Course: PG Pathshala-Biophysics

Paper 10: TECHNIQUES USED IN MOLECULAR BIOPHYSICS II (Based on

Spectroscopy)

Module:25. IR spectra of functional groups, IR bands of peptide and protein

Content Writer: Dr. Imtaiyaz Hassan, Jamia Millia Islamia, New Delhi

Introduction:

IR spectroscopy is used for the determination of structure of organic compounds and

biological macromolecules. When IR radiation (400-4000 cm-1) is passing through a sample,

a spectrum is generated which provides the structural features of any substance under

investigation. At the top IR spectra represents 100% transmittance or zero absorbance. The

“bands” represent wavenumbers of the absorbed radiation. One of the most important

applications of IR spectrum is determination of the bonding pattern in a molecule. The region

of IR spectrum below 1500 cm-1 is considered as “fingerprint region” which provide the

characteristic of whole molecule. The fingerprint region of IR spectra is used to identify

different molecules. IR helps in identification of the bonds/functional groups in an organic

molecule.

Objective:

In this module we discuss following section in detail,

1. Understanding of IR spectrum

2. Regions of the IR Spectrum

3. Factors affecting IR band

4. The IR spectra of various functional groups

5. IR spectrum of biological macromolecules

6. Summary

1. Understanding of IR spectrum

Chemical bonds of any organic and inorganic compounds in different environments will

absorb different intensities of light at different frequencies. The IR spectroscopy is used to

collect the absorption information of a molecule as a function of wave number (frequency)

which is described in the form of IR spectrum. The maximum absorption of IR radiation

occurs at particular frequency in the form of "peaks" or "signals" which may be directly

correlated to bonds within the compound under investigation. The interaction of the IR

radiation with the bond provides a unique qualitative probe to identify the functional group of

a molecule. All the functional group of organic compounds comprised of multiple bonds

bearing multiple IR bands/peaks. A typical IR spectrum is shown in the Figure 1 which

indicates characteristic features of different types of bonds or signatures.

Figure 1: Showing a characteristic IR spectrum.

2. Regions of the IR Spectrum

Characteristic IR absorption frequencies of organic functional groups are given in the Table 1

and Table 2. A typical IR spectrum is mainly divided into two different regions. The left half

lies above 2000 cm-1 possesses some typical characteristic information e.g. absorptions just

below 3000 cm-1 indicates C-H stretching of alkane (saturated carbons), and signals just

above 3000 cm-1 demonstrate unsaturated carbon atoms. Presence of exchangeable protons

can be observed in the form of a broad peak (3100 and 3600 cm-1) of alcohol, amide, amine,

or carboxylic acid groups. Presence of alkyne or nitrile groups can be easily at the

frequencies from 2800 to 2000 cm-1. However, the right half of the spectrum (below 2000

cm-1) having many peaks of varying intensities. A sharp peak at 1700 cm-1 is observed for the

carbonyl group and one or two intense peaks around 1200 cm-1 is for the C-O bond.

In detailed analysis the IR spectrum is divided into four primary regions. Atoms that bear low

molecular mass will allow the faster oscillation because of higher energy. Stronger bonds will

have higher energy oscillations:

Triple bonds > double bonds > single bonds in energy

The functional group region of the IR spectrum allows identification of functional groups.

The absorption bands in this region lie in the range of 4000 – 1500 cm-1. The lower energy

portion i.e. mid-IR region in (1500 – 400 cm-1) generally comprise of a complex set of peaks

due to the multifarious vibrations of several atoms. This region is unique for any particular

molecules and thus known as the fingerprint region which is useful for the identification of

any compound if their spectrum is known. Table 1 and 2 are showing signature frequencies of

organic functional groups. IR absorption data for some other functional groups are given in

the Table 3.

