Chrom Basic Principles

• Chromatography is a physical method of separation that distributes components in a mixture between two phases which moves in a definite direction

  
• Chromatography


  Based on the principle of partition of solutes in a mixture between two phases

  
• Separation Principle


  • Differential affinities of the various components of the analyte towards the stationary and mobile phase
  • Affinity is dictated by adsorption and solubility of the molecules
  • Substances will move with the mobile phase at different rates depending upon their Partition or Distribution coefficients

  
• Phases


  The phases are chosen such that components of the sample have different solubilities in each phase

  
• Purpose of Chromatography

  • Analytical - to determine chemical composition of a sample mixture
  • Preparative - to purify sufficient quantities of a substance

  
• Chromatographic Terms

  • Chromatograph - equipment that enables a sophisticated separation
  • Eluent - fluid entering column/solvent that carries the analyte
  • Eluate - mobile phase leaving the column
  • Stationary Phase - immobilized phase
  • Mobile Phase - moves in a definite direction (liquid (LC), gas (GC))
  • Retention time (tR) - time takes for a particular analyte to pass through the system
  • Dead or Void Time (tm) - the time between the sample injection and the appearance of the non retained solute peak or mobile phase
  • Ts - time duration of the analyte retained in the stationary phase
  • Sample (Analyte) - substance analyzed in chromatography
  • Solvent - any substance capable of solubilizing another substance

  
• Chromatogram


  • Visual output of the chromatograph
  • Graphical presentation of detector response, concentration of analyte in the effluent, or other quantity used as measure of effluent concentration
  • Display series of peaks referred as chromatographic peaks
  • Peaks are described in terms of width, height, and area
  • Used to identify and quantify the solute

  
• Classification of Chromatography

  • Based on shape of chromatographic beds: Planar Chromatography, Column Chromatography
  • Based on the physical state of mobile and stationary phase: Liquid Chromatography, Gas Chromatography, Supercritical Fluid Chromatography
  • Based on mechanism of separation: Ion-exchange Chromatography, Partition Chromatography, Affinity Chromatography, Adsorption Chromatography, Size exclusion Chromatography

  
• Planar Chromatography


  • Stationary phase is present on a plane
  • The plane can be a paper (paper chromatography) or thin layer of a substance spread on a glass, metal or plastic plate (thin layer chromatography, TLC)
  • Known as open bed chromatography

  
• Paper Chromatography


  1. Liquid partition chromatography

  2. The end of the paper is dipped in solvent mixture consisting of aqueous and organic components

  3. The solvent soaks in paper by capillary action because of fibrous nature of paper
• Mechanism of Separation in Paper Chromatography

  • Mobile phase rises up by capillary action
  • Testing sample is concentrated as a minute spot at the bottom of the filter paper
  • Sample mixture gradually rises up with the mobile phase which is liquid
  • Compounds in the mixture will be separated according to their ability of solubility
  • More polar substances will move slower and less polar substances will travel faster

  
• Procedure for Paper Chromatography

  1. A small spot of sample is applied to a strip of chromatography paper about 2 cm away from the base of the plate

  2. This sample is absorbed onto the paper and may form interactions with it

  3. The paper is then dipped into a solvent, taking care that the spot is above the surface of the solvent, and placed in a sealed container

  4. The solvent moves up the paper by capillary action and dissolved the sample mixture, which then travel up the paper with the solvent

  5. Different compounds in the sample mixture travel at different rates

  6. Analysis - spot corresponding to different compounds may be located by their color, UV light, Ninhydrin or by treatment with iodine vapors

  
• Thin Layer Chromatography (TLC)


  • Used to identify unknown compounds and determine the purity of the mixture
  • Stationary phase consists of a thin layer of adsorbent material, usually silica gel, aluminum oxide, or cellulose immobilized onto a flat carrier sheet
  • Mobile phase consisting of the liquid solution (volatile organic solvent) that is drawn up the plate via capillary action

  
• Advantages of TLC over Paper Chromatography
• Relative Front/Retention Factor Value (Rf value)


