A highly Sensitive (LOD; 0.04-0.4 ng/ml) method is developed for detection and quantification of acidic compounds (C3 -C10) containing mono and dicarboxylic acids on GC-MS. These compounds (C3 -C10) existed in trace amount, as secondary organic aerosols i.e. important constituents of Aerosols. Membrane extraction technique was utilized for selective enrichment (1-4300 times) of target compounds. Good repeatability (RSD% ≤ 10%) from selective organic phase (10% TOPO in DHE) was achieved with three phase HF-LPME. Aerosols containing samples, after Ultrasonic Assisted extraction were detected and quantified Through GC-MS. Effective derivatization of each target compound was performed with BSTFA reagent. Gas Chromatography, having capillary column and interfaced with mass spectrometry was used for separation, detection and quantification of target compounds.
Method Development and Application -hollow fiber Supported liquid membrane extraction of Fatty acids (C3-C10) containing mono and dicarboxylic acids and Detection of aerosols Samples after ultrasonic assisted extraction.
Impact of Atmospheric aerosols on human health and effect on radioactive stability in Earth’s atmosphere is getting importance now a days and this phenomenon has been well understood. . Atmospheric aerosols can harm respiratory and cardiovascular system of human.
Impact of Secondary organic aerosols as biogenic and anthropogenic antecedent is identified (Adams and sinfold, 2002) [1, 17]. Low molecular dicarboxylic acids (C3-C9) are also vital tracers of SOA . Short chain fatty acids are found as secondary organic aerosols which are also supposed to derive from long chain fatty acids . Importance of organic aerosol has been well established now a days and carboxylic acids are of great interest for environmental studies . Several studies and mechanisms were proposed to understand the production of these SOA precursors . Short chain carboxylic acids are found extensively in troposphere . Secondary organic aerosols (SOA) are formed in the atmosphere by gas particles conversions. Organic matter present in aerosol contains more than 90% of troposphere’s aerosols [5, 15].
Dicarboxylic acids found in nature as polymeric compounds such as suberin and cutin . Short chain dicarboxylic acids are found in vegetables [Siddiqui, 1989] and in soil containing micro organisms of durum wheat . Dicarboxylic acids are found in plant oils which have greater interest for cosmetic and pharmaceutical industries . Short chain dicarboxylic acids having aliphatic chain possess strong cyclotoxicity and antineoplastic activities .
Many analytical techniques are used to determine the composition of SOA so keeping in view these techniques new method for determination of fatty acids (common in SOA) has been developed. Membrane extraction is used in this method due to its increasing importance for high selectivity and high enrichment factor .
Dicarboxylic acids formed of bio oxidation of fatty acids so these are considered as metabolic part of fatty acid . Dicarboxylic acids and their derivatives can be used to make polymers and their condensation with diols in solution produces high molecular weight polyester . Additionally these dicarboxylic acids use less temperature in the reaction for the preparation of polyesters .
1.1. Analytes Description:
Properties (physical, chemical, etc.) of Compounds (C3-C10) containing mono and dicarboxylic acids are discussed in section; 1.1.1-1.1.12. These compounds (C3-C10) are the target analytes in this diploma project. These target analytes are extracted through Liquid phase micro extraction and detected by GC-MS system. Fig. 1.1-1.12 represents structures of target analytes (section; 1.1.1-1.1.12).
1.1.1- Adipic Acid
Adipic acid is a product of lipid per oxidation. Adipic acid does not undergo hydrolysis in the environment perhaps due to the lack of hydrolysable functional groups (Harris 1990) .
1.1.2- Malonic Acid:
Malonic Acid is a metabolite of plants and tissues and Malonyle-CoA . Malonic Acid is an intermediate for preparation of fatty acids from plants and other tissues . Malonic acid is also present in aerosols . Malonic acid is an important constituent of short chain fatty acids . Malonic acid present in beet rots as a Calcium salt .
1.1.3- Succinic Acid:
Succinic acid is found in atmosphere as water soluble compound and as a compound of Secondary organic aerosols . Succinic acid is a solid exists as crystals, anciently called spirit of amber. Succinic acid is an important intermediate in citric acid cycle which is very important constituent of living organism .
1.1.4- Glutaric acid:
Glutaric acid is found as SOA in aerosols . Glutaric acid is sparingly soluble in water , can be used to prepare a plasticizer for polyester .
1.1.5- Pimelic Acid:
Pimelic acid is a last dicarboxylic acid relative to carbon number which has IUPAC name. Derivatives of Pimelic acid are used for biosynthesis of amino acid typically lysine . Pimelic acid is produced, when Nitric acid is heated with Oleic acid as a secondary sublimation product which is not crystallized .
