Supercritical fluid chromatography

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					Supercritical fluid
   • Relatively new technique as an instrumental method

   •First suggested by Lovelock in 1958

   •First commercial instruments 1981 (packed columns)
    and 1985 (capillary column).

            gas and liquid
    • Is a hybrid of
    chromatography that combines some of the best
    features of both.
   SFC can be used to analyze classes of
    compounds that are not readily
    amenable to
       either gas chromatography (GC) or liquid
        chromatography (LC).

   These compounds includes

      nonvolatile or thermally labile and
     contain no chromophore that can be
      used for spectrophoto detection.
    Environment friendly method

   Supercritical fluids (SCFs) are increasingly
    replacing the organic solvents that are
    used in industrial purification and
    recrystallization operations because of
    regulatory and environmental pressures
    on hydrocarbon and ozone-depleting
            Fatty acid/lipid analysis

   Fatty Acid content of natural products is typically determined by GC    or
    HPLC techniques which both involve chemical modification of the

   •GC requires the derivatization of the fatty acids to
    methyl esters (FAME) leaving doubt as to whether the esters found are
    naturally occurring or formed through the derivatization process

   •SFC is capable of analyzing fatty acids without      derivatization
    as well as the natural occurring methyl esters

   SFC in the separation and analysis of lipids for separations of paraffin wax,
    free fatty acids, mono-, di- and triacylglycerols
    Phase diagram


            Supercritical fluid

     Critical temperature (Tc)

   Temperature above which a distinct liquid
    phase does not exist regardless of pressure.

   Where liquid and vapor have same density

   No phase change occurs above this point
               Supercritical fluid
   When a substance is brought above
       a particular temperature
       and pressure
   Both liquid and gas phases
       disappear
   And a new state generated called the

     supercritical       fluid is formed.
Principle of SFC
             Supercritical Fluid
   The mobile phase is a supercritical
    fluid (a fluid above its critical T and
    critical pressure)

   Supercritical fluid properties:
       density
       viscosity,
       refractive index which vary with T & P
            SFC Mobile Phases
   Mobile phases should have critical parameters
    that are easily reached using chromatographic
    pumps and ovens common to currently used

   Advantages of supercritical fluids over carrier
    gasses and liquid mobile phases are in its
    solubility properties, physical properties, and
    detector compatibility.
         SFC Separations

   SFC is a hybrid of gas and liquid
    chromatography that combines some of
    the best features of each

   As in HPLC, variation of the mobile phase
    composition affects separation
   In SFC, mobile phase affinity for the analyte is
    a function of mobile phase density

   Density is controlled by controlling system
    pressure / tmeperature

   Highly polar samples are not easy to handle
    (high critical parameters & high reactivity)
       SFC Advantages vs HPLC
   Supercritical fluids have low viscosities
       - faster analysis (5 to 10 X faster)
       - less pressure drop across the
    column - the use of open tubular
    columns is feasible
   Column lengths from 10 to 20 m are
   Can be used with a wide range of
    sensitive detectors
   Resolving power is ~5X that of HPLC
               SFC Advantages
   • 1. SCFs have solvating powers similar to liquid
    organic solvents, but with higher diffusivities, lower
    viscosity, and lower surface tension.

   • 2. Since the solvating power can be adjusted by
    changing the pressure or temperature separation of
    analytes from solvent is fast and easy.

   • 3. By adding modifiers to a SCF (like methanol to
    CO2) its polarity can be changed for having more
    selective separation power.
   4. In industrial processes involving food or
    pharmaceuticals, one does not have to
    worry about solvent residuals as you
    would if a "typical" organic solvent were

   5. Candidate SCFs are generally cheap,
    simple and many are safe. Disposal costs
    are much less and in industrial processes,
    the fluids can be simple to recycle.
        Important Properties of
          Supercritical Fluids
   – remarkable ability to dissolve large, non-volatile
        e.g. supercritical CO2 can dissolve n-alkanes
    containing over 30 carbon atoms

   – dissolved analytes are easily recovered

       equilibrate with atmosphere at relatively low temperatures

       e.g. analyte in supercritical CO2 can be recovered by reducing
        the pressure and allowing the CO2 to evaporate
   – no need for organic solvents
    environmentally friendly

   – inexpensive, innocuous and non-toxic

   – higher diffusion coefficients and lower
    viscosities relative to liquids faster and
    higher resolution separations
    Supercritical Fluid Extraction
Supercritical Fluid
based on the fact
near the   critical
point of the
solvent, its
properties change
rapidly with only
slight variations
of pressure/Temp.

