Chemotherapy, the term most normally used for antineoplastic therapy presents, was foremost used by a German physician Paul Ehlrich in 1900 ‘s for the intervention of bacterial infections with antibacterial. Ehlrich found that harmful bacteriums can be stained utilizing certain dyes and developed the thought of these molecules as “ Magic slugs ” . For medicine Gerhard Domagk was awarded Nobel award in 1939 for happening the usage of dye Prontosil as antibiotic. Prontosil breaks down to organize sulphanilamide in vivo and this was found by research group headed by J.Trefouel. This find started the research of sulphanilamide derived functions which proved to be a critical milepost in development of antibiotics.
Sulphapyridine ( intervention of Pneumonia ) , Sulphacetamide ( intervention of Urinary piece of land infections, Succinoylsulphathiazole ( intervention of GI piece of land infections ) and Sulphathiazole used for handling conflict lesions in World War II ) .
Sulphonamide drugs are bacteriostatic, i.e. they suppress the growing of bacteriums. Sulphanilamide is a competitory inhibitor of PABA ( p-aminobenzoic acid ) , which is required by bacteriums to synthesize folic acid ( a cofactor ) . The competitory suppression of PABA by sulphanilamide due to structural similarity, and inhibits the synthesis of nucleic acids that are required for growing by bacteriums and hence, suppresses bacterial growing. The sulphonamides gained the position of “ admiration drugs ” in the World War II period, and were the chief intervention against bacterial infections until penicillin came into general usage, after its successful usage in worlds was established in 1941. Apart from penicillin, tetracycline derived functions like Aureomycin and Terramycin were discovered. The freshly discovered antibiotics were more effectual and had fewer side effects [ 1 ] .
P Amino benzoic acid Sulphanilamide
Folic acid
Figure 1. Structure of p-aminobenzoic acid, sulphanilamide and folic acid
Sulphonamides are man-made antibacterial agent, active against figure of infections. They are active against gm positive beings chiefly pneumococci and meningococci. [ 1 ] . They are considered as antibacterial, carbonaceous anhydrase inhibitor, anticancerous, anti inflammatory agents [ 2 ] .
Structure activity relationships
Figure 2. General construction of sulphonamides.
The parity amino group has to be unsubstituted ( R1 should be either H or an acyl group ) .
The aromatic and sulphonamide group are both indispensable for activity.
The two groups, amino and sulphonamide, has to be straight attached to aromatic group.
Merely para substituted aromatic pealing exhibits activity, other permutation diminishes or eliminates the activity.
The sulphonamide N must be primary or secondary to bring forth the action.
Substitutions can merely be made at R2 place [ 1 ] .
Applications of sulphonamides:
The sulphonamides were the drug of pick before penicillin came in 1942. After the debut of penicillin, the sulphonamides took a back place due to the big figure of side effects. With the debut of less toxic parallels like sulphadoxine, the sulphonamides have once more become the drug of involvement. Soon the sulphonamides are prescribed in the undermentioned indicant
Urinary piece of land infections
Eye lotions
Infections of mucose membrane
Gut infections [ 1 ] .
The major side consequence of sulphonamides is the crystalurea that occurs due precipitation of drug in liver and kidney. This limits their frequent usage. However, the sulphonamide derived functions like sulphanylureas are still utile in the diabetes mellitus type II [ 3 ] .
Some of the researches carried out with sulphonamides reveal that:
they are CAR agonists, a atomic receptor which detects potentially toxic endo- and exogenic compounds and induces their riddance from the organic structure [ 4 ] .
They possess anticancer drugs due to their functionality to interact with cellular marks [ 5 ] .
In this undertaking work I will be fixing three sulphonamide drugs, sulphanilamide, sulphapyridine and sulphathiazole from a common precursor p-acetamidobenzenesulphonyl chloride and will happen out its pureness by spectroscopic methods like NMR, mass and IR spectrometry. The p-acetamidobenzenesulphonyl chloride can be synthesised from phenylacetamide, by responding the latter with chorosulphonic acid. Through electrophillic aromatic permutation reaction, the acetamido group directs wholly to the para place. However in this experiment I have ordered the p-acetamidobenzenesulphonyl chloride from Alfa Aesar chemical provider.
NUCLEAR MAGNETIC RESONANCE
Bing the universal method for finding of intermolecular interaction and non necessitating any mark specific cognition, NMR has batch of things to offer in construction finding. Furthermore, the ligand-target interaction is screened in a consecutive forward mode to avoid any false leads. High sensitiveness to weak interactions and elaborate structural elucidation for ligand binding manners are the chief advantages of NMR spectrometry [ 6 ] . This technique is based on the magnetic belongingss of the atomic karyon. Nuclear magnetic minute of karyon and external magnetic field ( B0 ) creates a atomic energy degree diagram. As per the quantum mechanics the magnetic energy of the karyon is limited to discrete values, Ei. These are known as Eigenvalues. Eigenvalues are related to eigenstates which are the provinces in which atom can be, besides known as stationary provinces. Using the high frequence sender, the atoms can be made to switch through eigenstates within energy degree diagram. The energy absorbed is detected, amplified and reproduced in the signifier of spectrum, known as resonance signal. We can obtain a spectrum like this for compounds holding atomic karyon with non zero magnetic minutes, such as 1H, 19F, 14N, 15N, etc. 12C, which is so of import in organic chemical science, has zero magnetic minute with even mass and even atomic figure, therefore the NMR surveies are merely limited to 13C.
