Mechanism, Development and Comparison of Infrared and Raman

Spectra in the Pharmaceutical Diagnosis and Living Cell Detection

Mengying Cao

1,*,† a

and Haorong Xu

2,† b

School of Petrochemical Engineering, Liaoning Petrochemical University, Fushun, China

Hangzhou New Channel School, Hangzhou, China

†

These authors contributed equally

Keywords: Infrared Spectra, Raman Spectra, Pharmaceutical Diagnosis, Living Cell Detection, Mechanism.

Abstract: Nowadays, applications of new detection methods for pharmaceutical diagnosis and living cell detection

have attracted more and more attention. Among these detection methods, there is a large improvement in the

Raman spectroscopy, which leads to a wide range of applications in different fields. The infrared

spectroscopy is still act as a main and important techniques in the different areas. This research compares

the similarities and differences of these two technologies. At the same time, this research will concern about

the mechanism of Raman and infrared spectroscopy and their applications in some domains, especially in

the various disease and pharmaceutical fields. Applications of Raman spectroscopy for detection of living

cells is systematically analyzed, including detection mechanism and specific detection process. For

pharmaceutical diagnosis, the advantages and disadvantages of Raman spectroscopy are present in this

research. This research provides a new idea for the applications of Raman spectroscopy and infrared

spectroscopy in the field of disease detection.

1 INTRODUCTION

Raman spectroscopy is a vibrational spectroscopy

technique with “fingerprint” identification of

molecular composition and structure. It can

distinguish samples of various substances and also is

one of the main analytical techniques used in optical

metrology.

Raman spectroscopy is not interfered by aqueous

solvents. As a result, it can be used in biomedical

analysis better than traditional infrared spectroscopy.

Raman spectroscopy is a promising diagnostic tool

that can help uncover the molecular basis of diseases

and provide objective, quantifiable molecular

information for diagnosis and therapeutic evaluation.

Raman scattering occurs when the polarizability

changes during the interaction of light with

molecular vibration/rotation and molecular motion.

When light interacts with a molecule, it can be

excited to a transient virtual state, which

immediately returns to the vibrationally excited state

of the electron’s ground state. Due to this

https://orcid.org/0000-0002-2845-1677

https://orcid.org/0000-0001-6426-2358

interaction, a small amount of energy is transferred

or removed from the molecule, and the resulting

scattered light is red-shifted or blue-shifted, which

contains encoded vibrational molecular information.

This causes light to be scattered at the optical

frequency at which it moves on the incident light.

By monitoring the intensity distribution of inelastic

scattered light as a function of frequency, a unique

spectral fingerprint of tissue samples was obtained.

Because each sample has a unique composition,

spectral profiles generated by Raman active

functional groups of nucleic acids, proteins, lipids

and carbohydrates. Raman scattering in tissue

provides rich information about the vibrational

structure of proteins, gag, lipids and DNA.

The Raman spectrum is usually recorded in the

so-called fingerprint region, which contains

relatively weak but highly specific Raman peaks.

More recently, additional attention has been paid to

the use of the high wave-number region, which

contains less specific Raman bands but shows a

higher degree of signal intensity. An important

advantage of Raman spectroscopy is the low

intensity of the water wave segment, which makes

the analysis of biomaterials very difficult in infrared

540

Cao, M. and Xu, H.

Mechanism, Development and Comparison of Infrared and Raman Spectra in the Pharmaceutical Diagnosis and Living Cell Detection.

DOI: 10.5220/0011230700003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 540-546

ISBN: 978-989-758-595-1

spectroscopy. However, Raman spectroscopy and

infrared spectroscopy are complementary, Raman

cannot replace infrared. It can complement each

other to provide more comprehensive and accurate

information about molecular vibration state and

molecular structure. But in some routine detection,

using Raman instead of infrared or using Raman as a

supplement to infrared, can improve the work

efficiency and detection speed. Many active

components in medicinal materials have different

pharmacological effects due to their different

functional groups and configurations. Raman

spectroscopy has certain advantages in structural

analysis and isomer identification of drug active

components due to its high selectivity and no need

for separation in mixture analysis.

Raman spectroscopy has powerful analytical

capability and can provide quantitative information

about chemical composition in biological samples. It

uses inelastic scattering of light to provide the

spectral characteristics of the internal structure and

conformation of the cell, thus reflecting the material

changes of the sample. In the process of tissue and

cytopathic disease, the structure, content and

conformation of each component in the cell will

change to varying degrees. It follows that it may

have a new role in the diagnosis of disease.

Raman spectroscopy can detect substance

changes in samples. And similarly, it can reflect

substance changes in the body caused by early

cancer. Cancer has been threatening people’s life

and health. In the context of an increasingly high

incidence of cancer worldwide, early diagnosis of

cancer is particularly important. There are numerous

lives behind the huge numbers in cancer reports.