Table 1: Characteristic IR absorption region of different functional groups

Functional

Group

Type of

Vibration

Characteristic

Absorptions (cm-1) Intensity

Alcohol

O-H (stretch, H-

bonded) 3200-3600 strong, broad

O-H (stretch, free) 3500-3700 strong, sharp

C-O (stretch) 1050-1150 strong

Alkane

C-H stretch 2850-3000 strong

-C-H bending 1350-1480 variable

Alkene

=C-H stretch 3010-3100 medium

=C-H bending 675-1000 strong

C=C stretch 1620-1680 variable

Alkyl Halide

C-F stretch 1000-1400 strong

C-Cl stretch 600-800 strong

C-Br stretch 500-600 strong

C-I stretch 500 strong

Alkyne

C-H stretch 3300 strong, sharp

stretch 2100-2260 variable, not present in

symmetrical alkynes

Amine

N-H stretch 3300-3500 medium

C-N stretch 1080-1360 medium-weak

N-H bending 1600 medium

Aromatic

C-H stretch 3000-3100 medium

C=C stretch 1400-1600 medium-weak, multiple

bands

Carbonyl

C=O stretch 1670-1820 strong

Ether

C-O stretch 1000-1300 (1070-1150) strong

Nitrile

CN stretch 2210-2260 medium

Nitro

N-O stretch 1515-1560 & 1345-1385 strong, two bands

Table 2: IR absorption region of carbonyl group containing function groups

http://www2.ups.edu/faculty/hanson/Spectroscopy/IR/IRfrequencies.html

Functional

Group

Type of

Vibration

Characteristic Absorptions

(cm-1)

Intensity

Carbonyl

C=O stretch 1670-1820 strong

(conjugation moves absorptions to lower wave numbers)

Acid

C=O stretch 1700-1725 strong

O-H stretch 2500-3300 strong, very broad

C-O stretch 1210-1320 strong

Aldehyde

C=O stretch 1740-1720 strong

=C-H stretch 2820-2850 & 2720-2750 medium, two peaks

Amide

C=O stretch 1640-1690 strong

N-H stretch 3100-3500 Un-substituted have two

bands

N-H bending 1550-1640

Anhydride

C=O stretch 1800-1830 & 1740-1775 two bands

Ester

C=O stretch 1735-1750 strong

C-O stretch 1000-1300 two bands or more

Ketone

acyclic stretch 1705-1725 strong

cyclic stretch 3-membered - 1850

4-membered - 1780

5-membered - 1745

6-membered - 1715

7-membered - 1705

strong

,-unsaturated stretch 1665-1685 strong

aryl ketone stretch 1680-1700 strong

Table 3: IR absorption spectra of different functional groups

Source: https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/Spectrpy/InfraRed/infrared.htm

3. Factors affecting IR band

In general, the most important factor that determines the frequency where a chemical bond

absorbs is the bond order and the types of atoms joined. The intensity of IR frequencies are

affected by two primary factors namely types of vibration (stretching or bending) and the

electronegativity difference. In general a greater the change in dipole moment a larger the

peak intensity is observed. Stretching induces more dipole moment than bending. The ability

Functional Class Characteristic Absorptions

Sulfur Functions

S-H thiols 2550-2600 cm-1

(wk & shp)

S-OR esters 700-900 (str)

S-S disulfide 500-540 (wk)

C=S thiocarbonyl 1050-1200 (str)

S=O sulfoxide

sulfone

sulfonic acid

sulfonyl chloride

sulfate

1030-1060 (str)

1325± 25 (as) & 1140± 20 (s) (both str)

1345 (str)

1365± 5 (as) & 1180± 10 (s) (both str)

1350-1450 (str)

Phosphorous Functions

P-H phosphine 2280-2440 cm-1

(med & shp)

950-1250 (wk) P-H bending

(O=)PO-H phosphonic acid 2550-2700 (med)

P-OR esters 900-1050 (str)

P=O phosphine oxide

phosphonate

phosphate

phosphoramide

1100-1200 (str)

1230-1260 (str)

1100-1200 (str)

1200-1275 (str)

Silicon Functions

Si-H silane 2100-2360 cm-1

(str)

Si-OR 1000-11000 (str & brd)

Si-CH3 1250± 10 (str & shp)

Oxidized Nitrogen Functions

=NOH oxime

O-H (stretch)

C=N

N-O

3550-3600 cm-1

(str)

1665± 15

945± 15

N-O amine oxide

aliphatic

aromatic

960± 20

1250± 50

N=O nitroso

nitro

1550± 50 (str)

1530± 20 (as) & 1350± 30 (s)

of a molecule to absorb radiation during a particular vibration is also depends on its electrical

geometry.

4. IR spectra of various functional groups

Different functional groups have a different absorption signature and intensities on the IR

spectrum. Recognition of absorptions bands of common functional groups is helpful for the

interpretation of IR spectra for its identification. Here we discuss locations and intensities of

absorptions produced by each common functional group in details.

Alkanes: In alkanes, “both C-C and C-H bonds stretches and bends lie in the region 1360-

1470 cm-1 e.g. in octane CH2-CH2 bond lies in the region 1450-1470 cm-1 whereas CH2-CH3

bond is seen in 1360-1390 cm-1 (Fig. 2). The sp3C-H lies in the region between 2800-3000

cm-1”.

