Fundamentals of

HPLC

SPINCO BIOTECH PVT LTD

HYDERABAD
 Course Outline

• Concept and scope of HPLC

• Separation Mechanisms

• Column Properties

2
 Concept of Chromatography

• Chromatography is an analytical method that the compounds are

physically separated prior to measurement

• The main purpose of chromatography is to separate and quantify

the target sample in the matrix

Liquid
Chromatography

Gas
Chromatography
Chromatography

Supercritical-fluid
Chromatography

3
 Why use HPLC?

• Simultaneous Analysis
• High Resolution
• High Sensitivity (ppm-ppb)
• Good repeatability
• Small sample size
• Moderate analysis condition
- no need to vaporize the sample like GC
• Easy to fractionate the sample and purify
• No destructive

4
 Scope of HPLC
Field Typical mixtures
Antibiotics, sedatives, steroids, analgesics, crude drugs,
Pharmaceuticals
cosmetics
Amino acids, proteins, peptides, carbohydrates, lipids,
Biochemical
enzymes, medicines, hormone
Mycotoxins, additives, saccharides, amino acids, vitamins,
Food products
fatty acid, coloring agents, antibacterials
Condensed aromatics, surfactants, propellants, dyes,
Industrial chemicals
polymers, plasticizers
Forensic chemistry Drugs, poisons, blood alcohol, narcotics
Inorganic ions, organic acids, agricultural chemicals,
Environmental field
pesticides, herbicides, phenols,
Clinical medicine Bile acids, drug metabolites, urine extracts, estrogens

5
 History of chromatography
M. Tswett : first developer of chromatography

Petroleum ether

Chlorophylls

CaCO3

6
 Separation Mechanism

Compounds are separated because the molecules

move at different rates in the column.

1
2

column

7
 Separation Mechanism

Due to different interaction between stationary phase

and different sample, the molecules move at different
rate, therefore separation can be done.
Mobile Phase

1
2

Stronger Weaker
interaction interaction

Stationary Phase

8
 Separation Modes

• Normal phase chromatography

• Reversed phase chromatography

• Ion chromatography

• Size exclusion chromatography

• Affinity chromatography

9
 Normal Phase Mode (1)

Stationary phase: polar

Silica gel type: For general use Si Modified Si

Cyano type: For general use Si Si-CH2CH2CH2CN

Amino type: For sugar analysis Si Si-CH2CH2CH2NH2

OH
Diol type: For protein analysis Si Si-CH2CH2CH2OCHCH2
OH

10
 Normal Phase Mode (2)

Mobile phase: Non-polar

Organic solvents:
Hexane,
Benzene,
Methylene chloride,
Chloroform,
Carbon tetrachloride
Diethyl ether,
THF
etc.

11
 Normal Phase Mode

• First developer used

– CaCO3 as Separation Column
– Petroleum ether as Developed Solvent

Stationary phase: Polar property

Mobile phase : Non-polar property

This combination is defined as

Normal Phase Mode

12
 What is the interaction?

Hydrogen bonding
Non-polar

Silica gel (polar)

13
 Hydrogen bonding

 If the sample has

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

Hydrogen bonding becomes strong.

 If the sample has bulky group, due to steric hindrance

Hydrogen bonding becomes weak.

23/2/8
 Retention Time and
Hydrogen bonding

Strong
1
HO
SiOH
SiOH

Weak

Very Weak OH 2 1

or
2

15
 Solvent Polarity

Hexane Hexane/IPA=98/2 Hexane/IPA=95/5

1 : Dioctyl phthalate
2 : Dibutyl phthalate
3 : Diethyl phthalate
4 : Dimethyl phthalate
16
 Stationary Phase for
Reversed Phase Column

• C18 (ODS) type

• C8 (octyl) type
CH3
• C4 (butyl) type
Si O Si C18H37
• Phenyl type
• TMS type CH3
Non-polar
• Cyano type

17
 Mobile Phase for Reversed Phase HPLC

• Water / buffer + Organic solvent

– Organic solvents:
– Methanol
– Acetonitrile
– THF
– Buffer:
– Phosphate buffer
– Acetate buffer
– etc

• Ratio of aqueous and organic solvents is important

18
 Reversed Phase Mode

Stationary phase: Non-polar property

Mobile phase : Polar property

This combination is defined as

Reversed Phase Mode

19
 Separation Mechanism

• Due to different interaction between packing material and different

sample, separation can be done.

