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HPLC(High performance liquid
chromatography)
㈜ 우존
신명선
2
Mobile Phase / Stationary Phase

Mobile pha
se
Strong
Weak

Stationary
phase
A site in which a moving
phase (mobile phase) an
d a non-moving phase (s
tationary phase) make co
ntact via an interface that
is set up.
The affinity with the mobil
e phase and stationary p
hase varies with the solut
e.  Separation occurs d
ue to differences in the s
peed of motion.
Liquid Chromatography

Chromatography in which the mobile ph
ase is a liquid.
–


The liquid used as the mobile phase is called t
he “eluent”.
The stationary phase is usually a solid o
r a liquid.
In general, it is possible to analyze any
substance that can be stably dissolved
in the mobile phase.
3
Interaction Between Solutes, Station
ary Phase, and Mobile Phase

4
Differences in the interactions between the solutes
and stationary and mobile phases enable separatio
n.
Solute
Degree of adsorption,
solubility, ionicity, etc.
Stationary p
hase
Mobile phase
Column Chromatography and Planar
Chromatography
Separation column
Paper or a substr
ate coated with p
articles
Packing material
Column Chromatography
Paper Chromatography
Thin Layer Chromatography (TLC)
5
Output concent
ration
Separation Process and Chromatogra
m for Column Chromatography
Chromatogram
Time
6
Intensity of detector signal
Chromatogram
tR
Peak
t0
tR : Retention time
t0 : Non-retention time
h
A
A : Peak area
h : Peak height
Time
7
Flow Channel Diagram for High Perform
ance Liquid Chromatograph
Detector
Column
Column oven
(thermostatic colum
n chamber)
Sample injection unit
(injector)
Pump
Eluent
(mobile phase)
Drain
Data processor
Degasser
8
HPLC Hardware: Part 1
Solvent Delivery System, Degasser,
Sample Injection Unit, Column Oven
9
Solvent Delivery Pump

Performance Requirements
–
–
–
–
–
Capacity to withstand high load pressures.
Pulsations that accompany pressure fluctuatio
ns are small.
Flow rate does not fluctuate.
Solvent replacement is easy.
The flow rate setting range is wide and the flo
w rate is accurate.
10
Solvent Delivery Pump:
Schematic Diagram of Plunger Pump
Motor and cam
Pump head
Check val
ves
Plunger
Plunger seal
10 -100µL
11
Solvent Delivery Pump:
Dual Plunger Type
Check valves
Plunger heads
Type
Type
12
Gradient System

Isocratic system
–

Constant eluent composition
Gradient system
–
Varying eluent composition


HPGE (High Pressure Gradient)
LPGE (Low Pressure Gradient)
13
14
Aim of Gradient System (1)

In isocratic mode
CH3OH / H2O = 6 / 4
Long analysis time!!
Poor separa
tion!!
(Column: ODS type)
CH3OH / H2O = 8 / 2
High- / Low-Pressure Gradient System
Low-pressure gradi
ent unit
Mixer
High-pressure gradient
Mixer
Low-pressure gradient
15
Online Degasser
Regulator
Helium c
ylinder
Polymeric film tube
Vacuum chamber
To pump
To pump
To draft
Drain valve
Eluent container
Helium purge method
Eluent container
Gas-liquid separation membrane method
16
Manual Injector
From pump
To column
LOAD position
From pump
To column
INJECT position
17
Autosampler
(Pressure Injection Method)
From pump
To column
From pump
To column
Sample Loop
LOAD
INJECT
18
19
Connectors

Male nut (SUS)
Ferrule (SUS)
–

Sealing possible up to 4
0 MPa
Ferrule
Male nut
Male nut (PEEK)
–
–
Can be connected witho
ut any tools
Resists pressures of up t
o approx. 25 MPa
Male nut (PEEK)
Dead Volume
(Extra-column volume)

Dead volume can cause peaks broadening.
Male nut
Dead volume
Tube
Excellent connection
Poor connection
20
Tubing

Material
–
–
–
Stainless steel (SU
S)
PEEK (polyether eth
er ketone)
Fluororesin

O.D. (outer diamete
r)
–

1.6 mm
I.D. (inner diameter)
–
–
–
–
0.1
0.3
0.5
0.8
mm
mm
mm
mm etc.
21
Mobile Phase

Water
–
–
“Ultrapure water” can b
e used with confidenc
e.
Commercial “distilled w
ater for HPLC” is also a
cceptable.

