BASIC CONCEPTS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HPLC
1
Everything you need to know

How Do You Measure Trace
Compounds in Mixture?
• What is 1 ppm, for example?
- One ppm (parts per million) means
one millionth or one in one million.
- 1 ppm is equivalent to one 1 yen
coin in one ton of gravel.
• What does it contain?
… Qualitative analysis
How much does it contain?
… Quantitative analysis
• If the substance cannot be
measured as it is …
® Separate the substance of
interest (target compound)!
3
1

Start by Separating Compounds
• By separating target compounds from other compounds
quantities can be measured more accurately!
4
Separation
If compounds are mixed
up, the quantity of each
compound cannot be
determined …
… but if you separate
them, it is easy to
determine each
quantity!
So, how do you separate the compounds?

Chromatography as a Measure for Separation
• Origin of Chromatography
Experiment by Tswett (at the beginning of 1900's)
5
Chromatography
Petroleum ether
Calcium carbonate
Chlorophyl To record
colors

What is Chromatography?
• Method for separating the solute from the stationary or mobile
phase based on the difference in interaction
6
Solute
(Chlorophyl)
Mobile phase
(Petroleum ether)
Interaction
(difference in absorption capability)
Stationary phase
(Calcium carbonate)
In the example
shown on the
previous page,

State of Matter and Types of
Chromatography
Mobile phase
Gas Liquid Solid
Stationary
phase
Gas
Liquid
Solid
7
Gas
chromatography
Liquid
chromatography

History of Chromatography
1900's Column liquid chromatography
1940's Thin-layer chromatography (TLC)
Paper chromatography
1950's Gas chromatography (GC)
Amino acid automatic analyzer
1960's Gel permeation chromatography (GPC)
And in 1969,
8

Birth of High Performance Liquid Chromatography
1969 Birth of high performance liquid chromatography
J.J. Kirkland of DuPont develops column packing material with
controlled surface porosity
This resulted in a big leap forward in developing an HPLC system
9
The pellicular packing material consists of a thin solid phase layer on a hard
core, which provides efficient separation even at high flowrates. Subsequent
progress resulted in the development of full-porous packing materials.
Glass bead
(core portion)
30 to 40 µm
1 to 2 µm thick solid phase layer
Pellicular packing material
10 µm
Full-porous packing material
2 to 5 µm
Full-porous packing
material(Curre
ntly the most
popular type)

Basic Terms
• High Performance Liquid Chromatography (HPLC)
High Speed Liquid Chromatography
High Pressure Liquid Chromatography
¥ High Performance Liquid Chromatography
• Chromatography: Method of analysis
• Chromatograph: Instrument
• Chromatogram: Resulting graph
• Chromatographer: Person performing analysis
10

Separation Process and
Chromatogram
11
Time
Outlet
concentration
Chromatogram

Chromatogram
12
tR
t0
Detector
signal
intensity
Time
Peak tR: Retention time
h
A
t0: Dead time
A: Peak area
h: Peak height

Typical Separation Modes
• Normal-phase/absorption chromatography
• Reversed-phase chromatography
• Ion exchange chromatography
• Size exclusion chromatography
14

Polarity of Compound
• Polarity
• Localization of electrons within
a molecule can cause molecules
to have negative and/or positive
poles.
• Water is a polar compound,
whereas methane is non-polar
compound.
• Compatibility of solvents
- Similar solvents are miscible
- Polar and non-polar solvents are
not miscible like oil and water
15
C
H H
H
H
Methane Acetic acid
C
C
H
H
O
O
-
H
O
H H
Water
d-
d+

Normal Phase and Reversed Phase
Stationary
phase
Mobile phase
Normal
phase
High polarity
(hydrophilic)
Low polarity
(hydrophobic)
Reversed
phase
Low polarity
(hydrophobic)
High polarity
(hydrophilic)
16

Normal-Phase/Absorption Chromatography
• Stationary phase: High polarity (hydrophilic)
- Silica gel or polar functional group which is chemically bonded
on the surface of silica gel
• Mobile phase: Low polarity (hydrophobic)
- Non-polar solvent such as hexane
17

Retention mechanism in Normal-
Phase/Absorption Chromatography
18
SiO2
OH
HO
OH
OH
Strong
Weak
Steric hindrance
Very weak
Intensity of hydrophilic interaction (hydrogen bonding)

Reversed-Phase Chromatography
• Stationary phase: Low polarity
- Octadecyl group bonded silica gel (called "ODS" or "C18")
• Mobile phase: High polarity
- Water, methanol, acetonitrile, etc.
- Sometimes salt is added.
19

