Liquid Chromatography Mass Spectrometry LC-MS

8/13/2019 Liquid Chromatography Mass Spectrometry LC-MS

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Preparedby :

Mohammed H. Rida &.


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History:

Classification based on Mobile Phase:

The Basic Liquid Chromatograph units :

combination of LC and MS , LC-MS Instrumentation

LC-MS applications:

OUTLINE


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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.


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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).


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Since the stationary phase is the fixed one then those soluteswhich have:-

stronger interactions with the stationary phase will tend to move

slower (have higher retention times The time a solute spends in acolumn)

than others which have lower or no interactions with thestationary 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, forexample in terms of polarity.

when their properties are so different, a solute will not tend tostay and interact with the stationary phase and will thus prefer tostay in the mobile phase

and move faster; a polar solvent and a non polar stationaryphase


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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)


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General classification Specific method Stationary phase Type 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


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A small volume of the sample is first introduced at the topof the chromatographic column. Elution involves :-

passing a mobile phase inside the column whereby solutes

are carried down the stream but on a differential scale dueto interactions with the stationary phase.

As the mobile phase continues to flow, solutes continue tomove downward the column.

Distances between solute bands become greater with timeand as solutes start to leave

the column they are sequentially detected. The followingschematics represent the process at various times:


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The dark colors at the center of the solute zones in the

above figure represent higher concentrations than areconcentrations at the sides.

This can be represented schematically as:


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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.


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The mobile phase supply system consists of number

of reservoirs (200 ml to 1,000 ml in capacity).

The Mobile Phase Supply System


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The Gradient Programmer and the LC Pump

-the solvent mixing occurs at high pressure,and then passed to the pump

-is the simplest but most expensive.

-each solvent requires its own pump.


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


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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

Th S l V l


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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.


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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 closelysimilar 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.


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Detectors

UV DetectorFixed Wavelength Detector

Multi-Wavelength Detectors

Diode Array Detector

Electrical Conductivity Detector

Fluorescence DetectorRefractive Index Detector

Tridet Multi Functional Detector


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The Tridet Multi Functional Detector

trifunctional detector

that detected solutes bythe UV detector, the

electrical conductivity

detector and the

fluorescence detector

simultaneously in a

single low volume cell.


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* 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 chromatographicpeaks, reducing the need for perfect chromatography.

Mass spectral data complements data fromother LC detectors


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Two-dimensional abundance data

and three-dimensional mass spectral data from a mass spectrometer


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Instrumentation

Mass spectrometers work by :-

1- ionizing molecules

2- sorting and identifying the ions according to their mass-to-

charge (m/z) ratios.


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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)


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Figure. Applications of various LC/MS ionization techniques


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1a- Electro Spray Ionization (ESI)

- to generate analyte ions in solution before the analytereaches 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.

*Electrosprayis especially useful for analyzing large bio molecules

such as proteins, peptides,and oligonucleotides


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Figure. Electrospray ion source


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1b- Atmospheric pressure Chemical Ionization (APCI)

- In APCI, the LC eluent is sprayed through a heated (typically

250C400C) vaporizer at atmospheric pressure.

-The heat vaporizes the liquid.

-The resulting gas-phase solvent molecules are ionized byelectrons 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.


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Figure 6. APCI ion source


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1c- Atmospheric pressure photo ionization (APPI)

Atmospheric pressure photo Ionization (APPI)

for LC/MS is a relatively new technique. As in APCI, avaporizer 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.


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Figure. APPI ion source


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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)


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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


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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.


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Figure .Time-of-flight mass analyzer


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2c- Ion trap

An ion trap mass analyzer consists of a circular ring

electrode plus two end caps that together form achamber.

Ions entering the chamber are trapped there by

electromagnetic fields.

Another field can be applied to selectively eject ionsfrom the trap.

Ion traps have the advantage of being able to perform

multiple stages of mass spec trometry without additional

mass analyzers.


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Figure. Ion trap mass analyzer


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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 ionsgenerate 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.


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Figure. FT-ICR mass analyzer


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Applications:-

LC/MS is suitable for many applications, from

pharmaceutical development to environmentalanalysis.

Its ability to detect a wide range of compounds with

great sensitivity and specificity has made it popular

in a variety of fields.


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Differentiation of similar octapeptides


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Determining the molecular weight of green fluorescent protein


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Full scan mass spectrum of ginsenoside Rb1

showing primarily sodium adduct ions

Figure. MS identification and quantification of individual

b di i f i l t l l d i t


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benzodiazepines from an incompletely resolved mixture


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Identification of a minor metabolite of deoxycholic acid

Liquid Chromatography Mass Spectrometry LC-MS

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