HPLC

Everything you need to know

1
Basic Concepts of HPLC

2
 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
- 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
Start by Separating Compounds
• By separating target compounds from other compounds
quantities can be measured more accurately!

Separation

If compounds are mixed … but if you separate

up, the quantity of each
compound cannot be them, it is easy to
determined … determine each
quantity!

So, how do you separate the compounds?

4
Chromatography as a Measure for Separation
• Origin of Chromatography
    Experiment by Tswett (at the beginning of 1900's)

Petroleum ether Chromatography

Chlorophyl To record
colors
Calcium carbonate

5
What is Chromatography?
• Method for separating the solute from the stationary or mobile
phase based on the difference in interaction

In the example
shown on the Solute
previous page, (Chlorophyl) Interaction
(difference in absorption capability)

Stationary phase Mobile phase

(Calcium carbonate) (Petroleum ether)

6
State of Matter and Types of
Chromatography
Mobile phase

Gas Liquid Solid

Gas

Stationary
Liquid
phase
Gas Liquid
chromatography chromatography
Solid

7
 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

1 to 2 µm thick solid phase layer

Glass bead
30 to 40 µm (core portion)
10 µm 2 to 5 µm

Full-porous packing
material(Curre
Pellicular packing material Full-porous packing material ntly the most
popular type)

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.
9
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
concentration

Chromatogram
Outlet

Time 11
 Chromatogram

tR
Detector signal intensity

Peak tR: Retention time

t0 t0: Dead time
h
A A: Peak area
h: Peak height

Time

12
Separation Modes of
HPLC

13
Typical Separation Modes
• Normal-phase/absorption chromatography
• Reversed-phase chromatography
• Ion exchange chromatography
• Size exclusion chromatography

14
 Polarity of Compound
• Polarity • Compatibility of solvents
• Localization of electrons within - Similar solvents are miscible
a molecule can cause molecules - Polar and non-polar solvents are
to have negative and/or positive not miscible like oil and water
poles.
• Water is a polar compound,
whereas methane is non-polar
compound.

H d-
H O
O
C H C C
H H O
-
H H d+ H
H
Methane Water Acetic acid
15
Normal Phase and Reversed Phase

Stationary
Mobile phase
phase

Normal High polarity Low polarity

phase (hydrophilic) (hydrophobic)

Reversed Low polarity High polarity

phase (hydrophobic) (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
Intensity of hydrophilic interaction (hydrogen bonding)

OH HO
SiO2 Strong
OH
Weak
Very weak
OH

Steric hindrance 18
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
Intensity of hydrophobic interaction

C18 (ODS) OH

Weak
Strong

CH3

20
 Ion Exchange Chromatography
Intensity of electrostatic interaction

R
Anion exchange N+ R
R

++++
Cation exchange SO3- + +
++++

21
Retention in Ion Exchange Mode

Packing material

Equilibrium with
mobile phase anions

22
Retention in Ion Exchange Mode

Packing material

Mobile phase anion is

displaced by analyte ion.

23
Retention in Ion Exchange Mode

Packing material

Mobile phase anion is

displaced by analyte ion.

24
Retention in Ion Exchange Mode

Packing material

Analyte ion is displaced

by mobile phase anion,
then adsorbed to next
ion exchange group.

25
Retention in Ion Exchange Mode

Packing material

Analyte ion is displaced

by mobile phase anion,
then adsorbed to next
ion exchange group.

26
 Retention in Ion Exchange Mode

Packing material Packing material Packing material

Equilibrium with mobile Mobile phase anion is Analyle ion is displaced

phase anions displaced by analyte ion. by mobile phase anion,
then adsorbed to next
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.
27
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

Packing material

Size of analyte molecules

determines whether they enter
pores or not.

29
Relationship Between Molecular Weight and
Elution Time in Size Exclusion Chromatography

(logarithmic scale) Exclusion Limit

Molecular Weight

Permeation Limit

Elution Volume

30
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

Detector

Column
Pump Column oven

Sample injector Waste bottle

Mobile phase
(eluent)
Data processor

34
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
 Solvent Delivery pump
Plunger Pump Front View

Check valve

36
Plunger and Plunger Seal

Plunger
Plunger seal

37
Solvent Delivery Pump
Plunger Pump Schematic Diagram

Pump head
Motor/cam

Check
valve

Plunger
Plunger seal 10 to 100 µL

38
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
39
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
40
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

& Check
valve

Plunger 10 to 100 µL
Plunger seal
41
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
42
Solvent Delivery Pump
Plunger Pump Schematic Diagram

Motor/cam Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
43
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
44
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
45
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
46
Solvent Delivery Pump
Plunger Pump Schematic Diagram

Motor/cam Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
47
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
48
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
49
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
50
Solvent Delivery Pump
Plunger Pump Schematic Diagram

