Chapter -4 GC-MS analysis of hexane and benzene extracts of P. granatum

Transcription

1 Chapter -4 GC-MS analysis of hexane and benzene extracts of P. granatum If you wish to reach the highest, begin with the lowest

2 4. GC-MS analysis of Hexane and Benzene extracts of P. granatum A. GC-MS of RP-1 Compounds isolated from the hexane fraction of Ethanol extract Methyl heptanes H 3 C Fig Methyl decane H 3 C Fig Methyldec-1-ene Fig Methyl decane H 3 C Fig methylhexane 138 Fig methylhexane

3 H 3 C Fig Heptyl cyclobutane Fig methylpentane H 3 C Fig methylbutan-1-ol H 3 C OH Fig Propylcyclobutane Fig Methyl octanoate O O Fig dec-7-enoic acid O H 3 C Fig.4.12 OH B. GC-MS of RP-2 Compounds isolated from the hexane: benzene fraction of Ethanol extract.

4 1. 2-methylpentan-3-ol OH C H 3 Fig.4.13 CH methyldecane H 3 C Fig methyloctan-1-ol H 3 C OH Fig methyloctane H 3 C Fig ,3-dimethylpentane H 3 C Fig ,5-dimethylhexane or 2,4-dimethylpentane H 3 C H 3 C Fig Octylcyclobutane

5 Fig Methylhexane H 3 C C H 3 Fig Methylheptane H 3 C Fig Pentylcyclobutane Fig Decanoic acid O H 3 C Fig OH C. GC-MS of RP-3 Compounds isolated from the benzene fraction of Ethanol extract. 1. n- Undecane H 3 C Fig Methyl Undecane H 3 C Fig Butylcyclopropane

6 Fig Methyl Pentanol O H Fig Methyl Nonane Fig Methyl Hexane H 3 C Fig Cyclobutane-3-ene-Pentane Fig Methyl Pentane H 3 C Fig ,4 - Dimethyl Pentane

7 H 3 C Fig Methyl Hexane Fig Methyl Hex-2-ene H 3 C Fig Methyl Heptane H 3 C Fig But-2-ene H 3 C Fig Henicos-5-ene H 3 C Fig GAS CHROMATOGRAPHY Gas chromatography produces highly defined separation in organic chemistry, biochemistry, environmental and biological fields. Besides this it finds extensive application for the detection, identification and quantification of fatty acids

8 methyl esters. Among its uses the most common are drug testing and environmental contaminant identification. GC analysis is a common confirmation test. It separates all of the components in a sample and provides a representative spectral output. The technician injects the sample into the injection port of the GC device. The GC instrument vaporizes the sample and then separates and analyzes the various components. Each component ideally produces a specific spectral peak that may be recorded on a paper chart or electronically. The time elapsed between injection and elution is called the "retention time." The retention time can help to differentiate between some compounds. The size of the peaks is proportional to the quantity of the corresponding substances in the specimen analyzed. The peak is measured from the baseline to the tip of the peak. [1] Gas chromatography and mass spectrometry are, in many ways, highly compatible techniques. In both techniques, the sample is in the vapor phase, and both techniques deal with the same amount of sample (typically less than 1 ng). GC can separate volatile and semi-volatile compounds with great resolution, but it cannot identify them. MS can provide detailed structural information on most compounds such that they can be exactly identified, but it cannot readily separate them. Therefore, it was not surprising that the combination of the two techniques was suggested shortly after the development of GC in the mid-1950s. It was in 1950s that the use of a mass spectrometer was was developed as the detector in gas chromatography by Roland Gohlke and Fred McLafferty. [2] Gas chromatography ("GC") and Mass spectrometry ("MS") make an effective combination for chemical analysis. In GC-MS technique, the gas chromatograph is directly coupled to fast speed mass spectrometer. The Gas chromatography produce highly defined separation of complex mixture and mass spectrometer analyses them. [3][4]. PRINCIPLE Chromatography is a very important analytical tool because it allows the chemist to separate components in a mixture for subsequent use or quantification. Most samples that chemists want to analyze are mixtures. The gas chromatograph makes it possible to separate the

