Chromatography Introduction - PowerPoint Presentation

Slide1

Chromatography

Slide2

Introduction

Mikhail

Tswett

, Russian, 1872-1919

In 1906

Tswett

used chromatography to separate plant pigments, He called the new technique

chromatography

because the result of the analysis was

'written colors”

Most materials in our surroundings are mixtures of two or more components.

Mixtures are either homogeneous or heterogeneous:

Homogeneous mixtures are uniform in composition, but heterogeneous mixtures are not.

Air is a homogeneous mixture and oil in water is a heterogeneous mixture.

Homogeneous and heterogeneous mixtures can be separated into their components by several physical methods.Slide3

The choice of separation techniques is based on the type of mixture and difference in the chemical properties of the constituents of a mixture.

Various types of separation processes but we will concern on chromatography in this lectureSlide4

Chromatography

Chromatography is a widely used experimental technique for the separation of a mixture of compounds into its individual components.

The word chromatography means "separation of colors"

but

today chromatography is used for both colored and colorless substances.

Chromatography is a physical method of separation in which the components to be separated are distributed between two phases,

One of which is

stationary phase

, while the other the

mobile phase

moves through it in a definite direction.Slide5

The stationary phase may be a solid packed in a glass plate or a piece of chromatography paper.

The sample is carried by a particular solvent called mobile phase. The mobile phase may be a gas or liquid.

The mobile phase is then passed through the stationary phase.

Affinity

: natural attraction or force between things.

Adsorption

: refers to the adhesion or

stickyness

of a substance to the surface of another substance, as opposed to the term “

absorption

” which refers to a substance penetrating into the inner structure of another substance.

https://www.youtube.com/watch?v=djIzXvwIz5USlide6

Illustration of chromatography

Components

Affinity to Stationary Phase

Affinity to Mobile Phase

Blue

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

Insoluble in Mobile Phase

Black

     

 

Red

 

    

Yellow        

Mixture

Components

Separation

Stationary Phase

Mobile Phase

Analyze

Identify

Purify

QuantifySlide7

The separation process is based on the fact that porous solids adsorbs different substances to different extremes depending upon their

polarity.A mixture to be separated is first applied to an immovable porous solid (like paper, or alumina, or fine silica sand) called the stationary phase.

The components of the mixture then get “washed” along the porous solid by the flow of a solvent called the mobile phase.

Each component of a mixture to be separated will be attracted differently to the porous stationary phase depending on its polarity and the polarity of the stationary phase chosen.

Remember that

“Like dissolve Like”.Slide8

If the stationary phase is polar then polar components will be attracted or stick more to it but non-polar components will move across the surface easily.

If the stationary phase is nonpolar then nonpolar components will be more attracted to it and the polar compounds will move along more quickly

.

The separation depends on several factors;

(a) solubility: the more soluble a compound is in a solvent, the faster it will move up the plate.

(b) attractions between the compound and the silica, the more the compound interacts with silica, the lesser it moves,

(c) Structure of the compound, the larger the compound the slower it moves up the plate.Slide9

Advantages of chromatography

Can

separate

complex mixtures with

great precision

. Even very similar components, such as proteins that may only vary by a single amino acid.

Can

purify

basically any soluble or volatile substance if the right adsorbent material, carrier fluid, and operating conditions are employed.

Exact

quantitative analysis

is done even from trace compounds.Small material consumption.The quantization has a broad linearity range.Analyses of several compound can be done during one run.Chromatography is a fast analysis method.Well established instrumentation with high level automation is commercially available.Slide10

Is this a pure substance or mixture?

TLC can be used to determine whether a sample is a single substance or a mixture.

A single pure substance gives a single spot. However, care should be taken since compounds of similar properties, isomers for example, may not separate.

A sample that results in many spots after development is a confirmation of a mixture of different compounds.

In that sense, TLC can also be used to determine the number of components in a mixture.Slide11

Are these compounds identical?

TLC can be used to confirm whether two or more compounds are identical or not. The compounds to be analyzed are spotted on the same plate and the plate developed. If the compounds have the same

Rf

value, this can be taken as an indication that they have a similar chemical nature.

However, it is important to keep in mind that many chemical compounds have the same

Rf

and TLC cannot be used as the only analytical tool to decide on the identity of a chemical compound. Slide12

TLC

PCSlide13

Classification of chromatography according to mobile phase:

1- Liquid chromatography: mobile phase is a liquid. (LLC, LSC).

