Infra Red Spectrometer Used To Identify Chemical Bonds Biology Essay

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This experiment was carried out to understand how an Infra red Spectrometer can be used to identify the chemical bonds and structure of thirteen organic liquids while relating them to their IR spectra obtained with the aid of an attenuated transmission accessory (ATR) and perform a quantitative analysis of fatty acid methyl esters (FAME) in diesel/biodiesel blends by measuring the absorbance of the ester.

The chemical nature of petrol, diesel and lubricating oil were obtained with petrol showing a combination of alcohol and aromatics and the likelihood of an ester; diesel containing aromatics, lubricating oil was similar in composition to diesel with only the varying CH2/CH3 ratio distinguishing them. Biodiesel was seen to be very similar to Oleic acid methyl ester. Vegetable oil is also seen to be similar to Biodiesel.

The region of the IR spectra which showed strong absorbance was in the 3200-2800cm-1 range occupied by the C-H stretches.

The FAME content in the diesel sample gotten from quantitative analysis is 2.8(±0.5)%.

The concentration of the unknown sample obtained with the aid of the linear calibration plot is .

1.0 INTRODUCTION

Infrared (IR) spectroscopy is a potent tool which makes use of distinctive wavelengths upon which chemical bonds in molecules absorb infrared radiation. This absorbed radiation causes changes in the energy level of diatomic bonds which can be related to stretching, vibration or rotation. The quantisation energy levels of bonds restrict the wavelengths at which the bonds can absorb Infrared. In IR spectroscopy, the wavenumber (ν) is plotted against the percentage transmission of incident radiation through a sample to obtain the IR spectrum. The wavenumber can be defined as the number of complete waveforms that can be fitted into a distance of 1cm i.e. 1/wavelength and is directly proportional to the level of radiation. Percentage Transmittance can be defined as the percentage of radiation that passes through sample to the detector without being absorbed. The relationship between Absorbance (A) and Transmittance (T) according to Beer Lamberts Law is shown as A = log10(I0/I) = -log10 T = ɛxCxL

Where; Io is the incident intensity

I is the transmitted intensity

É› is the absorption coefficient

C is the concentration of absorbing compound

L is the cell pathlength.

Infrared spectra can be acquired from samples as small as a few µg. This makes it possible to perform qualitative analysis of all three phases of matter i.e. solids, liquids and gases. Every compound produces different signature spectrum i.e. every band matches its peak position (wave number), and intensity (Rubinson and Rubinson, 2000). It is for this reason that infrared can be used as a fingerprint for the identification of the molecular components of samples by matching the spectrum of an unknown to a spectral library. In the absence of a suitable library, a basic interpretation from first principles can be carried out (Coates, 2000). Another qualitative use of infrared spectra apart from compound identification or comparison is to obtain structural information of functional groups (Rubinson and Rubinson, 2000).

Infrared spectra of organic compounds are divided into three regions;

The Functional group region between 4000-1300cm-1

The Fingerprint region between 1300-910cm-1

The aromatic region between 910- 650cm-1

This experiment was carried out to understand how an Infra red Spectrometer can be used to

Identify the chemical bonds and structure of thirteen organic liquids while relating them to their IR spectra obtained with the aid of an attenuated transmission accessory (ATR).

Perform a quantitative analysis of fatty acid methyl esters (FAME) in diesel/biodiesel blends by measuring the absorbance of the ester.

2.0 EXPERIMENTAL

The main apparatus used was a Thermo Scientific Nicolet is10 Fourier Transform Infrared (FTIR) Spectrometer which was set to take 36 scans per sample at a resolution of 4cm-1. Data obtained from the spectrometer is processed with the aid of Thermo Scientific OMNIC software. Two accessories (ATR and Transmission cell) were placed in the FTIR for use in different parts of the experiment.

2.1 QUALITATIVE ANALYSIS

The Attenuated Total Reflectance (ATR) accessory is placed in the FTIR Spectrometer for this procedure. A beam of IR radiation which is directed into an optically dense crystal (for example; a diamond crystal) at an angle reflects through it based on the internal reflection phenomenon thereby producing an evanescent standing wave at the crystal surface. The evanescent wave passes through 1-4µm of the sample situated on the crystal surface. Attenuated energy from the evanescent waves is passed back to the IR beam through the crystal and goes to a detector which creates a spectrum.

