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Chromatography and Spectroscopy

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It is the separation technique in which the mixture to be separated is in mobile phase which is made to move in contact with a selectively absorbent stationary phase. It can be an analytical method, examining the number and nature of the components in a very small amount of a mixture, but does not actually isolate them. Or it can be a preparative method, which investigates a large quantity of the mixture to obtain useable amounts of each component.



      It is the physical technique for the study of matter and its properties by examining the light, sound or particles emitted, absorbed or scattered by the matter by the application of electromagnetic radiations, which is represented in form of spectrum.  


Operation Technique:




A mixture is separated into its components by distribution between two phases; one phase is immobile which may be in the form of a porous bed, bulk liquid, layer or film is generally named as stationary phase, while the other phase is a mobile phase (fluid) that percolates through or over the stationary phase. Then separation occurs due to the repeated sorption/desorption by the movement of sample over the stationary phase in the direction of mobile phase. Effective separations require an adequate difference in the strength of the physical interactions of the sample components within the two phases, along with transport properties of the system that control sample movement within and between phases. ( REF _Ref277195303 \r \h  \* MERGEFORMAT 1)


Factors affecting separation:


  Intermolecular interaction between the two phases which can be predicted by the equilibrium constant.(thermodynamic properties)

  Extent of dispersion of solute molecules over the stationary phase



       The radiations which the matter absorbs or emit are analyzed by a device called spectrometer which separates it into various frequency components, measures intensity and plot a graph of intensity versus wavelength or frequency called spectrum.

Spectroscope is a device which separates the radiation into its component colors. It is simply a box with a slit at one end for the inlet of radiation and a light-separating device at the other end.





Types of chromatography:



Based on phases and distribution process. ( REF _Ref277173799 \r \h  \* MERGEFORMAT 1)



  For practical utility transport processes at least one phase must be reasonably fast that’s why solid-solid chromatography is impractical which is a very slow process.

  Two distinct phases are required to set up the distribution component of the separation mechanism, which explains why gas-gas chromatography does not exist and liquid-liquid separations are restricted to immiscible solvents.


Classification According To The Physical State: ( REF _Ref277174898 \r \h  \* MERGEFORMAT 4)


Gas Chromatography:


 Mobile phase: gas

Stationary phase: a non volatile liquid or a solid.


GC has five basic components and they are as follows:


1)      Pneumatic system:

Supplies gas (like helium and neon) and also controls its flowrate.


2) Injection system:

A sample of volatile mixture is injected into the carrier gas and the heated injector port, vaporises the sample if needed.

3) Column:

Carrier gas carries the sample to the coiled column where separation occurs. The separated components in order of their increasing interaction with the stationary phase emerges from the column

4) Oven

The column is placed in a thermostatically controlled oven.

5) Detector

It is an integral detector or a link to a mass spectrometer. There are two types of detectors

  thermal conductivity detector

  flame ionization detector




For each compound in a mixture one peak is observed on the chromatogram. In the particular set of operating conditions relating to the column; the retention time will increase with the size and polarity of the compound. To find the concentration of a particular compound, the peak height should be measured.

GC is used to analyse blood samples for the presence of alcohol. It is also used to analyse samples taken from athletes to check for the presence of drugs. In each case, it separates the components of the mixture and indicates the concentrations of the components. Water companies test samples of water for pollutants using GC to separate the pollutants, and mass spectrometry to identify them.


High Performance Liquid Chromatography:

HPLC is separates and detect components of the mixture and is widely used in drug testing, testing for vitamins in food and growth promoters in meat.


Basic Components:


1) Solvent Reservoir: stores the solvent.

2) The Pump System controls the flow and measures the volume of solvent (the mobile phase).

3) The Injector System: The sample to be separated is injected into the liquid phase at this point.

4) The Column is made of steel and packed usually with porous silica particles (the stationary phase). Different materials can be used depending on the nature of the liquid.

5) The Detector: When the components reach the end of the column they are analyzed by a detector. The amount passing through the column is small, that’s why solutes are analyzed as they leave the column. Therefore we link HPLC to a spectrometer.


The time a compound take to reach the detector allows the component to be identified. Like in GC, once the retention time of a solute has been determined for a column using a particular set of operating conditions, the solute can be identified in a mixture. A chromatogram is obtained for the sample.


Classification According To The Shape Of The Chromatographic Bed:( REF _Ref277174898 \r \h  \* MERGEFORMAT 4)


Column Chromatography:


Mobile phase (elute): solvent or mixture of different solvents

Stationary phase: a finely divided solid, such as silica gel or alumina


 A small volume of the sample is placed on top of the column. The sample should b soluble in the chosen mobile phase solvent. If the sample is highly soluble in the mobile phase solute will move quickly with the solvent resulting in non separation of certain components. The difference in the rate of movement through the medium is calculated to the retention time of sample.

