Introduction to High Performance Liquid Chromatography

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In This Section, We Will Discuss:

The differences between High Performance Liquid Chromatography and Gas Chromatography.

The components of the high performance liquid chromatograph (HPLC).

The separation process.

The chromatogram.

The most common modes of HPLC.

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You’ve Got a Problem to Solve

I need a quantitativeseparation ofcarbohydrates in someof our products as soon as possible.

I’ll need a separationtechnique.

I’ll geton it!

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Separation Techniques

I have two separation techniques in my lab,High Performance Liquid Chromatographyand Gas Chromatography. Which should I use?

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Comparison of HPLC and GC

Sample Volatility Sample Polarity

HPLC• No volatility requirement

• Sample must be solublein mobile phase

GC• Sample must be volatile

HPLC

GC

• Separates both polar andnon polar compounds

• PAH - inorganic ions

• Samples are nonpolarand polar

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Comparison of HPLC and GC

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Comparison of HPLC and GC

Sample Thermal Lability Sample Molecular Weight

HPLC• Analysis can take placeat or below roomtemperature

GC• Sample must be able to survive high temperature injection port and column

HPLC

GC

• No theoretical upper limit

• In practicality, solubility islimit.

• Typically < 500 amu

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Comparison of HPLC and GC

Sample Preparation Sample Size

HPLC• Sample must be filtered

• Sample should be insame solvent as mobilephase

GC

• Solvent must be volatileand generally lower boiling than analytes

HPLC

GC

• Sample size based uponcolumn i.d.

• Typically 1 - 5 L

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Comparison of HPLC and GC

Separation Mechanism Detectors

HPLC• Both stationary phase and mobile phase take part

GC

•Mobile phase is a sample carrier only

HPLC

GC

• Most common UV-Vis• Wide range of non-destructive detectors

• 3-dimensional detectors• Sensitivity to fg (detectordependent)

• Most common FID,universal to organiccompounds

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How can We Analyze the Sample?

Carbohydrates1. fructose2. Glucose3. Saccharose4. Palatinose5. Trehalulose6. isomaltose

Zorbax NH2 (4.6 x 250 mm)

70/30 Acetonitrile/Water

1 mL/min Detect=Refractive Index

1

23

4

5

mAU

time

6

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SeparationsSeparation in based upon differential migration between the stationary and mobile phases.

Stationary Phase - the phase which remains fixed in the column, e.g. C18, Silica

Mobile Phase - carries the sample through the stationary phase as it moves through the column.

Injector

Detector

Column

Solvents

Mixer

Pumps

High Performance Liquid Chromatograph

Waste

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SeparationsInjector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

High Performance Liquid Chromatograph

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SeparationsInjector

Detector

Column

Solvents

Mixer

Pumps

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

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time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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SeparationsInjector

Detector

Column

Solvents

Pumps

Mixer

Chromatogram

Start Injection

mAU

time

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The Chromatogram

Injection

to

tR

mAU

time

tR

to - elution time of unretained peak

tR- retention time - determines sample identity

Area or height is proportionalto the quantity of analyte.

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HPLC Analysis Parameters

Mobile Phases

Flow RateComposition

Injection Volume

Column Oven Temperature

WavelengthTime Constant

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Modes of High Performance Liquid Chromatography

Types of Compounds Mode StationaryPhase

Mobile Phase

NeutralsWeak AcidsWeak Bases

ReversedPhase

C18, C8, C4cyano, amino

Water/Organic Modifiers

Ionics, Bases, Acids Ion Pair

C-18, C-8 Water/Organic Ion-Pair Reagent

Compounds notsoluble in water

NormalPhase

Silica, Amino,Cyano, Diol

Organics

Ionics Inorganic Ions Ion Exchange

Anion or CationExchange Resin

Aqueous/Buffer Counter Ion

High Molecular WeightCompoundsPolymers

Size Exclusion

Polystyrene Silica

Gel Filtration- AqueousGel Permeation-Organic

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HPLC Applications

Chemical

Environmental

PharmaceuticalsConsumer Products

Clinical

polystyrenesdyesphthalates

tetracyclinescorticosteroidsantidepressantsbarbiturates

amino acidsvitaminshomocysteine

Bioscienceproteinspeptidesnucleotides

lipidsantioxidantssugars

polyaromatic hydrocarbonsInorganic ionsherbicides