Why Mass Spectrometry: An

argumentwildlifeUrban and Civil

Nov 16, 2013 (3 years and 9 months ago)

87 views

Why Mass Spectrometry: An
Introduction to the IU MSF

Jonathan A. Karty, Ph.D.

jkarty@indiana.edu

http://msf.chem.indiana.edu

Why Mass Spectrometry


Information is composition
-
specific


Very selective analytical technique


Most other
spectroscopies

can describe
functionalities, but not chemical formulae


MS is VERY sensitive


mg/L to
ng
/L sensitivity possible


Picomole

sensitivity is common in the MSF


Mass spectrometers have become MUCH
easier to use in the last 15 years

Three Questions


Did I make my compound?


Molecular weight is an intrinsic property of a substance



Did I make anything else?


Mass spectrometry is readily coupled to chromatographic
techniques



How much of it did I make?


Response in the mass spectrometer is proportional to
analyte concentration (R =
α
[M])


Each compound has a unique response factor,
α

Common MS Applications


Quick product identification (TLC spot)


Confirmation of elemental composition


Much more precise then EA


Selective detector for GC/HPLC


MS provides retention time AND molecular weight
information about each
analyte


Reaction monitoring


Crude reaction mixture MS


Stable isotope labeling


Stability studies

Important Concepts to Remember


Mass spectrometers analyze gas
-
phase ions, not
neutral molecules


Neutral molecules don’t respond to electromagnetic fields


If you cannot make a stable ion, MS is impossible



MS is not a “magic bullet” technique


MS can tell you composition of an ion (
C
x
H
y
O
z
)


Connectivity of the atoms in that ion is much more
challenging



Units


1
Da

= 1 u = 1.6605*10
-
27

kg (1/12 of a
12
C atom)


1
Th

= 1
Da
/e = 1.0364*10
-
8

kg/C

Molecular Weight Calculations


The molecular weight is computed by summing the
masses of all atoms in the compound/ion.


Erythromycin (M+H)
+
: C
37
H
68
N
1
O
13
+

= 12.011*37 + 1.008*67 +
14.007 + 15.999*13 =
734.93

Da



Yet
734.5

is observed by ESI
-
MS

Isotopic Distributions


Isotopes: same number of protons, different numbers
of neutrons


12
C has 6 of each,
13
C has 6 protons and 7 neutrons


Periodic table assumes a natural distribution of stable
isotopes (weighted average)



Carbon isotopes


C


12
C is 98.9% abundant,
13
C is 1.1% abundant


(0.989 * 12.0000) + (0.011 * 13.0034) =
12.011


For C
40
: 64.2%
13
C
0
, 28.6%
13
C
1
, 6.2%
13
C
2


Spectrum looks like 100% @ 480, 44.5% @ 481, 9.6% @ 482



Many elements have a variety of isotopes


Sn

has 7 naturally occurring isotopes


F, P, Na, I, Co, Au have only 1 natural isotope


Unregistered
485
484
483
482
481
480
479
100
90
80
70
60
50
40
30
20
10
0
C
40

mass spectrum

Monoisotopic

Masses


Monoisotopic

masses are considered for mass
spectrometry


Monoisotopic

masses are computed using the
most
abundant

isotope of each element (
12
C,
35
Cl,
14
N,
16
O,
79
Br,
11
B,
120
Sn etc)



For erythromycin,
monoisotopic

mass = 734.468


12
C
37
1
H
68
14
N
1
16
O
13


12.000 * 37 + 1.0078 * 68 + 14.0031 + 15.9949 * 13



Remember to include any ionizing reagent


Electron loss, proton addition, etc.



C
37
H
68
NO
13
+

Mass Spectrum

Average mass =
734.93

u

13
C
0
,
2
H
0
,
18
O
0

13
C
1
,
2
H
0
,
18
O
0

13
C
2
,
2
H
0
,
18
O
0

13
C
0
,
2
H
0
,
18
O
1

13
C
1
,
2
H
1
,
18
O
0

Observed isotope pattern is the convolution of
isotope patterns for all atoms

Isotopic Envelopes


Isotopic distributions can indicate/preclude the
presence of an element


Cl

has a unique 3:1 pattern (M:M+2)


B has a unique 1:4 pattern (M
-
1:M)


M+1 / M+ ratio can be used to count carbon
atoms in a molecule


[(M+1) / M+] / 0.011
≈ # carbon atoms


For morphine: (0.1901 / 1) / 0.011 = 17.28


17


Significant deviation from expected isotope pattern
should be a warning sign


Multiple compounds with similar masses in sample


Partial isotopic enrichment (e.g.
deuterated

solvents used)


Multiple ionization mechanisms at work


Intensity (%)
0
20
40
60
80
100
Mass [amu]
362
364
366
368
370
372
374
376
378
Intensity (%)
0
20
40
60
80
100
Mass [amu]
131
132
133
134
135
136
137
138
139
Two Complex Isotope Patterns

C
2
H
3
Cl
3

trichloroethane

C
12
H
27
SnBr

tributyltin

bromide

Monoisotopic

mass: 131.93

Average mass:
133.43

Monoisotopic

mass: 370.03

Average
mass: 369.96

Instrumentation in the MSF


One of 3 mass spectrometry facilities in the
department


MSF is in Chemistry A411 and A454


1 GC
-
EI
-
Q
-
MS (A454)


1 LC
-
ESI/APCI
-
Q
-
MS (A454)


2 LC
-
ESI
-
TOF
-
MS (A411)


1 MALDI
-
TOF
-
MS (A411)


1 EI/CI
-
BE
-
MS (A411, staff only)


Self
-
run experiments are $7
-
$10 per sample


Staff
-
run experiments are $16
-
$30


Training for Walk
-
Up MS will start after 11/7/10

Agilent 6890/5973 GC
-
MS


6890 GC


30 m long DB
-
5 (non
-
polar) column installed


Helium mobile phase


Split/
splitless

injector
with
autosampler


5973 MS


Electron ionization


Quadrupole

MS (10
-
800)


