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Chapter 1
HPLC Theory

Content
z
z
z
z

History
The Chromatographic principle
The HPLC system
HPLC parameters & terms
V0,

z
z
z
z

k, , N & R

The Resolution equation

Stationary phase, physical properties
Understanding the van Deemter curve
Column dimensions

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Definition of Chromatography
z

Analytical Science of Chemical Separations

Separate

one compound from another at the

molecular level in a liquid or gas state
CHROMATO = Color / GRAPHY = writing
z

First documented by the Russian Scientist

Tswett, 1903-1906
Sample

- Ground-up plant extract.

Poured into a glass tube packed with calcium
carbonate (column) - colored bands develop as
the extract percolated down through the column.
Compounds had separated.

Tswetts Chromatographic System

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Principle of Chromatography
Stationary phase
A V X
X A
V
V X
V X A
X
A V
A VX A

Mobile phase
A

Injection

A
V
A

X
V

X
X

X
V

V
A

Interaction

A
A

X X
X X
X X

V
A

A
A

V
V

Elution

Type of Chromatography as a
Function of Molecular Weight
Gas
Chromatography
Liquid Chromatography
Distribution/Adsorption
Liquid Chromatography
Ion Exchange
Size Exclusion Chromatography
Gel Permeation/Gel Filtration

~50

~500
~10.000
Molecular weight (MW)

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~2.000.000

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The Chromatographic System



HPLC Column
Data System
Solvent




AutoSampler

Pump

Detector
Waste

What is a Chromatogram?

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Terminology Mobile Phase

z
z
z

The mobile phase is a liquid for HPLC.

Total representative sample needs to be
soluble in the liquid.
Carrier of the sample, moving it through the
stationary chromatographic packing material.

Terminology Stationary Phase

z

The chromatographic packing material which

is held in a fixed position within column
hardware.
Performs the chemical separation.
Also

called the packing material, the

chromatographic material, or the adsorbent.

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Stationary vs. Mobile Phases

z

The stationary phase and the mobile phase

will have OPPOSITE properties to set up a
competition for sample analytes.

Chromatographic Parameters
z
z
z
z
z
z
z
z

V = Retention volume
V0 = Void volume
tr = Retention time
k = Retention factor or capacity factor
= Selectivity or separation factor
N = Plate count
W = Peak width
R = Resolution

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Elution Volume and Peak Width

V0 = Elution volume of unretained compound

equals roughly one column volume
V1 = Elution volume of compound 1
V2 = Elution volume of compound 2
W2= Peak width of compound 2 in terms of volume,
measured where the tangent crosses the baseline

k = Retention Factor
A Measure of Retention
V1

V2

V3

V0
TIME

k1 =

.5

V1-V0
V0

k1 = 1-0.5
0.5 = 1
k2 = 2-0.5
0.5 = 3
k3 = 5-0.5
0.5 = 9

 Describes how far the peak is from V0

 No dimension, independent of flow & column size

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The Retention Factor, k

k depends on the equilibrium of the analyte

between the stationary phase and the mobile phase

k is an equilibrium constant

Mobile phase (M)

A(M) B(M)

A(S)

Stationary phase (S)

B(S)

The Capacity Factor, k

 The capacity factor depends on the polarity of the

mobile phase and column stationary phase.

The polarity of the mobile phase can be altered by

changing the percentage of organic in a mobile

phase consisting of water and organic solvent.

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Changing k by Changing
Mobile Phase Polarity
80 % MeOH

50 % MeOH

60 % MeOH

40 % MeOH

Column : Symmetry C18 4.6x50mm

Mobile phase : Methanol/phosphate buffer
Sample : Caffeine
Acetaminophen
Aspirin
Salicylamide

30 % MeOH

= Selectivity
A Measure of Peak Separation
V1

V2

1.5

V3

V0
TIME

.5

k2
V2-V0
1,2 = k or 1,2 = V -V
1
1
0

1,2 =

1.5-0.5 = 2
1-0.5

5-0.5
2,3 = 1.5-0.5 = 4.5

Ratio between k-values for two adjacent peaks

Defines the position of two peaks relative to each other
Depends on the chemistry of the system

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= Selectivity
Mobile Phase
 depends on the

chemistry of the
mobile phase,i.e.
which organic solvent
is selected

40% Methanol
60% Water

33% Acetonitrile
67% Water

25% THF
75% Water

Column : Bondapak C18

R = Resolution
Degree of Magnitude of Separation

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R = Resolution
Degree of Magnitude of Separation
V2

V1

V3

V0
TIME

R=

.5

V2 - V1
(W1 + W2)

