2 biochemistry practical topic

Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta

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Practical Chromatography

What is it?

Some materials appear homogenous, but are actually a combination

of substances. For example, green plants contain a mixture of

different pigments. In addition, the black ink in the pens that are

used in this experiment is a mixture of different colored materials. In

many instances, we can separate these materials by dissolving them

in an appropriate liquid and allowing them to move through an

absorbent matrix, like paper.

Chromatography is a method used by scientists for separating

organic and inorganic compounds so that they can be analyzed and

studied. By analyzing a compound, a scientist can figure out what

makes up that compound. Chromatography is a great physical

method for observing mixtures and solvents.

The word chromatography means "color writing" which is a way that

a chemist can test liquid mixtures. While studying the coloring

materials in plant life, a Russian botanist invented

chromatography in 1903. His name was M.S. Tswett.

Chromatography is such an important technique that two Nobel prizes

have been awarded to chromatographers. Over 60% of chemical

analysis worldwide is currently done with chromatography or a

variation there on.


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Chromatography is used in many different ways. Some people use

chromatography to find out what is in a solid or a liquid. It is also

used to determine what unknown substances are. The Police and

other detectives use chromatography when trying to solve a crime. It

is also used to determine the presence of cocaine in urine, alcohol in

blood, PCB's in fish, and lead in water.

Chromatography is used by many different people in many different

ways.

Chromatography is based on differential migration. The solutes in a

mobile phase go through a stationary phase. Solutes with a greater

affinity for the mobile phase will spend more time in this phase than

the solutes that prefer the stationary phase. As the solutes move

through the stationary phase they separate. This is called

chromatographic development.

How it works

In all chromatography there is a mobile phase and a stationary phase.

The stationary phase is the phase that doesn't move and the mobile

phase is the phase that does move. The mobile phase moves through

the stationary phase picking up the compounds to be tested. As the

mobile phase continues to travel through the stationary phase it takes

the compounds with it. At different points in the stationary phase the

different components of the compound are going to be absorbed and


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are going to stop moving with the mobile phase. This is how the

results of any chromatography are gotten, from the point at which the

different components of the compound stop moving and separate

from the other components.

In paper and thin-layer chromatography the mobile phase is the

solvent. The stationary phase in paper chromatography is the

strip or piece of paper that is placed in the solvent. In thin-layer

chromatography the stationary phase is the thin-layer cell. Both these

kinds of chromatography use capillary action to move the solvent

through the stationary phase.

What is the Retention Factor, RF?

The retention factor, Rf, is a quantitative indication of how far a

particular compound travels in a particular solvent. The Rf value is

a good indicator of whether an unknown compound and a known

compound are similar, if not identical. If the Rf value for the unknown

compound is close or the same as the Rf value for the known

compound then the two compounds are most likely similar or

identical.

The retention factor, Rf, is defined as Rf = distance the solute (D1)

moves divided by the distance traveled by the solvent front (D2)

Rf = D1 / D2 where


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D1 = distance that color traveled, measured from center of the band

of color to the point where the food color was applied

D2 = total distance that solvent traveled

The Different Types of Chromatography

There are four main types of chromatography. These are Liquid

Chromatography, Gas Chromatography, Thin-Layer Chromatography

and Paper Chromatography.

Liquid Chromatography is used in the world to test water samples

to look for pollution in lakes and rivers. It is used to analyze metal

ions and organic compounds in solutions. Liquid chromatography

uses liquids which may incorporate hydrophilic, insoluble molecules.

Gas Chromatography is used in airports to detect bombs and is

used is forensics in many different ways. It is used to analyze fibers

on a person's body and also analyze blood found at a crime scene. In

gas chromatography helium is used to move a gaseous mixture

through a column of absorbent material.

Thin-layer Chromatography uses an absorbent material on flat

glass or plastic plates. This is a simple and rapid method to check the

purity of an organic compound. It is used to detect pesticide or

insecticide residues in food. Thin-layer chromatography is also used

in forensics to analyze the dye composition of fibers.


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Paper Chromatography is one of the most common types of

chromatography. It uses a strip of paper as the stationary phase.

Capillary action is used to pull the solvents up through the paper and

separate the solutes.

Therefore, Chromatography basically involves the separation of

mixtures due to differences in the equilibrium distribution of sample

components between two different phases. One of these phases is a

mobile phase and the other is a stationary phase.

Concentration of component A in stationary phase

Distribution Coefficient = --------------------------------------------------

Concentration of component A in mobile phase

Different affinity of these two components to stationary phase causes

the separation.

Kinds of Chromatography

1. Liquid Column Chromatography

2. Gas Liquid Chromatography

3. Thin-layer Chromatography


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LIQUID COLUMN CHROMATOGRAPHY

A sample mixture is passed through a column packed with solid

particles which may or may not be coated with another liquid. With

the proper solvents, packing conditions, some components in the

sample will travel the column more slowly than others resulting in the

desired separation.

