Reversed Phase Chromatography

Basic Principles

CUP
LAAQ-B-LC001B 1
 Polarity of Substances

• Polarity • Miscibility of solvents

– Property of a substance
whereby the positions of the
electrons give rise to positive
and negative poles
– Water: Polar
Methane: Nonpolar
– Solvents of similar polarities
can be easily dissolved
together.
– Polar and nonpolar molecules
have a similar relationship to
that of water and oil.

H –
H O
O
C H C C –
H H O
+ H
H H
H
CUP
LAAQ-B-LC001B Methane Water Acetic acid 2
 Nonpolar (Hydrophobic) Functional Groups and Polar
(Hydrophilic) Functional Groups

• Nonpolar Functional • Polar Functional Groups

Groups – -COOH
– -(CH2)nCH3 • Carboxyl groups
• Alkyl groups – -NH2
– -C6H5 • Amino groups
• Phenyl groups – -OH
• Hydroxyl groups

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LAAQ-B-LC001B 3
 Partition Chromatography

• A liquid (or a substance regarded as a liquid)

is used as the stationary phase, and the
solute is separated according to whether it
dissolves more readily in the stationary or
mobile phase.
• Liquid-liquid chromatography

CUP
LAAQ-B-LC001B 4
 Normal Phase / Reversed Phase

Stationary
Mobile phase
phase
Normal High polarity Low polarity
phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (hydrophilic)

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LAAQ-B-LC001B 5
 Reversed Phase Chromatography
• Stationary phase: Low polarity
– Octadecyl group-bonded silical gel (ODS)
• Mobile phase: High polarity
– Water, methanol, acetonitrile
– Salt is sometimes added.

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LAAQ-B-LC001B 6
 Separation Column for Reversed Phase
Chromatography
• C18 (ODS) type • Phenyl type
• C8 (octyl) type • TMS type
• C4 (butyl) type • Cyano type

CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2

Si -O-Si
CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH2 CH3

C18 (ODS)

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LAAQ-B-LC001B 7
 Effect of Chain Length of Stationary
Phase

C8

Medium
C18 (ODS)

Strong C4

Weak

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LAAQ-B-LC001B 8
 Hydrophobic Interaction

H2O H2O H2O

H2O
H2O
H2O Nonpolar solute H2O
H2O
H2O If a nonpolar H2O
H2O H2O substance is added... H2O H2O
Network of hydrogen bonds …the network is broken and...

H2O H2O
H2O
H2O H2O …the nonpolar substance
H2O H2O is pushed to a nonpolar
location.
Nonpolar solute

CUP
LAAQ-B-LC001B Nonpolar stationary phase 9
 Relationship Between Retention Time
and Polarity

C18 (ODS) OH

Weak
Strong
CH3

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LAAQ-B-LC001B 10
 Basic Settings for Eluent Used in
Reversed Phase Mode
• Water (buffer solution) + water-soluble organic
solvent
– Water-soluble organic solvent: Methanol
Acetonitrile
Tetrahydrofuran etc.
– The mixing ratio of the water (buffer solution) and organic
solvent has the greatest influence on separation.
– If a buffer solution is used, its pH value is an important
separation parameter.

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LAAQ-B-LC001B 11
 Difference in Solute Retention Strengths for Water
and Water-Soluble Organic Solvents

Tightly packed network Loose network

H2O H2O CH3OH CH3OH

H2O H2O
H2O CH3OH
H2O H2O CH3OH CH3OH

CH3OH
Nonpolar solute CH3OH
Nonpolar solute

Nonpolar stationary phase

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LAAQ-B-LC001B 12
 Relationship between Polarity of Eluent and
Retention Time in Reversed Phase Mode

Eluent: Methanol / Water

60/40

70/30

80/20
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LAAQ-B-LC001B 13
 Consideration of Analytical
Conditions

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LAAQ-B-LC001B 14
 Guidelines for Setting Mobile Phase Conditions (1)
Neutral (Nonionic) Substances

