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Part I Basic Principles of HPLC


Chromatography

and HPLC
Various HPLC modes and applications
Normal

phase
Reversed phase
Reversed phase ion pairing
Ion exchange (IC)
SEC (GPC/GFC)
Chiral separation
HPLC

system modes

Isocratic elution
Gradient elution

What is chromatography?
Chromatography

is one of the separation

technique.
The

main purpose of chromatography is


to separate and quantify the target
sample in the matrix.

Why mixed sample


can be separated?
direction of flow

river bed

What is the difference?

Strong

Weak

Interaction is different
by gravity

How can the separation


be carried out?
Separation

can be carried out by the column.

column
packing material, 3-5um

Interaction between packing


material and sample
Due

to difference interaction between packing


material and sample, separation can be done.

sample A
packing
material

sample B

Separation can be done


by the column
mixed sample

column

What kind of chromatography?

High

Performance Liquid chromatography


(HPLC)
Gas Chromatography (GC)
Thin-Layer Chromatography (TLC)
Capillary Electrophoresis(CE)

Advantage of Chromatography

Simultaneous Analysis
High

Resolution

High

Sensitivity (ppm-ppb)

Small

Injection Volume (1-100uL)

10

Advantage of HPLC
Moderate

analysis condition

- no need to vaporize the sample like


GC
Easy

to fractionate the sample


and purify
Good repeatability (C.V. < 1%)

11

Flow Diagram of HPLC

pump

injector

column
oven

detector

12

Separation modes of HPLC


Normal

Phase mode (NP)


Reversed Phase mode (RP)
Reversed Phase Ion Pairing mode (IPC)
Ion Exchange mode (IC)
SEC mode ( GPC / GFC )
Chiral separation mode

13

Normal Phase Mode

M. Tswett :
first developer of chromatography
Ber. Deut. Botan. Ges., 24, 384 (1906)
Adsorptionsanalyse und chromatographische
Methode. Anwendung auf die Chemie des
Chlorophylls.

14

Normal Phase Mode


Petroleum ether

Chlorophyll's
CaCO3

Chromatograp
Chromato
h
Colors

15

Normal Phase Mode


First

developer used

CaCO3

as Separation Column

Petroleum

We defined

ether as Developed Solvent

this combination as

Normal Phase mode


Column
Solvent

:
:

polar property
non polar property

16

Normal Phase HPLC Columns


Silica

gel type : general use


Cyano type : general use
Amino type
: for sugar analysis
Diol type
: for protein analysis
-Si-CH2CH2CH2CN

Si
Silica gel

-Si-CH2CH2CH2NH2

Si

-Si-CH2CH2CH2OCH(OH)-CH2(OH)

Modified Si

17

What is the interaction?


Hydrogen bonding

Silica gel (polar)

Non-polar

18

Hydrogen Bonding

Compounds contain

-COOH
-NH2
-OH

: Carboxyl group
: Amino group
: Hydroxyl group

Hydrogen bonding strong interaction


Note: Compounds with bulk group, hydrogen bonding may not
form due to stereo hindrance

Compounds do not contain these groups

No hydrogen bonding weak interaction

Retention Time and


Hydrogen bonding
Strong
SiOH

HO

SiOH
Weak
OH

Very Weak

or

19

Mobile phase solvents for normal


phase HPLC

Primary solvents (non-polar)

Hydrocarbons (Pentane, Hexane, Heptane, Octane)


Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
Methylene chloride
Chloroform
Carbon tetrachloride

Secondary solvents

20

Methyl-t-butyl ether (MTBE), Diethyl ether, Tetrahydrofuran (THF), Dioxane,


Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol, ethanol, methanol

A primary solvent is used as mobile phase. Addition of secondary


solvents is to to adjust retention time.
Solvents which possess UV transparency are often preferred for easy
detection.