2- Gas chromatography : mobile phase is a gas.

(GSC, GLC).Slide14

Classification according to the packing of the stationary phase

Planar chromatography:

A separation technique in which the stationary phase serves as a plane.

The plane can be either a paper (paper chromatography) or a layer of solid particles

sorbent

(silica gel, cellulose, aluminum oxide, ion exchange resin) spread on a

support

such as a glass- or a plastic- plate (thin layer chromatography).

Thin layer chromatography (TLC):

the stationary phase is a thin layer supported on glass, plastic or

aluminium

plates.

Paper chromatography (PC): the stationary phase is a thin film of absorbent paper. Column chromatography (CC): stationary phase is packed in a glass column.Slide15

For

qualitative analysis the different mobilities of substances are used, the distances passed by different substances are different. The distance between the starting line and the center of the spot of substance characterizes the substance. Retention factor, RF, provides better way to indentify substances .

For

quantitative determination

the

intensity

of the spot is used: the bigger the amount of substance in the mixture, the more intensive is the spot. Also the

size

of the spot can give quantitative information – the bigger the spot, the bigger the content of this compound in the mixture. Intensity of the spots is evaluated by comparing with the intensities of

analyte

spots with known amounts visually or using

densitometer.Slide16

Paper chromatography

Paper chromatography uses

paper

as the stationary phase and a liquid solvent as the mobile phase.

In paper chromatography, the sample is placed on a spot on the paper and the paper is carefully dipped into a solvent.

The solvent rises up the paper due to capillary action and the components of the mixture rise up at different rates and thus are separated from one another.Slide17

Principle of paper chromatography

Capillary Action:

the movement of liquid within the spaces of a porous material.

The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.

Solutes dissolve into solvents that have similar properties.

(Like dissolves like)

This allows different solutes to be separated by different combinations of solvents.

Separation of components depends on both their

solubility

in the mobile phase and their

differential affinity

to the mobile phase and the stationary phase.Slide18

TLC

Thin layer chromatography

is a technique used to separate and identify compounds of interest.

A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for support.

The silica gel acts as the stationary phase and the solvent mixture acts as the mobile phase.

In the ideal solvent system the compounds of interest are soluble to different degrees. Separation results from the partition equilibrium of the components in the mixture.Slide19

Principle

Different compounds in sample mixture travel different distances according to how strongly they interact with the stationary phase as compared to the mobile phase.

In principle, the components will differ in solubility and in the strength of their adsorption to the adsorbent and some components will be carried farther up the plate than others.

This liquid, or the

eluent

, is the mobile phase, and it slowly rises up the TLC plate by capillary action.

 Slide20

Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase.

For instance, if normal phase silica gel

is used as the stationary phase it can be considered

polar

. Given two compounds which differ in polarity, the more polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound moves higher up the plate (resulting in a higher

Rf

value). If the mobile phase is changed to a more polar solvent or mixture of solvents, it is more capable of dispelling solutes from the silica binding places and all compounds on the TLC plate will move higher up the plateSlide21

The specific

Retention factor (Rf

)

of each chemical can be used to aid in the identification of an unknown substance.

Rf

values can be calculated and compared with the reference values to identify the amino acids. Slide22

The stationary phase in TLC

silica gel

Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups.

The surface of the silica gel is polar and, because of the -OH groups, can form hydrogen bonds with suitable compounds around it.

The other commonly used stationary phase is

alumina -

aluminium

oxide

. The

aluminium

atoms on the surface of this also have -OH groups attached.Slide23

Electrophoresis

Chromatography

Migration of particles in an electrical field gradient

on a gel

The rate of movement of a solute through a stationary phase

Diffinition

is generally carried out on liquid and solid compounds

can be used for liquid, solid and gaseous compounds

samples

the electrical property of the element is used

Interactions between sample with mobile and stationary phase is used to carry out the investigations and separation

Principle

Difference between chromatography and Electrophoresis:Slide24

Experiment Slide25

Terms

Eluent

:

The mobile phase (usually for solvents)

Elution:

Motion of the mobile phase through the stationary phase

Elution time:

The time taken for a solute to pass through the system. A solute with a short elution time travels through the stationary phase rapidly,

i.e. it elutes fast.

