Advances in chromatography by frndzzz

VIEWS: 43 PAGES: 136

									  Advances in

    Head space analysis in gas
    Chiral chromatography,
    Ion pair chromatography
    Affinity chromatography
    Supercritical fluid chromatography
    Advantages and disadvantages of
    HPTLC over HPLC
         Gas chromatography

   Mobile phase is Gas

   Stationary phase is liquid/ solid
   Vaporization of sample

    • Gas-solid
         Physical absorption

    • Gas-liquid
         Liquid immobilized on inert solid
   • Carrier gas
    • He (common), N2, H2
    • F=25-150 mL/min packed
   column
    • F=1-25 mL/min
    • Column
          2-50 m coiled stainless
    • steel/glass/Teflon
   Oven: 0-400 °C ~ average boiling point of
   Detectors
    • FID, ECD, MS
     Flame ionization detector
    Rugged
    Sensitive
    Wide dynamic range
    Signal depends on C atoms in organic
    mass sensitive, not concentration

   Weakly sensitive
    • carbonyl, amine, alcohol, amine groups

   Not sensitive
    • H2O, CO2, SO2, NOx
      Electron capture detector
   • Electrons from radioactive source
    Organic molecules capture electrons
    Simple and reliable
   Sensitive to electronegative groups
    • halogens, peroxides
   Insensitive to amines, alcohols
   Largely non-destructive
   Limited dynamic range
    Thermal conductivity detector
    Change in gas thermal conductivity
    Difference between carrier gas and
    Thermal conductivity of He, H2 much
    larger than organics
    • Organics cause T rise in filament
   Rugged

   Wide dynamic range
   Packed
    •  liquid coated silica particles (<100-300
      mm diameter) in glass tube
    • best for large scale but slow and

   Capillary/Open Tubular
    • wall-coated (WCOT) <1 mm thick liquid
      coating on inside of silica tube
    • support-coated (SCOT) 30 mm thick
      coating of liquid coated support on inside
      of silica tube
    • best for speed and efficiency but only
      small samples
        Head space analysis
   The 'headspace' is the gas space in a
    chromatography vial above the

   Headspace GC is used for the
    analysis of volatiles and semi-volatile
    organics in solid, liquid and gas

   For analyses of alcohols in blood and
    residual solvents in pharmaceutical
Head space vial
   Other common applications includes:
    • industrial analyses of monomers in
     polymers and plastic

    • Flavour compounds in beverages and
      food products

    • Fragrances in perfumes and cosmetics.
   Very light volatiles in samples that
    can be efficiently partitioned:-

    • into the headspace gas volume from
     the liquid or solid matrix sample.

    • Higher boiling volatiles and semi-
      volatiles are not detectable with this
      technique due to their low partition in
      the gas headspace volume.
   Complex sample matrices, which
    would otherwise require sample
    extraction or preparation, or be
    difficult to analyse directly, are ideal
    candidates for headspace since they
    can be placed directly in a vial with
    little or no preparation.

   This saves both time and money.

                   A headspace sample is normally
                   prepared in a vial containing the
                   1. Sample
                   2. The dilution solvent
                   3. A matrix modifier

Volatile components from complex sample
mixtures can be extracted from non-volatile
sample components and isolated in the headspace
 or gas portion of a sample vial. A sample of the gas
 in the headspace is injected into a GC system for
separation of all of the volatile components.
Phases of the Headspace Vial

   G = the gas phase (headspace)
    The gas phase is commonly referred
    to as the headspace and lies above
    the condensed sample phase.

   S = the sample phase
    The sample phase contains the
    compound(s) of interest. It is usually
    in the form of a liquid or solid in
    combination with a dilution solvent
    or a matrix modifier.
           Partition coefficient
   The partition coefficient is the equilibrium
    distribution of an analyte between the
    sample phase and the gas phase.

   Samples must be prepared to maximise
    the concentration of the volatile
    components in the headspace.

