VIETNAM NATIONAL UNIVERSITY - HCMC

INTERNATIONAL UNIVERSITY

Instructor: Dr. Nguyễn Tấn Khôi

Subject: Analytical Chemistry

- Nguyễn Thị Mỹ Trang

- Trần Lê Bảo Ngọc

- Nguyễn Thị Thanh Vân

- Nguyễn Trương Quỳnh Nga

- Nguyễn Hữu Lộc

Group’s members

- Phùng Đan Tâm

- Huỳnh Quốc Thái

-Nguyễn Ngọc Trân

I. Introduction

II. Classification

- Column and thin layer chromatography

- Gas-liquid chromatography

III. Chromatography for biochemistry

OUTLINE

1. What is chromatography?

- Quality method that identify the components in mixture

by separate them  

•Mobile phase : may be a liquid, a gas or a supercritical fluid

, that continuously flows through the column and carries the

analytes.

= solvent + the sample being separated

•Stationary phase :a solid or a liquid which is immobilized

on the support particles, or on the inner wall of the column

tubing.

= column packing material

2. The principle of chromatography

The physical characteristics of a molecule are exploited in

chromatography to affect a separation. (molecular mass,

boiling point, ionic strength or molecular size)

3. Purpose of Chromatography

Analytical: determine chemical composition of a sample

Preparative: purify and collect one or more components

of a sample

4. Chromatogram

Graph showing detector response as a function of elution time.

(tR)A

tM

wB

tM = dead time tR = retention timewB = peak width at base

- Is the time between injection and the maximum of the detector

response for the analyte.

- Is dictated by:

Rf = D1 / D2

*where

D1 = distance that color traveled, measured from center of the

band of color to the point where the food color was applied

D2 = total distance that solvent traveled

RETENTION FACTOR

1.Base on the nature of adsorptive force( the mechanism of separation)

1.1 Adsorption

1.2 Partition

1.3 Ion Exchange

1.4 Size Exclusion

Liquid – chromatography

Gas – chromatography

2. Classification based on mobile phase:

2.1 Liquid chromatography

Liquid chromatography (LC) is a separation technique in

which the mobile phase is a liquid.

Liquid chromatography can be carried out either in a

column or a plane.

Classification of liquid chromatography

High Performance liquid chromatography (HPLC)

Paper chromatography (PC)

Column chromatography (CC)

Thin layer chromatography (TLC)

Column chromatography

The separation and

purification technique in a

vertical glass column.

The stationary phase is a powdered

absorbent

The mobile phase is a solvent

poured on top of column.

The analyzed mixture is loaded on

top of column.

Thin layer chromatography is used to:

Monitor the progress of a reaction

Identify compounds in a given substance

Determine the purity of a substance

Thin layer chromatography (TLC):

General

A stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate

Developing solution that travels up the stationary phase

Principle

Components of samples separate phase depend on how much

they adsorb on the stationary phase and how much they dissolve

in the mobile phase.

Process

Clip

2.2 Gas – chromatography

Gas chromatography

A common type of chromatography used in analytic chemistry

for separating and analyzing

Widely used for the determination of organic compounds that

can be vaporized without decomposition

Gas chromatographic

Gas-Liquid chromatographic

the stationary phase is an involatile liquid held on

particles of a solid support.

-Firstly, the process is carried out between a liquid stationary

phase and a gas moving phase

-Secondly, the various component of analytes are separated as

they progress

-Thirdly, detector is used to monitor the outlet stream from the

column

The mechanism

MASS SPECTROMETRY

INTRODUCTION

Different elements can be uniquely identified by their mass

I. INTRODUCTION

I. INTRODUCTION

Mass spectrometry is an analytical tool used for

measuring the molecular mass of a sample

I. INTRODUCTION

Biotechnology

Pharmaceutical

Clinical

Geological

I. INTRODUCTION

II. MS PRINCIPLESII. MS PRINCIPLES

Mass-to-charge (m/z) ratio:

The unit of mass indicated as the Dalton:

1Da= 1/12 of mass of single atom C , C = 12 mass units

m/z : An abbreviation is used to indicate physical quantity by

diving the mass of an ion in Dalton into the number of charges

carried by the ion.

