Abstract:

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.

1. Introduction:

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. [1]. 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 [2]. Short chain fatty acids are found as secondary organic aerosols which are also supposed to derive from long chain fatty acids [1]. Importance of organic aerosol has been well established now a days and carboxylic acids are of great interest for environmental studies [1]. Several studies and mechanisms were proposed to understand the production of these SOA precursors [1]. Short chain carboxylic acids are found extensively in troposphere [2]. 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 [3]. Short chain dicarboxylic acids are found in vegetables [Siddiqui, 1989] and in soil containing micro organisms of durum wheat [4]. Dicarboxylic acids are found in plant oils which have greater interest for cosmetic and pharmaceutical industries [6]. Short chain dicarboxylic acids having aliphatic chain possess strong cyclotoxicity and antineoplastic activities [18].

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 [24].

Dicarboxylic acids formed of bio oxidation of fatty acids so these are considered as metabolic part of fatty acid [42]. Dicarboxylic acids and their derivatives can be used to make polymers and their condensation with diols in solution produces high molecular weight polyester [39]. Additionally these dicarboxylic acids use less temperature in the reaction for the preparation of polyesters [39].

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) [5].

1.1.2- Malonic Acid:

Malonic Acid is a metabolite of plants and tissues and Malonyle-CoA [28]. Malonic Acid is an intermediate for preparation of fatty acids from plants and other tissues [7]. Malonic acid is also present in aerosols [8]. Malonic acid is an important constituent of short chain fatty acids [8]. Malonic acid present in beet rots as a Calcium salt [42].

1.1.3- Succinic Acid:

Succinic acid is found in atmosphere as water soluble compound and as a compound of Secondary organic aerosols [29]. 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 [42].

1.1.4- Glutaric acid:

Glutaric acid is found as SOA in aerosols [8]. Glutaric acid is sparingly soluble in water [41], can be used to prepare a plasticizer for polyester [41].

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 [41]. Pimelic acid is produced, when Nitric acid is heated with Oleic acid as a secondary sublimation product which is not crystallized [20].

1.1.6-Suberic Acid:

Suberic acid is produced from suberine [8]. Suberic acid can also be obtained by vigorous reaction condition of natural oil with nitric acid [8].

1.1.7-Azelaic 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 [11].

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 [30].

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 [30].

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 [41].

1.1.11-Phthalic Acid:

Phthalic acid is an aromatic dicarboxylic acid it is found as white crystalline state in pure form [41]. 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 [31].

1.1.12-Syringic Acid.

Syringic acid is found as humic substance in environment [40].

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 [34].

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 [23].

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) [23].

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 [21].

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 [20].

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” [19].
When UAE is utilized it increases speed of mass transport by vibration of mechanical transport from the sample matrix through a process called “cavitation” [21].

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 (1.2.2.1-1.2.2.2), in order to understand its practical use in analytical chemistry.

1.2.2.1- 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 [21].

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” [21].

The life time of transient bubble is so short that no mass transport or diffusion of gas is possible with in a sample [21]. 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 [21].

1.2.1.2 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 [21]. 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 [24]. Small quantity of solvent (usually in micro liters) is required comparative to old techniques of extractions (soxlet) [24]. 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 [24].

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 [24].

1.3.1.1 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 [22].

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 [22].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 [24]. 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 [22].

The process of three phase extraction [22] 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 [22].

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 [22]. 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 [22].

Extraction efficiency (E) can be calculated from Eq. 1.3[22].

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 [22]. 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 [22]. 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 [22].

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 [22].

When an acidic analyte is ionized in aqueous solution, total extractable fraction of analyte (α) is given in Eq. 1.5 [24].

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 [24].

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) [24].

∆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 [24].

The driving force for the extraction in neutral conditions of three phase LPME is the concentration gradient (∆C) from sample to accepter [12]. 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 [22].

1. Exhaustive extraction. 2. Kinetic extraction. 3. Equilibrium extraction.

1.3.4.1 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 [22]. 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 [12]. Mass transfer between organic phase and liquid phase is dependent on concentration gradient [12]. 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 [22]. 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 [12]. 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 [12].

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 (∆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 [16].

1.3.7 Trapping of Analyte in Three phase lpme[24]:

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 [24].

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 [22] and low volatility to prevent solvent losses during extraction process [16]. 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 [22]. Mixture of organic solvents can also be used as mobile phase [16]. 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 [17]. When Donor solution containing analytes is stirred at high speed, probability of fresh solution contact with membrane phase is enhanced [9]. 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.

1.3.10Volume 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 [17]. 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 [17].

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 [17]. 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 [17].

1.4. Detection and quantification of Analytes:

1.4.1-GC-MS analysis:

GC-MS is a powerful detection technique for environmental trace analysis due to its high sensitivity [14]. 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 [14]. 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 [14].

When gaseous analytes come to mass spectrometer they are converted to their respective molecular ions. Electron ionization in mass spectrometer strikes molecules to fragments [18]. These molecular ions are specific for each analyte and sensitivity and selectivity can be improved through selected ion chromatogram (SIM) [14]. Signal to noise ratio (SNR) is improved through extracted ion chromatogram (XIC) which is selected through SIM mode [14]. SIM mode is used for qualitative and quantitative analysis [14].

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 [14].

1.5. Derivatization:

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[14]. Both derivatizing reagents are applied separately and compared prior to GC-MS analysis.

1.5.1- Silylation:

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 [14]. BSTFA is found very efficient to convert hydroxyl groups to respective Silyl ester [18].

Advantage with BSTFA is that its derivative can be injected directly without purification and it can be used for very sensitive detection [18]. BSTFA is non polar and its efficiency can be improved by using BSTFA in Acetonitrile [32]. 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 [18].

2. Method:

2.1 Membrane extraction:

Three phase HF- LPME method is used for extraction. Section 2.1 describes the method for three phase hollow fiber liquid phase micro extraction technique.

2.1.1 Equipment and reagents for Membrane Extraction:

Hollow fiber Accurel PP polypropylene (Q3/2) is purchased from Membrana (Wuppertal, Germany). The wall thickness of membrane is 200 µm, Inner diameter 600 µm and pore size is 0.2 µm. Before extraction a 7.5 cm membrane was cut carefully with a fine cutter. After cutting membrane was washed in acetone and dried overnight.

A magnetic stirrer, containing multiple stations, model (Ika-werke, Germany) was used for agitation of donor solution. Micro Syringe 50 µl (Agilent, Australia) was used to push accepter solution inside the lumen of membrane and for holding of membrane. pH meter (Mettler Toledo) was used to measure pH for donor and accepter solution. Volumetric flask (Kebo, Germany) was used for extractions (contain donor solution).

