1

Introduction to
HPLC

SPC-CSC
 2

Chromatography

An analytical method used for the

separation, identification, and determination
of the chemical components in a complex
mixture.
 3

Classical Chromatography

Mikhail Tswett :
- Father of chromatography

First publication: Berichte der Deutschen

botanischen Gesellschaft,
24, 316-323; 384-393 (1906)

Title: Adsorptionsanalyse und chromatographische

Methode. Anwendung auf die Chemie des
Chlorophylls.
 4

Classical Chromatography
Petroleum ether
Chromato graph

Color
Chlorophyll's

CaCO3

“Like light rays in

the spectrum . . .”
 5

Chromatography

Two Common Types of Chromatographic

Techniques
 Gas Chromatography (GC)
GC
 High Performance Liquid Chromatography
(HPLC)
HPLC
 6

Applications for HPLC

 Pharmaceuticals  Food
 Antibiotics  Preservatives
 Vitamins  Vitamins
 Antipyretic &  Sugars
Analgesic drugs  Organic acids
 Environmental  Medical
 Inorganic ions  Amino acids
 Pesticides  Drugs
 Polymers  Metabolites
 Antioxidants
 Plasticizers
 7

PUMP

INJECTOR COLUMN DETECTOR

DATA
PROCESSOR

MOBILE
High Performance
Pressure LC LC
PHASE
 8

Chromatographic Data

Retention
Time

Peak Peak
detector Height Area

0 10
Time(min)
 9

Modes of HPLC

 Normal Phase mode

 Reverse Phase mode
 Reverse Phase Ion Pairing mode
 Ion Exchange mode
 SEC mode (GPC / GFC)
 Chiral separation mode
 10

Normal Phase Mode

 First technique used

 CaCO in Separation Column
3
Petroleum ether as Eluting Solvent
 We define this combination as
Normal Phase mode
Column : polar property
Solvent : non-polar property
 11

Reverse Phase Mode

Column : Non-polar property

Solvent : Polar property
water /methanol / acetonitrile
 12

Reverse Phase HPLC Columns

 C18 type Non-polar property

 C8 (octyl) type
--C18H35
 C4 (butyl) type
 Phenyl type Si
 TMS type
 Cyano type
 13

Principle of separation
“like dissolves like”
OH

C18 (ODS)
Weak

Strong
OH
 14

Effect of stationary phase

 Analytical Conditions
ODS C8 TMS 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
 15

Effect of Mobile Phase Composition

Acetonitrile 1: Azelaic acid

1 concentration
2 2: Benzoic acid
30%

3 3: Nitrobenzoic acid

15%

min
 16

Normal vs Reverse Phase

 Normal Phase
 good separation for stereo isomer

(Vitamin E etc..)
 variable retention time

 Reverse Phase
 good repeatable retention time

 rugged stationary phase

 17

HPLC System

 Isocratic elution system

 Single solvent of constant composition

 Gradient elution system

 Multiple solvents of variable composition

 High pressure gradient system

 Low pressure gradient system

 18

Isocratic Elution System

injector oven
pump detector

column

Single Solvent data

processor
 19

Gradient Elution System

A
column detector
injector
pump oven

concentration
B

pump
B

Time
 20

Isocratic Elution Mode

MeOH / H2O = 6 / 4
Long Analysis Time

Bad Separation

MeOH / H2O = 8 / 2

( column : ODS type )

 21

Gradient Elution Mode

95%

concentration
MeOH
30%
 22

High/Low pressure gradient system

High pressure Low pressure

gradient system gradient system

Degasser
mixer
low pressure
gradient device
 23

High/Low pressure gradient system

 High pressure gradient system

 excellent gradient accuracy
 complicated system (more than

two pumps)
 Low pressure gradient system
 simple system
 degasser is required
 24

QUALITATIVE/QUANTITATIVE
ANALYSIS
 25

QUALITATIVE ANALYSIS

Detector A (254nm)

butyl
Test Mix I - 2
Level_2_vp.dat
Name
Detector Response

250 250

propyl
ethyl
methyl
200 200

150 150
mAU

mAU
100 100

50 50

0 0

0 1 2 3 4 5 6 7 8 9
Minutes

Retention Time
 26

QUALITATIVE ANALYSIS

standard

Basic Question:
Does the sample
TR-std have a peak with a
TR-spl within +X%
sample of the TR-std?

