1.

A liquid mobile phase is pumped under pressure through a stainless steel

column containing panicles of stationary phase with a diameter of 3-10 pm
3. Separation of a mixture occurs according to the relative lengths
2. The analyte is loaded onto the head of the column via a loop
of time spent by its components in the stationary phase
valve
4. Componnents can be detected by variety detectors
1
5. Data processed by integrator/computer

Applications

The combination of high-pressure liquid chromatography (HPLC)

with monitoring by UV/ visible detection provides an accurate,
precise and robust method for quantitative analysis of
pharmaceutical products and is the industry standard method for
this purpose
Monitoring of the stability of pure drug substances and in drugs
in formulations with quantitation of any degradation products.
Measurement of drugs and their metabolites in biological fluids.
Determination of partition coefficients and pKa values of drugs
and of drug protein binding.

Strengths

Easily controlled and precise sample introduction ensures

quantitative precision.
HPLC is the chromatographic technique which has seen the
most intensive development in recent years leading to improved,
columns, detectors and software control
The variety of columns and detectors means that the selectivity
of the method can be readily adjusted.
Compared to GC there is less risk of sample degradation
because heating is not required in the chromatographic process
Readily automated.

Limitations
There

is still a requirement for reliable and

inexpensive detectors which can monitor
compounds that lack a chromophore
Drugs have to be extracted from their
formulations prior to analysis
Large amounts of organic solvent waste is
generated, which is expensive to dispose of
4

Modes of HPLC
Normal

Phase mode
Reverse Phase mode
Reverse Phase Ion Pairing mode
Ion Exchange mode
SEC mode ( GPC / GFC )
Chiral separation mode
5

Reverse Phase HPLC Columns

C18

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

Non-polar property
-Si-C18H35
Si

What is the interaction?

Hydrophobic
interaction

polar solvent

Non-polar
7

Hydrophobicity
If

the sample has

CH3CH2CH2--- : Carbon chain
: Aromatic group

If the sample has

-COOH
: Carboxyl group
-NH2
: Amino group
-OH
: Hydroxyl
group

Hydrophobicity
becomes strong.

Hydrophobicity
becomes weak.
8

Retention Time and

Hydrophobicity
OH

C18 (ODS)

Weak

Strong
OH

Increase of solvent polarity

20 %

30 %

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

40% / H2O

Solvent : MeOH
10

Effect of stationary phase

C8
Medium

C18 (ODS)

sample
Strong

C4

sample
Weak
sample
11

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

12

Structural Factors Which Govern

Rate of Elution 0f Compounds
from HPLC Columns

Elution of neutral compounds

For a neutral compound it is the balance between its polarity and Iipophilicity
which will determine the time it takes for it to elute from an HPLC column; the
pH of the mobile phase does not play apart. In the case of a reverse-phase
column, the more lipophilic a compound is the more it will be retained

Ionizable compounds
Control of elution rate of ionisable compounds by adjustment of pH of mobile
phase. Untuk asam Kapp = K/(1+10pH-pKa)
untuk basa Kapp = K/(1+10pKa-pH)

Mobile-phase conditions may be selected in straight-phase chromatography

where the ionisation of the analytes is suppressed, and basic compounds are run
in a basic mobile phase and acidic compounds are run with an acidic mobile
phase

The pH of the mobile phase can only be set within the range of ca 2-8.5 pH
units because of the tendency for extremes of pH to dissolve silica gel and
break the bonds between silane-coating agents and the silica gel support
13

Reverse Phase
Ion-Pair Chromatography
Ion-Pair Reagent

14

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)

15

Type of Ion-Pair Reagents

Hexane Sulfonate

Pentane 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

16

Concentration of
Ion-Pair Reagents

17

Causes of Tailing Peaks

Built-up

of garbage on the column inlet

Extra column effects (dead volume)
Sample Overload
Incorrect solvents for the sample
Secondary retention effects
Silanol

group
Residual heavy metal

18

Dead Volume
Dead

volume will cause a broad peak.

Sample

Injector Connection
Column Connection
Detector Connection

Good

Dead Volume

19

Incorrect solvent for sample

Better do not select a high soluble solvent as a

sample solvent.

Methanol as a sample solvent

20 uL

Ethanol as a sample solvent

20 uL

Caffeine

Ethanol as a sample solvent

10 uL

Caffeine

Caffeine

Better inject small

amount of sample.
20

Solubility effect
Low soluble solvent

High soluble solvent

21

Secondary retention effects

Silanol

Group

Even

modified silica gel (e.g. ODS, C8), residual

silanol group are still remained on the surface
area.
Silanol group will strongly absorb amine
compounds, therefore tailing will be happened.
C18
OH
C18
silica core
OC18
C18

Silanol group
negative charge

22

End capping
To

suppress the silanol group effect, an

end capping type of column will be
selected.

