50

Chapter 4

Part-A: Gas Phase

Studies of the Ionic

Liquids Through

Mass Spectrometry

52

4(A).1.Introduction and Literature Survey

Chemical analysis plays an increasingly important role in our complex natural

and post-industrial world by helping to determine the nature of materials of all types.

Among analytical methods, mass spectrometry (MS) has the distinctive capability of

providing high sensitivity and high selectivity in the detection and quantitation of a

wide variety of chemical and biological compounds. Sir J. J. Thomson was pioneered

the term Mass Spectrometry, in 1910, the first mass spectra was recorded by Thomson

[190]. Organic Chemists never gave any importance to the mass spectrometry upto

1950. After that the knowledge of gaseous ion chemistry, improved the role of mass

spectrometry [191]. Today, organic mass spectrometry has become an integral part of

organic chemistry. Along with infrared, ultraviolet and nuclear magnetic resonance

spectroscopy, it is an indispensable tool for molecular structure elucidation. There are

many other applications in the field of chemistry. The rapid growth of mass

spectrometry has been due to the ingenious and intuitive applications in the areas of

drugs and pharmaceuticals, physical sciences, chemical sciences, polymer sciences,

life and environmental sciences [192-196]. It finds much of its importance in

increasing, not replacing, the effectiveness of other techniques. In addition to that the

linking up ability of mass spectrometry with chromatography (i.e. hyphenated

technique), its parallel unit (i.e. tandem mass spectrometry) and the use of computers

to analyze mass spectral data have added new dimensions to mass spectrometry [197-

199].

Molten salts, fused salts or ILs with their widespread applications demand an

inherent requirement to determine the nature and purity of synthesized ILs, and to

characterize and quantify the variety of compounds dissolved within them. These pose

an interesting analytical problem, because the properties of ILs do not always readily

53

lend themselves to conventional analytical techniques. A research article by Jackson

and Duckworth [200] gives an excellent account of the various aspects of mass

spectrometry in the study of ILs. It is particularly noteworthy that this gave an

excellent coverage for the early attempts to obtain electron ionization (EI) mass

spectra and chemical ionization (CI) mass spectra of ILs, which were unsuccessful,

presumably because ILs have negligible vapor pressure and cannot be readily

transferred to the gas phase. Because of their inherently low vapour pressure, fast

atom bombardment (FAB) MS has been the most extensively used MS technique for

characterizing ILs. The ILs may be dissolved in a matrix such as glycerol, or analysed

without dissolution. FAB-MS spectra of ILs are typically characterized by a dominant

peak for the unbound cation followed by clusters of the form , where A is

the cation and B is the anion. One may also perform mass analyses of ILs by

dissolving them in a suitable solvent and performing electrospray mass spectrometry.

By referring this, we report and discuss here an ESI-MS/MS studies of 1-ethyl-3-

methylimidazolium bromide,1-methyl-3-propylimidazolium bromide,1-butyl-3-

methylimidazolium bromide, and N-butyl-pyridinium bromide,3-3‘-bis-(indolyl)-

phenylmethane, 3-3‘-bis-(indolyl)-4-chlorophenylmethane, 3-3‘-bis-(indolyl)-4-

methoxyphenylmethane, 3-3‘-bis-(indolyl)-3,4-(dimethoxy)-phenylmethane, 3-3‘-bis-

(indolyl)-cinnamyl-phenylmethane, and the template used TBAB. Herein, we include

pyridinium salt in the category of ILs although it has a melting point higher than

1000C. The particulars of instrumentations and the mechanism involved in ESI, triple

quadrupole system followed by the detailed discussion on the results obtained by

means of pattern of fragmentation is presented in following sections.

54

4 (A).1.2. Electrospray ionization (ESI)

ESI ―is a soft ionization technique that accomplishes the transfer of ions from

solution to the gas phase. The technique is extremely useful for the analysis of large,

non-volatile, chargeable molecules such as proteins and nucleic acid polymers.‖ In

which the solution is composed of a volatile solvent and the ionic analyte is at very

low concentration, typically In addition, the transfer of ions from the

condensed phase into the state of an isolated gas phase ion starts at atmospheric

pressure and leads continuously into the high vacuum of the mass analyzer. This

results in a marked softness of ionization and makes electrospray the ―wings for

molecular elephants‖. Another reason for the extraordinary high-mass capability of

ESI is found in the characteristic formation of multiply charged ions in case of high-

mass analytes. Multiple charging folds up the m/z scale by the number of charges and

thus compress the ions into the m/z range of standard mass analyzers.