• Eluent Composition
– Water / acetonitrile
– Water / methanol
• Separation Adjustment
– Changing the mixing ratio of the water and organic
solvent
– Changing the type of organic solvent

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LAAQ-B-LC001B 15
 pH of Eluent and Retention of Ionic
Solutes

COOH
Acidic
Increased
hydrophobicity
pH of eluent

COO
Alkaline
Increased
hydrophilicity
+
H
CUP
LAAQ-B-LC001B 16
 Guidelines for Setting Mobile Phase Conditions
Acidic (Anionic) Substances

• Eluent Composition
– Acidic buffer solution / acetonitrile
– Acidic buffer solution / methanol

Increase retention strength by making

the eluent acidic and suppressing
ionization!

CUP
LAAQ-B-LC001B 17
 Analysis of Basic Substances (1)
Problems Encountered with Alkaline Eluents

With alkaline eluents, although the

ionization of basic substances is
suppressed, and the retention
N+ N strength increases...
H

OH Si
O

OH Si OH
…silica gel dissolves in alkalis,
so the packing material
OH deteriorates rapidly.
OH
OH
CUP
LAAQ-B-LC001B 18
 Analysis of Basic Substances (2)
Influence of Residual Silanol Groups

Basic substances interact with the

residual silanol groups, causing
delayed elution and tailing.

Si
O

Si -O-Si-O
Residual silanol group N+
O H
Si

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LAAQ-B-LC001B 19
 Analysis of Basic Substances (3)
Addition of Sodium Perchlorate

ClO4 Ion pair

N+
H

Si
O

Si Basic substances form ion pairs with perchlorate

ions, thereby balancing the charge and
increasing the retention strength.

CUP
LAAQ-B-LC001B 20
 Guidelines for Setting Mobile Phase Conditions (3)
Basic Substances (Cationic Substances)

• Eluent Composition
– Acidic buffer solution containing anions with a low
charge density (e.g., perchlorate ions) / acetonitrile
– As above / methanol
Making eluent acidic
 Suppresses dissociation of residual silanol groups
 Prevents tailing!

Adding perchlorate ions

 Forms ion pairs  Increases retention strength!
 Suppresses tailing!
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LAAQ-B-LC001B 21
 Reversed Phase Ion Pair
Chromatography
• Increase the retention strength by adding an ion pair
reagent with the opposite charge to the target substance
into the eluent.
Ion pair formation Ion pair formation

Ion exchange-like effect Ion exchange-like effect

CUP
LAAQ-B-LC001B
Basic Substance Acidic Substance 22
 Representative Ion Pair Reagents
• Anionic Compounds
– Tetra-n-butylammonium hydroxide (TBA)
• Cationic Compounds
– Pentanesulfonic acid sodium salt (C5)
– Hexanesulfonic acid sodium salt (C6)
– Heptanesulfonic acid sodium salt (C7)
– Octanesulfonic acid sodium salt (C8)

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LAAQ-B-LC001B 23
 Points to Note Concerning the Use of
Ion Pairs
• Selection of Ion Pair Reagent
– In general, the retention strength increases with the length of the
alkyl chain.
• pH of Eluent
– The retention strength changes according to whether or not
ionization takes place.
• Concentration of Ion Pair Reagent
– In general, the retention strength increases with the ion pair
concentration, but there is an upper limit.
• Proportion of Organic Solvent in Eluent
– Optimize the separation conditions by considering the type and
concentration of the ion pair reagent.

CUP
LAAQ-B-LC001B 24
 HPLC Separation Modes

Separation Modes Other Than

Reversed Phase Chromatography

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LAAQ-B-LC001B 25
 HPLC Separation Modes
• Adsorption (liquid-solid) chromatography
• Partition (liquid-liquid) chromatography
– Normal phase partition chromatography
– Reversed phase partition chromatography
• Ion exchange chromatography
• Size exclusion chromatography

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LAAQ-B-LC001B 26
 Adsorption Chromatography
• A solid such as silica gel is used as the
stationary phase, and differences, mainly in
the degree of adsorption to its surface, are
used to separate the solutes.
• Liquid-solid chromatography
• The retention strength increases with the
hydrophilicity of the solute.

