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    Chromat 2014 PDF

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    Wahyu Dipa Pratama
    Chromatography is a technique used to separate components of a mixture based on how they interact with a stationary and mobile phase. There are several types of chromatography including gas chromatography (GC), liquid chromatography (LC), thin layer chromatography (TLC), and size exclusion chromatography. GC uses a gas mobile phase to separate volatile analytes through a column, while LC uses a liquid mobile phase to separate components in a complex sample. The document discusses various chromatographic principles, parameters, quantification methods, and detectors used with GC and LC.

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    Chromat 2014 PDF

    Uploaded by

    Wahyu Dipa Pratama
    60 views34 pages
    Chromatography is a technique used to separate components of a mixture based on how they interact with a stationary and mobile phase. There are several types of chromatography including gas chromatography (GC), liquid chromatography (LC), thin layer chromatography (TLC), and size exclusion chromatography. GC uses a gas mobile phase to separate volatile analytes through a column, while LC uses a liquid mobile phase to separate components in a complex sample. The document discusses various chromatographic principles, parameters, quantification methods, and detectors used with GC and LC.

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    Chromatography is a technique used to separate components of a mixture based on how they interact with a stationary and mobile phase. There are several types of chromatography including gas chromatography (GC), liquid chromatography (LC), thin layer chromatography (TLC), and size exclusion chromatography. GC uses a gas mobile phase to separate volatile analytes through a column, while LC uses a liquid mobile phase to separate components in a complex sample. The document discusses various chromatographic principles, parameters, quantification methods, and detectors used with GC and LC.

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    © All Rights Reserved
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    60 views34 pages

    Chromat 2014 PDF

    Uploaded by

    Wahyu Dipa Pratama
    Chromatography is a technique used to separate components of a mixture based on how they interact with a stationary and mobile phase. There are several types of chromatography including gas chromatography (GC), liquid chromatography (LC), thin layer chromatography (TLC), and size exclusion chromatography. GC uses a gas mobile phase to separate volatile analytes through a column, while LC uses a liquid mobile phase to separate components in a complex sample. The document discusses various chromatographic principles, parameters, quantification methods, and detectors used with GC and LC.

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    Chromatography

    ENVH 432
    Reading: Chapters 23-25 in Harris,
    Quantitative Chemical Analysis
    Chromatography
    • The separation of analytes for individual
    detection in a complex matrix

    GC

    LC

    Sample  Column  Peaks with area


    (or Extract) proportional to
    concentration
    Chromatography
    • Chromatographic Principles
    • Peak Separation and Quantification
    • Gas Chromatography
    • Liquid Chromatography
    • Detectors
    Principles of Chromatography
    • Stationary phase is fixed in place in a column or on a
    planar surface
    • Mobile phase (or Eluent) is moving over or through the
    stationary phase, carrying the analyte along with it
    • Chromatography: components of a mixture are
    separated based on rates at which they are carried
    through the stationary phase by a gaseous or liquid
    mobile phase
    • Elution: process of an analyte being washed through a
    stationary phase by the movement of a mobile phase
    • Chromatogram: the plot of analyte signal (function of
    concentration) versus elution time
    Thin Layer Chromatography (TLC)
    • Sample mixture applied to bottom of plate
    • TLC plate: Thin layer of silica (or other solid stationary
    phase) on metal plate backing
    • Liquid mobile phase carries sample through solid
    phase by capillary action
    • Separate fractions can be scraped off and further
    analyzed
    • TLC used to optimize conditions for liquid column
    chromatography or to purify
    synthesized standards

    TLC Separation of Chlorophyll


    Analyte Quantification Peak Height

    • Chromatogram
    • Retention Time
    • Resolution Threshold
    – Peak Shape Noise

    – Peak Width
    • Signal-to-Noise ratio Peak Area:
    • Threshold Integral of signal

    Peak Width
    Quantitation methods in
    chromatography
    • External standard calibration
    – Uses calibrant external to sample
    – Requires precise control of injection volumes
    • Internal standard calibration
    – Uses a standard in each sample and calibrant
    at a constant mass/sample
    – Does not require precise control of injection
    volumes
    IS
    Analyte Quantification Analyte

    External Standard

    Area of Analyte
    Calibration

    Concentration

    Internal Standard

    Relative Area
    Calibration
    Relative Area = Area of Analyte
    Area of Internal Standard