A review article attempted by Eberlin [201] gives an account of the important

applications of ESI in organic transformations. This review also enlightens the

significance of the conjugation of ESI-MS with its tandem version. More recently, a

review article by Traldi et al [202] has provided a comprehensive account of

developments in the ESI process. Though Fenn et al [203] have documented spray

action for ESI, but whole array of orders involved during the process of ionization,

modern aids in electrospray and formation of droplet was systematically described by

Hoffmann and Stroobant. Hoffmann and Stroobant‘s book entitled ―Mass

Spectrometry: Principles and applications‖ [204], gives considerable details of the

mechanism of ESI. A highly complex mechanism due to series of physical and

chemical processes result ions of the analytes starting from their solutions. The

Schematic of the mechanism of ion formation in ESI is shown in figure 4(A).1.

55

Mechanically an ESI is produced by applying a strong electric field, under

atmospheric pressure, to a liquid passing through a capillary tube with a weak flux

(normally 1–10 μlmin−1

). The electric field is obtained by applying a potential

difference of 3–6 kV between this capillary and the counter-electrode, separated by

0.3–2 cm, producing electric fields of the order of 106Vm−1

(Figure 4(A).1).This field

induces a charge accumulation at the liquid surface located at the end of the capillary,

which will break to form highly charged droplets. A gas injected coaxially at a low

flow rate allows the dispersion of the spray to be limited in space. These droplets then

pass either through a curtain of heated inert gas, most often nitrogen, or through a

heated capillary to remove the last solvent molecules. H-ESI is now very popular

Strategy for mass spectrometer, its superiority and attachment ability draw more and

more attention for various improvements.

Figure 4(A).1: Diagram of electrospray sources, using skimmers for ion focalization and a

curtain of heated nitrogen gas for desolvation [top], or with a heated capillary

for desolvation (bottom)

56

With this great development in the range and scope of mass spectrometry, a

book by Christopher G. Herbert and Robert A. W. Johnstone [205] entitled ―Mass

Spectrometry Basics‖ is a particularly important source of information on the design

and construction of high resolution mass spectrometers and gives an excellent account

of the salient features of the mass spectrometers and technical advances made in the

field.

Figure 4(A).2: Pneumatically assisted ESI

The schematic diagram of the process of pneumatic electrospray for the

formation of ionic aerosols before their generalization as inlets in organic chemistry

applications is shown in figure 4(A).2, in which the sample solution flows or is

pumped along a capillary tube, the end of which is held at a high positive or negative

electrical potential. Because of the electrical charge, the surface of the solution at the

outlet of the capillary also becomes charged and is repelled by the existing electric

field of the same sign. If the capillary tube is narrow enough, the liquid inside is

forced out of the end of the capillary, and the surface of the liquid is rounded with a

high radius of curvature. This point of liquid leads to repelling a steady stream of

charged droplet into a desolvation chamber. If the charged capillary tube is also

surrounded by an uncharged concentric capillary, nitrogen or other gas can be blown

through the annular space between the capillaries, which can be used to aid droplet

formation, as with concentric capillary pneumatic nebulizers.

57

As we are familiar with the process of formation of the spray, this could be

initiated at an ‗onset voltage‘ that, for a given source, depends on the surface tension

of the solvent. If one examines with a microscope the nascent drop forming at the tip

of the capillary while increasing the voltage, as schematically displayed in Figure

4(A).3, at low voltages, the drop appears spherical, then elongates under the pressure

of the accumulated charges at the tip in the stronger electric field; when the surface

tension is broken, the shape of the drop changes to a ‗Taylor cone‘ and the spray

appears. Gomez and Tang [204] were able to obtain photographs of droplets formed

and dividing in an ESI source. A drawing of a decomposing droplet is displayed in

Figure 4(A).3.

Figure 4(A).3: Decomposition of droplet

After the interpretation of the results, Gomez and Tang concluded that

breakdown of the droplets can occur before the limit given by the Rayleigh equation

is reached because the droplets are mechanically deformed, thus reducing the

repulsion necessary to break down the droplets. Figure 4(A).4 shows a model of the

deformation of droplet in an electrospray source.

Figure 4(A).4: A decomposing droplet in an ESI source: q- charge, є0- permittivity of the

environment, γ- surface tension, and d- diameter of supposed spherical droplet.