C18
C18
OH
O-TMS
TMS treatment
C18
C18
silica core
silica core
OH
O-TMS
TMS : trimethylsilyl group
C18
C18
C18
C18
[Non-End capping type]
[End capping type] 23

Secondary retention effects

Residual

heavy metal

Normal

silica gel has heavy metal as an impurity.

This heavy metal will have an interaction with
chelate compounds, therefore tailing will be
happened.

C18
O-TMS
+
C18
silicaMcore
O-TMS
C18
M+
C18

O
O

High pure type of

silica gel are available.
24

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.

25

HPLC system
Isocratic
Single

solvent of constant composition

Gradient
Multi

elution system
elution system

solvents of changeable composition

High

pressure gradient system

Low pressure gradient system

26

Isocratic Elution System

pump

oven

injector

detector

column

Single Solvent
27

Gradient Elution System

B
pump

oven

detector

B concentration

pump

injector

column

Time

28

Isocratic Elution Mode

MeOH / H2O = 6 / 4

Long Time Analysis

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

29

95%

30%

MeOH concentration

Gradient Elution Mode

30

High / Low pressure

gradient system
High pressure
gradient system

mixer
low pressure
gradient device

Low pressure
gradient system

Degasser

31

High / Low pressure

gradient system
High

pressure gradient system

excellent

gradient accuracy
complicate system (more than two pumps)
Low

pressure gradient system

simple

system
degasser is required
32

33

Quantity of oxygen
for saturation (uL)

Solubility of oxygen in 1 mL of
water-ethanol solvent mixture

200

100

50

Ethanol volume (%)

100
34

What's happen
in mixing two solvents?

H2O

Bubble will
form!!!

30
140

Mixing 50

140-50=90

EtOH 250
( 30 + 250 ) / 2 = 140

35

O2 concentration (mL)

Effect on pressure on solubility

of O2 in 1 mL of water

0.6

0.4
0.2

10

20

Pressure (atm)

36

System Suitability Test

It is compulsory for pharmaceutical company to

do System Suitability Test to comply GLP or
GMP.
Repeatability test (5 times)
RSD

less than 1%

Column
k,

Performance check

N, HETP, , Rs

This trend will be spread to other industry such

as food, environment etc.
37

Calibration method

External

calibration method
Internal calibration method
Standard additive method
Correction Normalization method

38

External Standard Calibration

Preparation of Standards
Target Compounds

Dilution

Dilution

Dilution

Dilution

39

Concentration

External Standard Calibration

200
180
160
140
120
100
80
60
40
20
0
0

1000

2000

3000

4000

Peak Area
40

External Standard Calibration

[Concentration]

Calculation of Results
Y = bX + a
125 ppm
2500
[Peak Area]

b : SLOPE
a : Y intercept
2500

41

Internal Standard Calibration

Preparation of Standards

Internal
Standard

Target Compounds

Dilution

Dilution

Dilution

Dilution

42

Internal Standard Calibration

Analysis of Vanillin

43

Internal Standard Calibration

[Terget Conc. / IS Conc.]

Analysis of Vanillin
4
3.5
3
2.5
2
1.5
1
0.5
0
0

10

15

[Tergat Area / IS Area]

44

Internal Standard Calibration

[Target Conc. / IS Conc.]

Calculation of Results
Y = bX + a
b : SLOP
a : Y intercept

1.67
5.0
[Target Area / IS Area]

T 2500
500
IS

Y = Target Conc. / IS Conc.

1.67 = Target Conc./ 100 ppm
Target Conc. = 167 ppm
45

Advantage of external standard

calibration method
Only

the target compound separation can be

focused.

Target

Target

46

Disadvantage of external
standard calibration method
Injection

error will directly influence the

quantitative result.
10 uL injection

100 ppm

11 uL injection

110 ppm
47

Advantage of internal standard

calibration method
Injection

error can be eliminated.

10 uL injection
1000
IS

11 uL injection
2000

2000 / 1000 = 2

1100
IS

2200
T

2200 / 1100 = 2
48

Advantage of internal standard

calibration method
Recovery

in the pretreatment process can

be estimated.
addition of IS (100 ppm)

standard IS (100 ppm)

IS
1000

to actual sample
T
IS
950

Recovery = (950/1000)x100 = 95%

49

Disadvantage of internal
standard calibration method
Separation

IS

is slightly difficult.

IS

IS

50

Disadvantage of internal
standard calibration method
It

is difficult to look for the IS compound.

The

chemical structure of IS compound is

similar with one of target compound.
IS sample is not existent in the actual
sample.