Milli-Q water was obtained from Millipore gradient system (Millipore, USA). Hydrochloric acid (37%, Fluka) and Sodium hydroxide monohydrate (Fluka) were used to prepare further solutions. Dihexyl ether (97%) was purchased from Sigma Aldrich. TOPO (99%; Aldrich) was used to prepare solutions in DHE (%, w/v).

2.1.2Set up for Membrane Extraction:

2.1.2.1 Donor solution:

The pH donor solution was adjusted to 2. All aqueous solutions were prepared in mill Q water and pH was adjusted by adding HCl (0.1M). All Samples were spiked in a dried 100 ml volumetric flask (Germany). This flask was then, filled up to mark with donor solution. Further 5 ml of donor solution was added in same flask in order to dip membrane inside donor solution. Total volume of donor solution was adjusted to 105 ml. A clean magnet was dropped in flask and then, this spiked solution inside the flask was allowed to stir for 30 minutes and at a fixed revolutions/min (800 rpm) of magnetic stirrer.

2.1.2.2 Accepter solution:

Accepter solution was prepared in milli Q water and pH 12 was adjusted by Sodium hydroxide (0.5 M, 5 M). The accepter solution was injected inside lumen of dried membrane through a micro syringe. Specific amount of (24 µl) accepter solution was injected inside lumen of hollow fiber via a BD micro syringe. Specific volume (24 µl) of accepter solution was fixed after several adjustments, for best compatibility with a 7.5 cm hollow fiber, to achieve good repeatability and enrichment.

2.1.2.3 Membrane solvent:

Membrane containing accepter solution was dipped for 15 s (Approximately) into the organic solvent (pure DHE or topo% solutions in DHE), to impregnate the fiber with organic solvent and to establish a membrane phase. The solvents, immobilized in the pores of hollow fiber were; pure DHE, 1% topo in DHE (w/v), 5% topo in DHE (w/v), 10% topo in DHE (w/v), 15% topo in DHE (w/v) and 19% topo in DHE (w/v). All solutions (topo in DHE) were prepared and mixed by manual shaking, although 15% topo in DHE and 19% topo in DHE solutions were prepared by vigorous shaking and were put inside sonicator for efficient mixing.

2.2. Sample preparations:

All primary solutions were prepared in methanol. Primary solutions were prepared by transferring specific weight of analytes to a sample vial, having air tight caps. This solution was diluted with methanol to prepare a solution of concentration (100 μg/ml). Table 2.1 represents properties (physical, chemical) of analytes. A (abbreviation) name was given respective to TMS ester of each analyte, new name consists of three words only. Molecular weight (Mw), Molecular (Molec) formula, Source (chemicals were purchased from), pKa values of individual analytes (dissociates in water) and purity (as labeled on each chemical) of each analyte is listed in Table 2.1.

Table. 2.1- Analytes source (purchased from)and purity.

Sr. No

Chemical name

Abbreviation

Mw

Molec formula

Purchased

from

pka. Values

Purity

(%)

1

Malonic Acid

Mal

104.06

C3H4O4

Aldrich

2.83, 5.69 (36)

99

2

Succinic Acid

Suc

118.09

C5H6O4

Fluka

4.19, 5.48 (36)

99.9

3

Glutaric Acid

Glu

132.04

C5H8O4

Aldrich

4.34, 5.42 (36)

99

4

Adipic Acid

Ad

146.14

C6H10O4

Fluka

4.34,5.44 (36)

99.5

5

Pimelic Acid

Pim

160.17

C7H12O4

Aldrich

4.48, 5.42 (36)

98

6

Suberic Acid

Sub

174.2

C8H14O4

Aldrich

4.52, 5.40 (36)

98

7

Azelic Acid

Aze

188.22

C9H16O4

Aldrich

4.55, 5.41 (36)

98

8

Cis-Pinonic Acid

Pin

184.23

C10H16O3

Sigma Aldrich

N/A

98

9

Pinic Acid

Pnc

186.2

C9H14O4

Sigma Aldrich

N/A

99

10

Syringic Acid

Syg

198.17

C9H10O5

Sigma

N/A

-

11

Pthalic Acid

Pth

166.03

C8H6O4

Sigma Aldrich

2.98,528(41)

99.5

12

4- Hydroxy benzoic Acid

Hyd

138.03

C7H6O3

Aldrich

4.52,9.23 (35)

99

Primary solution (100 μg/ml, solution A), containing individual analytes, was used to prepare multi component standard mixture (5 μg/ml). This solution was then named as ‘solution B’. Solution B was used to prepare further (dilute) solutions of different strengths (Table. 2.2).

Diluted multi component standard mixtures (containing all target analytes) with different solution strengths (2000, 1000, 500, 250, and 50 ng/ml) were prepared from ‘solution B’ for computing calibration curve. All standard solutions were prepared and diluted with Methanol solvent. Method for the preparation of dilute solutions, were presented in table 2.2.

Table 2.2- Sample preparation of different strength of analyte mixture.

Sr No

Amount of solution A(µl)

Methanol(µl)

Total volume

Solution strength

1

800

1200

2 ml

2000 ng/ml

2

400

1600

2 ml

1000 ng/ml

3

200

1800

2 ml

500 ng/ml

4

80

1920

2 ml

250 ng/ml

5

40

1960

2 ml

100 ng/ml

6

20

1980

2 ml

50 ng/ml

2.2.1 Sample preparation for extraction and detection by GC-MS:

15 µl of standard solutions, having 2000 ng/ml strength, was spiked to the donor solution in a volumetric (100 ml, Fig. 1.13) flask before insertion of hollow fiber, this solution was stirred for 20 min to mix sample solution thoroughly into the donor solution. After stopping extraction, 24 µl (almost) of sample was collected from the accepter solution via a syringe and this solution was transferred to a GC flask (pear shape glass vial), same amount of (0.1M) HCl was transferred to this vial to neutralize basic pH of the accepter solution. This vial, containing neutralized sample, was then put under stream of nitrogen, at a specific temperature (30-40 °C) to evaporate whole of the solvent. Extreme care was required to evaporate solvent under Nitrogen stream. After solvent drying, 20 µl of internal standard (in Acetonitrile) along with 10 µl of BSTFA reagent were poured in the same vial. This sample vial was put in an oven at 80 °C for one hour. Derivatization was accompanied during this hour and then sample was injected directly to the GC-MS system.

2.3- Chromatographic analyses:

Chromatographic analysis were performed on a 6890 series gas chromatographic system interfaced with Agilent 5973-N Mass selective detector. Gas chromatographic system was equipped with auto sampler and 7683 injector. EI source was used at -70 eV to produce ions. EI was operated in positive mode. Full Scan mode from 50 m/z to 600 m/z was used to study fragmented parts of each analyte. Quantitative analyses were done by selecting characteristic molecular ions of each analyte by selected ion monitoring (SIM) technique [14]. Extracted ion chromatogram (XIC) method was used, through data analysis window to get the information about retention time of characteristic ions. Peak area of each characteristic ion was manually selected and calculated for quantification purpose [14].

