis Of
Aspirin-Phenacitin-Caffeine (APC) Tablet

A. Determination of Spectrum in Each Separate Component

About 30 mg of asprin, phenacitin and caffeine were weighed and poured
into different conical vials. About 0.5 mL of deuterated chloroform (CDCl3) with a
clean, dry Pasteur pipette into the samples. The conical vials were swirled until
the samples were completely dissolved. The solutions were then transferred into
separate NMR tubes using clean, dry Pasteur pipette carefully. Once solutions has
been transferred into the NMR tubes, clean pipettes were used to add enough
deuterated chloroform (CDCl3) to bring the solution to total height to about 4-cm
from the bottom. The NMR tubes were then capped and were ensured the caps
were on tightly. The NMR tubes were inverted several times to mix the contents.
The samples were then ready to record its NMR spectrum. The NMR tubes were
inserted into its holder (one at a time) and the depth was adjusted using the
gauge provided.

B. Operations of Instrument

1. Click on topspin program.
2. Insert NMR tube into spinner.
3. Adjust the height wing gauge and do not adjust gauge position.
4. Use tissue to wipe clean NMR tube.
5. Insert sample into NMR autosampler > click enter.
6. Wait for sample to be injected into instrument.
7. Click create datasheet. Dont change data directory.
8. Click on use current method > add title on NMR spectrum title which
are determination of APC tablet on (H) NMR.
9. Click tune to autotune instrument. Click spin to turn on the sample
rotation.
10. Wait for sample to spin > sample icon at bottom.
11. Click shim to autoshim > click prosol.
12. Click gain to select auto adjust receiver gain
13. Click go to start acquisition data.
14. Click prac spectrum to process the fid data to a ppm spectrum.
15. Type sx ej to eject sample.

EXPERIMENTAL RESULT

Experiment 1: Fourier Transform Infrared (FTIR) Spectroscopy Analysis Of
Aspirin-Phenacitin-Caffeine (APC) Tablet

1. Outcome of Objectives:

A. Compound: Aspirin
Molecular formula : C9H8O4
Molecular structure:



Reference Functional Frequency Molecular
Wavelength (cm-1) Group Range (cm-1) Function
2872.09 Alkane 2853 2962 C-H stretch
1753.69 Carboxylic Acid 1710 -1780 C=O stretch
1690.21 Ester 1630 1780 C=O stretch
1458.07 Aromatic 1450 1600 C=C stretch
755.74 Aromatic; ortho- 735 770 C-H (out-of-plane
disubstituted bending)
1955 Benzene 1667 2000
Overtone
2699.29 Carboxylic Acid 2500 3000 O-H strecth
(Hydrogen
bonded)
1187.64 Ester 1020 1275 C-O stretch
1479.42 Alkane 1375 1650 C-H bend


B. Compound: Caffeine
Molecular formula : C8H10N4O2
Molecular structure:



Reference Functional Frequency Molecular
Wavelength (cm-1) Group Range (cm-1) Function
2955.68 Alkane 2853 2962 C-H stretch
1698.26 Amide 1630 1780 C=O stretch
1660.14 Amide 1630 1690 C=O stretch
Alkene 1620 1680 C=C stretch
1239.69 Aromatic amine 1200 1350 C-N stretch
2250.0 Nitrile 2220 2260 C=N
1359.83 Alkane 1350 1490 C-H bend
1431.07








C. Compound: Phenacitin
Molecular formula : C10H13NO2
Molecular structure:



Reference Functional Frequency Molecular
Wavelength (cm-1) Group Range (cm-1) Function
2928.01 Alkane 2853 2962 C-H stretch
3286.61 Amine 1710 -1780 N-H
3071.72 Aromatic ~3030 C-H stretch
1883.17 Benzene 1667 2000
Overtone
1659.63 Amide 1630 1690 C=O stretch
1509.03 Aromatic 1450 1600 C=C stretch
838.07 Aromatic; para- 800 860 C-H (out-of-plane
disubstituted bending)
3131.63 Aromatic ~3030 Ar-H stretch
1245.08 Ether 1000 1300 C-O stretch


D. Compound: ASA (Acetylsalicylic Acid)
Molecular formula : C9H8O4
Molecular structure:




Reference Functional Frequency Molecular
Wavelength (cm-1) Group Range (cm-1) Function
2871.09 Alkane 2853 2962 C-H stretch
1754.67 Carboxylic Acid 1710 -1780 C=O stretch
1693.48 Ester 1630 1780 C=O stretch
1457.46 Aromatic 1450 1600 C=C stretch
755.42 Aromatic; ortho- 735 770 C-H (out-of-plane
disubstituted bending)
2032.38 Benzene 1667 2000
Overtone
2699.32 Carboxylic Acid 2500 3000 O-H strecth
1219.91 Ester 1020 1275 C-O stretch
1187.87
1419.53 Alkane 1375 1650 C-H bend
3050 Aromatic ~3030 C-H stretch