58

The solvent contained in the droplets evaporates, which causes them to shrink

and their charge per unit volume to increase. Under the influence of the strong electric

field, deformation of the droplet occurs. The droplet elongates under the force

resulting from the accumulation of charge, similarly to what occurred at the probe tip,

and finally produces a new Taylor cone. From this Taylor cone, about 20 smaller

droplets are released. Typically a first-generation droplet from the capillary will have

a diameter of about 1.5 μm and will carry around 50,000 elementary charges. The

offspring droplets will have a diameter of 0.1 μm and will carry 300 to 400

elementary charges. The total volume of the offspring droplets is about 2% of the

precursor droplet but contain 15% of the charge. The charge per unit volume is thus

multiplied by a factor of seven. The precursor droplet will shrink further by solvent

evaporation and will produce other generations of offspring. These small, highly

charged droplets will continue to lose solvent, and when the electric field on their

surface becomes large enough, desorption of ions from the surface occurs. Charges in

excess accumulate at the surface of the droplet. In the bulk, analytes as well as

electrolytes whose positive and negative charges are equal in number are present at a

somewhat higher concentration than in the precursor droplet. The desorption of

charged molecules occurs from the surface. This means that sensitivity is higher for

compounds whose concentration at the surface is higher, thus more lipophilic ones.

When mixtures of compounds are analyzed, those present at the surface of droplets

can mask, even completely, the presence of compounds which are more soluble in the

bulk. When the droplet contains very large molecules, like proteins for example, the

molecules will not desorb, but are freed by evaporation of the solvent. This seems to

occur when the molecular weight of the compounds exceeds 5000 to 10,000 Da.

59

4 (A). 1.3. Triple Quadrupole Mass Spectrometers

The triple-quadrupole MS (TQMS) is a tandem arrangement, as illustrated in

(Figure 4(A).5). J. Gross [206] in his comprehensive book entitled ―Mass

Spectrometry: A Textbook‖ a set of two editions reported excellently step by step

modifications in the field with the help of citing standard literature. A somewhat

different type of book made its appearance in 2007, which was by Watson and

Sparkman [207] describing instrumentation, applications and strategies for data

interpretation involved in mass spectrometry.

Triple quadrupole mass spectrometers, QqQ, have almost become a standard

analytical tool for LC-MS/MS applications, in particular when accurate quantization

is desired. Ever since their introduction, they have continuously been improved in

terms of mass range, resolution, and sensitivity.

Figure 4(A).5: Schematic of a triple quadrupole mass spectrometer

To operate triple quadrupole mass spectrometers in the MS/MS mode, Q1

serves as MS1, the intermediate RF-only device, q2, acts as ―field-free region‖ for

metastable dissociations or more often as collision cell for CID experiments, and Q3 is

used to analyze the fragment ions exiting from q2. Typically, the mass selected ions

emerging from Q1 are accelerated by an offset of some ten electron volts into q2

where the collision gas (N2, Ar) is provided at a pressure of 0.1–0.3 Pa. In triple

60

quadrupole instruments Q1 and Q3 are operated independently as MS1 and MS2,

respectively, making MS/MS a straightforward matter. The experimental setups for

product ion, precursor ion, and neutral loss scanning are summarized in Table 4(A).1.

Table 4(A). 1: Scan modes of triple quadrupole instruments

Scan Modea

Operation at Q1 Operation at q2 Operation at Q3

product ion,

define m1

no scan, select m1 metastable or CID scan up to m1 to

collect its fragments

precursor ion,

define m2

scan from m2 upwards

to cover potential

precursors

metastable or CID no scan, select m2

constant neutral loss,

define ∆m

scan desired range metastable or CID scan range shifted by

∆m to low mass a Masses for reaction, m1

+ = m2

+ + n

4(A).2.Experimental

All compounds were synthesized in the laboratory. The details of the synthetic

methodology and spectral characterization (FT-IR and 1H-NMR) have been reported

in chapter 2 and 3 of the thesis. We carried out the mass analysis with the help of TSQ

Quantum Access triple stage quadrupole mass spectrometer system by Thermo

Scientific (USA). The system also consists of Finnigan Surveyor Auto-sampler Plus,

and Surveyor MS pump Plus. The system was tuned by infusing a low concentration

of tuning and calibrating solution that contains polytyrosine-1, 3, 6 directly into the

H-ESI source using a syringe pump at a flow rate of 2 µL/min. so as to achieve the

best ion beam intensity and stability, the singly charged, positive ions for poly-

tyrosine monomer, trimer and hexamer: m/z 182, 508 and 997 respectively were

observed. The system was then flushed with the mobile phase Methanol: Formic acid

(0.1%) of solute in the composition of 75:25. A Full Scan positive mode analysis of

the sample in the Q1MS mode was carried out to find out the Parent masses of the

various ILs. The parent masses of the ILs are summarized in Table 4(A).2-4(A).6.The

system was then operated in the MS +MS/MS mode for optimization by introducing

61

sample in the manual loop injection. The instrumental MS/MS conditions were: Spray

voltage: - 4500 V, Vaporizer temperature: - 150V, Sheath gas pressure: - 40,

Auxiliary Gas Pressure: -15, Capillary Temperature: - 300V, and the collision

Pressure: - 1.5 m Torr. The sample was injected and data acquisition was carried out

for all the ILS. The masses of the compounds in the sample were identified in this

mode. Argon was used as a collision gas and nitrogen as a nebulizing gas.

