FTIR Spectroscopy

E J G, Caro1
1
Cubao, Quezon City 1016

Email: ejgvcaro@mymail.mapua.edu.ph

Abstract. Classification tests have traditionally been used in biochemistry to determine the
identity of possible functional groups of unknown compounds by determining the reactivities
of the unknown compound’s functional groups. In recent times, spectroscopy has become the
most prevalent form of chemical identification. Fourier Transform Infrared spectroscopy makes
use of the stretching and bending of the bonds of the compounds as it absorbs infrared light.
The report aims to describe the conduct of an FTIR spectroscopy of two unknowns labelled C
and H. By considering the resulting IR spectrum and the results of the classification tests
showed the identity of the unknowns being ascorbic acid and lactic acid.

1. Background of the experiment

Many methods of determining the identity of unknown compounds have been invented over the years.
In the early days, most methods of determining the identity of unknown compounds involved various
tests of chemical and physical properties, the most prominent being that of the solubility tests which
involves testing and observing the reactions of the unknown with a given reagent. The resulting
physical and chemical changes then give clues on what functional groups are in the unknown, thereby
giving clues on the identity of the compound(1). However, this process is time-consuming, expensive
in terms of reagents, and the information provided can prove to be inadequate and even inaccurate in
certain circumstances(2). In order to solve this problem, a new more powerful method of identification
is needed.
The most prominent form of chemical identification is spectroscopy, which pertains to the
observation of the reaction of a given unknown compound to a given electromagnetic wave. By
observing how much light is absorbed or reflected, the structure of the compound can be
determined(3). There are 3 commonly used types of spectroscopy; FTIR, NMR, and MS spectroscopy,
this report primarily focuses on FTIR spectroscopy, however in most chemical identification requires
at least 1 result from 2 different types of spectroscopy in order to fully determine the chemical
structure of an unknown compound(4).
FTIR spectroscopy is the most common form of spectroscopy. This method uses an
interferometer and the Fourier transform to allow for quick and easy analysis of liquid and solid
compounds. It uses infrared red light which then induces vibrations in the chemical bonds of the
compound(5). All of these vibrations provide valuable information about the chemical structure of the
compound due to the fact that every type of bond produces a peak with a different wave number,
intensity, and shape. The resulting FTIR spectrum is divided into two portions, the diagnostic region
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and the fingerprint region, the latter portion indicates all the functional groups present in the compound
and the former is an IR spectrum unique to the compound. By combining these two sections, the
identity of the unknown compound can be determined by cross-referencing the IR spectrum
database(6).

2. Methods
It is worth noting that the experiment detailed in this report is a continuation of a previous report
which tackled the same unknowns and subjected both through a series of classification tests. Before
the FTIR was conducted, the results of the previous experiment were recounted and reconsidered for
the analysis of the results of the spectroscopy.

2.1. Preparation of the FTIR Machine

Before the samples could be loaded into the FTIR machine, it was first cleaned with hexane in order to
remove any traces of the previous compounds that have been analysed before. Once sufficiently
cleaned, the background was run first in order to account for the external vibrations which could affect
the resulting spectrum. Once all preliminary preparations are done the samples can then be loaded into
the machine.
2.2. Loading of Samples and Analysis
The first sample to be loaded was the solid sample. A few milligrams of the sample were placed into
the sample port of the machine and was then sealed and analysed. Once the analysis of the first sample
was complete and the spectra was properly saved, the machine was again cleaned with hexane. After
cleaning, a few millilitres of the second sample, which is liquid, was loaded into the machine. After
saving the resulting spectra, the machine was cleaned for a final time and the spectra of both samples
were printed and taken for analysis.

3. Results and Discussion

It is worth noting that this analysis is a continuation from a previous report, specifically, the same
samples from the classification tests were used in the FTIR spectroscopy. As such the results from the
classification tests would also be considered in this analysis and will be summarised in this report for
convenience.
3.1. Results of the Classification Tests
Recalling, there are two compounds that were analysed using classification tests, unknown H and
unknown C . The following table enumerates the observed physical properties of the unknowns:
Table 1. Physical Properties of Both Unknowns

Physical Unknown H Unknown C

Property

State of Matter Liquid Solid,

crystalline

Colour Clear, White

colourless

Odour Odourless Odourless

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The classification tests done on the unknowns also gave clues as to the possible identities of
the unknown compounds. The following table enumerates all of the relevant results of the
classification tests:
Table 2. Classification results of both unknowns.