Fig 2: IR spectra of octane

Alkenes: In alkenes “C=C and vinyl C-H bonds are present. C=C stretch is recorded at 1620-

1680cm-1 and it becomes weaker as substitution increases e. g. in 1-Octene the vinyl C-H

stretch occurs at 3000-3100 cm-1 (Fig. 3). There is a great difference between alkane, alkene

or alkyne C-H, if the band is slightly above 3000 it is vinyl sp2 C-H or alkynyl sp C-H”.

Fig 3: FTIR spectra of 1-octene

Alkynes: Alkynes contain “C=C and vinyl C-H bonds. C≡C stretch lies in 2100-2260 cm-1

and the strength depends upon the asymmetry of bond. It is strongest for terminal alkynes and

weakest for symmetrical internal alkynes e.g. in case of 1-octyne, C-H strech for terminal

alkynes occurs in the region 3200-3300 cm-1 (Fig. 4). Internal alkynes (R-C≡C-R) do not bear

this band”.

Fig. 4: FTIR spectra of 1-octyne

Aromatic Compounds: The stretching frequency for these bonds is slightly lower in energy

than normal C=C bond. In case of ethyl benzene a pair of sharp bands that are lower in

frequency and stronger lie at 1500 & 1600 cm-1. C-H bonds that lie off the ring are similar to

vinyl C-H and so lies at 3000-3100cm-1 (Fig. 5).

Fig 5: FTIR spectra of ethyl benzene

Unsaturated Systems: The substitution of alkenes and aromatic compounds are

distinguished through out-of-plane bending vibration region. Other peaks are also apparent in

this region. These peaks are used for the strengthening of hypothesizing the functional group

as shown below.

Ethers: The “C-O-C asymmetric band and vinyl C-H bonds are present in ethers. Eg. In the

FTIR spectra of di-isopropyl ether a strong band for the anti-symmetric C-O-C stretch occurs

at 1050-1150 cm-1” (Fig. 6).

Fig 6: FTIR spectra of di-isopropyl ether

Alcohols: A strong, broad O-H stretch from 3200-3400 cm-1is seen in the spectra of alcohols.

For example in case of 1-butanol, the C-O stretch extends from 1050-1260 cm-1 (Fig. 7). It

should be noted that the band position changes depending upon the alcohols substitution:

primary-1075-1000; secondary-1075-1150; tertiary-1100-1200 and phenol-1180 – 1260 cm-1.

C-H bonds (~2900-3000 cm-1).

R

C

H

C

R

C

H

CH2

R

C

H

C

R

C

R

CH2

R

C

R

C

R

H

R

H

R

H

985-997905-915

cm-1

960-980

665-730

885-895

790-840

R

R

R

R

R

RR

cm-1

730-770690-710

735-770

860-900750-810680-725

800-860

Fig. 7: FTIR spectra of 1-butanol.

Amines: Primary amines show “the –N-H stretch for NH2 as a doublet between 3200-3500

cm-1symmetric and anti-symmetric modes. Spectrum of 2- aminopentane shows NH2

deformation band from 1590-1650 cm-1 (Fig. 8),. There is a “wag” band at 780-820 cm-1 i.e.

not diagnostic. The spectra of secondary amine for e.g. pyrrolidine comprises N-H band for

R2N-H at 3200-3500 cm-1as the only sharp peak weaker than –O-H. Tertiary amines (R3N)

have no N-H bond” and so no N-H band in the spectra is seen (Fig. 9).

Fig. 8: FTIR spectra of 2-aminopentane.

Fig. 9: FTIR spectra of pyrrolidine.

Aldehyde and ketones: The C=O (carbonyl) stretch in aldehydes is recorded from 1720-

1740cm-1For eg in 3-cyclohexene-1-carboxaldehyde (Fig.10). A unique “Fermi doublet” is

seen in the spectra which is sp2 C-H stretch and appears at 2720 & 2820 cm-1. The IR spectra

of ketones have characteristic CO stretch. For e.g. in 3-methyl-2-pentanone the C=O stretch

occurs at 1705-1725 cm-1 (Fig. 11).

Fig. 10: FTIR spectra of cyclohexyl carboxaldehyde.

Fig. 11: FTIR spectra of 3-methyl-2-pentanone.

Carboxylic Acid and Esters: In case of “esters C=O stretch is recorded at 1735-1750 cm-1

as compared to ethers and alcohols which bars band at 1150-1250cm-1. For example in ethyl

acetate, a strong band for C-O is observed at a higher frequency (Fig. 12). In carboxylic

acids, a C=O band is seen between 1700-1725 cm-1.The O-H bond is highly dissociated and

has a broad band from 2400-3500 cm-1 covering up about half the IR spectrum the C-H

stretch also occurs in the region around 3000 cm-1 but this is usually mostly obscured by the

broad O-H absorption. For example in case of 4-phenyl butyric acid a broad absorption band

between about 2400 and 3400 cm-1 is due to the O-H stretch in carboxylic acids (Fig 13)”.

