Lab Liquid Chromatography

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Lab Liquid Chromatography Powered By Docstoc
					     Lab: Liquid
         Prelab discussion

*** this is an AP lab, but we’ll refer
            back to it in IB
        Liquid Chromatography
 • Analytical technique to separate
   components of a mixture

 • The components will be separated into
   bands that flow through the system at
   different rates.

chromato- which means "color" and -graphy which means "the field of study"
    Liquid Chromatography
• Components become separated between
  2 phases: stationary phase and mobile
  phase of chromatography system.
  – Moving phase = gas or liquid (solution to
    be separated is usually combined with
    mobile phase)
  – Stationary phase = solid (usually is polar,
    whereas solvent = nonpolar)
       Reverse Phase Liquid
       Chromatography (RPC)
• Stationary phase = nonpolar solid
• Mobile phase = polar solvent

• The mixture will be injected into an RPC column
  – More polar components attracted to polar solvent
    (move quickly through column with the solvent
  – Less polar components will move more slowly (spend
    more time absorbed onto nonpolar column medium)
       (= a band of colors for this lab: red and blue to make purple
       Kool-Aid )
         – See figure in lab handout
            Grape Kool-Aid
• Purpose: separate components of grape
  Kool-Aid using liquid chromatography
  – Kool-Aid is made up of many components:
    most importantly 2 dyes (red & blue)
    • RPC column = Sep-Pak C18 (nonpolar)
       – Column is a piece of equipment you will push sample
    • Part 1: use dilute isopropyl alcohol as the solvent
      (ELUANT: solvent used to extract something from
      another) & take measurements
      Measurements & Calcs.
• Use Sample Data given
a. Bandwidth, W (volume in mL)
      Calculate the W for each run, determine the
      average (Do this for BOTH dyes: red & blue)
b. VRave (average retention volume)
  Calculate the V Average Rave by using the Average
      Start Volume and adding it to 0.5 x Wavg that you got
      in a (above).
c. k’ capacity factor
d. …
e. …
***the lab walks you through the calculations
  Part 2: observations…
• use 4 eluants (solvents) of varying polarities to
  separate more components of Kool-Aid (citric acid,
  salt, polar dyes, nonpolar oils) 
          Polarity of isopropyl alcohol
• Isopropyl alcohol differs in polarity from water in that it is completely
  surrounded by hydrogen, making an even distribution in electrons so
  there is no difference in charge, and likewise very little polarity
  compared to water, whose hydrogens are all on one side.

• Since the electrons are pulled from the hydrogen, this side becomes
  very negative which makes water polar. Also, since water basically
  has two –OH groups
  on one side and
  isopropyl alcohol only
  has one (the
  hydrocarbons do not add to a charge
  since the hydrogen’s charges cancel
  each other out around their respective
  tetrahedrons), water is much more polar.
• The four eluting solvents (of varying polarity)
  decrease in polarity from one run to the next
  (water and then isopropyl alcohol starting from
  5%, 18%, 28%, to 70% being least polar since it
  has the most isopropyl alcohol and least amount
  of H2O).
• The water removed the most polar compounds
  from the column, and as the eluting solvent
  decreased in polarity, the less polar compounds
  emerged since ”like dissolves like.”

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