Liquid Chromatography 1

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Transcript of Liquid Chromatography 1

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    Liquid Chromatography 1 and Solid-Phase Extraction

    Lecture Date: April 9th, 2008

    Reading Material

    Skoog, Holler and Crouch: Ch. 28

    Cazes: Ch. 22, 26

    For those using LC in their work, see:L. R. Snyder, J. J. Kirkland, and J. L. Glajch, Practical HPLCMethod Development, 2nd Ed., Wiley, 1997.

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    Basic LC Terminology

    Adsorption chromatography

    The stationary phase is an adsorbent (like silica gel or any othersilica-based packing) The separation is based on repeated adsorption-desorption

    steps.

    Normal-phase chromatography The stationary bed is strongly polar in nature (e.g., silica gel),

    and the mobile phase is nonpolar (such as n-hexane ortetrahydrofuran).

    Polar samples are retained on the polar surface of the columnpacking longer than less polar materials.

    Reversed-phase chromatography The stationary bed is nonpolar (hydrophobic) in nature,

    The mobile phase is a polar liquid, such as mixtures of waterand methanol or acetonitrile.

    The more nonpolar the material is, the longer it will be retained.

    Size exclusion chromatography (SEC) column filled with material having precisely controlled pore

    sizes, and the sample is simply sieved or f iltered according toits solvated molecular size.

    Larger molecules are rapidly washed through the column;smaller molecules penetrate inside the pores of the packingparticles and elute later.

    Also called gel permeation chromatography (GCP)although the stationary phase is not restricted to a "gel"

    Ion-exchange chromatography (IC) the stationary bed has a charged surface of opposite charge

    to the sample ions. Used almost exclusively with ionic or ionizable samples. The stronger the charge on the sample, the stronger it will be

    attracted to the ionic surface and thus, the longer it will taketo elute

    The mobile phase is an aqueous buffer, where both pH andionic strength are used to control elution time

    Basic LC Terminology

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    Analytical Appl ications of LC

    The branches of the LC family:Note this means analyte polarity

    Basic Mechanisms used in LC Separations

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    High Performance Liquid Chromatography (HPLC)

    HPLC utilizes a high-pressure liquid mobile phase (ca.100-300 bar) to separate the components of a mixture

    These analytes are first dissolved in a solvent, and thenforced to flow through a packed small-particlechromatographic column, where the mixture is resolvedinto its components

    HP = high pressure and high performance

    Resolution depends upon the extent of interaction

    between the solute components and the stationaryphase

    Differences between HPLC and Classical LC

    Small ID (2-5 mm), reusable stainless steel columns

    Column packings with very small (3, 5 and 10 m)particles and the continual development of newsubstances to be used as stationary phases

    Relatively high inlet pressures and controlled flow of themobile phase

    Precise sample introduction without the need for largesamples

    Special continuous flow detectors capable of handling

    small flow rates and detecting very small amounts Automated standardized instruments

    Rapid analysis

    High resolution

    From now on, LC refers to HPLC

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    Advantages and Disadvantages of LC

    Advantages: Speed (minutes) High resolution Sensitivity Reproducibility Accuracy Automation

    Disadvantages: Cost Complexity

    Low sensitivity for some compounds Irreversibly adsorbed compounds not detected Co-elution difficult to detect

    More on Reversed-phase (RP) LC

    RP is the most widely used mode of HPLC (75%?)

    Separates molecules in solution on basis of theirhydrophobicity Non-polar stationary phase

    Polar mobile phase

    In practice: non polar functional group bonded to silica Stationary phase

    functional group bonded to silica

    this corresponds to a volume (Van deemter)

    Alkyl groups ( C4, C8, C18) retention increases exp. with chain length

    Mobile Phases Polar solvent (water) with addition of less polar solvent (acetonitrile

    or methanol)

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    The Packed Column and the Stationary Phase

    Packed LC columns, usually made of stainless steel andcarefully filled with material, are the heart of the LCexperiment

    The stationary phase fills the column its properties arecritical to the separation

    Review of Molecular Interactions

    The basis of separations (and most of chemistry)

    Name Energy (kcal/mol) Description

    Covalent 100-300Hold molecules together, orbital

    overlap

    Ionic 50-200 Electrostatic attraction

    Polar Hydrogen bonding Dipole-dipole

    -stacking

    3-10Vary from electrostatic-typeinteractions (e.g. hydrogen

    bonds) to much weaker

    Non-Polar Van der Waals

    (dispersion)1-5 Weak, induced dipole

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    Retention Mechanisms in LC

    HPLC is a dynamic adsorption process. Analyte molecules, whilemoving through the porous packing bead, tend to interact with thesurface adsorption sites. Depending on the HPLC mode, the differenttypes of the adsorption forces may be included in the retention process

    Hydrophobic interactions are the main ones in reversed-phaseseparations

    Dipole-dipole (polar) interactions are dominant in normal phase mode.

