Chromatography lc ms

Liquid Chromatography Mass Spectrometry LC-MS

1. Prepared by :-
Mohammed H. Rida &……………….

2. •History:
•Classification based on Mobile Phase:
•The Basic Liquid Chromatograph units :
•combination of LC and MS , LC-MS Instrumentation

• LC-MS applications:
OUTLINE

3. It was Mikhail Tswett, a Russian botanist, in 1903
who first invented and named liquid
chromatography.
Tswett used a glass column filled with finely divided
(calcium carbonate) to separate plant pigments. He
observed the separation of colored zones or bands
along the column.
The development of chromatography was slow and
scientists waited to early fifties for the first
chromatographic instrument to appear in the market
(a gas chromatograph).
liquid chromatographic equipment with acceptable
performance was only introduced about two decades
after gas chromatography.

4. CHROMATOGRAPHY
The separation of a mixture by distribution of its
components between a mobile and stationary
phase over time.
mobile phase = solvent (also called eluent)
penetrates or passes through a solid or
immiscible stationary phase
stationary phase = column packing material
-In a chromatographic separation of any type,
different components of a sample are transported
in a mobile phase (a gas, a liquid, or a
supercritical fluid).

5. Since the stationary phase is the fixed one then those solutes which
have:-
 stronger interactions with the stationary phase will tend to move slower
(have higher retention times” The time a solute spends in a column”)
than others which have lower or no interactions with the stationary phase
will tend to move faster.
chromatographic separations are a consequence of differential migration
of solutes.
maximum interactions between a solute and a stationary
phase take place when both have similar characteristics, for example in
terms of polarity.
when their properties are so different, a solute will not tend to stay and
interact with the stationary phase and will thus prefer to stay in the
mobile phase
and move faster; “a polar solvent and a non polar stationary phase”

6. According to the nature of the mobile phase,
chromatographic techniques can be :-
* classified into three classes:
a. Liquid chromatography (LC)
b. Gas chromatography (GC)
c. Supercritical fluid chromatography (SFC)

7. General classification Specific method Stationary phaseType of equilibrium
Liquid Chromatography(LC)
“Mobile phase: liquid”
Liquid-liquid or
partition
Liquid adsorbed on a
solid
Partition between
immiscible
Liquid- bonded
phase
Organic species
bonded to
a Solid surface
Partition between
liquid and
Bonded surface
Liquid -solid or
adsorption
solid Adsorption

8. A small volume of the sample is first introduced at the top
of the chromatographic column. Elution involves :-
passing a mobile phase inside the column whereby solutes
are carried down the stream but on a differential scale due to
interactions with the stationary phase.
As the mobile phase continues to flow, solutes continue to
move downward the column.
Distances between solute bands become greater with time
and as solutes start to leave
the column they are sequentially detected. The following
schematics represent the process at various times:

9. The dark colors at the center of the solute zones in the
above figure represent higher concentrations than are
concentrations at the sides .
This can be represented schematically as:

10. Chromatograms
The plot of detector signal versus retention time of solutes in a
chromatographic column is referred to as a chromatogram.
The areas under the peaks in a chromatogram are usually
related to solute concentration “quantitative analysis”.
The retention time of a solute is a characteristic property
of the solute which reflects its degree of interaction with both stationary and
mobile phases. Retention times serve “qualitative analysis”
parameters to identify solutes.

14. The mobile phase supply system consists of number
of reservoirs (200 ml to 1,000 ml in capacity).
The Mobile Phase Supply System

15. The Gradient Programmer and the LC Pump
-the solvent mixing occurs at high pressure,and then passed to the pump-the solvent mixing occurs at high pressure,and then passed to the pump
-is the simplest but most expensive.-is the simplest but most expensive.
-each solvent requires its own pump.-each solvent requires its own pump.

16. solvents are premixed at low pressure and then passed to the pump. solvents are premixed at low pressure and then passed to the pump.

17. The LC Pump
-pneumatic pump,
The pneumaticpump can provide extremely high
pressures and is relatively inexpensive,
-the total air pressure on the piston, diameter (y), is transferred to
a piston controlling the liquid pressure,
of diameter (x).
Because the radii of the pistons differ, there will be a
net pressure amplification of

18. The Sample Valve
- liquid samples are usually injected onto the column by a
syringe via a injector.
- Sample are placed on an LC column directly with either an
internal or external loop sample valve the valve being connected
directly to the column.

20. Column Ovens:
The effect of temperature on LC separations is often not nearly so
profound as its effect in GC separations, but can be critical when closely
similar substances are being separated.
Due to the lesser effect of temperature on solute retentionin LC (compared
to that in GC), temperature is not
nearly so critical in governing absolute retention time but is often
essential in achieving adequate resolution, particularly between
closely eluting solutes such as isomers.

21. Detectors
•UV Detector
•Fixed Wavelength Detector
•Multi-Wavelength Detectors
•Diode Array Detector
•Electrical Conductivity Detector
•Fluorescence Detector
•Refractive Index Detector
• Tridet Multi Functional Detector

22. The Tridet Multi Functional Detector
trifunctional detector
that detected solutes by
the UV detector, the
electrical conductivity
detector and the
fluorescence detector
simultaneously in a
single low volume cell.

