Mass spectrometry

Structure of a mass spectrometer

The Spectrometer of mass, initially conceived by the British Joseph John Thomson, comprises a source of Ionization followed one or more analyzers which separate the ions produced according to their report/ratio m/z , of a detector which counts the ions and amplifies the signal, and finally of a computing system to treat the signal. The result obtained is a spectrum of mass representing the reports/ratios m/z ions detected according to the x-axis and the relative abundance of these ions according to the axis of ordinates.

The mass spectrometer is thus composed of four parts:

the system of introduction of the sample: the sample can be introduced directly into the source, in liquid form (direct infusion) or solid (canes direct introduction, deposit on plate MALDI,…) or by association with a separative method (chromatography in liquid phase, Gas chromatography, capillary electrophoresis,…).

the source of ionization: it consists in vaporizing the molecules and ionizing them. A source of ionization can be used either in positive mode to study the positive ions, or in negative mode to study the negative ions. Several type of sources exist and are used according to the required result and of the analyzed molecules.

electronic ionization (I.E.(internal excitation)), chemical ionization (CI) and chemical desorption-ionization (DCI)
bombardment by fast atoms (FAB), atoms metastable (MAB) or ions (SIMS, LSIMS)
the coupling inductive plasma (ICP)
the chemical Ionization with atmospheric pressure (APCI) and the photoionization with atmospheric pressure (APPI)
the electronebulisation or électrospray (ESI)
the laser ionization-desorption assisted by matrix (MALDI), activated by a surface (SELDI) or on silicon (DIOS)
ionization-desorption by interaction with metastable species (DART)

the analyzer: makes it possible to separate the ions according to their report/ratio masses/load ( m/z ). There exist analyzers low resolution: the quadripole or quadrupole (Q), the trap with ions 3D (IT) or linear (BED), and of the analyzers high-resolution, allowing to measure the exact mass of the analytes: the magnetic sector coupled to an electric sector, the time of flight (TOF), ionic cyclotronic resonance with transform of Fourier (FTICR) and Orbitrap.

These analyzers can be coupled between them to carry out experiments of mass spectrometry out of tandem (MS/MS). In general, a first analyzer separates the ions, a cell of collision makes it possible to split up the ions, and a second analyzer separates the ions fragments. Certain analyzers, like the traps with ions or the FT-ICR, constitute several analyzers in one and make it possible to split up the ions and to analyze the fragments directly.

the detector and system of treatment: the detector transforms the ions into electrical signal. The more numerous the ions are, the more the current is important. Moreover, the detector amplifies the signal obtained so that it can be treated by means of computer.

For what is used the mass spectrometry?

Identification:

According to the type of ionization used, a spectrum of mass can be characteristic of a molecule. Thus by comparing it with banks of spectra, it is possible to identify the molecule.
During the use of an analyzer high-resolution (TOF, magnetic sector, FTICR, Orbitrap), the mass spectrometry makes it possible to measure with precision the monoisotopic mass of an ion and to deduce its rough Formule from it.

structural Analysis:

the parity of the measured mass is function of the parity of the number of nitrogen atoms which a molecule (Règle of the nitrogen) has.
Each atom has one or more isotopes which are different masses by definition. Thus, the proportion of each isotope observed on a spectrum of mass, i.e. the isotopic solid mass, is characteristic of the presence of certain atoms and their number in the measured ion (in particular: Cl, Br, which presents isotopes M and M+2 in notable quantity).
the ions can split up in a mass spectrometer: in the source of ionization, in the analyzer or a cell of collision. As fragmentations respect precise laws of Chimie in gas phase, the study of these fragments makes it possible to determine the structure of the ions.

Quantification:

a mass spectrometer is a universal and very sensitive detector. Its linear range goes from 3 to 7 orders of magnitude, from where the possibility of obtaining a reliable quantification on a broad field.

The source of ionization

Ionizations I.E.(internal excitation) and Ci, which require a certain level of vacuum, are preferentially used in coupling with gas chromatography (Ci functioning starting from a source I.E.(internal excitation)). On the other hand, two sources with atmospheric pressure (électrospray and APCI) known as with " ionization douce" , are mainly used in coupling with the chromatography in liquid phase.

