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?
4

Comparison of HPLC and GC

Sample Volatility
HPLC

Sample Polarity
HPLC

No volatility requirement

Separates both polar and

non polar compounds

Sample must be soluble

in mobile phase

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

Sample Molecular Weight

HPLC

Analysis can take place

at or below room
temperature

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

No theoretical upper limit

In practicality, solubility is
limit.

GC
Typically < 500 amu

Comparison of HPLC and GC

Sample Preparation
HPLC

Sample Size
HPLC

Sample must be filtered

Sample should be in
same solvent as mobile
phase

GC
Solvent must be volatile
and generally lower
boiling than analytes

Sample size based upon

column i.d.

GC
Typically 1 - 5 L

Comparison of HPLC and GC

Separation Mechanism
HPLC

Detectors
HPLC

Both stationary phase

and mobile phase take
part

GC
Mobile phase is a
sample carrier only

Most common UV-Vis

Wide range of nondestructive detectors
3-dimensional detectors
Sensitivity to fg (detector
dependent)

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

mAU

1 mL/min Detect=Refractive Index

time

10

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

Mixer

Mobile Phase - carries the sample

through the stationary phase as it
moves through the column.

Pumps

Column

Detector

Solvents

Waste

High Performance Liquid Chromatograph

11

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

High Performance Liquid Chromatograph

12

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

13

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

14

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

15

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

16

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

17

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

18

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

19

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

20

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

21

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

22

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

23

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

24

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

25

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

26

Separations
Injector

Mixer

Chromatogram

mAU

Pumps

Start Injection

time

Column

Detector

Solvents

27

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
28

HPLC Analysis Parameters

Mobile Phases

Flow Rate
Composition
Injection Volume
Column
Oven Temperature
Wavelength
Time Constant
29

Modes of High Performance Liquid

Chromatography

Types of Compounds

Mode

Stationary
Phase

Mobile Phase

Neutrals
Weak Acids
Weak Bases

Reversed
Phase

C18, C8, C4
cyano, amino

Water/Organic
Modifiers

Ionics, Bases, Acids

Ion
Pair

C-18, C-8

Water/Organic
Ion-Pair Reagent

Compounds not
soluble in water

Normal
Phase

Silica, Amino,
Cyano, Diol

Organics

Ionics Inorganic Ions

Ion
Exchange

Anion or Cation
Exchange
Resin

Aqueous/Buffer
Counter Ion

High Molecular Weight

Compounds
Polymers

Size
Exclusion

Polystyrene
Silica

Gel FiltrationAqueous
Gel PermeationOrganic

30

HPLC Applications
Bioscience
Chemical
polystyrenes
dyes
phthalates

tetracyclines
corticosteroids
Pharmaceuticals
antidepressants
barbiturates

Environmental
polyaromatic hydrocarbons
Inorganic ions
herbicides

proteins
peptides
nucleotides

Consumer Products
lipids
antioxidants
sugars

Clinical
amino acids
vitamins
homocysteine

31