Liquid Chromatography - Florida International Universityfaculty.fiu.edu/~almirall/Lecture22.pdf ·...

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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)




Autosampler

Detectors Solvents

Controller

Injector Pump

Columns



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

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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 µm!!)

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)

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

N ≈ 3000 L dp

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

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



Smaller Particles = Decreased Equilibration Time/Mass Transfer

Multiple Paths



P = f uxηL

Πr2 dp2

Column Pressure:

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

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O

Si Si

Si

Si

O

Si

O

O O

OH

OH

O-

OH

Silanol (Si-OH) Groups

~ 8 µmol/m2

Strongly retain compounds, and lead to tailing!

Siloxane Bonds

Solute (e.g. Analyte) or Solvent Hydrogen

Bonds


Silica (pH ≤ 8)

*Note: Water “deactivates” silica


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)


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 (εo) 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


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

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HPLC Solvents

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

dissolved O2 5. Equilibration/washing of column


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)


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


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

HPLC Injection Syringe (Sample)

To Waste

To Column

Solvent

Sample Loop

Load Position

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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



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

Sample

Lamp

Elliptical Mirror Mirror

Grating Polychromator

Photodiode Detector

Photodiode Array (PDA) Detector


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

Fluorescence Derivatization

Solvent Eluent Strength (εo) 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


UV Absorbance of Solvents







Refractive Index Detector

Incident Beam

Deflected Beam

Reference

Sample

Deflected Beam

Incident Beam

Reference Out Reference In

Sample Out

Sample In







Electrochemical Detector

Working Electrode

Reference Electrode

Counter Electrode

Sample In Sample Out

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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

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