LECTURE 4: CHROMATOGRAPHY

Content:

- Chromatographic separation- classifying analytical separations; column chromatography, planar chromatography- gas chromatography; introduction, application- hplc; introduction, application

Simple separation VS Chromatographic separation

Principle of simple separation (exp partitioning between phases):

- The separation occurs only one direction- Increase efficiency by using fresh extracting

phase.

The principle of chromatographic separation: extracting the solutes back and forth between fresh portion of the two phases. The two phases used are called the mobile phase and the stationary phase.

Chromatography

• Chromatography is the separation of a mixture based on the different degrees to which they interact with two separate material phases:

• The two phases are:

1) The stationary phase

2) The mobile phase

• The stationary phase – a phase that is fixed in place either in a column or in a planar surface. The stationary phase is either a porous solid used alone or coated with a stationary liquid phase.

• The mobile phase – a phase that moves over or through the stationary phase, carrying with it the analyte mixture.

It is also called the eluting fluid. The mobile phase can be a gas, a liquid, or a supercritical fluid.

Column VS Planar chromatography

• Principle of column chromatography:

The stationary phase is placed in a narrow column through which the mobile phase moves under the influence of gravity or pressure.

• Principle of planar chromatography

The stationary phase coats a flat glass, metal, or plastic plate and is placed in a developing chamber. A reservoir containing the mobile phase is placed in contact with the stationary phase, and the mobile phase moves by capillary action.

Exp paper chromatography and Thin layer chromatography.

Next presentation!(G12)

• Elaborate the following procedures in using gas chromatography

– Extraction– Standard solution– Calibration curve– Peak area determination

Your references1. Practical manual2. Text book

Chromatographic Techniques

• Classified according to the type of equilibration process involved. Equilibration process control by the type of stationary phase.

1. Adsorption Chromatography- The stationary phase is a solid.- Sample components are adsorbed on the solid

stationary phase.- The mobile phase may be a liquid or a gas.- The sample components are distribute between the

two phases through a combination of sorption and desorption process.

- Example: Thin layer chromatography.

Chromatographic Techniques

2. Partition Chromatography- The stationary phase is a liquid supported on an

inert solid.- The mobile phase may be a liquid or a gas.- In normal-phase C: a polar SP and non-polar

MP is used for nonpolar solutes.- In reversed-phase C:nonpolar SP and polar MP

is used for polar solutes.- Reversed phase is the most widely used.

Chromatographic Techniques3. Ion Exchange and 4. Size Exclusion Chromatography

Stationary Phase Mechanism

Ion Exchange Ion exchange resin ion exchange

equilibriaSize Exclusion Chromatography

A sievelike structure penetration of

a certain size

GAS CHROMATOGRAPHY (GC)

• A chromatographic technique in which the mobile phase is a gas.

• Parts of GC are:- The mobile phase- The stationary phase- Sample introductions- Temperature control- Detectors

Principle of Gas Chromatography

• Sample should be converted to vapor state (if it is not already a gas).

• Separation occurs as the vapor constituents equilibrate between carrier gas and the SP.

• The sample is automaticaly detected by detector.• Measuring the retention time and comparing this time

with that of a standard of a pure substances make it possible to identify the peak.

• Since the area of the peak is porpotional to the concentration, and so the amount of the substance can be quantitatively determined.

• The peak height can be compared with a calibration curved prepared in a same manner.

Assignment-Group

• Based in the given diagram, explain functions of each part of the GC.

Importance parts of GC

There are three parts most important for GC

1. The columns

2. The detectors

3. The mobile phase gas supply

The Columns

• Commonly used columns are packed columns and capillary columns.

Packed Column

• About 1-10 m long and 0.2-0.6 cm in diameter.

• Short columns made of glass and longer columns made of stainless steel or can also made of Teflon.

Functions of GC Columns

• To contain the stationary phase and the passing way of the mobile phase.

• The site where the separation of analyte occurs.

• To provide analysis in terms of resolution, sensitivity and retention time.

The Detectors

• Over 40 detectors have been developed since the introduction of GC.

• Commonly used detectors

1. Thermal Conductivity Detector (TDC)

- The original detector

2. Flame Ionization Detector (FID)

- The most sensitive and widely used detector for organic compounds.

3. Flame Photometric Detectors (FPD)

Functions of Detectors

• To respond to compounds analysed.

• To automatically detect the sample as it emerges from the column.

The mobile phase gas supply

• Usually an inert gas that available in pure form such as argon, helium or nitrogen.

• A highly dense gas is more effective.• The choice of gas determine by the type of

detector.

Functions:- To bring along gas and injected compounds

throughout the column up to detector.- To provide equilibration between the carrier gas

and the stationary phase for compounds separation.

• Sample Introduction

- Must consider three rules:

1. All constituents injected into GC must be volatile.

2. The analyte must be present at an appropriate concentration.

3. Injecting the sample must not degrade the separation (thermally stable).

Volatile Sample

• A volatile compound is a compound that easily evaporated because of their low molecular weight.

• In GC, the sample constituents need to be volatiled in order to move through the column.

• Nonvolatile solutes will condense on the column, degrading the column’s performance.

• Exp. of volatile compounds are from the monoterpenoids group (limonene, linalool,

champor, menthol etc.)

Applications of GC

• Widely used for the analysis of diverse array of samples in environmental, clinical, pharmaceutical, biochemical, forensic, food science and petrochemical laboratories.

Liquid Chromatography

1. High performance Liquid Chromaography

2. Size Exclusion

3. Ion Exchange

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

• Analyze sample in liquid form.• The sample carried through a chromatographic

column by a liquid mobile phase.• Parts of HPLC are:

- HPLC column- The mobile phases- The stationary phases- Sample introductions- HPLC plumbing- Detectors

Ion Exchange Chromatography

The principle

- To separate inorganic ions, both cations and anions.

- Separate based on exchange of ions in the stationary phase.

- The stationary phase consists of beads made of polystyrene polymer crosslinked with divinylbenzene.

Determination of analyte using HPLC.

Exercise:

The calibration curve of an external standards

y = 8403.7x

R2 = 1

0

10000

20000

30000

40000

50000

50 100 150 200 250

standards (ppm)

Sign

al (a

rbitr

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units

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