lecture i. 1- chromatographic methods a. braithwait, e.j. smith (1995) 2- modern thin layer...
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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