The spectra were collected using the ATR using the procedures presented in the laboratory manual.

2.2 QUANTITATIVE ANALYSIS

Transmission Cell Accessory (BS EN14078) was placed in the FTIR Spectrometer to produce an infra-red spectrum from the sample. FAME content of diesel was measured to show the amount of biodiesel present in the diesel blend.

The Diesel sample whose FAME content needed to be known was held in a potassium bromide cell with a known pathlength of 0.05cm. Oleic acid methyl ester (a suitable FAME with a known concentration) was analyzed in order to calibrate the spectrometer. Series of calibration standards using a solvent of spectrometric grade cyclohexane were obtained with the Transmission Cell Accessory using the steps in the laboratory manual.

3.0 RESULTS AND DISCUSSION

3.1 Interpretation of Spectra

Full IR spectra of all liquids (a-h) within the range 4000-600cm-1 which were obtained from the experiment and magnified spectra of hydrocarbon liquids (a-e and i-k) within the wavenumber range of 3200-2800cm-1 are analyzed showing their similarities and differences. The liquids i-k containing the hydrocarbons are compared to those of a-e to get an idea of their chemical nature.

The full list of the organic liquids can be found in the laboratory manual.

Fig 1: showing the spectra of iso-octane, n-octane and n-hexadecane within the range 4000-

600cm-1

CHn

1)CH2

3)CH3

4)CH3

4)CH3

3)CH3

2)CH2

1)CH2

3)CH2

2)CH2

1)CH2

CHn

CHn

Fig 2: showing the spectra of iso-octane, n-octane and n-hexadecane within the range 3200-

2800cm-1

Fig1. and Fig 2. show the spectras with the % Transmission plotted against wavenumbers (cm-1) for three alkanes. The C-H in the alkanes is seen to absorb mainly in the range of 2970-2850cm-1 with C-H bending occurring in the 1500-1350cm-1 range. Figure 2 shows the spectrum in the 3200-2800cm-1 range.

iso-octane http://www.chem.purdue.edu/gchelp/molecules/isooct.gif (C8H18):

Has a Molecular formula of CH3-(CH2)6-CH3

The bands, vibrations and wavenumbers are identified in the spectrum are as follows:

CH2 symmetrical stretching vibrations at weak intensity

CH2 Asymmetrical stretching vibrations at weak intensity

CH3 sharp peak with symmetrical stretching vibrations at 2950cm-1

The CH2/CH3 ratio is low which means that the number of CH2 bonds in the alkane chain is small. Thus the C-CH2-C peaks appear as small shoulders on the large C-CH3 peaks. C-H Bending occurs in the range 1500-1350cm-1

n-octane; (C8H18):

This is a straight-chain alkane.

CH2 symmetrical stretching vibrations at medium intensity

CH2 Asymmetrical stretching vibrations at medium intensity

CH3 sharp peak with symmetrical stretching vibrations at 2920cm-1

CH3 Asymmetrical stretching vibrations at medium intensity

The CH2/CH3 ratio is higher than the iso-octane which conforms to their molecular structures. C-H Bending occurs in the range 1500-1350cm-1

n-hexadecane C16H34

This is a straight chain alkane with Molecular formular of CH3-(CH2)28-CH3

CH2 symmetrical stretching vibrations at a strong intensity

CH3 sharp peak with symmetrical stretching vibrations at 2920cm-1

CH3 Asymmetrical stretching vibrations of weak intensity

There is a high CH2/CH3 ratio which is signified by high CH2 absorption. This conforms to the CH2/CH3 ratio in its molecular structure. The C-CH3 peaks are similar to that of n-octane because they have the same number of C-CH3 groups. C-H Bending occurs in the range 1500-1350cm-1

All three alkanes show Stretching of C-H to be stronger than the C-H bending.

Fig 3: showing the spectra of benzene, Ethyl-benzene and n-octanol within the range 4000-

600cm-1

4)CH3

3)CH3

4)CH3

1)CH2

C=C-H

C=C-H

C-O

O-H

C-H

C-H

3)CH3

1)CH2

CHn

C=C

CHn

C=C

Fig 4: showing the spectra of benzene, and Ethyl-benzene within the range 3200-2800cm-1

Benzene ; C6H6

An aromatic ring structure dominated by C=C, C-H bonds. From Fig 3 and Fig 4 we can deduce that Benzene has C=C-H stretching bonds with weak intensity within the range of 3100-3000cm-1 in the stretching region of the spectra. It also has medium intensity C=C bands at 1500cm-1 and C-H medium intensity and strong intensity bending bands respectively around 1050cm-1 and 670cm-1(out-of-plane bend).