4(A).3. Results and Discussion

This section is divided in two parts out of which first having a discussion on

ESI-MS/MS studies of the products obtained from Menschutkin quaternization

reaction (synthesis of ILs) of 1-methylimidazole and Pyridine with alkyl halide(s) by

employing molten TBAB as a template. In the similar way the second section covers

the detail study of the compounds which were synthesized via condensation reaction

between indole and aromatic aldehydes.

4(A).3.1. ESI-MS/MS studies of ILs

To our knowledge, only a few published reports deal with mass spectral

analysis of ILs [196, 202, 204-210]. The details of mass spectrums of the template

used, and all the four synthesized ILs are presented in figures 4(A).6 - 4(A).10 and

discussed below. In addition to this, we present here a likely detailed pattern of

fragmentation, which is evidenced earlier in the literature by using different ionization

methods. This involves the migration of β-proton with subsequent removal of the

neutral species i.e. alkenes, ring expansion through tropylium ion, loss of alkenes, the

McLafferty rearrangement, and the removal of heterocycle i.e. aziridine and pyridine.

The aqueous chemistry of tetra-alkyl-ammonium salts is very important. Such salts

also form clathrate-hydrates [215]. The properties like heat capacity, partial molal

volumes and entropies have led to concepts like hydration, solute-solute hydrophobic

62

interaction, micellization water structure making effect etc. [216, 217]. Therefore it

was felt that the mass spectral study of this compound would be interesting. The mass

spectrum is presented and analyzed.

The ESI-MS/MS spectral data obtained for tetra-butyl-ammonium ion is

depicted in Table 4(A).2 (entry 1a-1j) and our obtained pattern is shown in figure

4(A).6. The thermal decomposition mass spectrum for the said compound (TBA

cation) is reported in the literature [206] and shows close resemblance with the

present profile. The literature data have been explained on the basis of decomposition

of tri-n-butyl ammonium system. We observed the peaks at m/z = 242.315, 186.234,

142.129, 130.179, 100.204, 72.239, 58.245 (Table 4(A). 2), respectively which are in

harmony with the reported pattern. However, we observe additional peak resolutions

at 446.348, 414.253, 349.848 m/z respectively. These are being attributed to cation-

cation-cation or cation-anion-cation association in gas phase.

63

Table 4(A).2: ESI-MS/MS data for tetra-butyl-ammonium bromide

Compound Entry Observed m/z$

Calculated m/z*

Formula

Tetra-butyl-ammonium

bromide

1a 446.348 446.541 [C28H68N3]+

1b 414.253 414.479 [C26H60N3]+

1c 349.848 351.237 [C17H40N2Br]+

1d 242.315 242.285 [C16H36N]+

1e 186.234 186.222 [C12H28N]+

1f 142.129 142.159 [C9H20N]+

1g 130.179 130.160 [C8H20N]+

1h 100.204 100.113 [C6H14N]+

1i 72.239 72.081 [C4H10N]+

1j 58.245 58.066 [C3H8N]+

$- Experimentally observed values

* - Values calculated using Isotopic mass of the element [206]

The mass spectrum of tetra-butyl-ammonium ion (m/z – 242.165) can be

accounted in terms of loss of neutral species (butene and propene), migration of a

proton, and the McLafferty rearrangements as shown in scheme 4(A).1 and referring

to Table 4(A).2. The imminium ion obtained at m/z = 58.245 involves two

consecutive classical McLafferty rearrangements, as shown in scheme 4(A).1. The

peak observed at m/z=72.081 is due to removal of ethylene from the C6H14N+

(m/z =

100.113) ion.

64

N+

C4H9

C4H9

C4H9

H9C4CH2

CHCH2

CH3

N+

H

C4H9

C4H9

H9C4N+

H

C4H9

C4H9

H

CH2

CHCH2

CH32

1g [C8H20N]+

(m/z - 130.160) 1d [C16H36N]+ (m/z - 242.285) 1e [C12H28N]

+ (m/z - 186.222)

H2

N+

CH2

C4H9

H9C4

1f [C9H20N]+ (m/z - 142.159)

CH3

CCH2

H

N+

CH2

CH3

H9C4

CH3

CCH2

H

N+

CH2

CH3

CH3

CH2 CH2N

+

CH2

CH3

H5C2

1i [C4H10N]+ (m/z - 72.081) 1h [C6H14N]

+ (m/z - 100.113)

1j [C3H8N]+ (m/z - 58.066)

CH2

CHCH2

CH33

N+

H

C4H9

H

HH2

N+

C4H8

H

H

1i [C4H10N]+ (m/z - 72.081)

CH2 CH31. 2.