   To determine the concentration of an
    analyte in the headspace, it is necessory
    to calculate the partition coefficient (K).
      Calculating the Partition
   Partition Coefficient (K) = Cs/Cg

    where :-

    Cs is the concentration of analyte in
    sample phase;

    Cg is the concentration of analyte in gas

K values of common solvents in
  air-water systems at 40°C given in
      Solvent            K- value

      Cyclohexane         0.077
       n-hexane           0.14
  Tetrachloroethylene     1.48
1,1,1-trichloromethane    1.65
        o-xylene          2.44
        Toluene           2.82
        Benzene           2.90
   Dichloromethane        5.65
    n-butyl acetate       31.4
     ethyl acetate        62.4
  methyl ethyl ketone     139.5
       n-butanol           647
      Isopropanol          825
        Ethanol           1355
        dioxane           1618
Instrumentation for head space
   There are three types of headspace

• 1. Syringe Injection

• 2. Balanced Pressure

• 3. Pressurised Loop
1. Syringe Injection
   The syringe is heated and agitated in
    an oven for a pre-defined period of
    time (step 1).

   The heated syringe then removes an
    aliquot of the headspace (step 2)

   Injects it directly into the GC (step
   The syringe must be heated above the
    temperature of the oven:-

    • to avoid the risk of condensation

    • and hence carry-over from one sample to the

   After injection, the syringe is flushed with
    nitrogen or other inert gas.
    2. Balanced Pressure System

   This technique uses a seamless
    injection directly from the vial into
    the carrier gas stream without
    additional moving parts other than a
    valve and a needle.

   It is illustrated in the figure below.
   The sample reaches equilibrium (step 1)

   During these initial steps, a needle is
    inserted into the vial and is then
    pressurised with a carried gas (step 2)

   After the vial is pressurised and
    equilibrium has been reached, the valve is
    switched for a specific amount of time to
    redirect the sample into the transfer line
    and onto the column (step 3)
    3. Pressurised Loop System

   This technique typically uses a six-
    port valve, as shown in the figure
   It initially thermostats and
    pressurises the vial (step 1).

   After pressurisation, the valve is
    turned and the loop is filled with the
    sample (step 2).

   Once the loop has been filled, the
    valve is turned again to redirect the
    gas flow and flush the sample into
    the transfer line leading to the
    analytical column (step 3).
Principle of HS analysis


       Sample transfer
    Multiple headspace extraction
   When two consecutive aliquots taken
    from the same head space vial under
    identical conditions:-

    • Theoretically the two analysis should
      give the same results (peak areas) but

         The peak area is smaller in second analysis
          than first analysis
            Principle of MHE
   Although the partition coefficient k
    remains constant, the peak area of
    the second analysis is lesser than
    peak area obtained in the first

   If continue successive aliquots
    withdrawn from head space vial,
    eventually total concentration
   Thus the sum of the amounts of
    analyte removed in each extraction
    will be equal to the total analyte
    present in the original sample.

   This is called multiple headspace
    extraction method

   Advantage:

    • Complete extraction
    • Minimizing matrix effect
   During the consecutive analyses, the
    obtained peak areas are

           A1, A2, A3…so on

    The sum of these peak areas is
    proportional to the total sample
    present in the sample phase of
    headspace vial:

      ΣAi = A1 + A2 + A3 +…….

3 extraction
                 2 extraction   1 extraction
          Calibration of MHE

   The actual amount of the analyte is
    established by proper calibration.

   There are three ways to do this
    • 1.External standard
    • 2.Internal standard
    • 3.Standard addition
         1. External standard
   External standard is used

   MHE measurements with this
    standard sample will give the sum of
    peak area (ΣAex) corresponding to
    the analyte present in the standard
   Finally the analyte concentration in
    sample is calculated using following
            2. Internal standard
   In this case both analyte and internal
    standard undergoes multiple gas

   The calculation of the amount of
    analyte is carried out by the sum of
    the respective peak areas used

       fi is the response factor
   Response factor can be calculated

       Where superscript c indicates that these values refer
       to the separate calibration measurement
          3. Standard addition
    The known amount of the standard added
    to the sample

   The measurements are carried out under
    identical conditions

   No response factor is needed

   Peak area and amount is proportional,
    from this relationship we can determine
    the unknown sample concentration
           1. Single addition
   What happens when known amount
    of analyte added.