51

Calibration Method
External

standard calibration

Separation

is not difficult
Injection error will directly influence the
quantitative result

Internal

standard calibration

Injection

error can be eliminated

Recovery in the pretreatment procedure can be
estimated
Separation is slightly difficult
Difficult to look for the IS compound
52

Standard additive
calibration method
Original
Sample
Target

T
53

Standard additive
calibration method
x104

100 ppm=10x104
??? ppm= 7x104

17
12

10x104

Peak Area

??=70 ppm
7x104

T
-70

50
100 ppm
Added amount

54

Accurate Quantitation

[ Detector Response ]

Select Workable Range

ty
i
r
a
e
n
Li

c
e
p
Ex
t
i
s
Sen

R
d
te

e
g
n
a

y
t
i
iv

[ Concentration of Solute ]

55

Derivatization for detection

Postcolumn

Derivatization - automatization
Precolumn Derivatization - good separation
Sample itself
Column

Reagent
Detector

Derivatized Sample
Column

Detector
56

o - phthaldialdehyde (OPA)

Primary amine group

Detection : Fluorescence (Ex=350nm, Em=460nm)
Reaction Temperature : Ambient
Reaction speed : Fast
Reagent stability : Less stable
Derivatized product stability : Less stable

57

58

Preparation of Sample
Removal

of insoluble material

Filtration

Centrifuge

Control

(Precipitation)

of concentration

Concentration
Dilution

Extraction
Liquid

phase extraction
Solid phase extraction

Solvolysis

(Hydrolysis)
Derivatization for detection
59

Filtration
Membrane filter of 0.45 m mesh will be
used before injection in order to remove the
insoluble material.

60

Ultrafiltration

Separation of high
molecular weight
compounds such as
protein and
low molecular
weight compound

61

Centrifuge

62

Liquid-Liquid Extraction
Extracting

solvent

n-Hexane
Carbon

tetrachloride
Cyclohexane
Chloroform
Dichloromethane
1,1-Dichloroethane
Diethyl ether
Ethyl acetate
63

Liquid-Liquid Extraction
pH

control

Analyte

un-ionized for successful extraction

carboxyl

group (-COOH)
amine group (-NH2)
Mixing

and emulsion formation

Manual

or mechanical mixing
Minimize emulsions by less vigorous mixing
Centrifuge
64

Removal of Proteins
Serum 100 uL
Acetonitrile for denaturation of proteins
IS (20 ug/mL)

100 uL

Shake (10 sec)

Centrifugation (12,000 rpm

3 min)

Supernatant (10 uL Injection of HPLC)

65

Extraction of steroids from

serum
Serum 1mL
Hydrocortisone acetate (IS) 0.1 ug

Extraction (CH2Cl2 10 mL)

1.25N NaOH 1 mL

Organic phase

Filtration

0.1N HCl 1 mL

Organic phase

H2O 1 mL

Mobile phase 100 uL

Evaporation

Organic phase

Dry with Na2SO4

Aqueous phase

HPLC
66

Liquid Phase Extraction

67

Solid Phase Extraction

68

Benefit of SPE
Solid

phase extraction (SPE)

Convenient
Inexpensive
Time

- saving
Reduction of organic solvent volume
alternative to liquid - liquid extraction, the method
traditionally used for cleaning and concentrating
analytical samples
69

Extraction Tubes

Normal Phase

(silica gel, almina,

florisil, CN, NH2, Diol)
Reversed Phase (C18, C8, C4, Phenyl)
Ion Exchange (SAX, WAX, SCX, WCX)
70

71

Preventive Maintenance

Mobile Phase

Connection

Solvent grade
How to change mobile phase
Degassing
Selection of pipe
Dead volume

Injector
Column
Detector

72

Mobile phase
- solvent grade Pure

grade

water
Distilled water
Deionized water

Absorption

Water

pure
water

deionized
water

Wavelength (nm)

Due to existence of impurity,

deionized water shows higher
absorption.
73

What's happening in use of

deionized water ?
Ghost

peak will be appeared in gradient

elution!

H2O / MeOH
gradient

a
r
G

t
n
e
di

e
v
r
cu

ODS Column

Ghost Peak

74

Difference of analytical
and HPLC grade

Methanol

Acetonitrile

Hexane
75

Cut off point

If

the solvent has back ground

absorption,
Linear range will be smaller.
Noise will be bigger.
Baseline stability will be poor.