Suberic acid is produced from suberine . Suberic acid can also be obtained by vigorous reaction condition of natural oil with nitric acid .
Azelaic acid is an important constituent of secondary organic aerosols because it produces short chain fatty acids upon photo oxidation and also because it can be produced during oxidation of unsaturated acid that is found in Oleic acid .
1.1.8- Cis-pinonic Acid:
Cis-pinonic acid is also produced in atmosphere by photo oxidation of α-pinene in the existence of Ozone .
1.1.9- Pinic Acid:
Pinic acid is derivative of α-pinene. Pinic acid can be generated by photo oxidation of α-pinene with Ozone as given in this chemical reaction; (C10H16 + 5/3 O3 —-> C9H14O4 + HCHO). Pinic acid is present in a crystalline form used to prepare plasticizers .
1.1.10- 4-Hydroxybenzoic Acid
4-Hydroxy benzoic acid is exists as crystals. It is used to derive parabens and can be used as antioxidant .
Phthalic acid is an aromatic dicarboxylic acid it is found as white crystalline state in pure form . Phthalic acid is found abundantly in atmosphere and it has toxic properties. Aromatic acids are generally emitted through anthropogenic sources like reminiscent of solvent evaporation and Automobile exhaust .
Syringic acid is found as humic substance in environment .
1.2. Detection of Ultrasonic Assisted Extraction samples(UAE):
A detection procedure by GC-MS is established with reference standard injections and UAE samples. A theoretical description is given in section 1.2 for “Ultrasonic assisted extractions”. Unknown real Samples from Aerosols containing mono and dicarboxylic acids (C 3-C 10) are provided after Ultrasonic assisted extraction .
1.2.1- Ultrasonic Assisted Extraction:
‘Ultrasonic’ is derived from ultrasound. Ultrasound refers to a sound that has a higher frequency than a normal human can hear. This technique is used in chemistry in several aspects and due to application in chemistry it is known as Sonochemistry .
Ultra Sound is used in sample preparation in analytical chemistry like extraction, filtration, dissolution and sample purification. When Ultrasonic technique is used for assistance in extraction, this assistance in extraction is called “Ultrasonic assisted extraction” (UAE) .
There are many advantages by using UAE because it require less organic solvents ,non destructive, less expensive and less time consuming comparative to other sample preparation techniques like soxhlet .
The normal range of ultrasound frequencies used in laboratory ranges from 20 KHz to 40 KHz. Use of UAE is simple. A sample solution inside a vessel in an appropriate solvent can be placed inside ultrasonic bath at desired temperature and sound waves stir the sample .
The mechanism of US is as “when a sound source produces a high frequency waves, sample molecules starts vibrating and shift this vibration to other molecules of sample in a longitudinal direction when gas and liquid is used as a sample, while in solid sample both longitudinal and transverse waves can be produced” .
When UAE is utilized it increases speed of mass transport by vibration of mechanical transport from the sample matrix through a process called “cavitation” .
1.2.2- Theory of Ultrasonic Assisted extraction:
There are two theoretical aspects of sonication i.e. physical and chemical aspects in sample preparation. Physical and chemical aspects are described in section (126.96.36.199-188.8.131.52), in order to understand its practical use in analytical chemistry.
184.108.40.206- Physical aspects of UAE:
During Ultrasonic assisted extraction, a bubble in a liquid cannot take energy (due to US) and implodes. On the other hand due to Ultra sound in liquid extractions, the cavitational pressure is shifted relatively higher so formation of bubble is difficult .
Ultrasonic intensity produces cavitations in a liquid sample during extraction (UAE). Two types of US cavitation is produced known as “transient cavitation” (produce transient bubble) and “permanent cavitation” .
The life time of transient bubble is so short that no mass transport or diffusion of gas is possible with in a sample . Transient bubble is believed to be produced at US intensity (10 W/cm2) and permanent bubble at intensity (1-3 Watt/cm2). Sonochemical effects are intense inside the bubble because energy (numerous amounts) is produced during bubble eruption and production .
220.127.116.11 Chemical aspects of UAE:
When US radiation strikes a water molecule it produces free radicals OH* and H* due to collapsing cavitations’ bubble which exhibits high temperature and pressure inside and also many other radicals can be produced in solution . Radical OH* is believed to be more stable and can begin many new reactions while H* radical is not stable. Second Sonochemical effect is pyrolytic reactions that occur inside bubble and can degrade compounds under analysis [21, 23].