24. * combination of LC and MS offers the possibility to take advantage of
both LC as a powerful and versatile separation technique and MS as a
powerful and sensitive detection and identification technique.
a mass spectrometer
is more sensitive and far more specific than all other LC detectors.
-It can analyze compounds that lack a suitable chromophore.
-It can also identify components in unresolved chromatographic
peaks, reducing the need for perfect chromatography.
Mass spectral data complements data fromother LC detectors

25. Two-dimensional abundance data
and three-dimensional mass spectral data from a mass spectrometer

27. Instrumentation
Mass spectrometers work by :-
1- ionizing molecules
2- sorting and identifying the ions according to their mass-to-
charge (m/z) ratios.

28. Ion Sources
- ionize the analyte molecules and separate the
resultingions from the mobile phase.
ionization techniques are:
• 1a- Electro spray ionization (ESI)
• 1b- Atmospheric pressure chemical ionization (APCI)
• 1c- Atmospheric pressure photo ionization (APPI)

29. Figure. Applications of various LC/MS ionization techniques

30. 1a- Electro Spray Ionization (ESI)
- to generate analyte ions in solution before the analyte
reaches the mass spectrometer.
* The LC eluent is sprayed (nebulized) into a chamber at atmospheric
pressure in the presence of a strong electrostatic field and heated
drying gas.
* The electrostatic field causes further dissociation of the
analyte molecules
* The heated drying gas causes the solvent in the
droplets to evaporate.
*Electrospray is especially useful for analyzing large bio molecules
such as proteins, peptides,and oligonucleotides

31. Figure. Electrospray ion source

32. 1b- Atmospheric pressure Chemical Ionization (APCI)
•- In APCI, the LC eluent is sprayed through a heated (typically
250°C –400°C) vaporizer at atmospheric pressure.
•-The heat vaporizes the liquid.
•-The resulting gas-phase solvent molecules are ionized by
electrons discharged from a corona
needle.
•-The solvent ions then transfer charge to the analyte
molecules through chemical reactions (chemical ionization).
• -The analyte ions pass through a capillary sampling orifice
into the mass analyzer.

33. Figure 6. APCI ion source

34. 1c- Atmospheric pressure photo ionization (APPI)
• Atmospheric pressure photo Ionization (APPI)
for LC/MS is a relatively new technique. As in APCI, a
vaporizer converts the LC eluent to the gas phase.
• A discharge lamp generates photons in a narrow range of
ionization energies.
• The range of energies is carefully chosen to ionize as
many analyte molecules as possible while minimizing the
ionization of solvent molecules.
• The resulting ions pass through a capillary sampling orifice
into the mass analyzer.

35. Figure. APPI ion source

36. 2-Mass Analyzers
Although in theory any type of mass analyzer
could be used for LC/MS, four types:
• 2a- Quadrupole
• 2b- Time-of-flight
• 2c- Ion trap
• 2d- Fourier transform-ion cyclotron resonance
(FT-ICR or FT-MS)

37. •A quadrupole mass analyzer consists of four parallel rods
arranged in a square.
• The analyte ions are directed down the center of the square.
• Voltages applied to the rods generate electromagnetic
fields.
• These fields determine which mass-to-charge ratio of ions
can pass through the filter at a given time.
•Quadrupoles tend to be the simplest and least expensive
mass analyzers.
2a- Quadrupole

39. 2b- Time-of-flight
•In a time-of-flight (TOF) mass analyzer, a uniform electromagnetic force is
applied to all ions at the same time, causing them to accelerate down a
flight tube.
• Lighter ions travel faster and arrive at the detector first,
so the mass-to-charge ratios of the ions are determined by their arrival
times.
•Time-of-flight mass analyzers have a wide mass range and can be very
accurate in their mass measurements.

40. Figure .Time-of-flight mass analyzer

41. 2c- Ion trap
• An ion trap mass analyzer consists of a circular ring
electrode plus two end caps that together form a
chamber.
• Ions entering the chamber are “trapped” there by
electromagnetic fields.
• Another field can be applied to selectively eject ions
from the trap.
• Ion traps have the advantage of being able to perform
multiple stages of mass spec trometry without additional
mass analyzers.

42. Figure. Ion trap mass analyzer

43. 2d- Fourier transform-ion cyclotron resonance
(FT-ICR or FT-MS)
• An FT-ICR mass analyzer (also called FT-MS)
is another type of trapping analyzer.
• Ions entering a chamber are trapped in circular orbits by powerful
electrical and magnetic fields.
•When excited by a radio-frequency (RF) electrical field, the ions
generate a time dependent current.
•This current is converted by Fourier transform into orbital
frequencies of the ions which correspond to their mass-to-charge
ratios.
• FT-ICR mass analyzers can perform multiple stages of mass
spectrometry without additional mass analyzers.

44. Figure. FT-ICR mass analyzer

45. Applications:-
LC/MS is suitable for many applications, from
pharmaceutical development to environmental
analysis.
Its ability to detect a wide range of compounds with
great sensitivity and specificity has made it popular
in a variety of fields.

46. Differentiation of similar octapeptides

47. Determining the molecular weight of green fluorescent protein

48. Full scan mass spectrum of ginsenoside Rb1
showing primarily sodium adduct ions

49. Figure. MS identification and quantification of individual
benzodiazepines from an incompletely resolved mixture

50. Identification of a minor metabolite of deoxycholic acid