TR-spl
 27

QUALITATIVE ANALYSIS

Identification of individual
components in the sample
STANDARDS of known
composition are needed
TR (retention time) is the qualitative data
Directly compare the TR of the standard
and the unknown
 28

QUANTITATIVE ANALYSIS

Determination of the amount /

concentration of individual
components separated in the
sample
Peak area or peak height is the
quantitative data ( concentration)
STANDARDS of known
composition & concentrations are
needed
 29

QUANTITATIVE TERMS

Calibration/Standardization: generation of a curve that

shows the relationship between concentration and peak
area/height (per component)
External/Internal standardization method: two of the
most popular calibration methods
 30

QUANTITATIVE TERMS

External Standard Method

Internal Standard Method
 31

EXTERNAL STANDARD METHOD

Stock
Solution

Preparation of standard Target Compounds

Working standards

Dilution Dilution Dilution Dilution

1 2 3 4 5
Increasing solute concentration
 32

EXTERNAL STANDARD METHOD

3-point calibration curve
for peak # 1

y = mx + b
concentration
Increasing

Y = AREA or HEIGHT
X = CONC.
 33

EXTERNAL STANDARD METHOD

Calibration curve
Chromatogram for sample (for peak 1)

y = mx + b
 34

INTERNAL STANDARD METHOD

Preparation of standard solutions

Internal
Target Compounds IS Standard
Stock

Working Dilution Dilution Dilution Dilution

standards

1 2 3 4 5
Increasing solute concentration

Constant IS concentration
 35

INTERNAL STANDARD METHOD

y = mx + b
concentration
Increasing

Y = AREA/ HEIGHT RATIO

(target/IS)
Constant IS X = CONC. RATIO
conc. (target/IS)
 36

INTERNAL STANDARD METHOD

Calculation of Results
Y = mX + b
2500 m : Slope

[Target Area / IS
500 b : Y intercept
5.0

Area]
IS T 1.67
[Target Conc. / IS Conc.]

Y = mX + b X = Target Conc. / IS Conc.

? 1.67 = Target Conc./ 100 ppm
Area T = m . Conc T + b Target Conc. = 167 ppm
Area IS Conc IS
 37

INTERNAL STANDARD METHOD

 Properties of internal standard

1. Must be similar in chemical nature to the target
analytes
2. Must elute near the analytes of interest
3. Must not be present in the actual sample to be
analyzed.
4. Must be available in pure form
 38

 When do we use internal standard

calibration?
 Is external calibration enough?
 39

Advantage of External Standard

calibration method

 Only the target compound separation

can be focused.

Target Target
 40

Disadvantage of External Standard

calibration method

 Injection error will directly influence the

quantitative result.
10 uL injection 11 uL injection

100 ppm 110 ppm

 41

Advantage of internal standard

calibration method

 Injection error can be eliminated.

10 uL injection 11 uL injection
1100 2200
1000 2000
IS T
IS T

2000 / 1000 = 2 2200 / 1100 = 2

 42

Disadvantage of internal standard

calibration method
 Separation is slightly difficult.
IS
T IS T

T
IS
 43

Disadvantage of IS Method

 It is difficult to look for the IS

compound.
 The chemical structure of IS compound
should be similar to target compound.
 IS sample should not exist in the actual

sample.
 44

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
 45

Accurate Quantitation -
Select Appropriate Working Range

nge
x ra
M a

ng e
Ra
d
Response ]

t e
[ Detector

pe c
Ex
i t y
s i t iv
Sen
[ Concentration of Solute ]
 46

Preventive Maintenance
 47

Common problems in HPLC

 High back pressure
 Poor baseline stability and excessive noise
 Appearance of ghost peaks
 High background absorption of solvent
 Peak tailing
 Pump leakage
 Column failure
 48

Mobile Phase

 Water
Use high purity water
Distilled water
Deionized water

Absorptio
(18.2 mega-ohm) deionized
Or deionized, distilled
water water
pure

n
water
Wavelength (nm)
Due to existence of impurity,
deionized water may show higher
absorption.
 49

Mobile Phase

 Problem with deionized water is ghost peak

appear in gradient elution! (during trace analysis)

r ve
H2O / MeOH
t cu
e n
gradient adi
G r
ODS Column

Ghost Peak
 50

Mobile Phase
 Solvents
 Use hplc grade
Difference Between Analytical and HPLC Grade Solvents