Scheme 4(A). 1: Proposed pattern of fragmentation for tetra-butyl-ammonium bromide

The peak observed at m/z 446.348 is assigned to association of tri-n-butyl-

ammonium ion and two ions of di-n-butyl-ammonium ion (i.e. cation-cation-cation

association). The m/z 414.253 peak can be viewed as a combination of tetra-butyl-

ammonium cation (m/z=242.285), N-butyl-N-methyl imine cation (m/z= 100.113)

and N-ethyl-N-methylimine cation or butylimine cation (m/z= 72.081). A

combination of di-n-butyl-ammonium ion and N, N-dibutyl-imminium cation with a

65

bromine (anion) results in a species having molecular weight m/z=349.221. All these

mean that there is a clusterization of ammonium ions in the form of triplet cation-

cation-cation (+ + +) or triplet cation-anion-cation (+ - +) associations in gas phase.

The studies of excess thermodynamic properties of mixed aqueous solutions of

electrolytes have assumed great interest in recent years, since these provide useful

information on the interaction between ions of the same or different charge either

triplet or high order interactions [218-220]. In light of this, we studied the gas phase

behavior of tetra-butyl-ammonium bromide closely and which indicates the existence

of the triplet ion (+ + +) or (+ - +) interactions in gas phase. i.e. cation-cation-cation

or cation-anion-cation triplet forming species even in gas phase. We believe that

interactions get attenuated in presence of water and lead to the concepts involving

aggregation, micelles, and hydrophobic interactions.

66

Figure 4(A). 6: ESI-MS/MS spectrum of tetra-butyl-ammonium bromide. The inset describes the resolution of the parent TBA cation of tetra-butyl-

ammonium bromide

67

Table 4(A).3: ESI-MS/MS data for 1-ethyl-3-methylimidazolium bromide

Compound Entry Observed

m/z $

Calculated

m/z*

Formula

1-ethyl-3-

methylimidazolium

bromide

2a 303.113 302.111 [C12H22N4Br] +

2b 111.215 111.092 [C6H11N2]+

2c 83.227 83.061 [C4H7N2]+

$- Experimentally observed values

* - Values calculated using isotopic mass of the element [206]

N+

NCH3

CH3

NN+

CH3

CH3Br

-

NN+

CH3

CH3

Br-

NN+

CH3

CH3

[C12H22N4Br]+ (m/z = 302.111) [C6H11N2]+ (m/z = 111.092)

NN+

CH3

H

CH2 CH2NN

+

H CH3

[C6H11N2]+ (m/z = 111.092) [C4H7N2]

+ (m/z = 83.061)

Scheme 4(A).2: Proposed pattern of fragmentation for 1-ethyl-3-methylimidazolium bromide

The mass spectral data of 1-ethyl-3-methylimidazolium bromide are tabulated

in Table 4(A).3 (entry 2a-2c) and described in figure 4(A).7. It involves peaks at m/z

303.113, 111.215 and 83.227. The 303.113 peak is assigned to formation of the class

C2A+. We propose a pattern of fragmentation as shown in scheme 4(A).2 for 1-ethyl-

3-methylimidazolium bromide which involves migration of β-hydrogen, forming an

imidazolium cation having mass 83.061 (entry 2c).

68

Figure 4(A). 7: ESI-MS/MS spectrum with proposed pattern of fragmentation of compound 1-ethyl-3-methylimidazolium bromide

N NCH2

CH3

H3CBr

69

Table 4(A). 4: ESI-MS/MS data for 1-methyl-3-propylimidazolium bromide

Compound Entry Observed

m/z$

Calculated

m/z*

Formula

1-methyl-3-

propylimidazolium

bromide

3a 329.121 329.134 [C14H26N4Br] +

3b 125.225 125.108 [C7H13N2]+

3c 83.265 83.061 [C4H7N2]+

$- Experimentally observed values

* - Values calculated using isotopic mass of the element [206]

N+

NC3H7CH3

NN+

H7C3 CH3

Br-

NN+

H7C3 CH3

Br-

NN+

H7C3 CH3

[C14H26N4Br]+ (m/z = 329.134) [C7H13N2]+ (m/z = 125.108)

NN+

H7C3 CH3

CH2 CH3NN

+

H CH3

[C7H13N2]+ (m/z = 125.108) [C4H7N2]

+ (m/z = 83.061)

Scheme 4(A). 3: Proposed pattern of fragmentation for 1-methyl-3-propylimidazolium

bromide

The mass spectra of 1-methyl-3-propylimidazolium bromide reveals the peak

at m/z 83.265 is the daughter ion of the parent having m/z 125.225 (figure 4(A).8). In

the Table 4(A).4 entry 3a is the associated triplet (+ + -) having m/z = 329.121. We

suggest a scheme on the basis of the peaks observed which are accounted in terms of a

pattern of fragmentation as shown in scheme 4(A).3. It shows loss of propene

molecule through the migration of β-hydrogen.