Early detection, diagnosis and treatment will lead to

a greater chance of survival for patients. The Raman

spectrum of the tissue can be measured using a

microscope or custom optical fiber. In simple terms,

a single mode fiber is used to couple the laser to a

microscope and illuminate the sample with a

microscope objective. Confocal imaging based on

Raman spectrum can be achieved by using optical

fiber to collect backscattered light. A single optical

fiber acts as a pinhole to couple the light to a high-

throughput spectrometer, which is then dispersed to

a charge-coupled device (CCD) camera.

At present, the diagnosis of cancer mainly

depends on X-ray, CT examination, B ultrasound,

MRI examination and tumor marker detection. And

biopsy is still the best indicator of cancer

confirmation. Conventional imaging results can only

provide the basis for diagnosis, but they are not

sensitive and economical, and will bring great pain

to patients. By contrast, Raman spectroscopy has

high chemical specificity and can obtain abundant

molecular information without staining or labeling

the specimen. Raman spectroscopy, as a non-

invasive means of detection, can directly detect

biological samples, which is not only more sensitive,

but also relieves the pain and economic burden of

cancer patients.

Raman, an Indian physicist, irradiated benzene

liquid with a mercury lamp in 1928 and discovered a

new radiation spectrum line: this is a new molecular

radiation, called Raman scattering. Raman won the

Nobel Prize in physics in 1930 for the discovery of

this new molecular radiation and many light

scattering research achievements. At the same time,

Landsberg and Mandelstad of the former Soviet

Union reported the discovery of a similar

phenomenon in quartz crystals, namely Raman

scattering caused by optical phonons, called merger

scattering.

Roquette and Cabens in France and Wood in the

US confirmed the results of Raman’s observational

study. Because the Raman effect is too weak, it is

difficult to observe and study the weak Raman

scattering signal, let alone measure and study the

higher order Raman scattering effect. And the

volume of the tested sample must be large enough,

colorless, no dust, no fluorescence and so on. By the

mid-1940s, the progress of infrared technology and

commercialization of Raman spectroscopy

applications declined.

After 1960, the appearance of ruby laser makes

the study of Raman scattering into a new period.

Because the laser has good monochromaticity,

strong directivity and high-power density, using it as

excitation light source greatly improves the

excitation efficiency. It is an ideal light source for

Raman spectroscopy. With the improvement of

detection technology and the reduction of the

requirements for tested samples, Raman

spectroscopy has been widely used in physics,

chemistry, medicine, industry and other fields.

In the mid-1970s, the appearance of laser Raman

probe brought the possibility of microanalysis. Since

the 1980s, Spex company of the United States and

Rrinshow company of the United Kingdom have

launched a confocal laser Raman spectrometer,

bitman probe, because of the use of notchfilter to

filter out the excitation light, so that stray light is

suppressed. It is not necessary to use double or even

triple monochromator, and only need to use a single

monochromator. The efficiency of the light source is

greatly improved, so that the power of the incident

Mechanism, Development and Comparison of Infrared and Raman Spectra in the Pharmaceutical Diagnosis and Living Cell Detection

541

light can be very low. And the sensitivity is greatly

improved.

2 CONVENTIONAL INFRARED

SPECTROSCOPY

Infrared spectrum is also called infrared absorption

spectrum. It is the characteristic absorption spectrum

curve generated by resonance absorption between

infrared photon and molecular vibration and rotation

quantized energy level.

2.1 Mechanism

In organic molecules, the atoms that form chemical

bonds or functional groups are in a state of constant

vibration at frequencies comparable to those of

infrared light. When organic molecules are irradiated

with infrared light, chemical bonds or functional

groups in the molecules can occur vibration

absorption. Different chemical bonds or functional

groups absorption frequency is different. It will be in

different positions in the infrared spectrum, so as to

obtain the information of what kind of chemical

bonds or functional groups in the molecule.

The infrared spectrum is usually divided into

three regions: near infrared region (0.75~2.5 μm),

middle infrared region (2.5~25 μm) and far infrared

region (25~1000 μm). Generally speaking, the near

infrared spectrum is produced by the double

frequency and combination frequency of molecules.

The mid-infrared spectrum belongs to the

fundamental frequency vibration spectrum of

molecules. Far infrared spectrum belongs to the

rotational spectrum of molecules and vibration

spectrum of some groups.

2.2 Development

In the 1960s, a linear relationship between the

content of substances and the absorption peaks of

several different wavelength points in the near

infrared region is demonstrated, which made this

technique widely used in the analysis of agricultural

products. In the middle and late 1960s, the classical

near infrared spectroscopy (NIR) was exposed to the

weakness of low sensitivity and poor anti-

interference. With the emergence of various new

analytical techniques, people ignored the application

of this technique in analytical testing.