DIAGRAM OF SIMPLE LIQUID COLUMN CHROMATOGRAPHY

FOUR BASIC LIQUID CHROMATOGRAPHY

The 4 basic liquid chromatography modes are named according to

the mechanism involved:


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1. Liquid/Solid Chromatography (adsorption chromatography)

A. Normal Phase LSC

B. Reverse Phase LSC

2. Liquid/Liquid Chromatography (partition chromatography)

A. Normal Phase LLC

B. Reverse Phase LLC

3. Ion Exchange Chromatography

4. Gel Permeation Chromatography (exclusion chromatography)

LIQUID SOLID CHROMATOGRAPHY

The separation mechanism in LSC is based on the competition of the

components of the mixture sample for the active sites on an

absorbent such as Silica Gel.

Example:


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WATER-SOLUBLE VITAMINS

1.

Niacinamide

2.

Pyridoxine


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3. Riboflavin

4. Thiamin


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LIQUID-LIQUID CHROMATOGRAPHY

In Liquid-Liquid Chromatography the stationary solid surface is

coated with a 2nd liquid (the Stationary Phase) which is immiscible in

the solvent (Mobile) phase. Partitioning of the sample between 2

phases delays or retains some components more than others to

effect separation.


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ION-EXCHANGE CHROMATOGRAPHY


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Separation in Ion-exchange Chromatography is based on the

competition of different ionic compounds of the sample for the active

sites on the ion-exchange resin (column packing).

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF

AMINO ACIDS


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GEL-PERMEATION CHROMATOGRAPHY


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Gel-Permeation Chromatography is a mechanical sorting of

molecules based on the size of the molecules in solution. Small

molecules are able to permeate more pores and are, therefore,

retained longer than large molecules.

SOLVENTS

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

Retention Time

Time required for the sample to travel from the injection port through

the column to the detector.


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SELECTIVITY ()

Ratio of Net Retention Time of 2 components.

(Equilibrium Distribution Coefficient)


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RESOLUTION EQUATION


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HEIGHT EQUIVALENT TO A THEORETICAL PLATE

Length of a column necessary for the attainment of compound

distribution equilibrium (measure the efficiency of the column).


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EXAMPLES OF THEORETICAL PLATE, SELECTIVITY AND

HEIGHT EQUIVALENT TO A THEORETICAL PLATE


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GENERAL FACTORS INCREASING RESOLUTION

1. Increase column length

2. Decrease column diameter

3. Decrease flow-rate

4. Pack column uniformly

5. Use uniform stationary phase (packing material)

6. Decrease sample size

7. Select proper stationary phase

8. Select proper mobile phase

9. Use proper pressure

10. Use gradient elution


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Practical

Theory of Paper chromatography

When you look at a leaf, the green pigment chlorophyll is usually the

only pigment that appears to be present.

Actually, chlorophyll is only one of many types of pigments present in

the leaf and one of several that are involved in the process of

photosynthesis. Once removed from the leaf, the photosynthetic

pigments can be separated from one another and identified using a

process called chromatography.

Theory of paper chromatography

A small sample of a mixture is placed on porous paper which is in

contact with a solvent. The solvent moves through the paper due to

capillary action and dissolves the mixture spot. The components of

the sample start to move along the paper at the same rate as the

solvent.

Components of the mixture with a stronger attraction to the paper

(stationary phase) than to the solvent will move more slowly that the

components with a strong attraction to the solvent (mobile phase).

The difference in the rates with which the components travel along

the paper, over time, leads to their separation.

Particular mixtures will have chromatographic patterns that are

consistent and reproducible as long as the paper, solvent, and time


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are constant. This makes paper chromatography a qualitative method

for identifying some of the components in a mixture.

Objectives

o Prepare a leaf pigment solution.

o Prepare a paper chromatogram.

o Separate pigments of spinach leaves by paper chromatography

o Calculate the Rf values for various photosynthetic pigments

Materials

1. Chromatography Jar

2. Mortar & Pestle

3. Leaf

4. Chromatography paper

5. Chromatography solvent (90% Isopropyl Alcohol)

6. Ruler

7. Capillary tube

8. Calculator

Solution Preparation:

1. Place a large piece of spinach into your pestle and add

approximately 5ml of 90% isopropyl alcohol.

2. Thoroughly macerate the spinach/alcohol mixture to develop a

thick liquid, Chromatogram Preparation:


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1. Obtain a chromatography jar, a piece of fresh leaf, and a length of

chromatography paper (just long enough to fit from top to bottom of

the jar.).

2. Cut the tip of the paper such that it forms a point of a triangle.

3. Draw a line across the paper 1 cm up from the triangle. This is your

“start line”.

4. Using a capillary tube transfer a drop of the green pigment solution

to the center of your start line.

5. Pour approx. 1 cm of chromatography solvent into the

chromatography jar.

6. Open chromatography jars and hang the papers into the jar so the

tip of the triangle dips into the solvent. Do not submerge pigment lines

below the solvent level. Recap the jars immediately.

7. Allow the solvent to rise for about 15 minutes or until the solvent

line nears the top of your papers.

8. When the solvent line is about 1cm from the top of your paper.

Remove the papers and mark the farthest point of the solvent's

progress before this line evaporates.