    Ionic interactions are responsible for the retention in ion-exchangechromatography.

    Retention in LC is competitive:

    Analyte molecules compete with the eluent molecules for theadsorption sites. So, the stronger analyte molecules interact withthe surface, and the weaker the eluent interaction, the longeranalyte will be retained on the surface.

    Retention Mechanisms in LC

    Remember the elution order! Normal-phase vs. reversed-phase LC

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    Physical Properties of Stationary Phase Particles

    HPLC separations are based on the surface interactions,and depends on the types of the adsorption sites (surfacechemistry). Modern HPLC adsorbents are the small rigidporous particles with high surface area.

    Key parameters: Particle size: 3 to 10 m

    Particle size distribution: as narrow as possible, usually within 10%of the mean

    Pore size: 70 to 300 Surface area: 50 to 250 m2/g

    Bonding phase density (number of adsorption sites per surfaceunit): 1 to 5 per 1 nm2

    Electron microphotograph of spherical and irregular silica particles. [W.R.Melander, C.Horvath,

    Reversed-Phase Chromatography, in HPLC Advances and Perspectives, V2, Academic Press, 1980]

    The Most Popular Particle: Silica

    Macroporous spherical silica particle. [K.K.Unger,

    Porous silica, Elsevier, 1 979]

    Different morphology for different applications:

    Different chemistry:

    Si OH Si OH O

    H

    H

    Si

    OH

    OH

    Free Silanol Adsorbed Water Geminal Silanol

    Si

    O

    Si

    O

    DehydratedOxide Siloxane

    O

    H

    O

    H

    Si

    Si

    O

    Bound andReactiveSilanols

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    Chemical Modifications to Silica

    Silica (or zirconia, or alumina) by itself cannot do the job needed by

    modern LC users it must be functionalized and modified to suit theanalytical problem

    Residualsilanols

    SiO

    SiO

    SiO

    SiO

    SO

    Si

    Si OSi

    OSiOH

    S

    OHOH

    OH ii

    O

    O

    Diagram from Crawford Scientific

    Functionalizedgroups

    Chemical Modifications to Silica

    Groups are usually attached via reactionof an organosilane (which can be pre-polymerized in solution)

    Besides attaching groups, it is alsopossible to polymerize the silica (or theattached group)

    Purpose: stability at low pH, morecoverage

    High-carbon load

    Monomeric phases are morereproducible (easier reactions to control)

    Monomeric phases are also knownas sterically-protected

    Endcapping: fully react the silicasurface, remove silanols and theiracidity, more coverage

    Diagram from K. A. Lippa et al., Anal. Chem.2005, 77,7852-7861

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    Common LC Stationary Phases

    Name Structure Description

    SilicaNormal phase, for separating polar, non-ionic

    organics

    PropylReversed-phase, for hydrophobic interaction

    chromatography (proteins, peptides)

    C8Reversed-phase, like C18 but less retentive,

    used for pharmaceuticals, steroids,nucleotides

    C18Reversed-phase, retains non-polar solutes

    strongly. When bonded to 300A silica can beused for large proteins and macromolecules

    CyanoReversed-phase and normal-phase, more

    polar than C18, unique selectivity

    AminoReversed-phase, normal-phase, and weak

    anion exchange. RP used to separatecarbohydrates

    Si C3H7

    Si C8H17

    Si C18H37

    Si CH2CH2CH2CN

    Si CH2CH2CH2NH2

    Si OH

    Common LC Stationary Phases

    Name Structure Description

    PhenylReversed-phase, retains aromatic

    molecules. Also used for HIC(proteins)

    Diol

    Both reversed-phase and normal-phase utility. Used for RP SEC,

    also used for NP separations as amore rob