9 volatile components of a very small sample and to determine the amount of each component present. The essentials required for the method are an injection port through which samples are loaded, a column on which the components are separated, a regulated flow of a carrier gas (often helium) which carries the sample through the instrument, a detector, and a data processor. In gas chromatography, the temperature of the injection port, column, and detector are controlled by thermostatic heaters. [5] The method was introduced by James and Martin [6] who used a liquid phase of silicone grease with or without 10% stearic acid to separate normal saturated acids up to C 12, which were detected then Cooper and Heywood [7] expanded the method to acids up to C 22. The method has been used for the separation and identification of geometrical and positional isomers [8] [9]. The pioneering work by Thomson, Aston, Dempster and Nier led to the construction of mass spectrometers for precise measurement of ionic masses using magnetic and electrostatic analyzers. Today mass spectrometers are widely used to identify unknown compounds by way of determining their molecular weight and molecular formula at the expense of negligible amount of sample. The mass spectrum of a compound is given in the form of a bar graph representing the abundance of various ions with respect to their mass (m) to charge (z) ratio (m/z). In addition to the molecular ion, which infers the molecular weight of the sample, the other ions present in the mass spectrum are very characteristic of the compound, thus the mass spectrum of a compound becomes the fingerprint of it, in most of the cases [10]. Today, we all know that a single conventional analytical technique often does not suffice to recognize and quantify all analyses of interest in a sample. During the eighties, the use of multidimensional approaches slowly began to make headway to solve many complex problems. The main driving force behind these developments is the determination of trace level concentration of drugs and their metabolites in biomedical, biological, food, agricultural and environmental samples. Now trace-level determinations have becomes 100 fold stringent. GC-MS Instrumentation

10 GC-MS is an integrated composite instrument combining GC, which is excellent in its ability to separate and quantify components with MS, which is excellent in its ability to identify each component. Thus GC-MS is an integrated composite instrument, it consist of 1. Gas chromatograph 2. Interface (connector) 3. Mass spectrometer (MS) 4. Computer Fig The insides of the GC-MS, with the column of the gas chromatograph in the oven on the right.

11 Outline Diagram of Gas Chromatograph and Mass Analyzer This combination of GC-MS consists of determination of a mass spectrum for each chromatographically separated component of a mixture. Samples need not to be isolated or rigorously purified. The exit pressure of the gas chromatograph is approximately atmospheric. On the other hand, the mass spectrometer operates at high vacuum, 10-6 mm Hg. Therefore, the pressure must be reduced during transfer of effluent from the gas chromatograph to the mass spectrometer and for this purpose an interface is introduced [11]. The GC-MS is composed of two major building blocks: the gas chromatograph and the mass spectrometer. The gas chromatograph utilizes a capillary column which depends on the column's dimensions

12 (length, diameter, film thickness) as well as the phase properties (e.g. 5% phenyl polysiloxane). The difference in the chemical properties between different molecules in a mixture will separate the molecules as the sample travels the length of the column. The molecules take different amount of time (called the retention time) to come out of (elute from) the gas chromatograph, and this allows the mass spectrometer downstream to capture, ionize, accelerate, deflect, and detect the ionized molecules separately. The mass spectrometer does this by breaking each molecule into ionized fragments and detecting these fragments using their mass to charge ratio. These two components, used together, allow a much finer degree of substance identification than either unit used separately. It is not possible to make an accurate identification of a particular molecule by gas chromatography or mass spectrometry alone. The mass spectrometry process normally requires a very pure sample while gas chromatography using a traditional detector (e.g. Flame Ionization Detector) detects multiple molecules that happen to take the same amount of time to travel through the column (i.e. have the same retention time) which results in two or more molecules to co-elute. Sometimes two different molecules can also have a similar pattern of ionized fragments in a mass spectrometer (mass spectrum). Combining the two processes makes it extremely unlikely that two different molecules will behave in the same way in both a gas chromatograph and a mass spectrometer. Therefore when an identifying mass spectrum appears at a characteristic retention time in a GC-MS analysis, it typically lends to increased certainty that the analyte of interest is in the sample. Purge and Trap GC-MS For the analysis of volatile compounds a Purge and Trap (P&T) concentrator system may be used to introduce samples. The target analytes are extracted and mixed with water and introduced into an airtight chamber. An inert gas such as Nitrogen (N 2) is bubbled through the water; this is known as purging. The volatile compounds move into the headspace above the water and are drawn along a pressure gradient (caused by the introduction of the purge gas) out of the chamber. The volatile compounds are drawn along a heated line onto

