ĐẠI HỌC KHOA HỌC TỰ NHIÊN THÀNH PHỐ HỒ CHÍ MINH

KHOA HÓA HỌC

BỘ MÔN HÓA PHÂN TÍCH

LIQUID CHROMATOGRAPHY
FUNDAMENTAL
 CONTENTS
WAYS TO CONDUCT LIQUID
1 CHROMATOGRAPHY
Column/thin layer/high performance
Liquid chromatography

2 HPLC INSTRUMENTATION

HPLC SEPARATION
3 FUNDAMENTALS
Normal phase chromatography
Reverse phase chromatography

2 LIQUID CHROMATOGRAPHY – PART 1

Liquid Chromatography
Introduction
LIQUID
GAS CHROMATOGRAPHY
CHROMATOGRAPHY ▪ MP : Mixture of solvent
▪ MP : Caries gas ▪ SP : packed column
▪ SP : Capillary column ▪ Analysis: Almost all
▪ Analysis: compounds ( except gases)
Thermal stability ▪ Retention mechanisms:
High Volatility Reverse phase, Nomal phase,
Ion, HILIC,…

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Liquid Chromatography
Ways to conduct liquid chromatography (LC)

1
COLUMN 2 3
HPLC
CHROMATOGRAPHY
TLC HIGH PERFORMANCE
LIQUID
THIN LAYER
CHROMATOGRAPHY
CHROMATOGRAPHY

PREPARATIVE + ANALYTICAL +
PREPARATIVE
SEMIQUANTITATIVE PREPARATIVE

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Column Chromatography
Wet packing technique

❑Uniform length from top to

bottom
❑Uniform width or diameter
across that column
❑The material is slurried with
solvent and generally added
Particles ~ 30-200µm
to the column in portions.
Poorly Well ❑Mobile phase moves by
packed packed gravity
column column

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Column Chromatography
Wet packing technique

The sample which is

usually a mixture of
components is dissolved
in minimum quantity of
the mobile phase.

How to confirm the presence of

an analyte in collected fractions?
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Column Chromatography
How to confirm the presence of an analyte in collected fractions?
COLORING COMPOUNDS COLORLESS COMPOUNDS

THIN – LAYER CHROMATOGRAPHY

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Column Chromatography
Pros and Cons

Pros Cons
❑ Cheap ❑Low resolution
(No instrument) ❑Time and large solvent
❑ Crude sample cosuming
❑ High Capacity ❑Hard to control MP
(Large sample) velocity
❑Require other technique to
❑ Simple
confirm identity

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Column Chromatography
Applications

SEPARATING COMPOUND MIXTURES REMOVE IMPURITIES IN SPE

PREPARATIVE CHROMATOGRAPHY IN SAMPLE PREPARATION
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Thin-layer Chromatography

SEPARATION IS BASED ON
DIFFERING POLARITIES

A flat surface (glass, plastic, or aluminum) is coated with a thin layer of

adsorbent material (usually silica gel, aluminum oxide, …)
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Thin-layer Chromatography

11 LIQUID CHROMATOGRAPHY – PART 1 https://www.youtube.com/watch?v=rMGQavOMAmc&t=196s

Thin-layer Chromatography
What is the capillary action?

The solvent travels through

the system by capillary
action, thus solvent velocity
is determined by the nature
and packing structure of
the adsorbent
TLC Plate
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Thin-layer Chromatography
Detection of Components
❑ BANKING TLC ❑ UV LAMP ❑ CHEMICAL REAGENT

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 Thin-layer Chromatography
Retention factor (Rf)

Solvent front

Distance traveled by component (D.c)

Rf = Distance traveled by solvent (D.s)
D.c D.s
0<Rf<1
Rf = 0 → not migrate
origin
Rf=1: unretained
Rf value is a constant for each component only under identical experimental conditions (mobile phase,
temperature, thickness of layer and nature of adsorbent)

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Thin-Layer Chromatography
Pros and Cons