76

Cut off point

for HPLC grade solvent
Wavelength
Acetonitrile
1-Butanol

190 195 200 205 210 215 220 230 235

1.000 0.150
0.070 0.040
0.020
1.000 0.500 0.200

Chloroform

1.000 0.880 0.670

1.000 0.650

Cyclohexane
Ethanol

0.350

Ethyl Acetate
Ethyl Eter

0.750

Heptane

1.000

Hexane

0.250
1.000

Methanol

1.000
0.200
0.080
0.300

Methylene Chloride

Pentane
2-Propanol
THF

1.000

0.300

0.100
1.000

0.250
1.000 0.600

240

245

250

254
0.010
0.025
1.000 0.320 0.150
0.014
0.040
1.000
0.070
0.014
0.014
0.150
0.025
1.000 0.700 0.200
0.100
0.014
0.130
0.025
0.300
0.100
77

Tetrahydrofurn

Butylated hydroxytolune (BHT) as an oxidation

inhibitor is containing in the analytical grade of THF.
BHT has strong UV absorption. UV-detector is not
suitable for analytical grade of THF.
BHT is not available in HPLC grade. To prevent the
explosion , THF should be kept in the dark and cool
place, after seal is opened.

78

Chloroform

3% ethanol is containing analytical grade of

Chloroform to prevent a generation of phosgene
which is strong toxic gas. Ethanol will influence
the separation of normal phase mode.
HPLC grade of chloroform is not containing
ethanol. Once seal is opened, ethanol should be
kept in the dark and cool place.

79

How to change
the mobile phase
Buffer solution
Water
Aqueous Organic Solvent
( methanol, acetonitrile etc. )

2-propanol

Non-aqueous Organic Solvent

( hexane, chloroform )

80

Pipe

20 cm

How to change
the mobile phase

Do not
touch
directly

Suction Filter

rinse of suction filter

81

Degassing
Off line degassing

Vacuum pump
or Aspirator

On line degassing
He purging
Vacuum Chamber
He

Pump

Pump

Ultrasonic bath
Teflon tube
Vacuum Chamber
82

Selection of pipe
Material
Stainless

Steal

Teflon
PEEK

(polyether ether ketone)

Size
0.1

mmI.D. x 1.6 mmO.D.

0.3 mmI.D. x 1.6 mmO.D.
0.5 mmI.D. x 1.6 mmO.D.
0.8 mmI.D. x 1.6 mmO.D.
83

Pipe Material

Stainless Steel (whole connection can be applied)

Withstand up to thousand kg/cm2 pressure
Corrosion in the acidic condition ( particularly less than pH 2) or
high concentration of salt conditions
PEEK (whole connection can be applied)
Withstand up to 250 kg/cm2 pressure and whole pH range
(pH 1-14)
Week against high soluble organic solvent such as chloroform
Teflon (back pressure coil, reaction coil, drain tube)
Strong chemical resistance
Withstand up to only 5 kgf/cm2 pressure
84

Pipe Size

0.8 mmI.D.
connection for from pump to injector
drain pipe
0.5 mmI.D.
connection for from pump to injector
reaction coil
0.3 mmI.D.
connection for from injector to column
connection for from column to detector
back pressure coil
0.1 mmI.D.
resistor coil
85

Connector

Male Nut SUS / Ferrule

Mainly for pump, injector connection

withstand up to 400 kgf/cm2

Once ferrule is fixed,
can not adjust again

Ferrule
Male Nut SUS

Male Nut PEEK ( handy connector)

Mainly for column , detector connection

easy to handle
withstand up to 250 kgf/cm2
easy to make a dead volume

Male Nut PEEK

86

Reasons for Column Failure

Plugged

Fit or Column Packing

Adsorbed Sample & Solvent Impurities
Mechanical Shock, forming Voids
Chemical Attack of Packing Material

87

Plugged Fit or Column Packing

Buffer

solution must be filtered

by 0.45 um membrane filter.
Sample must be filtered
by 0.45 um membrane filter.
Connect

the column reversibly and flow the

washing solvent slowly.
Open the column end and wash the filter
(or change the filter).
88

Adsorbed Sample &

Solvent Impurities
Impurities

LC column

Wash

will cause a tailing peak.

a column using high soluble solvent.

Remove this parts and

repack the packing material.
only 1 cm will
be polluted
89

Mechanical Shock, forming

Voids
Do

not give the shock to column such as drop

of column or sudden release of pressure.
pressure
These mechanical shock will form voids.
Do not open drain valve
during operation
LC-10AD :::::::

000

xxxx

xxxx

xxxx

xxxx

000

xxxx

xxxx

xxxx

xxxx

xxxx

90

Voids
Voids

will cause split peaks.

void

Every peak will be split.

Hard to repair!!!
91

Chemical Attack of
Packing Material

Caution : Must check the valve in terms of

every items of polymer based column
92

Detector
- bubbles problems -

To solve bubbles problems, resistor coil will be

very effective.
0.3 mmID x 2 mL pipe as a resistor coil is used.
Before attaching this coil, must check whether
detector's cell can withstand pressure.

93

Points to be considered for

maintenance
Pump
check

a leak of plunger seal

wash a behind plunger seal for buffer
Injector
wash

an injection port after every injection

select a suitable washing solvent
Column
do

not keep a buffer solution

Detector
check

a life time of lamp or electrode

94