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LAAQ-B-LC001B 27
 Partition Chromatography

• A liquid (or a substance regarded as a liquid) is

used as the stationary phase, and the solute is
separated according to whether it dissolves
more readily in the stationary or mobile phase.
• Liquid-liquid chromatography

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LAAQ-B-LC001B 28
 Normal Phase and Reversed Phase

Solid phase Mobile phase

Normal High polarity Low polarity

phase (hydrophilic) (hydrophobic)
Reversed Low polarity High polarity
phase (hydrophobic) (hydrophilic)

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LAAQ-B-LC001B 29
 Normal Phase (Partition)
Chromatography
• Partition chromatography in which the stationary
phase has a high polarity (hydrophilic) and the
mobile phase has a low polarity (hydrophobic)
• Essentially based on the same separation
mechanism as adsorption chromatography in
which the stationary phase has a hydrophilic base,
such as silica gel

CUP
LAAQ-B-LC001B 30
 Stationary Phase and Mobile Phase Used in Normal
Phase Mode

• Stationary Phase
– Silica gel: -Si-OH
– Cyano type: -Si-CH2CH2CH2CN
– Amino type: -Si-CH2CH2CH2NH2
– Diol type: -Si-CH2CH2CH2OCH(OH)-CH2OH
• Mobile Phase
– Basic solvents: Aliphatic hydrocarbons,
aromatic hydrocarbons, etc.
– Additional solvents: Alcohols, ethers, etc.

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LAAQ-B-LC001B 31
 Relationship between Hydrogen Bonding and
Retention Time in Normal Phase Mode

SiOH HO
Strong
SiOH
Weak
Very weak
OH

Steric hindrance
CUP
LAAQ-B-LC001B 32
 Relationship Between Eluent Polarity and Retention
Time in Normal Phase Mode

Eluent: Hexane/methanol

100/0

98/2

95/5
CUP
LAAQ-B-LC001B 33
 Comparison of Normal Phase and
Reversed Phase
• Normal Phase
– Effective for separation of
structural isomers
– Offers separation
selectivity not available
with reversed phase
– Stabilizes slowly and is
prone to fluctuations in
retention time
– Eluents are expensive
CUP
LAAQ-B-LC001B
• Reversed Phase
– Wide range of applications
– Effective for separation of
homologs
– Stationary phase has long
service life
– Stabilizes quickly
– Eluents are inexpensive and
easy to use
34
 Ion Exchange Chromatography

R
Anion exchange N+ R
R

++++
Cation exchange SO3- + +
++++
Electrostatic interaction
(Coulomb force)
CUP
LAAQ-B-LC001B 35
 Stationary Phase Used in Ion Exchange
Mode
• Base Material
– Resin is often used.
– Silica gel is also used.

• Cation Exchange Column

– Strong cation exchange (SCX) -SO3-
– Week cation exchange (WCX) -COO-
• Anion Exchange Column
– Strong anion exchange (SAX) -NR3+
– Week anion exchange (WAX) -NHR2+

CUP
LAAQ-B-LC001B 36
 Dependence of Exchange Capacity of Ion Exchanger
on pH of Eluent

Strongly acidic Strongly basic

cation exchanger anion exchanger
Exchange capacity

Exchange capacity
Weakly acidic
cation exchanger Weakly basic
anion exchanger

0 7 14 0 7 14
pH pH
Cation exchange mode Anion exchange mode
CUP
LAAQ-B-LC001B 37
 Relationship between Retention Time and Salt
Concentration of Eluent in Ion Exchange Mode

Resin Resin Resin

The exchange groups An eluent ion is The solute ion is driven
are in equilibrium with driven away away by an eluent ion
anions in the eluent. and a solute ion and is adsorbed by the
is adsorbed. next exchange group.

Solute ions and eluent ions compete for ion exchange groups.