The successful application of multiple correction

technology in spectral analysis in 1970s promoted the

promotion of near-infrared spectroscopy technology.

In the late 1980s, with the development of computer

technology, the digitization of analytical instruments

and stoichiometry have been fully developed. The

good results obtained by stoichiometry in solving

spectral information extraction and background

interference have led to the application of NIR

spectroscopy in various fields.

Figure 1: Schematic illustration Raman spectroscopy in measuring process.

In the 1990s, the application of NIR spectroscopy

in the industrial field expanded rapidly, and the

literature on the research and application of NIR

spectroscopy increased almost exponentially,

becoming one of the fastest developing and most

eye-catching analytical techniques. Because of its

good transmission characteristics in conventional

optical fibers, this technology is also applied in the

field of online analysis.

3 COMPARISON OF RAMAN

AND INFRARED SPECTRA

3.1 Similarities

For a given bond, the infrared absorption frequency

is equal to the Raman shift and represents the energy

of the first vibrational level. For a given compound,

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

542

the infrared absorption wave number and Raman

displacement of some peaks are exactly the same,

and both of them are in the infrared region, both of

which reflect the molecular structure information.

Raman spectroscopy, like infrared spectroscopy, is

also used to detect the vibration and rotational

energy levels of matter molecules.

The vibration which has symmetry relation with

the center of symmetry is invisible in infrared, but

visible in Raman. The vibration with no symmetry

relation to the center of symmetry is visible by

infrared, but not by Raman.

3.2 Differences

As shown in Figure 1, the incident light and

detection light of infrared spectrum are infrared

light, while the incident light of Raman spectrum is

mostly visible light, and the scattered light is also

visible light. Infrared spectroscopy measures

absorption of light, while Raman measures

scattering of light. When photons interact with

molecules, they do so through electric dipole

moment transitions.

Therefore, molecules that have no polarity or

symmetry, have essentially no infrared absorption

effect, because there is no electric dipole moment.

Raman spectrum is not absorption spectrum, but

after the incident photon resonates with the

molecular vibrational and rotational quantized

energy level, the photon emits at another frequency.

The energy difference between the incoming and

outgoing photons is equal to the vibrational and

rotational transition energy levels of the molecules

involved in the interaction.

Unlike infrared absorption spectroscopy, Raman

spectroscopy is a photon-molecular interaction of

higher order, which is much weaker than infrared

absorption spectroscopy. However, because the

mechanism of its generation is the electric

quadrupole moment or magnetic dipole moment

transition, it does not require the molecule itself to

have polarity, so it is particularly suitable for the

detection of those symmetric molecules without

polarity.

Infrared spectroscopy measures the absorption of

light, expressed by wave number or wavelength,

while Raman spectroscopy measures the scattering

of light, and the horizontal axis is Raman

displacement. Infrared spectroscopy mainly reflects

the functional groups of molecules while Raman

spectroscopy mainly reflects the framework of

molecules and is mainly used to analyze biological

macromolecules.

4 LIVING CELLS DETECTION

4.1 Mecahnism

Raman spectroscopy has powerful analytical

capability and can provide quantitative information

about chemical composition in biological samples. It

uses inelastic scattering of light to provide the

spectral characteristics of the internal structure and

conformation of the cell, thus reflecting the material

changes of the sample. When photons of

monochromatic beam interact with molecules,

elastic collision and inelastic collision can occur, as

shown in Figure 3.

In the inelastic scattering process, energy

exchange occurs between photon and molecule, and

photon loses part of energy due to scattering by

molecule, resulting in the change of photon

frequency. But the difference between the frequency

of the scattered light and the frequency of the

incident light does not change because the frequency

of the incident light changes. Among them, the

change in the frequency of light is collectively called

the Raman shift. Raman scattering light can carry

information of substance because it is affected by its

structure.

According to the Boltzmann distribution law,

due to thermal equilibrium, the number of molecules

in the second highest energy level is always smaller

than the number of molecules in the lower energy

level, so the intensity of stokes Raman scattering

light is always greater than the intensity of anti-

Stokes Raman scattering light.

The Raman displacement of material is

independent of the incident light frequency, but

related to the vibration and rotational energy level

structure of the molecule, which are inherent

characteristics of the molecule. Therefore, any

substance with Raman activity has its own specific

Raman shift. If it wants to identify a substance, it

just measures it and find out its characteristic Raman

spectrum.

4.2 Detection of Living Cells

When the sample is irradiated by a certain frequency

of laser beam, it will emit Raman scattering of light,

which provides a lot of molecular information.

Through the interpretation of Raman spectrum, the

molecular type, spatial structure, chemical bond and

other information can be obtained. Microscopic

Raman spectroscopy technology can provide Raman

spectra of complete living cells, from which the

structure and changes of several major biological

Mechanism, Development and Comparison of Infrared and Raman Spectra in the Pharmaceutical Diagnosis and Living Cell Detection

543

macromolecules in complete living cells, such as

proteins, DNA, lipids and carbohydrates. The

traditional measurement of Raman spectrum is

divided into point scanning and line scanning.