HPLC grade

Methanol Acetonitrile Hexane

 51
Mobile Phase
Use solvents with low background absorption
Cut-off Point for HPLC Solvents
Wavelength 190 195 200 205 210 215 220 230 235 240 245 250 254
Acetonitrile 1.000 0.150 0.070 0.040 0.020 0.010
1-Butanol 1.000 0.500 0.200 0.025
Chloroform 1.000 0.320 0.150
Cyclohexane 1.000 0.880 0.670 0.014
Ethanol 1.000 0.650 0.350 0.040
Ethyl Acetate 1.000
Ethyl Eter 1.000 0.070
Heptane 0.750 0.200 0.014
Hexane 1.000 0.250 0.080 0.014
Methanol 1.000 0.300 0.150 0.025
Methylene Chloride 1.000 0.700 0.200 0.100
Pentane 1.000 0.300 0.100 0.014
2-Propanol 1.000 0.250 0.130 0.025
THF 1.000 0.600 0.300 0.100
 52

Changing the mobile phase

Buffer solution

Water

Aqueous Organic Solvent

( methanol, acetonitrile, etc. )
2-propanol

Non-aqueous Organic Solvent

( hexane, chloroform, etc. )
 53

Changing the mobile phase

Pipe

Old mobile phase New mobile phase

~20 cm
Do not touch directly

Suction Filter
Rinse suction filter
in fresh mobile phase
 54

Degassing
Eliminates dissolved gases which can contribute to
baseline noise and pump problems.

Off line degassing On line degassing

He purging
Vacuum Chamber
Vacuum pump Pump Pump
or Aspirator He

Ultrasonic bath
Teflon tube
Vacuum Chamber
 55

Reasons for Column Failure

 Plugged Frit or Column Packing

 Adsorbed Sample & Solvent Impurities
 Mechanical Shock, forming Voids
 Chemical Attack of Packing Material
 56

Plugged Frit or Column Packing

Back-Pressure too High!

 Shortens column lifetime

 Separation sometimes affected

 Caused by particulates in the sample

and or mobile phase

 57

Adsorbed Sample/Solvent Impurities

 Accumulated impurities/contaminants
retained in the column can cause peak
tailing.
 58

Prevent Column Clogging

Avoid Clogging!

 Mobile phase/buffer
solutions “must” be
filtered using by 0.45 or
0.2 um membrane filter.
 Samples must be filtered
using 0.45 or 0.2 um
membrane filter.
 Don’t leave buffer inside
the column when not in
use
 59

Remedies for Clogging/Contamination

 Washing
For reversed phase column, connect the
column in reverse and flow the washing
solvents slowly (in sequence).
 1. Wash with mobile phase without
buffer salts
 2. Wash with methanol or acetonitrile
 3. Wash with THF or isopropanol
 4. Wash with hexane
 5. Wash with THF or isopropanol
 6. Wash with methanol or acetonitrile
 60

Remedies for Clogging/Contamination

 Repacking
only ~1 cm might be polluted

Last resort:
Remove the contaminated part and
repack with clean packing material.
Always use the same size and type of
material used originally in the column.

colum
n
 61

Causes of Tailing Peaks

 Build-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
 62

Incorrect solvent for sample

 Avoid selecting a high soluble solvent as

sample solvent.
Methanol as a sample solvent Ethanol as a sample solvent

20 uL Caffeine 20 uL Caffeine

Ethanol as a sample solvent

 Better inject small
10 uL Caffeine amount of sample.
 63

Mechanical Shock, forming Voids

 Do not subject the column to shock,

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

000 0 000 0

xxxx xxxx 7 8 9

xxxx xxxx
4 5 6

xxxx xxxx 1 2 3

xxxx xxxx
0 ･ xxxx
 64

Voids

 Voids will cause split peaks.

void Every peak will be split.

Hard to repair!!!
 65

Precautions for the Packing Material

Silica gel Polymer

pH range 2 - 7.5 wider range
Organic solvent all solvent limited compatibility
Pressure less than 250 kgf/cm2 low pressure
Temperature better to set possible to set
at less than 60degC at high temperature

Reminder: Always read the literature or information

sheet that comes with the column!
 66

Washing for Injection Port

 Use of Needle Port Cleaner

 67

Caution

 Do not use pointed or beveled needle tip.

 Must use square end type.

 Do not use more than pH 10 solution.

 Must change rotor seal.
 68

Maintenance Tips

 Pump
 check for leaks
 wash plunger seal after using buffers
 Injector
 wash injection port after every injection
 select a suitable washing solvent
 Column
 do not store in buffer solution
 Always wash
 Detector
 check life-time of lamp or electrode