Classification Test Unknown H Unknown C

Baeyer’s Test Positive; lack of brown precipitate Negative; unknown does not contain
indicates aldehydes double or triple bonds

Jones Test N/A Positive; aldehyde

Hinsberg Test Negative; unknown is not an Positive; secondary amine

amine

Iodoform Test Positive; possibly a secondary N/A

alcohol without adjacent methyl
group

Lucas’s Test Positive; secondary alcohol N/A

Silver Nitrate N/A Negative; unknown does not contain

Test alkyl halides and sulfonyl chloride

Tollens’ Test Negative; for aldehydes Positive; for aldehydes

Sodium N/A Positive; carboxylic acid

Bicarbonate
Test

Chloroform and Negative; unknown is not an Negative; unknown is not an

Aluminium aromatic hydrocarbon aromatic hydrocarbon
Chloride Test

The classification tests results from Table 2 coupled with the initial observations in Table 1
resulted in the conclusion that compound H is a colourless and odourless liquid organic substance at
least has an aldehyde and a secondary alcohol as its functional groups and compound C is a a white,
crystalline, and odourless with an aldehyde group, a secondary amine, and a carboxylic acid functional
group as its functional groups. These test results are however subject to a relatively high probability of
error, thus a process such as FTIR is required to check whether or not the gathered data in the
classification tests are accurate or not.
3.2. Sources of Error
Before analysing the IR spectra of the sample, it is important to consider the sources of error in the
experiment. The first concerns the purity of the sample. Due to the fact that no check for purity was
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done for the unknown compounds such as chromatography or a boiling point test, the purity of the
sample cannot be determined and could therefore have a huge influence in the resulting spectra.
Another possible source of error is the residual external vibrations that the background scan of the
FTIR machine failed to consider, this could lead to an inaccurate reading of the vibration resulting in
unclear peaks which could affect the interpretation of the diagnostic region and could render the
fingerprint region unusable for a database cross-check to determine the actual identity of the
compound.
3.3. Results and Analysis of the FTIR Spectra
The following figure is the resulting FTIR spectrum of unknown H:

Figure 1. FTIR spectrum of unknown H.

From Figure 1, the peaks of interest can then be tabulated and evaluated. It is worth reminding
that the spectrum taken from the device is prone to error due to not being checked for impurities and
residual external vibrations that were not taken into account by background scan. The following table
enumerates all of the relevant peaks as well as the functional group associated with the peak.
Table 3. Peaks of interest from Unknown H

Wavenumber Intensity Functional Group

(cm-1)

3404.54 broad, strong O-H stretch (Alcohol)

2987.53 broad, medium O-H stretch (Carboxylic Acid)

1718.79 narrow, strong C=O stretch (Carboxylic Acid)

By taking all of the peaks from figure 1, it can be said that the compound has at least 1 alcohol
group and a carboxylic acid group. However, the results of unknown H from Table 1, should also be
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considered. Both the FTIR spectrum and the classification test, specifically the Lucas's Test, agree on
the fact that the unknown has an alcohol group. Due to the fact that the Sodium Bicarbonate Test was
not conducted for unknown H, the presence of a carboxylic acid was initially not detected. However
the FTIR did heavily suggest the presence of a carboxylic acid by showing the O-H and C=O stretch,
thus it can be said that the unknown did have a carboxylic acid group.
Now there is enough information in order to assume what is the possible identities of unknown
H. From the results of the classification tests and the resulting FTIR spectrum, it can be said that the
unknown has two functional groups; an alcohol(possibly secondary) and a carboxylic acid. The
simplest compound that can fits this description is lactic acid.

Figure 2. FTIR of lactic acid.

In terms of physical properties, both the unknown and lactic acid are odourless, white solids
at room temperature. In order to check the answer in more detail, the sample’s spectrum from Figure 1
to a standard spectrum in Figure 2(7). It can be seen that the two spectra match in terms of relevant
structure in both the fingerprint and diagnostic regions. There are still some notable differences in the
spectra of the two. This difference can be attributed to residual external vibrations that were not taken
into consideration by the FTIR machine. Thus it can be confirmed that unknown H is most likely lactic
acid.
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Figure 3. FTIR spectrum of unknown C.

The spectrum of unknown C, illustrated in Figure 3, is more complicated to analyse than the
spectrum of unknown H. An example of the possible complications of the analysis are the spiked
peaks from ~3500cm-1 to ~3200cm-1, which cannot be fully described as an O-H stretch of an alcohol
or an amine stretch. It is ultimately decided that the peaks mentioned represent an alcohol’s O-H
stretch because of its resemblance to the stretching of multiple O-H groups where some of which are
participating in hydrogen bonding(8). The following table enumerates all of the interpretations of the
relevant peaks in the diagnostic region of Figure 3.

