Introduction to Separations

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Introduction to Separations

• Extraction Theory
• Types of Chromatography
• The Chromatogram and other Terminology
• Effectiveness of Chromatographic Separations
• Band Broadening (the van Deemter Equation)

Gas Chromatography A Practical Course by
Gerhard Schomburg (QD79.C45 S3913 1990) HPLC A
Practical User's Guide by McMaster. (QD79.C454
M36 1994) HPLC and CE Principles and Practice,
by Weston and Brown. (QD79.C454 H63 1997)
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Extraction Theory

• Extractions work based on the partitioning of the
  analyte from one phase (the sample phase) into
  the extractant phase (often an organic solvent).
• A chelator can be used to enhance this effect by
  making the analyte more soluble in the extractant
  phase.

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Evaluating Extractions..

• Distribution Constant (K)
• an equilibrium constant for the partitioning of
  an analyte between two phases.
• Higher K values mean more effective extractions

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Some thoughts

• You want to extract acetic acid from an aqueous
  solution into hexane. Should you do it at pH 3 or
  pH 8?
• You want to extract lindane (hexachlorocyclohexane
  ) from aqueous solution into an organic solvent.
  What might a good solvent be? Acetonitrile,
  isopropanol, hexane, methylene chloride or
  acetone.
• Is it better to do one extraction of a sample of
  water (say 100 mL water) with 100 mL of hexane
  once, or to extract it ten times with 10 mL of
  hexane each time and then combine the hexane?

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Chromatography

• Chromatos (color). Tswett, 1903 used
  chromatography to separate plant pigments
• In modern consideration of what chromatography
  is, we.
• Add the sample to a mobile phase (eluent) and it
  enters the column.
• The sample is separated into its components while
  on the column, usually after interacting with a
  stationary phase.
• The eluent (or mobile phase) then elutes from the
  column.
• The eluent (now containing separated analytes in
  bands) is normally passed through a detector for
  quantitative analysis. It can also be collected
  if we are just using chromatography for
  purification purposes..

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Types of Chromatography...

• Adsorption Analytes are separated by their
  adsorption onto the surface of a solid stationary
  phase.
• Partition Analytes are separated by their
  adsorption on a thin film of liquid coated onto
  stationary phase particles.
• Ion-exchange chromatography Ions are separated
  based on electrostatic attraction to a charged
  functional group on the stationary phase.
• Size Exclusion (or molecular exclusion) Analyte
  molecules are separated based on their ability to
  travel through small pathways in the stationary
  phase.
• Affinity Chromatography A specific molecule
  bound to the stationary phase is designed to
  react to some specific analyte and bind to it.

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Chromatography and the Chromatogram

• The chromatograph is the instrument, the
  chromatogram is the output!!! The latter is a
  plot of signal versus time after injection.
• Think of chromatographic separations as a process
  in which analytes spend more or less time
  (depending on how much they like it) in the
  stationary phase in the chromatograph. Those that
  spend more time in the stationary phase elute
  later. Those that spend less time elute more
  rapidly. All spend the same amount of time in the
  mobile phase
• The difference in these times (which are really a
  series of equilibrium reactions) determines
  elution order.

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Terminology of Chromatography

• tm time it takes for the unretained peak (just
  mobile phase) to elute. This is sometimes called
  the dead time or void time. The volume of eluent
  (mobile phase), that elutes during this time is
  equal to the volume of empty space in the column.
  It is also the time the analyte spends in the
  mobile phase.
• tr retention time time it takes for the analyte
  peak to elute after injection. Its total time in
  the chromatographic system (column).
• tr(or ts) adjusted retention time Tr - Tm
• The amount of time the analyte spends in the
  stationary phase.

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Migration of Solutes

• Distribution Constant (Kc)
• Equilibrium constant for analytes equilibrating
  between mobile and stationary phases.
• So, for different analytes, the difference
  between Kc values gives an indication of the
  differences in their retention times.

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Other Terms (many, many more)

• Average linear velocity of mobile phase
• Average linear velocity of solutes
• These can both be related to the distribution
  constant and the column volume, etc. (see pages
  925-926)

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Retention Factor (k)

• The ratio of the time the analyte spends in the
  stationary phase to the time it spends in the
  mobile phase. Greater k values mean the analyte
  spends more time in the stationary phase.

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• The selectivity factor (?) is the ratio of
  retention factors for different analytes in the
  same sample.
• A higher selectivity factor ratio means a greater
  separation between two peaks.
• Always greater than unity (1)
• So, if ? 1, you have no separation
• Larger ? values mean greater separation of peaks.

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Plate Theory of Chromatography

• The number of theoretical plates is equivalent
  to defining a term related to the width of a
  chromatographic peak as it travels through the
  column. The more plates, the narrower the width
  and the greater likelyhood that neighboring peaks
  will be separated.
• Also, if we consider a column to be a length of
  tubing, with some portion of the tubing (a
  cross-section) as a plate. The smaller in height
  each plate is, the greater the number of plates
  in the column!
• So, we define the Number of plates in a column
  (N) and the Height Equivalent of a Theoretical
  Plate (HETP).
• More plates a better separation.

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Experimental Determination of the of Plates.

• It is impossible, because plate theory is
  theoretical, to physically measure plates.
• However, we can make comparisons between analytes
  on a given column.
• You can increase the of plates by making them
  smaller (decreasing H) or by making the column
  longer (increasing L). Both have tradeoffs.

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Column Efficiency Mobile Phase Flow Rate

• Column length being constant, we can increase N
  by decreasing H (HETP)
• There is an optimal mobile phase (eluent) flow
  rate to allow solutes to equilibrate between the
  two phases in any column, and that is one way to
  minimize H.

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Reducing Band Broadening (reducing H) and the van
Deemter Equation.

• H is plate height in cm.
• B/? refers to logitudinal diffusion
• Cs? is the stationary phase mass transfer term
• Cm? is the mobile phase mass transfer term.

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• Longitudinal Diffusion refers to the general
  nature of a bubble of solute in a solvent to
  diffuse into that solvent (essentially expand to
  become larger). Solutes migrate from more
  concentrated to less concentrated regions in the
  solvent. The longer a chromatographic band stays
  in the mobile phase, the more broad it will get.
  A good reason to separate your peaks as soon as
  possible by optimizing the separation.
• Equilibration Time refers to the fact that the
  longer a band is on the column, the more mobile
  lt--gt stationary phase equilibrations there will
  be. The stationary phase is also not infinitely
  thin, so some analyte molecules take longer to
  equilibrate in and out of this phase than others.
  As a result, the band tends to spread out due to
  the fact that not all equilibration steps take
  exactly the same time
• Multiple paths refer to the fact that some
  individual molecules in the band may take a
  different path through the column (closer to the
  side, perhaps) than other molecules. Their
  velocities get spread out over some narrow
  range.
• Overcome by using a tubular column over a packed
  when possible
• Show drawing on board in class
• Tubular columns have fewer paths, greater N,
  lower HETP and better resolution, with a tradeoff
  in lower capacity and higher cost. They are
  longer than packed columns.

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As a peak moves down the column.
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Column Resolution

• Resolution is essentially the discriminating
  ability of a chromatographic separation.
• Resolution is how far apart bands are, relative
  to their widths.
• Greater resolution greater separation with the
  compromises
• The longer an analyte is on the column, the more
  the band will spread. You want to limit band
  broadening with shorter separations
• The longer a run takes, the less productivity or
  throughput you will have.

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General Elution Problem
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Overloading too much sample (fronted peak)!
Tailing stationary phase degradation...