1.3. Liquid Phase microExtraction(lpme):
The application of membrane extractions in analytical chemistry has taken the intentions of analysts during recent time. The goal of utilizing membrane extraction is to achieve high enrichment, selective extraction and environmental friendly procedure . Small quantity of solvent (usually in micro liters) is required comparative to old techniques of extractions (soxlet) . Clean extracts are obtained and after extraction, recovered compounds are shifted to another analytical instrument like Gas chromatography or liquid chromatography directly for further quantitative analysis .
1.3.1 Hollow fiber membrane extraction:
Two types of membrane are used in LPME. One membrane is flat sheet porous and second membrane is polypropylene hollow fiber. In this project polypropylene hollow fiber is used as a membrane support in membrane extractions due to limited cost and to reduce carry over problems .
18.104.22.168 HF-LPME Technique:
When a hollow fiber is used in LPME, this technique (LPME) is called hollow fiber liquid phase micro extraction (HF- LPME). In HF- LPME technique, a hollow fiber is used containing a thin film of immobilized liquid membrane inside the pores while the fiber is dipped into an aqueous phase containing objective analytes. Target analytes can transport through the membrane into a liquid filled inside the lumen of the fiber, which is termed as accepter solution .
Extraction of target analytes (C3-C10) was carried through three phase HF- LPME during whole of the project. Donor solution was contained analytes in aqueous medium, a suitable organic solvent i.e. Dihexyl ether (TOPO mixture) was used in pores of hollow fiber as a stationary liquid membrane support (SLM). Accepter solution was in aqueous medium .Target analytes were recovered into accepter phase after evaporation of water. Acetonitrile solvent was added in dried GC vial along with derivatizing reagent. After derivatization these samples were injected into a Gas chromatographic system.
1.3.2 Basic Principle of LPME:
Basic principle is same for all LPME techniques (two phase or three phase LPME), the variation is only from accepter region . In three phase liquid phase micro extraction technique (HF- LPME) a donor aqueous solution is filled in a vial or flask containing sample analytes. A short piece of hollow fiber is used and accepter solution is injected inside fiber through a micro syringe after injecting accepter solution one end is closed and other end contains syringe needle. Fiber containing solutions is inserted in an appropriate organic solvent having less polarity (Dihexyl ether) to create a stationary liquid membrane (SLM). Donor solution pH is adjusted such that it can restrain the ionization of target analytes .
The process of three phase extraction  can be explained as follows in Eq 1.1.
Where ‘A’ is a target analyte, ‘K1’, ‘K2’, ‘K3’ and ‘K4’ are first order extraction rate constants. In order to obtain combined distribution coefficient, at equilibrium recovery, Eq. 1.2 is derived .
D accepter/sample = C eq accepter / C eq sample
= C Org sample/ C eq accepter
=α D .Korg/sample / α a. Korg/accepter………….1.2
In Eq. 1.2, C eq accepter, C eq sample and C Org sample are the concentration of analytes at equilibrium, in accepter phase, in aqueous sample phase and in organic membrane phase respectively.Here Korg/sample, Korg/accepter are the partition ratio’s between Organic phase and sample phase and between accepter phase and organic phase respectively . α D and α a are the extractable fraction of total concentration of target analyte in sample and accepter respectively.
If conditions are similar between sample and accepter, other than ionization of analytes in sample phase, from Eq. 1.2, equilibrium is independent from partition ratio of stationary liquid membrane in three phase lpme i.e. it depends mainly on ionization of analytes in sample .
Extraction efficiency (E) can be calculated from Eq. 1.3.
V sample, V accepter and V mem , in Eq. 1.3, are the volume of donor sample phase, aqueous accepter phase and organic immobilized membrane liquid phase respectively. D accepter/sample and D Org/sample are individual distribution coefficients relative to accepter phase to sample phase and Organic phase (SLM) to sample phase respectively . Eq. 1.3 is derived for three phase lpme. It is evident; from the interpretation of Eq. 1.3 that efficiency is mainly controlled by individual distribution coefficients. Individual distribution ratios are directly dependent on partition coefficients, so by increasing the partition ratios efficiency can be improved . Partition coefficients can be improved by properly adjusting the pH of donor and accepter and by using an appropriate organic solvent. Volume of sample and organic phase should also be kept minimum, according to Eq. 1.3 in order to develop efficiency .
1.3.3-Mass transfer in LPME:
Enrichment factor (Ee) for three phase LPME is given in Eq. 1.4.
Ee = C accepter/C initial
= V sample. E / V accepter ………….. 1.4
In Eq. 1.4, C accepter is the concentration of target analyte, present in final stage inside accepter solution .
