HPLC :

High
Performance (Pressure)
Liquid
Chromatography

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ether Chromatography

colors
chlorophyll

CaCO3

2
Light stone water flow
Heavy stone

base

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 flow

Mobile phase Interaction

power

Stationary
phase

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Chromato (-graphy) : Method
Chromato (-graph) : Instrument
Chromato (-gram) : Picture
Chromato (-grapher) : Person

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Mobile phase: Liquid

Stationary phase: Solid or Liquid

Samples dissolved in mobile phase can be analysed.

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Reservoir Pump Injector Column Detector

Data
Processor

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 Oven
Detector

Column

Pump
Degasser Autosampler

Waste

Data processor
Reservoir
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Problems caused by dissolved air in mobile phase
o Unstable delivery in pump
o Bigger noise and large baseline-drift in detector cell

In order to avoid causing the problems, mobile phase

should be degassed.

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Aspirator Membrane filter
(Size: 0.45 um)

Aspirator

Ultrasonic cleaning unit 10

 Pump head
Motor & Cam

Check valves

Plunger
Plunger seal
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 from Pump from Pump

to Column
to Column
LOAD
from Pump from Pump

to Column
to Column
INJECT
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Partial-filling
o The volume of the sample loaded is limited to half the
sample loop volume.
Complete-filling
o In order to replace all the mobile phase in the loop,
excess sample (two to five loop volumes) must be
used.

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from Pump to Column from Pump to Column

Sample Loop
Sample Loop

LOAD INJECT 14
Isocratic system
o Fixed (un-changeable) mixing ratio during analysis

Gradient system
o Changeable mixing ratio during analysis
• HPGE (High Pressure Gradient)
• LPGE (Low Pressure Gradient)

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 Low pressure
gradient unit

Gradient mixer

high pressure atmospheric pressure

HPGE LPGE
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in isocratic mode
Methanol / water = 6 / 4
Long analysis time

Methanol / water = 8 / 2
Poor separations

(Column : ODS type)

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 Gradual change of the mixing ratio during analysis

95%

Methanol concentration
in mobile phase

30%
Short analysis time
&
Excellent separation 18
Flow mode
o Isocratic flow / Binary gradient
Flow rate
o Isocratic flow: A.Flow
o Binary gradient: T.Flow, B.Conc
Pressure
o P.Max, P.Min

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Air circulation heating

Block heating
o Aluminum block heater

Jacket heating

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Oven temperature

T.Max

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UV/UV-VIS

PDA (Photodiode array)

Fluorescence

Refractive Index

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 Cell C : Concentration

Ein Eout

D2 / W Lamps
l

A
A = e·C·l = –log (Eout / Ein)
(A : Absorbance)
C 23
 Cell
Grating

D2 / W Lamps
1 nm / element

512 photodiodes

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 Spectrum

Chromatogram
Absorbance

Retention time 25
 Excitation wavelength

+ hn 1 *

* hn 2 +
Emission wavelength
Excitation state

Quasi-excitation state
hn1
hn 2
Fluorescence
Ground state 26
Photodiode
Reference

angle

W Lamp
Sample

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PC workstation software

Integrator)

System controller

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 tR
Peak
tR : Retention time
A : Area
Signal

h h : Height
A

Time
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1. Adequate sensitivity
2. Good Stability and Reproducibility
3. A linear response to analytes that extends over several orders
of magnitude
4. A short response time that is independent of flow rate
5. High reliability and ease of use
6. Similarity in response toward all analytes or alternatively a
highly predictable and selective response toward one or more
classes of analytes
7. Nondestructive of sample
8. Minimal internal volume in order to reduce zone broadening
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Material Size
o Stainless steel (SUS) O.D. (Outer Diameter)
o PEEK (Polyether ether ketone) o 1.6 mm
I.D. (Inner Diameter)
o 0.1 mm
o 0.3 mm
o 0.5 mm
o 0.8 mm etc.

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Male nut (SUS) Ferrule
Ferrule (SUS)
o Pressure: up to 40 MPa
Male nut

Male nut (PEEK)

o can be connected without
any tools
Male nut (PEEK)
o Pressure: up to 25 MPa

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Water Organic solvent
o Ultrapure water o HPLC grade
o HPLC grade o Super-high grade may be
used in some application.
o Some solvents such as THF
and chloroform include
Stabiliser, which cause a
problem.

