Prepared by :-

Mohammed H. Rida &……………….

•History:

•Classification based on Mobile Phase:

•The Basic Liquid Chromatograph units :

•combination of LC and MS , LC-MS Instrumentation • LC-MS applications:

OUTLINE

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.

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).

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”

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)

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

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:

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:

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.

The mobile phase supply system consists of number of reservoirs (200 ml to 1,000 ml in capacity).

The Mobile Phase Supply System

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.

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

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

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.

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.

Detectors

•UV Detector

•Fixed Wavelength Detector

•Multi-Wavelength Detectors

•Diode Array Detector

•Electrical Conductivity Detector

•Fluorescence Detector

•Refractive Index Detector

• Tridet Multi Functional Detector

The Tridet Multi Functional Detector

trifunctional detector that detected solutes by the UV detector, the electrical conductivity detector and the fluorescence detectorsimultaneously in a single low volume cell.

* combination of LC and MS offers the possibility to take advantage ofboth 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

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

Instrumentation

Mass spectrometers work by :-

1- ionizing molecules 2- sorting and identifying the ions according to their mass-to- charge (m/z) ratios.

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)

Figure. Applications of various LC/MS ionization techniques

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

Figure. Electrospray ion source

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.

Figure 6. APCI ion source

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.

Figure. APPI ion source

2-Mass Analyzers

Although in theory any type of mass analyzercould 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)

•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 expensivemass analyzers.

2a- Quadrupole

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.

Figure .Time-of-flight mass analyzer

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.

Figure. Ion trap mass analyzer

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.

Figure. FT-ICR mass analyzer

Applications:-

LC/MS is suitable for many applications, frompharmaceutical 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.

Differentiation of similar octapeptides

Determining the molecular weight of green fluorescent protein

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

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

Identification of a minor metabolite of deoxycholic acid