Ethyl-benzene

A ring structure with additional C-H bonds. From the structure, Ethyl-benzene consists of the benzene compound and an ethyl group. Hence From Fig 3 and Fig 4 we see that it has C-H stretching bands listed below as well as weak intensity C=C-H bands within 3100-3000cm-1, medium intensity C=C bands around 1500cm-1 and C-H bending around 1450cm-1 and in the range of 700-650cm-1.

CH2 symmetrical stretching vibrations of weak intensity

CH3 symmetrical stretching vibrations at 2920cm-1

CH3 Asymmetrical stretching vibrations of weak intensity

The benzene ring does not have C-H stretch bands. Comparing the spectra of the ring structures from Fig 4 showing Benzene and Ethyl-benzene, we can see that the C=C-H stretch bands of Benzene are at a higher absorption than that of Ethyl-benzene. This may be due to the presence the C-H stretch bands of the ethyl group attached to the ring in the ethyl-benzene.

n-octanol , C8H18O

A straight chain alcohol which has a molecular structure of CH3-(CH2)7-OH.

In line with the molecular structure, the spectrum in Fig 3 shows the following: There is a broad O-H stretching band between 3500-3100cm-1 of medium intensity, a C-O band of strong absorption intensity at a 1200-1000cm-1 range. C-H stretching bands of strong intensity are also present between 3000-2800cm-1range because they dominate the molecular structure. These C-H bands are as follows:

CH2 symmetrical stretching vibrations at medium intensity

CH3 sharp peak with symmetrical stretching vibrations at 2910cm-1

CH3 Asymmetrical stretching vibrations of weak intensity

Fig 5: showing the spectra of Oleic acid methyl ester, TCE and Petrol within the range 4000-600cm-1

CHn

C-O

C-O

C-H

C=O

4)CH3

C-Cl

CHn

1)CH2

3)CH3

O-H

C=C-H

3)CH3

1)CH2

Oleic acid methyl ester

The structure shows the presence of C=O, C-O and C-H bonds. These are also identified in the spectra in Fig 5. The C-O band is absorbed at a medium intensity within a range of 1300-1100cm-1. The CHn bending occurs at a medium intensity within the 1500-1400cm-1 range. The C=O stretch band occurs at a strong intensity with a sharp peak around 1740cm-1. The C=C-H stretch band occurs at a weak intensity around 3000cm-1. The C-H stretch bands identified are listed below:

CH2 symmetrical stretching vibrations at medium intensity absorption at 2850cm-1

CH3 sharp peak with symmetrical stretching and strong intensity absorption at 2920cm-1

Tetrachloroethylene (TCE)

The structure of this liquid shows that a major bond of C-Cl exists and this is reinforced by the spectra in Fig 5. The spectrum shows the C-Cl bond having strong intensities of absorption between the 950-750cm-1 range. Although C=C is present in the structure it is not absorbed in the spectra and is therefore not detected.

Petrol

In order to identify the bonds and understand the chemical nature of petrol its spectra in Figure 5 and 6 were compared with spectra from Figures 1-4. The various comparisons are shown below.

The first broad peak has a weak intensity of absorption and falls within the range of 3300-3100cm-1. The band which closely resembles this is the O-H band from n-octanol in Figure 3. It can therefore be assumed that this weak intensity band is an O-H group.

Three stretching peaks of intensities ranging between medium to strong occur within the 3000-2800cm-1 range. Upon comparison with the alkanes which show strong peaks in this region, it can be inferred that the peaks are C-H bands with the following properties;

CH2 symmetrical stretching vibrations at a medium intensity around 2870cm-1

CH3 symmetrical stretching vibrations at a strong intensity around 2925cm-1

CH3 Asymmetrical stretching vibrations of strong intensity around 2960cm-1

Bending of varying intensity from strong to weak occurs within the 1500-1350cm-1 region and this corresponds to that of the alkanes and can be therefore be assumed to be a CHn bend.

A weak band is located between 1100-1000cm-1 and upon comparison it closely resembles the C-O in the ester and n-octanol. It can therefore be assumed that petrol has a C-O group.

