Analytical Method II

• What is chromatography?

  It involves the separation of soluble components in a solution by specific differences in physical-chemical characteristics.
• How does chromatography separate complex mixtures?

  It separates them based on different physical interactions between the individual compounds and the stationary phase of the system.
• What are the two forms of chromatography mentioned?

  • Planar Chromatography
  • Column Chromatography
• What is paper chromatography used for?

  It is used for drug screening as a semiquantitative screening test.
• What does the Rf value represent in chromatography?

  Rf stands for retention factor; it is the relative distance of migration from the point of application.
• Why is the extraction of the drug in chromatography pH dependent?

  The pH must be adjusted to reduce the solubility of the drug in the aqueous phase.
• What is the sorbent used in thin layer chromatography?

  The sorbent is thin plastic plates impregnated with a layer of silica gel or alumina.
• What types of samples can be analyzed using thin layer chromatography?

  Examples include blood, urine, and gastric fluid.
• What is gas chromatography used for?

  It is used for the separation of steroids, barbiturates, blood, alcohol, and lipids.
• What is the principle behind gas chromatography?

  It is useful for compounds that are naturally volatile or can be easily converted into a volatile form.
• How are specimens treated in gas chromatography?

  Specimens are vaporized and swept onto the columns.
• What is the role of the flame ionization detector in gas chromatography?

  It is used to detect the sample during the chromatography process.
• What determines the elution order of volatile compounds in gas chromatography?

  The elution order is based on their boiling point.
• What is the mobile phase in gas chromatography?

  The mobile phase is usually an inert gas like Nitrogen, Helium, Hydrogen, or Argon.
• What is the purpose of a molecular sieve in gas chromatography?

  It removes unwanted oxygens, hydrocarbons, and water vapor that may interfere in the test sample during analysis.
• What are the two types of columns used in gas chromatography?

  Packed column and capillary column.
• What is the difference between packed and capillary columns in gas chromatography?

  Packed columns are made of glass or stainless steel, while capillary columns are made of fused quartz.
• What is the stationary phase in gas chromatography?

  The stationary phase is packed in the inner wall of the column and is made up of silicone grease or wax which can withstand high temperature.
• What is the temperature range maintained during the analysis in gas chromatography?

  The temperature of the column is kept between 150 to 300 degrees Celsius.
• How does separation occur in gas chromatography?

  Separation occurs based on the interaction of molecules between the mobile phase and stationary phase.
• What is mass spectrometry (MS) based on?

  It is based on fragmentation and ionization of molecules using a suitable source of energy.
• How is mass spectrometry used in conjunction with gas chromatography?


  Substances are first separated by gas chromatography before being analyzed by mass spectrometry.
• What information can mass spectrometry provide?

  It can detect structural information and determine molecular weight.
• What is the gold standard for drug testing?

  GC-MS (Gas Chromatography-Mass Spectrometry).
• What is tandem mass spectroscopy (MS/MS) used for?

  It can detect 20 inborn errors of metabolism from a single blood spot.
• What is liquid chromatography based on?

  It is based on the distribution of solutes between a liquid mobile phase and a stationary phase.
• What is High Performance Liquid Chromatography (HPLC) known for?

  It uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution technique.
• What are the uses of HPLC?

  It is used for the fraction of drugs, hormones, lipids, carbohydrates, and proteins; separation and quantitation of various hemoglobins associated with specific diseases, and rapid HbA1c.
• What are the five separation mechanisms used in liquid chromatography?

  1. Gel/Gel Permation/Gel Filtration/Size Exclusion/Molecular Sieve Chromatography
  2. Ion Exchange Chromatography
  3. Partition Chromatography
  4. Affinity Chromatography
  5. Adsorption Chromatography
• How does gel filtration chromatography separate molecules?

  It separates molecules based on differences in their size and shape.
• What is hydrophilic gel used for in chromatography?

  It is used for the separation of enzymes, antibodies, and proteins.
• What is hydrophobic gel used for in chromatography?

  It is used for the separation of triglycerides and fatty acids.
• What is ion exchange chromatography based on?

  It is based on the exchange of sample ions and mobile phase ions with charged groups of the stationary phase.
• What does partition chromatography separate compounds based on?

  It separates compounds based on their partition between liquid mobile phase and a liquid stationary phase coated on a solid support.
• What is affinity chromatography used for?

  It is used to separate a few solutes from other unretained solutes using immobilized biochemical ligands as the stationary phase.
• What is the principle behind adsorption chromatography?

  Separation is based on the differences between adsorption and desorption at the surface of solid particles.
• What does fluorometry measure?

  It measures the amount of light intensity present over a zero background.
• What is the principle of fluorometry?

  It determines the amount of light emitted by a molecule after excitation by electromagnetic radiation.
• What light source is used in fluorometry?

  Mercury ARC or Xenon lamp at 365-366 nm.
• What are the light detectors used in fluorometry?

  Photomultiplier tube or phototube.