Applications Of Infrared Spectroscopy Biology Essay

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As change in energy between these three region causes absorption of various bonds and molecules making different types of vibration. Like heavy atoms like some organometallic or inorganic molecules only absorbs smallest energetic far infrared region (FIR), organic atoms absorbs mid infrared region (MIR).11 Near infrared region (NIR) excite harmonic or overtone vibrations.11citation

More on protein

Infrared spectroscopy (IRS)is definitely most important classical analytical technique fordetermination of structuresvarious molecules. Infrared spectroscopy (IRS) has greater sensitivity to chemical composition, bonding and design ofmolecules hence study of protein by IR is very useful method. As differentiation and classification of many microorganisms is now possible by IRS techniques.1


IRS can be identified from small soluble to large membrane protein mostly in a short calculating time usually 1 microsecond (µs), less amount sample require usually 10 to 100 micrograms (μg) and low cost. Some advantages of IRS in protein study is widely recognized in academic world but development and further advances of IRS in this field still need to come be exploit commercially. 1

Absorption of infrared spectrum happens when frequency of light and frequency of vibration of molecule matches and this frequency of vibration depend on strength and polarity of the vibrating bonds which actually influenced by inter- and intramolecular effects.1In short individual molecule starts specific bonds to vibrate after infrared radiation (IR) absorption as similar to that of its diatomic oscillator.11

After absorption of near, mid or far-infrared radiationcauses an excitation of vibrational and dipole moment changes of molecules and any absorption of these particular band is depends on bond type like single, triple or aromatic, vibrating masses, electron loosing or giving position, inter or intramolecular forces, polarity and other vibration coupling.1

All molecules containing polar bonds absorb infrared radiation as well as absorption capacity increases as polarity of these bond increases.1

Mostly all molecules absorb infrared radiation hence big molecules gives overlapping bands and this concept of IR is called marker free technique. But a study change this idea by encoding Nitrile group (C≡N) probe genetically by infrared label for binding to myoglobin. 1,14

The spectrum of infrared (transmittance %)is plotted against the wavenumber (ū) (inverse of the wavelength (ʎ)) directly proportional to the transition energy (E) and has the unit cm−1. 1

Wavelength (ÊŽ) = velocity of light (c) / frequency (v) [cm/sec]

Hence,wavenumber(Å«)=frequency(v)/velocity of light(c) [sec/cm]

Energy of quantum E is corresponds to radiation of frequency (u)

E is given by Bohr equation as follow,

E = hu= hūc

h is the planck constant (h = 6.626 X 10-34Js) and c is the velocity of light remains the constant.

Therefore energy of radiation is directly proportional to wavenumbers and inversely proportional to the wavelength, this can be shown as follow, E α ū α 1/ʎ

13, 15

Information that can be derived from Infrared Spectroscopy.

1.Chemical structure of the vibrating group.

2. Chemical properties of neighbouring groups in a molecule

3.Redox state

4.Bond parameters

5.Bond angles and conformation

6.Hydrogen bonding

7.Electric fields

8.Conformational freedom


Near-infrared spectroscopy (NIRS) was not that much popular until 1960 but all credit for successful implementation of NIRS is goes to KarlNorris who acknowledged the capacity and start the use of Near-infrared in quality controls of some types of food, agriculturalcomponents in the industry.11 The revised published document of European Medicines Agency (EMA) in February 2009 shows use of NIR by the pharmaceutical industry current Good Mnufacturing Practices ( cGMP ) recommends the usage of NIR in pharmaceutical and analytical procedure of dosage forms.11

Near-infrared spectroscopy (NIRS) is very important non invasive 11 analytical method about Green Chemistry and Process Analytical Technology (PAT). 10

With the aim of dispatch any batch of pharmaceutical drug at the market which analyzed by NIRS or any analytical technique must have the assurance that this lot will be providing safe and bioavailable drugs to the patient. This is only possible when there is enough assurance about results generated by analytical methodsisof high quality and reliable.10

Therefore, any analytical method, the validation step is must towards the end of development of that method with permission of sufficient guarantees that the future outcomes from those instruments must be precise and concise to the true value. 10

Revised document of European Pharmacopoeia (2.2.40) recently published PAT concepts dealing with NIRS. 10

The first validation method of NIRS method is traditional chemometric method involves two steps with the use of 'a calibration set of data' known as root mean square error of calibration (RMSEC) and the use of 'cross validation technique' known as root mean square error of the cross validation (RMSECV).'