Motor/cam Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
51
Solvent Delivery Pump
Plunger Pump Schematic Diagram
Motor/cam
Pump head

Check
valve

Plunger 10 to 100 µL
Plunger seal
52
 High-Pressure/Low-Pressure Gradient System

Low-pressure gradient unit

Mixer
Mixer

High-pressure gradient Low-pressure gradient

53
Isocratic and Gradient
• Gradient elution if the mobile phase
• Isocratic elution (fixed composition is gradually changed
composition) during analysis …
Water/methanol = 6/4

Ratio of methanol in
95%

mobile phase
30%
Water/methanol = 2/8

Reducing analysis time

while maintaining
necessary separation
(Column: C18 column)
54
 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
Pressure regulator valve Vacuum chamber
membrane tube
He cylinder

To solvent
To solvent delivery pump
delivery pump
To draft

Exhaust valve

Mobile phase Mobile phase

He purge type Gas-liquid separation

membrane type 56
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

From pump

To column
LOAD position
From pump

To column
INJECT position
58
Manual Injector

Switchfrom
Switch from INJECT to LOAD
INJECT LOAD to INJECT
INJECT
LOAD
From
From pump
pump

LOAD position

To column
To column

INJECT position
59
 Autosampler (Direct injection
type)
From pump To column From pump To column

Needle

Sample vial
LOAD INJECT
Measuring
pump
60
 Column Oven
• Purposes • Types
- To obtain the - Air circulating type
repeatability of the - Block heating type
retention time • Aluminum block heater
- To improve separation - Thermal insulation column
jacket type
• Water bath

61
 Detector
• Ultraviolet-visible • Electric conductivity
absorbance detector (UV- detector
VIS) • Electrochemical
• Photodiode array UV-VIS detector
detector • Mass spectrometer
• Fluorescence detector
• Refractive index detector
• Evaporative light scattering
detector

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

Grating Sample cell

l I0 I Photodiode

I0 I0 Photodiode

Reference cell

D2 / W lamp

64
 Principles of UV Detection
Detection cell C: Concentration

I0 I

A
l

A = e·C·l = –log (I / I0) C

(A: Absorbance, e: Molar extinction coefficient)
65
Photodiode Array Detector
Absorbance

Absorbance
Wavelength Wavelength
Time
Time
Absorbance

Absorbance

Time Wavelength
66
Comparison of Major Detectors
Sensi-
Selectivity Gradient
tivity
Light absorbing
UV-visible detector ng ™
substance
Fluorescent
Fluorescence detector pg ™
substance

Refractive index detector None µg ´

Non-volatile
Evaporating light scattering detector µg ™
substance

Electro conductivity detector Ionic substance µg r

Substance with redox

Electro chemical detector pg r
properties
Notes: The above only applies in general terms. There are also exceptions.
™: Good; r: Average; ´: Poor 67
Mass Spectrometer (LCMS)
Atmospheric
pressure High vacuum

Mass analyzer
API probe

Detector

RP TMP1 TMP2
(High vacuum pump)
68
 Advantages of LCMS
• Detection at a specific • Peak identification and
m/z® High selectivity structural analysis using MS
spectra
A
A'
A
Time A', B' B m/z

B' B
Time

Time Total ion chromatogram m/z

Chromatograms for
MS spectra
specific m/zs
69
 Qualification and
Quantitation in HPLC

70
"Qualification" and "Quantitation"
• "Qualification" • "Quantitation"
Investigates the types Investigates the
of compounds. quantities of target
- Structural analysis compounds.
- Identification
(compares to standard
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
Area
Conc. A1 Calibration curve
C1
A4

A2

Peak area
A3
C2

A2
A3
C3
A1

A4
C4 C1 C2 C3 C4
Concentration
74
 Quantitation (2)
Calibration Curve in Internal Standard Method
Conc. Area
Internal standard
Analyte

Analyte area / Internal standard area

A1 AIS Calibration curve
C1 CIS A4 /AIS

A2 AIS A3 /AIS
C2 CIS

A2 /AIS
A3 AIS
C3 CIS
A1/AIS

A4 AIS
C4 CIS C1/CIS C2 /CIS C3 /CIS C4 /CIS
Analyte conc. / internal standard conc.
75
Quantitation (3)
Advantages of Internal Standard Method
• Determination results are independent from injection volume or
pretreatment recovery rate
IS
X

AX / AIS
Recovery
100 %

Same
area ratio
IS
X
Recovery
90 %

CX / CIS

76
Selection of Reagents and
Preparation of Mobile
Phase

77
 Selecting Solvent
• Water • Organic solvent
- Using so-called - Using HPLC grade is always
"ultrapure water" is a safe choice.
always a safe choice. - Special grade may also be
- Commercially-available acceptable for some
HPLC-grade distilled detection conditions.
water is acceptable as - Take care of solvents
well. containing stabilizers
(tetrahydrofuran,
• Acids and salts chloroform, etc.).
- In general, use special
grade products or better.

