# Why Mass Spectrometry: An

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Nov 16, 2013 (4 years and 5 months ago)

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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
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

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

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

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!