All RMSE criteria (R2cal) are built on the calculated root mean squared error



wheren sample number,ytandÅ·tis reference and predicted value for sample t respectively. 10

Second validation method of NIRS is ICH Q2 validation which measures precision (repeated and intermediateprecision), accuracy, linearity and a valid concentration range. 10, 16

NIRS method validated by ICH Q2 guideline includes water content determination in active pharmaceutical ingredient (API) in many drug products or drug production steps as well as quantification of adjuvant in vaccines, excipients of antibacterial and antifungal agents. 10

Near-infrared (NIR) spectroscopy is now considered as resourceful analyticalmethod in most of the field including the pharmaceutical sciences. The application of NIRS used for control and measurement of physicochemical parameters of the end medicinal products like thickness, disintegration time, size, porosity, hardness and compression strength also used in various steps of manufacturing processes of tablet, capsule like mixing, granulation or drying to determine the end-point of these processes. 11

following fi

gure 1 shows the implementation of NIR to in-,on-and at-line processes control in various steps of manufacturing process.

Figure 1 Usage of NIR spectroscopy in various critical stages of pharmaceutical manufacturing process from API, excipients to final products. 11


Identification and verification of special vibration mode of residual solvent like methanol and ethanol inpharmaceutical products cannot be clearly visualized in original spectra.

Therefore 2 Dimensional (2D) nearinfrared spectroscopy (NIRS) is very useful for difficult analysis and easily distinguish between exact bands of ethanol and methanol which are between 5000-4500 cm−1 and not clearly seen.11

Molecules analyzed by NIRS possess change in dipole moment; characteristic feature of NIR is distinguished between overtones and combination bands of OH, CH and NH. Combination bonds are the sum of two vibrations and overtone is due to excitation of electron at high energy level. Total spectrum of NIR contains hydroxyl (OH) stretching region (first overtone), combination of hydroxyl group (OH) stretching and second overtone of carbonyl (C= O) found in 4800-5300 cm−1 region,first overtone of alkyl(CH) stretching found in 5900-5350 cm−1, second overtone of alkyl (CH) stretching found in 6500-9000cm−1, also upto 4 overtones from the absorptions of methoxy (C H), aromatic (C H), amines (primary,

secondary, and tertiary NH) or SH. 11

discussion of NIR

Advantages an disadvantages of NIR

The benefits of NIRS method are many like it is noninvasive, nondestructive techniques, ability to penetrate glass container, less measurement time less than 1 minute, no sample preparation with minimal drying and grinding, can be obtained several spectra of same product to increase accurate result and high frequency of spectra. It has low cost analysis but requires high initial financial investment for the instrument. Not any existing analytical techniques like colorimetry, HPLCis reliable for quantification of polymer molecules like Polyethylene glycol bound to a protein but NIRS is effective method to evaluate the of polymer moiety conjugate to protein.

NIRS, also recommended by food drug administration (FDA) and Pharmacopoeias and increasingly adopted analytical technique as Process Analytical Technology (PAT) in pharmaceutical industry.11

NIRS also has some demerits like less sensitivity for determination of molecules having concentration less than 0.1% (w/w), difficult to transfer calibration between two instruments of same manufacturer and for chemometric calculations and development of calibration requires trained personnel like of good chemists, pharmaceutical technologists and mathematician

One of the difficulties of NIR with aqueous environment solutions of API for quantification. Due to high absorption of water broad peak appears on a spectrum that causes limited detection for other smaller quantity of compounds which are present in the analyzed sample.11

Still, NIRS certainly play an important role in pharmaceutical industry mainly for an analysis of many continuous pharmaceutical processes, end products and even initial raw materials.11 Apart from pharmaceutical processes NIR also effectively applied in biotechnology field by analyzing and detecting genetically modified organisms by comparing DNA fragments, protein molecule.11

Obsessive compulsive disorder (OCD) is an anxiety disorder described by disturbing thoughts that produce, apprehension, fear, uneasiness or worry,repetitive behaviors meant to reduce the related anxiety or worry; or obsessions and compulsions mostly seen in elderly patients and 2 to 3% prevalence in the total population.8


This is due to some dysfunction, injury or disease. After 50 years OCD is not commonly seen but most commonly seen psychiatric condition and frequently associated due to structural cerebraldamage or did not receive an effective treatment for OCD since past many years.

Moreover older patient with untreated OCD are probably experienced multiple area disability which enable them to live independently in the society and decrease a quality of life. Hence long lasting OCD has side effect on older patent's mental and physical health, by experiencing cardiovascular dysfunction and rising coronary heart disease. 17


Subcortical structures and frontal cortex be part of the cause in the Pathophysiology of obsessive-compulsive disorder (OCD) is suggested by functional neuro-imaging studies of human brain. NIRS can measures functional imaging of brain activity i.e. fluctuation in the redox state of cytochrome c oxidase and change in the concentration of deoxy-hemoglobin (deoxy-Hb) and oxy-hemoglobin (oxy-Hb) by their NIR region spectra between 700 and 1000 nm. Neurovascular coupling increase activation of brain which lead to elevation of cerebral blood flow without a similar rise of oxygen consumption thus increasing concentration of oxy-Hb and a decrease in the deoxy-Hb.8

