1
Introduction to
HPLC
SPC-CSC
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