Some cardinal points to observe in NMR spectrum are
1 ) Different protons in the compound produce different resonance signals, and these are separated by chemical displacement.
2 ) The figure of protons giving rise to peculiar signal is represented by country under signal, which is given by Integration.
3 ) The spectral lines appear as vests, doublets, threes, or fours, depending on the spin-spin yoke.
Therefore we can state that chemical displacement tells us the chemical environment of the karyon responsible for that signal and integrating concludes the figure of protons present. The spin-spin yoke relates to the positional relationship of the karyon as the yoke invariable is straight relative to the figure and type of bonds dividing them. And the signal multiplicity is due to the figure of protons on the neighbouring group. Besides the NMR spectrum is temperature dependent for many compounds due to the presence of resonance signifiers ( high barrier of rotary motions along the bonds ) . 13C NMR is besides a critical technique in organic chemical science and biochemistry and besides 19F, 15N, 31P outputs of import information.
Instrumentality: There are normally 2 types of NMR instrument, uninterrupted moving ridge and Fourier transform. Earlier the uninterrupted moving ridge was used but after the debut of FT in 1970, CW are largely replaced by FT. In CW NMR the sample is introduced to the strong magnetic field and the beginning ‘s frequence is scanned.
In FT-NMR the sensitiveness of NMR is increased by entering big figure of spectra and so adding them together. In this add-on the noise increases 10 creases but the signal strength is increased by 100 creases, and therefore the sensitiveness is increased.
The typical FT-NMR system contains a 3 constituents:
Superconducting magnet and investigation,
spectrometer, and
computing machine terminus.
The superconducting magnet has ports that are filled with liquid He which creates an environment giving zero opposition.
Figure 3. Picture of NMR spectrometer
Applications:
1 ) . A really of import tool for structural elucidation of a compound.
2 ) . Can be used to happen out the pureness of the compound.
3 ) . Applied successfully in new drug find.
4 ) . Can be used for reaction and procedure monitoring.
5 ) . Used for analyzing cell metamorphosis non-invasively.
7 ) . Used in Quality confidence.
8 ) . Information about molecular gesture.
9 ) . Food analysis.
Strengths:
1 ) . It is extremely sensitive technique.
2 ) . A non-invasive technique [ 7 ] .
Infra Red Spectroscopy
Compounds with covalent bonds, organic and inorganic, absorb electromagnetic radiations in the infra ruddy part of electromagnetic spectrum. Wavelength of this part is longer than seeable visible radiation, runing from 400 to 800 nanometers, nevertheless shorter than micro-cook radiations. From chemical science point of position, the vibrational part of the infra ruddy is of peculiar involvement. The soaking up of infra ruddy radiation causes the molecule to excite to the higher province. Merely certain frequences of infra ruddy radiation are absorbed by the molecule and correspond to alter in energy from 8 to 40kJ/mole. The radiation in this scope encompasses the stretching and vibrational frequences of the covalent bonds. During soaking up, merely the frequences of infra ruddy radiations fiting the vibrational frequence of the molecule are absorbed. The captive energy increases the amplitude of vibrational gestures of the bonds in molecule. The matching of the frequence of radiation with the bond gesture does non intend that the bonds will absorb energy. The bonds which have a dipole minute that changes as a map of clip can absorb infra ruddy radiations. Even the symmetric bonds do non absorb Infra ruddy radiation.
Stretching and flexing manners are the sort of vibrational gestures that give rise to the soaking up or in other words make the molecule infra red active. Any molecule holding three or more atoms and at least two of which are indistinguishable, the stretching could be symmetric and asymmetric. The different bending manners are scissoring, wagging, swaying and writhing.
Combination set is the connection of two vibrational frequences to give a new frequence within molecule, provided, such a quiver is infra ruddy active.
Difference bands consequences from difference from two interacting frequences.
Instrumentality:
Instrument used to mensurate the soaking up spectrum is called the infra-red spectrometer. There are two types of spectrometers normally in usage, diffusing and Fourier transform instruments. Both these instruments measure the soaking up spectrum in 4000 to 400 cm-1.
FTIR spectrometer provides spectrum much faster than Dispersive type.
Diffusing infra ruddy spectrometers: A beam of infra ruddy radiation is produced from a hot wire, which is divided into two equal strength analogue beams. In one beam, the sample is placed and mention in the other. Radio beams are so passed through the monochromator, and are dispersed in uninterrupted spectrum of Infra ruddy frequences. Quickly revolving sector ( the beam chopper ) alternately passes the two beams through diffraction grates. The diffraction grating varies the wavelength of the radiation making the thermocouple sensor. The sensor senses the difference in the strengths of sample and mention beam. This signal is amplified and drawn by the recording equipment as spectrum of the sample.