The broadness of the O-H band is due to hydrogen bonding between molecules.

Fig. 12: FTIR spectra of ethyl acetate

Fig. 13: FTIR spectra of 4-phenyl butyric acid.

Acid anhydrides: “Due to the coupling of the anhydride via ether oxygen, splits the carbonyl

band into two having a separation of 70 cm-1. Eg. in propionic anhydride several bands are at

1740-1770 cm-1 and 1810-1840 cm-1 (Fig. 14). Mixed mode C-O stretch at 1000-1100 cm-1 is

seen in the given spectra.”

Fig 14: FTIR spectra of propionic anhydride.

Amides: An “amide 1ᵒ (-NH2) the N-H stretch occurs from 3200-3500 cm-1 as a doublet and

if the amide is 2ᵒ (-NHR) the N-H stretch occurs at 3200-3500 cm-1 as a sharp singlet. The

FTIR spectra of isobutyramide display features of amines and carbonyl compounds. C=O

stretch at 1640-1680 cm-1” is seen in the Figure 15.

Fig.15: FTIR spectra of isobutyramide

Nitro group (-NO2) and Nitriles (the cyano- or –C≡N group): “Two bands symmetric and

asymmetric are seen at 1300-1380 cm-1 and 1500-1570, respectively in the FTIR spectra of 2-

nitro propane (Fig. 16). Since this group is a strong resonating and electron withdrawing

group, it is itself vulnerable to resonance effects. The principle group in nitriles is the carbon

nitrogen triple bond recorded at 2100-2280 cm-1” (Fig.17).

Fig. 16: FTIR spectra of 2-nitropropane

Fig. 17: FTIR spectra of propionitrile.

5. IR spectra of biological macromolecules:

IR spectrum may also be employed to investigate biological systems. A wealth of

information in the IR spectrum of biological molecules is hidden which may be exploited

extensively. Figure 18 is showing typical IR spectra of biological components

highlighting the most prominent absorption features.

Fig. 18: IR spectra of biological components highlighting the most prominent absorption

features.

IR spectroscopy of proteins: FTIR spectroscopy gives the details of secondary structure

content of proteins as compared to X-ray crystallography and NMR spectroscopy that

give the details of the tertiary structure. FTIR spectroscopy works by irradiating the IR on

a sample andanalyzing which wavelengths in the IR of the spectrum are absorbed by the

sample. Characteristic bands found in the IR spectra of proteins and polypeptides include

the amide. These bands arise from the amide bonds that link the amino acids. The

absorption associated with the amide band results in stretching vibrations of the C=O

bond of the amide. Since the C=O and the N—H bonds are involved in the hydrogen

bonding that takes place between the different elements of secondary structure, the

locations of both the amide are sensitive to the secondary structure content of a protein

(see Figure 19).

Fig. 19: FTIR spectra of a typical protein showing amide peaks.[(Krimm & Bandekar Adv

Protein Chem 1986;38:181-364]

The amino acid and peptide “absorption bands lies in the 3400 cm-1 due to O–H and N–H,

bond stretching. The broad absorption bands in the region 3030-3130 cm-1 are due to

asymmetric valence vibrations of the ammonium (NH3+) group. The symmetric absorption

vibrations in 2080-2140 cm-1 or 2530-2760 cm-1 can be due to amino acid chemical

structures. The deformation vibrations of ammonium group lies at 1500-1600 cm-1, along

with the absorptions characteristic of the carboxylate ion. The asymmetrical deformation

bands from 1610-1660 cm-1 is related to carboxylate (COO-) group, and it generally

represents weak absorption. The bands in the 1724-1754 cm-1 region correspond to the

carbonyl (C=O) vibration” Figure 20.

Figure19: The amide region of the IR spectra for the protein, lysozyme, in its native (red) and

adsorbed (blue) states (substrate: ZnSe).

[Source:https://www.reading.ac.uk/AcaDepts/sd/pharmacy/public_html/staff/green]

6. Summary:

IR is one of the extensively used analytical techniques which may be employed in any state

as liquids, solutions, pastes, powders, films, fibres, gases, This technique has wide range of

application including analysis of functional groups of organic and inorganic molecules such

as proteins, lignin, chitin, polymers, etc. Each molecule has a particular IR spectrum

signature which is dependent on the chemical structure and the configuration of attached

atoms to this. Based on signature spectrum IR spectroscopy is used to investigate the

chemical structure of any compound.

End of Module

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