ANACHEM 2 PREFI

• Chromatography


  Physical method of separation that distributes components in a mixture between two phases which moves in definite direction

  
• Tswet


  Russian botanist (father of chromatography) credited for the development of chromatography
• Tswet Experiment


  1. Tall glass open column filled with sand-like particles

  2. Ground up plant extract

  3. Poured into the column and saw colored "bands" develop as the extract percolated down thru the column

  4. Different compounds have separated
• Chromatography (Greek)


  Chroma - color; Graphy - writing/study of
• Chromatography


  • Based on 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 - dictated by adsorption and solubility of the molecules
  • Substances will move with the mobile phase at different rate depending upon their Partition or Distribution coefficients
  • The phases are chosen such that components of the sample have different solubilities in each phase

  
• 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 [i.e. liquid (LC), gas (GC)]
  • Retention time (tR) - time takes for a particular analyte to pass through the system (fromthe column inlet to the detector) under set conditions
  • Dead or Void Time (tm) - the time between the sample injection and the appearance of the non retained solute peak or mobile phase that move through the column that reach the detector
  • 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 (1) width, (2) height, (3) 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
  • Liquid partition chromatography
  • The end of the paper is dipped in solvent mixture consisting of aqueous and organic components
  • The solvent soaks in paper by capillary action because of fibrous nature of paper

  
• Mechanism of Separation in Planar Chromatography

  1. Mobile phase rises up by capillary action

  2. Testing sample its concentrated as a minute spot at the bottom of the filter paper

  3. Sample mixture gradually rises with the mobile phase which is liquid

  4. Compounds in the mixture will be separated according to their ability of solubility

  5. More polar substances will move slower and less polar substances will travel faster

  
• Procedure for Planar 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 solute sample

  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
• 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

  
• 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)

  
• 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 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
  • Average rate of movement of the mobile phase molecule, 𝐮
• Retention Factor or Capacity Factor, k'

  • Widely used parameter in describing the migration rate of solutes on a column
  • The ratio between the adjusted retention time and the dead time
  • The longer A component is retained by the column, the greater is the capacity factor
• 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) on the column (measure of the retention of two species)
  • The greater the selectivity factor, the greater the separation between two components
  • Species A elutes faster than species B (small retention time)
  • 𝜶 = 1; solutes have identical retention time
  • The selectivity factor is always greater than one. 𝜶 > 1 means efficient separation
  • The greater the ratio of partition coefficient (K) between the mobile phase and stationary phases, the greater the separation between components
  • If t'RB will have a large value and t'RA will have a small value, the greater the separation between the two components
• Band Broadening


  • Term used to describe the overall dispersion or widening of a sample peak as it passes through a column (or separationsystem)
  • Efficiency of a chromatographic column is reflected by the amount of band broadening that occurs as solute/analyte passes through the column
  • Band Broadening reflects a loss of column efficiency
• Theories on Chromatographic Column Efficiency

  • Theoretical Plate Theory
  • Kinetic Rate Theory

  
• Theoretical Plate Theory


  • Chromatographic column contains a large number of separate layers, called theoretical plates
  • It is important to remember that the plates do not really exist
  • Measurement of column efficiency and band broadening: Number of theoretical plate in a column, N (the more plate, the better), The Plate Height (Height Equivalent to a Theoretical Plate, HETP or H) - It is the height equivalent of one theoretical plate, It is the length of the column that represents one theoretical plate, the smaller the value for H, the more efficient is the column, the more theoretical plates are packed into a length of column, Alternative expression for N and H, in terms of standard deviation of a peak, Experimental Evaluation of N and H - the number of theoretical plates that a real column possesses can be found by examining a chromatographic peak after elution

  
• Kinetic Rate Theory


  • Column efficiency and band broadening are affected by rate of elution and the paths available to solute molecules as they travel between particles of stationary phase
  • van Deemter equation for plate height - tell us how flow rate and column affect the plate height. All factors that cause band broadening such as (A)mobile phase mass transfer (Eddy Diffusion), (B) longitudinal diffusion, and (C)stationary phase mass transfer or resistance to mass transfer
  • A) Mobile Phase Mass Transfer (Eddy Diffusion) - The stationary phase is possibly made up various particle sizes and randomly arranged to constitute the packing material. Subsequently, mobile phase molecules eddied around these particles, following multiple paths, resulting to different flow rates. Consequently, solute molecules would elute at different rate, causing bands to broaden
  • B) Longitudinal Diffusion - This results in the migration of a solute from the concentrated center of band to more dilute regions on either side. Band broadening due to diffusion is very common to gas chromatography as gas molecules easily dispersed
  • C) Stationary Phase Mass Transfer or Resistance toMass Transfer - The thickness of the stationary phase may contribute to band broadening. A solute gets absorb into the matrix of the stationary phase, the rate at which it can easily diffuse out to the surface and dissolve into the mobile phase, depends on the thickness of the stationary phase (analyte may tend to have high affinity for stationary phase)

  
• Minimizing band broadening in chromatography

  1. Low or moderate flow rate of mobile phase

  2. Increasing linear velocity of gas molecules (increasing flow rate)

  
• Longitudinal diffusion


  • Migration of solute from concentrated center of band to more dilute regions on either side
  • Band broadening due to diffusion is very common to gas chromatography as gas molecules easily dispersed

  
• Stationary phase mass transfer or resistance to mass transfer

  • Thickness of stationary phase may contribute to band broadening
  • Solute gets absorbed into matrix of stationary phase, rate at which it can diffuse out depends on thickness of stationary phase
• Van Deemter plot


  Plot of plate height (H) vs. average linear velocity (u) of mobile phase

  
• Column resolution, Rs


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

  
• Baseline resolution is achieved when Rs = 1.5
• Obtaining high column resolution


  1. Increase number of theoretical plates (N) by reducing height equivalent to a theoretical plate

  2. Control capacity factor (k')

  3. Optimize k' and increase selectivity factor (α)

  
• Gas chromatography and high performance liquid chromatography

  Chromatographic techniques used for separation of volatile compounds

  
• Types of gas chromatography

  • Gas-solid chromatography
  • Gas-liquid chromatography

  
• Basic operation in gas chromatograph

  1. Inject volatile sample

  2. Vapor carried through column by carrier gas

  3. Separated analytes flow through detector

  4. Detected response displayed as chromatogram

  
• Capillary columns


  • Higher resolution
  • Shorter analysis time
  • Greater sensitivity
  • Lower sample capacity
• Protecting gas chromatography columns


  1. Installation of guard columns and retention gaps

  2. Use of high-quality carrier gases
• Stationary phase in gas chromatography


  Separates different components in a solution causing each to exit the column at a different time

  
• Choosing stationary phase


  • Related to chemical nature of solute and stationary phase
  • Must have different k (retention factor) for each solute
  • Retention factor must not be too large or small
  • Apply "like dissolves like" rule
  • Polar solutes need polar stationary phases
  • Non-polar solutes need non-polar stationary phases
  • Intermediate solutes need something in between
• Temperature and pressure programming in gas chromatography

  1. Varying temperature to improve elution of all compounds

  2. Varying pressure to reduce retention times of late-eluting compounds

  
• Carrier gas/mobile phase in gas chromatography

  Transports compounds but not involved in separation mechanism

  
• Sample injection in gas chromatography

  1. Using calibrated micro syringes or autosamplers

  2. Slow injection or oversized sample causes band broadening