70

Figure 4(A).8: ESI-MS/MS spectrum with the proposed pattern of fragmentation of compound 1-methyl-3-propylimidazolium bromide

N NCH2

H2C

H3CCH3

Br

71

Table 4(A).5: ESI-MS/MS data for 1-butyl-3-methylimidazolium bromide

Compound Entry Observed

m/z$

Calculated

m/z*

Formula

1-butyl-3-

methylimidazolium

bromide

4a 359 358.170 [C16H30N4Br] +

4b 139.174 139.124 [C8H15N2]+

4c 83.209 83.061 [C4H7N2]+

4d 41.486 42.034 [C2H4N]+

$- Experimentally observed values

* - Values calculated using isotopic mass of the element [206]

N+

NC4H9CH3

Br-

NN+

H9C4 CH3

Br-

NN+

H9C4 CH3

[C16H30N4Br]+ (m/z = 358.170) [C8H15N2]+ (m/z = 139.124)

NN+

H9C4 CH3

CH2 C3H6NN

+

H CH3

[C8H15N2]+ (m/z = 139.124) [C4H7N2]

+ (m/z = 83.061)

NN+

H9C4 CH3

NN+

H CH3

N

H

CH N+

CH3

[C4H7N2]+ (m/z = 83.061) [C2H4N]+ (m/z = 42.034)

Scheme 4(A).4: Proposed pattern of fragmentation for 1-butyl-3-methylimidazolium bromide

The closer scrutiny of the mass spectrum of [bmim][br] [figure 4(A).9] shows

peaks at m/z 139.174 which is the daughter ion of the parent which have m/z = 359

(the associated triplet of + - + ions). Similarly the peak at m/z 139.174 gives the

daughter ion at m/z 83.209 (table 4(A).5). The occurrence of these peaks as shown in

figure 4(A).9 can be accounted by scheme 4(A).4. It includes pattern of

fragmentation, where we have utilized the rearrangement in which β-hydrogen

72

migrates with loss of neutral species (i.e. Butene), resulting in an ion having m/z

83.061 (entry 4c). Further interesting pattern of fragmentation is observed in the case

of the peak at m/z 41.486, which involves removal of neutral species alkenes propene,

followed by loss of three member nitrogen heterocycle (i.e. Aziridine).

73

Figure 4(A).9: ESI-MS/MS spectrum with the proposed scheme of fragmentation of compound 1-butyl-3-methylimidazolium bromide

N NCH2

H2C

H3CCH2

CH3

Br

74

Table 4(A).6: ESI-MS/MS data for n-butyl-pyridinium bromide

Compound Entry Observed

m/z$

Calculated

m/z*

Formula

n-butyl-pyridinium

bromide

5a 351.3 351.143 [C18H28N2 Br] +

5b 136.127 136.113 [C9H14N]+

5c 80.16 80.05 [C5H6N]+

5d 57.421 57.07 [C4H9]+

$- Experimentally observed values

* - Values calculated using isotopic mass of the element [206]

N+

CH3

CH2CH3

N+

H

[C9H14N]+ (m/z - 136.113) [C5H6N]

+ (m/z - 80.05)

N+

CH3

N[C4H9]

+

[C9H14N]+ (m/z - 136.113) (m/z - 57.07)

Scheme 4(A).5: Proposed pattern of fragmentation for n-butyl-pyridinium bromide

The results depicted in Table 4(A).6 (entry 5a-5d) for n-butyl-pyridinium

bromide show the peaks at m/z =351.3 (an associated triplet of + - + ions), 136.127,

80.16 and 57.421 respectively. We suggest a pattern of fragmentation as described in

scheme 4(A).5. It seems that the rearrangement concept utilized earlier helps us to

identify the presence of ions having m/z value very close to 5b, 5c and 5d ions.