NIST 02 library installed

Agilent 1200/6130 LC
-
MS


1200 HPLC


Binary pump


0.05
-
2
mL
/min


Autosampler

with 6
-
position column selector


Diode array UV
-
VIS
detector


6130 MS


Dual mode
electrospray
/APCI source


Can perform all 4 modes of
ionization in 1 experiment


50
-
3,000 m/z
quadrupole


Easy Access Software

Waters
CapLC
-
LCT LC
-
MS


CapLC


Flow rates 1
-
40
uL
/min


C18, C8, and C4 columns


Single wavelength UV
-
VIS
detector


LCT


Electrospray

ionization


Time
-
of
-
flight MS (TOF)


100
-
6,000 m/z


Capable of accurate mass
spectrometry


5
ppm

error for formula
confirmation

Bruker

Autoflex

III


MALDI
-
TOF mass
spectrometer


200
-
150,000 m/z


Can make both positive
and negative ions from
same spot


Easy to interpret mass
spectra for large
polymers


Matrices available for
wide array of
analytes

Thermo MAT
-
95XP


Magnetic sector mass
spectrometer


Accurate mass spectrometry
is its main function


Electron ionization and
Chemical ionization sources


Trace GC available for low
MW species or mixture
analysis

Accurate Mass Spectrometry


Accurate mass spectrometry can unambiguously
confirm chemical composition



Mass accuracy is often reported as a relative value


ppm

= parts per million, 1
ppm

= 0.0001%



5
ppm

error is used by many journals as a standard
to confirm a chemical formula


Instrumentation in the IU MSF routinely achieves this
level of accuracy


5
ppm

at mass 300: 300 * (5/10
6
) =
±
0.0015
Da


Formula Matching Basics


Atomic weights are not integers (except
12
C)


14
N = 14.0031
Da
;
1
H = 1.0078
Da


16
O = 15.9949
Da
;
127
I = 126.9045
Da


Table of isotopes link on MSF website



Difference from integer mass is called “mass defect”


Related to nuclear binding energy (E = mc
2
)



Sum of the mass defects depends on formula


H, N increase mass defect


Eicosane

(C
20
H
42
) =
282.3286



O,
Cl
, F, Na decrease it


Morphine (C
17
H
19
NO
3
) =
285.1365


More Formula Matching


Accurate mass measurements narrow down possible
formulas for a given molecular weight


534

entries in NIST’08 library @ mass 285


Only
3

formulas within 5
ppm

of 285.1365


46

compounds with formula C
17
H
19
NO
3



Mass spectrum and user info complete the picture


Isotope distributions indicate / eliminate elements


User
-

supplied info eliminates others (e.g. no F)


Suggested formula has to make chemical sense

Formula Matching Example

Only 9 ways to combine up to 40 C, 50 H, 5 N, 5 O, and 2 Cl to get a mass within
20 ppm (0.0061 u) of 306.0820, only 3 have 2 Cl

Elemental Composition Report
Tolerance = 20.0 PPM / DBE: min = -1.5, max = 50.0
Selected filters: None
Monoisotopic Mass, Even Electron Ions
370 formulas evaluated with 9 results within limits
Elements Used:
C: 0-40 H: 0-50 N: 0-5 O: 0-5 Cl: 0-2
Error
20
ppm



Mass
intensity
Calc. Mass
mDa
PPM
i-FIT
306.082
100
306.0816
0.4
1.3
39.7
C17 H18 N Cl2


306.0776
4.4
14.4
376
C12 H18 N3 O2 Cl2


306.0875
-5.5
-18
701.7
C10 H22 N O5 Cl2


306.0798
2.2
7.2
1945.8
C18 H13 N3 Cl


306.0857
-3.7
-12.1
2205.2
C11 H17 N3 O5 Cl


306.0766
5.4
17.6
9102.8
C18 H12 N O4


306.078
4
13.1
9195.6
C19 H8 N5


306.0879
-5.9
-19.3
9289.5
C17 H12 N3 O3


306.0838
-1.8
-5.9
9543.2
C12 H12 N5 O5
error in:
Formula
Zoloft C
17
H
18
Cl
2
N

Accurate MS Notes


Accurate MS is possible with the MAT
-
95 and
LCT mass spectrometers


Bruker

MALDI
-
TOF can do 20
ppm

mass accuracy


Only MSF staff can perform accurate mass
measurements


All accurate MS submissions MUST include a
nominal (low res) mass spectrum to
demonstrate purity PRIOR to accurate MS


Sample LCT Accurate Mass Report

Sample MAT
-
95 Accurate Mass Report

Upcoming Lectures


All in C033 from 5:30
-
6:15


Small molecule EI and GC
-
MS (10/14)


Small molecule ESI/APCI and LC
-
MS (10/18)


Biomolecule
/polymer analysis by ESI and MALDI
(10/20)


Possible special interest seminars in November


Quantitation

and other advanced topics for small
molecule MS


Intro to Bioinformatics


These will only occur if sufficient interest is expressed


For a more in
-
depth treatment of all topics covered,
take C613 in the spring!