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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
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
2- After finished the reaction, dry slide in air
3- Put the sample in mobile phase container ,allow to run
4- Dry the paper
5- Visualize spots by spraying with ninhydrin
6- Calculate Rf for each amino acid spot
C-calculation
Calculate Rf for each amino acid sample spot from law Rf=x/y
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
dissolve completely then add the following to it + [0.5ml
aniline+10ml H3PO4+ 50 ml acetone]
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
4- After finished the reaction, dry slide in air
5- Put the sample in mobile phase container ,allow to run
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
Calculate Rf for each sugar sample spot from law Rf=x/y
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
dissolve completely then add the following to it + [0.5ml
aniline+10ml H3PO4+ 50 ml acetone]
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
4- After finished the reaction, dry slide in air
5- Put the sample in mobile phase container, allow to run
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
Calculate Rf for each sugar sample spot from law Rf=x/y
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
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
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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)
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
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Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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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
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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
Practical Chromatography Course –Dr Ehab Aboueladab-Lecturer of Biochemistry-Mansoura University-Branch Damietta
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Results Names of amino acids found in the mixture.
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