E. Compound: Unknown A (Benzoic Acid)
Molecular formula : C7H6O2
Molecular structure:


Reference Functional Frequency Molecular
Wavelength (cm-1) Group Range (cm-1) Function
2834.94 Alkane 2853 2962 C-H stretch
1686.42 Carboxylic Acid 1630 1780 C=O stretch
934.51 Aromatic 900 920 C-H stretch
1583.55 Aromatic 1450 1600 C=C stretch
707.67 Aromatic; 690 710 C-H (out-of-plane
monosubstituted bending)
1910.0 Benzene 1667 2000
Overtone
2834.94 Carboxylic Acid 2500 3000 O-H strecth
1454.08 Alkane 1375 1650 C-H bend
3025.00 Aromatic ~3030 C-H stretch


Experiment 7: Nuclear Magnetic Resonance (NMR) Spectroscopy Analysis Of
Aspirin-Phenacitin-Caffeine (APC) Tablet

1. Outcome of Objectives:

A. Compound: ASA (Acetylsalicylic Acid)
Molecular formula : C9H8O4
Molecular structure:



H Signal Chemical Shift Integral #H Multiplicity
Theoretical Experimental
HA 2.1 2.4 2.278 3 H Singlet
HB
HC
HD 6.5 8.0 7.196 1 H Doublet
HE 6.5 8.0 7.409 1 H Triplet
HF 6.5 8.0 7.665 1 H Triplet
HG 6.5 8.0 7.942 1 H Doublet
HH 11.0 12.0 Missing 1 H Singlet

B. Compound: Phenacitin
Molecular formula : C10H13NO2
Molecular structure:


H Signal Chemical Shift Integral #H Multiplicity
Theoretical Experimental
HA 2.1 2.4 2.023 3 H Singlet
HB
HC
HD 5.0 9.0 9.794 1 H Singlet
HE 6.5 8.0 6.834 2 H Doublet
HF
HG 6.5 8.0 7.452 2 H Doublet
HH
HI 3.2 3.8 3.981 2 H Quartret
HJ
HK 0.7 1.3 1.278 3 H Triplet
HL
HM


C. Compound: Caffeine
Molecular formula : C8H10N4O2
Molecular structure:



H Signal Chemical Shift Integral #H Multiplicity
Theoretical Experimental
HA 2.2 2.9 2.491 3 H Singlet
HB
HC
HD 2.2 2.9 Missing 1 H Singlet
HE 2.2 2.9 Missing 3 H Singlet
HF
HG
HH 2.2 2.9 Missing 3 H Singlet
HI
HJ

D. Compound: Unknown A (Benzoic Acid)
Molecular formula : C7H6O2
Molecular structure:


H Signal Chemical Shift Integral #H Multiplicity
Theoretical Experimental
HA 11.0 12.0 12.966 1 H Singlet
HB 6.5 8.0 7.942 2 H Doublet
HF
HE 6.5 8.0 7.473 2 H Triplet
HC
HD


E. Compound: Aspirin
Molecular formula : C9H8O4
Molecular structure:



H Signal Chemical Shift Integral #H Multiplicity
Theoretical Experimental
HA 2.1 2.4 2.257 3 H Singlet
HB
HC
HD 6.5 8.0 7.218 1 H Doublet
HE 6.5 8.0 7.388 1 H Triplet
HF 6.5 8.0 7.644 1 H Triplet
HG 6.5 8.0 7.942 1 H Doublet
HH 11.0 12.0 Missing 1 H Singlet


DISCUSSION

Aspirin, C9H8O4 which is also acetylsalicylic acid, is a drug used to reduce
fever and relieve mild to moderate pain from conditions such as muscle aches,
toothaches, common cold, and headaches. It may also be used to reduce pain and
swelling in conditions such as arthritis. Aspirin is known as a salicylate and a
non-steroidal anti-inflammatory drug. Phenacetin is a synthetic, white
crystalline solid that is slightly soluble in water and benzene, soluble in acetone
and very soluble in pyrimidine. It was formerly known as pain-relieving and
fever-reducing drug, which was widely used. It is used in research as the
preferred marker for detecting CYP1A2-based inhibition potential in vitro.
Human ingestion of phenacetin can result in a bluish discoloration of the skin
due to a lack of oxygen in the blood (cyanosis), dizziness and respiratory
depression. It is reasonably anticipated to be a human carcinogen.

The nature of caffeine reveals that it is a bitter white crystalline alkaloid.
It is a common ingredient in a variety of drinks (soft and energy drinks) and is
also used in combination with various medicines. Caffeine is the most versatile
compound in the sense that almost every human being is exposed to this
compound via various beverages and medicines. Caffeine is widely used in many
soft drinks as flavoring agent and is deliberately added to make people addicted
to these drinks. Caffeine is a naturally occurring alkaloid and it can be found in at
least 63 plant species and is present in their leaves, seeds, and fruits. It is a well-
established fact that caffeine acts as a stimulant to the central nervous system
and heart and also increases the activity of brain through its adenosine
antagonist action. Nowadays, it is most commonly used in various
pharmaceuticals. Caffeine is used in the treatment of mild respiratory depression
caused by narcotics and for the treatment of circulatory failure.