Pros Cons
❑ Easy to use ❑Low resolution
❑ Quick, inexpensive ❑Controlling MP veclocity
❑ Possible multiple anhd composition
analysis ❑Semi-quantitative method
❑ Small amount of ❑Require other technique to
qualitative
sample

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Thin-Layer Chromatography
Applications

❑Purity of Sample
❑Examination of reaction
❑Identification of compounds
❑Separating the components of an extract

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High Performance Liquid Chromatography
Injection Separation Detection

Time t

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High Performance Liquid Chromatography
Van Deemter Equation
❖ Mass Transfer
❖ Eddy diffusion

Different paths
❖ Longitudinal Diffusion
Flow

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High Performance Liquid Chromatography
Instrumentation

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High Performance Liquid Chromatography
Instrumentation

MOBILE PHASE PURITY

(HPLC GRADE)

DISSOLVED GASES

SUSPENDED
PARTICLES

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High Performance Liquid Chromatography
Instrumentation
SUSPENDED PARTICLES

Membrane filter

21 LIQUID CHROMATOGRAPHY – PART 1 Solvent filter

 High Performance Liquid Chromatography
Instrumentation
DISSOLVED GAS

Problems for gas bubbles in HPLC systems

• PEAK RETENTION TIMES Ultrasonic baths

AND AREAS CHANGE
(IN PUMP)
He

• PEAK DEFORMATION (IN

COLUMN)

• SPIKING AND
SAWTOOTH NOISE
(IN DETECTOR)
Degasser
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High Performance Liquid Chromatography
Pump

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High Performance Liquid Chromatography
Pump

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High Performance Liquid Chromatography
Elution

Isocratic elution
Method with a mobile phase of constant composition
Useful for analyzing one compound or several compounds that have similar
retentions

Gradient elution
Method with two or more components in a mobile phase and its composition is being
changed depending on analysis time
Useful for simultaneous analysis of compounds that have wide range of retentions
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High Performance Liquid Chromatography
Elution

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High Performance Liquid Chromatography
Gradient Elution

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High Performance Liquid Chromatography
6-Port Valve Injection

28 LIQUID CHROMATOGRAPHY – PART 1 https://www.youtube.com/watch?v=eCj0cRtJvJg&t=107s

High Performance Liquid Chromatography
6-Port Valve Injection
Vinjection > 3 Vloop

29 LIQUID CHROMATOGRAPHY – PART 1

High Performance Liquid Chromatography
Column

Length: 100 – 250 mm

Particles: 3-5 µm

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High Performance Liquid Chromatography
Column

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High Performance Liquid Chromatography
Guard Column What are guard column?
Packed with the same material as analytical
column
Particle size: 3-5 µm
Length: 10-30 mm
Connected between the injector and analytical
column using low deep volume tubing
How guard column work?
Retain sample and solvent impurity that could
be irreversible adsorbed onto analytical column
Filter solid particles from blocking the frit of
32 LIQUID CHROMATOGRAPHY – PART 1 the column.
High Performance Liquid Chromatography
UV Detector

A=

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High Performance Liquid Chromatography
UV Detector
Selection of detector wavelength

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High Performance Liquid Chromatography
UV Detector
UV Cutoff

OP PESTICIDES SEPARATION ON
C18 COLUMN, UV DETECTION.
GRADIENT: MeOH 35-65%

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HPLC Separation Mode

Normal Phase Reversed Phase

Anion Cation
Size Exclusion
36 LIQUID CHROMATOGRAPHY – PART 1 Ion Exchange
HPLC Separation Mode
Normal Phase
❑ Stationary Phase: Polar (hydrophylic)
Silica, alumina
❑ Mobile Phase: Non-polar (hydrophobic)
Hexane, Chloroform
❑ More attracted to Polar analytes
❑ Less attracted to Non-polar analytes
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Reversed Phase
❑ SP: Non-polar (hydrophobic)
C18; C8
❑ Mobile Phase: Polar (hydrophylic)
Methanol, H2O
❑ More attracted to Non-polar analytes
❑ Less attracted to olar analytes
 HPLC Separation Mode
Normal Phase – Adsorption Chromatography
POROUS STRUCTURE OF CHROMATOGRAPHIC PARTICLE