Fourier transform spectrometers: the optical tract produces interferogram, which is complex signal. However, its wave like form consists of all the frequences doing up the infra ruddy spectrum. An interferogram is intensity versus clip secret plan which is them converted by Fourier transform to a secret plan indistinguishable to intensity versus frequence. The secret plan made by Fourier transform is virtually indistinguishable to the secret plan received by Dispersive type spectrometer.
The chief advantage of utilizing FTIR spectrometer is its velocity ; it can obtain spectra in less than a 2nd. Therefore the instrument can enter figure of spectrum of the same sample which are stored in the computing machine memory and subsequently converted by Fourier transform. It generates a spectrum with better signal to resound ratio. Hence, FTIR is more fast and sensitive than scattering instrument.
Uses:
As every bond has different soaking up form and all the bonds exist in different environment in different compounds, no two compounds show the same spectrum. Hence, the infra ruddy spectrum is used as fingerprint of a molecule.
Determination of molecule ‘s structural information [ 8 ] .
Mass Spectroscopy
It is the technique to find the molecular mass of the compound. This is done by dividing the ions harmonizing to mass/charge ratio.
Principle:
Figure 4. Principle of mass spectroscopy
Adopted from M.Sc. OMED 0104 talk notes, Dr. Birthe Neilson, 2010
The sample is introduced to the mass spectrometer through the recess which could be HPLC, GC, or Sample home base. The sample is so ionized, and the ions are separated harmonizing to their mass/charge ratio. The detached ions are so detected by the sensor and the signals are recorded in the information system.
Vacuum is created inside the spectrometer so that average free way of molecules can be increased and the hit between the ions can be avoided.
Ionization: the molecules are ionized so that there way can be controlled utilizing electric or magnetic Fieldss. Some of the methods used to bring forth ionisation are
Electron expulsion Ma†’M+ + e-
Electron gaining control M + e- a†’M-
Protonation M + H+ a†’MH+
Deprotonation MH a†’ M- + H+
Cationizaion M + cation+ a†’ M ( cation ) +
There are two types of ionisation methods, soft and difficult. Soft ionisation is good for molecular weight finding and as the difficult method causes atomization, is good for structural information [ 9 ] .
Different ionisation techniques used are:
Electron ionisation: it is besides known as electron impact. This technique is largely utile for analyzing volatile organic molecules.
Chemical ionisation: in this technique the reagent gas is ionised foremost and the charge is transferred to the sample molecule by chemical procedure. Normally used reagent gas is methane.
Matching gas chromatography to Mass spectrometry: by matching the gas chromatography to mass spec, more complex volatile sample mixtures can be analysed.
Field and Plasma desorption ionization: these methods are non in usage today but these two techniques were the first 1s to be used for ionization of non-volatile molecules.
Fast atom barrage: secondary ion mass spectroscopy, SIMS, is a technique used to analyze chemical nature of stuffs and surfaces.
Matrix assisted laser desorption ionisation: In MALDI the sample is assorted with the matrix compound that absorbs the energy from the optical maser and aids in ionizations of the sample through negatron transportation and chemical procedures. MALDI is a good established method in analyzing polar high molecular weight compounds such as proteins and nucleic acids.
Electrospray ionisation: The chief advantage of this technique is that the molecular weight measuring of each multiply charged ion is made and mean of all the values is taken, which gives the truth of +/- 0.01 % .
Atmospheric force per unit area chemical ionisation: an electrical discharge is developed around the gaseous sample molecule by evaporating the solution watercourse. Ionization is done by chemical procedures. This method can non be applied for larger molecules [ 10 ] .
After the ionisation the charged ions are separated by mass analysers. Some of the mass analysers used are:
Time of flight ( TOF )
Magnetic sector/ dual concentrating magnetic sector
Quadrupole mass analysers
Quadrupole ion trap ( QIT )
Ion cyclotron resonance ( ICR )
EXPERIMENTAL
Purpose: The purpose of this undertaking is to synthesize three sulphonamide drugs, sulphanilamide, sulphapyridine and sulphathiazole, and to characterize the synthesised compounds by NMR, mass and FTIR spectrometries.
Instrumentality:
1 ) . Infra ruddy spectrometry: Infra-red analysis was performed with KBr phonograph record utilizing Perkin Elmer Paragon 1000 FTIR spectrometer ( scope 4000 to 400 cm-1 ) .
2 ) . Mass spectrometry: Analysis was performed utilizing the ESI method and acetonitrile as dissolver.
3 ) . NMR analysis was performed by JEOL 500 MHz NMR spectrometer at a frequence of 270.16 MHz and at the pulse length of 3.76 micro-secs with the relaxation hold of 1sec.
p-acetamidobenzenesulphonyl chloride
Chemical construction:
Figure 5. Structure of p-acetamidobenzenesulphonyl chloride
IUPAC Name: 4 acetamidobenzenesulphonyl chloride
p-acetamidobenzenesulphonyl chloride was ordered from Alfa Aesar, batch no. 10148484. FTIR, Mass and NMR spectra were used to prove the pureness of the natural stuff as the pureness of concluding merchandise depends majorly on the pureness of the natural stuff.
Physical belongingss: light brown pulverization.