9. Allow the filter papers to dry, and then make a sketch of the

chromatogram. Some possible colors and the pigments they

represent are:

o Faint yellow - carotenes


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o Yellow - xanthophylls

o Bright green - chlorophyll a

o Yellow-green - chlorophyll b

o Red - anthocyanin

10. Measure the distance from the start point to the front line and

each of the pigment lines. Record these measurements in the data

table. Calculate the Rf values for each pigment according to the

following formula;

Calculation of Rf

Distance the pigment travels from the original spot of solvent

Rf = ----------------------------------------------------------------------------------

distance to the solvent front


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Practical

Theory of Thin Layer Chromatography

Thin layer chromatography (TLC) is among the most useful tools for

following the progress of organic chemical reactions and for assaying

the purity of organic compounds. TLC requires only a few ng (nano

grams) of sample for a successful analysis and can be accomplished

in a matter of minutes. Like all chromatographic methods, TLC takes

advantage of the different affinity of the analyte with the mobile and

stationary phases to achieve separation of complex mixtures of

organic molecules.

Theory of Chromatography

Stationary Phase

Silica gel, the most commonly used stationary phase, has the

empirical formula SiO2. However, at the surface of the silica gel

particles, the dangling oxygen atoms are bound to protons. The

presence of these hydroxyl groups renders the surface of silica gel

highly polar. Thus, polar functionality in the organic analyte interacts

strongly with the surface of the gel particle and nonpolar functionality

interacts only weakly. Polar functionality in the analyte molecules can

bind to the silica gel in two ways: through hydrogen bonds and

through dipole-dipole interactions. The total strength of the interaction


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is a sum of these two components. It should be noted that the shape

of the organic analyte is also a factor in predicting the strength of its

interaction with silica gel. Thus, an analyte that displays multiple polar

groups in position to interact with the surface of the stationary phase

with interact more strongly than an analyte that displays the same

polar functionality in a way that does not permit multidentate binding.

Modes of Interaction of Analyte with Silica Gel

For silica gel chromatography, the mobile phase is an organic solvent

or mixture of organic solvents. As the mobile phase moves past the

surface of the silica gel it transports the analyte past the particles of

the stationary phase. However, the analyte molecules are only free to

move with the solvent if they are not bound to the surface of the silica


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gel. Thus, the fraction of the time that the analyte is bound to the

surface of the silica gel relative to the time it spends in solution

determines the retention factor of the analyte. The ability of an

analyte to bind to the surface of the silica gel in the presence of a

particular solvent or mixture of solvents can be viewed as a the sum

of two competitive interactions. First, polar groups in the solvent can

compete with the analyte for binding sites on the surface of the silica

gel. Therefore, if a highly polar solvent is used, it will interact strongly

with the surface of the silica gel and will leave few sites on the

stationary phase free to bind with the analyte. The analyte will,

therefore, move quickly past the stationary phase. Similarly, polar

groups in the solvent can interact strongly with polar functionality in

the analyte and prevent interaction of the analyte with the surface of

the silica gel. This effect also leads to rapid movement of the analyte

past the stationary phase. The polarity of a solvent to be used for

chromatography can be evaluated by examining the dielectric

constant (ε) and dipole moment (δ) of the solvent. The larger these

two numbers, the more polar is the solvent. In addition, the hydrogen

bonding ability of the solvent must also be considered. For example

methanol is strong hydrogen bond donor and will severely inhibit the

ability of all but the most polar analytes to bind the surface of the

silica gel.


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The TLC Experiment

The first step in conduction a TLC experiment is to select the elution

solvent. For most organic molecules, a good starting point is 2 parts

ethyl acetate to 3 parts hexanes. Place about 10 mL of the elution

solvent in a 100 mL beaker covered with a watch glass. To ensure

that the atmosphere in the elution chamber is saturated with solvent

vapor, place a piece of filter paper, torn into a square, along the

inside wall of the beaker. Be sure that the bottom of the filter paper

touches the solvent. Using a pencil, draw a line on the TLC plate

about 5 mm from the bottom. Cross the line in three places with short

pencil lines. These three intersections are the locations onto which

you will place the sample. Prepare a solution of you sample in the

least polar solvent in which it is soluble. About 1 mg (a speck) of

sample dissolved in two to three drops of solvent is all that is

required. The sample is introduced onto the TLC plate using a micro

capillary. Dip the end of the micro capillary into the sample solution. A

small volume of the solution will flow into the micro capillary. Now you

can spot the capillary onto the pencil lines on your TLC plate. Be sure

that the spots on the TLC plate are no more than 3 mm in diameter.

Let the spotting solvent evaporate for a few seconds and then place

the TLC plate in the elution chamber with the sample spots at the


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bottom. Note that the sample spots should be above the level of the

elution solvent. If this is not

the case, use a pipette to remove a small amount of the elution

solvent from the chamber. Now let the sample elute to a point where

the solvent front is about 5 mm from the top of the TLC plate. To

visualize the spots on you TLC plate you will use UV light, iodine or a

series of chemical stains. You may need to adjust the polarity of the

solvent if the retention factor (RF) of you analyte is too large or too

small. The Rf is calculated by dividing the distance traveled by the

analyte by the distance traveled by the solvent. The ideal solvent

gives the analyte an Rf of 0.3.