The chromatography columns vary in size and polarity. The type of column and suitable solvent for the separation of mixture into its constituents is chosen by hit and trial method.


Type of column for chromatography:


Packed column

            The stationary bed coated with liquid stationary phase fill the inside of the column

Tubular column

            The stationary phase fills the column with a path in the centre of the column for the mobile phase (iupac 1993)


Planar chromatography:


In this technique of separation the stationary phase is present as a plane or on a plane. When the mixture of sample is placed on the plane, different compounds travel different distances according to the interactions between the mobile and stationary phase. The specific Retention factor (Rf) of each component is used in the identification of an unknown substance.


Rf      =        Distance the solute moves

Distance the solvent front moves


There are two types of planar chromatography:

Paper chromatography: (the plane is a paper)

Thin layer chromatography: (the plane is a glass plate)


Paper chromatography:


Mobile phase: solvent

Stationary phase: chromatographic paper


 In this type of chromatography the mobile phase is solvent and water held in the fibre of chromatographic paper is the stationary phase. With the help of the dropper the mixture is spotted on the strip of chromatography paper. Strip is suspended in the tank containing the solvent with the lower end of the strip dipped inside in the solvent. But the sample spot is kept above the solvent. The solvent is drawn up the paper by capillary action. The solvent moves up the paper with the components of mixture at different rates due to difference in the interaction between the two phases.






Thin Layer Chromatography:



Mobile phase: solvent

Stationary phase: a thin layer of finely divided solid, such as silica gel or alumina, supported on glass or aluminium.


Thin layer chromatography is similar to paper chromatography as it involves spotting the mixture on the plate and the solvent moves up the plate in the chromatography tank. But separation is more efficient as very small paricles are in stationary phase. The plate is able to separate a number of samples concurrently within a relatively short analytical run.

It is particularly useful in forensic work, for example in the separation of dyes from fibres.


Classification According To The Mechanism Of Separation: ( REF _Ref277174898 \r \h 4)


Adsorption chromatography: ( REF _Ref277175234 \r \h  \* MERGEFORMAT 2)


Mobile phase: liquid or gas

Stationary phase: solid


It is one of the oldest types of chromatography. The mobile phase is adsorbed onto the surface of a stationary phase. The equilibrium between the mobile and stationary phase accounts for the separation of different solutes. Separation occurs due to the differences between the adsorption affinities of the components of sample for the surface of an active solid.


Forces responsible for chromatographic adsorption are:


Forces of attraction:

            Dipole-dipole attraction

Hydrogen bonding

Polarizability forces

Weak covalent bond

Vander Waal forces


Forces causing solute movement:





Partition chromatography:

In this type of chromatography a thin film is formed on the surface of a solid support by a liquid stationary phase. Solute forms equilibrium between the mobile phase and the stationary liquid. Separation occurs to the relative solubilities of component in both phases.


Ion exchange chromatography:

In this type of chromatography, resin is used as a stationary phase. Anions and cations covalently attach onto the resin. Electrostatic forces are responsible for the attraction of solute ions of the opposite charge in the mobile liquid phase to the resin. Separation occurs due to difference in the ion exchange affinities of the components of the sample.


Molecular Exclusion Chromatography:

Also known as gel permeation or gel filtration. In this type of chromatography there is no an attractive interaction between the stationary phase and solute. Liquid or gaseous phase passes through a porous gel which are separated based on molecular size. The pores are normally small and exclude the larger solute molecules, but allow smaller molecules to enter the gel, causing them to flow through a larger volume. This causes the larger molecules to pass through the column at a faster rate than the smaller ones.


Affinity Chromatography:


This is a selective chromatography technique based on highly specific interaction between one specific kind of solute molecule and the second molecule that is immobilized on a stationary phase. Usually used for purification of proteins. For example, an antibody, to some specific protein, can be taken as the immobilized molecule. When a mixture of protein is passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. Then by changing the ionic strength or pH protein is extracted.


Types of spectroscopy:

      Different types of spectroscopy based on different energy sources.


Absorption spectroscopy:

In Absorption spectroscopy the power of a beam of light is measured before and after interaction with a sample and then compared. Specific absorption techniques tend to be referred to by the wavelength of radiation measured such as ultraviolet, infrared or microwave absorption spectroscopy.


Infrared spectroscopy gives information about bond vibration in molecules. 

UV/visible spectroscopy reveal electronic energy levels in molecules. 

Microwave spectroscopy reveals motion and rotation of molecules.

The result of this kind of measurement is an absorption spectrum, a plot of energy absorbed as a function of wavelength. 