Electronic ionization (I.E.(internal excitation))

Electrons emitted by a filament meet the molecules which enter the source: at the time of the meeting, if the kinetic energy of the electrons is sufficient, an electron is torn off molecule M , transforming it into a radical ion M+° . This one can then split up according to its internal energy. I.E.(internal excitation) leads thus to provided an enough spectrum, with many fragments, very rich in structural information.

Chemical ionization

In addition to device I.E.(internal excitation) above, a reactive gas is introduced into the source and is ionized by electronic impact. A series of reactions follows which gives rise to ions being able to react with the molecules of analyte arriving in the source. This type of reactions ion-molecules mainly produces (in positive mode) ions + and +, thus making it possible to reach the molecular mass of the analyte.
Le methane, isobutane and the ammonia are among the most used chemical gases of ionization.

The électrospray

See also: Ionization by electronebulisor (ESI)

Its principle is the following: with atmospheric pressure, the droplets of aqueous solutions are formed at the end of a fine silica capillary, metallized on the surface and carried to a high potential. The intense electric field confers a density of important load to them. Under the effect of this field and thanks to the possible assistance of a coaxial draft, the liquid waste is transformed into cloud of fine droplets (spray) in charge according to the mode of ionization. Under the effect of a second heated draft, the droplets evaporate gradually by losing solvent molecules by complex mechanisms of desolvatation and evaporation. Their density of load becoming too important, the electric repulsions reaching the level of the surface tensions, the droplets explode while releasing from the microgouttelettes made up of protonic or déprotonées molecules of the analyte, carrying a variable number of loads.
Les ions thus formed is then guided using electric potentials applied to two cones of sequential sampling acting as barriers with the parts maintained downstream under a high vacuum (<10e-5 Torr). During this course with raised pressure, the ions undergo multiple collisions with the solvent and gas molecules, which supplements their desolvatation. While varying the electric potentials applied in the source it is possible to cause more or less important fragmentations.
L' favors this method of ionization as for the APCI is obtaining multichargés ions, for the macromolecules, polymers. It makes it possible in addition to generate an ionization " douce" : mainly are formed of the molecular ions.

The APCI

The liquid samples are directly introduced into a pneumatic atomizer. Under the effect of a nitrogen or air blast, the liquid is transformed into fine fog. A heating ensures then the desolvatation of the compounds. The latter are then ionized chemically with atmospheric pressure: in general, the vaporized mobile phase plays the part of gas of ionization and the electrons are obtained starting from discharges of electrode crowns. The ionization of the compounds is very favoured at the time of these techniques because the frequency of the collisions is high with atmospheric pressure.
L' APCI is a technique similar to chemical ionization (CI), it calls upon reactions ion-molecules in gas phase, but with atmospheric pressure and leads primarily to the formation of ions ⁺ or ^-.

The MALDI

Principle

A pulsated laser beam is used, generally in the field them ultraviolet, to desorb and to ionize a mixture stamps/sample cocristallized on a metal surface, the target.
the molecules of matrix absorb the energy transmitted by the laser in the form of photons UV, are excited and ionized. The energy absorptive by the matrix causes its dissociation and its passage in gas phase. The ionized molecules of matrix transfer their load to the sample. The expansion of the matrix involves the sample within the dense gas phase where it will finish ionizing.
L' ionization of the sample thus takes place either in the solid phase before desorption, or by transfer of load at the time of collisions with the excited matrix after desorption. It leads to the formation of monochargés and multichargés ions of ⁿ⁺ type, with a clear preponderance for monochargés.

The matrix

It is a small molecule, able to form crystals containing the analyte. It has as a property to strongly absorb with the wavelength of the laser. It ensures the stability of the sample thus, preserving it of a too important degradation by the photons.
Il is possible to use varied matrices, from which much is derived from the Cinnamic acid (acid gentisic (DHB), α-cyano-4-hydroxycinnamic (α-HCCA), sinapinic (SA),…). However no general rule governs really their choice for an application, even if l'α-HCCA is often used for the analysis of Peptide S, while SA is appropriate well for the study of the Protéine S.