Determination of runing point: The sample was loaded in the capillary tubing and placed in the thaw point setup, and the temperature was recorded when the sample thaws.
Consequence: The runing point of the sample was 146.5oC
Literature Value: approximately 146oC [ 11 ] .
RESULTS AND DISCUSSION:
FTIR spectrometry:
Sample preparation- the howitzer and stamp were washed with propanone to take any hints of the H2O as K bromide dissolves in H2O. Approximately 100 milligrams of KBr was ground up to organize all right pulverization. A little sum of sample was added to it. The dye set was besides washed with the propanone, arranged and the sample loaded into it. The imperativeness was used to organize the KBr discs to lade in the slit. The vacuity was applied and the force per unit area applied for 3 min. The force per unit area was so released and the phonograph record was taken out and loaded in the FTIR spectrometer.
Graph 1 – FTIR spectrum of p-acetamidobenzenesulphonyl chloride
The following tabular array summarizes the consequences obtained by FTIR analysis for p-acetamidobenzenesulphonyl chloride
Table 1 – Table for FTIR analysis of p-acetamidobenzenesulphonyl chloride
No.
PEAK cm-1
Assignment
1
3307
N-H stretch ( 20 amide )
2
1681.7
C=O stretch
3
1585.5
Aromatic ring C=C “ external respiration ”
4
1402.6
-CH3 crook distortion
5
1369
S=O stretch
The extremum at 3307 shows the presence of secondary amide, and the extremum at 1681.7 is because of C=O stretching. The presence of benzine ring is confirmed by the extremum at 1585.5. Medium -CH3 distortion has resulted into a extremum at 1402.6. and presence of S=O stretch is apparent from the extremum at 1369.
1H NMR spectrometry:
0.0013gm of sample was weighed and dissolved in DMSO D6 ( premixed with TMS ) . The solution was loaded in the NMR tubing, labelled and submitted for proton and C-13 NMR analysis.
Graph 2 – 1H NMR spectrum of p-acetamidobenzenesulphonyl chloride
Consequences derived from proton NMR spectrum:
Table 2 – Table for 1H NMR analysis of p-acetamidobenzenesulphonyl chloride
Chemical SHIFT ( ppm )
Integration
SPLITTING Form
Assignment
2.0
3
vest
-CH3
7.55
4
multiplet
Ar-H
10.07
1
vest
-NH
The proton nuclear magnetic resonance spectrum shows all the extremums as expected from the construction. The extremum at around 7.5 is a multiplet due to non-equivalent protons attached to the ring. All the other extremums are at expected places. The extremum at 2.0 is the methyl group, which appears as vest due to no next H atoms. The extremum at 10.07 is due to amide proton nowadays in the molecule. The extremum at 2.5 is due to DMSO-D6, the dissolver used and the little extremum at 0 is the Tetramethylsilane ( TMS ) , the mention criterion used.
NMR C-13 spectrometry:
Graph 3 – C-13 NMR spectrum of p-acetamidobenzenesulphonyl chloride
Table 3 – Table for C-13 NMR analysis of p-acetamidobenzenesulphonyl chloride
Chemical SHIFT ( ppm )
ASSIGNMEENT
21.0
-CH3
117.9
AROMATIC C
126.1
AROMATIC C
139.6
AROMATIC C
142.3
AROMATIC C
168.4
-C=O
The first extremum a 21.0 is due to methyl C in the molecule. Since we have plane of symmetricalness in the molecule, therefore we will be acquiring merely 4 extremums for the aromatic ring. The extremums at 117.9 and 126.1 are the C atoms with the H attached to them, at ortho and meta places. The extremums at 139.6 and 142.3 are due to the Cs at place 1 and 4, as they do non hold any protons attached to them therefore they will give a weak signal. And besides due to attachment to nitrogen and sulfur they will be shifted more downfield. The extremum at 40 is due to DMSO D6. The most downfield extremum at 168.4 is due to -C=O, as O is negatively charged and strongly deshields the carbonyl C.
Mass spectrometry:
Graph 4 – Mass spectrum of p-acetamidobenzenesulphonyl chloride
The mass spectrum of p-acetamidobenzenesulphonyl chloride has been obtained by negative ESI manner, hence the expected mass of molecular ion is
[ M+-1 ] = [ 233-1 ] = 232The extremum at 232 is due to the merchandise, p-acetamidobenzenesulphonyl chloride.
The extremum at 214 is the base extremum, and it is due to the hydrolysis of the sample as the sample was kept in waiting line for analysis.
Ar-SO2Cl Ar-SO3H
The extremum at 234 is due to the isotope ( perchance Cl-37 ) .
As we have received all the consequences as expected from the molecular construction, the sample is pure and can be used for farther experiment.