NIRS investigate neurobiological function in 12 OCD patients (7 males, 5 females) with 12 sex- and age-matched control patients. The prefrontal lobe measures concentrations of oxyhemoglobin (oxy-Hb) after every 0.1 s, using 24 channels near infrared spectroscopy machine (Hitachi ETG-100, Hitachi Medical Corporation, Tokyo, Japan) by calculating the absorption of 2 wavelengths of NIR (760 and 840 nm).8

This study shows OCD group patients showed Oxy-Hb in the prefrontal cortex were very less than control group in particular in the left lateral prefrontal cortex. Hence this revealed that OCD patients have less prefrontal hemodynamic responses.8

Also when NIRS in response to the Stroop color-word task investigate cerebral hemodynamic changes in 12 OCD children patients and 12 healthy sex- and age-matched controls then it is found that oxy-Hb changes in the prefrontal cortex among the OCD children patients showed much decreased amount than control group.8

For this study 12 healthy intelligent quotient control subjects (7 males, 5 females) were taken through publishing local print advertising and did not have any history or symptoms of OCD or other neurological problem. All subjects were Japanese, right handed right-handed and Japanese. Nara Medical University obtained ethical approval for the study as well as about the study written informed consent was obtained by all subjects.8


Using positron emission tomography (PET) in OCD patient find high abnormality in localized regions of the brain, including the caudate nucleus, anterior cingulate cortex and orbitofrontal cortex. Consistent abnormality shown by using single photon emission computed

tomography (SPECT) as OCD patent shows dysfunction of the caudate nucleus and orbitofrontal cortex as well as abnormality of prefrontal cortex shown by functional magnetic resonance imaging (fMRI). These methodologies like SPECT, PET and fMRI have some demerits like it needs large apparatuses hence cannot be use at bedside for diagnostic & prevents also not provide high time resolution. In contrast, multi-channel NIRS has developed as high resolution bedside functional mapping of the cerebral cortex.8

NIRS is actually suitable for other psychiatric subjects with some advantages over other techniques used like NIRS is very insensitive to motion artifact and can be used the study that cause a motion in the subject. Secondly, without any distraction and without changing natural sitting subjects can be easily examined. Third reason is as compare to other neuroimaging modalities cost of NIRS is very low with relatively easy implementing of the procedure. Fourth reason is NIRS gives high temporal resolution which is very effective in characterizing prefrontal activity and assessed brain function in many psychiatric disorders like depression, schizophrenia, attention deficit hyperactivity disorder (ADHD), bipolar disorder and dementia.8

Still scientist finding an effective treatment for OCD in older patients and possibly surprising as they identified that there is significant lack of research for this disease association.17

In addition, Partial Least Squares Regression analysis was applied to estimate the age of blood

stains on colored backgrounds. The age of blood stains up to 1 month old was estimated successfully with

a root mean squared error of prediction of 8.9%. These findings are an important step toward the practical

implementation of blood stain identification and age estimation in forensic casework, where a large

variety of backgrounds can be encountered.


In this study, we identified NIR spectral features of bloodstains

which can be used for blood stain identification and age

estimation, even when blood is deposited on colored backgrounds.


At the forensic casework, detection of a blood stain on plain and dark background is more important and this identification is required to prove the presence of blood in the stain. These identified stains can be further used for DNA analysis and construction.

Other identification test using chemical for identification like luminescence, fluorescence, change in color most likely lost the blood strain patternand interferes with later teat, DNA analysis or may harmful for investigators. Therefore there is need for non-destructive method which used for identification easily and quick without sampling or lengthy laboratory examination. Application of visible reflectance can easily identified bllod stain non-destructively but this identification severly hampered by absorption by dark background. As NIR less absorbrd by many dark colored substrate therefore NIR solve this problem. Unlike visible spectroscopy absorption, NIR absorbes combination and overtone of molecules bending and stretching vibration of -CH, -NH, -OH group between 720 to 2500 nm, hence it gives more information regarding chemical structures of the sample. After identification of blood successfully it is important to find out age of the bloodstain sample help in progressive aspect crime and crime scene investigators. Electron paramagnetic resonance, RNA degradation measurements, high performance liquid chromatography and atomic force

Microscopy are investigated for determining age of the blood but most requires preparation odf sample and need to be carry out on lab scale. Same as blood stain identification Near Infrared Spectroscopy (NIRS ) and visible spectroscopy can be used for non destructive investigation of time after bleeding.5