21

Increase of solvent polarity


0%

1 : Dioctyl phthalate
2 : Dibutyl phthalate
3 : Diethyl phthalate
4 : Dimethyl phthalate

2%

Solvent : Hexane

5% / MeOH

22

Reversed Phase mode

Column
Solvent

:
:

Non-polar property
Polar property

- Normal Phase mode Column


Solvent

:
:

polar property
non polar property

23

Reversed Phase HPLC Columns


C18

(ODS) type
C8 (octyl) type
C4 (butyl) type
Phenyl type
TMS type
Cyano type

Non-polar property
-Si-C18H37
Si

24

What is the interaction?


Hydrophobic
interaction

Non-polar

polar solvent

25

Hydrophobicity
If

the sample has more


CH3CH2CH2--- : Carbon chain
: Aromatic group

If

the sample has more

-COOH
-NH2

: Carboxyl group
: Amino group

-OH

: Hydroxyl group

Hydrophobicity
is stronger.

Hydrophobicity
is weaker.

Retention Time and


Hydrophobicity
OH

C18 (ODS)
Strong
OH

Weak

26

Mobile phase solvents for


reversed phase HPLC
Water (buffer)

+ Organic solvents

When buffer is used, the concentration and pH are


important factors
Methanol (MeOH), acetonitrile (A CN) or THF are
common organic solvents for r.p HPLC.

Optimization

of water (buffer) and organic


solvents ratio is very important.

27

28

Increase of solvent polarity


20 %

30 %

1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate

40% / H2O

Solvent : MeOH

29

Effect of stationary phase


C8
Medium

C18 (ODS)

sample
Strong

C4

sample
Weak
sample

30

Effect of stationary phase


ODS

C8 TMS

Analytical Conditions

Column : Shim-pack CLC-ODS


Mobile phase : MeOH : H2O = 7 :3
Flow rate : 1.0 mL/min
Temperature : 40 C
Injection volume : 10 uL
Detection : UV-254 nm
Peaks
1. Methyl benzoate
2. Ethyl benzoate
3. n-Propyl benzoate
4. n- Butyl benzoate

31

Normal vs Reversed Phase


Normal

Phase

good

separation for stereo isomers


(Vitamin E etc..)
changeable retention time
Reversed
good

Phase

repeatable retention time


rugged stationary phase

Reversed Phase
Ion-Pair Chromatography

32

Ion-Pair Reagent

33

Ion-Pair Reagents
Anion

Compounds

Tetra-n-butylammonium

Cation

hydroxide (TBA)

Compounds

Butanesulfonic

acid sodium salt (C4)


Pentanesulfonic acid sodium salt
(C5)
Hexanesulfonic acid sodium salt (C6)
Heptanesulfonic acid sodium salt
(C7)
Octanesulfonic acid sodium salt (C8)
Decanesulfonic acid sodium salt (C10)

Ion Pairing
Separation of Carboxylic Acids

Column: Bonded C18


Mobile Phase: H2O/MeOH
1:1 with TetrabutylAmmonium Hydroxide

34

Ion Pairing
Separation of Amino Compounds

Analytical Conditions

Peaks
1. Nicotinic acid
2. Nicotinamide
3. Pantothenate
4. Pyridoxine
5. Riboflavin
Phosphate

6. Thiamine
7. Caffeine
8. Folic acid
9. Biotin
10. Riboflavin

35

Column : Shim-pack CLC-ODS


Mobile phase : [A] : [B] = 9 : 1
[A]
100 mM phosphate buffer (pH=2.1)
0.8 mM sodium octane sulfonate
[B] acetonitrile
Flow rate : 1.5 mL/min
Temperature : 40 C
Injection volume : 10 uL

Ion Paring
Important Considerations
Type

of Ion-Pair reagents
Concentration of Ion-Pair reagents
pH

of solvent
RCOO- + H+

R-COOH

(pKa=4.5)
R-NH2 + H+

R-NH3+
(pKa=6.0)

36

37

Type of Ion-Pair Reagents


Hexane Sulfonate

Mobile Phase: H2O/MeOH


1:1,with 0.005M ion pairing
reagent and 1% HOAc
1 Maleic Acid
2 Phenylephrine
3 Phenylpropanolamine
4 Naphazoline
5 Phenacetin
6 Pyrilamine