A Factor four capillary column (30 m x 0.25 mm) having phase thickness 0.25 µm and 5% phenyl cross linked (Varian, Germany) was used. Column was fitted with a retention gap.

Helium (99.9995% pure) was used as carrier gas. Table 2.3 shows, schedule of oven (GC) temperature that was programmed for Gas chromatographic analysis.

Table 2.3– GC Temperature parameters.

Ramp

Rate C/Min

Temperature C

Hold Min.

Time total

-

60

2

2

Ramp 1

2.5

120

0

24

Ramp 2

10

220

0

34

Ramp 3

20

300

0

38

Gas (He; 99.999% pure) flow rate (1.5 ml/min) was used with Splitless injection mode. Fixed amount (volume) of sample (2 µl), was injected to the GC Injector (285 °C) throughout this project. Acetonitrile (HPLC grade), Acetone (HPLC grade) and Methanol (HPLC grade) were purchase from Fisher Scientific (USA).

2.3.1 Sample preparation for GCMS analysis:

15 µl of standard solution of a specific concentration was transferred, via a micro pipette to a conical glass vial. This sample was put in an oven at 80 °C for 20 min to evaporate methanol. After solvent evaporation, 20 µl internal standard (in Acetonitrile), along with 10 µl of BSTFA reagent were shifted to the previously dried vial. After pouring BSTFA and Acetonitrile, vial was tightly capped and put in oven at 80 °C for 60 min. After 60 min of derivatization reaction, same vial was put to the auto sampler and sample was injected directly to the GC system. Standard solutions were injected in duplicate, standard concentrations (Injected) were ranged from 16.66 - 666.6 ng/ml (absolute quantity) to check linearity. Table 2.3 shows different standard amounts that were used to obtain regression line (calibration curve).

Table 2.4- Concentration (ng/ml) of standard solutions in Injection vial.

Sr.No

1

2

3

4

5

6

Sample Injected

666.6 ng/ml

333.3 ng/ml

166.6 ng/ml

83.3 ng/ml

33.3 ng/ml

16.66 ng/ml

2.3.2Set up for retention time to confirm mass spectra of analytes (GC-MS Analysis):

10 µl of each analyte, containing individual analyte (solution A), were poured to a vial and this vial was put in oven at 800 C for 20 minutes to evaporate methanol. Respective retention time and fragmented ions were listed in table 2.5.

2.3.3 Derivatization:

N-O bis(trimethylsilylyl)trifluroacetamide with 1% Trimethyl chloroSilane (BSTFA) and N-methyl-N-trimethylsilyl-trifluoroacetamide with 1% Trimethyl chloro Silane (MSTFA) were purchased from Sigm. Both reagents were used for derivatization and were compared for best selectivity. Trimethyl silyl (TMS) derivatives were produced after reaction with target analytes, where as Acetonitrile was used as a solvent in reaction medium. Acetone and Hexane were also used as a solvent for derivatization reaction, discussed above. The TMS derivatives are presented in table 2.3[18, 32, 19, 15, and 37]. These TMS esters (table 2.3) were purposed to produce through derivatization reaction before injecting to GC-MS system.

2.3.3 Selection of internal standard:

1-Phenyl dodecane (97%) was purchased from Acros Organic (Geel, Belgium). Internal standard was used to get consistent and reproducible results [32]. Derivatization reagent does not react with internal standard. Peak area of analytes (A a) was divided by internal standard peak area (A is) i.e. A a/A is. Peak area was calculated from detector response.

2.3.4 Dryingprocedure (solvent):

Drying of sample’s solvent was carried through evaporation. Two methods were utilized for evaporation till dryness. First method was; to dry in oven at 80 °C and second method was; to dry under gentle stream of nitrogen at 30-35 °C. Both methods were applied and results were compared. First method of drying was applied for samples containing Methanol and second method was applied for samples containing water solvent. Sample before evaporation after membrane extracted was neutralized by using 24 µl of 0.1 M HCl (the same amount was used as accepter solution). Drying procedure under nitrogen all solutions after extraction were evaporated till dry by the same procedure.

Table 2.5 -Structure of TMS derivatives of derivatized target analytes.

Sr. No.

TMS ester (Name)

TMC derivative of analytes

Structure of TMS derivative

Molecular Weight

1

Mal

C 9 H24 O4 Si2

248.09

2

Suc

C10 H22 O4 Si2

262.11

3

Glu

C 11 H24 O4 Si2

276.12

4

Ad

C 12 H26 O4 Si2

290.14

5

Pim

C 13 H28 O4 Si2

304.15

6

Sub

C 14 H30 O4 Si2

318.17

7

Aze

C 15 H32 O4 Si2

332.18

Sr. No.

TMS ester (Name)

TMC derivative of analytes

Structure of TMS derivative

Molecular Weight

8

Pin

C 12 H26 O4 Si2

328.73

9

Pnc

C 15 H30 O4 Si2

330.17

10

Syg

C 13 H21 O4 Si2

297.10

11

Pth

C 14 H22 O4 Si2

310.11

12

Hyd

C 13 H22 O4 Si2

282.11

*[ Note: table 2.5 contains structures of TMS derivative of target analytes, these structures are sketched according to proposed reaction and for understanding of fragments produced from these proposed structures].

2.4 Aerosols samples Ultrasonic Assisted Extraction:

Samples were provided after ultrasonic extraction for derivatization and quantification. Solvent from the Ultra sonic extracted mixture (containing Samples) was evaporated (section; 2.3.4) till dryness. Dried sample was derivatized (section; 2.3.1 2.3.3). Quantification of unknown samples (1-23) was carried through in the same procedure.

2.5 Limit of detection (LOD):

Limit of detection of an analyte is defined as signals from minimum concentration of an analyte which can be distinguished from signals of blank [19] or background signals [25]. LOD information is very important for trace analysis. LOD is calculated [19] from the standard deviation of response from standards used in calibration curve and slope of analyte curve (b) given in equation [25].

LOD = b + 3 s y/x …………………….. 2.1

In Eq. 2.1, “s y/x” is residual standard deviation of the regression line.

2.6 Limit of Quantification (LOQ):

Limit of quantification is smallest concentration of analyte that can be determined quantitatively with a certain degree of assurance [19].It is calculated from a linear response from the analyte area/height. Eq. 2.2 describes limit of quantification which is calculated from regression line from standards [25].