Figure 2: The Raman shift under different experimental conditions.

Point-to-point scanning imaging involves

focusing a laser into a point, moving the sample

under the laser with an automatic sample stand, and

sequentially collecting Raman spectra through an

array of points across the designated area of interest

on the sample. Linear focusing scanning imaging is

similar to point-to-point scanning imaging. The

difference is to shine a laser on a line rather than a

point on the sample. This method can collect spectra

simultaneously from multiple locations on the

sample, and can use more laser power, but reduces

the exposure time and does not damage the sample.

Proteins are the main components of cells and

the material basis of many cell functions, such as

cell catalytic reaction, material transport, immune

function, genetics and metabolism. Raman

spectroscopy can not only provide the structural

characteristics of amino acids, but also be used for

quantitative analysis of secondary structure of

proteins. As shown Figure 2, an obvious Raman

shift can be observed when some chemical reactions

occur.

Lipids are an important part of cell membranes.

Raman spectroscopy can be used to study the

composition of cell membrane, the location of lipids

in various states and their interactions with various

ions. Elucidating the composition and spatial

structure of lipid membranes in living cells can be

applied in many fields such as membrane

pathophysiology and membrane pathophysiology.

The following is a microscopic Raman analysis

for the lipidomes of individual organelles. There are

hundreds of thousands of chemically different lipids.

Although they usually show only minor differences

in chemical structure, they tend to block chemically

selective probes from labeling specific lipids.

Figure 3: The used Raman spectrometer in laboratory.

4.3 Use of Raman Spectra

Raman spectrometers have evolved from ordinary

academic laboratory instruments to powerful

commercial solution-based systems. Update

instruments are easy to use because they do not

require the user to constantly adjust or have complex

optical knowledge, and interact with the Raman

spectrum library.

At present, there are two main methods to

analyze Raman data: univariate and multivariate.

The first approach uses the area, intensity, or center

of gravity characteristics of the Raman spectrum to

understand sample chemistry. Figure 4 shows the

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544

basic composition of Raman spectrometer. Although

univariate data analysis can be used directly, it

requires that the components have sufficient Raman

bands to distinguish and be unique. Overlapping

bands in biological and pharmaceutical area make

multivariate data analysis techniques necessary.

High resolution system that enables rapid

measurement and is suitable for in vivo clinical

applications, and it is often used in detecting living

tissue such as tumor.

Figure 4: The typical Raman spectrometer.

5 RAMN SPECTRA IN

PHARMACEUTICAL

DIAGNOSIS

The wide origin, variety and complexity of Chinese

medicinal materials bring many difficulties to the

inspection and management of Chinese medicinal

materials. In recent years, rapid and nondestructive

identification of Chinese medicinal materials by

Raman spectroscopy has attracted more attention. By

using Raman spectroscopy, 12 different habitats and

different planting modes and different collecting time

of traditional Chinese medicine radix scutellariae are

analyzed. The results show that the characteristic

peak frequency and intensity of Raman spectroscopy

to identify different ways of planting of radix

scutellariae samples than traditional method. This is

more direct, fast, and do not destroy samples, more

accurate science. Many active components in

Chinese medicinal materials have different

pharmacological effects due to their different

functional groups and configurations. Raman

spectroscopy has been widely used in the structural

analysis and isomer identification of traditional

Chinese medicine (TCM) due to its strong selectivity

and the absence of separation in mixture analysis.

With the appearance of portable Raman

spectrometer, drug regulatory departments have

considered it as an important tool for drug

counterfeiting. According to the existing literature,

Raman spectroscopy is quick and can significantly

improve the efficiency of drug market regulation.

Raman spectrum can give fingerprint information

about compound structure, and can distinguish some

pharmaceutical excipients by Raman band, which has

certain advantages over infrared spectrum in some

aspects. It can be used for quality control of

pharmaceutical excipients. The characterization and

structure of these probes is shown in the Figure 5.

Figure 5: Structure and characterization of SERS probes

6 CONCLUSIONS

This paper shows the comparison between infrared

and Raman spectra. And application of Raman

spectroscopy for the pharmaceutical diagnosis and

living cell detection is also analyzed. Raman

spectroscopy has broad prospects in cell sorting and

nondestructive detection of cancer detection.

However, Raman spectroscopy usually has weak

signal. If the general fluorescence signal is stronger

than Raman signal, the fluorescence summit is

superimposed on Raman peak, and its interference to

signal judgment is relatively large. Fortunately,

methods to remove strong fluorescence background

to solve the influence of fluorescence background on

the extraction of Raman spectral components have

been developed.

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