sample A
packing
material
sample B

20
 What Is the Interaction?
Hydrophobic Interaction

A Less polar analyte

B More polar analyte

B B
A
A
B
A B
A
A B
A B

Support Nonpolar Interstitial area

particle bonded phase (mobile phase)

Less polar (more hydrophobic) analytes are more attracted and

spend more time associated with the hydrophobic bonded
phase, therefore, they are eluted last.
21
 Hydrophobicity

• If the sample has more

– CH3CH2CH2--- : Carbon chain

– : Aromatic group

– Hydrophobicity is stronger

• If the sample has more

– -COOH : Carboxyl group

– -NH2 : Amino group
– -OH : Hydroxyl group

Hydrophobicity is weaker
22
 Retention Time and Hydrophobicity

OH
1

C18 (ODS)
Weak

Strong
1 2
OH

23
 Mobile Phase for Reversed Phase HPLC

• Water / buffer + Organic solvent

– Organic solvents:
– Methanol
– Acetonitrile
– THF
– Buffer:
– Phosphate buffer
– Acetate buffer
– etc

• Ratio of aqueous and organic solvents is important

24
 Increase of Solvent Polarity

H2O/MeOH=20/80 H2O/MeOH=30/70 H2O/MeOH=40/60

1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate
25
 Effect of Stationary Phase

C8

Medium
C18 (ODS)

sample
Strong
C4

sample
Weak

sample

26
 Effect of Stationary Phase

 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

ODS C8 TMS

27
 Polarity of Common
organic functional groups & solvents

Functional groups Organic solvents

Non-polar
• Aliphatic hydrocarbons • Hexane
• Olfins • Carbon tetrachloride
• Aromatic hydrocarbons • Ether
• Halids • Benzene
• Sulfides • Methylene chloride
• Ethers • THF
• Nitro compounds • Iso-propanol
• Esters, Aldehydes, Ketones • Chloroform
• • Ethyl acetate
Alcohols, amine
• Acetonitrile
• Sulfones
• Methanol
• Sulfoxides
• Water
• Amides
Polar
• Carboxylic acids

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 Scope of HPLC

106

Gel Size Gel

105
permeation exclusion filtration

Molecular weight
104

103
Normal Reversed Ion
phase phase exchange
102

Nonionic polar

Nonpolar Ionic
Water-insoluble Water-soluble

Increasing polarity
29
 Choice of LC mode

Mode Solvent type used Compound type

Neutral or non-ionised
Reversed compounds which can be
H2O/Buffer, ACN, MeOH
Phase dissolved in water/organic
mixtures
Same as above with addition
Ion-Pair RP Ionic or ionizable compounds
of ion-pair reagent
Mixture of isomers and
Normal
Organic solvents compounds not soluble in
Phase
organic/water mixtures
Ion Inorganic ions, proteins,
H2O/Buffer
exchange nucleic acids, organic acids.
High molecular weight
SEC H2O, THF, CHCl3, DMF
compounds

30
 Which Column Should I Choose?

31
 Understand Columns

HPLC column is the heart of the separation

• Controlling a separation means understanding and controlling

the chemistry and physics going on inside of the column

• Different columns can vary in plate number, band symmetry,

retention, band spacing, and lifetime.

32
 Parameters of Columns

• Container
– length of column
– Diameter of column
– Material
• Packing materials
– Packing material
– Particle size
– Pores
– Shape of packing material
– Surface area
– Carbon content
– Chemistry of bonding
– Silanol
– End-capping
– Purity
– …

33
 Column Dimension

Inner diameter Length Particle size

Type
(cm) (cm) (µm)

Analytical 0.3 - 0.46 3 - 25 3 - 10

Semimicro 0.1 – 0.21 10 – 25 3–8

Semipreparative 0.8 – 1.0 10 - 25 5 - 10

Preparative 2.0 – 5.0 10 – 25 10 - 20

34
 Packing Materials

Silica based
Most popular
Narrow pore size and diameter distribution
Higher mechanical strength
Highest efficiency
No dimensional variation (e.g. swelling) with change in
solvents
Surface acidity cause problems when separating basic
compounds

35
 Packing Materials

Polymer based
Most are divinylbenzene-cross-linked polystyrene.
Longer lifetime
Lower backpressure
Wider pH range
Can be used for separating highly basic solutes at high pH,
Particles swell can be more noticeable in gradient elution –
packed bed can shift – low efficiency
Higher temperature