                      Molecular basis of SFE
            SFC components

   1. SFC Columns:
   2.SFC mobile phase
   3.SFC injection
   4.SFC pump
   5. SFC detector
                 1. SFC Columns:

     There are two types of analytical columns used in SFC.
     Capillary columns of fused silica coated with cross-linked
     chemically bonded stationary phases, that are used in
     GC are equally applicable in SFC.

     Also packed columns developed for highperformance
    liquid chromatography (HPLC) are being used with SFC.

     The columns are conventionally stainless steel.
         2. SFC Mobile Phase:
   CO2 is the primary mobile phase used in SFC.

   The advantage of CO2 as the mobile phase is
    low cost, low interference with chromatographic
    detectors, nontoxicity, low critical temperature
    (31.1 oC), inflammability and that it can permit a
    flame ionisation detector to be used, with all the
    benefits in terms of ease of use, linearity and
    sensitivity that we have come to expect of this
    device in GC applications.
            2. SFC Mobile Phase:
   Disadvantage of carbon dioxide is its inability to elute very   polar or
    ionic compounds.

   By adding a small portion of a   second fluid, modifier, this can be

    Modifiers are generally an organic fluid (such as alcohols and
    cyclic ethers and water) which are completely miscible with carbon

   Modifiers improve the solvating ability of the SCF and sometimes enhance
    separation selectivity.

   A modifier fluid is commonly used, especially in packed column SFCs.
   Supercritical fluids can be used to extract analytes from samples.

   The main advantages of using supercritical fluids for extractions
    is that they are
         extract the analytes faster and
         more environmentally friendly
         than organic solvents.

For these reasons

    supercritical fluid CO2 is
   the reagent widely used as the supercritical solvent.
            3. SFC Injection:
   For packed SFC a typical LC injection valve is
    commonly used.

   In capillary SFC small sample volumes must be
    quickly injected into the column and therefore
    pneumatically driven valves are used.

   The ovens used in SFC are generally
    conventional GC or LC ovens.
                  4. SFC Pump
   The type of high pressure pump used in SFC is
    determined by the column type.

   For packed columns reciprocating pumps are generally
    used while for capillary SFC syringe pumps are used.

   Reciprocating pumps allow easier mixing of the mobile
    phase or introduction of modifier fluids. Syringe pumps
    provide consistent pressure for a neat mobile phase.
                5. SFC Detector:
   Optical detectors, flame detectors and spectroscopic detectors can
    be used.

   However, the mobile phase composition, column type and flow rate
    must be taken into account when the detector is selected.

   Some care must also be taken such that the detector components
    are capable of withstanding the high pressures of SFC.

   In practice, SFC operates at low to moderate temperatures and
    seems most suited to the analysis of heat-sensitive compounds of
    high molecular weight such biological fluids.

   The technique is therefore an alternative to high-temperature GC
    and to HPLC.
Multicomponent separation

 Applicable to a class of compounds that is not
 readily amenable to either gas-liquid or liquid

    These compounds are nonvolatile or thermally
    unstable and, non-photometric detected.

 Separation of these compounds is possible with
supercritical-fluid chromatography at temperatures below
  100 oC.
    Natural Products separation and

   Lipophilic – amphiphilic compounds with
    properties between volatiles and hydrophilic

       Carotenoids,
       Tocopherols
       Vitamins
       Phenolics
        Pesticides Separation and

   Chromatography has been used for the
   analysis of pesticide residues in canned
   foods, fruits and vegetables.

       Pyrethroids
       herbicides
       fungicides
       and carbamates have been analyzed.
Drugs Separation and Analysis

   Modern drug substances are commonly
    nonvolatile and thermally or chemically labile
    therefore analysis by HPLC is common over GC
   e.g...
       Phenothiazine
       Antipscychotics
       Beta blockers
       Felodipine
       Anticancer drugs like Cyclophosphamide,

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