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May-July. 2012, Vol. 2, No. 3, 1462-1471
e- ISSN: 2249 –1929
Journal of Chemical, Biological and Physical Sciences
An International Peer Review E-3 Journal of Sciences
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Section C: Physical Sciences
CODEN (USA): JCBPAT
Research Notes
Negative ion mode mass spectrometry- an overview
Pavani Challamalla, Somsubhra Ghosh*, N. Parthiban, K. N. V Rao and David Banji.
Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh, India
Received: 1 May 2012; Revised: 20 May 2012; Accepted: 28 May 2012
ABSTRACT
Negative ion mode Mass spectrometry is the most comprehensive and versatile tool. Recent advances
in negative ion mode mass spectrometry have improved sensitivity, identification and elucidation of
organic and inorganic compounds. All compounds will not produce negative ions but many important
compounds of environmental or biological interest can produce negative ions under the right
conditions. For such compounds, negative ion mode mass spectrometry is more efficient, sensitive and
selective than positive ion mass spectra. In this review, discuss the overall framework of the negative
ion mode mass experiments into its key components, such as principle, ionization techniques,
instrumentation, fragmentation and applications.
Keywords: Negative ion, Mass Spectrometry, Ionization, Fragmentation, Mass Analyser.
INTRODUCTION
During the last few years, improvement techniques were carried out for the ionization in negative ion
mode mass spectrometry and its applications. For so many years, there was little use of negative ion
mode mass spectrometry for the investigation of structural and analytical problems. Negative ions could
be attributed to several factors associated with irreproducibility, low the mechanisms of negative ion
reactions in the gas phase were very limited. This neglect of sensitivity and in appropriate
instrumentation, the production of negative ions from a given sample is usually dependent both on the
electron energy and the ion source temperature; slight variations in either of these parameters can lead to
marked changes both in the total number of negative ions formed and in the relative abundances of ions
given by the sample. A further problem until quite recently was that most commercial mass spectrometers
could not readily be operated in the negative ion mode .At the electron energies normally employed to
obtain positive ion spectra, the sensitivity for the production of negative ions is two or three orders of
magnitude less than that for positive ion production. However, in many instruments, modifications to the
electron multiplier electronics are necessary if any negative ions are to be detected (1).
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PRINCIPLE
In negative ion mode mass spectrometry, the sample may be in the form of gas, liquid and solid. Solid
must be converted to the vapour state before entering into the ionization chamber to obtain the stream of
molecules that must flow into the ionization chamber with gases, where the sample molecules must be
converted to negatively charged particles or radical anions through absorption of electrons (electron
capture) by the ion source such as inductively coupled plasma torch from this electrons are generated. In
such way there is a bombardment between the molecule and electrons takes place this leads to capturing
of electrons.
M
e
M.-
For the production of negative ions and positive ions some ionization techniques such as electro spray
ionization, atomic pressure ionization, matrix assisted laser desorption ionization, plasma desorption
ionization techniques are used, but negative chemical ionization technique is only used for negative ions.
The negatively charged atom or molecule are kept in a magnetic field or subject it to an electric field and
measure its speed or radius relative to its mass to charge ratio by using analyzers and detect ions using
detectors(2).
Fig. 1: Shows Schematic diagram of Mass Spectrometer.
IONIZATION TECHNIQUES
The introduction of alternative methods of ionization, in particular chemical ionization revived interest in
negative ions& electron capture spectra, which are providing increasingly useful in a wide variety of
applications. Concurrently a growing body of information on the thermodynamics, kinetics and
mechanisms of negative ion reactions is being built up from results obtained by the use of techniques such
as high pressure source mass spectrometry, ion cyclotron resonance mass spectrometry. In negative ion
mode mass spectrometry, ion sources are operated at pressures of up to 1 torr (133pa) produce high
concentrations of low energy electrons which may directly react with sample molecules to form negative
molecular daughter ions and fragment ions alternatively , a reagent gas may be used to produce negative
chemical ionization mass spectra which ,in general exhibit less fragmentation than positive ion mode. The
sensitivity of the electron attachment process is very high for electronegative elements and it may be
enhanced for other compounds by making use of fluorinated derivatives. Under chemical ionization
conditions, reagent ions which react as bronsted bases frequently give (M-1) – ions in great abundance
,by detecting these ions we get structural information of particular compound(1). In negative mode proton
abstraction, electron capture or anion attachment is commonly observed (3).