SULPHANILAMIDE
Structure:
Figure 6. Structural representation of sulphanilamide synthesis
IUPAC Name: 4 aminobenzenesulphonamide
Synthesis of sulphanilamide:
5.006gm of p-acetamidobenzenesulphonyl chloride was added into a 150ml Erlenmeyer flask and 15ml of conc. ammonium hydrated oxide added to it in the goon. The mixture was stirred good with the stirring rod. The mixture was so heated on a warming mantle for 15 min. , with frequent stirring. The stuff became a pastelike suspension ; the flask was removed and placed in the ice bath. The mixture was good cooled and 6M Hydrochloric acid was added until the mixture was acidic to litmus paper ( bluish litmus turned ruddy ) . The mixture was further cooled and the merchandise was filtered over the Buchner funnel with vacuity. The merchandise is washed with 50ml of cold H2O and kept for drying nightlong. The dried petroleum merchandise was transferred to Round underside flask. 3ml of conc. Hydrochloric acid, 6ml of H2O and anti-bumping rocks were added to it. The solution is refluxed for 15min and so boiled for excess 10min. and cooled to room temperature. A clear solution was formed. To this 5ml of H2O and small bleaching C was added. The solution was so filtered by gravitation after agitating into a 200ml beaker. 4gm of Na hydrogen carbonate was dissolved in little sum of H2O and added to the filtrate with stirring until the solution became impersonal to litmus. Sulphanilamide precipitated after the add-on of Na hydrogen carbonate. The mixture was cooled on ice bath and filtered utilizing Buchner funnel. The merchandise was recrystallised by fade outing it in H2O, and so chilling it over ice bath. It was filtered utilizing Buchner funnel. The merchandise was allowed to dry.
Physical texture of sulphanilamide: White crystalline pulverization.
Determination of runing point: The sample was loaded in the capillary tubing and placed in the thaw point setup, and the temperature was recorded when the sample thaws.
Consequence: The runing point of the sample was 163oC.
Literature value- thaw point should be around 162oC-165oC [ 12 ] .
Theoretical output computation:
P-Aceamidobenzenesulfonyl chloride a†’ sulphanilamide
mol. wt. 233.67 mol. wt. 172.20
Gram molecules of merchandise:
5gm * 1mole = 0.021397 mol,
233.67
Therefore, mass of merchandise will be
0.021397 * 172.20 = 3.6845gms
Theoretical output of sulphanilamide is 3.6845gms
Practical output of sulphanilamide is 2.130gms.
Percentage output obtained for the merchandise is 2.130/3.6845*100 = 57.81 %
Result and treatment for sulphanilamide:
FTIR spectrometry: Sample preparation- the howitzer and stamp were washed with propanone to take any hints of the H2O as K bromide is dissolves in it. Approximately 100 milligrams of KBr was land to organize all right pulverization. Small sum of sample was added to it. The dye set was besides washed with the propanone, arranged and the sample loaded in it. The imperativeness was used to organize the KBr phonograph record to lade in the slit. The vacuity was applied and the force per unit area applied for 3 min. the force per unit area was released and the disc was taken out and loaded in the FTIR spectrometer.
Graph 5 – FTIR spectrum of sulphanilamide
Table 4 – Table for FTIR analysis of sulphanilamide
No.
PEAK cm-1
Assignment
1
3478.6
-NH2 stretch ( aminobenzine ) , symmetric
2
3375.8
-NH2stretch ( aminobenzine ) , asymmetric
3
3366.2
-NH stretch ( sulphonamide )
4
1595.7
Aromatic ring -C=C- stretch
5
1313.6
SO2 Stretch
The extremums at 3478.6 and 3375.8 is due to -NH2 stretch, the former one is the aniline NH2 and the latter is NH2 attached to SO2. The aromatic ring is indicate by the [ extremum at 1595.7. the SO2 generated the extremum at 1313.6.
1H NMR spectrometry:
Sample readying: 0.0011gm of sample was weighed and dissolved in DMSO D6 ( premixed with TMS ) . The solution was loaded in the NMR tubing, labelled and given for H1 and C-13 spectrometry in the NMR research lab.
Graph 6 – 1H NMR spectrum of sulphanilamide
The consequences from the 1H NMR analysis are as follows:
Table 5 – Table for 1H NMR analysis of sulphanilamide
Chemical SHIFT ( ppm )
Integration
SPLITTING Form
Assignment
5.8
2
vest
Ar-NH2
6.6
2
doublet
Ar-H
6.9
2
vest
-SO2NH2 ( amide )
7.4
2
doublet
Aromatic C
The extremums at 6.6 and 7.4 are due to the protons attached to the aromatic ring. Due to the plane of symmetricalness, we have received merely two extremums and they are with approximately a doublet splitting form. Each of these protons is chiefly coupled to a proton on an next C. The extremum at 5.8 is because of the -NH2 group attached to C-1.
The extremum at 6.9 is besides due to -NH2 group but it is shifted downfield being attached to a sulphonyl group. The H atoms of the aminoalkane and sulphonamide groups are exchangeable, so if my anticipation about their signals is right, the extremums should vanish with a D2O shingle. The extremum at 2.5 is of DMSO-D6.
1H NMR D2O shingle: The deuterated H2O was added to the sample NMR tubing and shaken good and the spectrum recorded once more.
Graph 7 – 1H D2O shingle NMR spectrum of sulphanilamide
As expected the two extremums due to amine and sulphonamide groups have disappeared, and this confirms that the sample is sulphanilamide. The excess extremum generated at 3.9 in this spectrum is due to the H2O signal.
