Introduction to High Performance Liquid

Chromatography
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 quantitative
separation of I’ll get
carbohydrates in some on it!
of our products
as soon as possible.

I’ll need a separation

technique.

3
Separation Techniques

I have two separation techniques in my lab,

High Performance Liquid Chromatography
and Gas Chromatography. Which should I use?
4
 Comparison of HPLC and GC

Sample Volatility Sample Polarity

HPLC HPLC
• No volatility requirement • Separates both polar and
non polar compounds
• Sample must be soluble
in mobile phase • PAH - inorganic ions

GC GC
• Samples are nonpolar
• Sample must be volatile
and polar

5
Comparison of HPLC and GC

6
Comparison of HPLC and GC

Sample Thermal Lability Sample Molecular Weight

HPLC HPLC
• Analysis can take place
• No theoretical upper limit
at or below room
temperature
• In practicality, solubility is
limit.

GC GC
• Sample must be able
to survive high • Typically < 500 amu
temperature injection
port and column

7
Comparison of HPLC and GC

Sample Preparation Sample Size

HPLC HPLC
• Sample must be filtered
• Sample size based upon
column i.d.
• Sample should be in
same solvent as mobile
phase

GC GC

• Solvent must be volatile • Typically 1 - 5 L

and generally lower
boiling than analytes

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Comparison of HPLC and GC

Separation Mechanism Detectors

HPLC HPLC
• Both stationary phase • Most common UV-Vis
and mobile phase take • Wide range of non-
part destructive detectors
• 3-dimensional detectors
• Sensitivity to fg (detector
dependent)

GC GC
• Most common FID,
•Mobile phase is a universal to organic
sample carrier only compounds

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How can We Analyze the Sample?

Carbohydrates
1. fructose
2. Glucose
3. Saccharose
4. Palatinose
5. Trehalulose
6. isomaltose 5

2
3
Zorbax NH2 (4.6 x 250 mm) mAU
4
70/30 Acetonitrile/Water 1
6
1 mL/min Detect=Refractive Index

time

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Separations
Separation in based upon differential
Injector
migration between the stationary and
mobile phases.
Mixer Stationary Phase - the phase which
remains fixed in the column, e.g. C18,
Silica

Pumps Mobile Phase - carries the sample

through the stationary phase as it
moves through the column.
Column

Detector

Waste
Solvents

High Performance Liquid Chromatograph

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph

12
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

13
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

16
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

18
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

19
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

20
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

22
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

25
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

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 The Chromatogram

to - elution time of unretained peak

tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time

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HPLC Analysis Parameters

Mobile Phases

Flow Rate
Composition

Injection Volume
Column
Oven Temperature
Wavelength
Time Constant
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Modes of High Performance Liquid
Chromatography

Types of Compounds Mode Stationary Mobile Phase

Phase
Neutrals Reversed C18, C8, C4 Water/Organic
Weak Acids Phase cyano, amino Modifiers
Weak Bases

Ionics, Bases, Acids Ion C-18, C-8 Water/Organic

Pair Ion-Pair Reagent

Compounds not Normal Silica, Amino, Organics

soluble in water Phase Cyano, Diol

Ionics Inorganic Ions Ion Anion or Cation Aqueous/Buffer

Exchange Exchange Counter Ion
Resin
High Molecular Weight Size Polystyrene Gel Filtration-
Compounds Exclusion Silica Aqueous
Polymers Gel Permeation-
Organic

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HPLC Applications

Bioscience
Chemical proteins
peptides
polystyrenes nucleotides
dyes
phthalates

tetracyclines
Pharmaceuticals corticosteroids
antidepressants
barbiturates Consumer Products
lipids
antioxidants
sugars
Environmental
polyaromatic hydrocarbons Clinical
Inorganic ions amino acids
herbicides vitamins
homocysteine

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