When an acidic analyte is ionized in aqueous solution, total extractable fraction of analyte (α) is given in Eq. 1.5 .
α = [AH]/ [A-][AH] = 1/[1+10(pH-pKa)] ……….. 1.5
In the context of Eq. 1.3, the overall distribution constant (D) at equilibrium can be rearranged as given in Eq. 1.5 .
D = 1+10 s (pH-pKa) . KD /1 + 10 s (pH-pKa). KA ………….. 1.6
‘s’ is equal to 1 for acidic analytes (Eq. 1.6). ‘pKa’ is dissociation constant and pH refers to donor or accepter solution(Eq. 1.6) .
âˆ†C = αD .Cs – αa CA.KA/KS ………… 1.7
Eq. 1.5-1.7 are derived from Henderson-Hasselbalch relation, in this equation α represents the extractable fraction of analytes .
The driving force for the extraction in neutral conditions of three phase LPME is the concentration gradient (âˆ†C) from sample to accepter . The concentration gradient between two phases, between donor and accepter, is described in Eq. 1.7. K represent partition ratio of uncharged analyte between the membrane and aqueous phase. CA and Cs are the concentrations of analytes in accepter and sample phase respectively.
1.3.4 End point for extraction:
Three end points are normally considered for extraction .
1. Exhaustive extraction. 2. Kinetic extraction. 3. Equilibrium extraction.
22.214.171.124 Exhaustive extraction:
Exhaustive end point is the specific end point (time), when all amount of analytes are exhausted (which can be practically possible) present in donor . In this practical diploma work, Exhaustive end point will be applied in (LPME) extractions. Enrichment factor will increase by growing analyte concentration in accepter by the passage of time, at certain point it reaches a stable value . Mass transfer between organic phase and liquid phase is dependent on concentration gradient . Enrichment factor can be improved by increasing the value of αD preferably close to unity and decreasing the value of αA to zero. Such conditions for the αD and αA values are called “infinite sink” conditions, normally required for exhaustive extractions . Situation close to these values can be achieved for acids by selective tuning the pKa values. For example for acidic compound if pH of accepter is adjusted, 3.3 (pH) units above than the pKa of acidic analytes this Difference set the value of αA to 0.0005, at this point accepter can capture all analytes. At this set value (αA), enrichment factor increases linearly with time . Peak time of enrichment factor, when other parameters are constant, can be calculated by comparison of CA maximum. CA maximum (‘CA’ is considered as time dependent) can be obtained by careful calculation of CA maximum values at a certain time, before this value starts to decrease again .
1.3.5 Rate of LPME:
Two parameters, govern the rate of extraction (when extraction approaches to equilibrium conditions), are membrane controlled extractions or diffusion controlled extractions [13, 24]. The maximum concentration Ee can be obtained when concentration gradient (âˆ†C) is approaches to zero described in Eq. 1.8 [13, 24].
Ee (max) = (C a / C d) max
= αD/αA ………….. 1.8
In membrane controlled extractions, the rate limiting step is the diffusion of target analytes. When analytes pass through the organic phase, the mass transfer (Km) is given in Eq. 1. 9 [13, 16].
Km µ K.D m /h m ……….. 1.9
In Eq. 1.9; K is partition coefficient, Dm is membrane diffusion coefficient and ‘h m ‘ is the thickness of membrane [13, 16].
1.3.6 Addition of Trioctylphosphine oxide(TOPO):
Mass transfer can be improved for acidic analytes by using different concentrations (w/v) of TOPO in organic solvent typically for short chain carboxylic acids. Interaction of TOPO with polar acids in solution takes place efficiently due to hydrogen bonding .
1.3.7 Trapping of Analyte in Three phase lpme:
Concentration enrichment of analytes in three phase LPME can be achieved by stable mass transfer through the membrane to accepter phase. Back diffusion of analytes is prevented by trapping of analytes in accepter phase. In order to achieve high enrichment of acidic analytes pH of accepter phase is fixed enough basic so that when acidic analytes reached to the accepter solution becomes charged. Analytes could not be driven back to donor. So this trapping of analytes due to pH adjustment is called ‘’direct trapping’’. For high enrichment purpose, pH of accepter is usually adjusted 3.3 pH units higher than the pKa values of acidic target analytes while extracting from acidic donor. Buffer capacity of accepter should be sufficient such that during extraction protons from acidic donor cannot be neutralized by the concentration gradient between two aqueous phases during three phase lpme .