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Different ionization techniques: There are different ionization techniques which are described below in
details.
(a) Chemical ionization: It is very low energy process when compared to the electron ionization. The
lower energy yields less fragmentation. It is a simpler spectrum.
Mechanism: In a chemical ionization technique, ions are produced through the collision of the analyte
with ions of a reagent gas that are present in the ion source. Some common reagent gases include:
methane, ammonia and isobutane. Inside the source, the reagent gas is present in large excess compared to
the analyte. Electrons entering the source will ionize the reagent gas. The resultant collisions with reagent
gas molecules will create ionization plasma. Negative ions of the analyte are formed by reactions with
this plasma.
Benefits: It gives molecular weight information.
Limitations: Sample must be thermally volatile and stable.
(i) Negative chemical ionization: Negative ions are formed by the electron capture. Resonance electron
capture refers to the capture of an electron by a neutral molecule to produce a molecular anion. The
electron energy is very low, and the specific energy required for electron capture depends on the
molecular structure of the analyte. Electron attachment is an endothermic process, so the resulting
molecular anion will have excess energy. In negative –ion chemical ionization, a buffer gas (usually a
common chlorine gas such as methane) is used to slow down the electrons in the electron beam until some
of the electrons have just the right energy to be captured by the analyte molecules. The buffer gas can also
help stabilize the energetic anions and reduce fragmentation.
CH4 + e-
CH3- + H+
Benefits: Efficient ionization, high sensitivity
Greater selectivity for environmentally or biologically important compounds
Limitations: Not all volatile compounds produce negative ions (2)
(ii) Atmospheric pressure chemical ionization: In atmospheric chemical ionization , liquid is pumped
through a capillary and nebulised at the tip. A corona discharge takes place near the tip of the capillary ,
ionization by ionizing gas and solvent molecules present in the ion source. These ions then react with the
analyte and ionize it via charge transfer.
Benefits: The technique is useful for small, thermally stable molecules that are not well ionized by ESI (4)
(b) Electrospray ionization: ESI is a soft ionization technique. Electrospray process produces (M-H)_
ions at atmospheric pressure, which enables multiple samples to be run without the need to break
vacuum(5). Figure 2 shows ESI source schematic, a high negative potential (0.5-4Kv) is applied across an
electrospray needle and a counter electrode .The electric field causes the ions of interest to migrate to and
out of the electrospray needle, where a taylor cone is formed. As charge builds up, the taylor cone releases
charged droplets. Evaporation of the droplets forces the charged analytes closer together until the
Rayleigh limit is reached and the droplet breaks apart in what is termed a “Coulombic explosion”. This
process continues until no solvent remains, after which the charged analytes are accelerated towards the
opposite charge of the counter electrode and thus into the mass spectrometer. Coulombic explosion is
nothing but gas-phased ions are produced from the charged droplets. Negative ion mode measures (M H)- (add ammonia to solvent) by electrospray ionization(6). Acids , phenols, nitro compounds are easily
ionize in the negative ionization mode by ESI(7).
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Fig. 2: Shows Schematic diagram of negative electrospray ionization
(c) Matrix assisted laser desorption: Matrix- assisted laser desorption/ionization mass
spectrometry(MALDI-MS), has emerged as a powerful method that allows for the analysis and detection
of a wide range of biomolecules. In the most widespread form of MALDI-MS the sample consists of
analyte molecules dilutely embedded in a matrix of highly light absorbing, low-mass molecules. The
matrix molecules are resonantly excited by an ultraviolet (UV)—by far the most common case—or
infrared (IR) laser pulse of typically nanosecond duration, and the absorbed energy causes an explosive
breakup of the sample and ionization of a fraction of the analyte molecules. Consequently, a volume of
the matrix and the trapped analyte molecules are ejected into the gas phase. The ejected material contains
both neutral and charged species that interact with each other during the expansion of the plume in the ion
source. The standard MALDI ion source usually operates under high
vacuum conditions, typically a
pressure,1026 torr, and is combined with a TOF mass analyzer with axial extraction (8). MALDI has
rapidly becomes a powerful technique for the analysis of peptides and proteins is carried out in both
positive and negative mode (9).
