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Transcript of Liquid Chromatography - Florida International almirall/   Lecture # 22 –...

  • 1

    Lecture # 22 Liquid Chromatography

    Chapter 25

    Final Exam is on Tuesday, April 22 11:15 AM - 1:15 PM

    Liquid Chromatography

    Solvent (Eluant)

    Mixture (with analyte)

    Stationary Phase

    Eluate

    Z = Stationary Phase

    Mobile Phase (solvent)

    Elu

    tion

    e.g. Glass-Column Chromatography

    High-Performance Liquid Chromatography (HPLC)

    Liquid Chromatography

    High-Performance Liquid Chromatography (HPLC)

    Liquid Chromatography

    Autosampler

    Detectors Solvents

    Controller

    Injector Pump

    Columns

    High-Performance Liquid Chromatography (HPLC)

    Liquid Chromatography

    Autosampler Detectors

    Solvents

    Pump

    Column (in oven)

    Computer

    1 2

    3

    1 2 3

    1 2

    3 Packed Column:

    Multiple Paths

    Open Tubular

    HPLC Columns

    e.g. GC

    Gas Diffusion is 10x higher than liquid diffusion

  • 2

    HPLC Columns

    1. Guard Columns 2. Column Length (L): typically 5-30 cm 3. Inner Diameter: typically 1-5 mm

    a. 4.6 mm most typical (analytical) b. Capillary Columns (~ 25 m!!)

    4. Temperature (Heating) a. Decreases viscosity of solvent b. Decreases retention time, increases

    diffusion/resolution

    HPLC Columns Stationary Phase

    Silica

    Spherical Irregular Microporous (~300 m2/g)

  • 3

    HPLC Columns Stationary Phase

    Column Efficiency 1/ Particle Size (N = number of plates)

    N 3000 L dp

    L = Column Length (cm) dp = Particle Diameter (m)

    Van Deemter Equation: H A + B/ux + Cux

    HPLC Columns Stationary Phase

    Column Efficiency 1/ Particle Size (N = number of plates)

    Smaller Particles = Decreased Equilibration Time/Mass Transfer

    Multiple Paths

    HPLC Columns Stationary Phase

    Column Efficiency 1/ Particle Size (N = number of plates)

    P = f uxL

    r2 dp2

    Column Pressure:

    f = factor depends on particle shape/packing ux = linear flow rate = viscosity of solvent L = length of column r = radius of column dp = diameter of particle

  • 4

    O

    Si Si

    Si

    Si

    O

    Si

    O

    O O

    OH

    OH

    O-

    OH

    Silanol (Si-OH) Groups

    ~ 8 mol/m2

    Strongly retain compounds, and lead to tailing!

    Siloxane Bonds

    Solute (e.g. Analyte) or Solvent Hydrogen

    Bonds

    HPLC Columns Stationary Phase

    Silica (pH 8)

    *Note: Water deactivates silica

    HPLC Columns Stationary Phase

    Bonded Stationary-Phase

    Si O

    O

    O

    Si R

    CH3

    CH3

    Si O

    O

    O

    Si R

    CH2

    CH2

    CH

    CH3 CH3

    CH

    CH3 CH3 Prevents H3O+ hydrolysis of Si-O-SiR bond at low pH (< 2)

    HPLC Columns Stationary Phase

    Bonded Stationary-Phase

    Si O

    O

    O

    Si R

    CH3

    CH3

    Polar Phase R = (CH2)NH2 Amino R = (CH2)C=N Cyano R = (CH2)OCH2CH(OH)CH2OH Diol

    Non-Polar Phase R = (CH2)17CH3 Octadecyl (C18) R = (CH2)7CH3 Octyl (C8) R = (CH2)3C6H5 Phenyl

    normal-phase

    reversed-phase

    HPLC Solvents/Elution

    Adsorbed Solute/Analyte

    Desorbed Solute/Analyte

    Solvent

    Adsorbed Solvent (displaces solute/analyte)

    HPLC Solvents/Elution Eluotropic Series

    ranks solvents by their relative abilities to displace solutes from a given adsorbent

    Solvent Eluent Strength (o) UV Cut-Off (nm) Pentane 0.00 190 Hexane 0.01 195 Heptane 0.01 200 Toluene 0.22 284 Chloroform 0.26 245 Dichloromethane 0.30 233 Diethyl Ether 0.43 215 Ethyl Acetate 0.48 256 Methyl t-Butyl Ether 0.48 210 Acetonitrile 0.52 190 Acetone 0.53 330 Tetrahydrofuran 0.53 212 2-Propanol 0.60 205 Methanol 0.70 205

    Mor

    e P

    olar

    Less

    Pol

    ar

    HPLC Solvents/Elution

    Normal Phase Chromatography: polar stationary phase and a less polar solvent; a more polar solvent has higher eluent strength

    e.g. silica with hexane/ethyl acetate

    Reversed Phase Chromatography: stationary phase is non-polar or weakly polar and the solvent is more polar; a less polar solvent has a higher eluent strength.