The analyzer

The quadripolar analyzer

A quadripole consists of four parallel electrodes of hyperbolic or cylindrical section. The distant opposite electrodes of 2 $r_0$ are connected between them and are subjected to the same potential. The adjacent electrodes are carried to of the same value, but opposite potentials so that the variation of potential is equal to $\ phi_0$ .
Ce potential $\ phi_0$ results from the combination of tensions, one continues (U) the other alternative (V) high frequency F: $\ phi_0 = U - V \ cdot cos (2 \ pi ft)$
En applying this potential difference between each pair of electrodes, it creates for itself a quadripolar electric field. A point of coordinates (X, there, Z) located in the electric field will then be subjected to the potential: $\ phi = \ phi_0 \ cdot \ frac {x^2 - y^2} {r_0^2}$ The trajectory of a penetrating ion in the quadripole uniform according to axis Z and will thus be described by the equations of Mathieu according to the two other axes. It is possible to define according to the values U and V of the zones of stability such as coordinates X and of the ion remain strictly lower than $r_0$ there. One of them is exploited in mass spectrometry (see figure) (the ions which are in this zone will thus have a stable trajectory in the quadripole and will be detected). By keeping constant report/ratio U/V, one obtains a line of operation of the analyzer. A sweeping of U with constant U/V allows the successive observation of all the ions whose zone of stability is cut by the line of operation. The resolution between these ions is all the more large as the slope of the right-hand side is high. In the absence of continuous tension, all the ions of m/z reports/ratios higher than that fixed by the value of V applied will have a stable trajectory (X and there < $r_0$ ), the quadripole is then known as transparency and is used as focalisator of ions.
Les main advantages of the quadripolar spectrometer resides in its flexibility in use, its unit resolution on all its range of mass, its satisfactory scanning rate, like its adaptability with various interfaces allowing the coupling with the gas chromatography or liquid.

The quadripolar ionic trap

It is an ionic trap where the preparation, the analysis and the detection of the ions are carried out in the same space, following successive temporal sequences.
Le trap consists of three electrodes with hyperbolic section: an annular electrode framed by two electrodes (of entry and exit) which form the caps higher and lower device. A tension in radio frequency

V \ cdot cos (2 \ pi ft)

combined or not with a tension continues U is applied between the central electrode and the two electrodes caps. The resulting field is then three-dimensional.
Les fields of stability of the ions is again determined by the equations of Mathieu. That exploited is defined such as when the ions leave there, their radial trajectory remains stable contrary to that according to the axis of Z. A sweeping of the amplitude of the radio frequency V will thus involve the expulsion of the ions trapped according to this axis, towards the detector. The stable trajectories of the ions, within the resulting quadripolar field are three-dimensional, in the form of eight.

The time of flight

Principle

The analyzer in time of flight consists in measuring the time that an ion puts, subjected to a preliminary tension, traversing a given distance.

Indeed, the kinetic energy is worth:

$E_c = \ frac {1} {2} \ cdot m \ cdot v^2 = \ frac {1} {2} \ cdot m \ cdot \ frac {l^2} {t^2}$ m being mass, v speed, L the distance covered during the flight, and T the time of vol.
In addition, an ion of load Z subjected to an accelerating tension V gains a kinetic energy

E_c = Z \ cdot E \ cdot V

E being the Elementary charge.

One from of deduced thus $\ frac {m} {Z} = 2 \ cdot E \ cdot V \ cdot \ frac {t^2} {l^2}$

The report/ratio masses on load is directly measurable as from the time of vol.