75

Figure 4(A).10: ESI-MS/MS spectrum with the proposed pattern of fragmentation of compound n-butylpyridinium bromide

N+

CH3Br

-

76

Lattice energy of ammonium salts (particularly imidazolium and pyridinium)

is responsible for their very low melting nature, such compounds consist of a large

cation and an anion that is very difficult to fit into the lattice. Mass spectrums

obtained by us (Figures 4(A). 6-10) also indicate presence of associations for tetra-

alkyl-ammonium ions, imidazolium ions and pyridinium ions in the form of triplet

interaction species [C2A]+. We believe that such association may be due to cation -

cation affinity assisted by π-π stacking interaction due to the ring electrons. In the

studies of physico-chemical processes involved in the existence of ionic liquids

(imidazolium and pyridinium salts), Dupont and Suarez [221] have indicated the

presence of π-π stacking via weak C-H—π interaction in methyl and imidazolium ring

π system in solid phase. Such cation-cation π-π stacking interactions have also been

indicated in theoretical studies in gas phase [222]. Association of several imidazolium

and pyridinium ions also has been postulated [C2A]+ by Gross [223]. It is difficult to

quantify the energetics of such interactions, however the spectral, simulation and ab

initio studies do indicate the vital structural effects [224, 225]. We suggest that such

molecular arrangements can generate channels in which the bromide anion is

accommodated as chains. The said structural pattern in the form C2A+ may be

visualized in a form as given in figure 4(A).11.

N+

NRCH3

NN+

R CH3

Where R-= b) -C2H5, c) -C3H7, d) -C4H9

N+

CH3

N+

CH3

Br- Br

-

Figure 4(A).11: Visualized associated triplets of imidazolium and pyridinium ions with

bromide anion

77

4(A).3.2. ESI-MS/MS analysis of BIMs

It is well known that, the exceptionally large number of alkaloids and

medicinally important compounds are derived from indole. Its reactions, and

particularly the synthesis of complex derivatives, occupy central stage in heterocyclic

chemistry. As comparable to other indole derivatives, family of bis-(indolyl)-

methanes (BIMs from here) are well known for their biological activity. BIMs are

potential chemo-preventive, and have been phyto-chemically derived from Brassica

vegetables [148].

In no other class of natural products has the utility of mass spectrometry been

recognized so rapidly as in the field of indole and related alkaloids. While the first of

several pioneering studies by Biemann and collaborator appeared in 1960, number of

articles from various laboratories had been published in the intervening years, and

there is no doubt that mass spectrometry has now become an indispensable

component of alkaloid chemist‘s armamentarium of physical methods. As will be

shown in this chapter of the thesis, the recent structure elucidation of BIMs alkaloids

rests on mass spectroscopic evidence obtained with minute amount of material. A

comprehensive book by Budzikiewicz, Djerassi and Williams [226] manifests the role

of mass spectrometry in the structure elucidation of natural products, this book

emphasizes the importance of mass spectrometry to probe the structures of alkaloids

of indole family.

There are two principal reasons why indole alkaloids lend themselves so

readily to mass spectroscopic investigation. First, they contain two centers the indole

nucleus (aromatic π electrons and /or indole nitrogen atom) and a basic nitrogen atom,

which have a great capability of for stabilizing a positive charge. Second in most

instances they possess a carbocyclic framework, where certain bonds are especially

78

prone to rupture, thus giving rise to a very few intense fragment ions. Even slight

changes in this carbocyclic framework can lead to a completely different mass

spectral fragmentation pattern, which can be used either for identification purposes or

at least for assignment of an unknown alkaloid to a specific sub-group.

The advancement in the field of mass spectrometry provides a broad spectrum

of applications in structure elucidation of variety of natural products. Herein, we

successfully applied an ESI-MS/MS to the characterization of the products obtained

from the condensation reaction between indole and aldehydes by employing molten

[bpy][Br] as a template, and the ESI-MS/MS spectral data obtained are tabulated in

Table 4(A).7. The details of mass spectrums (figure 4(A).12-16) of all the five BIMs

are discussed in the following pages. In addition to this, we report here a detailed

pattern of fragmentation, which involves formation of new six membered ring and

the ring expansion through tropylium ion, loss of phenyl ring, and the removal of

heterocycle i.e. indole ring.

