Chromatography and conductometry, Biology tutorial


Chromatography and Conductometry are separation and analytical methods extensively utilized in chemistry and biological sciences. Most things which happen in nature are the mixture of substances that can only be divided or analyzed using techniques known.


Chromatograpy, firstly is a method for separating components of the mixture by differential distribution of components between the stationary stage and mobile (moving) stage. Originally utilized for separation of colored substances from plants (Greek, Chromos meaning coloured) is now most widespread method of separation and purification of colored/colorless organic compounds. Separation of 2 sample components in chromatography is based on the different distribution between two non-miscible phases. Stationary stage, a liquid or solid, is fixed in system. Mobile stage, a fluid, is streeming through chromatographic system. In gas chromatography mobile stage is gas, in liquid chromatography it is a liquid. Molecules of analytes (mixture to be separated) are distributed between mobile and stationary phase. When present in stationary phase, they are preserved, and are not moving through system. On the contrary, they migrate with velocity, v, of mobile phase when being there.

Types of chromatography:

Paper Chromatography is the most common kinds of chromatography in which filter paper acts as support for immobile liquid stage. Removing liquid flows between fibres of cellulose of filter paper but these aren't stationary phase. True stationary stage is very thin film of liquid generally water adhering to surface of fibers. Substrate to be divided is distributed between two liquids, stationary liquid which is held on fibers of paper and moving liquid in developing solvent. It utilizes strip of paper and capillary action is utilized pull solvents up through paper to separate solutes. Any substance which reacts or bonds with paper can't be estimated using the method. Components of mixture move up paper with solvent at different rates, Rf, because of the differing interactions with stationary and mobile phases.

Rf = (Distance the solute moves/Distance the solvent front moves)

Thin-layer Chromatography:

Surface of plate comprises of very thin layer of silica gel on the plastic or Aluminium backing. Silica gel is form of silicon dioxide (silica). It has benefit over paper chromatography in that its separations are extremely effective due to much smaller size of particles in stationary phase. Gas chromatography and high performance liquid chromatography are more difficult chromatographic methods.

Column Chromatography:

Column chromatography is often utilized by organic chemists to purify liquids (and solids). The impure sample is loaded onto column of adsorbent, like silica gel or alumina. The organic solvent or mixture of solvents (eluent) flows down through column. Components of sample separate from each other by dividing between stationary packing material (silica or alumina) and mobile elutant. In column chromatography, stationary phase is packed in the glass tube to form the cylinder or column of granules. Stationary stage may be silica gel or ion exchange resin or the variety of other substances which may have specific affinity for amino acid molecules. Stationary stage in polar compounds is attracted to polar column packing by hydrogen bonding or dipole-dipole attractions. More polar component relates more strongly with stationary stage. Polar compounds are moved slowly. Non-polar compounds come off column first, whereas polar compounds are going to come off column last. Generally, one begins with less polar solvent to remove less polar compounds, and then slowly increase polarity of solvent to remove more polar compounds.

Gas Chromatography (GC):

The gas is mobile phase and stationary phase can be either solid or non-volatile liquid. There are 5 fundamental GC components:

  • Pneumatic system -gas supply (flow control and measurement).
  • Injection system - heated injector port, where sample is vaporized if essential
  • Column - where separation happens
  • Oven -coiled column is completely contained in thermostatically controlled oven.
  • Detector - integral detector or link to mass spectrometer

How Gas Chromatography Works:

1) Carrier gas, examples of which are Helium and Neon flows through system. The valve controls flow rate.

2) The sample of volatile mixture is injected into carrier gas. Sample is vaporized in heated injector port.

3) Carrier gas carries vaporized sample in column. Columns are stainless steel or glass tubes. Packed columns have porous support material. Sample mixture undergoes the series of interactions between stationary and mobile stages as it is carried through system by carrier gas.

4) Coiled column is contained in thermostatically controlled oven.