Operation

An analyzer in time of flight is composed of a zone of acceleration where is applied the accelerating tension, and of a zone called tube of flight, free of field.
Les accelerated ions penetrates in the tube of coasting flight of any field. The separation of the ions thus will depend only on the speed acquired at the time of the phase of acceleration. The ions of the smallest report/ratio m/z will arrive at the detector the first. For each group of of the same ions report/ratio m/z , a signal is recorded on the level of the detector in the form of a function time/intensity.
Ce mode of detection comprises however certain limitations in term of resolution: thus two identical, of the same initial speed, but localized ions at two different points, will enter the tube of flight at different speeds and times. That more far from the detector in the beginning will be accelerated longer and will thus have a time of shorter flight, from where a dispersion in time and energy. The réflectron mode makes it possible to mitigate this phenomenon.

En mode réflectron, an electrostatic mirror imposes an electric field of direction opposed to that of the initial accelerating field, and thus of the movement of the ions. The latter thus see their modified trajectory: they penetrate in the réflectron and come out from it with a longitudinal speed of direction opposed at their initial speed. The energy ions arrive the first at the level of the réflectron and will penetrate there more deeply, they will be thus considered in a longer time. In this way, all the of the same ions report/ratio m/z are focused on the same plan, the detector of the réflectron being placed within focusing of these ions.
Moreover, the réflectron makes it possible to lengthen the distance from flight without to increase the size of the analyzer: the ions spend more time to reach the detector, and reduce also their dispersion in time, the resolution is some thus largely improved.

The FT-ICR

The analyzer with cyclotronic resonance of ion is composed of a cell ICR (of cubic configuration for example) which comprises in particular six plates under tension, isolated ones from the others. The application of a magnetostatic field B following axis Z subjects the ions to the force of Lorentz F = ez v ^ B. Their movement in the plan (xy) is then “cyclotronic”, i.e. circular uniform of frequency f= eB/(2π.m/z). The ions are in addition confined along axis Z by an electrostatic field imposed by the two plates parallel with the plan (Oxy), resulting from the application of a weak tension.
Once trapped in the cell, the ions thus have the same trajectory but not the same position at one given moment. It is thus advisable to give to the of the same ions m/z an overall movement by putting them in phase, by cyclotronic resonance. For that, the ions m/z are excited by an alternate field of frequency corresponding to their frequency cyclotron: to excite all the ions of a certain range of m/z, a tension containing all the corresponding frequencies cyclotron is forced. The ions then are accelerated, put in phase and see the ray of their orbit increasing.
Le running induced by the coherent movement of the of the same ions m/z will be measured on the plates of detections: it will be a sinusoid deadened of cyclotronic frequency. The current induces total measured will be thus the sum of sinusoids deadened of the cyclotronic frequencies corresponding to the ions of m/z excited by resonance. The frequency cyclotron being proportional to 1 (m/z), the reverse of the transform of Fourier of the current obtained makes it possible to lead to the spectrum of mass in m/z.

Cet analyzer with the one of the best resolutions which are (Rs>100 000), consequently spectrum ms has a greater capacity of peaks, which maximizes the quantity of information for the analysis of complex mixtures. However, the width of the peaks being proportional to (m/z) ², the resolution is better with the m/z lower than 5000 Th. The excellent precision of the FT-ICR to the measure of mass (5-10 ppm) raises or decreases ambiguities on the identification of the compounds. The range of mass depends on the value of the magnetic field, it extends until 27000 Da for a field from 7 T. On the other hand, the dynamic range is restricted enough, with 2-3 decades, because this analyzer by containment suffers from the same defect as the quadripolar trap, the possible coexistence of a limited number of ions. Consequently, the very minority peaks in the spectrum of mass will present a measurement of less precise mass. The FT-ICR even allows the analysis in MS/MS in the cell, with varied possibilities of activation of the selective ions and thus of fragmentation.

The orbitrappe

The orbitrappe is composed of a hollow electrode, inside which coaxialement an electrode in the shape of spindle is placed. The particular shape of these two electrodes allows the imposition of an electrostatic field quadro-logarithmic curve with the tension: U (R, Z) = k/2 (Z ² - R ² /2) + k/2 Rm ² ln (r/Rm) +C.
avec Rm ray characteristic of the central electrode, K curve of the field, and C a constant.
Le field is in particular quadripolar along axis Z of the electrodes. The ions are injected tangentially with the central electrode and are trapped around it by the electrostatic force which compensates for the centrifugal forces. The movement of the ions breaks up then as follows: a circular motion around the central electrode in the plan (xy) and an oscillatory movement of to and from according to axis Z. In particular, the ions of a given m/z will be on the same circular trajectory which oscillates axially with a frequency F. F is independent speed or of the energy of the ions and expresses itself like 1/2π√ (km/z). In the same way that for the FT-ICR, the current induced by these oscillations allows by a transform of Fourier to reach the m/z.