Fluorescence spectroscopy:

In fluorescence spectroscopy higher energy photons are used to excite a sample, which then emit lower energy photons. This technique has found its applications in the biochemical and medical field and can also be used for confocal microscopy, fluorescence resonance energy transfer and fluorescence lifetime imaging.

X-ray spectroscopy: ( REF _Ref277206326 \r \h 12)

In x ray spectroscopy, X-rays are used for excitation of the atoms, inner shell electrons in the atom are excited to outer empty orbitals or they may be completely removed, ionizing the atom. The inner shell "hole" will then be filled by electrons from surrounding outer orbitals. Energy available in this de-excitation process will be emitted as radiation (fluorescence) or will remove other less-bound electrons from the atom (Auger effect). The X-rays frequencies and auger energy are measured and used for determining the electronic structure of materials.

Flame spectroscopy: ( REF _Ref277206326 \r \h 12)

In flame spectroscopy, liquid solution samples are subjected into a burner or nebulizer/burner combination to desolvate, atomize, and sometimes to excite to a higher energy electronic state. Flame used during analysis requires fuel and oxidant usually in the form of gases. This method can be used for analyzing metallic element analytes in the part per million, billion, or possibly lower concentration ranges. Light detectors are used to detect light with the analysis information coming from the flame.

            The usage of flame for solvation, atomization and excitation of atoms is used in many spectroscopic techniques.


  Atomic Emission Spectroscopy - Atoms are excited from the heat of the flame to emit light. A total consumption burner with a round burning outlet is used. A high resolution polychromator / monochromator can be used to produce emission intensity vs. wavelength spectrum over a range of wavelengths for detecting the elements of sample.


  Atomic absorption spectroscopy (often called AA) - A pre-burner nebulizer is used to create mist and a slot-shaped burner that gives a longer path length flame. The nebulizer and flame are used to desolvate and atomize the sample and the excitation of the atoms is done by the use of lamps shining through the flame at various wavelengths. The amount of light absorbed after going through the flame determines the amount of analyte in the sample


  Atomic Fluorescence Spectroscopy: A burner with a round burning outlet is used. The flame is used to solvate and atomizes the sample, but a lamp shines light at a specific wavelength into the flame to excite the analyte atoms in the flame. The atoms of certain elements can then emit light in a different direction. The intensity of this fluorescing light is used for quantifying the amount of analyte element in the sample.


Plasma Emission Spectroscopy:

It is the method of analysis of various elements in a sample, by passing the sample solution through a plasma (high temperature ionized gas) source, thereby exciting the outer orbital electrons of the analyte with simultaneous emission of electromagnetic radiations (in the form of lights), which are analyzed by means of a spectrograph, which separates various wavelengths of analytes in the sample. It is somewhat similar to optical emission spectroscopy, but is preferred over it.

Plasma are divided into three classes, depending on the technique of production

  Direct current plasma (DCP)

  Argon plasma

  Microwave plasma

  Inductively coupled plasma (ICP)

Spark or arc (emission) spectroscopy:

This method is used for the analysis of metallic elements in solid samples. For analyzing non-conductive materials, the sample is ground with graphite powder to make it conductive. In traditional arc spectroscopy method, the sample solid is ground. An electric arc or spark is passed through the sample, heating the sample to a high temperature to excite the atoms in it. The excited analyte atoms glow, emitting light at various wavelengths that are detected by spectroscopic methods. The conditions producing the arc emission are not controlled quantitatively; the analysis for the elements is qualitative.Nowadays, the spark sources with controlled discharges under an argon atmosphere making this method quantitative, and is use in control laboratories of foundries and steel mills.

Raman spectroscopy: ( REF _Ref277206326 \r \h 12)

Raman spectroscopy uses the inelastic scattering of light to analyze vibrational and rotational modes of molecules and is a type of spectroscopy that is complementary to infrared spectroscopy. The resulting 'fingerprints' are an aid to analysis.

Coherent anti-Stokes Raman spectroscopy (CARS): ( REF _Ref277206326 \r \h 12)

CARS is a recent technique that has high sensitivity and powerful applications for ''in vivo'' spectroscopy and imaging.

Nuclear magnetic resonance spectroscopy (NMR): ( REF _Ref277206326 \r \h 12)

Nuclear magnetic resonance spectroscopy analyzes the magnetic properties of atomic nuclei to determine different electronic local environments of hydrogen, carbon or other atoms in an organic compound or other compound. It is used to help determine the structure of the compound.

Mass Spectrometry (MS): ( REF _Ref277206326 \r \h 12)

A mass spectrometer source produces ions. Information about a sample may be obtained by analyzing the dispersion of ions when they interact with the sample, generally using the mass-to-charge ratio.

Multiplex or Frequency-Modulated Spectroscopy: ( REF _Ref277206326 \r \h 12)

In this type of spectroscopy, each optical wavelength that is recorded is encoded with an audio frequency containing the original wavelength information. A wavelength analyzer can then reconstruct the original spectrum.