    Wo the original amount present
   Ao Area of original amount present
   Wa amount of analyte added to
    original sample, now area of peak is
   Based on the proportionality between
    analyte amount and peak area, we
    can write:
Determination of camphor content
  by standard addition method
   A = 1gm of camphor

   B = 1gm camphor+ 5 mg addition of
    known amount of camphor

   ΔA =   Increase in a peak area due
    to         addition of 5mg
    2. Handling of added standard
          (GPA and SPA)
   Advantageous:
    Additional amount is added into either Gas
    Phase and Sample phase

This is the two way process:

    Analyte moves from Sample to headspace
    and vice-versa

    At equilibrium both movements
    compensate each other
        Gedanken experiment
A. Sample phase addition:

   To understand this a series of headspace
    vials are taken

   Pure matrix (sample phase) added into

   Analyte of definite concentration added

   All vials kept for progressive period of
    time in thermostat
   Conclusion:
    • At start no analyte is present in

    • As the thermostatting time increases,
      the more and more analyte diffuse till
      the equilibrium

    • Then on the amount of the analyte in
      the headspace remains constant
      regardless of thermostatting
Diagram for equilibrium
         B. Gas phase addition
   Next we repeat the experiment that the
    analyte is added into the headspace/ gas

   No analyte is present in sample phase

   Analyte concentration decreases in
    headspace until the equilibrium reached

   Then on, the concentration remains
    constant regardless of thermosatting time
   In general we use the SPA technique
    for liquid samples

   GPA technique is used for solid
3. Determinations of multiple

  Original             Sample +
  sample          Added known amount

   If the increasing amount of the
    analyte is added to the original
    sample, the results are evaluated by
    regression analysis

To explain the evaluation of multiple
 measurements with increasing
 amounts of addition, we start the
 following equation:
   Reorganizing the above equation:-

        The above equation corresponds to the straight line equation

        y= ax + b
   Carrying out the no. of additions, the
    data (Wa vs A(o+a)) can evaluated by
    linear regression analysis; then from
    the slope a and intercept b of the
    linear plot,

    value of Wo, the amount of analyte
    in the original sample can be

    • Wo = b/a
        1. Analysis of solid samples
            (Adsorption system)

                                 As the liquid displacer/ modifier added,
Major                 Head
Displacers:           space
                                 the adsorbed analyte releasing into liquid

Benzyl alcohol        Liquid/    and now Partitioned into liquid and head space
Benzoic acid esters              When the modifier conc. Increases , the solid
Dimethyl acetamide               Analyte remains as suspension in liquid,

                       Solid     that’s why it is also known as SUSPENSION
1.   Methyl ethyl ketone
2.   Trichloroethane
3.   Toluene
4.   n-octane

                           B= only 400mg activated charcoal+ 4 compounds

                           A= only 400mg activated charcoal+ 4 compounds
                               + displacer (2ml benzoic acid esters)

                           1hr equilibrium at 80 0C, the head space vial
Recovery of the compounds
        2. Surface modification
   Disadvantage of suspension

    • Dilute with liquid reduce sensitivity
    • Impurities of solvent interfere analysis

   Here liquid displace is much better
    referred as “Liquid modifier”
 Example: determination of 1.9% isopropyl
 alcohol and 5.2% water in a drug powder

                                         Peak 2 is
              Ethyl cellosolve


1. Figure A shows a nonlinear behavior in low conc. Region due to increase adsorptivity

for low conc., very small peak for IPA

2. Figure B, glycerol added as modifier, better sensitivity observed and MHE plot is
     Ion-pair chromatography

   Similar to reverse phase

   Difference only the addition of ion
    pairing agent to the mobile phase in
    ion-pair chromatography

   When 2>pKa>8, then ion pairing
    agents can be used
Without ion-paring agent

As pH increases RT decreases
With positive ion pairing agent

Tetra butyl ammonium (TBA+)

RT increases as pH increases
Effect of ion pairing agent on

         More hydrophobic, strongly retained, less conc.
Retention as a function of ion-pair
     reagent concentration
Solvent strength relationship for ion
            pair HPLC
Effect of solvent strength on band
spacing of ion-pair chromatograph
Effect of temperature
 Branch of science which deals the
 structures of compound in three
 Isomers: same formula,
             but contain a different
             arrangements of atoms
             and different chemical
             and physical properties

   Enantiomers
   Diastereomers
   Meso compounds
   Optical rotation
   Racemic mixture
   Resolution
               Relationships of Stereoisomers
                                                 Isomers: Compounds with the
                                                    same molecular formula
                                                     Same atom            Different atom
                                                    connectivity          connectivity

                                              Stereoisomers                    Constitutional (or structural)
                       Interconvert through                 Not readily
                       rotation about a                     Interconvertible
                       single bond

                        Conformational                      Configurational
                     isomers or rotamers                       isomers
                                                                                         Constitutional (structural)
                                                           w/o            w/
                           chiral centers (opt. inactive)                 chiral centers (optically active)
                                mirror images at this carbon
Conformational isomers
                                Not mirror images at this carbon                       Chiral
                              Geometric isomers
       Not mirror images
                                                                   Diastereomers                Enantiomers
  Diastereomers                      Cis, Trans
                                    (E,Z) isomers
     cis and trans isomers
                                                                                                             mirror images
   Enantiomers are the 2 mirror image forms of a
    chiral molecule which are not superimposable

    • can contain any number of chiral centers, as
      long as each center is the exact mirror image
      of the corresponding center in the other

    • Identical physical and chemical properties, but
      may have different biological profiles.