1.3.8 Selection for Organic phase:
Choice of organic solvent has basic importance in method validation because this solvent directly affect partition coefficient. Organic phase solvent should have low solubility in water  and low volatility to prevent solvent losses during extraction process . Organic phase should have high distribution coefficient, between donor to organic phase and between organic to accepter phase, to achieve high enrichment. Organic phase should have adequate affinity to the hollow fiber. Organic phase should be immobilized sufficiently to cause efficient trapping of analytes in the pores through polarity matching . Mixture of organic solvents can also be used as mobile phase . In this project organic solvent is either pure DHE or DHE is also mixed with different amount of TOPO (section; 1.3.6) to achieve high stability of organic phase [22, 24].
1.3.9 Agitation of sample:
Extraction kinetics can be improved by agitation. Agitation increases analyte diffusion from donor to accepter. Organic membrane solution (DHE) is very stable inside pores of the membrane. Shaking by a magnetic stirrer helps analyte transfer from donor solution to the accepter solution . When Donor solution containing analytes is stirred at high speed, probability of fresh solution contact with membrane phase is enhanced . In order to enhance mass transfer all membrane extractions in this project are assisted through agitation by a magnetic stirrer. A membrane extraction assembly is shown in Fig. 1.13.
1.3.10 Volume of donor and acceptor solutions.
Volume of donor and accepter solution is very important because sensitivity can be improved by proper volume adjustment of accepter solution. Volume of accepter solution should be minimum comparative to donor to get better sensitivity . Volume of accepter solution should be enough to be injected, detected and quantified by GC or HPLC. Volume of the accepter solution should be enough to fill lumen of hollow fiber appropriately .
1.3.11 Adjustment of pH.
Proper adjustment of pH of donor and accepter is very important because high partition ratio can be obtained in three phase lpme by proper adjustment of donor and accepter solution . According to Eq. 1.7, Efficiency can be improved by increasing concentration gradient which depends mainly on pH. In this project three phase lpme is utilized on acidic analytes (C3-C9) containing carboxylic and hydroxyl groups so in donor solution pH is adjusted slightly lower than the pKa values of analytes to suppress ionization of these analytes .
1.4. Detection and quantification of Analytes:
GC-MS is a powerful detection technique for environmental trace analysis due to its high sensitivity . Aerosols are existed in trace level so their detection requires a sensitive device with low limit of detection. GC-MS suffers less matrix effect and is usually cost effective and highly selective . Analytes are separated according to their charge to mass (m/e) ratio after passing through mass spectrometer. Scan mode is used for identification of each analyte .
When gaseous analytes come to mass spectrometer they are converted to their respective molecular ions. Electron ionization in mass spectrometer strikes molecules to fragments . These molecular ions are specific for each analyte and sensitivity and selectivity can be improved through selected ion chromatogram (SIM) . Signal to noise ratio (SNR) is improved through extracted ion chromatogram (XIC) which is selected through SIM mode . SIM mode is used for qualitative and quantitative analysis .
Analytes (C3-C10) are polar and non volatile, so these analytes cannot be detected in pure form and separated by using Gas chromatographic column. A derivatization step is necessary to convert Analyte into volatile substances. Derivatization is made to convert carboxylic and hydroxyl functional groups to their respective ester functional group .
Two derivatization reagents; ‘’N, O-bis(trimethylsilyl) trifluoroacetamide’’ (BSTFA) and ‘’N-(tertbutyldimethylsilyl)-N-methyltrifluoroacetamide’’ (MSTFA) are commonly used for esterification of hydroxyl and carboxylic functional groups before injecting to GC-MS system. Both derivatizing reagents are applied separately and compared prior to GC-MS analysis.
Analytes containing carboxylic acids (C3-C10) are introduced to GC-MS after derivatization. Carboxylic acids are converted to their respective trimethyl silyl ester (TMS derivative) by BSTFA.
A nucleuphilic attack is taken place by a hetero atom to silicon atom when BSTFA reagent is used as a derivatization reagent . BSTFA is found very efficient to convert hydroxyl groups to respective Silyl ester .
Advantage with BSTFA is that its derivative can be injected directly without purification and it can be used for very sensitive detection . BSTFA is non polar and its efficiency can be improved by using BSTFA in Acetonitrile . Chemical structure of BSTFA is shown in Fig [1.14] below.
Due to the use of BSTFA reagent in the reaction, a common peak is appeared at m/z= 73, due to [Si(CH3)3]+ molecular ion and at m/z=145 due to [OH=Si(CH3)2]+ molecular ion . when Analytes containing dicarboxylic acids are used for MS analysis, Ion peak is appeared at m/z=147. Ion peak at m/z=147 is appeared due to the [(CH3)2Si=Si(CH3)2]+ molecular ion .
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