Pharmacy Courses

Interview Questions and Answers on Chromatography (Part-2)

What is the principle of HPLC?

A sample is injected through the injector in a flow of mobile phase and travels through a stationary phase (column). The analytes in the sample mixture move with the flow of the mobile phase and interact with the solid support. The rate of movement relies on the affinity of the analytes toward the stationary phase. The strongly interacting analytes with the stationary phase travel gradually, while the less interacting compounds elute quickly.

What is the principle of Gas Chromatography?

Gas chromatography follows the principle of the partitioning of volatile compounds with the mobile phase (gaseous) and stationary phase (liquid or solid). The separation speed of molecules through the column is based on the affinity for the stationary phase; the molecules which are partitioned in the gas first come out, whereas the other eluted later.

What is the principle of Thin Layer Chromatography (TLC)?

TLC is a separation technique where the molecules of mixtures separate using differential migration through a stationary phase, the solvent mixture flowing through the virtue of capillary forces. After chromatography is complete, solutes are detected on the surface of a TLC plate by visualizing the reagents and using a UV cabinet. TLC is a type of planar chromatography, such as paper chromatography, but the stationary phase in TLC is a thin-split Sorbent, which spreads as a thin layer of aluminum, glass, or plastic supporting flat plate.

What is the principle of Fluorescence Spectroscopy?

Fluorescence is the emission of light from a molecule, which returns from the lowest vibration level of an excited single state to its normal ground state. Excited by the molecules, it is achieved by exposing the light source of the specified wavelength. At what time the molecule/compound is fluorescent, a part of the absorbed light is converted to fluorescence light, an emission of light at a lower wavelength. 

The fluorescence intensity is directly proportional to the concentration of the fluorescent species. Obviously, the linear relationship among the concentration and the fluorescent, intensity falls just to the diluted solution at high concentrations, make it required to first set up a concentration to detect the concentration of fluorescent graph and unknown sample with known standards. Fluorometry use to concentration determining of fluorophores, it provides sensitive absorption more than the colorimetric method. 

What is the principle of UV Spectroscopy?

UV spectrophotometer principle adheres to the Beer-Lambert Law; this law expresses that when a beam of monochromatic light is gone through a sample solution an absorbing analyte. The diminishing rate of the radiation power alongside the thickness of the sample solution is really corresponding to the incident radiation and solution concentration.

What are the Factors Affecting Separation in Column Chromatography?

  • The dimension of the column
  • The particle size of the adsorbent
  • Nature of the solvent
  • Pressure
  • Temperature

Which type of plates are used in IR?

The IR plates that we use in the IR spectroscopy are made of polished sodium chloride (NaCl). Since it is transparent to infrared radiation it is used to take the IR spectra of liquid samples, these plates work similarly to potassium bromide (KBr) for solid samples. The major difficulty of using a liquid sample is to decide a solvent with which to dilute the sample. No solvent is ideal, however, if some information about the molecule is known, a solvent can be selected accordingly. We can use for CHCl3 or DCM liquid for dissolving the sample since it is suitable for plates. Water is not a suitable solvent as NaCl is water-soluble. If there is water in the sample the plates will be ruined.

Why is pH important for HPLC buffers?

As many we know the buffers are commonly used in mobile phases of HPLC to keep the pH stable. They are composed of a weak base or acid, combined with its conjugate acid or base. Because the retention, peak shape, column stability, reproducibility, and selectivity of ionizable analytes is very sensitive to the pH of the mobile phase, hence it is required to control the pH by adding a buffer in the mobile phase.

What is the difference between ascending chromatography and descending chromatography?

Both ascending chromatography and descending chromatography are types of paper chromatography used to separate molecules. The major difference between ascending and descending paper chromatography is that, In ascending chromatography, the analytes are in the mobile phase and travels from bottom to top (by capillary action). Similarly, in descending chromatography, the solvent travels from top to bottom. This method gives rapid separation as the mobile phase moves with gravity. However, the principle of separation in both types remains the same; ascending chromatography is technically easy to execute.

What is Affinity Chromatography?

Affinity chromatography is the method by which the interactions of reversible biospecific between molecular species, its legand is utilized in the separation of such molecular species from a biological environment. Affinity is used to tell how two molecules behave with one another. Polar substances have a preference for each other to the substance of non-polar and we generate antibodies with intimacy to antigen for help fight against the infections.

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