La precision of measurements of m/z is particularly good (1-2 ppm) and the resolution (until 100.000) competes with that of the FT-ICR, the more so as being proportional to 1/√ (m/z), it decreases less quickly with the m/z than in the case of the FT-ICR. The dynamic range is satisfactory (> 3 decades). The orbitrappe is mainly used in mass spectrometry out of tandem, associated with a linear trap

The analyzer with magnetic sector

The ion is ejected in a medium in which reign a uniform magnetic field perpendicular to the plan of the trajectory. Because of Force of Lorentz, the trajectory is curved, and the point of impact of the ion (thus its deviation) makes it possible to know its mass starting from the load.

Indeed, that is to say $\ vec B \,$ the magnetic field (leader $\ overrightarrow {OZ}$ ) of coordinates $\ begin {pmatrix} 0 \ \ 0 \ \ B \ end {pmatrix}$ and $\ vec v_0 \,$ orthogonal initial speed with $\ vec B \,$ , it directs $\ overrightarrow {OX}$ .

One has then: $q \ vec v \ wedge \ vec B = \ begin {pmatrix} qB \ dowry there \ \ - qB \ dowry X \ \ 0 \ end {pmatrix}$ .

From where, by writing the fundamental relation of dynamics: $\ left \ {\ begin {matrix} m \ ddot X =qB \ dowry there \ \ m \ ddot there = - qB \ dowry X \ end {matrix} \ right.$ .

That is to say: $\ left \ {\ begin {matrix} \ ddot X - \ omega_0 \ dowry there =0 \ \ \ ddot there + \ omega_0 \ dowry x=0 \ end {matrix} \ right.$ where $\ omega_0= \ frac {qB} {m}$ .

Let us pose $\ tilde V= \ dowry X + I \ dowry y$ . There is then $\ dowry \ tilde V + I \ omega_0 \ V=0$ tilde.

While solving, $\ tilde V (T) =v_0 e^ {- I \ omega_0t} =v_0cos (\ omega_0t) - v_0isin (\ omega_0t)$ .

And thus: $\ left \ {\ begin {matrix} \ dowry X (T) =v_0cos (\ omega_0t) \ \ \ dowry there (T) =-v_0sin (\ omega_0t) \ end {matrix} \ right.$ $\ left \ {\ begin {matrix} X (T) = \ frac {v_0} {\ omega_0} sin (\ omega_0t) \ \ there (T) = \ frac {v_0} {\ omega_0} cos (\ omega_0t) - \ frac {mv_0} {qB} \ end {matrix} \ right.$ (using the initial conditions).

It is indeed the parametric equation of a circle of radius $R_c= \ frac {mv_0} {\ left|qB \ right|}$ .

The spectrometer measures then the distances from impact when the particle carried out a half-circle. The distance to the point of origin thus corresponds to the diameter with the double of the ray given by the last formula. The load of the particle thus makes it possible to deduce its Masse from it.

The mass spectrometry out of tandem (MS/MS)

The mass spectrometry out of tandem consists in selecting an ion by the first mass spectrometry, splitting up it, then to carry out the second mass spectrometry on the fragments thus generated.
Elle can be realized using many apparatuses combining of the magnetic, electric, quadripolar sectors or of times of flight, but also within the same analyzer in the case of a trap door of ions.