13 a 'trap'. The trap is a column of adsorbent material at ambient temperature that holds the compounds by returning them to the liquid phase. The trap is then heated and the sample compounds are introduced to the GC-MS column via a volatiles interface, which is a split inlet system. P&T GC-MS is particularly suited to volatile organic compounds (VOCs) and BTEX compounds (aromatic compounds associated with petroleum). [12] Types of Mass Spectrometer Detectors The most common type of mass spectrometer (MS) associated with a gas chromatograph (GC) is the quadrupole mass spectrometer, sometimes referred to by the Hewlett-Packard (now Agilent) trade name "Mass Selective Detector" (MSD). Another relatively common detector is the ion trap mass spectrometer. Other detectors are also there such as time of flight (TOF), tandem quadrupoles (MS-MS), or in the case of an ion trap MS n where n indicates the number mass spectrometry stages. Analysis A mass spectrometer is typically utilized in one of two ways: Full Scan or Selective Ion Monitoring (SIM). The typical GC/MS instrument is capable of performing both functions either individually or concomitantly, depending on the setup of the particular instrument. Full scan MS When collecting data in the full scan mode, a target range of mass fragments is determined and put into the instrument's method. An example of a typical broad range of mass fragments to monitor would be m/z 50 to m/z 400. The determination of what range to use is largely dictated by what one anticipates being in the sample while being cognizant of the solvent and other possible interferences. A MS should not be set to look for mass fragments too low or else one may detect air (found as m/z 28 due to nitrogen), carbon dioxide (m/z 44) or other possible interferences. Additionally if one is to use a large scan range then sensitivity of the instrument is decreased due to performing fewer scans per second since each scan will have to detect a wide range of mass fragments.

14 Full scan is useful in determining unknown compounds in a sample. It provides more information than SIM when it comes to confirming or resolving compounds in a sample. During instrument method development it may be common to first analyze test solutions in full scan mode to determine the retention time and the mass fragment fingerprint before moving to a SIM instrument method. Selected ion monitoring In selected ion monitoring (SIM) certain ion fragments are entered into the instrument method and only those mass fragments are detected by the mass spectrometer. The advantages of SIM are that the detection limit is lower since the instrument is only looking at a small number of fragments (e.g. three fragments) during each scan. More scans can take place each second. Since only a few mass fragments of interest are being monitored, matrix interferences are typically lower. To additionally confirm the likelihood of a potentially positive result, it is relatively important to be sure that the ion ratios of the various mass fragments are comparable to a known reference standard. Types of Ionization After the molecules travel the length of the column, pass through the transfer line and enter into the mass spectrometer they are ionized by various methods with typically only one method being used at any given time. Once the sample is fragmented it will then be detected, usually by an electron multiplier diode, which essentially turns the ionized mass fragment into an electrical signal that is then detected. The ionization technique chosen is independent of using Full Scan or SIM. Electron Ionization By far the most common and perhaps standard form of ionization is electron ionization (EI). The molecules enter into the MS (the source is a quadrupole or the ion trap itself in an ion trap MS) where they are bombarded with free electrons emitted from a filament, not much unlike the filament one would find in a standard light bulb. The electrons bombard the molecules, causing the molecule to fragment in a characteristic and reproducible way. This "hard

15 ionization" technique results in the creation of more fragments of low mass to charge ratio (m/z) and few, if any, molecules approaching the molecular mass unit. Hard ionization is considered by mass spectroscopists as the employ of molecular electron bombardment, whereas "soft ionization" is charge by molecular collision with an introduced gas. The molecular fragmentation pattern is dependent upon the electron energy applied to the system, typically 70eV (electron Volts). The use of 70eV facilitates comparison of generated spectra with National Institute of Standard (NIST-USA) library of spectra applying algorithmic matching programs and the use of methods of analysis written by many method standardization agencies. Chemical Ionization In chemical ionization a reagent gas, typically methane or ammonia is introduced into the mass spectrometer. Depending on the technique (positive CI or negative CI) chosen, this reagent gas will interact with the electrons and analyte and cause a 'soft' ionization of the molecule of interest. A softer ionization fragments the molecule to a lower degree than the hard ionization of EI. One of the main benefits of using chemical ionization is that a mass fragment closely corresponding to the molecular weight of the analyte of interest is produced. Positive Chemical Ionization In Positive Chemical Ionization (PCI) the reagent gas interacts with the target molecule, most often with a proton exchange. This produces the species in relatively high amounts. Negative Chemical Ionization In Negative Chemical Ionization (NCI) the reagent gas decreases the impact of the free electrons on the target analyte. This decreased energy typically leaves the fragment in great supply. Interface (direct coupled) - When the inside diameter of the capillary column is 0.25 mm or 0.32 mm; the column is directly connected with the ion source. If