Organic Solvent
–
–
–
HPLC-grade solvent ca
n be used with confide
nce.
Special-grade solvent i
s acceptable dependin
g on the detection con
ditions.
Care is required regardi
ng solvents containing
stabilizers (e.g., tetrahy
drofuran and chlorofor
m)
22
Replacement of Eluent

Mutually insoluble solvents m
ust not be exchanged directl
y.
Water
2-Propanol
Hexane

Aqueous solutions containi
ng salt and organic solvent
s must not be exchanged
directly.
Buffer solution
Water
Water-soluble
organic solvent
23
Mixing, Filtration, and Offline Degas
sing of the Eluent
Membrane filter with pore
size of approx. 0.45 µm
Decompression
by aspirator
Decompression
by aspirator
Ultrasonic cl
eaning unit
24
Reversed Phase Chromatography P
art 1
Basic Principles
25
Polarity of Substances

Polarity
–
–

Property of a substance w
hereby the positions of the
electrons give rise to positi
ve and negative poles
Water: Polar
Methane: Nonpolar
H
–
O
C
H
–
–
H
H
H
Methane
+
Miscibility of solvent
s
H
Water
Solvents of similar polariti
es can be easily dissolve
d together.
Polar and nonpolar molec
ules have a similar relatio
nship to that of water and
oil. H
O
H C C
–
O
H
Acetic acid
26
Nonpolar (Hydrophobic) Functional Groups an
d Polar (Hydrophilic) Functional Groups

Nonpolar Function
al Groups
–
-(CH2)nCH3

–
Polar Functional Gr
oups
–
Alkyl groups
-C6H5


-COOH

–
Phenyl groups
-NH2

–
Carboxyl groups
Amino groups
-OH

Hydroxyl groups
27
28
Normal Phase / Reversed Phase
Stationary
phase
Mobile phase
Normal
phase
High polarity
Low polarity
(hydrophilic)
(hydrophobic)
Reversed
phase
Low polarity
High polarity
(hydrophobic)
(hydrophilic)
Reversed Phase Chromatography

Stationary phase: Low polarity
–

Octadecyl group-bonded silical gel (ODS)
Mobile phase: High polarity
–
–
Water, methanol, acetonitrile
Salt is sometimes added.
29
Separation Column for Reversed Ph
ase Chromatography



C18 (ODS) type
C8 (octyl) type
C4 (butyl) type
Si
-O-Si



Phenyl type
TMS type
Cyano type
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3
C18 (ODS)
30
Effect of Chain Length of Stationary
Phase
C8
Medium
C18 (ODS)
Strong
C4
Weak
31
Hydrophobic Interaction
H2O
H2O
H2O
H2O
H2O
H2O
H2O
Nonpolar solute
H2O
H2O
If a nonpolar
H2O
substance is added...
H2O
H2O
H2O
…the network is broken and...
Network of hydrogen bonds
H2O
H2O
H2O
H2O
H2O
H2O
H2O
H2O
Nonpolar solute
Nonpolar stationary phase
…the nonpolar substance
is pushed to a nonpolar
location.
32
Relationship Between Retention Tim
e and Polarity
OH
C18 (ODS)
Weak
Strong
CH3
33
Basic Settings for Eluent Used in Re
versed Phase Mode

Water (buffer solution) + water-soluble or
ganic solvent
–
–
–
Water-soluble organic solvent: Methanol
Acetonitrile
Tetrahydrofuran e
tc.
The mixing ratio of the water (buffer solution) a
nd organic solvent has the greatest influence o
n separation.
If a buffer solution is used, its pH value is an i
mportant separation parameter.
34
Difference in Solute Retention Strengths fo
r Water and Water-Soluble Organic Solvent
s
Tightly packed network
H2O
H2O
H2O
H2O
H2O
Loose network
CH3OH
H2O
H2O
CH3OH
CH3OH
CH3OH
CH3OH
Nonpolar solute
CH3OH
CH3OH
Nonpolar solute
Nonpolar stationary phase
35
Relationship between Polarity of Eluent a
nd Retention Time in Reversed Phase Mo
de
Eluent: Methanol / Water
60/40
70/30
80/20
36
Acid Dissociation Equilibrium
H+
If an acid is added...
...the equilibrium shifts to t
he left to offset the increas
e in H+.
A- + H+
HA
If an alkali is adde
d...
…the equilibrium shifts t
o the right to offset the d
ecrease in H+.
OH-
The equilibrium always shifts
in a way that offsets changes.
37
Acid Dissociation Constant and pH
-Based Abundance Ratio
1.0
A- + H+
The acid dissociation constant, Ka,
is defined as follows:
[A  ][ H  ]
Ka 
[HA ]
CH3COO-
CH3COOH
0.8
Abundance ratio
HA
0.6
0.4
0.2

[A ]
pH  pK a  log
[HA ]
 pH   log[ H  ] 


 pK   log K 
a 
 a
0.0
1
2
3
4
5
6
7
8
9
pH
pKa
Relationship Between Abundance Ratio
and pH Value of Acetic Acid and Acetic Acid Ions
38
pH of Eluent and Retention of Ionic
Solutes
COOH
Acidic
Increased
hydrophobicity
pH of eluent
COO
Alkaline
Increased
hydrophilicity
+
H
39
Analysis of Basic Substances (1)
Problems Encountered with Alkaline Eluen
ts
N+
H
N
OH
With alkaline eluents, although the ion
ization of basic substances is suppres
sed, and the retention strength increa
ses...
Si
O
OH
Si
OH
OH
OH
…silica gel dissolves in alkalis,
so the packing material deterior
ates rapidly.
OH
40
Analysis of Basic Substances (2)
Influence of Residual Silanol Groups
Basic substances interact with the re
sidual silanol groups, causing delaye
d elution and tailing.
Si
O
Si
-O-Si-O
Residual silanol group
O
Si
N+
H
41
Reversed Phase Ion Pair Chroma
tography