Retention Mechanism in Reversed-Phase
Chromatography
20
C18 (ODS)
CH3
Strong
Weak
OH
Intensity of hydrophobic interaction

Ion Exchange Chromatography
21
N+
R
R
R
SO3
-
+
+
+
+
+
+
+
+
+
+
Anion exchange
Cation exchange
Intensity of electrostatic interaction

Retention in Ion Exchange Mode
22
Equilibrium with
mobile phase anions
Packing material

23
Packing material
Mobile phase anion is
displaced by analyte ion.

24
Packing material

25
Packing material
Analyte ion is displaced
by mobile phase anion,
then adsorbed to next
ion exchange group.

26
Packing material
Analyte ion is displaced
ion exchange group.

27
Packing material
Equilibrium with mobile
phase anions
Analyle ion is displaced
ion exchange group.
Analyte and mobile phase ions compete for ion exchange groups.
Increasing the salt concentration of the mobile phase causes quick elution of analyte.
Packing material
Packing material

Size Exclusion Chromatography
•Chromatography method that separates
compounds by molecular size (bulkiness)
•Name varies depending on application field.
- Size Exclusion Chromatography (SEC)
- Gel Permeation Chromatography (GPC)
- Chemical industry field, synthetic polymers, non-aqueous
- Gel Filtration Chromatography (GFC)
• Biochemistry field, biological macromolecules, aqueous
28

Principle of Size Exclusion Chromatography
29
Packing material
Size of analyte molecules
determines whether they enter
pores or not.

Relationship Between Molecular Weight and
Elution Time in Size Exclusion Chromatography
30
Exclusion Limit
Permeation Limit
Elution Volume
Molecular
Weight
(logarithmic
scale)

Guidelines for Selecting the Separation Mode (1)
Necessary Information
• Soluble solvent
• Molecular weight
• Structural formula, chemical properties
- Does it ionize?
- Is the method suitable for detection?
- Can the sample be derivatized?
etc.
31

Guidelines for Selecting the Separation Mode (2)
Basic Policy
• The first priority is given to reversed-phase
chromatography that uses C18 columns!!
• Exceptions
- Polymers (> 2,000) ® Size exclusion
- Optical isomer ® Chiral column
- Stereoisomer, position isomer ® Normal-phase/absorption
- Inorganic ion ® Ion chromatography
- Sugar, amino acid, organic acid ® dedicated column
32

Hardware Components
of
HPLC System
33

HPLC Flow Line Diagram
34
Waste bottle
Pump
Sample injector
Column
Detector
Mobile phase
(eluent)
Data processor
Column oven

Solvent Delivery Pump
• Required performances
- Pressure resistance
- Minimal pulsation from pressure fluctuation
- Flowrate constancy
- Easy to switch solvents
- Wide range and accurate flowrate settings
35

36
Check valve
Solvent Delivery pump
Plunger PumpFront View

Plunger and Plunger Seal
37
Plunger seal
Plunger

Plunger Pump Schematic Diagram
38
Motor/cam
Plunger
Plunger seal
Check
valve
Pump head
10 to 100 µL

39
Plunger
Plunger seal
Check
valve
Pump head
10 to 100 µL
Motor/cam

40
Plunger
Plunger seal
Check
valve
Pump head
10 to 100 µL
Motor/cam

41
& Check
valve
Pump head
Plunger
Plunger seal
10 to 100 µL
Motor/cam

42
Check
valve
Pump head
10 to 100 µL
Plunger
Plunger seal
Motor/cam

43
Check
valve
Pump head
10 to 100 µL
Motor/cam
Plunger
Plunger seal

44
Plunger
Plunger seal
Pump head
10 to 100 µL
Motor/cam
Check
valve

45
Check
valve
Pump head
Plunger
Plunger seal
10 to 100 µL
Motor/cam

46
Check
valve
Pump head
10 to 100 µL
Plunger
Plunger seal
Motor/cam

47
Check
valve
Pump head
10 to 100 µL
Motor/cam
Plunger
Plunger seal

48
Plunger
Plunger seal
Check
valve
Pump head
10 to 100 µL
Motor/cam

49
Check
valve
Pump head
Plunger
Plunger seal
10 to 100 µL
Motor/cam

50
Check
valve
Pump head
10 to 100 µL
Plunger
Plunger seal
Motor/cam

51
Check
valve
Pump head
10 to 100 µL
Motor/cam
Plunger
Plunger seal

52
Plunger
Plunger seal
Check
valve
Pump head
10 to 100 µL
Motor/cam

High-Pressure/Low-Pressure Gradient System
53
High-pressure gradient
Mixer
Low-pressure gradient unit
Low-pressure gradient
Mixer