5) Separated components come out in order of increasing interactions with stationary stage. Least retarded component comes through first. Separation is attained when one compound is adequately retarded to stop overlap with another component of sample, as it emerges from column.

6) Two kinds of detector can be utilized: (1) thermal conductivity detectors that respond to changes in thermal conductivity of gas leaving column and (2) flame ionization detection (FID). In thermal conductivity, as carrier gas leaves column, it cools detector. When the solute emerges with carrier gas, it doesn't cool detector to same extent.

Once GC has separated the mixture, components can be recognized using known retention times. For unknown compounds solutes are gathered individually and analyzed using another method, like mass spectrometry. For every compound in mixture one peak is seen on chromatogram.

High Performance Liquid Chromatography:

Fundamental Components:

1. Solvent Reservoir.

2. Pump System controls flow and estimates volume of solvent (mobile phase). Flow rates of HPLC columns are slow - frequently in range of 0.5 -5 cm3 min-1

3. Injector System: Sample to be divided is injected in liquid phase at this point.

4. Column is composed of steel and packed generally with porous silica particles (stationary phase). Different materials can be utilized depending on nature of liquid. The long column is not required as separation in HPLC is very effective. Different components of sample are carried forward at different rates by moving liquid phase, because of their differing interactions with stationary and mobile phases.

5. Detector: When components reach end of column they are analyzed by the detector. Amounts passing by column are small, so solutes are analyzed as they leave column. HPLC is generally linked to spectrometer.

Applications of chromatography:

Thin layer chromatography is mainly useful in forensic work, for instance in separation of dyes from fibres. Gas Chromatography is utilized to analyze blood samples for presence of alcohol. It is also utilized to analyze samples taken from athletes to check for presence of drugs. In every case, it divides components of mixture and specifies concentrations of components. HPLC has several uses like drug testing, testing for vitamins in food and growth promoters in meat.


One of the most significant features of electrolyte solution is their ability to carry electric current. Electrolyte conductance is possible by movement of positive and negative ions that originate by dissociation of electrolyte. Conductometry handles with conductivity of electrolytes. Resistance of solution is estimated by applying alternating voltage to measuring cell. The conductivity of a solution depends on;

The number of ions: The more ions a solution contain, the higher its conductivity.

Ionic mobility in the general way: Mobility in turn relies on:

  • Kind of ion: the smaller an ion, the more mobile it is and better it conducts electrical current.
  • Solvent: more polar a solvent, the more entirely ionized are compounds dissolved in it. Water is the ideal solvent for ionic compounds.
  • Temperature: conductivity of solutions increases with increasing temperature at the rate which ranges from 1 to 9% per Kelvin, depending on ion.
  • Viscosity: ionic mobility reduces with increasing viscosity that means that conductivity also reduces.

Measuring setup:

Minimal measuring setup comprises of conductometer and conduntivity cell connected to it. Usually following items are also utilized: temperature sensor closed measuring/titrating vessel which can be thermostatted, and magnetic stirrer.

Conductometer is the instrument for estimating complex resistances by alternating voltages (in contrast to measurement of solely ohmic resistances of metallic conductors, liquids, together with measuring cell, comprise the network of resistances and capacities).

Conductivity measuring cell comprise of 2 electrodes which face each other and are as inert as possible. Platinum is usually used as electrode material. Smooth (shiny) electrodes must only be utilized for conductivities <20 US/cm. For higher conductivities, platinized electrodes are utilized. To avoid determining errors because of changes in electrical field, measurement is performed in strictly defined volume. Accordingly, different cells are utilized which are optimized for conductivity range to be covered. Measuring cells are distinguished by their cell constant c. For low conductivities, cells with the small cell constant are utilized while cells with the high cell constant serve to determine high conductivities.

Applications of conductometry:

  • Experimental determinations of conducting properties of electrolytic solutions are extremely significant as they can be utilized to study quantitative behavior of ions in solutions.
  • They can also be utilized to find out several physical quantities like degree of dissociation and dissociation constants of weak acids and bases.
  • They form foundation for conductometric titration methods.

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