Fig. 3: Shows Schematic diagram of MALDI
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al
(d) Plasma desorption ionization: Decay of 252Cf (californium) produces two fission fragments that
travel in opposite directions. One fragment strikes the sample knocking out 11-10
10 analyte ions. The fission
of the 252 C f nucleus is highly exothermic, and the energy released is predominantl
predominantly
y carried away by a
wide range of fission fragments, which are heavy atomic ion pairs. The ion pair of fragments depart in
opposite directions. A single fission fragment passing through a thin film can desorb many positive and
negative ions, electrons, and upto 2000 neutral molecules. It is the interaction of these fission fragments as
they pass through matter that forms the basis of the plasma desorption process. This ionization method is
especially useful for large biological molecules (10).
INSTRUMENTATION
It contains 3 major components which are described here in details. (i) Ion source: The important step in
obtaining mass spectrum is to ionize the sample. The minimum energy required to ionize an atom or a
molecule is called ionization potential. The technique used for the production of ions by the bombardment
of electrons from an electrically heated tungsten filament. The bombarding electrons have energy of about
70eV. Due to the bombardment, the molecules generally capture one electron to form a frag
fragment
ment ion. The
energy required for the capture of electron one is usually 10eV.With this energy, no ions are formed, i.e.,
no fragmentation of the parent ion takes place. But if the energy of the bombarding electron is around
70Ev, then additional energy is consumed in fragmenting the parent ion .This results in the formation of
fragment ions or the daughter ions (11).
Fig. 4: Shows Schematic Diagram of Mass Spectrometer (12)
(ii) Mass analyzers: The negatively charged ions produced in the ion chamber are accelerated by
applying potential. These ions then enter the mass analyser.Here the fragment ions are differentiated on
the basis of their m/z ratio.
Different types of mass analyzers:
a) Quadrupole mass analyzer
b)Time –of-flight analyzer
c)Ion trap mass analyzer
(a) Quadrupole Mass analyzer: A quadrupole mass analyzer consists of four circular cross sections are
arranged with their axes forming a squre array. A positive dc voltage is applied to one set of diagonally
paired rods, negative dc voltage to the other pair. A radiofrequency ac voltage is also applied to one pair
of rods, and to the other pair is placed in rf voltage 1800C out of phase with the first.
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The resulting electrical field within the space defined by the four rods are used for resolve ions injected
into the field. Ions entering this quadrupole electric field undergoing oscillatory trajectories, and at any
given combination of applied fields, only one m/z ratio attains a stable trajectory that carries it through the
analyzer;and other ions are collected on the rods. By varying the dc or rf fields, different m/z ratios are
brought to focus.
Fig. 5: Shows Schematic diagram of Quadrupole mass analyzer (13)
(b)Time –of-flight analyzer: Ion separation is based on the principle that ions of different masses,
possessing equal kinetic energy, have different velocities. If there is a fixed distance for the ions to travel,
the time of travel will vary with their mass, the lighter ions travelling faster and reaching the detector in a
shorter period of time. According to this concept, the kinetic energy of an ion accelerated through an
electrical potential V will be
Zv =mv2 /2
(Equation 1)
and the velocity of the ion is the length of the flight path L divided by the time t. It takes the ion to travel
over that distance:
v=L/t
(Equation 2)
substituting the equation 2 in equation 1
Zv=mL2/2t2
Thus, it follows that
m/z=2Vt2/L2
(Equation3)
The TOF mass analyzer requires very fast electronics to accurately measure ion flight times that may be
submicrosecond. Furthermore, the ions in a TOF system must be created in short, well defined pulses so
that the ions all start their journey toward the detector at the same moment.