Nuclear magnetic resonance spectroscopy, commonly referred to as nmr,
has become the preeminent technique for determining the structure of organic
compounds. Of all the spectroscopic methods, it is the only one for which a
complete analysis and interpretation of the entire spectrum is normally
expected. Although larger amounts of sample are needed than for mass
spectroscopy, NMR is non-destructive, and with modern instruments good data
may be obtained from samples weighing less than a milligram. Infrared (IR)
spectroscopy is one of the most common and widely used spectroscopic
techniques. Absorbing groups in the infrared region absorb within a certain
wavelength region. The absorption peaks within this region are usually sharper
when compared with absorption peaks from the ultraviolet and visible regions.
In this way, IR spectroscopy can be very sensitive to determination of functional
groups within a sample since different functional group absorbs different
particular frequency of IR radiation. Also, each molecule has a characteristic
spectrum often referred to as the fingerprint. A molecule can be identified by
comparing its absorption peak to a data bank of spectra. IR spectroscopy is very
useful in the identification and structure analysis of a variety of substances,
including both organic and inorganic compounds. It can also be used for both
qualitative and quantitative analysis of complex mixtures of similar compounds.

The identification of major functional groups and major peaks were done
and the method used was by observing spectrums obtained from FTIR and NMR.
In the FTIR experiment, the standards aspirin, phenacitin, caffeine and
acetylsalicylic acid (ASA) and unknown A were first mixed with KBr powder (for
KBr pelleting method) separately in an agate mortar and pestle in a ratio of 1:99.
The mixture was then grinded to a fine powder before being pressed into a pellet
using handpress and die pin. The pellet formed was ensured to be clear at center
and thin so that light rays coming from the instrument can penetrate the pellet.
The experimental procedures were followed to obtain the right size of pellet. The
pellet was then inserted into the FTIR instrument and students were taught the
operational procedure. The accurate information were keyed in and the
standards and unknown in the pellet form were then analyzed using FTIR
instrument. Spectrums for each standards and unknown were then obtained
from the output of the instrument. By observing the spectrums obtained and
using the FTIR chart as a guide, major functional groups of all standards and
unknown were identified (as explained in the results section).

In the NMR experiment to identify major peaks of standards aspirin,
phenacitin, caffeine and acetylsalicylic acid (ASA) and unknown A, 30 mg of the
standards and unknown were first weighed. The standards and unknown were
then mixed with deuterated chloroform. After the mixtures have been dissolved,
the mixtures of the standards and unknown were the transferred to separate
NMR tubes and enough deuterated chloroform were added to bring the total
solution height to 4-cm. The tubes were then inserted into the NMR instrument.
Since NMR instrument were unavailable for use, the instructor explained the
demonstration of how the instrument worked. Spectrums of NMR for all
standards and unknown A were obtained. Since no standards or unknown were
able to be analyzed, the instructor gave spectrums of standards and unknowns
from previous experiments. This also explained why some results are inaccurate.
By observing the spectrums and referring to 1-H NMR chart, major peaks of the
standards and unknown were identified (as explained in the results section).

The difference between FTIR method and NMR method is that FTIR
method was able to analyze and give major functional groups of samples
analyzed. But 1- H NMR method were able to analyze and give major peaks of
samples analyzed. And with the major peaks, molecular structure of samples
analyzed were able to be interpreted. Using both data from FTIR and NMR
spectrums, which gave the major functional groups and major peaks, unknown A
were identified to be benzoic acid. Benzoic acid (C7H6O2) is an organic aromatic
monocarboxylic acid. The cobalt can synthesize it or manganese catalyzed
atmospheric oxidation of toluene. Recently, benzoic acid has been prepared from
toluene by employing TiO2 nanotubes electrode. Benzoic acid reacts with
hydrogenating reagents to afford hexahydrobenzoic acid. The thermal
decomposition of the product in the presence of lime or alkali produces benzene
and carbon dioxide.


CONCLUSION

The major functional groups of standards aspirin, phenacitin, caffeine and
acetylsalicylic acid (ASA) were identified using FTIR spectra obtained. The major
peaks of standards aspirin, phenacitin, caffeine and acetylsalicylic acid (ASA)
were also identified using NMR spectra obtained. By observing both spectras, it
is revealed that unknown A appears to be benzoic acid.


REFERENCES

1. Aurnaud MJ (1987). The pharmacology of caffeine. Prog. Drug 31: 273.

2. Bolton S, Null G (1981). Caffeine, psychological effects, use and abuse.
Orthomol. Psychiatr., 10(3): 202 211.

3. http://cdn.intechopen.com/pdfs/37067/InTech-
Fourier_transform_infrared_spectroscopy_for_natural_fibres.pdf

4. http://chem.ch.huji.ac.il/nmr/whatisnmr/whatisnmr.html

5. https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/n
mr/nmr1.htm