LOQ = b + 10 s y/x ………………… 2.2

3-Results

3.1. Detection and quantification of Ultrasonic assisted extraction samples:

3.1.1 –Detection by GC-MS:

All individual analytes (100 µg/ml) were injected separately to GC-MS system at 33,333 ng/ml (in GC vial) concentration to confirm analyte’s presence in chromatogram. Each analyte was run on scan mode of Mass Spectrometer after derivatization and characteristic ions were determined as well as retention times of each analyte. Retention time, Characteristic ions and selected ions for each analyte are presented in table 3.1.

Table 3.1- Set up for retention time and characteristic ion study for each analyte including internal standard in Scan mode of MS.

Sr No

Analyte name

Retention time*min

Characteristic ion

Selected ion

1

Mal

13.89

147,73,233,75

233

2

Suc

18.70

147,73,148,75,247

147

3

Glu

22.85

147,73,261,75,158,

147

4

Ad

25.56

73,111,147,75

147

5

Pim

27.30

73,75,147,155,125,

147

6

Sub

28,65

73,75,187,217

187

7

Aze

29.87

73,75,201,129,147

201

8

Pin

25.84

73,171,75,83

171

9

Pnc

28.26

73,129,75,171,172

171

10

Syg

31.06

297,73,253,141

297

11

Pth

28.63

147 , 73 , 295

295

12

Hyd

27.61

267 , 223 , 193

223

13

IS

30.77

246

246

3.1.2 LOD,LOQ and linearity of Standard injections:

Multi component standards, concentration ranges from 16.66 -666.6 ng/ml (absolute injection concentration), were injected to GC-MS system after derivatization and results were presented in table 3.2. Total ion chromatogram for standard contains minimum concentration of analytes (16.66ng/ml) and UAE sample “24” is presented in Fig. 3.1. Calibration curves, for individual standards are presented in Fig. 3.2-3.3.

Fig. 3.1- TIC GC-MS chromatogram. (A) Standard analysis (16.66ng/ml); (B) UAE sample unknown

Fig. 3.2-Fig 3.3 shows peaks for selected ions. Chromatogram was superimposed by using standard and sample ion peaks, for comparative study, by using extracted ion chromatogram through MS computer window.

Fig. 3.2- SIM, GC-MS Overlay chromatograms for the standard analysis (16.66 ng/ml), UAE Sample (24). (A) SIM 147; (B) SIM 187

Fig. 3.3- SIM, GC-MS Overlay chromatogram for the standard analysis (16.66 ng/ml), UAE Sample (24). (C) SIM 147; (D) SIM 187

A regression line was drawn by computing six multi component standards (for “Mal” through five points). Fig. 3.4 - 3.5 represent calibration curves, calculated from characteristic ion with respect to each target analyte (Section 2.3.1), through XIC window.

Fig. 3.4- Calibrationcurves (a-e) for the standards (16.66-666.6 ng/ml)

Fig. 3.5- Calibration curves (g-k) for the standards (16.66-666.6 ng/ml)

Eq. 2.1 -2.2 were used to calculate LOD and LOQ from parameters of calibration curve.

Table 3.2- Slope and Regression coefficients of the Regression line, LOD and LOQ of individual analytes.

Sr. no

Analytes

Conc range(ng/ml)

Slope(m)

Regression coefficient

LOD(ng/ml)

LOQ(ng/ml)

1

Mal

16.66-666.6

0.0011

0.999

0.405

1.347

2

Suc

16.66-666.6

0.0082

0.9945

0.146

1.465

3

Glu

16.66-666.6

0.0039

0.995

0.236

0.776

4

Ad

16.66-666.6

0.0014

0.996

0.083

0.247

5

Pim

16.66-666.6

0.0008

0.99

0.079

0.238

6

Sub

16.66-666.6

0.0007

0.997

0.059

0.177

7

Aze

16.66-666.6

0.0005

0.996

0.040

0.132

8

Pin

16.66-666.6

0.0011

0.997

0.066

0.217

9

Pnc

16.66-666.6

0.0011

0.995

0.076

0.250

10

Syg

16.66-666.6

0.001

0.994

0.072

0.236

11

Pth

16.66-666.6

0.0007

0.993

0.060

0.200

12

Hyd

16.66-666.6

0.0016

0.995

0.099

0.328

3.1.3- Quantification of UAE Real samples (Aerosols):

Amount of analytes were quantified by GC-MS after UAE. Each sample (1-23), containing multiple analytes, was run duplicate in GC-MS system. Amount of each analyte (ng/ml) was presented in table 3.3, as quantified from computing with regression line (table 3.2). Quantification of unknown UAE samples is presented in Fig. 3.6- 3.8. Comparison between calculated amounts of analyte was presented (Fig. 3.6- 3.8) graphically (with respect to three other analytes) to study the variations in amount, for the same analyte in all real samples (1-23).

Table 3.3- Amount (ng/ml) of each analyte in separate samples (1-23) after UAE.

sample

Mal

Suc

Glu

Ad

Pim

Sub

Aze

Pin

Pnc

Syg

Pth

Hyd

1

<LOQ

46

62

56

46

87

104

47

70

52

135

122

2

<LOQ

845

103

134

45

114

130

49

94

56

118

163

3

<LOQ

97

82

114

61

110

132

51

73

60

132

132

4

<LOQ

258

112

142

83

103

127

48

72

57

117

143

5

<LOQ

47

63

89

53

89

90

51

56

54

73

126

6

<LOQ

119

78

124

47

102

104

67

95

53

161

197

7

<LOQ

95

78

126

47

104

110

61

124

163

126

158

8

<LOQ

151

89

115

52

99

110

50

63

51

106

130

9

<LOQ

315

111

134

68

124

169

53

104

51

126

183

10

<LOQ

38

62

97

52

96

99

53

65

56

105

141

11

<LOQ

78

60

91

46

93

101

42

51

53

75

119

12

<LOQ

69

83

369

138

308

547

92

100

59

93

208

13

<LOQ

66

87

315

174

211

465

102

75

53

85

177

14

<LOQ

156

62

131

80

101

176

50

52

43

59

78

15

<LOQ

175

101

238

86

150

173

67

90

49

103

184

16

<LOQ

54

72

171

81

141

156

63

67

50

83

144

17

<LOQ

99

92

212

97

144

167

71

105

53

83

167

18

<LOQ

4

52

84

67

96

98

82

69

57

69

167

19

<LOQ

76

74

180

63

112

84

97

77

61

99

171

20

<LOQ

59

75

219

83

115

125

123

94

57

144

145

21

<LOQ

208

121

245

76

149

291

113

175

55

508

268

22

<LOQ

56

75

137

64

103

93

71

132

58

172

174

23

<LOQ

86

106

159

79

122

166

100

137

62

84

226

Fig. 3.6- A comparitive study of analyte’s amount, calculated cocentration (Mal, Suc, Glu and Ad) vs UAE unknown samples (1-23).