Isocratic and Gradient
• Isocratic elution (fixed
composition)
• Gradient elution if the mobile phase
composition is gradually changed
during analysis …
54
(Column: C18 column)
Water/methanol = 6/4
Water/methanol = 2/8
95%
30%
Reducing analysis time
while maintaining
necessary separation
Ratio
of
methanol
in
mobile
phase

Degasser
• Problems caused by gases in the mobile phase
- Abnormal solvent delivery with a pump
- Noisy and /or fluctuated baseline (due to detector cell)
The mobile phase should be degassed to prevent these
problems.
55

Online Degasser
56
To draft
Gas-liquid separation
membrane type
He purge type
He cylinder
To solvent
delivery pump
Mobile phase
Pressure regulator valve
Exhaust valve
Mobile phase
membrane tube
Vacuum chamber
To solvent
delivery pump

Sample Injector
• Required performances
- Not to leave any sample remaining.
- Sample band broadening is minimal.
- Injection volume can be freely varied.
- Injection loss is minimal.
- Excellent durability and pressure resistance.
57

Manual Injector
58
INJECT position
LOAD position
From pump
To column
From pump
To column

Manual Injector
59
INJECT position
LOAD position
From pump
To column
INJECT
Switch from INJECT to LOAD
From pump
To column
LOAD
Switch from LOAD to INJECT
From pump
To column
INJECT

Autosampler (Direct injection
type)
60
From pump From pump To column
Sample vial
Needle
Measuring
pump
To column
LOAD INJECT

Column Oven
• Purposes
- To obtain the
repeatability of the
retention time
- To improve separation
• Types
- Air circulating type
- Block heating type
• Aluminum block heater
- Thermal insulation column
jacket type
• Water bath
61

Detector
• Ultraviolet-visible
absorbance detector (UV-
VIS)
• Photodiode array UV-VIS
detector
• Fluorescence detector
• Refractive index detector
• Evaporative light scattering
detector
• Electric conductivity
detector
• Electrochemical
detector
• Mass spectrometer
62

Requirements for "detection
conditions"
• Selectivity
- To detect only target compounds ideally
- To give larger response to target compounds and
smaller response to other compounds
• Sensitivity
- To have appropriate sensitivity.
• Applicability to separation conditions
• Ease of use, etc.
63

UV Detector
64
Sample cell
Reference cell
Photodiode
Photodiode
I0
I0
I0
Grating
D2 / W lamp
l I

Principles of UV Detection
65
Detection cell
A = e·C·l = –log (I / I0)
l
C: Concentration
(A: Absorbance, e: Molar extinction coefficient)
I0 I
A
C

Photodiode Array Detector
66
Wavelength Wavelength
Time
Time
Time Wavelength
Absorbance
Absorbance
Absorbance
Absorbance

Comparison of Major Detectors
Selectivity
Sensi-
tivity
Gradient
UV-visible detector
Light absorbing
substance
ng ™
Fluorescence detector
Fluorescent
substance
pg ™
Refractive index detector None µg ´
Evaporating light scattering detector
Non-volatile
substance
µg ™
Electro conductivity detector Ionic substance µg r
Electro chemical detector
Substance with redox
properties
pg r
67
Notes: The above only applies in general terms. There are also exceptions.
™: Good; r: Average; ´: Poor

Mass Spectrometer (LCMS)
68
API probe
Atmospheric
pressure High vacuum
RP TMP1 TMP2
(High vacuum pump)
Detector
Mass analyzer

Advantages of LCMS
• Detection at a specific
m/z® High selectivity
• Peak identification and
structural analysis using MS
spectra
69
A
B
A', B'
A
A'
B' B
m/z
m/z
Time
Time
Time Total ion chromatogram
Chromatograms for
specific m/zs
MS spectra

Qualification and
Quantitation in HPLC
70

"Qualification" and "Quantitation"
• "Qualification"
Investigates the types
of compounds.
- Structural analysis
- Identification
(compares to standard
compounds)
• "Quantitation"
Investigates the
quantities of target
compounds.
71

"Qualification" with HPLC
• Identification based on retention time
• Spectrum measurement by connected detector
- UV spectrum
- MS spectrum
• Identification by other analytical instrument after
preparation.
72

"Quantitation" with HPLC
• Quantitation based on peak area or height
Creating a calibration curve based on the standard
compounds in advance
- External standard method
- Internal standard method
- Method of standard addition (MSA)
73