16 the carrier gas flow is less than 2 ml/min, a turbo molecular pump is satisfactory as the vacuum pump. Jet separator A carrier gas jet separator is generally used in GC-MS. Its function is to remove carrier gas completely from the GC effluent to prevent losses in mass spectral resolution due to pressure broadening from ion - molecule collisions. The principle of the jet separator is the preferential diffusion of lighter carrier gas molecule away from the GC effluent stream as it emerges at high velocity from a narrow aerator. The main functions of molecular jet separator are: 1. It ideally passes the entire sample without decomposition, while the entire carrier gas is removed. 2. It increases the sample to carrier gas ratio, when its input and output concentrations are compared. 3. Reduces the outlet pressure from GC column to a pressure suitable for the ion source. The carrier gas is almost always a small molecule such as helium or hydrogen with a high diffusion coefficient, whereas the organic molecules have much lower diffusion coefficients. In operation, the GC effluent (the carrier gas with the organic analytes) is sprayed through a small nozzle, To avoid the problem thermo stable liquid phase of concentration 0.5 to 5% is used. The use of computer system in combination with GC-MS instrument is very important [13-14]. It facilitates the detection and identification of the compound present in minor quantity. The computer must have large care storage and be able to process the data at high speed. The main advantages in computerization of GC-MS are as follows: 1. The establishment of the automatic mass scale with the help of a reference compound or sensor measuring the magnetic field. 2. Collection of output data at faster rate on the GC-MS instrument. 3. The background spectra can be subtracted from the actual spectra for i.e. sample bleed from sample.

17 4. Presentation of mass spectra in normalized and is plotted as a bar graph just after the sample has been injected into the column. General Uses of GC-MS Identification and quantitation of volatile and semi-volatile organic compounds in complex mixtures Determination of molecular weights and (sometimes) elemental compositions of unknown organic compounds in complex mixtures Structural determination of unknown organic compounds in complex mixtures both by matching their spectra with reference spectra and by a priori spectral interpretation Common Applications Quantitation of pollutants in drinking and wastewater using official U.S. Environmental ProtectionAgency (EPA) methods Quantitation of drugs and their metabolites in blood and urine for both pharmacological and forensic applications Identification of unknown organic compounds in hazardous waste dumps Identification of reaction products by synthetic organic chemists Analysis of industrial products for quality control. [15-17] GC-MS Studies The Hexane, Hexane benzene fractions, were examined in GC-MS for its chemical composition by GC-MS engine model, GC Clarus 500; Perkin Elmer and Computer Mass Library (Wiley 138L) of 80,000 compounds with a GC column Elite 1 (100% Methyl Poly Siloxane). The other conditions were as follows: Injector: GC-Clarus 500; Perkin Elmer; Carrier gas flow Helium 1 ml/min; Split ratio 1:25; Sample injected 1μl; Oven temperature 110 o C 2 min hold; Up to 270 o C at the ratio of 5 deg/min 4 min hold; Injector temperature 250 o C; Total GC- time 38 min; MS inlet line temperature 200 o C; Source temperature 200 o C; Electron energy 70eV; Mass Scan ; MS time 39 min. Identification of components

18 The retention index is calculated for all volatile constituents using a homologous series of n-alkanes. The components of oil were identified by matching their mass spectra with those stored in the computer library such as Wiley, New York mass spectral (MS) library and their retention indices (RI) either with authentic compounds or with published data in the literature.

19 Present work: For the present work we have taken the oily fraction of P.granatum (rind) as isolated from the separation of hexane extract. The hexane eluate of ethanol extract of P.granatum yielded a waxy fraction which was rechromatographed on silica gel column. Its hexane eluates yielded waxy fractions, which were small in amount and due to the small quantity and very less separation seen on the TLC plates they were separated by Gas Chromatography and were analysed by mass spectrophotometer associated with it from Saurashtra University Rajkot. The compounds were identified by comparing their retention time and covet indexes with that of literature and by interpretation of mass spectra. SAMPLE- RP-1 CH3 CH3 CH (CH2)4 CH3

