BCH 4028

Instrumental Analytical Chemistry

Liquid chromatography
& Mass spectrometry

These notes are

from Agilent
Literature
 LC/MS
LC MS
provide information about the
high molecular high detection
resolving weight, structure,
power specificity
identity & quantity of
specific sample components
 Sample types

 sample types range from small

pharmaceutical compounds to large proteins
 suitable for the
analysis of large,
polar ionic, thermally
unstable & involatile
compounds
Sample types
 Selectivity & Sensitivity

High
selectively
Detect High
compounds sensitivity
of interest
in a complex Detect only
matrix those masses
characteristic
of the
compounds
being
monitored
 Interfacing LC & MS

LC MS

Pressure High pressure to Vacuum

separate a liq. phase Limited gas
Produces a high gas load load
Flow eq. to 1 L/min gas 1 mL/min
Temp. Near ambient temp. Elevated temp.
Mass range No Yes
limitation

Buffer Inorganic buffers Prefers volatile

buffers
 API-Electrospray

Analyze samples that become

multiply charged: proteins, peptides &
oligonucleotides
singly charged: benzodiazepines & sulfated
conjugates

Process:
nebulization Ion
& charging desolvation
evaporation
http://www.chm.bris.ac.uk/ms/theory/esi-ionisation.html
 API-Electrospray

charged droplets shrinks droplets

carries away uncharged
species
 Atmospheric pressure chemical ionization: APCI

Applicable to a wide range of polar &

nonpolar analytes that have moderate
molecular weights

Process:
nebulization &
desolvation ionization
nebulization occurs in a by corona
hot(typically 250 oC–400 oC) discharge
vaporizer chamber to
evaporate droplets
APCI
 Scan vs. SIM

Scan
– detects signals over a mass range
 max. qualitative info.

Selective Ion Monitoring

– only monitor a few m/z ratios
 max.
quantitative info.
Scan vs. SIM
 CID

MS/MS is accomplished by a process

called collision-induced dissociation in
which ions break apart as a result of
collisions with other molecules

EI can produce CID by using higher

voltages on the electrode
 CID

single ion can be selected

and fragmented
 Adapting LC methods

 Orthogonal spray ion source is very

tolerant of non-volatile components in
samples or mobile phase
 The flow is directed orthogonal to the lens
axis & ions are focused into the mass filter.
 Orthogonal spray orientation & high-
capacity drying gas system improve
instrument performance &  maintenance
requirements.
Adapting LC
methods: orthogonal
spray
 Applications

 Molecular weight determination

 Combinatorial chemistry
 Pharmaceutical applications
 Biochemical applications

Clinical applications
 Food applications
 Environmental applications
Molecular weight determination Applications

Examples

C-terminus: threonine vs. threonine amide

 API-ES

Determining molecular weight of green

fluorescent protein by API-ES

Between m/z 1000 and 1300 there are

6 peaks. Two of these adjacent peaks
are at 1118.85 and 1167.55.
What is the parent ion mass?
Pharmaceutical applications Applications

peaks unresolved
Environmental applications Applications

have 1 ring and

differ by 1 Cl

has 2 Cl and
a 2nd ring
attached to
the first by
O
 Further
reading
http://www.chem.agilent.com/scripts/Li
teratureresults.asp?iprodinfotype=4&ip
rodgroup=8

Videos
http://www.chem.agilent.com/Scripts/
Generic.ASP?lPage=8683&indcol=N&pro
dcol=Y