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 Un-dissolved solvents Buffer must not be
must not be used in replaced directly with
replacement. organic solvent.
Water Buffer

2-Propanol Water

Hexane Organic solvent 34

 Stationary phase Mobile phase

Normal High polarity Low polarity

phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (hydrophilic)

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 Polarity Solubility
o (+) and (-) charges o Similar solvents can be
exist in a molecule. easily soluble.
Water: polar o Polar/Nonpolar
Methane: nonpolar molecules are similar to
Water/Oil.

H
–
O C
H H H H
+ H
Water methane 36
Stationary phase: Low polarity
o ODS (Octadecyl silane) column

Mobile phase: High polarity

o Water, Methanol, Acetonitrile
o Buffer

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 C18 (ODS) OH

weak
strong
CH3

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Mobile phase: Methanol /Water

Methanol / Water

60 / 40

Methanol / Water

70 / 30

Methanol / Water

80 / 20
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 2
 tR 
N = 16 
W 
2
 tR 
= 5.55 
H  W1/ 2 
W1/2
 tR  H 
2
H1/2
= 2 π 
W  Area 
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Retention time

Spectrum

Another instrument analysis

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Peak area / Peak height

Calibration curve by standard sample

o External standard
o Internal standard

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Concentration Area
A1 Calibration curve
C1
A4

A2

Peak area
A3
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
Concentration 43
 Concentration
Internal Area
Target standard
A1 AIS Calibration curve

Area: Target / Internal standard

C1 CIS A4 /AIS

A2 AIS A3 /AIS
C2 CIS

A2 /AIS
A3 AIS
C3 CIS
A1/AIS

A4 AIS
C4 CIS C1/CIS C2 /CIS C3 /CIS C4 /CIS
Concentration: Target / Internal standard
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1. It is sensitive

2. It is readily adaptable to accurate quantitative determinations

3. It is suitable for separating nonvolatile species

4. It is suitable for separating thermally fragile species

5. Due to its widespread applicability to substances that are of

prime interest to industry, to many fields of science and to the
public.
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• Confidence in the Analytical Method and the Result
produced is important.
• What is confidence?
• The method should have been validated prior to the
Analytical Investigation i.e. thoroughly tested to show
that the method gives ACCURATE results for the type of
sample being analysed.
• If we are concerned about the ACCURACY of a result, then it
is obvious that concern should extend all the way from
SAMPLING to the publication of the final RESULT.

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• Thus what areas would you consider to be important in
producing an ACCURATE analysis?

• The sampling procedure should produce a representative

sample
• The sample should not become contaminated or alter
chemically during storage
• There should be no contamination of the sample within the
laboratory or during the analysis
• Any losses in extraction, separation and concentration
procedures should be minimized

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• There should be no interference in the final analysis from
other components in the sample.
• Results should be correctly calculated and archived for
future reference.

• Thus the overall methodology needed or employed to

minimize the potential errors is referred to as QUALITY
ASSURANCE.

• The measures employed to ensure the validity of

individual results is referred to as QUALITY CONTROL

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1. Sampling and sample storage procedures which ensure
that the sample is truly representative and that it reaches
the laboratory unchanged
2. Sampling and analysis in duplicate
3. Specifications within the analytical scheme for reagent
purity and apparatus cleanliness
4. Repeated checks on the instrument performance
5. Traceability in any standards used. This means that the
stated concentrations in any standard used must be
traceable back to primary international standards

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6. Inclusion in each analytical batch of additional of
additional samples of known composition. These will
confirm the RELIABILITY of the method and could include
the ff:
a. Blank Samples – samples made up as close as possible in
composition to the unknown, excluding the compound being
determined. These are introduced before stages in the in the
analysis when contamination is likely. A positive determination of
the analyte in the blank would indicate contamination.

b. “Spiked” Samples – these are samples to which a known quantity

of the compound being determined has been added. A valid
analysis of the spiked and unspiked sample will be able to
determine accurately the quantity added.
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Certified Reference Materials (CRMs) – these are
materials similar in type to the unknown sample and
have an accurately determined composition.

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