20 CH3 CH3 CH (CH2)7 CH3 CH3 CH3 CH2 CH (CH2)5 CH2=CH2

21 CH3 CH3 CH2 CH (CH2)6 CH3 CH3 CH3 CH2 CH (CH2)2 CH3

22 CH3 CH3 CH (CH2)3 CH3 (CH2)6 CH3

23 CH3 CH3 CH (CH2)2 CH3 CH3 CH3 CH CH2 CH2 OH

24 (CH2)2 CH3 O CH3 CO (CH2)6 CH3

25 O CH3CH2 CH=CH (CH2)5 C OH SAMPLE RP - 2 CH3 CH3 C CH (CH2) CH3 H OH

26

27 CH3 CH3 CH2 CH (CH2)4 CH3 CH3 CH3 CH2 CH (CH2)4 CH2OH

28 CH3 CH3 CH3 CH (CH2)2CH CH3 CH3 CH3CH2 C CH2 CH (CH2)4 CH3 CH3

29 CH3 CH3 CH (CH2)3 CH3 (CH2)7 CH3

30 (CH2)4 CH3 CH3 CH3 CH (CH2)4 CH3

31 O CH3 (CH2)8 C OH SAMPLE RP - 3 CH3 (CH2)9 CH3

32

33 CH3 CH3 CH2 CH CH2 CH2 OH CH2 CH2 CH2 CH3

34 CH3 CH2 CH (CH2)5 CH3 CH3 CH3 CH (CH2)3 CH3

35 -CH2 CH=CH CH2 CH3 CH3 CH3 CH (CH2)2 CH3

36 CH3 CH3 CH2 CH (CH2)2 CH3 CH3 CH3 CH3 CH CH2 CH CH3

37 CH3 CH3 CH (CH2)4 CH3 CH3 CH3 (CH2)2 CH=CH CH3

38 CH3 CH=CH CH3 CH3 (CH2)3 CH=CH(CH2)12 CH3

39 Identification of P.granatum (hexane fraction): RP-1 Peak 1: 2-Methylheptane Mass spectrum showed that the molecule was an aliphatic compound. Base peak obtained at m/z 43, showed the presence of ( CH 2 )2- CH3 moiety. The mass peak at m/z 113 confirmed the compound to be an aliphatic compound having 7 carbons. peak 1 was identified as 2-Methylheptane. Thus the Peak 2: 2-Methyldecane Mass spectrum showed that the molecule was a long chain aliphatic compound. Base peak obtained at m/z 43, showed the presence ( CH 2 )2- CH3 moiety. The mass peak at m/z 131 confirmed the compound to be an aliphatic compound having 10 carbons. peak 2 was identified as 2-Methyldecane. Thus the Peak 3: 8-Methyl, 3 dec-1-ene- Mass spectrum showed that the molecule was an aliphatic compound. The possible fragmentation with peaks at 86,71 and 57,43 Base peak obtained at m/z 57, showed the presence of ( CH 2 )n and the mass peak at m/z 133 confirmed the compound to be an unsaturated compound having 10 carbons. peak 3 was identified as 8-Methyl 3dec-1-ene. Thus the Peak 4: 3-Methyldecane Mass spectrum showed that the molecule was an aliphatic compound. Base peak obtained at m/z 57, showed the presence of ( CH 2 ) n The mass peak at m/z 134 confirmed the compound to be an aliphatic compound having 10 carbons. identified as 3-Methyldecane. Thus the peak 1 was Peak 5: 3-Methylhexane Mass spectrum showed that the molecule was a small aliphatic compound. 100% abundant fragment obtained at m/z 57, showed the presence of CH3 CH2CH2CH2 CH 2. The mass peak at m/z 93 confirmed the compound to be an aliphatic compound having 6 carbons. Thus the peak 1 was identified as 3-Methylhexane. Peak 6: 2-Methylhexane Mass spectrum showed that the molecule was a small aliphatic compound. Base peak obtained at m/z 43, showed the presence of ( CH 2 )n The mass peak at m/z 103 confirmed the compound to be an aliphatic compound having 6 carbons. identified as 2-Methylhexane. Thus the peak 1 was

40 Peak 7: Heptylcyclobutane Mass spectrum showed that the molecule was a small aliphatic compound. Base peak obtained at m/z 56, showed the presence of CH 2. The mass peak at m/z 93 confirmed the compound to be an aliphatic compound having 6 carbons. 1 was identified as Heptylcyclobutane. Thus the peak Peak 8: 2-Methylpentane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 85, which was due to the presence of CH3-(CH2)n. This confirmed the compound to be 2-Methylpentane. Peak 9: 3-Methylbutan-1-ol Mass spectrum showed that the molecule was a long chain aliphatic alcohol. Abundant fragment obtained at m/z 43 and 147 was due to the position of hydroxyl groups. [18] This confirmed the compound to be an aliphatic alcohol. Thus the peak 4 was identified as 3-Methylbutan-1-ol. Peak 10: Propylcyclobutane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 100, which was due to the presence of CH3CH2CH2-. [21] This confirmed the compound to be Propylcyclobutane Peak 11: Methyl octanoate- Mass spectrum showed the mass peak at 173. The mass fragments at 85, 60 and 43 confirm the long chain of compound. The peak at 73 was due to the cleavage of a fragment by McLefferty rearrangement. [19,20] Thus the peak 11 was identified as Methyloctanoate. Peak 12: 7 -dec-7-enoic acid- Mass spectrum identified the molecule as organic acid. The position of double bond was confirmed by the fragments at 83 and 69, the peak at 55 indicated the formation of CH3- CH2-CH=CH + ion. 10 Carbons were confirmed by the peaks at 97, 83, 69 and the compound was identified as 7 -dec-7-enoic acid Identification of P.granatum (Hexane: Benzene fraction): RP-2 Peak 1: 2-Methylpentan-3-ol 3-Methylbutan-1-ol Mass spectrum showed that the molecule was a long chain aliphatic alcohol. Abundant fragment obtained at m/z 43 and 147 was due to the position of hydroxyl