Quantitation (1)
Calibration Curve in External Standard Method
74
C1 C2 C3 C4
A1
A2
A3
A4
Concentration
Peak
area
Calibration curve
C1
C4
C3
C2
Conc.
Area
A1
A2
A3
A4

Quantitation (2)
Calibration Curve in Internal Standard Method
75
C1/CIS C2 /CIS C3 /CIS C4 /CIS
A1/AIS
A2 /AIS
A3 /AIS
A4 /AIS
Analyte conc. / internal standard conc.
Analyte
area
/
Internal
standard
area
Calibration curve
C1
C4
C3
C2
Conc. Area
A1
A2
A3
A4
Analyte
Internal standard
CIS
CIS
CIS
CIS
AIS
AIS
AIS
AIS

Quantitation (3)
Advantages of Internal Standard Method
• Determination results are independent from injection volume or
pretreatment recovery rate
76
Recovery
100 %
Recovery
90 %
CX / CIS
A
X
/
A
IS
X
IS
X
IS
Same
area ratio

Selection of Reagents and
Preparation of Mobile
Phase
77

Selecting Solvent
• Water
- Using so-called
"ultrapure water" is
always a safe choice.
- Commercially-available
HPLC-grade distilled
water is acceptable as
well.
• Acids and salts
- In general, use special
grade products or better.
• Organic solvent
- Using HPLC grade is always
a safe choice.
- Special grade may also be
acceptable for some
detection conditions.
- Take care of solvents
containing stabilizers
(tetrahydrofuran,
chloroform, etc.).
78

Content and Types of Water
79
* In-house purification
Distilled water, ion exchange water, ultrapure water, etc.
(Ultrapure water: treated by Reverse Osmosis Membrane, ion exchange membrane,
membrane filter, UV irradiation, etc.)
* Commercially-available products
Distilled water, ion exchange water, purified water, pure water, HPLC-grade
distilled water, etc.
• Water contains a variety of substances!
- Organic compounds and inorganic compounds
- Ionic, nonionic, etc.
• Just a word "water" itself is unclear…
- Therefore, it is necessary to be very clear about what type
of water you mean.
Basically, use water from ultra-pure water purification
system or HPLC-grade distilled water.

Water Handling
80
• Clean and pure water is easily contaminated
- Suppress various contamination factors.
Example: Working environment, tools/apparatus, handling, etc.
- Maintenance is very important for ultra-pure water
systems.
Example: Reverse-osmosis filter or ion-exchange cartridges, etc.
- Take care of storage containers as well.
Example: UV-absorbing additives can leach from plastic containers.
In particular, always use fresh water in wash bottles.

Effects by Water Contamination
• Effects upon analytical results
- Background noise increase
UV short-wavelength detection, electric
conductivity detection, evaporative light
scattering detection, LCMS, etc.
- Quantitation error
- Appearance of “ghost peaks”
• Effects upon columns and
equipment
- Column performance deterioration
- Clogging in flow lines
81
Example of ghost peaks detected at 200 nm UV
using reversed-phase water/acetonitrile gradient
Acetonitrile 100 %
Water
100 %

UV Spectra of Organic Solvents
82
Acetonitrile Hexane
Methanol
Special grade
Special grade Special grade
Wavelength (nm) Wavelength (nm) Wavelength (nm)
For HPLC
For HPLC
For HPLC
Fig. 1 UV Spectrum of Methanol Reagent Fig. 2 UV Spectrum of Acetonitrile Reagent Fig. 3 UV Spectrum of Hexane Reagent
Absorbance
/
AU
Absorbance
/
AU
Absorbance
/
AU

Methanol and Acetonitrile
• Absorbance
- HPLC-grade acetonitrile is
lower particularly at short
wavelengths.
• Viscosity
- Acetonitrile is lower,
particularly when mixed with
water.
• Elution strength in reversed
phase
- Acetonitrile is often stronger.
• Separation selectivity
- Varies depending on
compounds.
• Peak shape
- Generally no difference
- Sometimes can differ
depending on column or
compound.
• Price
- Methanol is cheaper.
83

Mobile Phase Replacement
• Never directly replace
solvents with an
incompatible solvent.
• Never directly replace an
aqueous salt solution with
an organic solvent.
84
Water
Hexane
2-propanol
Buffer solution
Water-soluble organic solvent
Water

Mobile Phase Filtering
85
• Filtration must be done when
the mobile phase contains
insoluble compounds.
• High salt concentration mobile
phases should be filtered.
Connected to an
aspirator
Approx. 0.45 µm pore
size membrane filter