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Fig. 6: Shows Schematic diagram of Q-TOF Mass analyzer
(c) Ion trap mass analyzer: Ion traps are ion trapping devices that make use of a three-dimensional
quadrupole field to trap and mass-analyze ions.It was invented by Wolfgang Paul (Nobel Prize1989) . It
Offers good mass resolving power. The ion trap consists of two hyperbolic end-cap electrodes and a
doughnut-shaped ring electrode (by which end cap electrodes are connected). An alternating current (AC)
or (DC) and an RF potential is applied between the ring and endcaps electrodes. In the ion trap analyzer
,ions of all m/z values are in the trap simultaneously, oscillating in concentric trajectories. Sweeping the
RF potential results in the removal of ions with increasing m/z values by putting them unstable trajectory
causes them to be ejected from the trap in the axial direction toward the detector.
Fig. 7:Schematic diagram of Ion trap mass analyzer
Fig. 8: Shows Schematic diagram of Ion trap mass analyzer
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(iii) Ion Detectors: The ions are separated by the analyzer. Which are detected and measured by
electrically or photographically? The ions pass through the collecting slit one after the other and fall on
the detector. The spectrum is recorded by using oscillograph. In this recording, 3 to 5 records of the same
peaks are made galvanometers having different sensitivities. The choice of ion beam detection depends on
a number of factors, most important factor is the ion beam intensity. Ion beams tend to range from a few
ions per second all the way upto a fraction of a nanoampere (109 -1010 ions per second).
Two types of ion detectors are used for negative ion detection. Such as
(I)Faraday cups
(II)Electron multipliers
(I) Faraday Cups: Faraday cups are simply a metal receptacle that is connected to an extremely sensitive
current meter (usually called an electrometer). The negative charges associated with ions colliding with
the “cup” must be balanced by a flow of electrons from “ground” through the electrometer and into the
cup. This current is measured by the electrometer, which amplifies filters and digitizes the signal (14).
(II) Electron multipliers: Electron multiplier tubes are used for both negative and positive ion detection
(15)
.The detector of a typical mass spectrometer consists of a counter that produces a current that is
proportional to the number of ions that strikes it. Clearly, each peak in the mass spectrum represents a
very small electrical signal, and the detector must be able to amplify this tiny current. Through the use of
electron multiplier circuits, this current can be measured so accurately that the current caused by just one
ion striking the detector can be measured. When an ion strikes the surface of the electron multiplier (lead
–doped glass coated with lead oxide), two electrons are ejected. The approximately 2-Kv potential
difference between the opening and end of the detector draws the electrons further into the electron
multiplier, Where each electron strikes the surface again ,each causing the ejection of two more electrons.
This process continues until the end of the electron multiplier is reached, and the electrical current is
analyzed and recorded by the data system. The signal amplification just described will be 2n , where n is
the number of collisions with the electron multiplier surface(2).
Figure No 9. Shows Schematic diagram of Electron multiplier detectors12
APPLICATIONS OF NEGATIVE ION MODE MASS SPECTROMETRY
(a) Negative ion mode mass spectrometry is an analytical technique, it gives information about the
molecular structure of organic and inorganic compounds.
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(b) It is an accurate method for the determination of molecular mass of the components and its elemental
composition.
(c) It also used to investigate reaction mixtures (gases, liquids and solids) and also used in understanding
reaction kinetics and mechanisms of unimolecular decomposition reactions (16).
(d) Using solid phase extraction (SPE) and liquid chromatography/tandem mass spectrometry
(LC/MS/MS), 46 pesticides in positive ion mode and 14 pesticides in negative ion mode were
analyzed (17).
(e) Sugar and sugar alcohols such as sorbitol and glucose were successfully analyzed by mass
spectrometry using atmospheric pressure ionization in negative ion mode (18).
(g) ) ESI in the negative ion mode has been employed to determine the structure of glycosides of
anthraquinones [67,68], coumarins [67], flavonoids [4,31,67,69,70], iridoids [67,71], aromatics [30,67],
cardenolides [29],isoflavonoids [57] and triterpenes [67,72] (19).
(h) Chemical warfare agents, explosives, dyes are analyzed by using both positive and negative ion mode
by using liquid chromatography-Mass spectrometry(20).