42
Increase the retention strength by adding an ion pai
r reagent with the opposite charge to the target sub
stance into the eluent.
Ion pair formationIon pair forma
Ion
exchange-like
Ion
effect
exchange
Basic Substance
Acidic Substance
Normal Phase and Reversed Phas
e
43
Solid phase
Mobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)
Stationary Phase and Mobile Phase Used i
n Normal Phase Mode

Stationary Phase
–
–
–
–

Silica gel: -Si-OH
Cyano type: -Si-CH2CH2CH2CN
Amino type: -Si-CH2CH2CH2NH2
Diol type: -Si-CH2CH2CH2OCH(OH)-CH2O
H
Mobile Phase
–
–
Basic solvents: Aliphatic hydrocarbons,
aromatic hydrocarbons, etc.
Additional solvents: Alcohols, ethers, etc.
44
Relationship between Hydrogen Bonding
and Retention Time in Normal Phase Mod
e
SiOH
Strong
HO
SiOH
Weak
Very weak
OH
Steric hindrance
45
Ion Exchange Chromatography
R
Anion exchange
N+ R
R
Cation exchange
SO3-
++++
+
+
++++
Electrostatic interaction
(Coulomb force)
46
Stationary Phase Used in Ion Excha
nge Mode

Base Material
–
–

Cation Exchange Column
–
–

Resin is often used.
Silica gel is also used.
Strong cation exchange (SCX)
Week cation exchange (WCX)
-SO3-COO-
Anion Exchange Column
–
–
Strong anion exchange (SAX)
Week anion exchange (WAX)
-NR3+
-NHR2+
47
Detection Condition Requirements

Sensitivity
–

Selectivity
–


The detector must have the appropriate lev
el of sensitivity.
The detector must be able to detect the tar
get substance without, if possible, detectin
g other substances.
Adaptability to separation conditions
Operability, etc.
48
Representative HPLC Detectors








UV-VIS absorbance detector
Photodiode array-type UV-VIS absorbance
detector
Fluorescence detector
Refractive index detector
Evaporative light scattering detector
Electrical conductivity detector
Electrochemical detector
Mass spectrometer
49
UV-VIS Absorbance Detector
Ein
Eout
A
C: Concentration
Detection cell
l
A = e·C·l = –log (Eout / Ein)
C
(A: absorbance, E: absorption coefficient)
50
Optical System of UV-VIS Absorban
ce Detector
Grating
l
Sample cell
Ein
Eout
Photodiode
Ein
Ein
Photodiode
Reference cell
D2 / W lamp
51
Optical System of Photodiode Array
Detector
Sample cell
D2 / W lamp
Grating
A single photodiode
measures the absorbance for
the corresponding wavelength
at a resolution of approx. 1 nm.
Photodiode array
52
Data Obtained with a Photodiode Ar
ray Detector
Spectrum
Absorbance
Chromatogram
Retention time
53
Fluorescence Detector
Excitation wavelength
*
+ hv1
*
hv2 +
Excited state
Fluorescence wavelength
Quasi-excited state
hv1
hv2
Fluorescence
Ground state
54
Optical System of Fluorescence Detec
tor
Xenon lamp
Fluorescence
grating
Photomultiplier tube
Fluorescence
Excitation grating
Excitation
Sample cell
light
55
Differential Refractive Index Detecto
r (Deflection-Type)
Light-receiving unit
Reference cell
Light
Sample cell
56
Evaporative Light Scattering Detector
Light-receiving unit
Drift tube
Nebulizer
Column eluate
Nebulizer gas
Drain
Assist gas
Light source
The column eluate is evaporated and the light scattered b
y the particles of nonvolatile substances is detected.
57
Principle of Electrical Conductivity Detec
tor
V
I
A
I A
K   k
E L
L
k  K
A
A
L
Electrode
K:
I:
E:
A:
L:
k:
Electrical conductivity [S]
Electric current [A]
Voltage [V]
Electrode surface area [cm2]
Distance between electrodes [cm]
Specific electrical conductivity [S•cm-1]
58
Electrochemical Detector
Electrode
HO
R
HO
2e-
O
R
+ 2H+
O
59
Cell Structure of Electrochemical De
tector (Amperometric Type)
Reference electrode
(Ag/AgCl)
Working electrode
(glassy carbon)
Eluent
Electrode couple
60
Mass Spectrometer (LCMS)
Atmospheric
pressure
API probe
High vacuum
Quadrupole MS analyzer
Electron multipl
ier tube
RP TMP1 TMP2
(high vacuum pumps)
61
62
Advantages of LCMS (1)

Quantitative analysis with excellent selectivit
y
m/z=100
A
TIC A:100
B
B:100
C:150 D:150
m/z=150
C
D