If the salt concentration of the eluent increases, the solutes are eluted sooner.
CUP
LAAQ-B-LC001B 38
 Ion Exclusion Chromatography

H+

H+

H+

Strong acid ions are repelled by Depending on the level of dissociation,

charge and cannot enter the pore. some weak acid ions can enter the pore.

CUP
LAAQ-B-LC001B 39
 HPLC Hardware

Detectors and Their Ranges of Application

CUP
LAAQ-B-LC001B 40
 Detection Condition Requirements
• Sensitivity
– The detector must have the appropriate level of
sensitivity.
• Selectivity
– The detector must be able to detect the target
substance without, if possible, detecting other
substances.
• Adaptability to separation conditions
• Operability, etc.

CUP
LAAQ-B-LC001B 41
 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

CUP
LAAQ-B-LC001B 42
 UV-VIS Absorbance Detector

C: Concentration
Detection cell

Ein Eout

A
l

A = e·C·l = –log (Eout / Ein) C

(A: absorbance, E: absorption coefficient)

CUP
LAAQ-B-LC001B 43
 Optical System of UV-VIS
Absorbance Detector
Grating
Sample cell
l Ein Eout Photodiode

Ein Ein Photodiode

Reference cell

D2 / W lamp

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LAAQ-B-LC001B 44
 Spectrum and Selection of
Detection Wavelength

The longer wavelength

is more selective.

200 250 300 350

Wavelength [nm]
CUP
LAAQ-B-LC001B 45
 Optical System of Photodiode
Array Detector
Sample cell
Grating

A single photodiode
D2 / W lamp measures the absorbance for
the corresponding wavelength
at a resolution of approx. 1 nm.

Photodiode array

CUP
LAAQ-B-LC001B 46
 Data Obtained with a Photodiode
Array Detector
Spectrum

Chromatogram
Absorbance

CUP
LAAQ-B-LC001B
Retention time 47
 Advantages of Photodiode Array
Detectors
• Peak Identification Using Spectra
– Complementation of identification based on
retention time
– Library searches
• Evaluation of Peak Purity
– Purity evaluation performed by comparison of the
shape of spectra from the peak detection start
point to the peak detection end point

CUP
LAAQ-B-LC001B 48
 Fluorescence Detector
Excitation wavelength

+ hv1 *

* hv2 +
Fluorescence wavelength
Excited state
Quasi-excited state
hv1
hv2
Fluorescence

CUP
LAAQ-B-LC001B
Ground state 49
 Optical System of Fluorescence Detector

Xenon lamp
Fluorescence
grating
Photomultiplier tube

Fluorescence

Excitation
Excitation grating Sample cell
light

CUP
LAAQ-B-LC001B 50
 Fluorescence Derivatization Reagents

• OPA Reagent (Reacts with Primary Amines)

S-R’
CHO
+ R-NH2 N-R
CHO R’-SH
o-phthalaldhyde
(OPA)

 ADAM Reagent (Reacts with Fatty Acids)

+ R-COOH

CHN2 CH2OCOR
9-anthryldiazomethane
(ADAM)
CUP
LAAQ-B-LC001B 51
 Differential Refractive Index
Detector (Deflection-Type)

Light-receiving unit
Reference cell

Light

Sample cell

CUP
LAAQ-B-LC001B 52
 Optical System of Differential Refractive Index
Detector (Deflection-Type)

Slit W lamp

Reference cell
Sample cell
The slit image moves if the
refractive index inside the
flow cell changes.

Photodiode

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LAAQ-B-LC001B 53
 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

by the particles of nonvolatile substances is detected.

CUP
LAAQ-B-LC001B 54
 Electrical Conductivity Detector

Pure water NaCl aqueous

solution

Cl- Na+

The bulb does not light with water. The bulb lights if there are ions.