Fig. 3.7- A comparitive study of Analytes, calculated cocentration (Pim, Sub, Aze, Pin ) vs UAE unknown samples (1-23).

Fig. 3.8- A comparitive study of Analytes, calculated cocentration (Pnc ,Syg,Pth,Hyd)vs UAE (unknown) samples (1-23).

Total presence of each analyte in whole of the real samples (average for all analytes) was plotted with respect to individual analyte, quantified after UAE, is presented in Fig. 3.4.

Fig.3.9- Comparison of Average concentration (ng/ml) of all analyte.

3.2. Membrane Extraction:

3.2.1 LOD,LOQ and linearity of Standard injections after column cutting:

After three month continuous running GC-MS system was started to cause problems after many troubleshooting instrument started work again. Approximately 1.5 meter column was cut and GC-MS instrument was tuned successfully. Again new standards were run on chromatographic system and new regression lines were obtained and are presented in table 3.4 with individual standard runs (2.3.1) and new LOD and LOQ were determined or individual analyte by using Eq 2.1 and 2.2.

Table3.4-Slope ofRegression line and Regression coefficients of calibration curve, LOD and LOQ of individual analytes (After column cutting and without retention gap)

Sr. No.

Analytes

Slope(b)

Regression coefficient

LOD(ng/ml)

LOQ(ng/ml)

1

Mal

0.0010

R² = 0.998

0.3

1.1

2

Suc

0.0145

R² = 0.999

0.4

1.4

3

Glu

0.0078

R² = 0.998

0.3

1.1

4

Ad

0.0029

R² = 0.998

0.2

0.6

5

Pim

0.0018

R² = 0.998

0.1

0.5

6

Sub

0.0013

R² = 0.998

2.7

9.0

7

Aze

0.0009

R² = 0.999

0.1

0.1

8

Pin

0.0021

R² = 0.999

0.1

0.2

9

Pnc

0.0019

R² = 0.998

1.2

4.1

10

Syg

0.0017

R² = 0.997

0.2

0.5

11

Pth

0.0011

R² = 0.998

0.1

0.3

12

Hyd

0.0032

R² = 0.998

0.2

0.7

3.2.2 Enrichmentfactor (Ee) of extracted analytes by three phase HF-lpme:

Enrichment factor was presented in table 3.5, after triplicate extraction, from duplicate injection and average values were calculated for each organic phase separately. Results, from different organic phase solvents (1% - 19% topo in DHE) and respective enrichment factor for individual analyte, were presented in table 3.5. While Fig. 3.13 graphically presents enrichment factor of each analyte ‘Ee’ values (table 3.5). Total ion chromatogram, from standard and sample is presented in Fig. 3.10.

Table 3.5- Enrich factor of each extracted analyte by three phase HF-lpme with different %age of topo in DHE

Sr. No.

Analytes (TMS)

0%topo(DHE)

1 % TOPO

5 % TOPO

10% TOPO

15% TOPO

19% TOPO

1

Mal

42

34

137

205

401

35

2

Suc

223

240

559

541

419

805

3

Glu

169

165

230

235

305

464

4

Ad

408

314

576

520

842

1327

5

Pim

239

236

433

382

1042

940

6

Sub

431

401

987

1192

2100

1188

7

Aze

1126

1061

4699

4449

4607

1349

8

Pin

139

170

280

490

320

135

9

Pnc

177

180

320

660

1216

660

10

Syg

200

200

209

297

521

207

11

Pth

225

220

358

263

1345

2741

12

Hyd

307

542

1575

1591

543

195

Fig. 3.10- TIC GC-MS chromatogram. (A) Standardanalysis 666.6 ng/ml;(B) Extraction sample (2000ng/ml standard spiked) y mixing 10% topo in DHE

Fig. 3.11-3.12 represents selected ion chromatogram, single ion representative of (respective) analyte. SIM chromatograms for standard (166.6 ng/ml) and extracted sample (10% topo in DHE) are overlaid as obtained through extracted ion chromatogram from data analysis window.

Fig. 3.11- SIM (GC-MS) Overlay chromatograms for the standard (1666.6 ng/ml) and Extractedsample (2000ng/ml, standard spiked) by using 10% topo in DHE. A m/z= 147; B m/z= 171; C m/z= 187; D m/z= 201

Fig. 3.12- SIM (GC-MS) Overlay chromatograms for the standard (1666.6 ng/ml) and Extracted sample (2000ng/ml, standard spiked) by using 10% topo in DHE. E m/z= 223; F m/z= 295; G m/z= 297

Fig. 3.13-Enrichment factor vs. Analytes at different topo contents (mixture in DHE)

3.2.3- Enrich efficiency (E) of extracted analytes by three phase HF-lpme:

Enrichment efficiency was calculated according to Eq. 1.4 and results are presented in table 3.6. Calculated results of each analyte are presented with respect to different organic phases (%age) (3.2.2). A corresponding graph in Fig. 3.14 is obtained from table 3.6.

Table 3.6- Extraction efficiency of each extracted analyte by three phase HF-lpme with different %age of topo in DHE

Sr.No.

Analytes (TMS)

0%topo (DHE)

1 % TOPO

5 % TOPO

10% TOPO

15% TOPO

19% TOPO

1

Mal

1

1

3

5

9

1

2

Suc

5

5

13

12

10

18

3

Glu

4

4

5

5

7

11

4

Ad

9

7

13

12

19

30

5

Pim

5

5

10

9

24

21

6

Sub

10

9

23

27

48

27

7

Aze

26

24

107

102

105

31

8

Pin

3

4

6

11

7

3

9

Pnc

4

4

7

15

28

15

10

Syg

5

5

5

7

12

5

11

Pth

5

5

8

6

31

63

12

Hyd

7

12

36

36

12

4

Fig 3.14-Enrichment Efficiency (%) vsindividual analytes at different topo contents

3.2.4 Repeatability between different membranes

Repeatability (%RSD) of each extracted analyte, as a result of triplicate extractions by three phase HF-LPME, is presented in Fig. 3.15 relative to each membrane phase (%topo in DHE).

Fig 3.15- A comparison of repeatability between different membranes for triplicate extractions

A Comparison of total enrichment factor of individual analyte, including all extracted analyte, to the organic phases is (table 3.12) presented in Fig. 3.16, enrichment factor is calculated averagely from triplicate extractions.

Fig. 3.16- A comparison between total enrichment and different %age of topo in DHE when used as membrane liquid for triplicate extractions.