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Analysis of Proteins by Thin-Layer Chromatography


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1. Equipment and Supplies

The following equipment is needed for a single development,

conventional

TLC analysis:

1) Amber glass storage bottles (250 ml)

2) Capillary pipettes (1 .0 and 0.2 l size)

3) Conventional TLC chamber with a lid

4) Glass vials with caps (1 and 4 ml)

5) Graduated cylinder (100 ml)

6) Oven

7) Reagent sprayer

8) Ruler (inch and metric)

9) Saturation pad (20 x 20 cm)

10) Spray box

11) Spray stand

2. Chemicals and Materials

1) 0.1 N hydrochloric acid

2) Eluent components

Butanol

Acetic acid

Water

3) Ethanol


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4) Cellulose plate, 20 x 20 cm

5) Methanol

6) Ninhydrin (Caution: toxic reagent, handle with care)

7) Amino acid standard solutions (1 mg/ml)

Glutamic acid Tyrosine

Hydroxyproline Proline

Lysine Threonine

Serine

8) Binding media reference materials (hydrolyzed)

Whole egg Egg white

Egg yolk Casein

3. Samples

Samples may be taken from facsimile paintings or unknowns. The

sample should be approximately 500 mg in weight and contain only

the paint layer or material of interest. The paint layer or material being

investigated should be separated from all other layers, such as the

ground, varnish layers, or support. Samples are hydrolyzed before

analysis,

Protocol K

Amino acid standard solutions are made with glutamic acid,

hydroxyproline, lysine, proline, serine, threonine, and tyrosine.

Each standard solution is made in a concentration of 1 mg/ml by


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weighing 2 mg of an amino acid into a 4-ml glass vial and adding 2

ml of 0.1 N HCI. These solutions can be used for 3-4 weeks after

preparation. Reference solutions of binding media are made from

whole egg, egg white, egg yolk, casein. These solutions are

prepared by hydrolysis following the same procedure as for the

samples (Protocol K).

The reference materials should be prepared in a concentration of 2.0-

2.5 g/l in 0.1N HCI.

4. Preparation Procedures

Preparation for TLC analysis includes prewashing the TLC plate,

making fresh eluent systems and detection reagents, and saturating

the TLC chamber.

The following preparation procedures are started 24 hours prior to

analysis:

1) Prepare cellulose TLC plates

The cellulose plate must be washed in methanol before analysis. This

procedure takes approximately 4 hours. Place 30-60 ml of methanol

in a clean conventional TLC chamber. Allow the chamber to

equilibrate with methanol for approximately 30 minutes. The cellulose

TLC plate is inserted vertically into the methanol, and the chamber is

covered with the lid. Allow the methanol to rise to the top of the

cellulose TLC plate. Remove the plate from the chamber and dry it in


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a fume hood. Store the cleaned cellulose TLC plate in a desiccator

containing silica gel.

2) Prepare eluent

Mix butanol, acetic acid, and water in an 80:20:20 volume ratio.

Seal the solution in an amber bottle to maintain freshness before use.

Prepare 60 ml of the eluent fresh daily for an analysis.

3) Prepare TLC chamber

Presaturate chamber with solvent system at least 4 hours before

analysis.

(Note: It is useful to presaturate the chamber overnight.) To do this,

place 30-60 ml of the eluent inside a clean, dry conventional TLC

chamber. Insert a saturation pad into the solvent system. Cover the

chamber with a lid.

4) Prepare ninhydrin detection reagent Weight 0. 158 g of

ninhydrin into a 250-ml amber bottle. Add 100 ml of ethanol. Mix

thoroughly. The reagent can be stored in a refrigerator for 4-5 weeks.

5. TLC Analysis Procedures

To analyze protein hydrolysates by TLC, the samples are spotted in

individual lanes at the baseline of a prewashed cellulose plate. The

plate is placed in a saturated conventional TLC chamber containing a

saturation pad and the eluent (butanol: acetic acid : water, 80:20:

20). The development of the plate is complete when the eluent front


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reaches a distance of 17 cm from the baseline. The plate is removed

and dried in a fume hood before spraying with the ninhydrin reagent.

This reagent reacts with the amino acid components to produce

colors that aid in the visualization of the separation zones or spots.

After 24 hours the plate can be documented

The following nine steps describe the procedure for analysis:

1) Draw the base line

Using a ruler and pencil, lightly draw a line 1 cm from the bottom

edge of the plate. Very lightly mark the lanes with short tick marks at

intervals of 1 cm along this baseline, for a total of 19 lanes. In the

upper left corner, number the plate with a reference number, used to

relate the TLC to information in the research notes. Beside the

number, place the date and the analyst's initials. Place a mark 17 cm

from the baseline as a reference to help determine the completion of

the development.

2) Apply the standard and reference solutions to the plate

All solutions are applied following the spotting procedure noted in

Protocol H.

