Alexandria University Faculty Of Science

Practical Training Course In Applications Of Plant Physiology

Prepared By Dr . Mohamed Mohamed Ibrahim Lecturer Of Plant PhysiologyFaculty Of Science , Botany Dept.

Alexandria University

E.Mail: m_ibramim 2004@yahoo.com

separation & identification of multiple components in a complex mixture.

chromatographic methods

for the

Thin-Layer Chromatography Thin-Layer Chromatography (TLC)(TLC)

Separation of Food Dyes by Thin Layer Chromatography (TLC )

• general, versatile method for separation of components in a mixture

• identification of "unknowns" by comparison with reference samples

• no restrictions on sample type - organic, inorganic, biological, or medical

• high sensitivity - detection of g amounts or less (10-6 g)

Types of Chromatographic Methods Essential Components

1. TLC - Thin Layer Chromatography 1. stationary phase

2. LC - Liquid Chromatography 2. mobile phase

HPLC - High Performance Liquid Chrom. 3. sample mixture

3. GC - Gas Chromatography

Thin-Layer ChromatographyThin-Layer Chromatography

ChromatograpChromatographyhy

Stationary Phase Mobile Phase

What does it do? stays put! flows around SP

What is it? fine solid with fluid (liquid or gas) (physical form) lots of surface

How does it effect retains sample by moves sample along sample movement? surface interaction

Analogous to… an obstacle course “motivating force”

Specifically for thin layer (250 m) 98% ethyl acetate TLC Lab #5 of silica gel (SiO2)x + 2% acetic acid

Thin Layer Thin Layer ChromatographyChromatography

TLC plate

O O O | | | O Si O Si O Si O H

| | | O O O | | | O Si O Si O Si O H

| | | O O O

silica gel - silicon dioxide (SiO2)x

(a common, inexpensive stationary phase)

bulk (SiO2)x

These exposed OH unitsgive silica gel a

relatively polar surface.

surface

5 x 10 cm. TLC slide250 m silica gel layer

impregnated with afluorescent indicator,on a plastic backing

Thin Layer Thin Layer ChromatographyChromatography

Four Stages in TLC

1. Sample Application Capillary used to spot solution of each sample.

2. Development This is when the separation actually occurs.

3. Visualization Our results will be viewed under UV light.

4. Interpretation of Results Comparison of retention factors.

TLC involves spotting a dilute solution (1%) TLC involves spotting a dilute solution (1%) of sample on one end of a small sheet that of sample on one end of a small sheet that has been coated with silica gel (SiOhas been coated with silica gel (SiO22) or ) or alumina (Alalumina (Al22OO33), known as the stationary ), known as the stationary

adsorbent phaseadsorbent phase..The sheet is placed upright inside a jar in a The sheet is placed upright inside a jar in a

small pool of solvent. As the solvent rises up small pool of solvent. As the solvent rises up the sheet by capillary action, the the sheet by capillary action, the components travel at different rates based components travel at different rates based on competing interactions with the mobile on competing interactions with the mobile

(solvent) and adsorbent phases(solvent) and adsorbent phases..SiOSiO22 is used for separation of more polar is used for separation of more polar

compounds while Alcompounds while Al22OO33 is used in the is used in the separation of non-polar compoundsseparation of non-polar compounds..

TLC plate

“finishing line” 1 cm.

A. Draw “guide lines” lightly with pencil

“starting line” 1 cm.

B. Dissolve solid sample in MeOH

C. Use TLC capillary to transfer and spot dissolved sample

ACE ASP CAF ACE ASP CAF #5 #5 #5 Ref. Ref. Ref.

11 . .Sample Application Sample Application (spotting)(spotting)

22 . .Development of Development of TLC PlateTLC Plate

TLC plate

TLC Developing Chamber (just a glass jar with solvent in it!)

A. Place spotted TLC plate in developing chamber

B. Developing solution is drawn up the plate by capillary action

C. Remove TLC plate when solvent reaches top line

Developingsolution

(mobile phase)

}

{keep capped}

NOTE: During this ~20 min.developing stage, compoundsin the original spots are beingpulled through the silica gel.

33 . .Visualization of TLC Visualization of TLC ResultsResults

A. Allow solvent to evaporate from surface of TLC plate.

C. Mark spots with a pencil while viewing under UV.

UVB. View results under UV light. look for grayish spots on the fluorescent green background

44 . .Interpretation of TLC Interpretation of TLC ResultsResults

A. Determine retention factors (Rf) for each spot detected.

B. Use Rf’s of reference spots to identify the other components.

ACE ASP CAF ACE ASP CAF #5 #5 #5 Ref. Ref. Ref.

distance spot has moveddistance solvent has moved

_______________________Rf = = XY

Y

- - - - - - - - - - - - - - - - - - - - - - -

X3

- - - - - - - - - - - - - - - - - - - - - - - - - - -

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

X1

X2

How do you interpretany other spots?

4

1

3

2

??

How do you identifythe unknown tablets?

Finally:

CommentsCommentsDo not let the plate sit in the solvent Do not let the plate sit in the solvent

chamber after the solvent front chamber after the solvent front reaches the top of the plate. Whyreaches the top of the plate. Why??

When spotting, the solution typically When spotting, the solution typically adsorbs very quickly and the spot adsorbs very quickly and the spot

can easily get larger than desired (1 can easily get larger than desired (1 mm diameter)mm diameter)..

Make 3-4 lanes on each plate, Make 3-4 lanes on each plate, marked with pencil and ruler and marked with pencil and ruler and experiment with large and small experiment with large and small

spots to find which works bestspots to find which works best..Record the Rf values for each Record the Rf values for each

substancesubstance..

Follow lab handout for all Follow lab handout for all experimental proceduresexperimental procedures..

Keep the layer of liquid with the Keep the layer of liquid with the most colormost color..

Do not evaporate the Do not evaporate the dichloromethane solutiondichloromethane solution

Solvents used are flammable, use Solvents used are flammable, use cautioncaution

Separation of components should be Separation of components should be easy to distinguisheasy to distinguish..

leaf pigmentsleaf pigments colorcolor

carotenescarotenes goldengolden

pheophytinpheophytin olive greenolive green

chlorophyll achlorophyll a blue greenblue green

chlorophyll bchlorophyll b yellow greenyellow green

luteinlutein yellowyellow

violaxanthinviolaxanthin yellowyellow

neoxanthinneoxanthin yellowyellow

Separation of Plant Pigments by Column Chromatography (CC)