A collection of strong intensity bands is observed between 900-600cm-1 and upon comparison to alkanes and aromatics in Figures 1-4 it corresponds to the out-of-plane C-H bend of benzene. Petrol may be assumed to possess an aromatic group.

Fig 6: showing the spectra of Petrol, Diesel, and Lube oil within the range 3200-2800cm-1

Fig 7: showing the spectra of Diesel, Lube oil and Biodiesel within the range 4000-600cm-1

C=C-H

CHn

CHn

C=C

C=C

C-O

2)CH2

4)CH3

C=O

4)CH3

3)CH3

1)CH2

1)CH2

1)CH2

CHn

3)CH3

3)CH3

Diesel fuel

The three peaks between 3000-2850cm-1 from the diesel spectra in Figure 6 and 7 are compared to the peaks in the same wavenumber region in Figures 1-4. They are identified as follows:

CH2 symmetrical stretching vibrations at a medium intensity around 2850cm-1

CH2 asymmetrical stretching vibrations at a weak intensity around 2870cm-1

CH3 symmetrical stretching vibrations at a strong intensity around 2920cm-1

CH3 Asymmetrical stretching vibrations of medium intensity around 2960cm-1

The diesel spectrum in Fig 7 is compared with the alkanes and aromatic spectra of Fig 1-4. The medium intensity band around 1500cm-1 corresponds to the aromatic C=C bond of benzene and ethyl-benzene in Fig 3. Diesel can therefore be assumed to contain aromatics.

Around 1400cm-1 a medium intensity band is observed and compared with the spectra in Fig. 1-4. The closest similarity is found in the Ethyl-benzene spectra in Fig 3 which brings the assumption that the band is a CHn bend.

Lubricating oil

Figures 6 and 7 contain the spectra of lubricating oil which is seen to be very similar to that of Diesel. They both have bands of similar C-H stretch absorptions, C-H bend and C=C stretch. The only difference being that the CH2/CH3 ratio of Lubricating oil is slightly lower than that of Diesel which is manifested in figure 6 with the asymmetric C-CH2-C peak appearing as a small 'shoulder' on the side of the symmetric C-CH3-C peak. All the bonds present are as follows:

CH2 symmetrical stretching vibrations at a medium intensity around 2850cm-1

CH2 asymmetrical stretching vibrations at a weak intensity around 2870cm-1

CH3 symmetrical stretching vibrations at a strong intensity around 2920cm-1

CH3 Asymmetrical stretching vibrations of medium intensity around 2960cm-1

The C=C medium intensity band around 1500cm-1

CHn bend at a medium absorption intensity around 1400cm-1

Biodiesel

Upon comparison of the Biodiesel spectrum in Figure 7 with the spectrum from Figure 1-6 it is observed that it bears very close similarities to that of Oleic acid methyl ester in Figure 5.

We therefore have,

C-O band absorbed at a medium intensity within a range of 1300-1100cm-1.

The CHn bend occurring at a medium intensity within the 1500-1400cm-1 range.

The C=O stretch band occurring at a strong intensity with a sharp peak around 1740cm-1.

The C=C-H stretch band occurring at a weak intensity around 3000cm-1.

The C-H stretch bands identified are listed below:

CH2 symmetrical stretching vibrations at medium intensity absorption at 2850cm-1

CH3 sharp peak with symmetrical stretching and strong intensity absorption at 2920cm-1

Vegetable oil

Upon comparison of Vegetable oil spectrum in Figure 8 with all the spectra from Figures 1-7 it is observed to be very similar to the spectrum of Biodiesel. Hence it has the following,

CH2 symmetrical stretching vibrations at medium intensity absorption at 2850cm-1

CH3 sharp peak with symmetrical stretching and strong intensity absorption at 2920cm-1

The C=O stretch band occurring at a strong intensity with a sharp peak around 1740cm-1

The C=C-H stretch band occurring at a weak intensity around 3000cm-1

The C-O stretch band absorbed at a medium intensity within a range of 1300-1100cm-1

Fig 8: showing the spectra of Vegetable oil within the range 4000-600cm-1

C=C-H

C=O

C-O

CHn

3)CH3

1)CH2

4.0 CALIBRATION CALCULATIONS

Actual weight of FAME =124.96g

Therefore, Actual concentration of FAME in 25ml of cyclohexane =

Actual concentrations of the calibration solutions are calculated and placed as shown in this example;

