474 PHG

1- Chromatographic methods
A. Braithwait , E.J. Smith (1995)
2- Modern thin layer chromatography (chromatographic science services vol, 52) N. Grinberg (1990)
3- Preparative chromatography technique
Hostettman 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 6 th ed Robert M. Silverstein, Francis X Webster (1996).

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-

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

I- According to Mobile phase
A- Liquid chromatography
B- Supercritical fluid chromatography
C- Gas chromatography
II- According to stationary phase
A- Gas – solid chromatograph
B- Liquid – liquid chromatograph
C- Gas – liquid chromatography

III- According mechanism of separation
A- adsorption chromatography (Solid St.
Phase)
B- Partition chromatography (two immiscible liquids)
C- Ion exchange chromatography
Which 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 chromatography
The 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- Column chromatography
All the major chr. Process are routinely carried out using column mode
For classical column chr. The following items are required
A
Adsorbent
B
Mobile phase
C
Column 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 rate
Decrease particle size increases the surface area and consequently increases separation power.
.

A- Silica gel

 is the most popular adsorbent with a general formula SiO
2
(H
2
O) n
The active sites of silica gel are the hydroxyl groups attached to silicon atoms "Silanol groups" .
H
O
Si
Free silanol


The selective adsorptivity of activated silica is attributed to the surface silanol group which form hydrogen bond solute with a particular
N
O
Si
H
O
Si
H
H
O
O
O
H
Si
O
O
H

Adsorbed compound
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 O
Si i
S
Siloxane

B- Alumina

It is a porous polymer of Al
2
O
3 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


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

1- Column material and diameter
2- Packing of column
3- Sample loading
4- Development
5- 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


1- Gradient elution

2- Isocratic elution

GRADIENT ELUTION
 The mobile phase composition is changed during the separation process.
polarity
ISOCRATIC ELUTION
 The mobile phase composition remains constant throughout the separation procedure.
70%
60%
50%
40%
30%
20%
10%
0 5` 10` 15`
Gradient
20`
25` 30`
Isocratic elution
Time

GRADIENT ELUTION TECHNIQUE
Advantages of gradient elution technique
1Shortening the time of analysis.
2- Reduces tailing, gives sharp peak.
3- Increases the sensitivity of analysis.
4Decreases the retention of the latereluting components so that they elute faster.
5- substances with different properties can be separated in one operation.

DISADVANTAGES OF ISOCRATIC ELUTION
A- Tailing

Is formation of diffusely bounded zone or band in the development
Process.

1.
2.
3.
4.
Factors leading to tailing strong interaction between solute and stationary phase application of excessive amount of the sample to the column poor column packing improper selection of mobile phase
B- Fronting

Fronting is represented by extended diffused front portion of the peak and Sharpe tail.
 occurs when the interaction between solute molecules is strong relative to those between solute and stationary phase