  • The rate of migration of the various substances being separated are governed by their relative solubilities in the polar stationary phase and non polar mobile phase
  • The migration rate of substances usually expressed as Rf (relative front value)
  • Rf = distance travelled by the substance / distance travelled by the solvent front
• Column Chromatography


  • Stationary phase is held in a narrow tube through which the mobile phase is forced under pressure
  • Mobile phase may be gas (gas chromatography), liquid (liquid chromatography), supercritical fluid (supercritical fluid chromatography)
  • When the stationary phase in LC consists of small-diameter particles, the technique is High Performance Liquid Chromatography (HPLC)

  
• Supercritical Fluid


  A highly compressed fluid that combines the properties of gas and liquid
• Elution in Column Chromatography

  1. Isocratic elution

  2. Gradient elution

  
• Chromatogram


  • The position on time axis may serve to identify the components of the sample
  • The peak is symmetrical like a Gaussian Peak in statistics
  • The areas under the peaks provide quantitative measure of the amount of each component

  
• Asymmetric Chromatographic Peaks

  • Peak tailing
  • Peak fronting
• Factors affecting clean separation or resolution in Column Chromatography

  • Migration rates of solutes
  • Band Broadening

  
• Migration Rates of Solutes


  • Depends on the magnitude of the equilibrium constant (also known as distribution constant, partition constant, or partition coefficient, K) by which the solutes distribute between the mobile and stationary phases
  • Average rate of solute migration, v = u * (1 / (1 + k'))
  • Average rate of movement of the mobile phase molecule, u

  
• Relationship between Retention Time and Distribution Constant

  • Retention time = u * (1 / (1 + k'))
  • Retention factor or Capacity factor, k' = (tR - tM) / tM
  • Ideally, separation is performed at 1 < k' < 10
• Selectivity Factor, α


  • Also known as relative retention, ratio of the distribution constant of the strongly retained specie (KB) and the less retained specie (KA)
  • Describe the separation of two species (A and B)
• Column Chromatography


  1. Migration Rates of Solutes

  2. Retention Factor or Capacity Factor, k'

  3. Selectivity Factor, α

  4. Band Broadening and Column Efficiency
• Retention Factor or Capacity Factor, k'


  Ratio between the adjusted retention time and the dead time for solute A
• Selectivity Factor, α


  Also known as relative retention, ratio of the distribution constant of the strongly retained specie (KB) and the less retained specie (KA)
• Selectivity Factor, α


  • Describe the separation of two species (A and B) on the column (measure of the retention of two species)
  • The greater the selectivity factor, the greater the separation between two components
  • The greater the ratio of partition coefficient (K) between the mobile phase and stationary phases, the greater the separation between components
• Band Broadening


  Term used to describe the overall dispersion or widening of a sample peak as it passes through a column (or separation system)
• Column Efficiency

  • Reflected by the amount of band broadening that occurs as solute/analyte passes through the column
  • Band Broadening reflects a loss of column efficiency
• Theoretical Plate Theory


  1. Number of theoretical plates in a column, N

  2. Plate Height (Height Equivalent to a Theoretical Plate, HETP or H)
• Theoretical Plate


  Chromatographic column contains a large number of separate layers, called theoretical plates
• Number of Theoretical Plates, N

  • The more plates, the better
  • Ideal value for N > 2000
• Plate Height, H


  • The height equivalent of one theoretical plate
  • The smaller the value for H, the more efficient is the column
• Kinetic Rate Theory


  1. Mobile Phase Mass Transfer (Eddy Diffusion)

  2. Longitudinal Diffusion

  3. Stationary Phase Mass Transfer or Resistance to Mass Transfer
• Van Deemter Equation


  Tells us how flow rate and column affect the plate height
• Column Resolution, Rs


  Quantitative measure of column ability to separate a mixture into a series of chromatographic peak

  
• Baseline resolution is achieved when R = 1.5
• To obtain high resolution, the number of theoretical plates (N), capacity factor (k'), and selectivity factor (α) must be maximized
• Basic Instrumentation