Apply 1.0 I of the reference or standard solution to a tick mark on

the origin of a lane using a capillary pipette. The total volume may be

applied in a series of smaller volumes to minimize the diameter of the

spot. An air gun may be used to rapidly evaporate the carrier solvent


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between applications. Take care not to get the air gun too close to the

pipette, as the sample will evaporate.

3) Apply unknown sample solutions

If possible, apply each unknown sample in two different volumes. For

example, in one lane apply 1.0 I of the unknown sample, and in a

second lane apply 0.2 I of the same solution. (The unknown sample

may or may not be very concentrated, and this procedure minimizes

the possibility of overloading the plate.)

4) Develop the TLC plate

Once the plate is spotted, either develop immediately or store in a

desiccator.

To develop the plate, quickly insert the spotted cellulose TLC plate

into the saturated chamber, with the baseline oriented toward the

bottom of the chamber and the front facing away from the saturation

pad. Replace the lid of the chamber. Do not leave the chamber open

for any length of time, as the vapor phase equilibrium will be lost.


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5) Completion of development

Develop the plate until the solvent front travels a distance of 17 cm.

Development usually takes about 4 hours.

6) Dry the plate

Remove the plate from the chamber, hang it vertically, and let it dry

for about 30 minutes at room temperature in the fume hood.


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7) Prepare to spray plate

Spraying of a ninhydrin reagent should always be performed under a

well ventilated fume hood or some other device to ensure effective

removal of the reagent cloud and solvent vapors, which are toxic.

Protective glasses, laboratory gloves, and a respirator should always

be worn during spraying. Set the plate on a clean, dry spray stand

inside a spray box. Fill the reagent sprayer with 15-20 ml of ninhydrin

detection reagent.

8) Spray plate with ninhydrin

Hold the reagent sprayer 8-10 cm from the surface of the TLC plate

and spray the plate slowly back and forth, then up and down, until the

plate is evenly covered (generally until the cellulose layer just begins

to turn transparent).


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9) Heat plate

Dry the plate for 15-20 minutes in the fume hood, and then place it

for 10 minutes in a preheated oven at 100 °C.

6. Data Analysis Procedures

After the separation zones are visualized with the detection reagent

Evaluation of the plate can include qualitative or semi quantitative

techniques. The migration distances, color, and intensity of the

separation spots are noted. The Rf value for each spot is calculated


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Practical

1-Separation of Amino Acid by TLC

A-Reagent

1- Mobile Phase [ butanol: formic Acid: water] ratio [70:1:29]

2- Silica gel

3- Standard Amino acids (1%)

4- Ninhydrine spray [0.2 g dissolve in 100ml acetone]

B-Procedure

1- Weight 1g of silica gel, then dissolved in 3 ml distilled water

and mix for 5 min. [note: wash glass plate by alcohol and

accurate is clean before add silica gel on it ]

2- Pour silica gel solution on slide from glass, dry in oven for 1h

3- Load of standard amino acid and unknown sample spots

4- Put the slide with sample in mobile phase container, allow to

run

5- After finished the reaction, dry slide in air

6- Visualize spots by spraying with ninhydrin

7- Calculate Rf for each spot

C-calculation

Calculate Rf for each amino acid sample spot from law Rf=x/y


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Practical

2-Separation of Amino Acid by paper chromatography

A-Reagent

5- Mobile Phase [butanol: glacial acetic Acid: water] ratio

[12:3:5]

6- Whattman filter paper number 1

7- Standard Amino acids (1%)

8- Ninhydrine spray [0.2 g dissolve in 100ml acetone]

B-Procedure

1- Load of standard amino acid and unknown sample spots


3- Put the sample in mobile phase container ,allow to run

4- Dry the paper


6- Calculate Rf for each amino acid spot

C-calculation



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Practical

3-Identification of sugars by paper chromatography and TLC

A-Reagent

1-Mobile Phase [Methanol: glacial acetic Acid: water] ratio

[60:10:30]

2-Whattman filter paper number 1

1. Standard sugars (1%) [Glucose, fructose, Maltose etc]

2. Diphenylamine spray [0.5 g dissolve in 50 ml acetone until

dissolve completely then add the following to it + [0.5ml

aniline+10ml H3PO4+ 50 ml acetone]

B-Procedure

3. Load of standard glucose, fructose, maltose and unknown

sample spots

4. After finished the reaction, dry slide in air

5. Put the sample in mobile phase container ,allow to run

6. Leave spot to dry in air ( paper or TLC)

7. Visualize spots by spraying with Diphenylamine reagent mixture

8. Dry in oven until the spots are appear

9. Calculate Rf for each sugar spot

C-calculation

Calculate Rf for each sugar sample spot from law Rf=x/y


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Practical

4-Identification of sugars in milk by paper chromatography and

TLC

A-Reagent

1. Mobile Phase [Methanol: glacial acetic Acid: water] ratio

[60:10:30] for paper chromatography

2. Mobile Phase [Ethyl acetate:isopropanol:pyridine:water]

ratio [26:14:2:7] for TLC

3. 10% Trichloroacetic acid (TCA) [dissolve10g/100ml water]