4- Discussions:

4.1 -Chromatographic Analysis:

4.1.1 Selection of Derivatization reagent:

Three solvents; Acetone, Acetonitrile and Heptane were used to inject TMS derivative of analytes (table 2.1) in chromatographic analysis during the start for this project. BSTFA and MSTFA (section; 2.3.3) were used as derivatizing reagents; these reagents were compared for efficiency. Derivatization with MSTFA was found unsuitable for chromatographic analysis, comparative to BSTFA. BSTFA reagent provides excellent results (Fig 2.1). Acetonitrile was used as a solvent, with this solvent derivatization was found satisfactory. Base line of chromatographic analysis was also found satisfactory with Acetonitrile. Acetonitrile was found more effective when BSTFA was used as derivatizing reagent (comparative to MSTFA). Chromatographic results were also acceptable with Acetone but more importantly, derivatization reaction was taken place effectively in Acetonitrile. All analytes were derivatized in Acetonitrile solvent. BSTFA reagent and Acetonitrile solvent (containing internal standard) were found to work effectively with 1:2 ratio (Fig. 3.1).

4.1.2- Optimum Drying procedure

Evaporation of the solvent (water, Methanol) was very important because primary solutions were prepared in methanol and derivatization reaction was taking place in Acetonitrile solvent. Similarly after extraction accepter solution was in aqueous medium. Derivatization is water sensitive [30]. Water need to dry prior to derivatization.

Samples after extraction were in basic solution analytes were existed in their corresponding salt [18, 32, 15]. Derivatization with BSTFA was difficult to take place with salt, solution containing extracted sample was neutralized by adding low concentrated acid (0.1M HCl).

Standard Solutions containing methanol were evaporated till dryness in oven at 80 ᵒC and several standards (16.66 - 1666.6 ng/ml) were run on GC-MS to check linearity after drying in the same scheme (2.3.4). Drying of standards (primary standards were in methanol) evaporated in oven (80 ᵒC) for 20 min, this drying procedure provide good linearity (Fig. 3.2-3.3).

Evaporation under stream of Nitrogen was performed to the samples after derivatization, to eliminate excess of BSTFA and to avoid unwanted peaks (side products) produced from BSTFA side reactions (Fig. 3.1, 1.6]. TMS derivatives of analytes were found to escape quickly through drying, this step of evaporation was eliminated and solution containing TMS derivatives of acids were injected directly (Containing BSTFA).

After membrane extractions, recovered samples (in accepter) were evaporated to dryness under gentle nitrogen stream prior to derivatization (Fig 3.10, 3.11, 3.12, 3.13).

4.1.3- Confirmation of TMS derivative of acids on their retention time on scan mode:

Primary standard solutions (33,333 ng/ml), containing Mal, Suc, Glu, Ad, PIm, Sub, Aze, Pin, Pnc, Syg, Pth and Hyd were run on GC-MS. Full scan detection mode was used on GC-MS system and EI (scan) spectra was used to find fragmented ions and proper retention times of each analyte. EI mass spectrum of analytes was very complex and many fragments were obtained in chromatogram, these fragments are hard to explain because many side products (Fig. 3.1, 3.10) were produced due to use of BSTFA reagent [29]. Detection of individual analytes at their characteristic ions is discussed below after selected ion monitoring study (Fig. 3.2-3.3, 3.11-3.13).

When standard containing ‘mal’ was injected (33,333 ng/ml), ion fragments at m/z =147, 73 and 233 were obtained (Table 3.1) in decreasing order of their intensity at 13.89 min (retention time). Base peak at m/z = 147 was produced due to [(CH3)2Si=Si (CH3)2] + ion which is also an abundant ion fragment for all of analytes containing dicarboxylic functional group. A third abundant fragment ion was appeared at m/z =233 due to [C8H17Si2O4] + molecular ion.

Suc, Glu, Ad and PIm retention time, at 18.70 min, 22.85 min, 25.56 min and 27.30 min respectively were confirmed by minimum three injections (each analyte was run separately). These four analytes shows many ion fragments, intense ions at respective m/z are listed in table 3.1, in decreasing order of their intensity. Most important and common ion fragment was at m/z=147 (already described), so this molecular ion was selected as SIM for (Suc, Glu, Ad and PIm) analytes.

Sub (analyte) retention time was found at 28.65 min (temperature programmed, section 2.3). Ion fragment at m/z=187 was third intensive ion peak, this molecular ion was selected to provide good separation from other interfering peaks.

Aze (analyte) retention time was 29.87min, selected ion was at m/z= 201.

Standard solution containing Pin and pnc were investigated to confirm their retention times. Pin and pnc analytes, retention time was found at 25.84 min and 28.26 min respectively. Pin and pnc produced a common fragment ion at m/z=171 (Table 3.1), so this ion peak was selected for SIM mode.

Syg retention time was 31.06 min, ion at m/z= 297 was selected for SIM mode. This ion was also a base peak for this analyte (table 3.1).

Pth retention time was found at 28.63 min, third intensive fragment ion was at m/z=295 (selected for SIM).

Pth and Sub analyte peaks were eluted at almost same retention (Fig. 3.7) time but since different ions were selected, so no interference was observed in-between.

Hyd (33,333ng/ml) analyte peak was appeared at 27.61 min. When this peak was beaten to fragments by scan mode the resulting ion peaks (Table 3.1) were found. Fragment ion at m/z=223 was selected.

Peak from internal standard (1-Phenyl dodecane) was eluted at 30.77 min and molecular ion at m/z=246 was selected for SIM analysis. 1-Phenyl dodecane was unaffected by derivatization.

There were some unwanted peaks in total ion chromatogram; these peaks were mainly eluted before 13.8 min (Fig. 3.1, 3.10). There could be several reasons and sources for unwanted chromatographic peaks (beyond the scope of this project). The important source (investigated) was the peaks eluted from column bleed.

There were some unwanted peaks, eluted in between analytes. Extra peaks were made ineffective (ignored) due to SIM mode i.e. all required peaks were well resolved from unwanted peaks.

During extraction (GC-MS analysis), a ghost peak was eluted continuously at around 12 min (Fig 3.8). This unwanted peak did not disturb analyte ‘mal’ (eluted at 13.05 min), but this peak was appeared to disturb column performance. This ghost peak at 12 min (might be) was eluted due to topo or due to some impurity in DHE etc.

4.1.4- Optimum temperature and SIM method validity:

Temperature programmed was selected and validated, considering elution of every analyte and through literature survey, after several chromatographic experiments by injecting standard solutions of known strength [29]. All analytes (including internal standard) were eluted before 240 ᵒC in 32 min before reaching to Column maximum temperature (325 ᵒC). Each analyte was well separated by selected temperature program (Table 2.2).

4.1.5- Calculation from XIC:

After selecting SIM method required ions are extracted through XIC from data analysis window. All quantification of analytes is done through this window.