CUP
LAAQ-B-LC001B 55
 Principle of Electrical Conductivity Detector

I A
V K   k
I
E L
L
k  K
A
A A K: Electrical conductivity [S]
I: Electric current [A]
E: Voltage [V]
A: Electrode surface area [cm2]
L Electrode L: Distance between electrodes [cm]
k: Specific electrical conductivity [S•cm-1]

CUP
LAAQ-B-LC001B 56
 Limiting Equivalent Ion Conductance, l
[S•cm2/mol], in Aqueous Solution (25ºC)

Cation l Anion l
H+ 349.8 OH– 198.3
Li+ 38.6 F– 55.4
Na+ 50.1 Cl– 76.3
K+ 73.5 Br– 78.1
NH4+ 73.5 NO3– 71.4
(CH3)3NH+ 47.2 CH3COO– 40.9
Mg2+ 53.0 C6H5COO– 32.3
Ca2+ 59.5 SO42– 80.0

CUP
LAAQ-B-LC001B 57
 Electrochemical Detector

Electrode
HO R

HO
2e-
O R
+ 2H+
O

CUP
LAAQ-B-LC001B 58
 Cell Structure of Electrochemical
Detector (Amperometric Type)

Reference electrode Working electrode

(Ag/AgCl) (glassy carbon)

Eluent

Electrode couple
CUP
LAAQ-B-LC001B 59
 Mass Spectrometer (LCMS)

Atmospheric
pressure High vacuum

Quadrupole MS analyzer
API probe

Electron
multiplier tube

RP TMP1 TMP2
(high vacuum pumps)

CUP
LAAQ-B-LC001B 60
 Atmospheric Pressure Ionization

Electrospray Ionization (ESI)

+
+ +
++- - + -- + +
+- -+- +
+-+-+-
+ +- + - ++ +
- + +
+

3) n E
Liquid
Liquid Samples
Sample

1)
+

2) of S

Io
C va
C
High
High Voltage

ou p
Neburaizing
Nebulizing Gas

Ev o l
ha
Voltage

lo ora
ap ve
rg

n
Gas

ed

ol nt

E x tio
at
D

cl n
io
ro

us
n
pl

io
et

n
Atmospheric Pressure Chemical Ionization (APCI)

Molecular ion reaction

Liquid Sample
Liquid Samples Heater
Heater
Neburaizing Gas
Nebulizing Gas Colona Discharge
Corona Discharge
CUP
LAAQ-B-LC001B Nnndle
Needle 61
 Advantages of LCMS (1)

• Quantitative analysis with excellent selectivity

m/z=100
A
TIC A:100 B
B:100
C:150 D:150

m/z=150
C D

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LAAQ-B-LC001B 62
 Advantages of LCMS (2)

• Peaks can be identified with MS spectra.

M/Z

M/Z

CUP
LAAQ-B-LC001B M/Z 63
 Comparison of Detectors

Possibility of
Selectivity Sensitivity
Gradient System
Light-absorbing
Absorbance ng Possible
substances

Fluorescence Fluorescent substances pg Possible

Differential
None µg Impossible
refractive index
Evaporative light
Nonvolatile substances µg Possible
scattering
Electrical
Ionic substances ng Partially possible
conductivity
Oxidizing / reducing
Electrochemical pg Partially possible
substances
CUP
LAAQ-B-LC001B Note: The above table indicates general characteristics. There are exceptions.
64
 Post-Column Derivatization

Reaction
chamber

Pump
Reaction
solution
CUP
LAAQ-B-LC001B 65
 Application Examples of Post-
Column Methods
• Amino Acids
– Orthophthalic acid, OPA
(fluorescence)
– Ninhydrin (visible absorption)
• Reducing Sugars
– Arginine (fluorescence)
• Carbamate Pesticides
– Alkaline hydrolysis - OPA
(fluorescence)
• Bromate Ions
– Tribromide ionization
(ultraviolet absorption)
– o-Dianisidine
(visible absorption)
• Cyanide Ions
– Chlorination - pyrazolone
(visible absorption)
• Transition Metal Ions
– 4-(2-Pyridylazo) resorcinol,
PAR (visible absorption)

