CHROMATOGRAPHY AND

MASS SPECTROSCOPY

ROHAN DEOKAR

WHAT IS CHROMATOGRAPHY???

Quite simply, it is a broad range of physical methods used to separate and or to analyze complex mixtures.

The mixture is dissolved in a fluid called the mobile phase/eluent, which carries it through a structure holding another material called the stationary phase.

The various constituents of the mixture travel at different speeds, causing them to separate.

The separation is based on differential partitioning between the mobile and stationary phases.

Subtle differences in a compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation.

HOW DOES IT WORK ?????

A mixture of various components enters the chromatography process, and the different components are flushed through the system at different rates.

These differential rates of migration as the mixture moves over adsorptive materials provide separation.

Repeated sorption/desorption acts that take place during the movement of the sample over the stationary bed determine the rates.

The smaller the affinity a molecule has for the stationary phase, the shorter the time spent in a column.

TERMS USED IN CHROMATOGRAPHY

The analyte/sample/solute is the substance to be separated during chromatography which may consist of a single component or a mixture of components.

Analytical chromatography is used to determine the existence and possibly also the concentration of analyte(s) in a sample.

A bonded phase/stationary phase is covalently bonded to the support particles or to the inside wall of the column.

The eluate is the mobile phase leaving the column. The retention time is the characteristic time it takes for a particular

analyte to pass through the system (from the column inlet to the detector) under set conditions.

BASIC OPERATION The process of a chromatographic separation

takes place within a hollowed center chromatography column made of glass/metal in a packed bed or open tubular column.

A packed bed column contains particles of the stationary phase while open tubular columns are lined with a thin film stationary phase.

The mobile phase/carrier fluid is a solvent which carries the mixture to be separated and can either be a liquid or a gas, depending on the type of process, while the stationary phase is a viscous liquid coated on the surface of solid particles.

The partitioning of solutes between the two phases lead to the desired separations.

The materials comprising the mobile and stationary phases vary depending on the type of chromatographic process being performed.

WORKING MECHANISM1. FEED INJECTION –

The feed is injected into the mobile phase. The mobile phase flows through the system by the action of a pump.

2. SEPARATION IN THE COLUMN -

As the sample flows through the column, its components will adsorb to the stationary phase to varying degrees. Those with strong attraction to the support move slowly than those with weak attraction.

3. ELUTION FROM THE COLUMN-

After the sample is flushed or displaced from the stationary phase, the different components will elute from the column at different times.

The components with the least affinity for the stationary phase (weakly adsorbed) will elute first, while those with the greatest affinity for the stationary phase (strongly adsorbed) will elute last.

4. DETECTION -

The different components are collected as they emerge from the column.

A detector analyzes the emerging stream by measuring a property which is related to concentration and characteristic of chemical composition.

THE CHROMATOGRAM Since the sample is separated in the

column, different peaks correspond to different components present.

The chromatogram show the results of separations of protein mixtures by ion exchange chromatography.

The lettered peaks correspond to different proteins (A = ovalbumin, B = conalbumin, C = cytochrome c, D = lysozyme).

The separation corresponding to the chromatogram on the left was performed at pH 5.85, while the one on the right was performed at pH 6.5.

Operation conditions such as pH and temperature have a significant effect on the output.

WHAT INFORMATION CAN BE ATTAINED ???

The level of complexity of the sample is indicated by the number of peaks which appear.

Qualitative information about the sample composition is obtained by comparing peak positions with those of standards.

Quantitative assessment of the relative concentrations of components is obtained from peak area comparisons.

Column performance is indicated by comparison with standards.

WHAT IS MASS SPECTROSCOPY?????

Mass spectrometry (MS) is an analytical technique that separates the components of a sample by their mass, producing spectra of the masses of the molecules of a sample material.

The spectra determine the elemental composition of a sample, the masses of particles and molecules, and to explain the chemical structures of molecules and protein characterization.

It is mainly used in identifying unknown compounds, determining isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation.

Other uses include quantifying the amount of a compound or studying the fundamentals of gas phase ion chemistry (the chemistry of ions and neutrals in a vacuum).