4.1.6- Limit of detection:

Limit of detection (LOD) for this method containing C3-C10 acids (when most of the work was done with ultrasonic assisted samples) was varied in the range of 0.040-0.405 ng/ml (table 2.3, 3.2), after computing the calibration curves passing through six (at least five) multi component standard solutions (16.66 - 666.6 ng/ml).

LOD, after column cut, was obtained from computing multi component standard solutions containing C3-C10 acids. LOD was found in the rage of 0.1-2.7 ng/ml (table 2.3, 3.4). Sensitivity was not as good as it was reported elsewhere [18]. UAE and membrane extraction was found to enrich the trace analytes many times, so this LOD values are acceptable and applicable with these enrichment techniques i.e. especially for membrane extractions (table 3.5).

4.1.7- Limit of quantification:

Limit of quantification (LOQ) was calculated from LOD values (Eq. 2.2). LOQ of analytes (C3-C10) was found in the range of 0.1 -1.3 ng/ml (table 3.2). LOQ for ‘mal’ was higher than other analytes. LOQ was found satisfactory for further analysis and calculations.

After column cut, LOQ was found in the range of 0.1 -9 ng/ml (table 3.4).

4.1.8- Linearity:

Current Method shows good linearity for all of the target analytes. Linearity was estimated through square of regression co efficient (R2), calculated through calibration curve from standards [38]. Linearity ranges from 0.990-0.999 for all of the analytes (table 2.3; Fig. 3.4-3.5).

4.1.9- Trouble shooting with GC-MS System:

GC-MS caused a lot of problems during half time of this project and many experiments, those were aimed for this project, could not put into practice. Problem was started from internal standard poor response; improper results and continuous declining of chromatographic peaks (peak height, area) of internal standard were observed. After poor response from IS, small peaks related to target analytes were started to disappear (especially ‘mal’). In this procedure, after some days, IS peak response reduced to less than 10 times (Approximately).

GC syringe (auto sampler) was replaced. Still, no improvement was observed to the quality of chromatographic peaks (It was suggested that syringe did not injected samples properly).

Retention gap coupled to GC capillary column was cut, half a meter from injector side. This treatment to the column did not work as well. New standards were prepared to confirm presence of target compounds.

GC oven temperature was left at 300 ᵒC, overnight; to elute any impurity trapped inside Column. Another half a meter retention gap was cut. GC-MS system was pumped down, new tuning was made and also air water check was run again to check leaks in column. Liner was removed and washed with methanol [37]. Problems were still not rectified by applying different techniques (normally used).

Problem with GC-MS was sustained for more than three and half months, during this time different troubleshooting procedures were utilized (discussed above). Finally Retention gap was removed and 1.5 meter column was cut i.e. one meter from GC Injector side and half a meter from detector side. Problem was rectified after column cutting, liner cleaning, pumping down etc. GC-MS system was started to work again for 20 days only.

Calibration curves of standards (multi component) were performed again. Chromatographic experiments (GC), those are practically possible after problem rectification, are described in section. 4.3 (before new problems were started to begin).

4.2-Quantification of Aerosols (real) samples after UAE:

Real samples were extracted by Ultra sonic assisted extraction (by else) before detection (table 3.3). Descriptions of individual detected analytes (after UAE) are given in next paragraphs of this section.

Malonic acid (mal) was detected below LOQ i.e. ‘mal’ could not be quantified through this method even though ‘mal’ peak was visible and larger than noise peak (S/N=3). Malonic acid existence below limit could be due to the reason, that when sample containing analytes was dried under stream of Nitrogen, Malonic acid has strong affinity with water, gaining water from environment or from the solvents if dried less can not be derivatized with BSTFA in Acetonitrile solvent, as BSTFA can only derivatize when analyte is dissolved in Acetonitrile.

Succinic acid concentrations were varied from 4-845 ng/ml, Glutaric acid; 52-111 ng/ml, Adipic acid; 56-369 ng/ml in the Aerosols sample. Malonic acid, Glutaric acid, Adipic acid and Succinic acid concentrations were represented graphically in Fig. 3.1. These analytes has been found to display some variations in concentration, from one sample to the other. Glutaric concentration (Fig. 3.1) was steady through out the samples (1-23), where as concentrations of Succinic and Adipic acid were inconsistently varied.

Pimelic acid concentration was varied in the range of 45-174 ng/ml, Suberic acid; 87-211 ng/ml, Azelic acid; 84-465 ng/ml and Cis-Pinonic acid; 48-113 ng/ml (Fig. 3.3), in real samples after UAE (1-24). In samples (12, 13) concentration of these analytes was high, typically in sample 12, especially Azelaic acid (45 ng/ml). Azelaic acid presence was dominant comparative to other three members (Fig. 3.2). Amount of these analytes were consistent (except; sample 12, 13) and did not show lot of variations in aerosols.

Concentration of analytes; Pinic acid, Syringic acid, Pthalic acid and 4-Hydroxy benzoic acid, were varied in the range of 51-175 ng/ml, 43-163 ng/ml, 59-508 ng/ml, 78-268 ng/ml respectively in the real samples (Aerosols) after UAE (Fig. 3.3). 4-Hydroxy benzoic acid was found dominantly in almost all samples except sample ‘21’ Pthalic acid was in higher concentration comparative to other analytes in Fig. 3.3. Syringic acid was found in lesser amount comparative to other three analytes. Calculated amounts of these analytes did not show many variations in between all real samples (except; sample 7, 21). In sample ‘7’ Syringic acid value was unexpectedly high i.e. may be due to sampling error.

From above discussions, it is obvious that all analytes were present in aerosols samples, although Malonic acid was detected below LOQ.

Average concentration of individual analyte was plotted in Fig. 3.9 including all real samples (1-23), results with respect to each analyte are discussed below.

Azelaic acid was found in 166 ng/ml amount, this amount was higher comparative to all other analytes. Concentrations of 4-Hydroxy Benzoic acid (162 ng/ml) and Adipic acid (160 ng/ml) were found close to Azelaic Acid. Malonic acid was descried earlier (can not be quantified). Syringic Acid existence was (59 ng/ml) lowest than all other quantified analytes.

4.3. Membrane Extraction:

4.3.1- Selection for donor pH:

pH of the donor solution was adjusted less than pKa values of analytes (table 3.1). Donor solution pH value was adjusted at 2.0; this pH value is enough to prevent dissociation of analytes into their ions. This pH value forced analytes to remain in their original (un- dissociated) state and as a result membrane organic phase captured these analytes.