4. Whattman filter paper number 1

5. Standard sugars (1%) [Glucose, fructose, Maltose etc]

6. Diphenylamine spray [0.5 g dissolve in 50 ml acetone until



B-Procedure

1- 2 ml from milk + 2 ml TCA and mix well, after precipitation ,

make centrifuge at 3000 rpm for 5 min

2- Collect only supernatant (sample) and discard the pellet

3- Load of standard glucose, fructose, maltose and unknown

sample spots


5- Put the sample in mobile phase container ,allow to run


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6- Leave spot to dry in air ( paper or TLC)

7- Visualize spots by spraying with Diphenylamine reagent

mixture

8- Dry in oven until the spots are appear

9- Calculate Rf for each sugar spot

C-calculation



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Practical

5-Identification of sugars in Fruit Juice by paper

chromatography and TLC

A-Reagent

1- Mobile Phase [Methanol: glacial acetic Acid: water] ratio

[60:10:30] for paper chromatography

2- Mobile Phase [Ethyl acetate:isopropanol:pyridine:water]

ratio [26:14:2:7] for TLC

3- Absolute Ethanol

4- Whattman filter paper number 1

5- Standard sugars (1%) [Glucose, fructose, Maltose etc]

6- Diphenylamine spray [0.5 g dissolve in 50 ml acetone until



B-Procedure

1- 2 ml from fruit juice + 3 ml Ethanol and mix well, after

precipitation , make centrifuge at 3000 rpm for 5 min

2- Collect only supernatant (sample) and discard the pellet

3- Load of standard glucose, fructose, maltose and unknown

sample spots


5- Put the sample in mobile phase container, allow to run


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6- Leave spot to dry in air ( paper or TLC)

7- Visualize spots by spraying with Diphenylamine reagent mixture

8- Dry in oven until the spots are appear

9- Calculate Rf for each sugar spot

C-calculation



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Practical

6-Separation of Lipid by TLC

A-Reagent

1- Mobile Phase [ petroleum ether : diethyl ether: glacial

acetic acid ] ratio [79:20:1]

2- Silica gel

3- Standard lipids (1%)

4- Iodine spray [0.2 g dissolve in 100ml acetone]

B-Procedure

1- Weight 1g of silica gel, then dissolved in 3 ml distilled water

and mix for 5 min.

2- Pour silica gel solution on slide from glass, dry in oven for 1h

3- Load of standard lipid and unknown sample spots

4- Put the slide with sample in mobile phase container, allow to

run



7- Calculate Rf for each spot

C-calculation



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Practical

7-Electrophoresis

Principle

Electrophoresis means the migration of charged

particles in a liquid medium under the influence of an electric field.

When an electric field is applied to a medium containing charged

particles or molecules (e.g. DNA or protein), the negatively

charged molecules migrate towards the positive electrode

(anode) and vice versa. After separation (according to difference

in charge and mass) permanent fixation of the fractions at the

position to which they migrate is done. Bands are then stained in

order to visualize them.

Components

1. Power supply: provide stable direct current, and has controls

for both voltage and current output. (cathode & anode)

2. Support medium: It is the heart of the system where

separation occurs there. Its function is to provide an inert porous

medium for the electrolytes solution. Zone Electrophoresis is

classified according to the support medium type. Support media may

be Thin sheet (of paper, cellulose acetate or silica) or Gel (of starch,

agarose or polyacrylamide that separate samples according to the


51

charge and size) e.g. cellulose acetate electrophoresis,

polyacrylamide gel electrophoresis.

3. Buffer: It serves as a multifunctional component in the

electrophoretic process as it: a) carries the applied current

(electrophoresis buffer) b) establish the pH at which electrophoresis is

performed (gel buffer). c) Determine the electric charge of the

sample (sample buffer). There are several considerations must be

done to select buffer: A) the buffer must be not interact with sample.

B) The ionic strength and concentration of buffer must be suitable for

sample. C) It must allow the sample to be charged not denaturated.

4. Stains: It is used to visualize and locate the separated protein

and nucleic acid fractions e.g. Coomassie Brilliant Blue (CBB).

(Tracking dye such as Bromophenol Blue (BB) is often used to see

the sample movement on gel (do not stain sample bands) that

enables us to terminate the process when the bands reach lower

buffer reservoir. It moves faster than any macromolecules).

Procedures

(e.g. Poly-Acrylamide Gel Electrophoresis) SDS-PAGE

Laemmle (1970)


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A) Gel preparation:

Fist: prepare the following solutions as follow:

Second: prepare the separating and stacking gel as follow:

Separating gel: (3.5ml from 1) + (2.5ml from 2) + (0.1ml from

4) + (0.05ml from 5) + (10μl from 6)

Stacking gel: (0.6ml from 1) + (1.25ml from3) + (0.05ml from 4)

+ (0.05ml from 5) + (5μl from 6)

Note: These gels are polymer. We can control their pores though the

concentration of their constituents .high concentration decrease pores

size, and become suitable for passing low molecular weight proteins

and vice versa.