Table 4(A).7: ESI-MS/MS data for the synthesized bis(indolyl)methane compounds

Entry Peak(s) R’ R Theoretical

Mass

Practical Mass Formulae

Obs. Mass Calc. Mass

1a

Ia

-H -H 322.157

321.09 [m-1] 321.149 C23H17N2+

ib 203.991 204.086 C15H10N+

ic 116.177 116.055 C8H6N+

id 77.132 77.039 C6H5+

2a

iia

-H -Cl 356.602

355.50 [m-1] 355.594 C23H16N2Cl+

iib 238.015 238.531 C15H9NCl+

iic 116.346 116.055 C8H6N+

2b

iiia

-H -OCH3 352.171

351.17 [m-1] 351.164 C24H19N2O+

iiib 233.996 234.102 C16H12NO+

iiic 77.204 77.039 C6H5+

2c

iva

-OCH3 -OCH3 382.187

381.2 0 [m-1] 381.179 C25H21N2O2+

ivb 244.929 245.117 C17H13N2+

ivc 116.954 116.055 C8H6N+

2d

va

-H -H 348.173

347.10 [m-1] 347.165 C25H19N2+

vb 230.026 230.102 C17H12N+

vd 103.186 103.056 C8H7+

79

NN

m-1

NNH H

H H

R

RR' R'

NN

H H

R

R'

m-1

BA

I)

NN

H H

m-1

NN

H H

m-1

NN

H H

AB

II)

Where:

Entry R R‘ m/z in Da.

1a -H -H 321.09

2a -H -Cl 355.50

2b -H -OCH3 351.17

2c -OCH3 -OCH3 381.20

2d -H -H 347.10

R

RR

R'R'

R'

Scheme 4(A).6: Proposed fragmentation pathway suggested for the formation of I] tropylium

cation, and II] aza-fulvenium cation through m-1 ion configuration

The close examination of mass spectra‘s of all the compounds, reveals the

peaks at 203.991, 238.015, 233.996, 244.929 and 230.026 Da. which are the daughter

ion peaks of their parent ions 1a, 2a, 2b, 2c and 2d respectively (Scheme 4(A).6).

Pattern of fragmentation suggested in scheme 4(A).7 is quite interesting and shows

formation of a 6-membered ring after subsequent loss of one of the indole ring in gas

phase.

80

NN

N N

H

N

H H

N

H H

H

R

R

RR

R

N

R

R'

R'

R'

R'

R'R'

- H2

Where:

Entry R R‘ m/z in

Da.

1a -H -H 203.991

2a -H -Cl 238.015

2b -H -OCH3 233.996

2d -H -H 230.026

I)

NH

NH

NH

NH N

II)R

R'

R'R

R'R

-H2

NH

NH

OCH3

OCH3

OCH3 OCH3

NH

NH

N NH

H

III)

Scheme 4(A).7: Proposed pattern of fragmentation, in which I and II] formation of 6-

membered cyclic structure after loss of one indole molecule for entry 1a, 2a,

2b and 2d, III] formation of an aza-fulvenium cation after loss of di-

substituted phenyl ring for entry 2c

81

The peaks at m/z 116.177 and 77.132 (1a), 116.346 (2a), 77.204 (2b), 116.954

(2c) are the characteristics of indole and benzene cation, while, the daughter ions

having m/z 103.186 is of 2d which is obtained after a loss of diindolylmethane species

from its parent species Va (Table 4(A).7).

The author is happy with these mass spectral studies. It has been shown

successfully, the occurrence of non-polar cation –non polar cation (low charge

density), -anion interactions even in gas phase for tetra-butyl-ammonium halides.

These leads to association in a gas phase in the form of dimer/trimer and higher

aggregation. The observations are important from the point of view of modeling,

computer simulation and clusterization studies.

Similarly, the analysis of mass spectrums of ILs yielded new information

about [C2A]+ ions in the form of π-π stacking and weak C-H-π interactions involving

imidazolium and pyridinium ionic species. The hypothesis advanced to account the

patterns for indole derivatives in the form of fragmentation pattern leads to a detection

of formation of tropylium ion with subsequent breakdown of phenyl and indole rings.

The experimental detection and analysis thus yielded satisfaction of doing a scientific

investigation.

82

Figure 4(A).12: ESI-MS/MS spectrum with the proposed pattern of fragmentation of compound 3-3‘-bis-(indolyl)-phenylmethane

NH

NH

83

Figure 4(A).13: ESI-MS/MS spectrum with the proposed pattern of fragmentation of compound 3-3‘-bis-(indolyl)-4-chlorophenylmethane

NH

NH

Cl

84

Figure 4(A).14: ESI-MS/MS spectrum with the proposed pattern of fragmentation of compound 3-3‘-bis-(indolyl)-4-methoxyphenylmethane

NH

NH

OCH3

85

Figure 4(A).15: ESI-MS/MS spectrum with the proposed pattern of fragmentation of compound 3-3‘-bis-(indolyl)-(3-4-dimethoxy)-phenylmethane

NH

NH

OCH3

OCH3

86

Figure 4(A).16: ESI-MS/MS spectrum with the proposed pattern of fragmentation of compound 3-3‘-bis-(indolyl)-cinnamyl-phenylmethane

NH

NH