NMR C-13 spectrometry:
Graph 8 – C-13 NMR spectrum of sulphanilamide
Table 6 – Table for C-13 NMR analysis of sulphanilamide
Extremums
Assignment
112.4
Aromatic C ( ortho places )
127.3
Aromatic C ( meta places )
130.0
Aromatic C ( C-SO2NH2 )
151.8
Aromatic C ( C-NH2 place )
Due to plane of symmetricalness there are merely four signals. The signal generated by C at 1 and 4 places are downfield because of negatively charged atoms attached to them. The extremum at 40 is due to the dissolver DMSO D6.
Mass spectrometry:
Sample readying: 0.00123 gram of sample was dissolved in 1ml ( 1000Aµl ) . 10 Aµl was taken from the solution and once more diluted with 990Aµl dissolver. The dissolver used is Acetonitrile. The prepared sample was run with 30 % H2O + 70 % CH3OH in the mass spectrometer utilizing ESI method for ionisation.
Graph 9 – Mass spectrum of sulphanilamide
The spectrum was taken as positive ESI, so the extremums will demo the mass as +1.
The extremum at 173 shows the presence of the merchandise, sulphanilamide.
The extremum at 105.1 should be formed by the remotion of SO2 group from the molecule.
The extremum at 155.1 is due to loss of NH2.
The extremum at 214 is due to the natural stuff left unreacted.This extremum was besides seen in the mass spectra of the natural stuff due to hydrolysis of p-acetamidobenzenesulphonyl chloride.
The extremum at 255.0 is besides an dross.
SULPHAPYRIDINE:
Structure:
Figure 7. Structural representation of sulphapyridine synthesis
IUPAC Name: 4-amino-N-pyridine-2-ylbenzenesulphonamide
Synthesis:
2.4115gm of 2-aminopyridine was dissolved in 10ml of anhydrous pyridine ( antecedently dried over KOH pellets ) in a unit of ammunition underside flask. 6.0018gm of p-acetamidobenzenesulphonyl chloride was added to the mixture. The solution was refluxed for 20min. in a warming mantle. The mixture was cooled somewhat and 50ml of H2O added to it with some H2O to assistance in the transportation. The mixture was stirred in the ice bath until the oil crystallised. The solid was filtered through the Buchner funnel. The rough merchandise was transferred to the unit of ammunition underside flask and 20ml of 10 % Na hydrated oxide is added. The solution was allowed to reflux for 40min, utilizing stop prick lubricating oil to forestall the articulations from stop deading. The mixture was cooled and neutralized with 6M hydrochloric acid. The sulphapyridine precipitated out in the reaction is filtered utilizing Buchner funnel. The sulphapyridine was recrystallised by fade outing in 100ml of 95 % ethyl intoxicant utilizing hot home base for heating. The solution was so filtered, cooled and crystallised merchandise separated by vacuity filtration.
Physical texture: xanthous white formless pulverization.
Determination of runing point: The sample was loaded in the capillary tubing and placed in the thaw point setup, and the temperature was recorded when the sample thaws.
Consequence: The runing point of the sample was 191oC. literature value is between 190 nad 193oC [ 13 ] .
Calculation of per centum output:
p-acetamidobenzenesulphonyl chloride + 2-Aminopyridine a†’ sulphapyridine
mol. wt. 233.67 mol.wt. 249.29
Theoretical output:
6.0018gm * 1mole = 0.02568 mol,
233.67
Therefore, the expected mass is 0.02568 * 249.29 = 6.40gms
Practical output =2.4115gm
Percentage output =37.68 %
RESULTS AND DISCUSSION:
IR spectrometry: The sample was triturated with the K bromide and pressed to organize the discs, which are so loaded in the FTIR spectrometer.
Graph 10 – FTIR spectrum of sulphapyridine
The consequences of the FTIR spectrum are as under:
Table 7 – Table for FTIR analysis of sulphapyridine
NO.
Extremum
Assignment
1
3427.9 & A ; 3349.1
-NH2 STRETCH
2
3254.1
-NH STRETCH ( SULPHONAMIDE )
3
3036.5
AROMATIC RING,
C-H stretch
4
1596.3, 1531.0, 1499.4, 1461.3
Confirm THE PRESENCE OF BENZENE RING “ RING Breathing ”
5
1138.8
SO2 STRETCH
6
765.9
o-SUBSTITUTED PYRIDINE RING,
C-H Bending
The IR spectrum of the merchandise reveals the followers inside informations about the molecule:
The molecule contains NH2 stretch, apparent by the extremums at 3427.9 and 3349.1.
The extremum at 3254.1 is because of the NH stretch of the sulphonamide group in the molecule.
There is a extremum at 3036.5 which is the feature of the aromatic ring C-H stretch, and presence of benzine ring is proven by the extremums present at 1596.3, 1531.0, 1499.4 and 1461.3 ( typical of “ pealing external respiration ” ) .
The SO2 group gives rise to the extremum at 1138.8.
The extremum observed at 765.9 is due to the ortho substituted pyridine ring, C-H bending.