41 groups. This confirmed the compound to be an aliphatic alcohol. Thus the peak 4 was identified 2-Methylpentan-3-ol [21] Peak 2: 2-Methyldecane The mass spectrum identified the molecule as an aliphatic long chain hydrocarbon. 100% abundant fragment obtained at m/z 71, 57.43, 41, which were due to the presence of CH3 CH2CH2CH2 CH 2. This confirmed the compound to be 2-Methyldecane. Peak 3: 6-Methyloctane-1-ol Mass spectrum showed that the molecule was a long chain aliphatic alcohol. Abundant fragment obtained at m/z 43 and 147 was due to the position of hydroxyl groups. This confirmed the compound to be an aliphatic alcohol. Thus the peak 4 was identified as 6-Methyloctane-1-ol Peak 4: 3-Methyloctane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 3-Methyloctanoate. Peak 5: 2, 3-Dimethylpentane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 2, 3-Dimethylpentane. Peak 6: 2, 5-Dimethylhexane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 2, 5-Dimethylhexane. Peak 7: Octylcyclobutane Mass spectrum showed that the molecule was a small aliphatic compound. Base peak obtained at m/z 56, showed the presence of CH 2. The mass peak at m/z 93 confirmed the compound to be an aliphatic compound having 6 carbons. 1 was identified as Octylcyclobutane. Thus the peak Peak 8: 2-Methylhexane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 2-Methylhexane. [22] Peak 9: 2-Methylheptane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56,

42 which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 2-Methylheptane. Peak 10: Pentyl cyclobutane Mass spectrum showed that the molecule was a small aliphatic compound. Base peak obtained at m/z 56, showed the presence of CH 2. The mass peak at m/z 93 confirmed the compound to be an aliphatic compound having 6 carbons. Thus the peak 1 was identified as Pentylcyclobutane. Peak 11: Decanoic acid Mass spectrum showed that the molecule was a long chain aliphatic alcohol. Abundant fragments obtained at m/z 43. And the mass peak was seen on 174. This confirmed the compound to be an aliphatic compound. Thus the peak 2 was identified as 2-Methyl- Undecane. Identification of P.granatum (Benzene fraction): RP-3 Peak 1: n-undecane Mass spectrum showed that the molecule was a long chain aliphatic compound. Abundant fragments obtained at m/z 43, were due to the CH 2. This confirmed the compound to be an aliphatic compound having 11 carbons. Thus the peak 1 was identified as n-undecane. Peak: 2. 2-Methyl-Undecane Mass spectrum showed that the molecule was a long chain aliphatic alcohol. Abundant fragments obtained at m/z 43. And the mass peak was seen on 170 which confirmed the compound to be an aliphatic compound. Thus the peak 2 was identified as 2- Methyl-Undecane. Peak 3: Butyl Cyclopropane : Mass spectrum showed that the molecule was a small aliphatic compound. Base peak obtained at m/z 56, showed the presence of CH 2. The mass peak at m/z 93 confirmed the compound to be an aliphatic compound having 6 carbons. 1 was identified as Butylcyclopropane. Thus the peak Peak 4: 3-Methyl Pentan-1-ol Mass spectrum showed that the molecule was a long chain aliphatic alcohol. Abundant fragment obtained at m/z 43 and 145 was due to the position of hydroxyl groups. [22] This confirmed the compound to be an aliphatic alcohol. Thus the peak 4 was identified as 3-Methyl Pentan-1-ol.