86
water methanol
50%
Actual curve
Air bubble generated
water methanol
50%
Actual curve
Air bubble generated
Acetonitrile is more sensitive than methanol due to endothermic property
Saturated Air Solubility

Sample Injection into HPLC
System
• Prepare a solution.
- Only liquids can be
injected.
- Use composition that is
soluble with mobile
phase.
• Avoid column
deterioration as much
as possible.
- Avoid alkaline sample
solution for silica-based
column.
• Quantitative analysis
- Measure solid samples
accurately using an
analytical balance.
- Dilute and mix liquid
samples accurately
using pipettes and
volumetric flasks.
88

Substances That Should not be Injected into
Columns
• Insoluble substances (microparticles, precipitate,
etc.)
• Substances that will be precipitated in mobile
phase
• Substances that will irreversibly adsorb to packing
material
• Substances that will chemically react with or
dissolve the packing material
89

Filtration and Centrifuge
• Basically, filtration must be done for every
type of samples before injection.
• It is convenient to use an approx. 45 µm
pore size disposable filter.
• Centrifuge may be applied for samples
that are hard to filter.
90
Filter
Syringe

Deproteinization
• Precipitation method
- To add organic solvent (acetonitrile, etc.).
- To add acid (trichloroacetic acid, perchloric acid, etc.).
- To add heavy metal or neutral salt.
• Ultrafiltration method
91

Solid Phase Extraction
92
(1)
Conditioning
(2)
Sample loading
(3)
Washing
(4)
Elution
Solvent
with low
elution
strength
Target
component
Unnecessary
component
Solvent
with high
elution
strength

Phenomena Generated by
Column Degradation
• Peak shape
deterioration
- Peak broadening
- Shoulder peak or peak
splitting
• Decrease of retention
- Peaks elute quickly.
• Change of separation
selectivity
- Separation pattern is
changed.
94
Broad peak
Peak splitting
Shoulder peak

Precautions for Column Handling
• Always use solvents that can go through the
column and maintain appropriate pH levels.
• Never let the packing material dry out.
• Never inject solids or microparticles.
- Always filtrate samples.
• Keep pressure as low as possible.
- Never exceed maximum pressure limits.
- Never apply sharp pressure changes.
• Never apply strong impacts.
95

Guard Column and Pre-Column
96
* Pre-column: Traps impurities and substances that interfere with analysis
in the "mobile phase".
* Guard column: Traps contaminants and insoluble substances in the
"sample".
Pre-column Guard column

Column Rinsing
• Flush with a mobile
phase that has a high
elution strength.
- For reversed-phase
columns, increase the
organic solvent ratio.
(ex.) acetonitrile 100 %,
etc.
- For ion-exchange columns,
increase salt
concentration or change
pH.
• Secondary adsorption
must also be considered.
- To remove basic
compounds from a
reversed-phase column
Flush with a mixture of
acetonitrile and aqueous
acid solution containing
sodium perchlorate.
97

Theoretical Plate Number N: Column
Performance Index
98
2
R
2
2
/
1
R
2
R
2
R
π
2
54
.
5
16
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Area
H
t
W
t
W
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W
W1/2
H1/2
H

l Prepare fresh mobile phase / check amount of mobile
phase needed
l Purging pump/rinsing of flow line tubes
l Flushing column/rinsing
l Checking column pressure and stability
l Checking maximum and minimum pressure of the
method and column (take a daily record)
l Record number of injections, temperature, pressure
l Have spare parts available
DAILY PRACTICES

102
1. Clinical
The analysis of whole blood, plasma, serum and urine is a most insightful
method in clinical research.
In clinical applications, analytical instruments unfold a multitude of
benefits:
l Identification of over- and undersupply of vitamins, minerals and trace
elements
l Uncovering of fraud in sports by detecting doping agents in blood or
urine.

104
2. Environmental
l A key technology to measure the status of environmental conditions, this
means to diagnose, evaluate and control pollution

105
3. Food and Beverages Industries
To ensure high product quality during several steps
in the manufacturing process, such as quality control
of raw materials (e.g. natural products)
• Analysis of degradation of vitamins in baby food
• Quantification of food additives or pesticide residues

106
4. Pharmaceuticals
A surge of regulations to monitor side effects from interfering substances and
contaminations in drugs.
Applications include:
• Drug discovery (biomarker discovery, screening and drug
synthesis)
• Drug development (pre-clinical development, toxicology,
pharmacology, formulation, process development)
• Manufacturing and QA/QC (process for production, quality
control and assurance)
• Biopharmaceutical (protein characterization, impurity analysis
for quality assurance)
• Herbal medicine/natural products

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

BASIC CONCEPTS OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

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