    Concentration
    Chromatographic Parameters
    • Stationary Phase (solid or solid-liquid)
    – Adsorption
    – Partition GC

    – Molecular exclusion HPLC


    – Ion-Exchange
    – Affinity (protein lock to antibody key)
    • Mobile Phase (gas or liquid)
    – Flow rate
    – Composition
    • Column Dimensions
    – Length, width (ID), particle size or film thickness
    Peak Resolution
    Change Column phase or
    Signal

    dimensions (e.g., Length)


    Change Flow
    • Mobile phase (gas or liquid)
    1 Change Gradient
    • Temperature or pressure
    • Mobile Phase Composition
    Time (minutes)

    Gas Chromatography

    Oven Temperature (C)


    1

    2
    2

    Time (minutes)
    GC vs HPLC
    GC HPLC
    • Highly efficient separations • (Slightly) less efficient
    • Analytes must be (semi) separations
    volatile • Small and large molecules,
    • Analytes must be thermally polar and non-polar
    stable • Useful for thermally
    • Non-polar, small molecules unstable analytes
    • Limited control over mobile • Many choices available for
    and stationary phases mobile and stationary
    phases
    Gas Chromatograph (GC)
    Chromatogram
    Gas Chromatography
    • Mobile Phase (gas)
    – N2, He, H2
    • Stationary Phase
    – Packed Columns
    • Silica (short & wide)
    – Open Tubular Columns
    • Liquid-bonded siloxanes
    R O Si R
    • Long & narrow CH3
    R O Si R
    CH3
    GC: Partition
    Chromatography
    • Liquid film with siloxane
    backbone

    • Choose attached functional


    groups that match the
    polarity of your analytes

    • For separation of nonpolar


    trihalomethanes, EPA
    Method 551.1 recommends
    GC columns with
    1% Diphenylpolysiloxane or
    6% cyanopropylphenyl-
    polysiloxane phases
    GC Detectors
    • Quantitative
    – Thermal Conductivity Detector (TCD)
    – Flame Ionization (FID)
    – Electron Capture (ECD)
    – Flame Photometric (FPD)
    • Qualitative and Quantitative
    – Infrared Spectrometer (FTIR)
    – Mass Spectrometer (MS or MS/MS)
    GC Detector
    Detector Detection Limit Linear range
    TCD 400 pg/mL (propane) > 105
    FID 2 pg/s > 107
    ECD 5 fg/s 104
    FPD 1 pg/s P, 10 pg/s S >104, >103
    FTIR 200 pg to 40 ng 104
    MS 25 fg to 100 pg 105

    Reference: Table 24-5 in Harris, Quantitative Chemical Analysis


    GC Detector Pros/Cons
    • TCD and FID
    – Generic detectors that give relatively uniform
    response to all carbon-containing compounds
    – Not very sensitive
    • ECD
    – Very sensitive for electron-capturing species, e.g.
    halogens and aromatics
    – More sensitive than full scan MS
    – Not structure specific
    – Wide variation in response from analytes
    • FPD: specific to N, S, P analytes
    GC Detector Pros/Cons
    • FTIR
    – Gives info on functional groups
    – Not as sensitive as MS
    • MS or MS/MS
    – Gives structural info
    – Responds to all compounds
    – Can be very sensitive (SIM, SRM)
    – expensive
    High Performance Liquid
    Chromatography (HPLC)
    High Performance Liquid Chromatograph
    (HPLC)

    Fluorescence
    detector

    Diode array (UV)


    detector

    Column heater + Solvents


    micro switching
    valve
    Solvent
    degasser
    Autosampler

    High pressure
    Sampler pump unit
    cooling unit
    HPLC Parameters
    • Stationary Phase (solid or solid-liquid)
    – Adsorption
    – Partition
    – Molecular exclusion
    – Ion-Exchange
    – Affinity (protein lock to antibody key)
    • Mobile Phase (liquid)
    – Flow rate
    – Composition
    • Column Dimensions
    – Length, width (ID), particle size
    Choice of Separation Mechanism
    Adsorption Chromatography
    • Liquid-solid chromatography
    • Normal Phase
    • Separation of neutral species
    on the basis of polarity
    • Stationary phase examples
    – Bare Silica
    – Calcium carbonate
    • Adsorption process similar to trapping air samples on
    charcoal or sample clean-up with granular activated
    carbon (GAC)
    Partition Chromatography
    • Liquid-liquid chromatography
    • Siloxane bonded-phase
    coverings to silanol backbone
    – Reverse Phase
    • C18 most common (ODS)
    • Separation of neutral species on the basis of
    hydrophobicity
    – Ion-Pair Chromatography for separation of ionic species
    – Chiral Separation of optically active isomers
    (enantiomers)
    Ion-Exchange Chromatography (IC)