36
 Silica Gel Properties
Particle Size

Larger particles:
Lower back pressure
More economical
Smaller particles:
Higher efficiencies
Particle size Longer life time
Reduced run time

Analytical column : 3 - 5 µm
(meet the requirement for most HPLC separation)
Semi-preparative columns : 5 - 10 µm
Preparative columns : 10 - 20 µm
37
 Silica Gel Properties
Spherical and Irregular Shape

Spherical type Irregular type

Stronger Higher backpressure

Longer life Lower cost
Higher efficiency Broad band

38
 Silica Gel Properties
Porous and Non-porous

Pore Size : 70-120 Å (analytical column)

•Normally silica gel has small pores.

•For analytical use, 70Å -120Å of pore size
is very common.
•For large size of the sample such as
protein or synthetic polymer, 300Å, 500Å
and 1000Å (even bigger) are available.

39
 Silica Gel Properties
Surface Area

70 – 120 Å pores 150 - 400m2/g small molecule

Narrow pore separations

150 – 1000 Å pores 10 – 150 m2/g macromolecular

Wide pore separations

40
 Silica Gel Properties
Carbon Content

• If the carbon content is high, the packing material has strong retention
capability. 12% -20% is the most common range of carbon content.
• Consistent carbon content is important to batch to batch reproducibility.

41
 Silica Gel Properties
Chemistry of Bonding
• A delicate process is needed to control diameter, shape, and
porosity during preparation.

OH CH3 OH
Si Si
SiO2 X Si C18H37 SiO2 CH3
O O O O
CH3 O Si O Si O Si C18H37
O Si O Si OH
- HCl Si O O
Si O O CH3
O Si O Si
Si O OH Si O OH

OH O

CH3 Si CH3
C18H37
42
 Silica Gel Properties
Silane Coupling Reagent

• Mono functional silane coupling reagent

– Cl-Si(CH3)2-R

• Trifunctional silane coupling reagent

– X3-Si-R

• Trifunctional alkoxysilane coupling reagent

– (EtO)3-Si-R

• As far as reproducibility is concerned, mono functional

silane coupling reagent is commonly used.
–
R = C18, C8, C4, Phenyl, C3H7CN, C3H7NH2, Diol

43
 Silica Gel Properties
Residual Silanol

• 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 occur.

C18
OH Residual Silanol
silica gel C18
OH
C18
C18

44
 Silica Gel Properties
End Capping
• Endcapping is a process to fully react (silanize) the residual silano
groups on the silica support surface with a small silane such as
trimethylchlorosilane, or dimethyldichlorosilane, etc.
• Endcapping increases coverage of the silica support to minimize
unwanted interactions with solutes.

C18 C18
OH O-TMS
C18 TMS treatment C18
silica gel silica gel
OH O-TMS
C18 C18
C18 C18

[Non-End capping type] [End capping type]

45
 Comparison of End Capping Type and
Insufficient End Capping Type

Peaks
1. Propranolol
2. Acenaphthene
3. Ammitriptyline

Shim-pack VP-ODS Other column

46
 Silica Gel Properties
Purity - Trace Metal
• The purity of the silica support is of strong
concern in separating basic and highly polar
M+
compounds. Surface metal
• Some silicas are contaminated with certain
metals, which can complex with chelating OH
solutes, causing asymmetrical or tailing peaks,
or completely retaining compounds. M+ Si
Internal metal
• Other metals in the silica lattice (especially Al) (activated silanol)
activate surface silanol groups so that they are
highly acidic.
C18
M+ O-TMS
O
silica core C18
O-TMS O
M +
C18
C18
47
 Shim-pack VP-ODS Column

• Packing characteristics of Shim-pack VP-ODS

 1)Silica particles : Entirely porous, spherical silica
 particles with a high purity
Shim-pack
 2) Particle size : 5 µm VP-ODS
 3) Pore size : 120 Å
 4) Pore volume : 1.25 mL/g
 5) Specific surface area : 410 m2/g
 6) carbon content : 20%
 7) End-capping : Used Others
 8) Trace metal content : 30 ppm max
 9) ODS functional group : Mono functional

1. N-acetylprocainamide
2. Phenol
48
 Quality Certificate of
Shim-pack VP-ODS

49
 Use Your Column Wisely

what every HPLC user should know

Outline

• Good practice
• Use guard column
• Hydrophobic collapse
• Reasons of column failure

50
 Good Practice

• For new columns, it is best to condition under low flow rate (0.2-0.3
ml/min) for about 30 mins before operating under analytical flow rate.
Same applies for columns that have not been used for long time.