Mobile phase:

The part of the chromatography system that is mobile. Commonly a solvent mixture (as in column chromatography or thin layer chromatography or a gas (as in gas chromatography).Slide26

Instruments, chemicals and glassware:

Eluent

.

[Mix n-

butanol

, acetic acid (purity 98 – 100 %) and distilled water in volume ratio 5:1:5. Stir for 10 minutes, then let the layers separate. Use upper layer as

eluent

].

Developing Solution:

Dissolve 0.3 g of

ninhydrin

in 100 ml n-butanol. Add 3 ml of glacial acetic acid. 0.02 M solutions of different amino acids (e.g. leucine, methionine, alanine and serine) in H2Odd. Chromatographic paper Elution chamber, Glass capillaries for spotting the samples. Drying oven at ~ 60° C.Slide27

Procedure

Rubber gloves must be used during this work

to avoid contamination

of chromatographic paper with amino acids from skin, and

for protecting skin

from solvents and

ninhydrin

while working with the sprayer or sprayed paper.

While the paper is being prepared for chromatographic analysis it should be kept on a piece of filter paper.

3. Mark the

starting line

to the paper - 8-9 mm from the edge of the plate - with graphite pencil (very slight line!). Also mark the

locations where the samples will be spotted. The distance between neighboring spots should be about 8 mm

and the spots should be at least 5 mm away from the paper’s edge. Usually the spot of unknown substance is applied to the center of the starting line. only use a pencil (do not use a pen, as the dye may separate and run along with your sample), Slide28

Before applying samples to the paper and filling the elution chamber fit the length of chromatographic paper with the height of elution chamber.

5. The spots of individual amino acids and sample solutions are applied to the chromatographic paper. Use separate clean and dry glass capillary for each solution. Dip the capillary into solution – some solution is drawn into the capillary. With the filled capillary touch the prepared location on chromatographic paper. The spot on the paper should not be bigger than 2-3 mm. (You can exercise spotting on a sheet of filter paper.)

6. After application of samples let the spots dry. Meanwhile measure with a graduated test-tube 5 ml of

eluent

into the elution chamber. Cover the chamber with lids and let the chamber atmosphere saturate with

eluent

vapors for at least

10 min.Slide29

Elution is stopped when the solvent front has traveled up the plate until 7-10 mm from the lid.

Remove the paper from elution chamber and place it on a sheet of filter paper. After 2-3 minutes mark the

eluent

front with pencil and dry the paper in oven.

When the plates are removed from the chamber, quickly trace the solvent front (the highest solvent level on the plate) with a pencil.

Monitor the run.

The time that it takes for the solvent to rise up the plate is dependent upon your choice of solvent. Generally a run will take only a few minutes, but you must monitor the solvent front to be sure that it does not reach the end of the plate. This could result in your sample being carried to the end of the plate and an unusable TLC run. A good guideline to follow is to allow the solvent front to reach a point about 1 cm from the end of the slide. Slide30

9.

When the paper is dry, take it into the fume hood and spray it with solution of ninhydrin until the paper is slightly damp. Chromatographic paper and the paper supporting it should lie at 45° angle while spraying. The chromatographic paper is again put in the drying oven (60 C) for 15 min to speed up the reactions.

10. Remove chromatographic paper from drying in the oven, draw the contours and centers of the chromatographic bands. Calculate RF values by the method described above.

11. Compare retention of standard substances and components in sample and determine which amino acids were present in the sample.Slide31
Slide32

Determination of amino acids using

thinlayer

chromatography

Adding fluorescence indicators to the sorbent layer during the process of preparation of the plates or spraying the plates with fluorescent solutions and then observing under

ultraviolet lamp

.

I

2

vapor as indicator .

Ninhydrin

spray.(Visualizing Agents for amino acids )

Development of

Ruhemann’s

purple from ninhydrin and amino acid.Slide33
Slide34

This value is constant for any given compound under a given set of conditions and can be used to identify an unknown compound. Slide35

Rf

values for some aminoacids

RF

Amino acid

0.03

Glycine

0.05

Arginine

0.51

Tyrosine

0.55

Tryptophan

0.22

Proline0.41CysteineSlide36

الامتحان النصفي

يوم الاثنين

10/4/2017

الساعة 10:00

From lab1:

“Qualitative tests for amino acids”

to lab5:

“Qualitative tests for

carbohydates

”

Slide37

That’s all

Thanks