43 Peak 5: 3-Methylnonane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 3-Methylnonane. Peak 6: 2-Methylhexane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 2-Methylhexane. Peak 7: 2 pent-2-en-1-ylcyclobutane- Mass spectrum showed that the molecule was a small aliphatic compound. Base peak obtained at m/z 56, showed the presence of CH 2. The mass peak at m/z 93 confirmed the compound to be an aliphatic compound having 6 carbons. peak 1 was identified as 2 pent-2-en-1-ylcyclobutane. Thus the Peak 8: 2-Methylpentane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 3-Methylhpentane. Peak 9: 2, 4 - Dimethyl Pentane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 2,4-Dimethylpentane. Peak 10: 3-Methylhexane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 3-Methylhexane. Peak 11: 2-3-methylhex-2-ene Mass spectrum showed that the molecule was an aliphatic compound. The possible fragmentation with peaks at 86,71 and 57,43 Base peak obtained at m/z 57, showed the presence of CH 2 and the mass peak at m/z 133 confirmed the compound to be an unsaturated compound having 10 carbon. peak 3 was identified as 2-3-methylhex-2-ene ene. Thus the Peak 12: 2-Methylheptane The mass spectrum identified the molecule as an aliphatic hydrocarbon. 100% abundant fragment obtained at m/z 56, which was due to the presence of CH3CH2CH2CH2 CH 2. This confirmed the compound to be 3-Methylhexane.

44 Peak 13: 2 -but-2-ene ene Mass spectrum showed that the molecule was an aliphatic compound. The possible fragmentation with peaks at 86, 71 and 57,43 Base peak obtained at m/z 57, showed the presence of CH 2 and the mass peak at m/z 133 confirmed the compound to be an unsaturated compound identified as 2 -but-2-ene. having 10 carbon. Thus the peak 3 was

45 Peak 14: Henicos-5-ene- Mass spectrum showed that the molecule was an aliphatic compound. The peaks at 98, 83, 56 and 41 indicate the long chain aliphatic compound and the presence of a single unsaturation. Thus the compound was identified as Henicos-5-ene. Experimental Work The powdered fruit rind of P. granatum was extracted and then fractionated with various organic solvents as discussed in chapter 3 Processing of Ethanol extract: The ethanol extract was fractionated by liquid-liquid extraction with different solvents in their increasing order of polarity and various fractions were collected. The non polar solvent hexane yielded a fraction which was coated on silica and was again fractionated by hexane which yielded oily fraction which was very less in quantity. There TLC examination in the system Hexane: Benzene (9:1) showed long spot indicating the mixture of components having Rf values very close to each other and could not be separated by TLC or even column chromatography and it was decided to analyze them by GC-MS analysis. GC-MS analysis revealed the presence of 12, 11 and14 different compounds from the three samples RP-1, RP-2, RP-3 respectively. The compounds were identified by comparing their retention time and covet indexes with that of literature and by interpretation of mass spectra. Mass Spectrum: m/z (rel. int. %) Investigation of hexane fraction RP-1 Peak 1: (R. time: 2.125) (1.23%) Line.1 M + 113, 85, 71,57,43, 41, 27 Peak 2: (R. time: 2.375) (22.79%) Line.2 M + 135, 105, 86, 71,57,43, 41, 27 Peak 3: (R. time: 2.157) (19.03%) Line.3 M + 133, 105, 86, 71,57,43, 41, 26 Peak 4: (R. time: 2.675) (22.38%) Line.4 M + 134, 119, 98, 86, 71, 57, 43, 41, 27 Peak 5: (R. time: 2.917) (2.97%) Line.5 M + 93, 85, 71,57,43, 41, 27 Peak 6: (R. time: 2.967) (3.29%) Line.6