Triple quadripole

Triple quadripole results from the association of two quadripolar analyzers in series, separated by a cell from collision often consisted of a shorter quadripole. This combination of quadripoles makes it possible to work in simple ms or tandem. To carry out an acquisition in ms, it is enough to apply only one alternating voltage to the one of the analyzers to return it " transparent" like the cell of collision, this one then not containing a gas.
Lors of an acquisition in MS/MS, the cell of collision is filled with an inert gas (argon for example) under a relatively high pressure ( $10^ {- 2}$ torr). The kinetic energy of the selected ion is converted at the time of its successive collisions into internal energy. The dissociation of the ion will be carried out when its energy interns will have become higher than the energy of activation necessary to fragmentation. This technique of dissociation activated by collision (CAD) can be amplified by increasing the kinetic energy of the ions selected by application of a potential difference between the source and the cell of collision.
L' analyzes MS/MS can be carried out according to four different modes according to required information: the mode going down more is used to obtain structural information, the two modes (ascending and loss of neutral) are of a more restricted use and make it possible to highlight ions having common characteristics. Fourth mode (Multiple Reaction Monitoring or MRM), derived from the mode going down, is dedicated to the quantification.

in downward mode , the ion studied is selected by focusing the first analyzer on his report/ratio m/z . The fragments formed in the cell of collision are separated by the second analyzer and are analyzed. The spectrum obtained presents at the same time the precursory ion (or ion relative) and its ions fragments (or produced ions).
in ascending mode , the first analyzer sweeps a range of mass while second is focused on only one report/ratio m/z . All the ions generated in source and able to give a of the same fragment report/ratio m/z thus will be thus detected.
in loss mode of neutral , the two analyzers simultaneously sweep a range of mass and with a constant shift of mass. The established spectrum will present then all the ions parents able to split up by generating a neutral of mass equal to the imposed shift.
in mode MRM , the ion relative studied is selected by the first analyzer and is split up in the cell of collision, as in downward mode. On the other hand, the second analyzer is focused on the produced ion. This operating process has a double selectivity, on the level of the selections of the ion relative and produced ion. Moreover the two analyzers being fixed at constant tensions, the sensitivity of detection is improved compared to other modes of sweeping, making to MRM a mode of choice for the quantification.

$MS^n$ out of quadripolar trap (" trap door of ions")

Within a quadripolar trap (" trap door of ions"), the analysis out of tandem is carried out initially by selection of ions whose value m/z is selected. These trapped ions then will split up by collision (internal acquisition of energy, vibrationnelle excitation) using a tension RF (radio frequency) correspondent at their frequency of resonance, and the formed produced ions are in their turn trapped. A selective ejection in mass of the produced ions (fragments) can then be realized for their analysis.
L' obtaining ions of higher generations is possible by simple renewal of the process (selection of a produced ion, fragmentation, selection of a produced ion of 2nd generation, fragmentation, etc…). This sequence is called $MS^n$ , N being the number of generations of ions. Thus the $MS^2$ is the MS-MS and so on…

Tof Tof

Hybrid: Tof quadripole

These apparatuses make it possible to combine the strong points of the analyzers quadripoles and Tofs. It consist of a double quadripole (1st analyzer and cell of collision) and of an analyzer in time of flight like second analyzer. The advantages compared to triple quad are a better sensitivity and a better resolution.

See too

Biophysics
Spectrometry
IA-Farmhouse

External bonds

ProtéoWiki - Gate of mass spectrometry: Encyclopedia of proteomic
Course of Olivier Laprévote in mass spectrometry
Seen relevant sites on the mass Spectrometry (English)

Random links:

Mass spectrometry

Structure of a mass spectrometer

For what is used the mass spectrometry?

The source of ionization

Electronic ionization (I.E.(internal excitation))

Chemical ionization

The électrospray

The APCI

The MALDI

Principle

The matrix

The analyzer

The quadripolar analyzer

The quadripolar ionic trap

The time of flight

Principle

Operation

The FT-ICR

The orbitrappe

The analyzer with magnetic sector

The mass spectrometry out of tandem (MS/MS)

Triple quadripole

MS^n out of quadripolar trap (" trap door of ions")

Tof Tof

Hybrid: Tof quadripole

See too

External bonds

$MS^n$ out of quadripolar trap (" trap door of ions")