38 LIQUID CHROMATOGRAPHY – PART 1

HPLC Separation Mode
Normal Phase – Adsorption Chromatography
NPC is called adsorption chromatography

Water is adsorbed
onto the
adsorption sites on
the SP → decrease
in solute retention
→ deactivation of
the SP

39
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HPLC Separation Mode
Normal Phase – Adsorption Chromatography

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Normal Phase – Adsorption Chromatography

Mobile Phase Strength

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Normal Phase Chromatography
Separation of geometric isomers

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 HPLC Separation Mode
Reversed Phase Chromatography
POROUS STRUCTURE OF CHROMATOGRAPHIC PARTICLE

The longer C chain, the denser of C chains

on the surface the more rentention
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Reversed Phase – Adsorption Chromatography
Mobile Phase Strength

ACN: H2O
MeOH:H2O

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HPLC Separation Mode
Reversed Phase Chromatography

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Reversed Phase Chromatography
Effect of pH on The Retention

HA ՞ H+ + A-
B + H+ ՞ BH+

46 LIQUID CHROMATOGRAPHY – PART 1

(a) What pH would be best for the separation of benzoic acid,
4-nitrophenol, and 3-methylbenzoic acid?
(b) What pH would be best for the separation of benzoic acid, 3-methylbenzoic acid, and
4-methylaniline?
(c) What pH would be best for separation of 4-nitrophenol, 4-methylaniline, and codeine
on a typical C18-silica column?

47 LIQUID CHROMATOGRAPHY – PART 1

(a) Nonpolar aromatic compounds were separated by HPLC on an octadecyl (C18) bonded
phase. The eluent was 65 vol% methanol in water. How would the retention times be
affected if 90% methanol were used instead?
(b) Octanoic acid and 1-aminooctane were passed through the same column described in
(a), using an eluent of 20% methanol/80% buffer (pH 3.0). State which compound is
expected to be eluted first and why.

CH3CH2CH2CH2CH2CH2CH2COOH
Octanoic acid
CH3CH2CH2CH2CH2CH2CH2CH2NH2
1-Aminooctane

48 LIQUID CHROMATOGRAPHY – PART 1

(a) Nonpolar aromatic compounds were separated by HPLC on an octadecyl (C18) bonded
phase. The eluent was 65 vol% methanol in water. How would the retention times be
affected if 90% methanol were used instead?
(b) Octanoic acid and 1-aminooctane were passed through the same column described in
(a), using an eluent of 20% methanol/80% buffer (pH 3.0). State which compound is
expected to be eluted first and why.

CH3CH2CH2CH2CH2CH2CH2COOH
Octanoic acid
CH3CH2CH2CH2CH2CH2CH2CH2NH2
1-Aminooctane

49 LIQUID CHROMATOGRAPHY – PART 1

 The figure shows reversed-phase retention data for three compounds.

(a) Identify whether compounds A, B, and C are weak acids or bases. For each compound, what is
pKa and the retention factor of the fully protonated form?
(b) Over what pH range would a method be least rugged with regard to retention of component
C?
(c) Each different symbol in the plot indicates a different buffer (circle = pH 2.48 phosphate; plus
= pH 5.01 acetate; and so on). Why are different buffers used for this experiment?
 A mixture of 14 compounds was subjected to a reversed- phase
gradient separation going from 5% to 100% acetonitrile with a gradient
time of 60 min. All peaks were eluted between 22 and 50 min.
(a) Is the mixture more suitable for isocratic or gradient elution?
(b) If the next run is a gradient, select the starting and ending
%acetonitrile and the gradient time.

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