    • Different optical rotations (One enantiomer is
      (+) or dextrorotatory (clockwise), while the
      other is (-) or levorotatory (counter
               Enantiomers of 2-hydroxy propane

   Mirror image of each other but not

         CH3                                      CH3

    H             OH                    HO               H

         C2H5                                     C2H5
Lactic acid
Ordinary light
Plane Polarized Light
Rotation of light by Optically active compound
Optical rotation
Example: Tartaric acid
Meso-tartaric acid
   stereoisomers that are not enantiomers
    Diastereoisomers may be chiral or achiral

    • Have different chemical and physical
      characteristics, and can be separated by
      non-chiral methods.
    • Has at least 2 chiral centers;
    • the number of potential diastereomers
      for each chiral center is determined by
      the equation 2n, where n=the number of
      chiral centers
         Racemic mixture/Racemate
     1:1 mixture of enantiomers.
   Contain both enantiomer in equal
   One is R and other S
   R rotate PPL in clockwise direction
   S rotate PPL in anticlockwise direction
   Finally optical rotation is Zero
   Both enantiomers are not easy to separate
    due to same physical chemical properties
Racemic drugs
Importance of enantiomers as drugs
       Chiral chromatrography
   Single enantiomer always better

   enantioselective analysis by
    chromatographic methods has
    become the focus of intensive
    research of separation scientists.
           Stationary Phases
   New chiral stationary phases pioneered by

   Chiral agents are derivatized and
    immobilized on silica support

   And served as the basis of chiral
    separation in chiral chromatography
   chiral stationary phases contribute to
    nature of molecular recognition.

          differential retention of
    Since the
    enantiomers in the chromatographic
    system employing chiral stationary phases

   can be attributed only to chiral
    discrimination by the chiral sites

   chiral stationary phases can be sensitive
    to very subtle differences between the
classification of types of
chiral stationary phases
A. Chiral affinity by proteins
   (serum albumin, a1-acid glycoprotein, ovomucoid and

B. Stereoselective access to helical chiral polymers
   (derivatized or free polysaccharides).

C. Steric interactions between pi-Donor pi-acceptor type
   of chiral aromatic amide groups (Pirkle).

D. Host-guest interactions inside chiral cavities
   (cyclodextrins, crown ethers and imprinted polymers).

E. Ligand exchange (copper ions complexed with chiral moieties).

Most widely used A-D
           SILICA GEL
   use of protein immobilized to the surface of silica gel, or other
    support, as the chiral discriminator.

   Many small chiral biomolecules have shown stereoselective affinity
     serum albumin and to a1-acid
   the mobile phases are mostly aqueous buffers containing a limited
    percentage of organic modifiers.

                                     even very
    When the protein stationary phases are efficient,
    small differences in binding affinity of the
    enantiomers to the protein give rise to resolution between them.
   Polysaccharides such as cellulose and amylose
    consist of D glucose units linked by 1-4 glucosidic
    bonds, forming the natural polymers with a
    highly ordered helical structure.

   The three hydroxyls on each glucose unit
    can be derivatized to form strands around the
    chiral glucose.

   The derivatized glucose unit can in principle
    act as a chiral site discriminating between
    enantiomers that interact differently with the
   The acetate ester, bezoate ester, or
    phenylcarbamate derivatives of
    glucose, have shown better performance.
   Mobile phases are usually organic, normal phase
    type solvents, however, aqueous solvents can
    also be used in many versions of the stationary
Figure illustrates the structure of a glucose
unit of the amylose based stationary phase,
derivatized with dimethylphenylcarbamate.
   The structure provides the possibilities of p-p
    interaction of aromatic groups with the aromatic
    amide at the chiral site with the amide groups.