Table 4. Peaks of interest from Unknown C

Wavenumber Intensity Functional Group

(cm-1)

3523.88 broad, strong O-H stretch (Alcohol)

3404.32 broad, strong O-H stretch (Alcohol)

3308.91 broad, strong O-H stretch (Alcohol)

~3200.00 broad, strong O-H stretch (Alcohol)

1752.23 narrow, medium C=O stretch (Ester)

1654.56 narrow, strong C=C stretch (Alkene)

In this case, the results from the classification tests contradict the spectra. The classification
tests indicate that the unknown has a secondary amine, an aldehyde, a carboxylic acid, and is an
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alkane. These conclusions are deemed to be inaccurate as the FTIR spectrum does not support the
presence of such structural properties. The FTIR spectrum indicated the presence of 4 different alcohol
groups, an ester, and an alkene. The simplest compound that fits this description is ascorbic acid, in
order to check if this conclusion is correct, Figure 3 was compared to a standard FTIR for ascorbic
acid.

Figure 4. FTIR of ascorbic acid

By comparing Figure 3 and Figure 4, which is a standard FTIR for ascorbic acid(9), it can be
seen that there are some resemblance. Both spectra feature the spikes from ~3500cm-1 to ~3200cm-1
which indicate the O-H stretches of multiple alcohol groups. The fingerprint region of the sample also
resembles the standard ascorbic acid fingerprint region. Just like in the unknown H, the differences can
be attributed to the residual external vibrations, however this may not be the only factor at play and the
sample may have been contaminated which could have contributed to the inconsistencies in the spectra
and the contradictory results from the classification tests. It is likely the case however that the faulty
results of the classification tests are due to improper conduct of the classification tests.

4. Conclusion
By taking into account both the results of the classification tests from a previous experiment and the
results from the FTIR spectra of the unknown compounds, it can be concluded that the identity of
unknown H is lactic acid, as is supported by the classification tests and the IR spectrum, and the
identity of unknown C is ascorbic acid due to the similarities between the standard IR spectrum of
ascorbic acid and the IR spectrum of unknown C. It was also determined that the classification tests
done were inaccurate in some parts, thus the results cannot be used in full confidence in determining
the identity of the unknown.

5. Recommendation
It is recommended to conduct the classification tests again to properly determine the reactivity of the
unknown compounds. It is also recommended to subject the unknowns to FTIR spectroscopy again in
order to gain a clearer spectrum and to minimise the sources of error such as external vibrations and
sample purity. Finally, in order to fully confirm the identities of both unknowns, it is recommended to
subject the samples to NMR or MS spectroscopy
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References

[1] Smith S. Organic functional group test: A Level Chemistry [Internet]. Online Chemistry Tutor.
2016 [cited 2023 Jan 30]. Available from:
https://www.onlinechemistrytutor.net/organic-tests-level-chemistry/
[2] Lisa NicholsButte College. 6.4A: Overview of chemical tests [Internet]. Chemistry LibreTexts.
Libretexts; 2020 [cited 2023 Jan 30]. Available from:
https://chem.libretexts.org/Courses/SUNY_Oneonta/Chem_221%3A_Organic_Chemistry_I_(
Bennett)/2%3ALab_Textbook_(Nichols)/06%3A_Miscellaneous_Techniques/6.04%3A_Chem
ical_Tests/6.4A%3A_Overview_of_Chemical_Tests
[3] Chu S, Graybeal JD, Stoner JO, Hurst GS. spectroscopy. In: Encyclopedia Britannica. 2023.
[4] Louise Saul MS. The different types of spectroscopy for chemical analysis [Internet].
Azooptics.com. 2018 [cited 2023 Jan 30]. Available from:
https://www.azooptics.com/Article.aspx?ArticleID=1382
[5] No title [Internet]. Sigmaaldrich.com. [cited 2023 Jan 30]. Available from:
https://www.sigmaaldrich.com/PH/en/technical-documents/technical-article/analytical-chemist
ry/photometry-and-reflectometry/ftir-spectroscopy
[6] Griffiths, P.R.; Holmes, C. 2002. Handbook of Vibrational Spectroscopy, Vol 1. Chichester:
John Wiley and Sons.
[7] Faisal, Aseel & khalaf, Hussein. 2019. Synthesis of polylactic acid using Zn powder under
microwave irradiation. IOP Conference Series: Materials Science and Engineering. 571.
012085. 10.1088/1757-899X/571/1/012085.
[8] Smith BC. Infrared spectral interpretation: A systematic approach, second edition. 2nd ed.
Boca Raton, FL: CRC Press; 2021.
[9] Senapati, Sadhana & Das, Smrutiprava & Patnaik, A. (2012). Kinetics and Mechanism of
Oxidation of L-Ascorbic Acid by Pt(IV)(aq) in Aqueous Hydrochloric Acid Medium.
Advances in Physical Chemistry. 2012. 10.1155/2012/143734.