Human blood contains oxy-hemoglobin (HbO2) gives red color to blood, when blood comes to the open atmosphere then oxidation of HbO2 starts and covert into methemoglobin (met-Hb) and then hemichrome (HC), these conversion cause change in blood color from red to dark brown. HbO2 presents in bloodstains is biphasic. Initialy rapid oxidation of HbO2 begins, but decreases after a few hours. Oxidation of HbO2 is strongly temperature dependent but not on humidity, whereas conversion of met-Hb into HC intensely depends on humidity. Comprehensing this decay rate and factors of color change is vital for interpreting identification test results into forensic practice which enable determination of age of bloodstain at the crime scene.18

NIRS successfully demonstrate NIR region shows spectral changes in wavelength band from 1460 to 1860 nm were beneficial to determine the age of a bloodstain on any background like on glass, gauze or cotton. Use of NIR reflectance spectra estimate not only age of blood stains but compared these results qualitatively with different components of blood and distinguish blood from non-blood stains with high specificity and sensitivity.5


To depict the spectral structures of pure blood, first measurements were done on pure single drop of blood from male volunteer (volunteer #1) directly deposited on sample holder without substrate.5

Using a Fourier transform near infrared spectrometer (FT-NIRS) and NIR reflectance spectra from 800 to 2778 nm of the pure blood stain was repeatedly calculated after 5 min for the first 5 hours and then hourly for next 4 days , 240 measurements in total.

In forensic case work blood sample always deposited on substrate hence this study used five colored cotton sample of diameter 3 cm as shown in figure 2, on which both non-blood and blood samples were deposited. For this following sample sets made:5

Figure 2 pictures of bllod sampleas on blood stains on white , black, red, green and blue with reference sample shown with bottom row.5

1. Blood on white cotton, for this three blood sample taken from female volunteer (volunteer #2) , next to this plain cotton reference created . Reflectance spectra of this sam[ple measured repeatedly next for 77 days with many frequencies for two hours in the beginning up to monthly in the end, hence 54 measurement in total. 2. Blood on colored cotton, three blood sample taken from female volunteer (volunteer #2) and each three samples deposited on blue, black, green, red cotton, next to this plain cotton reference created as shown in figure 2. Reflectance spectra of this sample measured repeatedly next for 28 days, 66 measurement in total. 3. Non blood on white cotton, for this 30 different non-blood samples as shown in figure 3 were carefully chosen as they can give false positive result like blood stain sample when detect by using luminol (bleach) or visible spectroscopy. Non-blood substances measured repeatedly after 1 week.5

Figure 3 Pictures of non-blood samples holding cotton substrate numbering as follow: 1. tea, 2. coffee, 3. red wine, 4. red grape juice, 5. black currant soda ('cassis'), 6. coke, 7. cherry coke, 8. red berry juice, 9. tomato juice, 10. ketchup, 11. curry, 12. tabasco, 13. ketjap manis, 14. Worstershire sauce, 15. soy sauce, 16. balsamic, 17. Maggi aroma, 18. red wine, 19. cherry marmalade, 20. forest fruit marmalade, 21. apple syrup, 22. chocolate sauce, 23. red cabbage, 24. red beet, 25. bleach, 26. lipstick, 27. lipstick, 28. lipstick, 29. red food colorant, and 30. red food colorant. 5

NIR reflectance spectra of all blood stain samples on cotton and reference samples were measured between 400 and 2500 nm using a FOSS NIR systems 6500 spectrophotometer. 32 scans have taken from different position to obtain a an average spectrum, in between samples were stored in a laboratory at room temperature approximately 22.8 oC. 5

2.3. Preprocessing

All measured NIR reflectance spectra were transformed to absorbance spectra i.e. log(1/R), where R is the reflectance plotted against wavelength(nm). All absorbtion spectra (log (1/R)) of sample deposited on differentn cotton background substract with absorption spectrum of reference sample of same color.

All analysis of data done by custom-made scripts written in MATLAB (The Mathworks Inc., Natick, Massachusetts, USA).5

3. Data analysis

3.1. Bloodstain characterization

To characterize the absorbance spectra of pure blood, the

absorbance maxima were compared to reference absorption

maxima (see Table 1) of the following blood components:

oxyhemoglobin, methemoglobin, albumin, globulin, triglycerides,

glucose, cholesterol, and urea. Reference spectra of fibrinogen were

not found, as research is often done on serum, which does not

contain fibrinogen. Other components, normally present in lower

concentrations in blood, were not taken into account [20].


This NIR techniques help to the investigators when crime was committed and to determined is identified stain is relevant to the case as well as distinguishing blood stains from other non-blood stains likely to give false results can be done at 100% sensitivity and 100 % specificity, this all analysis can be done non-destructively. As the aging of blood stains is alter by various factors like temperature, lighting conditions and humidity hence, more study and research is require to study effect of NIR spectra and meet the validating data to legal ansd scientific communities.5


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