   The discrimination is affected by the steric fit in
    the cavity.
   An example of the separation of enantiomers of
  cannabidiol, one of the substances in Marijuana is
           shown in the following Figure.

the type of hydrogen bonding groups on the enantiomers can be very
functional in the chiral recognition that is responsible for the separation
            C. CHIRAL CAVITY

   Resposible for stereoselective guest-host
    interactions govern the resolution.

   such stationary phases is the proper fit of the
    molecule to the chiral cavity in terms of size and

   This category of stationary phases includes crown
    ethers, imprinted polymers and cyclodextrins.

    A majority of pharmaceutical applications were
    accomplished using cyclodextrins, and therefore,
    the discussion is concentrated on them.
   The monomers are arranged so that
    a shape of a hollow truncated cone is

    A relatively hydrophobic chiral cavity
    is formed, comprised of essentially
    methylene and 1,4 glucoside bonds,
    with which the intercalated solute
   Cyclodextrins
    Cyclodextrins are macrocyclic molecules
    containing 6, 7 and 8 glucopyranose
    units (a-, b-, g- cyclodextrin
    respectively), as shown in following figure
     D. p-DONOR p-ACCEPTOR -
           PIRKLE TYPE
   Chiral stationary phase preceded all the others described

   The pioneering work of Pirkle had such an impact on the
    field that the whole category of donor-acceptor type
    stationary phases was named after him.

    The structure of these type of stationary phases is based
    on single strands of chiral selectors,
    connected via amidic linkage onto aminopropyl
    silica as shown in Figure
   The preliminary work used
    • p-donor type anthryl groups, which
     were subsequently changed
    • p-acceptor dinitrobenzoylphenyl
     (DNBP) derivatives of amino acids.
   Since the DNBP group is a p-acceptor

   Chiral compounds should possess a p-
    donor group such as an aromatic ring
    with alkyl, ether or amino substituents, in
    order to be separated.

    Moreover, solutes should be able to form
    hydrogen bonds or enter into dipole-
    stacking with the amide group attached
    to an aromatic system on the stationary

This chiral stationary phase will interact with
the disteriomer to form transient
diasteriomeric complex

 •π-π interactions
 •Dipole interation
 •Inclusion complexing (Crystal-matrix
Mechanism of chiral recognition
   Flurbiprofen examples using
          HPLC and SFC
   HPLC (normal phase)       SFC (normal phase)

 = 1.76                   = 1.35
Run time = 20.5 minutes   Run time = 10 minutes
Flow rate = 1.5 mL/min    Flow rate = 0.4 mL/min
    Application of chiral chromatography
   Stereoselective pharmacokinetics study of
    chiral drugs and their metabolites in living
    organisms, mainly in humans,

   Stereoselective interactions of drugs with

   Studies on stereoselective fate of different
    drugs may be exemplified by
    investigations on:
    • metabolism and pharmacokinetics of ibuprofen
      and other drug in healthy volunteers
   pharmacokinetics of BOF- 4272 (xanthine
    oxidase inhibitor) in rats and dogs (Chiralcel)
    and metoprolol (teicoplanin CSP

   Metabolism of flobufen in human hepatocytes
    (Chiralcel OD-R column) or in guinea pigs (1-allyl
    terguride column)

   Metabolism of pentoxifylline (hemorheological
    agent) (cellobiohydrolase column for preparative
    enantioseparation of chiral metabolite and
    indirect mode after its derivatization with
    diacetyl-L-tartaric acid)

   Disposition of venlafaxine
    (antidepressant) enantiomers in rats,
    pharmacokinetics of nefopam
    (analgesic) and desmethylnefopam
    (for both vancomycin CSP and MS

   Metabolism of citalopram
    (antidepressive) enantiomers in
    CYP2C19/-CYP2D6 phenotyped
    panels of healthy Swedes (Cyclobond
    I 2000 column)
         In food and agriculture
   Chromatographic enantioseparations
    found applications in solving different
    problems of agriculture and food industry.

    Only recent results may be mentioned
    here, like studies of :
     • wine malolactic fermentation (use of
      multidimensional enantioselective GC-MS with
      2,3-di-O-methyl-6- O-tert-butyldimethylsilyl)-
      b-cyclodextrin as chiral selector for
      determination of ethyl lactate and other
      chiral compounds enantiomeric ratios)
    In environmental protection
   Enantioselective chromatography
    also assists environmental protection
    in determinations of enantiomers of
    different environmental pollutants

To top