Mass spectrometry:
Sample readying: 0.00115 gram of sample was dissolved in 1ml ( 1000Aµl ) . 10 Aµl was taken from the solution and once more diluted with 990Aµl dissolver. The dissolver used is Acetonitrile. The prepared sample was run with 30 % H2O + 70 % CH3OH in the mass spectrometer utilizing ESI method for ionisation.
Graph 11 – Mass spectrum of sulphapyridine
The information gained from the mass spectrum analysis of sulphapyridine obtained by positive ESI is listed below:
Expected mass of molecular ion is M+1=249+1=250
The extremum at 250.2 is the base extremum and is due to the ionised molecule itself ( M+1 ) .
The extremum at 251.2 and 252.2 could be due to the presence of and isotope, could be the heavy hydrogen atom or C-13.
The extremum at 156 is due to loss of pyridine ring, and [ H2N-Ar-SO2 ] + is the disconnected molecule.
NMR Spectroscopy: 0.0014gm of sample was weighed and dissolved in acetone-d6 ( premixed with TMS ) . The solution was loaded in the NMR tubing, labelled and submitted for proton and C-13 spectrometry in the NMR research lab
RESULT AND DISCUSION:
NMR H1 spectrum:
Graph 12 – 1H NMR spectrum of sulphapyridine
The consequences from the H1 NMR spectrum for the merchandise are as under:
Table 8 – Table for 1H NMR analysis of sulphapyridine
Chemical SHIFT ( ppm )
Integration
SPLITTING Form
Assignment
5.5
1
Singlet
-NH2
6.6
2
Doublet
Ar-C
6.9
1
Three
H AT 4th POSITION IN PYRIDINE Ring
7.6
2
Doublet
Ar-C
7.3
1
Doublet
H at 3rd POSITION IN PYRIDINE Ring
8.1
1
Doublet
H at 6th POSITION ADJACENT TO N IN PYRIDINE Ring
9.5
1
Singlet
SO2-NH
NMR C-13 spectrum:
Graph 13 – C-13 NMR spectrum of sulphapyridine
The consequences obtained by C-13 NMR spectrum are listed below:
Table 9 – Table for C-13 NMR analysis of sulphapyridine
Extremums
Assignment
112.7
Ar-C
113.7
Ar-C
119.0
HETROAROMATIC C
119.3
HETROAROMATIC C
139.0
HETROAROMATIC C at place 2
130.1
HETROAROMATIC C at 3rd place
148.5
HETROAROMATIC C at 6th place
In this spectrum we should hold received entire of nine extremums. There are 11 C in the molecule but due to the presence of plane of symmetricalness in the aromatic ring we expect nine signals. Merely seven extremums were observed. The Cs at places 1 and 4 in the benzine ring would be expected to give weak signals ( no H attached to them )
So these signals are likely obscured by the “ noise ” as the base line.
SULPHATHIAZOLE:
Structure:
Figure 8. Structural representation of sulphathiazole synthesis
IUPAC Name: 4-amino-N- ( 1,3-thiazol-2-yl ) benzenesulfonamide
Synthesis:
2.5010gm of 2-aminothiazole was weighed. 6.5061gm of
p- acetamidobenzenesulphonyl chloride was weighed. 10ml of anhydrous pyridine ( dried over KOH pellets ) was added to 2-aminothiazole in unit of ammunition underside flask.
p-acetamidobenzenesulphonyl chloride was added easy and temperature was checked throughout the add-on. The temperature was ever kept below 40oC. Addition is done with uninterrupted twirling. The mixture was allowed to reflux for 25 min. Mixture was cooled and poured into 75ml of H2O. The solution was stirred with glass rod. Crystals were isolated by vacuity filtration and washed with cold H2O, and left to dry overnight. Weight of the solid merchandise was 5.6967gm. The solid was dissolved in 57ml of 10 % NaOH. The solution was allowed to reflux for one hr. After reflux, solution was cooled and conc. HCl was added till the pH reached 6 ( litmus paper was used to set the pH ) . Solid Na ethanoate was added till the litmus turned bluish ( ruddy litmus was dipped in the solution ) . The solution was brought to furuncle and cooled in ice bath. The merchandise was filtered by vacuity was filtration. The merchandise obtained e dissolved in H2O and boiled. The mixture was filtered at Buchner funnel and filtrate was cooled easy. The precipitated merchandise was filtered by vacuity filtration and allowed to dry overnight.
Physical texture: Yellow white pulverization.
Determination of runing point: The sample was loaded in the capillary tubing and placed in the thaw point setup, and the temperature was recorded when the sample thaws.
Consequence: The runing point of the sample is 200.5oC.
Literature value- is between 200 and 204oC [ 14 ] .
Calculation of per centum output:
p-acetamidobenzenesulphonyl chloride + 2-Aminothiazole a†’ sulphathiazole
mol. wt. 233.67 mol.wt. 255.31
Theoretical output:
6.5061gm * 1mole = 0.02788 mol,
233.67
Therefore, theoretical mass is 0.02788 * 255.31 = 7.12gms
Practical output =2.4095gm
Percentage output =33.84 %
RESULT AND DISCUSSION:
FTIR Spectroscopy:
Graph 14 – FTIR spectrum of sulphathiazole
Table 10 – Table for FTIR analysis of sulphathiazole
No.