    e.g. C18 or C8 with water/methanol/acetonitrile

    OH

    OH

    OH

    OH

  • 5

    HPLC Solvents/Elution

    HPLC Solvents

    1. HPLC Grade Solvents 2. Intake Filter 3. Guard Column 4. Purged (sparged) to remove

    dissolved O2 5. Equilibration/washing of column

    HPLC Solvents/Elution

    Isocratic elution: performed with a single solvent (or constant solvent mixture)

    Gradient elution: continuous change of solvent composition to increase eluent strength

    Isocratic vs. Gradient Elution

    HPLC Solvents/Elution Isocratic vs. Gradient Elution

    Isocratic Mixtures of Solvent A (Aqueous Buffer) and Solvent B (CH3CN)

    HPLC Solvents/Elution Isocratic vs. Gradient Elution

    Isocratic Mixtures of Solvent A (Aqueous Buffer) and Solvent B (CH3CN)

    HPLC Solvents/Elution Isocratic vs. Gradient Elution

    Gradient Elution with Solvent A (Aqueous Buffer) and Solvent B (CH3CN)

    HPLC Injection Syringe (Sample)

    To Waste

    To Column

    Solvent

    Sample Loop

    Load Position

  • 6

    HPLC Injection Syringe (Sample)

    To Waste

    To Column

    Solvent

    Sample Loop

    Inject Position

    HPLC: Detection of Analytes

    1. Mass Spectrometry ($$$) 2. Spectrophotometric Detector (UV/Visible

    and Fluorescence)

    3. Refractive Index Detector 4. Evaporative Light-Scattering Detector 5. Electrochemical Detector

    HPLC: Detection of Analytes Spectrophotometric Detection (UV/Visible and Fluorescence)

    Light Sources: Mercury Lamp (254 nm) Deuterium (< 400 nm)/Tungsten (> 400 nm) Laser (Fluorescence)

    Detector Light

    Source

    1 cm

    Eluate Out

    Eluate In

    Microcell

    Spectrophotometric Detectors

    HPLC: Detection of Analytes Spectrophotometric Detection (UV/Visible and Fluorescence)

    Light Sources: Mercury Lamp (254 nm) Deuterium (< 400 nm)/Tungsten (> 400 nm) Laser (Fluorescence)

    Photodiode Array (PDA) Detectors: records the spectrum of each solute

    Sample

    Lamp

    Elliptical Mirror Mirror

    Grating Polychromator

    Photodiode Detector

    Photodiode Array (PDA) Detector

    Spectrophotometric Detectors

  • 7

    HPLC: Detection of Analytes Spectrophotometric Detection (UV/Visible and Fluorescence)

    Light Sources: Mercury Lamp (254 nm) Deuterium (< 400 nm)/Tungsten (> 400 nm) Laser (Fluorescence)

    Photodiode Array (PDA) Detectors: records the spectrum of each solute

    Fluorescence Derivatization

    Solvent Eluent Strength (o) UV Cut-Off (nm) Pentane 0.00 190 Hexane 0.01 195 Heptane 0.01 200 Toluene 0.22 284 Chloroform 0.26 245 Dichloromethane 0.30 233 Diethyl Ether 0.43 215 Ethyl Acetate 0.48 256 Methyl t-Butyl Ether 0.48 210 Acetonitrile 0.52 190 Acetone 0.53 330 Tetrahydrofuran 0.53 212 2-Propanol 0.60 205 Methanol 0.70 205

    Spectrophotometric Detectors

    UV Absorbance of Solvents

    HPLC: Detection of Analytes

    1. Mass Spectrometry ($$$) 2. Spectrophotometric Detector (UV/Visible

    and Fluorescence)

    3. Refractive Index Detector 4. Evaporative Light-Scattering Detector 5. Electrochemical Detector

    Refractive Index Detector

    Incident Beam

    Deflected Beam

    Reference

    Sample

    Deflected Beam

    Incident Beam

    Reference Out Reference In

    Sample Out

    Sample In

    HPLC: Detection of Analytes

    1. Mass Spectrometry ($$$) 2. Spectrophotometric Detector (UV/Visible

    and Fluorescence)

    3. Refractive Index Detector 4. Evaporative Light-Scattering Detector 5. Electrochemical Detector

    Electrochemical Detector

    Working Electrode

    Reference Electrode

    Counter Electrode

    Sample In Sample Out

  • 8

    HPLC: Detection of Analytes

    Approximate Limit Useful with Detector of Detection (ng) Gradient? Mass Spectrometry 0.1 1 Yes UV/Visible 0.1 1 Yes Fluorescence 0.001 - 0.01 Yes Refractive Index 100 1000 No Evaporative Light Scattering 0.1 1 Yes Electrochemical 0.01 1 No

    Assigned Problems in Chapter 25:

    Problems: 25-1, 25-2, 25-4, 25-6, 25-9, 25-10