Pentane Sulfonate

Concentration of
Ion-Pair Reagents

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39

Washing column in case of TBA

If amine modifier or TBA ion pair reagent is used as a


mobile phase, the column must be washed when analysis is
finished.
In order to remove these amine modifier or TBA ion pair
reagent, sodium perchlorate is effectively working. Because
sodium perchlorate has strong affinity to amine compounds.
Following solution may be recommended for column
washing
0.1% phosphoric acid including 100 mM sodium
perchlorate : methanol = 1 : 1

40

Ion Exchange Mode


R

Ion Attraction Force

N+ R
R

SO3

Sample
+ + + +
+
+
Sample
+
+
+ + + + +

41

Ion Exchange Mode


Biological

Fields
(protein, peptide, amino acid analysis)
Ion chromatography
Cation

Exchangers

Strong

Cation Exchange (SCX)


Weak Cation Exchange
(WCX)
Anion

(R-SO3-)
(R-COO-)

Exchangers

Strong Anion

Exchange
Weak Anion Exchange

(SAX)
(WAX)

(R4N+)
(DEAE)

Protein Analysis
using WCX column
Analytical

Conditions

Column

: Shim-pack WCX-1
Mobile phase :
[A] 20 mM phosphate buffer (pH=6.0)
[B] A+0.25M sodium sulfate
[A] - [B] 30 min linear gradient
Flow rate : 1.0 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. albumin
2. myoglobin
3. -chymotrypsinogen A
4. liponuclease A
5. lisozyme

42

Ion Exchange mode


Important Considerations
pH

of Buffer solution
Concentration of Buffer solution
Elution Method
Isocratic

elution
pH gradient elution
increasing Ionic strength gradient

43

44

What is SEC ?
Size Exclusion Chromatography (SEC)
GPC

(Gel Permeation chromatography)

mainly

GFC

polymer science field

(Gel Filtration Chromatography)

mainly

biological science field

45

Principle of SEC
No Interaction Force
Difference of Traveling Time

46

Elution order

SEC
Column

47

Purpose of GPC / GFC


GPC
Molecular Weight measurement

polymer
GFC
Separation of

protein

of

Molecular Weight (LogMW)

Relationship between
MW and RT
Exclusion limit
Permeation limit

Time

48

49

Calibration Curve
Inject

the standard polymer sample one


by one to know the relationship between
molecular weight and retention time.
No.
1
2
3
4
5
6
7
8
9
10
11

time(min)
22.0
22.6
23.4
25.0
27.4
31.0
33.8
38.4
39.8
42.8
46.8

mol. wet.
5500000
1800000
860000
400000
160000
50000
20000
4000
2000
600
80

50

Actual Sample Injection


To calculate

the MW,
GPC software is required.

Protein Separation
using GFC column
Analytical

Conditions

Column

: Asahipak GFA-50
Mobile phase :
0.1 M sodium phosphate
0.1 M NaCl (pH=7.0)
Flow rate : 0.5 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. glutamate dehydrogenase
2. lactate dehydrogenase
3. enolase
4. adenylate kinase
5. cytochrome C

51

52

Chiral Separation Mode

C*
NH2

M
ir
ro
r

*C
COOH

HOOC

*Chiral Center

NH2

(L) form
(D) form
Physical properties are all the same
except optical rotation.

53

Separation of Chiral Compound


Direct

separation using Chiral Column


Diastereomeric derivatization
(L) + (R)

(L)-(R)

Enantiomer

Diastereomer

(R) + (R)

(R)-(R)

Diastereomer can be separated


by reverse / normal phase column.

Direct separation
using Chiral Column
Stationary

phase has chiral moiety.