Extremum
Assignment
1
3464.8 & A ; 3359.0
-NH2 stretch
2
3100.0
AROMATIC RING,
C-H STRETCH
3
1593.6, 1570.0, 1499.4, 1419.5
CONFIRMS BENZENE RING
“ Ring BREAHING ”
4
1143.2
SO2 STRETCH
5
1088.3
C-S
6
830.9
Para DISUBSTITUTED RING, OUT OF PLANE C-H BENDING
The IR spectrum of sulphathiazole exhibits 2 crisp soaking ups at 3464.8 and 3359.0, which are the feature of -NH2 stretch. The little extremum at 3100.0 ( its non marked ) corresponds to the aromatic ring C-H stretch, and the benzine ring is confirmed by the presence of extremums at 1593.6, 1570.0 ( non marked ) , 1499.4, 1419.5. in these the extremums at 1593.6 and 1499.4 are due to aromatic ring C=C quiver and other two extremums at 1570.0 and 1419.5 are the C=C bending manners. The 1143.2 extremum is due to SO2 and the 1088.3 extremum appears due to C-S bond in the thiazole ring. The extremum at 830.9 is the declarative that the aromatic ring is para-disubstituted ring, with out of plane C-H bending.
1H NMR spectrum: 0.0015gm of sample was weighed and dissolved in DMSO D6 ( premixed with TMS ) . The solution was loaded in the NMR tubing, labelled and given for H1 and C-13 spectrometry in the NMR research lab
Graph 15 – 1H NMR spectrum of sulphathiazole
Table 11 – Table for 1H NMR analysis of sulphatiazole
Chemical SHIFT ( ppm )
Integration
SPLITTING Form
Assignment
5.8
2
Singlet
-NH2
6.5
2
Doublet
AROMATIC H ortho places
6.7
1
Doublet
H at place 4 in THIAZOLE Ring
7.2
1
Doublet
H at place 3 in THIAZOLE Ring
7.4
2
Doublet
AROMATIC H meta places
12.4
1
Singlet
-NH GROUP
The extremum at 5.8ppm is due to the aminoalkane group in the molecule. The H atoms in the benzine pealing give rise to the extremums at 6.5 and 7.4. The former is due to I± H and due to plane of symmetricalness, so the integrating is 2. The most downfield extremum is due to the sulphonamide. The sulphonyl group and N are all negatively charged atoms, so the sulphonamide H atom has shifted downfield more than expected.
NMR C-13 spectrum:
Graph 16 – C-13 NMR spectrum of sulphathiazole
C-13 NMR spectra of Sulphathiazole provides the following inside informations:
Table 12 – Table for C-13 NMR analysis of sulphathiazole
Extremums
Assignment
107.3
HETROAROMATIC C at place 5
112.3
Ar-C
124.1
HETROAROMATIC C at place 2
127.6
Ar-C
134.0
C-NH2
152.1
HETROAROMATIC C at 4th place
In the above spectra we have received 6 extremums alternatively of 7. The signal of the staying C-atom is perchance weak and is hidden within the quivers.
Mass spectrometry:
Sample readying: 0.00112 gram of sample was dissolved in 1ml ( 1000Aµl ) . 10 Aµl was taken from the solution and once more diluted with 990Aµl dissolver. The dissolver used is Acetonitrile. The prepared sample was run with 30 % H2O + 70 % CH3OH in the mass spectrometer utilizing ESI method for ionisation
Graph 17 – Mass spectrum of sulphathiazole
The information gained by the positive ESI mass spectrum of sulphathiazole is as under:
The extremum at 256 is the base extremum and is due to the ionized molecule of sulphathiazole.
The extremums at 257 and 258 could be due the presence of isotopes.
Peak at 258 is besides due to the presence of isotopes.
Decision:
Sulphanilamide, sulphapyridine and sulphathiazole were synthesized from
p-acetamidobenzenesulphonyl chloride. The synthesized merchandises were so assessed and analyzed by different spectroscopic techniques FTIR, NMR, and mass spectroscopy.
In FTIR analysis all the expected characteristic extremums were observed and the constructions of all compounds were confirmed.
In mass spectroscopy utilizing electrospray ionisation method the merchandises synthesized have shown the expected consequences. However, in the mass spectrum of Sulphanilamide, the extremum at 214 m/z indicates the opportunities of some sum of get downing stuff left unreacted as this extremum is common in both, get downing stuff and concluding merchandise. The other two compounds gave the spectrum as expected and did non demo any dross. In NMR spectroscopic analysis, the C-13 spectrum of sulphapyridine and sulphathiazole showed less figure of extremums than expected. The signals of the losing extremums were weak and therefore remain concealed in the vibrational base line. All the extremums that were received were in right place as expected, so the compound can be regarded as pure.
Further Work:
The C-13 spectrum of two compounds, sulphapyridine and sulphathiazole had some of the extremums losing. So, the farther work involves the C-13 spectrometry of the compounds at assorted parametric quantities and understanding what are the different effects of these parametric quantities on the spectrum and which are best for analyzing these compounds.