    • Separation of ionic
    analytes on the basis
    of charge, on ion-
    exchange stationary
    phases
    Ion-Exchange Chromatography
    • Anion exchangers:
    RX+A- + M+B- ⇔ RX+B- + M+ + A-
    – contain bound positive groups
    • Strongly basic (SAX): Aryl–N(CH3)3+Cl-
    • Weakly basic (WAX): R–NH(R)2+Cl-
    – exclude neutrals and positive ions (elute first)
    • Cation exchangers:
    RX-C+ + M+B- ⇔ RX-M+ + C+ + B-
    – contain bound negative groups
    • Strongly acidic (SCX): Aryl–SO3-H+
    • Weakly acidic (WCX): R–CO2-H+
    – exclude neutrals and negative ions
    Ion-Exchange Chromatography (IC)

    • Suitable for both inorganic analytes and organic


    analytes (retains charged and elutes neutrals)
    • Eluent suppressor technique heightens
    sensitivity for inorganic analytes, decreases
    background signal
    • Examples:
    – Post-Column Reaction with UV detection of bromate
    as a disinfection byproduct of ozone water treatment
    – Chelating microcolumn used for metal (Al) speciation
    in soils and sediments
    – Analysis of organic acids in foods and beverages
    Size-Exclusion Chromatography (SEC)

    • Separation of molecules on
    the basis of differences in
    molecular size, not by
    partitioning behavior
    • Ideal for separation of heavy
    natural product molecules from
    lighter analytes and salts
    • Gel-Permeation SEC for nonpolar analytes
    – aqueous solvents and hydrophilic packings
    • Gel-Filtration SEC for polar or ionic analytes
    – hydrophilic solvents and aqueous packings
    Size-Exclusion Chromatography (SEC)
    Affinity Chromatography
    • Lock and Key mechanism
    – Enzymes and substrates
    – Antibodies and antigens
    – Receptors and hormones

    • Isolates a single compound from a complex mixture


    – Interfering compounds elute first
    • Specific binding of compound to stationary phase
    – A molecular imprinted polymer is polymerized in the presence
    of a template molecule
    HPLC Mobile Phase
    • Composition of solvent
    – water, phosphate buffer, methanol, acetonitrile
    • Isocratic elution
    – single solvent or constant solvent mixture
    • Solvent gradient
    – Increase eluent strength to elute the strongly retained
    analytes
    – Reversed phase column: start with 100% aqueous
    phase (A) and gradually increase the % of organic
    solvent phase (B) running through the column as
    mobile phase
    – Normal phase…..starts with non-polar solvent,
    gradually increase % of polar solvent (e.g. methanol)
    HPLC Detectors
    • Quantitative
    – Ultraviolet (UV) – Refractive Index
    – Conductivity – Light-Scattering
    – Fluorescence Post-column
    – Electrochemical Derivatization

    • Qualitative and Quantitative


    – Photodiode array (DAD): UV spectrum of
    each peak
    – Mass spectrometer (MS) and MS/MS
    HPLC detectors
    Detector Limit of detection (ng) specificity

    Evaporative light 0.1-1 none


    scattering
    Ultraviolet 0.1-1 low

    Refractive Index 100-1,000 none

    Electrochemical 0.01-1 low

    Fluorescence 0.001-0.01 high

    Mass spectrometry 0.0001-1 Very high

    Adapted from Harris, D.C Quantitative Chemical Analysis


    Chromatographic Image Resources
    • Skoog, D.A.; West, M.W.; Holler, F.J. Fundamentals of
    Analytical Chemistry, 7th Ed, Saunders College
    Publishing, 1997.
    • www.chem.ubc.ca/courseware/121/tutorials/exp3A/colu
    mnchrom/
    • www.agilent.com
    • www.waters.com
    • www.dionex.com
    • www.wikipedia.org
    – Chromatography, Gas chromatography, Liquid chromatography,
    Size-exclusion chromatography (Isaac Yonemoto)

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