Instrumental Analysis
LEC # 16
**Mass spectroscopy is for detecting the molecular weight/mass of the
analyte by 3 steps:
1) Evaporation of analyte: by heat, vacuum, steering by N. gas or all
of these together.
2) Ionization of analyte : to give a positive charge to an analyte and
this happen by two ways >>>
a) Hard Ionization: compartment (‫ (تقسيم‬of analyte by high energy
electrons which lead to fragmentation of analyte and this
process called electron impact. (fragmentation could be not
100% )
b) Soft ionization: fragmentation here may or may not occur (not
essential) .
3) Analysis: the analyzer knows the molecular weight of the analyte
/fragment. Molecular Ion: is the analyte with a +ve charge and a
free radical.
** Types of ionization:
1) Electron impact: the filament produces electrons which are
accelerated towards the anode (positively charged), the vapor
sample will face these electrons and compartment occurs to
produce a molecular ion. Fragmentation here may occur
- If our vapor sample was ethanol >>>
2) Electron spray ionization: this type is a soft ionization method and
it’s used in the mass spectroscopy that used in HPLC because this
type of ionization deal with organic solvents ( related to the mobile
phase in HPLC)
Method: when the mobile phase /solvent enter the detector a highly
positively charged cone (high voltage of 1.5 K Volt) will charge the
solvent positively. The charged solvent will come out from taylor cone
(which has a tip with an hole in micro meter scale) in shape of droplets
under pressure (spraying) , then evaporation of the solvent droplets
occur by heat , vacuum and steering by N. gas , the condensation of
these little +ve charged solvent molecules occur ( accumulate on each
other) which lead to a strong repulsion interaction which cause a further
fragmentation into smaller droplets so the rate of evaporation will
increase (because of the increase of the surface area) >>>>> this will
continue happening until all the mobile phase/solvent vaporized and
leave the positive charge to the analyte to become a molecular ion ,
these analytes will be accelerated towards the highly negative charged
cathode.
** The drug will take the charge according to the functional group it
contains (when the mobile phase evaporates will give the positive charge
to the electronegative group – if it has one amine group >>>> it will take
one positive charge).
3) Matrix assisted laser desorption ionization (MALDI) :
-It is used for detecting large molecules (peptides).
-It is composed of two components: matrix (major) and analyte (minor)
on a disk when the matrix is exposed to a laser with a certain wave
length (lambda max of the matrix) which causes the excitation and
ionization of it (become +ve) >>> desorption occur between matrix and
analyte and both of them will have supersonic acceleration towards the
detector this acceleration will make these two components to collide
with each other to give the analyte a positive charge.
**Analyzer: it is used in detecting the mass of analyte/fragments.
- Quadruple: a type of analyzer , that has four rods two of them
connected to the DC current, while the other two are connected to
the AC current with a radiofrequency energy, two of them are
anodes and the other two are cathodes.
When the ions from ionization chamber comes to this analyzer it will
exposed to two repulsion and two attraction forces from the rods, this
will cause the bioscillation movement )‫ (حركة لولبية بين األقطاب‬.
We can control the electrical field range to allow the analyte with a
certain molecular weight to reach the detector than the other components
(like the non resonant ion in the figure).this will cause the neutralization
of the other components and the disappearance of them.
The detector detects the molecular mass depending on the analyte’s
speed and time of analyte spent in the quadruple.
*high resolution mass spectroscopy HRMS: is a very sensitive detector,
can calculate the mass to 4 digits
*Molecular weight of the fragment is lower than the analyte’s molecular
weight.
*detector signal is directly proportional with the concentration of
analyte.
__________________________________________________________
**fragmentation types:
1) Homolytic and Heterolytic α-cleavage
Homolytic α-cleavage:
- The oxygen atom is electronegative atom with +ve charge, so it
will take electrons to be neutralized either from the α bond of
H(first one) or from α bond of CH3(second one).
The product in the second rxn is major than the first one because the
radical in the second rxn is more stable than the first one.
Heterolytic α-cleavage:
Here the oxygen will take the 2 electron (not only one electron like
homolytic cleavage) from the adjacent bond.
2) Cleavage with proton transfer:
Firstly, the oxygen will take the electrons from the α bond of R
(homolytic cleavage, R leave as radical), but here the oxygen will also
take another electron from H (we can’t predict which proton the O+
will take).
- In mass spectra:
It is the relation between mass to
charge ratio with the relative
abundance (intensity).
 Mass spectrum of ethanol:
- Base peek: highest relative abundance (highest intensity)
(from homolytic cleavage with the largest radicle).
- CH3CH2+ (from heterolytic cleavage).
- CH3CHO+H (from homolytic cleavage, when the proton leave as
radical).
- Molecular ion peek: is the highest molecular weight, for the
analyte without fragmentation (M+., ethanol).
So, from the spectra we can know the molecular weight,
the fragment we have, predict how the fragmentation happen,
and predict the analyte we have.
 General rules:
1. Compounds containing only C, H, O, with any type of charge
will always have even molecular weight (MWt).
2. Compounds having CHO & a halogen atom X will also have an even
MWt, because the difference between H & X will be even.
3. Compounds containing C, H, O & one atom of N will always have an
odd number of hydrogens & odd MWt.
4. Even number of nitrogens will mean even MWt and hydrogen atoms
number.
5. Compounds formed entirely from elements of monoisotopic nature
will show intense M+• plus minute peaks at M+1 & M+2, due to minor
isotopes of higher mass.
•Br, Cl, S & Si have 2 or more isotopes with > 1% relative abundance.
Br has two isotopes, one with 79 MWT and the other with 81 MWT.
(With relative abundance of 1:1 in nature).
- How could I know if the compound has Br??
In spectra it appears as molecular ion beside it M++2 with two
molecular weight difference.
Done by: Anood Saleh.