(i) By using negative chemical ionization we can measure the MDA(3,4-Methylene dioxyamphetamine),
MDMA(3,4-Methylene dioxy methamphetamine), MDEA(3,4-Methylene dioxy ethyl amphetamine) in
blood(21).
CONCLUSION
Mass spectrometer is operated in positive and negative ion mode. Generally now a days positive ion mode
is used in many cases and Negative ion mode spectroscopy is also being used in some specific and
sensitive cases, in such cases negative ion mode is more efficient than the positive ion mode .In future it
can be used in other fields also.
REFERENCES
(1) K. R. Jennings, Philosophical Transactions A., 1979,293:125-133.
(2) D. Pavia, Spectroscopy, 2007, page no.401-419.
(3) K. S Graham, Applications of liquid chromatography: Mass spectrometry to
cytochrome
p450 inhibition screening and the measurement of plasma free Metanephrines, University
of Wales Swansea 2005.
(4) J. J. Pitt, Clin Biochem Rev, 2009, 30(1): 19-34.
(5) Available at: http://www.colby.edu/chemistry/instruments/ElectrosprayIntro.pdf, Electrospray
ion mass spectrometry.
(6) D. F Smith, Petroleomics applications of fourier transformion cyclotron resonance mass
spectrometry:Crude oil and bitumen analysis, The Florida state university college of arts
and sciences, Oct, 2007.
(7) K. A. Schug, Pseudo-molecular ion formation by aromatic acids in negative ionization mode
electrospray ionization mass spectrometry, Oct, 2002.
(8) R. Ekman, J. Silberring, A. M.W. Brinkmalm, A. Kraj, D. M. Desiderio, N. M. Nibbering,
1470 J. Chem. Bio. Phy. Sci. Sec. C, 2012, Vol.2, No.3, 1462-1471.
Negative ion...
Somsubhra Ghosh et al.
Mass spectrometry: Instrumentation,interpretation and applications, (WileyInterscience, New
York) 2008.
(9)
M. Dashtiev,
E. W¨afler, U. R¨ohling, M. Gorshkov, F. Hillenkamp, R. Zenobi.,
International Journal of Mass Spectrometry, 2007, 268:122-130.
(10) Available at: http://www.freepatentsonline.com/5204530.html, Noise reduction in negativeion quadrupole mass spectrometry, April 20, 1993.
(11) Y. R. Sharma, Elementary Organic Spectroscopy; Principles And Chemical Applications, S.
Chand & Company Ltd, 2007, 279-290.
(12) D. Wishart, Mass spectrometry: Methods & theory.
(13) K. A. Connors, A text book of Pharmaceutical Analysis, Wiley- India, New Delhi, 2007, 306
-307.
(14) Available at: http://www.whoi.edu/cms/files/wjenkins/2005/9/Notes07_5343.pdf, Principles
of mass spectrometry, AMS, and ion optics.
(15) M. Fabrizi, C. Pagura, A. Tolstogouzov, S. Daolio, M. Ferretti, E. Magnone and G. L.
Olcese, Rapid Commun. Mass Spectrom; 1998,12, 675–682.
(16) G. D. Rao, A text book of pharmaceutical analysis, volume -2,30 th july, 2002, page
no.140 - 146.
(17) Chin-Kai Meng , Determination of pesticides in water by SPE and LC/MS/MS in both
positive and negative ion modes,September ,2008.
(18) H. Kumaguai, Application of Liquid Chromatography/Mass Spectrometry to the Analysis of
Sugars and Sugar-Alcohol, 2001.
(19) M. Kubota, K. Yoshida, A. Tawada, M. Ohashi, J. Mass Spectrom. Soc. Jpn., 2002, 50 (2),
58-66.
(20) G. M. Cabrera, Phytochemistry: Advances in Research, 2006,1-22.
(21) ) F. T. Peters, N. Samyn, C.T. Lamers, W.J. Riedel, T. Kraemer, G.D. Boeck, H.H. Maurer,
Clinical Chemistry, 2005, 51(10), 1811-1822.
*Correspondence Author: Somsubhra Ghosh
Nalanda College of Pharmacy, Nalgonda, Andhra Pradesh, India
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