46 M + 103, , 71,57,43, 41, 27 Peak 7: (R. time: 3.083) (19.29%) Line.7 M + 122, 105, 84, 70, 56, 42, 41, 27 Peak 8: (R. time: 3.358) (0.62%) Line.8 M + 106, 85, 71, 57, 43, 41, 27 Peak 9: (R. time: 3.433) (1.16%) Line.9 M + 100, 88, 85, 70, 57, 43, 41, 27 Peak 10: (R. time: 3.550) (6.55%) Line.10 M + 123, 100, 84, 56, 55, 44, 27 Peak 11: (R. time: ) (0.51%) Line.11 M + 256, 239, 227, 213, 199, 185, 157, 143, 129, 115, 98, 97, 83, 69, 55, 41, 27 Peak 12: (R. time: ) (0.19%) Line.12 M + 264, 235, 209, 165, 137, 123, 98, 97, 83, 69, 55, 41, 27 Investigation of hexane: benzene fraction RP-2 Peak 1: (R. time: 2.125) (1.59%) Line.1 M + 120, 85, 71, 57, 43, 41, 27 Peak 2: (R. time: 2.383) (17.22%) Line.2 M + 152, 135, 86, 71, 57, 43, 41, 27 Peak 3: (R. time: 2.525) (19.24%) Line.3 M + 147,119, 98, 86, 71, 57, 43, 41, 27 Peak 4: (R. time: 2.658) (22.14%) Line.4 M + 127, 103, 86, 71, 57, 43, 41, 27 Peak 5: (R. time: 2.925) (3.87%) Line.5 M + 104, 98, 85, 71, 57, 43, 41, 27 Peak 6: (R. time: 2.975) (3.66%) Line.6 M + 117, , 71,57,43, 41, 27 Peak 7: (R. time: 3.083) (20.80%) Line. M + 139, 135, 105, 84, 70, 56, 42, 41, 27 Peak 8: (R. time: 3.358) (0.91%) Line.8 M + 99, 85, 71, 57, 43, 41, 40, 27 Peak 9: (R. time: 3.442) (1.66%) Line.9 M + 108, 100, 85, 57, 43, 41, 27 Peak 10: (R. time: 3.550) (8.53%) Line.10 M + 127, 100, 84, 56, 55, 41, 27

47 Peak 11: (R. time: ) (0.40%) Line.11 M + 256, 239, 227, 213, 199, 185, 174, 157, 143, 129, 115, 98, 85, 73, 60, 43 41, 27 Investigation of benzene fraction RP-3 Peak 1: (R. time: 2.125) (1.81%) Line.1 M + 157, 135, 105, 85, 71,57,43, 41, 27

48 Peak 2: (R. time: 2.383) (15.74%) Line.2 M + 150, 135, 107, 86, 71,57,43, 41, 27 Peak 3: (R. time: 2.417) (2.22%) Line.3 M + 96, 70, 67,42, 41, 27 Peak 4: (R. time: 2.525) (18.06%) Line.4 M + 135, 105, 86, 71, 51, 43, 41, 27 Peak 5: (R. time: 2.683) (20.45%) Line.5 M + 140,135, 105, 86, 71, 57,43, 41, 27 Peak 6: (R. time: 2.975) (10.94%) Line.6 M + 115, 100, 85, 71,57,43, 41, 27 Peak 7: (R. time: 3.092) (18.98%) Line.7 M + 135, 105, 87, 70, 56, 55, 41, 27 Peak 8: (R. time: 3.358) (0.90%) Line.8 M + 125, 85, 71, 51, 43, 41, 27 Peak 9: (R. time: 3.442) (1.77%) Line.9 M + 100, 85, 70, 57, 43, 41, 27 Peak 10: (R. time: 3.550) (8.87%) Line.10 M + 102, 84, 83, 70, 56, 41, 27 Peak 11: (R. time: 3.658) (0.14%) Line.11 M + 135, 98, 84, 83, 69,56, 55, 41, 27 Peak 12: (R. time: 3.783) (0.07%) Line.12 M + 135, 98, 84, 70, 56, 55, 41, 27

49 Peak 13: (R. time: 3.867) (0.02%) Line.13 M + 286, 244, 187, 124, 98, 97, 70, 56, 55, 41, 27 Peak 14: (R. time: 4.442) (0.01%) Line.14 M + 244, 159, 130, 98, 84, 83, 68, 56, 55, 41, 27 Table: 4.1 GC-MS details of sample: RP-1 Peak R.Time Area Area% Name Methylheptane Methyldecane Methyldec-1-ene Methyldecane Methylhexane Methylhexane Heptylcyclobutane Methylpentane Methylbutane-1-ol Proply cyclobutane Methyloctanoate dec-7-enoic acid

50 Table: 4.2 GC-MS details of sample: RP-2 Peak R.Time Area Area% Name Methylpentan-3-ol Methyldecane Methyloctane-1-ol Methyloctane ,3-Dimethylpentane ,5-Dimethylhexane Octylcyclobutane Methylhexane Methylheptane Pentyl cyclobutane Decanoic acid

51 Table: 4.3 GC-MS details of sample RP-3 Peak R.Time Area Area% Name n- Undecane methyl Undecane Butyl Cyclopropane methyl pentane-1-ol methyl nonane methyl hexane pent-2-en-1- ylcyclobutane methyl Pentane ,4- dimethyl pentane methyl hexane methylhex-2-ene methyl heptane But-2-ene

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