• Flush with stronger solvent after daily use. For example, ODS
columns may be flushed with acetonitrile after use daily to remove
strongly absorbed materials from column.

• Do not leave buffer solution in the column.

• Cap both ends of the column when it is not used to prevent the
packing material from drying out.

• Use guard column.

51
 Use Guard Column

Guard column serves as a sacrificial column top which can be

replaced when the problem occurs.
The guard column should contain the identical stationary phase
with analytical column.
The guard column should be packed with the same packing
technique as the analytical column.

• If the packing of guard column and analytical column is different: the

separation capability and protection capability decrease.
• If the packing material size of guard column is larger than that of analytical
column: peak may be broader.

Guard column Analytical column

52
 Hydrophobic Collapse

• Under low organic solvent (<5%) condition, hydrophobic alkyl chains

(C18) of the stationary phase are not wettable and appear to “fold” down
on the silica surface to avoid a highly aqueous, hydrophilic mobile
phase.
• In this folded or matted-down state, the alkyl chains are much less able
to interact with solutes, resulting in a loss of retention.

53
 Why Do Columns Die?

• Killer 1: Chemical attack of packing material

• Killer 2: Material that bind to the column

• Killer 3: Things that cause the pressure increases

• Killer 4: Column voids forming

54
 Killer 1

Chemical Attack of Packing Material (1)

Low pH (<2.0) – bonded phase loss

• Below pH 2, bonded phase comes off and free silanol are
formed, making the column more polar and increasing the
cationic-exchange character of the surface.

High pH (>8.0) - end voids

• At high pH, silica begins to dissolve, forming an end void
rapidly.
• To be safe it is best to keep pH below 7.5.

55
 Killer 1

Chemical Attack of Packing Material (2)

High temperature – bonded phase loss

• Increases the solubility of silica packing and accelerates end void
production.

High salt (>200mM) - end voids

• Tends to erode column beds by increasing the ionization and,
therefore, the solubility of the silica.

56
 Killer 2
Bound Material
• Bound compounds – material stuck to or coated on the surface of the
packing that changes the column’s running characteristics.

– Non-polar organics
• Affect specifically the later running peaks in a separation.
• Contaminated water is a notorious source of this problem, and is the usual
place to look for the culprit.

– Inorganic cations
• The metal ions form a pair bond couple and lock the silanol into the ionized
form. The partition changes.

– Charged organics
• Proteins and ion pairing reagents
• Pretreat the sample properly
• Use a dedicated column

57
 Column Cleaning (1)

• Washing the column will remove non-polars.

• Buffered mobile phase:

must wash out the buffer with the same mobile phase minus the
buffer first

• Aqueous organic solvent

Wash with Acetonitrile or methanol for at ~20 column volumes.

58
 Column Cleaning (2)

For reversed phase column

• 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

59
 Killer 3
Pressure Increases

• All the columns only can be used under a certain pressure

• Locate the point of the pressure increases

Column or system?

• There are three areas in a column where pressure increase can

occur:
• Inlet frit
• Outlet frit
• Column bed

60
 Precautions

• Buffer solution must be filtered

• by 0.2 or 0.45 µm membrane filter.

• Sample must be filtered

• by 0.2 or 0.45 µm membrane filter.

• Be sure do not jump from buffer to pure organic or from organic to

buffer. This can lead to buffer precipitation, plugging and pressure
problems.

• Always use a washout, intermediate solvent.

61
 Solution (1)

–Wash the column with suitable solvent

–Open the column end, carefully remove the filter/frit with the
column in an upright position, and sonicate it.

–Change the filter/frit.

–Wash the column reversely at low flow rate.

62
 Solution (2)

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

63
 Killer 4
Column Voids

• Center voids – it is fatal

• Symptom – voids will cause split peaks

• Reason – mechanical shock

void
Every peak likes rabbit ear.

column
64
 Avoiding Mechanical Shock
• Do not give shock to columns such as
• Drop on the ground
• Bang round in the drawer
• Release pressure suddenly
• Do not jump to immiscible solvents
• Do not reverse the column flow
• Do not jump flow rate suddenly.

These mechanical shocks will form voids.

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

Do not drop the column on Do not open drain valve

the ground during operation
65