Photoemission Mssbauer spectroscopy: ( REF _Ref277206326 \r \h 12)

In this technique is based on Mssbauer effect which uses gamma rays for analyzing the resonant absorption frequency. It is used for analyzing the properties of specific isotope nuclei in different atomic environments.

     Uses of chromatography:

         In chemical plant, chromatography is used to separate particles and contaminates like pesticides and insecticides.

         Government agencies use chromatography to separate toxic material from drinking water and for monitoring of air quality.

         In pharmaceutical companies chromatography is used for preparing pure raw materials and checking contaminants from manufactured compounds.

  • In organic chemistry many compounds are very close to each other in terms of atomic or molecular weight, composition and physical property. Therefore chromatography is used for identification of compounds.
  • In food industry, chromatography technique is used for proper food maintenance.


Uses of spectroscopy:


  Examining of the molecular structure.

  Estimation of the energy levels of the ions and complexes in a chemical system along with their compositions.

  Study of the structure making and structure breaking processes in solutions.

  Examining the intrinsic configuration and relative association and chemical shifts

  The identification of substances through the spectrum emitted from them or absorbed in them.

  Widely used in astronomy and remote sensing.





Combination of chromatography and spectroscopy:( REF _Ref277177082 \r \h  \* MERGEFORMAT 10)


For the identification of sample and detection the combination of chromatography with spectroscopy is used.


Gas chromatography-mass spectrometry ( Gc-ms): ( REF _Ref277176622 \r \h 7)


We use combination of gas chromatography with mass spectroscopy. Individually these devices are sensitive and bulky, difficult to handle, using their combination simplifies its structure and improved its operating time.

GC-MS is used for drug detection, fire investigation, environmental analysis, explosives investigation, and identification of unknown samples. 


Spectroscopy in conjunction with liquid chromatography: ( REF _Ref277177082 \r \h  \* MERGEFORMAT 10)

Modern liquid chromatography can separate very complex mixture but has problem in elucidating structure of the eluted component. The solute eluted from the column, in the past, were collected as fractions, concentrated and then examined by suitable spectroscopic techniques. Now elute is directly send to the spectrometer and data is collected concurrent with the separation process. There are two methods for it depending on the speed at which data can b acquired and on the type of the spectroscopic data obtained. The most common spectroscopic techniques employed for the elucidation of molecular structure are:

  Ultraviolet spectroscopy

  Infrared spectroscopy

  Raman spectroscopy

  Mass spectrometer(ms)

  Nuclear magnetic resonance spectroscopy(NMR)

If the spectroscopic data can be obtained rapidly then mass spectrometer (LC-MS) or Fourier Transform infrared spectrometer (LC-FTIR) is used. If the scanning rate of spectrometer is slow then nuclear magnetic resonance (NMR) or IR spectrometer are used.


Combination of liquid chromatography with Nuclear magnetic resonance spectroscopy (NMR): ( REF _Ref277177082 \r \h  \* MERGEFORMAT 10)

NMR is the only spectroscopic technique that in any circumstances, without the aid of the any supplementary spectroscopic technique can identify the unknown mixture. When it is employed with liquid chromatography; it forms a powerful analytical system that is used for separation and identification of the unknown mixtures.

Although, it is a very useful technique; but it has some serious difficulties in association of these two techniques.

1.     Intensity of NMR signal is dependent on the flow rate of solvent. As the flow rate increase the signal decreases.

2.     For high resolution of NMR the magnetic field must be homogeneous that requires the sample tube to spin at high speed; which make it impossible for flow in the NMR cell.

3.     It would sufficiently reduce the solvent consumption but that demands very small cell volume.


Combination of liquid chromatography with mass spectrometer: ( REF _Ref277177082 \r \h  \* MERGEFORMAT 10)


The LC/MS is another important technique in structure elucidation of eluted solutes but the system is not as comprehensive as LC/NMR as it requires other spectroscopic information. For example the IR spectrum of substance for identification of certain functional groups is used. But it also has certain advantages over other spectroscopic methods that make it ideal to combine with LC.

1.      Mass spectra is obtained rapidly

2.      Small amount of material is required to form the spectra.

3.      Data collected is highly informative with respect to molecular structure.



1)       Jack, C.;”Encyclopedia of Chromatography”;3rd  Ed; Volume 3,(40-42)  


2) [23-10-2010]

3) [23-10-2010]

4)  [23-10-2010]

5) [23-10-2010]

6) [23-10-2010]




9) [23-10-2010]

10)  Raymond, W.S.P;”liquid chromatography detector” ;2nd Ed; volume 33(181-191) [2-11-2010]

11) [2-11-2010]

12) [2-11-2010]

13) [2-11-2010]








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