78
 Content and Types of Water
• 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.
* 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.
Basically, use water from ultra-pure water purification
system or HPLC-grade distilled water. 79
 Water Handling
• 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.

80
 Effects by Water Contamination
• Effects upon analytical results
- Background noise increase
UV short-wavelength detection, electric Acetonitrile 100 %

conductivity detection, evaporative light

scattering detection, LCMS, etc.
Water
- Quantitation error 100 %

- Appearance of “ghost peaks”

Example of ghost peaks detected at 200 nm UV

using reversed-phase water/acetonitrile gradient
• Effects upon columns and
equipment
- Column performance deterioration
- Clogging in flow lines

81
 UV Spectra of Organic Solvents

Methanol Acetonitrile Hexane

Special grade Special grade

Absorbance / AU

Absorbance / AU
Absorbance / AU

Special grade
For HPLC
For HPLC
For HPLC

Wavelength (nm) Wavelength (nm) Wavelength (nm)

Fig. 1 UV Spectrum of Methanol Reagent Fig. 2 UV Spectrum of Acetonitrile Reagent Fig. 3 UV Spectrum of Hexane Reagent

82
 Methanol and Acetonitrile
• Absorbance • Separation selectivity
- HPLC-grade acetonitrile is - Varies depending on
lower particularly at short compounds.
wavelengths.
• Peak shape
• Viscosity - Generally no difference
- Acetonitrile is lower, - Sometimes can differ
particularly when mixed with depending on column or
water. compound.
• Elution strength in reversed • Price
phase - Methanol is cheaper.
- Acetonitrile is often stronger.

83
 Mobile Phase Replacement
• Never directly replace • Never directly replace an
solvents with an aqueous salt solution with
incompatible solvent. an organic solvent.

Water Buffer solution

2-propanol Water

Hexane Water-soluble organic solvent

84
Mobile Phase Filtering
• Filtration must be done when
the mobile phase contains
insoluble compounds.
• High salt concentration mobile
phases should be filtered.

Approx. 0.45 µm pore

size membrane filter

Connected to an
aspirator

85
 Saturated Air Solubility
Acetonitrile is more sensitive than methanol due to endothermic property

Air bubble generated

Actual curve

water 50% methanol 86

Sample Preparation

87
 Sample Injection into HPLC
System
• Prepare a solution. • Quantitative analysis
- Only liquids can be - Measure solid samples
injected. accurately using an
- Use composition that is analytical balance.
soluble with mobile - Dilute and mix liquid
phase. samples accurately
using pipettes and
• Avoid column volumetric flasks.
deterioration as much
as possible.
- Avoid alkaline sample
solution for silica-based
column.

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.
Filter

Syringe
90
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
(1) (2) (3) (4)
Conditioning Sample loading Washing Elution
Solvent
with low
elution
strength

Solvent
with high
elution
strength

Target
component

Unnecessary
component

92
Columns Maintenance

93
Phenomena Generated by
Column Degradation

• Peak shape • Decrease of retention

deterioration - Peaks elute quickly.
- Peak broadening • Change of separation
- Shoulder peak or peak selectivity
splitting
Broad peak - Separation pattern is
changed.

Shoulder peak

Peak splitting

94
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

Pre-column Guard column

* Pre-column: Traps impurities and substances that interfere with analysis

in the "mobile phase".
* Guard column: Traps contaminants and insoluble substances in the
"sample".
96
Column Rinsing
• Flush with a mobile • Secondary adsorption
phase that has a high must also be considered.
elution strength. - To remove basic
- For reversed-phase compounds from a
columns, increase the reversed-phase column
organic solvent ratio.
(ex.) acetonitrile 100 %,
Flush with a mixture of
etc.
acetonitrile and aqueous
- For ion-exchange columns, acid solution containing
increase salt sodium perchlorate.
concentration or change
pH.

97
Theoretical Plate Number N: Column
Performance Index

2
æt ö
N = çç R ÷÷
ès ø
2
æ tR ö
= 16çç ÷÷
èW ø
2
H æ tR ö
W1/2
= 5.54çç ÷÷
è W1 / 2 ø
H1/2 2
æ tR • H ö
= 2πçç ÷÷
W è Area ø

98
DAILY PRACTICES
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
WHAT TO DO?
HPLC Applications
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.

102
103
2. Environmental
l A key technology to measure the status of environmental conditions, this
means to diagnose, evaluate and control pollution

104
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

105
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

106
107
APPLICATIONS
Pharmaceuticals n Food
Antibiotics n Preservatives

Vitamins n Vitamins

Antipyretic & Analgesic n Sugars

drugs n Organic acids
Environmental n Medical
Inorganic ions n Amino acids
Pesticides n Drugs
Polymers n Metabolites
Antioxidants
Plasticizers