B) Pouring the gel in electrophoresis unit:

1. The separating gel is transferred to the gel glass sandwich.

Wait till polymerization (25min) (take care with air bubbles)

2. The stacking gel is then transferred over separating gel. The

comb is inserted into the top then removed after polymerization


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C) Sample application:

1. The sample is homogenized by sample buffer (Tris + SDS+ 2-

mercapto ethanol + BB + sucrose) then centrifuged.

2. 25μl of supernatant is applied side by side in the wells inside

the gel.

3. 15μl of protein marker is applied in the last well for comparisons

with unknown bands.

4. The upper and lower buffer reservoir is filled with

electrophoresis buffer (Tris + glycine + SDS)

D) Running of samples: The power is switched on. Wait till the

bands reach at lower end of gel (stopping gel) then switch off.

E) Detection and quantification:

1. The plate is removed, dried then stained to see the bands.

2. Compare the separating unknown bands with the known marker

bands.

(The process can carry out without SDS in certain samples: Native-PAGE)

Applications

1. Gel electrophoresis is used in quantitative analysis in

molecular biology and genetics.

2. Nucleic acids carry negative charge on their suger-phosphte

backbone so they migrate into gel with similar rates and


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separation is done due to different molecular size. Agarose gel

electrophoresis is suitable for DNA and RNA analysis.

3. Proteins have different charges, so they charged negatively

(denaturated) by SDS in order to migrate into the gel with

similar rates and separation is done due to different molecular

size. Polyacrylamide gel electrophoresis is suitable for protein

analysis

4. Other different types of electrophoresis have many applications

in many fields.

Factors affecting electrophoresis

1. The sample:

a) Charge: migration increase with charge increase

b) Size: migration decrease with size increase

2. The support media:

a) If adsorption, migration decrease

b) If molecular sieving, migration increase

3. The buffer:

a) Composition, bad buffer decrease migration

b) Concentration increase, migration decrease

c) pH affect ionization


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4. The electric field:

a) Voltage: migration increase with its increase

b) Current: migration increase with its increase

c) Resistance: migration decrease with its increase

5. The heat: migration increase with its increase due to fall in

resistance.

Types of electrophoresis

Electrophoresis has two mains types according to the support media:

(I) Thin sheet electrophoresis: it separates samples according to

the charge. Support medium is thin sheet.

1. Paper electrophoresis: used in past to separate charged

samples (support medium is thin sheet of paper)

2. Cellulose acetate electrophoresis: it suitable for separation of

radio-labeled substances especially for clinical investigations

(support medium is cellulose acetate that is prepared by

treating cellulose with acetic anhydride)

3. Thin layer electrophoresis (TLE): as in TLC but the plate is

placed in electrophoresis unit (support medium is silica)

(II) Gel electrophoresis: it separates samples according to the

charge and molecular size. Support medium is gel.

1. Continuous gel electrophoresis:


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a) Starch gel electrophoresis: it is prepared heating and

cooling starch in suitable buffer.

b) Agar/Agarose gel electrophoresis: Agar composed of

agaropectin and agarose. Separation based on charge only

c) Polyacrylamide gel electrophoresis (PAGE):

it is made from acrylamide monomers copolymerized with the

cross linker N,N`methylenebisacrylamide in presence of

ammonium persulphate and TEMED as catalyst. Separation based

on molecular size (molecular sieving). It has two types:

Native-PAGE: under non-denaturating conditions.

SDS-PAGE: under denaturating conditions.

2. Discontinuous gel electrophoresis:

3. Two dimensional gel electrophoresis

Gradient gel electrophoresis:

a) Isoelectric focusing

b) Pulse-field gel electrophoresis

c) Capillary electrophoresis

Native PAGE SDS- PAGE

1. used to determine total proteins

2. detergent not used to avoid deformation of proteins

1-used to determine fragments of protein 2-detergents are used to do fragmentation of proteins


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1D 2D

1-separation according to molecular weight 2-depends on gradient Acrylamide

1-separation according to isoelectric point of protein 2-depends on gradient pH

Material Function

Acrylamide and Bisacrylamide To form a net structure through which, proteins will sieve according to their size

Separating gel Provide an inert porous medium for separation

Stacking gel To press all sample bands in one line in order to run with each other

Sodium dodecyl sulfate (SDS) To provide a negative charge to proteins

Amm. Per sulfate (APS) Initiate the reaction between Acrylamide and Bisacrylamide

TEMED Increase or catalyst the reaction

Ampholyte To establish a pH gradient on

electrophoresis unit before running

proteins. When running proteins, they

move on till reaching a pH

corresponding to its isoelectric point


57


58


59

SEPARATION OF AMINO ACIDS BY THIN LAYER CHROMATOGRAPHY

MATERIALS NEEDED

• silica gel plate

• mobile phase: 1-butanol, glacial acetic acid and water (4:1:1)

• known solutions of amino acids

• unknown solutions of amino acids

• micropipette

• developing tank

• 2% ninhydrin solution

• heat gun

• pencil

• gloves

PURPOSE: To understand the concepts of chromatography and to

identify unknown amino acids.