4.3.2- Selection for accepter pH and Optimum Volume:

Accepter pH was adjusted at basic pH for trapping analytes in accepter to prevent back diffusion. For trapping of acidic analytes (C3-C10), pH of the accepter was selected at 12 (0.1M NaOH). This pH (accepter) was 3 pH units higher than pKa values of each analyte (except 4-Hydroxy benzoic difference was about 2.7 pH units, table 2.1). This pH of accepter solution was good enough to cause trapping of all analytes in the accepter.

Two techniques were applied to inject accepter solution in hollow fiber. 1st method was to inject accepter solution via B.D micro syringe contains (Approx) 45 µl of accepter solution, fiber was flushed with some of the accepter solution and remaining accepter inside lumen of fiber was then trapped by sealing one of the fiber’s open sides with aluminum strip while the other end of the fiber contained syringe needle.

Second technique was to calculate the actual accepter volume, the volume that lumen of a hollow fiber can contain easily, this volume was calculated averagely with five fibers having same length. Volume (24µl) of accepter solution was adjusted which was also capable to hold target analytes. This technique was found to work eficienly, because this technique was easy to handle and repeatability (%RSD) of analytes (enrichment) was found best in this case.

4.3.3- Selection for Hollow fiber length:

Length of Hollow fiber 7.5 cm was found adequate, because it has enough capacity to hold 23-26 µl of accepter solution easily and this length can be inserted into a (100 ml) volumetric flask vertically via a syringe (Fig. 1.13) i.e. the membrane assembly for this experiment.

4.3.4- Stirring speed:

Stirring speed was fixed at 800 rpm, this speed of magnetic stirrer was found reasonable for membrane extraction to speed up the mass transfer of analytes with three phase HF-LPME (Section 1.3.9).

4.3.5-Selection of Organic phase:

Dihexyly ether was used as organic phase solvent in three phase hf-lpme for enrichment of acidic analytes (C3-C10) presented in table 2.1. Every analyte was enriched when DHE was (table 3.5) used. When topo was mixed with DHE solvent; polarity of DHE solvent was changed. organic phase was originated to interact more effectively after topo mixed in DHE (w/v) [16].

Multi component standard (2000 ng/ml) was spiked to (105 ml) donor solution. results were calculated, after triplicate extractions. Extracted analytes were quantified from computing the results with the standards regression lines obtained after GC-MS (table 3.5, Fig. 3.13). It is obvious that organic phase has major advantage when three phase Hf-lpme was utilized.

4.3.6- Enrichment Factor (Ee) and Efficiency (%):

Enrichment factor (Ee) was obtained, after triplicate extraction (table 3.5). Enrichment factor was obtained with different organic phases while same amount of standard was spiked in all extractions. Enrichment factor (all the analytes) was found to vary in the range of 34-4699 times after three hours of extraction. Minimum Enrichment factor was obtained when pure DHE was used only as an Organic solvent (Fig 3.13).

Malonic acid has minimum enrichment comparative to every other analyte. The reason of minimum enrichment of Malonic acid could be explained by considering two reasons. One reason is its low pKa value than any other analyte i.e. it is more soluble in donor phase. Second reason is that it reacts with accepter solution and precipitates.

Maximum theoretical enrichment possible was calculated according to Eq. 1.8 and it was 4375 times for individual analytes. Maximum enrichment that was practically possible for analytes Malonic acid; 407 times (15% topo), Succinic Acid; 805 times (19% topo), Glutaric acid; 464 times (19% topo), Adipic acid; 1327 times (19% topo), Pimelic acid; 1042 times (15% topo), Suberic acid; 2100 times (15% topo), Azelaic acid; 4699 times (5% topo), Cis-Pinonic acid; 490 times (10% topo), Pinic acid; 1216 times (15% topo), Syringic acid; 521 times (15% topo), Pthalic acid; 2741 times (19% topo) and 4-Hydroxy Benzoic acid; 1591 times (10% topo). Values in parenthesis represent the different membrane phase (TOPO in DHE).

From this (enrichment) study it is apparent that selective enrichment of each analyte is possible with different Organic phases in three phase HF-LPME and this selective enrichment can lead to efficient detection of environmental samples.

According to Eq. 1.4 Enrichment factor and Efficiency are interrelated. Fig. 3.14 graphically represents Efficiency. Efficiency of extraction varied in the range of 1 -107% from Malonic acid (minimum) to Azelaic acid (maximum). .

When total enrichment of all target analytes is compared (Fig. 3.16) an interesting aspect was revealed that enrichment curve, started to increased from DHE- 1% topo (in DHE), suddenly increased from 1% - 15% topo (in DHE),maximum at for 15% TOPO (in DHE) and then this curve again started to decline after 15-19% topo (in DHE).

4.3.7- Repeatability:

Different organic phases were used in several extraction experiments (0-19% TOPO in DHE). Results were obtained (triplicate extractions) with many variations (bad repeatability) in between different extraction experiments with the same reaction conditions and the same spiked standards, for the same target analyte (Fig. 3.15). Repeatability was worse when only DHE (RSD%; 20-160%) was used as a membrane liquid. Results were better when topo was mixed with DHE from 1- 19% topo, remarkable difference was obtained for 5-19% topo in DHE as compared to DHE only. Sustainable results were obtained when 10% topo was used as membrane liquid, repeatability was found better (RSD% ≤ 10%) for all the analytes. Results were also acceptable with 19% topo (RSD% ≤ 20%).

10% topo in DHE was selected as a best organic phase composition because this organic phase mixture provides excellent repeatability (RSD% ≤ 10%).

5-Conclusion:

Present Diploma work was focused on detection and quantification short chain(C 3- C10) dicarboxylyic acid and mono carboxylic acid. These acid are found in aerosols in trace amount, HF-lpme is used to enrich these compounds so that their quantification become possible because these compounds are difficult to quantify through most of analytical instruments [6] but current method by using GC-MS provide reasonable detection and quantification limit for all of the analytes despite GC-MS system is having some internal problems throughout this project. Extra work is required to achieve validation for the membrane extraction but this was not achieved due to GC-MS problem. Most of the analytes were enriched many times which certainly represent the importance of membrane extraction. Use of topo shows a lot of improvement in enrichment factor. 10% topo in DHE was found excellent in terms of repeatability and enrichment. Enrichment can be improved for selective analytes and effectively evaporation of solvent especially accepter solution. There were some unwanted peaks in the chromatogram but these peaks were eluted at different retention times mostly before analyte started to elute, these unwanted peaks were well separated by SIM method.

Real samples after Ultrasonic assisted extraction were effectively quantified through this method except Malonic acid. Use of derivatizing reagent made possible for the detection of all target analytes and BSTFA was found excellent to derivatize all acids( C 3- C10). This method provide good LOD (0.040-0.4 ng/ml) , good linearity for standards (R2 ³ 0.99).