(R)
(R)

(L)
Strong interaction
Weak interaction

(R)

54

55

Gradient Elution in HPLC


Isocratic

elution mode

One

mobile phase with a constant


composition

Gradient
Multi

elution mode

mobile phases with changing


composition

56

Isocratic Elution System

pump

oven

injector

column

Single Solvent

detector

57

Gradient Elution System

B
pump

oven

B concentration

pump

injector

column

Time

detector

58

Isocratic Elution Mode


MeOH / H2O = 6 / 4

Long Time Analysis

Bad Separation
MeOH / H2O = 8 / 2
( column : ODS type )

59

95%

30%

MeOH concentration

Gradient Elution Mode

60

Part II Instrumentation of HPLC

Solvent delivery pump

Sample injector

Column oven

Detector

61

Flow Diagram of HPLC

pump

injector

column
oven

detector

62

Desirable Pump Performance


High

Pressure Resistance

Precise
Low

Flow Rate

Pressure Fluctuation

63

Pump pressure fluctuation


Low pressure-fluctuation pump

This pressure fluctuation


will affect detector noise.

P=0.5

High pressure-fluctuation pump

P=1.5

Methanol 1.0mL/min 40 kgf/cm2

64

Plunger reciprocating pump

motor and cam

pump head

check
valve
plunger
plunger seal

10 -100uL

65

Plunger reciprocating pump

Advantage
Low

pressure fluctuation
Very easy to replace the another
solvent
Disadvantage
Change

the plunger seal

Dual plunger
with parallel flow line
check valve

plunger head

66

Very low pressure fluctuation


Refractive index detector
Conductivity detector
Electrochemical detector
MS detector
The number of maintenance
parts is more.

Dual plunger
with tandem flow line
check valve

67

Low pressure fluctuation


UV / PDA detector
Fluorescence detector
The number of maintenance
parts is less. So this design is
suitable for routine analysis.

Sub plunger
Main plunger

68

Single plunger
check valve
High pressure-fluctuation
Low sensitivity analysis
using UV / PDA and
Fluorescence detector
The number of maintenance
parts is minimized.

69

HPLC Injectors

Manual injector

Auto injector

70

6 port valve system


inject position

load position
pump

column

sample loop

pump

column

71

Manual Injector

Rheodyne Manual Injector

72

Manual Injector

[LOAD]

Pump

Pump

Column

Column

[INJECT]

Response of Detector

Relationship between injection


volume and detector's response
Loop injection
Partial
injection
Half volume
of sample loop

3 times volume
of sample loop

Volume of Injection

73

74

Injection Method
Partial Injection Method
better

to inject less than half volume of


sample loop

Loop Injection Method


better

to inject more than 3 times


volume of sample loop

75

Dispersion and Dilution Effect


Less than half volume

dr
ai
n

dr

m
u
p

loop

dr
ai

co
lu
m

ai n

More than half volume

m
u
p

co
lu
m

in
a
dr

Sample Zone
Dilution Zone
Dispersion Zone

loop

76

Dispersion and Dilution Effect


Less than 3 times
dr
a

m
u
p

in

More than 3 times


p

co
lu
m

in
a
dr

dr
a

loop

m
u
p

in

co
lu
m

in
a
dr

loop
Sample Zone
Dilution Zone

How to inject the sample


- Rheodyne Manual Injector Insert

a syringe at INJECT position.


Turn knob to the LOAD position.
Inject a sample.
Turn a knob to the INJECT position.
Remove the syringe.
Wash an injection port.

77

78

INJECT / LOAD position

[INJECT ] position

[LOAD] position

79

End Position of Drain Pipe

Injection port
Must put the end of drain pipe
at higher than Injection port

80

Washing for Injection Port


Use

of Needle Port Cleaner

81

Caution
Do

not use pointed or beveled needle tip.

Must

Do

use square end type.

not use more than pH 10 solution.

Must

change rotor seal.