A mass spectrometer measure the m/z ratio of ion created

from the molecules.

II. MS PRINCIPLESII. MS PRINCIPLES

MS PROCEDURE

4 stages:

IONISATION DETECTION ACCELERATION DEFLECTION IONISATION ACCELERATION DEFLECTION IONISATION ACCELERATION DETECTION DEFLECTION IONIZATION ACCELERATION

MS PROCEDURE

MS PROCEDURE

•The sample ionisation

chamber.

•The electrically heated metal

coil gives off electrons which

are attracted to the electron

trap(+)

IONIZATION

MS PROCEDURE

MS PROCEDURE

and pass through three slitsare repelled away from

the positive ionisation

chamber

The positive ions

and pass through three slits

ACCELERATION

DEFLECTION

MS PROCEDURE

• Different ions are deflected by the magnetic field by different amounts.

• The amount of deflection depends on:+ The mass of the ion+ The charge on the ion

DETECTION

MS PROCEDURE

• Only ion stream B makes it

right through the machine to

the ion detector.

• When an ion hits the metal

box, its charge is neutralized

by an electron jumping from

the metal on to the ion

IV. INSTRUMENTS

MS instruments consist of three modules :

MS INSTRUMENTS

How MS is used when coupled with

GC and LC

I. Gas chromatography-mass spectrometry (GC/MS or GC-MS)

A gas chromatograph: separate different compounds. This stream of

separated compounds is fed directly into the ion source.

A metallic filament to which voltage is applied. This filament emits

electrons which ionize the compounds. The ions can then further

fragment, yielding predictable patterns.

Intact ions and fragments pass into the mass spectrometer's analyzer

and are eventually detected.

A mass spectrometer (left) directly coupled to a gas chromatograph (right)

II.Liquid chromatography mass spectrometry (LC/MS or LC-MS)

Separates compounds chromatographically before they are introduced to the

ion source and mass spectrometer.

The mobile phase is liquid, usually a mixture of water and organic solvents

(instead of gas), so the ions fragments cannot yield predictable patterns.

Most commonly, an electrospray ionization source is used in LC/MS. There

are also some newly developed ionization techniques like laser spray.

Chromatography for biochemistry

Some of application of Chromatography

Toxicology is an area where chromatography is used.

Separating and identifying different drugs of abuse.

In sports medicine, any illegal drugs will be picked up using

chromatographic techniques. (eg Gas chromatography)

*Marijuana, an indian hemp plant often trafficked illegally.

The area of toxicology involves testing for the

use of illegal substances, poisons and

alcohol.

Using samples from a suspect such as hair, a

toxicologist can confirm whether a person

has used illegal drugs weeks ago or only

yesterday.

In sports medicine

One of the roles of the masculinising hormone testosterone is to increase

muscle size and strength. Taking extra testosterone, or taking a chemical that the

body can use to create extra testosterone, could therefore enhance an athlete's

performance. For this reason taking it is banned by the World Anti-Doping

Agency (WADA).

The problem is that it is not always easy to measure these two

substances, particularly as they are only present in urine at very low

concentrations.

So, a testing that makes use of liquid chromatography-tandem mass

spectrometry. This method has incredibly high sensitivity and increases the

power with which officials can search for both testosterone and epitestosterone in

a sample.

Applications of LC-MS/MS techniques

food safety

environment

pharmaceutical

Applications in food safety

Applications in food safety using turbulent flow chromatography.

By injecting food samples directly into the LC/MS system, which

eliminates time-consuming and costly sample pre-paration steps,

food safety and quality laboratories can achieve significant

analytical improvements.

Turbulent flow chromatography technology also allows the broad

selection of stationary phases.

These features make the technology a versatile and important tool in

the food safety arena.

Applications in environment

LC-MS has become an invaluable technique for trace analysis of polar compounds in aqueous samples of the environment and in water treatment.

LC-MS is of particular importance due to the imetus it has provided for research into the occurrence and fate of polar contaminants, and of their even more polar transformation products.

Applications in pharmaceutical

The application of LC-MS in areas :

systematic toxicological analysis drugs of abuse

therapeutic drug monitoring doping in sport

pesticides peptides and further potential

forensic applications.

Chromatography for biochemistry