BACKGROUND

The discovery of chromatography in 1944 revolutionized the separation and

detection of amino acids and dipeptides. The separation is based on the liquid-

liquid partition of the compounds between two immiscible phases. Initially the

separations were primarily conducted on filter paper and were called paper

chromatography. In paper chromatography the hydrated cellulose fibers of the

paper act as the stationary phase. A polar solvent ascends in the vertically held

paper by capillary action and is the mobile phase. In thin layer chromatography

(TLC) a thin uniform layer of silica gel acts as the stationary phase. TLC is

replacing paper chromatography because the plates are easier to use than the

paper, they give a sharper separation and the amino acids or dipeptides can

easily be collected from the plate.

Many microscopic distributions of the compounds occur between the mobile and

the stationary phases. In time equilibrium is established between the two phases

and the more soluble compounds move farther along the plate; different

compounds move 2 Amino Acids different distances from the origin. The plate is

dried, sprayed with a ninhydrin solution and heated in order to locate the amino

acids. The ninhydrin reacts with the amino acids to form colored products. The

ratio of the distance moved by the amino acids to the distance moved by the


61

solvent front from the original spot on the paper is defined as the Rf value and is

characteristic of the compound.

Rf compound = distance traveled by compound /distance traveled by solvent

Rf values depend on several factors: type of silica gel plus binder used,

water content of the thin layer, concentration of solute, temperature, manner of

development and distance of the starting point from the solvent. Known

compounds are usually run on the same plate as the unknowns to assist in

identification of the unknowns rather than relying solely on published Rf values.

PROCEDURE

1. Put gloves on. If your developing tank isn't prepared, add enough solvent to a

depth of approximately 1 cm or less.

2. Get your silica gel plate. Carefully hold the plate by the sides to prevent

disturbing the silica gel layer. Draw a pencil line about 1.5 cm from the bottom

plate.

3. Mark one point on the line for each one of your known and unknown solutions.

(If you have four known solutions and 2 unknowns, mark six points.) Leave

margins of at least 1.5 cm on both sides. Number each point.

4. At point number 1 apply a very small drop of one of your known 1 2 3 4 5 6 7

solutions. Do not wet • • • • • • • 3 Amino Acids the silica beyond a diameter of 2-3

mm. Locating the center of large spots will be difficult later when the spot has

moved along the paper.

5. After the liquid has evaporated (only a few seconds), add a second drop to the

same spot. Record the name of the amino acid and the number of the spot.

6. Repeat this procedure for the remaining solution. Remember to record the

name of the amino acid or unknown number and the number of the spot.

7. Allow all the spots to dry completely.

8. Place your TLC plate in the developing tank with the mobile phase with the

spots toward the bottom.

9. Allow the solvent to ascend the silica gel to at least ¾ of its height, which will to

require 1 hour or less. (The farther the solvent ascends, the greater the

separation. Immediately remove the plate, if the solvent reaches the top.)

10. Remove the plate and quickly mark the farthest advance of solvent front with

a pencil, unless it reached the top of the paper.


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11. Dry the plate with a heat gun. Be careful to move the heat gun around and

not heat one point continuously. Do this procedure in the hood.

12. Spray the plate with the 2% ninhydrin solution in the hood.

13. Do not allow the ninhydrin solution to stream down the plate, because this

may move some of the compounds.

14. Dry the plate again with the heat gun. Do not over heat the plate. Long

heating times may cause browning of the plate over the entire surface.

15. Circle each colored spot with a pencil. The ninhydrin spots fade gradually, so

circle at once.

16. Measure the distance from the origin to the center of each colored spot and

calculate the Rf values for all spots. 4 Amino Acids

17. Record the Rf values and the color of each ninhydrin spot.

18. Identify the unknown amino acids.

QUESTIONS

1. Why does touching the silica gel with your hands potentially contaminate

your plate?

2. Why can an Rf value never be greater than 1?

3. What would happen if so much solvent was used (mobile phase) that the

original spots were covered with solvent?

4. What would happen if you made the line and points with an ink pen rather than

a pencil?

5. You dropped and mixed up your samples. You know that one contains

only valine, one contains valine and glycylvaline, one contains valine and alanine,

one contains only glycine and valine, and one contains glycine and glycylleucine.

How would you determine what your samples are using TLC and the data below?

Can you figure out what they all are?

Rf Values for Amino Acids and Dipeptides Compound Rf Color

Glycine 0.26-0.29 purple

Alanine 0.39-0.42 purple

Glycylvaline 0.62-0.66 gray

Valine 0.62-0.64 purple

Glycylleucine 0.76-0.80 light brown

Rf values taken two days after solvents were mixed and with solvent advance

100 mm in 43 minutes at a temperature of 31C. Calculating Rf Values


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Results Names of amino acids found in the mixture.

2 biochemistry practical topic

Education

word chromatography

thesekinds of chromatography

amobile phase

themobile phase

main types of chromatography

ingas chromatography

phase thanthe solutes

unknown compound