Column Temperature
Control Devices
Column temperature control devices are functioning to keep the
column temperature constant. The temperature fluctuation of
column will influence retention time reproducibility.
Column

Oven (heat /or cool)


Heated Column Jacket

Aluminum block

Insulated

Column Jacket

Water bath

82

83

Detectors for Detectors


Ultraviolet

/ Visible detector
Photodiode Array detector
Fluorescence detector
Conductivity detector
Refractive Index detector
Electrochemical detector
Mass spectrometer detector

(UV/VIS)
(PDA)
(RF)
(CDD)
(RID)
(ECD)
(MS)

84

Ultraviolet / Visible detector


C : concentration

Cell
Ein

Eout
l

Lambert-Beer's law

ACl= - log (Eout / Ein)


(A : absorbance)

85

Ultraviolet / Visible detector


Grating

Sample Cell

Ein

Eout

Photodiode

Ein

Ein

Photodiode

Reference Cell
D2 / W lamp

86

Lambert-Beer's law

Absorbance

ACl= - log (Eout / Ein)


2.5

linear range

Concentration

87

Spectrum
275 nm

208 nm

275 nm

208 nm

88

Additional Functions

Dual Wavelength
Ratio

mode

Plot mode
Wavelength Time Program mode
Wavelength Scan mode

89

Dual Wavelength Mode

90

Ratio Plot Mode

Wavelength Time
Program Mode

91

92

Wavelength Scan Mode

93

Photodiode Array detector


Sample Cell

D2 / W lamp

Grating
One element can
detect one absorbance
at one wavelength.

512 Elements Photodiode Array

94

Photodiode Array detector

W
av
ele
n

gth

Absorbance

Spectrum
Chromatogram

Time

95

Photodiode Array detector

UV spectra

of peaks are obtained, which are


supportive for identification.
By checking peak purity, one can know if any
impurity is present.

Use Spectrum When Determining


Separation Parameters
1

2
3

30%

1: Azelaic acid
2: Benzoic acid
3: Nitrobenzoic acid

Acetonitrile
concentration

15%

min

Identification by Spectrum
Elution sequence
30%
Peak 1

Peak 2

Peak 3

Acetonitrile

Azelaic acid

Benzoic acid

Nitrobenzoic acid

15%

98

Comparison by 3 Point Spectrum

Acetyl Salicylic Acid

up slope, peak top


down slope

99

Fluorescence detector
Excitation Wavelength

+ h 1

h 2+

A*

Emission Wavelength

h 2

h 1
A

Fluorescence
A

Cell design
of Fluorescence detector

100

101

Derivatization Reagents
OPA reagent for primary amines A
CHO
CHO

+ R-NH2

N-R

o-phthalaldhyde
(OPA)

ADAM reagent for fatty acids


+ R-COOH
CHN2

9-anthryldiazomethane
(ADAM)

CH2OCOR

102

Refractive Index detector


Optical System

103

Refractive Index detector

104

Chromatogram of sugars

Analytical Conditions

Column : Shim-pack CLC-NH2


Mobile phase : Acetonitrile / water
=7/3
Flow rate : 1.0 mL/min
Temperature : Ambient

Peaks
1. Glycerol
2. Xylose
3. Fructose
4. Glucose
5. Sucrose
6. Manose
7. Lactose

105

Conductivity detector
V
I

A
L

K (conductivity) = I [A] / E [V]


=A [cm2] / L [cm] * k
(k : specific conductivity)

k= (I/E)*(L/A)

electrode

106

Conductivity detector
Conductivity

is very affected to temperature.


Must keep the cell in the temperature control
devise.

107

Type of Ion Chromatography


Non-Suppresser type
simple

system (easy maintenance)


low conductivity mobile phase
Suppresser Type
low back grand current
difficult maintenance

108

Non-Suppresser Type

extremely low
ion exchanger
Low conductivity
mobile phase

109

Suppresser Type

110

Conductivity and Peak Response

Chromatogram of Anion
in the brine

Analytical Conditions

Column : Shim-pack IC-A3


Mobile phase :
8.0 mM p-hydroxybenzoic acid
3.2 mM Bis-Tris *
Flow rate : 1.5 mL/min
Temperature : 40 C
Injection Volume : 100 uL

Peaks

1.
2.
3.
4.
5.
6.