Biomedical applications include use for respiratory gas and trace analysis.

BASIC PRINCIPLE

The sample is vaporized into a gas and then ionized. Ions are separated according to their mass to charge ratio

and are accelerated through a potential difference and focused into a beam.

The ion beam passes through a magnetic field which bends the charged stream.

Lighter components or components with more ionic charge will deflect in the field more than heavier or less charged components.

A detector counts the number of ions at different deflections and the data can be plotted as a 'spectrum' of different masses.

The atoms or molecules can be identified by correlating known masses to the identified masses or through a characteristic fragmentation pattern.

Signal processing results are displayed as spectra of the relative abundance of ions as a function of the mass-to-charge ratio.

COMPONENTS OF THE SPECTROMETER

A mass spectrometer consists of three components: an ion source, a mass analyzer, and a detector.

1. ION SOURCE / IONIZER - The ion source is the part of the mass spectrometer that ionizes the

material under analysis (the analyte). There are a wide variety of ionization techniques, depending on the

phase (solid, liquid, gas) of the sample and the efficiency of various ionization mechanisms for the unknown species and determine the types of samples which can be analyzed.

Electron ionization and chemical ionization are used for gases and vapors while electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) are used for liquid and solid biological samples.

The ions are then transported by magnetic or electric fields to the mass analyzer.

2. MASS SELECTION - Mass analyzers separate the ions according to their mass-to-charge

ratio. The following two laws govern the dynamics of charged particles in electric and magnetic fields in vacuum:

  (Lorentz force law);   (Newton's second law of motion) F is the force applied to the ion, m is the mass of the ion, a is the

acceleration, Q is the ion charge, E is the electric field, and v × B is the vector cross product of the ion velocity and the magnetic field.

Equating the above expressions for the force applied to the ion yields the classic equation of motion for charged particles:

3. DETECTOR-

The final element of the mass spectrometer is the detector which records either the charge induced or the current produced when an ion passes by or hits a surface. Some detectors also give spatial information.

BASIC WORKING OF A MASS SPECTROMETER The following example of sodium

chloride describes the working operation.

In the ion source, the sample is vaporized and ionized (transformed into electrically charged particles) into sodium (Na+) and chloride(Cl-) ions.

Sodium atoms and ions are monoisotopic, with a mass of about 23 amu. Chloride atoms and ions come in two isotopes with masses of approximately 35 and 37 amu.

The analyzer part of the spectrometer contains electric and magnetic fields, which exert forces on ions traveling through these fields.

S The speed and direction of a

charged particle may be varied while passing through the electric field and magnetic field respectively.

The magnitude of the deflection of the moving ion's trajectory depends on its mass-to-charge ratio. Lighter ions get deflected by the magnetic force more than heavier ions.

The streams of sorted ions pass from the analyzer to the detector, which records the relative abundance of each ion type.

This information is used to determine the chemical element composition of the original sample and the isotopic composition of its constituents (the ratio of 35Cl to 37Cl).

ADVANTAGES AND DISADVANTAGES

Advantages include-1. Increased sensitivity since the analyzer reduces background

interference.2. Excellent specificity from characteristic fragmentation

patterns to identify unknowns or confirm the presence of suspected compounds.

3. Information about molecular weight and isotopic abundance of elements.

Disadvantages include-4. It fails to distinguish between optical and geometrical

isomers and the positions of substituent in o-, m- and p- positions in an aromatic ring.

5. Limited scope in identifying hydrocarbons that produce similar fragmented ions.

COMBINED TECHNIQUES An important enhancement to the mass resolving and mass

determining capabilities of mass spectrometry is using it in tandem with chromatographic separation techniques.

A common combination is gas chromatography-mass spectrometry (GC/MS or GC-MS) in which a gas chromatograph is used to separate different compounds.

This stream of separated compounds is fed online into the ion source, a metallic filament to which voltage is applied.

This filament emits electrons which ionize the compounds. The ions can fragment, yielding predictable patterns.

Intact ions and fragments pass into the mass spectrometer's analyzer and are eventually detected.

These technique are used in the fields of forensics and determination of respiratory gas analysis along with trace analysis.

THANK YOU !!!!!!