Learning Center

1.0 Spectroscopy (UV-VIS)

2.0 Electro-analytical

3.0 Chromatography

4.0 Planar chromatography (PC/TLC)

5.0 Instrumental separation techniques (GC/HPLC/CE)




1.0 Spectroscopy (UV-VIS):


Spectroscopy can be defined as interaction of electromagnetic radiation with matter. In particular, we are interested in quantitative determination of  inorganic salts, organic and biological species using spectroscopic principles. Its based on Beer's law which states that concentration of an absorbing analyte is linearly related to its absorbance. Analytical instruments based on spectroscopy principles are called as calorimeters, UV-VIS spectrophotometer, or spectrometers.


A = ε*c*l




A = Absorbance at a wavelength λ

ε = wavelength dependent molar absorptivity coefficient, typically reported for λmax

c = concentration (in molarity)

l = path length of the cuvette (typically equal to 1 cm)


UV-VIS Spectrophotometer are instruments designed to analyze chemicals within the ultraviolet and visible portions of the electromagnetic spectrum (200nm-800nm). They consist of many components such as a light source, diffracting medium, etc and they are explained below. Single beam instruments consist of a light source, a monochromator, sample compartment, and a  photodiode as a light detector, and an amplifier and data acquisition circuitry which communicates with the computer digitizing, displaying, and storing the spectrogram. The monochromator consists of a diffraction grating (mounted on a stepper motor driven rotating platform) and a slit which allows “scanning” of the entire wavelength range. One disadvantage of this design is that we need to subtract the solvent spectra from sample by subtracting the reference spectra from the sample spectra. In order to overcome this design flaw, Double beam instruments feature a beam splitter which splits the light into two beams, one going through sample solution  and other through reference solution; a ratio of two signals is calculated automatically by the data acquisition and amplification circuitry and the spectra is displayed on the computer. This design automatically compensates for drift in the light source, amplifier or detector.


Another type of instruments are called diode array detectors. These are single beam instruments with photodiodes replaced by diode array or Charged coupled devices (CCD) typically having between 1000-3500 pixels. This allows simultaneous capture of spectra at multiple wavelengths removing the need to “scan” the wavelength range by the stepper motor.    Reference spectra still has to be electronically subtracted like in traditional single beam instruments.  


Fig 1: Comparison of Single beam with double beam spectrometer. Source Link.


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2.0 Electro-analytical chemistry techniques:


This an umbrella term for a wide variety of quantitative and qualitative analytical techniques based on electrical properties of the analyte solution when analyzed in an electrochemical cell. The three main categories are Potentiometry, where difference in electrode potentials is measured, without drawing a significant amount of current; Coulometry, where cell's current is measured over time; an instrument called potentiostats are used to maintain the potential of a working electrode at a constant level relative to a reference electrode and voltammetry, where a cell's current is measured while changing its potential.

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3.0 Chromatography:


Chromatography stands for a collection of techniques used to separate individual chemicals from a mixture of chemicals. It was invented by Russian botanist, Mikhail Tswett, who first reported it to separate plant pigments such as chlorophyll and xanthophyll by passing a solution of these chemicals through a glass column containing calcium carbonate. The separated pigments show up as colored bands, thus, giving the name to the method; in greek, chroma means "color" and graphein means "to write".


The mixture to be separated is dissolved in the “mobile phase”, which could be a liquid, gas or a super critical fluid. Mobile phase is forced through an immiscible solid phase called “stationary phase”, which is generally packed into a glass or stainless steel column. When mobile phase is passed through stationary phase, in a process labeled as elution, the chemicals in the mixture distribute themselves between the mobile and stationary phase, and chemicals strongly retained (i.e. sorbed) by stationary phase travel slowly down the column than the ones strongly retained by mobile phase. Based on differences in this absorption rate, we effectively get separate discrete bands or regions which are enriched in one chemical over the other and this effect is the basis of chromatography which leads to separation of individual chemicals from a mixture. The time taken for the solute to travel through the column is called retention time, which is dependent on the polarities of the mobile and stationary phases, length of stationary phase etc. The simplest type of chromatography is shown in fig__, which is simply called “column chromatography”; the liquid mobile phase is passed through the stationary phase packed in a glass column under gravity, with no external pressure applied.



Chromatography is further subdivided into separate categories based on the type of stationary and mobile phases; and each of those have their own advantages/disadvantages in separating various types of chemical mixtures.




Fig 2: Separation of a chemical mixture (colored red and blue), into separate discrete bands using chromatography. Source: Link.


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4.0 Planar chromatography (PC/TLC):


4.1 Paper chromatography (PC):


In this type of chromatography, The mobile phase is a liquid which travels up a filter paper which serves as a stationary phase via capillary action. This technique has advantages of being very inexpensive, and due to that, its used for teaching purposes as well as a quick screening tool to qualitatively determine presence/absence of pollutants such as pesticides and other organic contaminants. Retardation factor Rf is defined as the ratio of the distance traveled by the center of a spot to the distance traveled by the solvent front.




Fig 3: A paper chromatogram indicating separation of different chemicals. Source: link.


4.2 Thin-layer chromatography (TLC):


This type is extremely similar to paper chromatography, except that stationary phase here consists of a thin layer of silica or alumina backed on a glass or aluminum foil plate. This results in higher quality separations while still competing with the convenience and inexpensive nature of paper chromatography. Retardation factor is calculated the same way as for paper chromatography.




Fig 4: An Aluminum backed silica thin layer chromatographic plate with separation of different chemicals. Source: Link.


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5.0 Instrumental separation techniques (GC/HPLC/CE)


5.1 Gas Chromatography (GC):


In this type of chromatography, the sample is vaporized into carrier gas like helium or nitrogen as mobile phase and run through coiled stainless steel columns containing the stationary phase. GC can only be used for chemicals which can be vaporized without decomposition, and hence its typically restricted to lower molecular weight chemicals. Advantages of GC include high rate of sample separation and analysis with low sample weight, and high resolution.



Fig 5: A diagram showing main components of a gas chromatograph. Source: Link.


5.2 High performance liquid chromatography (HPLC):


This type is based on column chromatography, except that the columns in this case are made out of stainless steel which can withstand high pressures (~6000 psi) compared to gravity separations of classic column chromatography. HPLCs can be further subdived into analytical type or preparative type based on amount of sample separated. So the chief components of a HPLC are a high pressure pump, a column, and a detector, which can be a UV-Vis spectrometer type, a mass spectrometer, or an electrochemical detector.