R1,2 =
R2,3 =

2- 1
=2
(0.5 + 0.5)
5- 2
(0.5 + 1.0)

=4

N = Theoretical Plates
A Measure of Efficiency

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Calculation of Column Efficiency

5 Method

18.70
N5 = 25
1.03

V = 16.63
R

= 8240 plates

16.63
N5 = 25
1.44

V = 18.70
R

= 3334 plates
W = 1.03

4.4%
height

Inject

Inject

4.4%
height

W = 1.44

Good Column

Bad Column

Efficiency Expressed as HETP

H = HETP (Height Equivalent to Theoretical Plate)
{Need H to be as Small as Possible so you can fit more plates into a column}

Plate

L
HETP = N
N= # Plates
(From Distillation Theory)

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L
5 Plates
(Larger H)

column
length

10 Plates
(Smaller H)

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Resolution Equation

-1

R = 1/4

k
1+k

Selectivity

Capacity
Efficiency

Resolution Equation
The Efficiency Term

R = 1/4

-1

k
1+k

What happens to R if N is increased 2x or 3x ?

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Resolution Equation
The capacity term

R = 1/4

-1

k
1+k

What happens to R if k = 1, 2, 10 or 20 ?

Resolution Depends on k
k Value
0
1
2
3
10
20

k Term
0
1/2
2/3
3/4
10/11
20/21

k Resolution?
0
0.50
0.67
0.75
0.91
0.95

Resolution

k Ideal Range ?

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Resolution Equation
The selectivity term

R = 1/4

-1

k
1+k

What happens to R if = 1.1 or 1.4 ?

Resolution Depends on
1

= 1 .1

1 .1 1
= 0.09
1 .1

= 1 .4

1 .4 1
= 0.29
1 .4

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, k, N Control Resolution

Initial
Increase N
Increase k
Increase

Stationary Phase Physical Properties

z
z
z

Column construction materials

Particle shape
Particle size
The

z
z

van Deemter plot

Particle pore size

Column dimensions

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Column Construction - Materials

z Stainless

Steel

z Glass
z Plastic
HDPE

(High Density Polyethylene

PEEK (Polyetheretherketone)

Column Construction
Filters / Frits
Stationary Phase

EndFittings

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Packing of the Column

Packed Bed (Stationary Phase)

Frit
Direction of flow
to pack column

Spherical Particle

Irregular Particle

Types of Packing Materials

z

Silica (inorganic)
Silicone

Dioxide

Similar to beach sand

Alumina (inorganic)
Aluminum

z
z

Oxide

Zirconia (inorganic)
Polymeric (organic)
Plastic

z
z

Hybrid (inorganic/organic)
Carbon

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Particle Size of Packing Material

Particle size is measured in:
z

Microns
For

= 10 6 meters

analytical columns: 2.5 5 m (10 m)

Highest Efficiency Columns are Packed with

Smallest Particle Sizes!

Particle Size Distribution

z

The range of particle sizes present

Example: 5 m (3 m - 7 m)

Narrower distribution gives higher

performance, but are more costly to produce

Microns

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4 5 6

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Common Particle for HPLC

z
z
z

Chromatographic Silica is by far the most

commonly used particle for HPLC
Many different types of silica based packing
materials are manufactured by many different
Chromatographic performance is greatly
determined by how the silica particle is made.

Surface Attribute of Particles

z

High surface area particles

Key

to high performance for all porous

chromatographic sorbents

Particles are highly porous [similar to the pores

in a sponge]
Pore

size is measured in atomic size units, called

Angstroms (10-10 meters)

Typical analytical packing material particles have

pores ranging from 60-125

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Comparison of Porosity and

Surface Area

Many small pores

60-125
300-500

Few large pores

High surface area

z
z

m2/g

Lower surface area

Good for small

molecules and
pharmaceuticals

250-4,000
50-150 m2/g

Good for larger

molecules and proteins

Typical Column Behavior

For N (Plate Count)
Plates vs. Flow Rate
N

10000
9000
8000
7000
6000
5000
4000
3000
2000
1000

Optimum
Only one flow rate
gives maximum efficiency

1.0

2.0
Flow Rate
{mL/min}

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3.0
Every column will have its own
specific curve depending on
dimensions and particle size

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Why Does the Flow Rate Change

the Plate Count?
van Deemter Equation
H = A + B + C

H = HETP (Height Equivalent to Theoretical Plate)

{Need H to be as Small as Possible so you can fitmore plates into a column}

= Linear Velocity (speed) of Mobile Phase (mm/sec)

A= Interparticle channels - velocity independent
B=Molecular diffusion axially inversely proportional to velocity
C=Mass transfer kinetics - directly proportional to velocity

Linear Velocity of the

Mobile Phase - u
Linear Velocity u = { Flow Rate } = cm /min.
cm
Area
3

Cross-sectional
Area of ID

Column

cm3 = mL

cm
mm
= cm or
or
min.
sec.
sec.

