474 PHGLecture I

RECOMMENDED BOOKS 1- Chromatographic methodsA. Braithwait , E.J. Smith (1995) 2- Modern thin layer chromatography

(chromatographic science services vol, 52) N. Grinberg (1990)

3- Preparative chromatography techniqueHostettman K., Hostettman M. And

Marston A. (1986)4- Organic structure analysis Crews P., Rodriguez J. And Jaspers M.

(1998)5-Spectroscopic identification of organic

compounds 6th ed Robert M. Silverstein, Francis X Webster (1996).

CHROMATOGRAPHY Chromatography is a technique by

which compounds of mixture are separated by differential migration of dissolved sample between two immiscible phases in a specified system.

Two immiscible phases: Mobile phase is liquid or gas Stationary phase is adsorbent or a

second liquid

CLASSIFICATION OF CHROMATOGRAPHY

I- According to Mobile phaseA- Liquid chromatographyB- Supercritical fluid chromatographyC- Gas chromatographyII- According to stationary phaseA- Gas – solid chromatographB- Liquid – liquid chromatographC- Gas – liquid chromatography

III- According mechanism of separation

A- adsorption chromatography (Solid St. Phase)

B- Partition chromatography (two immiscible liquids)

C- Ion exchange chromatographyWhich is used for separation of

charged molecules by using anaionic and

cationic resin

D- Affinity chromatography in bio fluids (antigen-antibodies reaction)

E- Gel filtration chromatography (steric exclusion, or molecular sieving )

The column is packed with material having controlled pore sizes and the sample is screened or filtered according to its molecular size, there is no interaction between solute and stationary phase. The large molecules rapidly washed through the column, the smaller molecules penetrate inside the pores and elute later.

Large molecules

small molecules

IV- according to the equipment and the operational procedures

A- Column chromatography- The stationary phase is packed in

tube and mobile phase pass through it by gravity or pressure

-Column chromatography- HPLC- GC- SEC

B- Planar chromatographyThe stationary phase is solid

coated onto glass, plastics foil (TLC) or supported by cellulose fibre of paper sheet (paper

chromatography)

TERMINOLOGY

Adsorbent: finely divided homogenous solid having uniform particle size and large surface area which is capable of attracting molecules to its surface.

Chromatogram : a record at the end of chromatographic separation

Development: description of the process of chr. (running of the mobile phase through the stationary phase)

Analyte; components of sample mixture

Eluent: solvent used for separation in chromatographic techniques

Effluent: liquid out of the column Elution: seeping out of the

components of the mixture in pure or partially mixed form.

Resolution: the ability of any chromatographic process to separate pure compound.

Retention time: time taken to elute a particular solute

Rate of flow: distance travelled by solute / distance travelled by solvent

Retention volume: volume of mobile phase required to elute a particular solute

Tailing: disadvantage of chromatography and solute is eluted in several fraction

Visualization: making the colourless bands visible

MODES OF CHROMATOGRAPHIC SEPARATION

I- Adsorption chr.I- Column chromatographyAll the major chr. Process are

routinely carried out using column mode

For classical column chr. The following items are required

AAdsorbent

BMobile phase

CColumn construction

A- ADSORBENT (STATIONARY PHASE)

The adsorption power of the adsorbent depends on

e.g. Washing or heating The larger the particle size the lower is

the back pressure the faster is the flow rate and more poor is the resolution (bad separation)

The average particle diameter in open column chr. Is 10-2000um

The Ideal adsorbent must fulfill the following requirements:

Insoluble in mobile phase. Inert to solutes (adsorptive). Colorless especially when work

with colored mixtures. Suitable particle size enough to

give good separation and reasonable flow rateDecrease particle size increases the surface area and consequently increases separation power..

A- Silica gel is the most popular adsorbent with

a general formula SiO2 (H2O)n

The active sites of silica gel are the hydroxyl groups attached to silicon atoms "Silanol groups" .

Free silanol

Si

O

H

The selective adsorptivity of activated silica is attributed to the surface silanol group which form hydrogen bond solute with a particular

Water is adsorbed by silica and inactivate it so we must activate it before using by heat at 190-200° C for two hours.

Excessive heating leads to formation of non active siloxane

Adsorbed compound

N

O O

H

OH

H

O

Si

O

Si

O

H

O

Si

H

Siloxane

Si

O

Si

B- Alumina

It is a porous polymer of Al2O3 available in various commercial varieties for both column and planar chr.

Activation by heating at 200-400° C for 12 hrs Types of commercial alumina :

1- Neutral alumna pH 7– 7.5.

2- Acidic alumina pH 4. It is prepared by washing aluminum oxide with 2N HCl then with distilled water.

3- Basic alumina pH 10. This type is prepared by washing with NaOH then distilled water.

C- Magnesium silicate (Acidic adsorbent)

It helps separate acetylated sugar steroids and essential oils.

D- Kieselguhr it is prepared from the siliceous skeleton remains of microscopic marine animals.

E- Charcoal colour, low sample recovery, non selective adsorptivity restrict of its use to limited application

B- MOBILE PHASE

It is moving solvent that percolate through the stationary phase

Ideal mobile phase- inert- low boiling point- Low toxicity- Low price- Low viscosity- Non volatile

C- COLUMN CONSTRUCTION

1- Column material and diameter2- Packing of column3- Sample loading4- Development5- Fractions collection

1- column material, dimensions Columns are made up of glass, stainless or

synthetic polymer Tube-like glass column with length 10-100

times the internal diameter

2- packing of the column There are two types of packing

a- wet packing (slurry)

- the specified amount of adsorbent is distributed in mobile phase in a beaker

- the slurry is poured into the column after closing the bottom by cotton and close the tape

- the solvent flow start by opening the tape (outlet) until the packing is settled.

b- dry packing- dry adsorbent is poured directly in the

column solvent pass through the adsorbent

3- sample loading (application of sample) There are two methods of sample loading

a- dry method The sample is adsorbed onto small

amount of stationary phase , dry, then delivered onto column top

2- wet method Dissolve the sample in a small

volume of the mobile phase and delivered onto the top of the column.

4- column development (elution) The process start by the continuous

passage of suitable phase (mobile ph.) through the stationary phase

ELUTION TECHNIQUES FOR COLUMN

1- Gradient elution 2- Isocratic elution

GRADIENT ELUTION ISOCRATIC ELUTION The mobile phase composition

is changed during the separation process.

The mobile phase composition remains constant throughout

the separation procedure.

Time

polarity

10%

20%

30%

40%

50%

60%

70%

0 5` 10` 15` 20` 25` 30`

Isocratic elution

Gradient

GRADIENT ELUTION TECHNIQUE

Advantages of gradient elution technique

1- Shortening the time of analysis.

2- Reduces tailing, gives sharp peak.

3- Increases the sensitivity of analysis.

4- Decreases the retention of the later-eluting components so that they elute faster.

5- substances with different properties can be separated in one operation.

A- Tailing Is formation of

diffusely bounded zone or band in the development Process.

B- Fronting Fronting is

represented by extended diffused front portion of the peak and Sharpe tail.

DISADVANTAGES OF ISOCRATIC ELUTION

Factors leading to tailing1. strong interaction

between solute and stationary phase

2. application of excessive amount of the sample to the column

3. poor column packing4. improper selection of

mobile phase

occurs when the interaction between solute molecules is strong relative to those between solute and stationary phase