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.

Youve Got a Problem to Solve

I need a quantitative separation of carbohydrates in some of our products as soon as possible.

Ill get on it!

Ill need a separation technique.

Separation Techniques

I have two separation techniques in my lab, High Performance Liquid Chromatography and Gas Chromatography. Which should I use?
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Comparison of HPLC and GC

Sample Volatility
HPLC
No volatility requirement Sample must be soluble in mobile phase

Sample Polarity
HPLC
Separates both polar and non polar compounds PAH - inorganic ions

GC
Sample must be volatile

GC
Samples are nonpolar and polar

Comparison of HPLC and GC

Comparison of HPLC and GC

Sample Thermal Lability HPLC
Analysis can take place at or below room temperature

Sample Molecular Weight HPLC

No theoretical upper limit
In practicality, solubility is limit.

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

GC
Typically < 500 amu

Comparison of HPLC and GC

Sample Preparation HPLC
Sample must be filtered Sample should be in same solvent as mobile phase

Sample Size HPLC

Sample size based upon column i.d.

GC
Solvent must be volatile and generally lower boiling than analytes

GC
Typically 1 - 5 L

Comparison of HPLC and GC

Separation Mechanism HPLC
Both stationary phase and mobile phase take part

Detectors HPLC
Most common UV-Vis Wide range of nondestructive detectors 3-dimensional detectors Sensitivity to fg (detector dependent)

GC
Mobile phase is a sample carrier only

GC
Most common FID, universal to organic compounds

How can We Analyze the Sample?

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

Zorbax NH2 (4.6 x 250 mm)

70/30 Acetonitrile/Water 1 mL/min Detect=Refractive Index

mAU
1

3 4 6

time

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Separations
Injector

Separation 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.

Mixer

Pumps

Column

Detector

Solvents

Waste

High Performance Liquid Chromatograph

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

High Performance Liquid Chromatograph

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

13

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

15

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

16

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

18

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

20

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

21

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

22

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

23

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

24

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

25

Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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Separations
Injector Chromatogram

Mixer

mAU

Pumps

Start Injection
Column

time

Detector

Solvents

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The Chromatogram

to - elution time of unretained peak tR- retention time - determines sample identity tR

tR mAU to Injection time

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Area or height is proportional to the quantity of analyte.

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
Neutrals Weak Acids Weak Bases

Mode
Reversed Phase

Stationary Phase
C18, C8, C4 cyano, amino

Mobile Phase
Water/Organic Modifiers

Ionics, Bases, Acids

Ion Pair
Normal Phase Ion Exchange Size Exclusion

C-18, C-8

Water/Organic Ion-Pair Reagent

Organics

Compounds not soluble in water Ionics Inorganic Ions

Silica, Amino, Cyano, Diol Anion or Cation Exchange Resin Polystyrene Silica

Aqueous/Buffer Counter Ion Gel FiltrationAqueous Gel PermeationOrganic

High Molecular Weight Compounds Polymers

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HPLC Applications
Bioscience Chemical
polystyrenes dyes phthalates proteins peptides nucleotides

tetracyclines Pharmaceuticals corticosteroids antidepressants barbiturates

Consumer Products
lipids antioxidants sugars

Environmental
polyaromatic hydrocarbons Inorganic ions herbicides

Clinical
amino acids vitamins homocysteine 31