F
Cl
NO2
Br
NO3
SO4

Bis-Tris : bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane

(1.4 ppm)
(10200 ppm)
(10 ppm)
(43 ppm)
(44 ppm)
(431 ppm)

111

Chromatogram of Cations
in Tap water

Analytical Conditions

112

Column : Shim-pack IC-C3


Mobile phase : 2.0 mM Oxalic Acid
Flow rate : 1.0 mL/min
Temperature : 40 C
Injection volume : 100 uL

Peaks

1. Na
2. NH4
3. K
4. Mg
5. Ca

(8.25 ppm)
(0.01 ppm)
(1.66 ppm)
(2.22 ppm)
(11.85 ppm)

113

Electrochemical detector
Working
electrode
Reference
electrode

AUX electrode

114

Principal of ECD detection


R

O + H+
e-

[ Applications ]
GC
Pt
Ag
Au

: phenol compounds
general use
: H2O2
: halogen ion
: sugar analysis

Electrode
Glassy Carbon (GC)
Pt, Ag, Au

115

Chromatogram of Catecolamines

Analytical Conditions

Column : Shim-pack CLC-ODS


Mobile phase :
80 mM phosphate buffer (pH=2.7)
100 mM NaNO3, 200 mg/l SOS
5 mg/l EDTA, 4 % acetonitrile
Flow rate : 1.0 mL/min
Applied Potential : + 0.8 V
Temperature : 40 C
Injection volume : 10 uL

Peaks
1. Noradrenalin ( 5 ppb)
2. Adrenalin (5 ppb)
3. Dopamine (5 ppb)

LCMS
Atmospheric Pressure Ionization

116

Pneumatically Assisted ElectroSpray(ESI)


Liquid Samples
Nebulizing gas

High Voltage

+
+-+-++

+
+

+
+
+-+-+++

+
+

on
si
lu
xc n
E tio
n
lo ora
ou p
C va
3) n E
Io
n
io
at
or nt
ap ve
Ev o l
2) of S

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

Atmospheric Pressure Chemical Ionization(APCI)


Ion molecular reaction
Liquid Samples
Nebulizing gas

Heater

Corona Discharge
Needle

117

Design of LCMS
Atmosphere

High Vacuum

API Probe

MS
detector

RP TMP1 TMP2
(Vacuum pump system)

What kind of compounds


can be analyzed ?
ESI

drugs and their metabolites


peptides
proteins
many kinds of natural product
(-OH, -NH2,-COOH, SO2, PO3 etc.)

APCI

pesticides
steroids
drugs

118

What kind of benefits


LC/MS users can get ?

119

Powerful qualitative capability


Selective

quantitative capability
M/Z

M/Z

M/Z

120

What kind of benefits


LC/MS users can get ?

Powerful qualitative capability

Selective quantitative capability m/z=100

A
TIC

B
A:100
B:100
D:150
C:150

m/z=150
C

121

Analysis of Pesticide
TIC

isoxation oxon

iprofenfos

EPN oxon

303
298
289
577

Analysis of Pesticide 1
(Mass Spectrum)
NO 2

O
P O
OC 2H5

EPN oxon

O
O

P
O

P OC 2H5
OC 2H5

OCH(CH 3)2
OCH(CH 3)2

isoxation oxon

iprofenfos

122

123

Mass Separation

TIC
HO
HO
HOH 2C

OH
O

HOH 2C

O
HO

Maltose

O
OH

OH

HOH 2C
HO
HO

OH

OH

Glucose
HO
HO

387
225
195

H 3C

Xylose

O
OH

OH

124

Comparison of each detector

UV/ VIS
LOD 1 ppb
GC
Yes

RF
10 ppt
Yes

RID
100 ppb
No

CDD
1 ppb
No

ECD
10 ppt
No

MS
1 ppb
Yes

LOD : Limit of detection (S/N=3)


GC : Gradient Compatibility
PDA sensitivity is slightly less sensitive than normal UV detector.