Example: For 0.5g/l nominal concentration i.e. 1cm3 of 4.998g/l standard solution diluted to 10cm3 we have;

= 2.9988g/l

Table 1 showing Actual Concentration and absorbance of solutions used for calibration

Nominal Concentration

(gL-1)

Actual Concentration

q (gL-1)

Absorbance

0.50

0.4998

0.0934

1.87

1.00

0.9996

0.173

3.46

2.00

1.9992

0.338

6.76

3.00

2.9988

0.500

10.00

5.00

4.9980

0.641

12.82

Unknown (diesel sample)

2.5(±0.4)

0.381(±0.001)

7.62(±0.001)

Example of calibration function calculation

Calculating the calibration function for a cell of standard pathlength 1cm for all concentrations we have;

For actual concentration of 0.4998gL-1,

Fig. 9: showing the calibration plot of absorbance against concentration for the FAME standard solutions

From the plot in Fig 9 the regression analysis is obtained as shown in Table 2.

Table 2: showing the results of regression analysis performed on calibration plot

 

Coefficients

Standard Error

Intercept

1.28090625

0.804173241

X Variable 1

2.479554322

0.287136592

Regression Statistics

Multiple R

0.980472218

R Square

0.961325771

Adjusted R Square

0.948434361

Standard Error

1.026880186

Observations

5

Therefore; R2 = 0.9613

Intercept (b) = 1.3±0.8

Gradient (a) = 2.5±0.3

A/L = a.q + b…………………………………………………………………eqn1

7.62(±0.001) = [2.5 (±0.3) Ã- q] + 1.3 (±0.8)

Calculating error associated with measurement using eqn1;

……………………………………………………………eqn2

Taking the numerator we have; 7.62(±0.001) - 1.3(±0.8) = 6.3±0.8

Converting to relative errors we have;

Therefore; q

The FAME content of the diesel sample is calculated using the equation;

………………………………………………………….eqn 3

Where X (dilution factor) = 10(±0.02)

d (density of FAME at 20oC) = 880.0kgm-3 = 880.0(±0.1) gL-1

L (the actual pathlength) = 0.05cm

a (gradient) = 2.5±0.3

b (intercept)= 1.3±0.8

Error in X is ±0.02 and is gotten from the use of a 10ml volumetric flask.

Error in d is ±0.1.

Combining eqn2 and eqn3, the equation is simplified to get;

= 2.84(±16.0%) = 2.8(±0.5)%

5.0 SOURCES OF ERROR

Absorbances in the form of slight peaks were obtained from spectrometer components, the ATR crystal and presence of carbon dioxide and water vapour in the air.

Error associated with weighing of FAME sample

Systematic error associated with using the volumetric tube.

"Instability in the wave number scale of the FT-IR spectra. The thermal expansion and contraction of the cavity of the reference laser in a typical commercial instrument is found to produce changes in the laser wavenumber of ±0.034 cm-1" (Weis and Ewing, 1998)

6.0 CONCLUSION

The FAME content in the diesel sample gotten from quantitative analysis is 2.8(±0.5)%.

The concentration of the unknown sample obtained with the aid of the linear calibration plot is.

The region of the IR spectra which showed strong absorbance was in the 3200-2800cm-1 range occupied by the C-H stretches.

Comparison of C-H stretch bands in the organic liquids spectra showed that the relative sizes of CH2 and CH3 peaks were influenced by the CH2/CH3 ratio which is signified by high CH2 or CH3 absorption. This phenomenon corresponded to the molecular structures of each compound.

Upon comparison of liquids a-h with i-k, an idea of the chemical nature of petrol, diesel and lubricating oil were obtained. Apart from the C-H stretch bonds present they had the following compositions

Petrol is seen to contain an O-H group which may indicate presence of alcohol, C-H bend similar to benzene indicating it may contain aromatics, and a weak C-O comparable to that of an ester or an alcohol.

Diesel is seen to contain a C=C group indicating the presence of benzene, and a CHn bend also indicating benzene. It is therefore largely aromatic.

Lubricating Oil is similar to diesel except it has a lower CH2/CH3 ratio.

Biodiesel is found to have a similar chemical nature to Oleic acid methyl ester.

Vegetable oil is also seen to be similar to Biodiesel.

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