CUP
LAAQ-B-LC001B 66
 Quantitative Analysis

Absolute Calibration Curve Method and

Internal Standard Method

CUP
LAAQ-B-LC001B 67
 Qualitative Analysis
• Identification based on retention time
• Acquisition of spectra with detector
– UV spectra
– MS spectra
• Transfer to other analytical instruments after
preparative separation

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LAAQ-B-LC001B 68
 Quantitative Analysis
• Quantitation performed with peak area or
height.
• Calibration curve created beforehand using a
standard.
– Absolute calibration curve method
– Internal standard method
– Standard addition method

CUP
LAAQ-B-LC001B 69
 Calibration Curve for Absolute
Calibration Curve Method
Area
Concentration
A1 Calibration curve
C1
A4

A2

Peak area
A3
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
CUP
LAAQ-B-LC001B
Concentration 70
 Calibration Curve for Internal
Standard Method
Concentration Area

Area for target substance / Area for internal standard

Target Internal
substance standard A1 AIS Calibration curve
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 of target substance /
CUP
LAAQ-B-LC001B
Concentration of internal standard
71
 Advantages of Internal
Standard Method (1)
• Not affected by inconsistencies in injection volume.

IS
X AX / AIS
10 µL
injected

Same area
ratio
IS
X
9 µL
injected
CX / CIS
CUP
LAAQ-B-LC001B 72
 Advantages of Internal
Standard Method (2)
• Not affected by the pretreatment recovery rate.
IS
X

AX / AIS
100%
recovery
rate

Same area
ratio
IS
90% X
recovery
rate
CX / CIS
CUP
LAAQ-B-LC001B 73
 Selection Criteria for Internal Standard

• It must have similar chemical properties to the target

substance.
• Its peak must appear relatively near that of the target
substance.
• It must not already be contained in the actual samples.
• Its peak must be completely separated from those of
other sample components.
• It must be chemically stable.

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LAAQ-B-LC001B 74
 Sample Pretreatment

Tasks Performed Before Injection

CUP
LAAQ-B-LC001B 75
 Objectives of Pretreatment
• To improve the accuracy of quantitative values
• To improve sensitivity and selectivity
• To protect and prevent the deterioration of
columns and analytical instruments
• To simplify measurement operations and
procedures
• To stabilize target substances

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LAAQ-B-LC001B 76
 Substances That Must Not Be
Injected into the Column
• Insoluble substances (e.g., microscopic particles
and precipitation)
• Substances that are precipitated in the eluent
• Substances that irreversibly adsorb to the packing
material
• Substances that dissolve, or chemically react,
with the packing material

CUP
LAAQ-B-LC001B 77
 Filtration and Centrifugal Separation

• In general, filter every

sample before injection!
• It is convenient to use a
disposable filter with a
pore diameter of approx.
0.45 µm.
• Centrifugal separation is
applicable for samples that
Filter Syringe
are difficult to filter.

CUP
LAAQ-B-LC001B 78
CUP
 Deproteinization
• Precipitation
– Addition of organic solvent (e.g., acetonitrile)
– Addition of acid (e.g., trichloroacetic acid,
perchloric acid)
– Addition of heavy metal or neutral salt
• Ultrafiltration

CUP
LAAQ-B-LC001B 79
 Solid Phase Extraction

(1) (2) (3) (4)

Conditioning Sample addition Rinsing Elution
Solvent with
low elution
strength

Solvent with
high elution
strength

Target
component
Unwanted
components

CUP
LAAQ-B-LC001B 80
 Pre-Column Derivatization

• OPA Reagent (Reacts with Primary Amines)

S-R’
CHO
+ R-NH2 N-R
CHO R’-SH
o-phthalaldhyde
(OPA)

 2,4-DNPH (Reacts with Aldehydes and Ketones)

R
NHNH2 NHN=C
R
+ C=O R’
R’ H+
O2 N NO2 O2 N NO2
2,4-dinitrophenylhydrazine
(2,4-DNPH)
CUP
LAAQ-B-LC001B 81