T e r m in o lo g y
L in e a r V e l o c i t y ( c m /s e c ) - - S p e e d a t w h ic h t h e
m o b ile p h a s e is f lo w in g t h r o u g h t h e c o lu m n - f u n c t io n o f t h e F lo w R a t e a n d C o lu m n In t e r n a l D ia m e t e r
L in e a r
V e lo c ity
(c m /s e c )

1 .0

0 .1

0 .3

0 .5

0 .8

0 .8

2 .4

4 .0

6 .2

7 .8

0 .6

1 .8

3 .0

4 .8

6 .0

0 .1 5

0 .4 5

0 .7 5

1 .2

1 .5

4 .6 m m ID
m L /m in
4 .0 m m ID
m L /m in
2 .0 m m ID
m L /m in

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Sample/Analyte BANDS and van Deemter A

Term (Interstitial Volume or Channels)
A
Larger particles, +/or irregular shape => larger interstitial channels/
more interstitial volume (between particles) => broader band,
broader peak, less efficiency ==> more A

A
Smaller particles, spherical, well packed => less interstitial volume =>
tight band, sharp peak ==> less A
No impact of linear velocity

H = A + B + uC

Particle Size

Efficiency

Sample/Analyte BANDS and Van Deemter

B Term (Diffusion of Band)
B
Slower linear velocity u, (or, flow rate), more axial
diffusion of band => broader peak ==> more B Term

B
Higher linear velocity u, less time for axial
diffusion of band => sharper peak ==> less B Term

H = A + B + uC
u

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Efficiency

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Sample/Analyte BANDS and

Van Deemter C Term (mass transfer)
Stationary phase
mass transfer
Packing
material

Sample
molecules
XX

Mobile phase
mass transfer
Sample
molecules

Influence of
particle size
X

Packing
material

Solvent
Solvent

Bandspreading
X

Bandspreading

X
X

Bandspreading

Sample/Analyte BANDS and

Van Deemter C Term (Mass Transfer)
C
High linear velocity, u, causes spreading of analyte molecules (band) due to
effect on mass transfer (into and out of the stationary phase not enough time to
adsorb/partition)
=> broad bands => broad peaks == > more C Term

C
Lower linear velocity, u, causes reduced spreading of analyte molecules
=> narrower bands => sharper peaks == > less C Term

H = A + B + uC
u

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Cu

Efficiency

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The Van Deemter Curve

H = A + B + uC
Height Equivalent to Theoretical Plate

HETP

Note: Minimum HETP point is related

to viscosity. As viscosity increases,
the minimum HETP point occurs
at slower linear velocity

PLATES

Note: Once past optimum,

as flow rate increases so does
linear velocity - HETP
Increases - plate count
DECREASES
Lowest HETP => Optimum Plate Count

HETP

Linear Velocity

{cm/sec}

Comparison of the Van Deemter

Plots for 5 m and 2.5 m Particles
Speed with maximum
efficiency (resolution)
should be achievable
with smaller particles

30
28
26

H (m)

24
22
20
18
16

Benefit of Smaller Particle

* Reduction in HETP
* Higher Linear Velocity Range

5 m XTerra Particle

14
12
10

2.5 m XTerra Particle

8
6
4
2
0

0.5

1.5

2.5

3.5

Linear Velocity (mm/sec)

Alden

(50/50, acetonitrile / water mobile phase)

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Effect of Temperature on Column

Efficiency -- Van Deemter

HETP, H [m]

35
30

5C
25

25C
60C

20
15
10
0

Linear velocity, u [mm/s]

As temperature is increased, lower HETP, higher N,

higher linear velocity, reduced viscosity, improved mass
transfer

Column Dimensions
z

Length: Millimeters or Centimeters

Internal Diameter: Millimeters

Internal diameter

Analytical :
1mm - 4.6mm
Preparative : 7.8mm - 50mm

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Length

2cm - 30cm
20mm 300mm

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Summary of Column Geometry

z

Diameter
Smaller

diameter INCREASES sensitivity.

Larger diameter INCREASES column Capacity.
z

Length
Longer

column has a HIGHER resolving power

and capacity.
Shorter Column has FASTER speed of separation
and LOWER capacity.

Summary of Theory
z

Defined
V0,

z
z
z

k, , N & R

Learned the effect of k, , and N on

resolution
Learned the importance of particle size and
flow on column efficiency (van Deemter plot)
Looked at the effect of column dimensions

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