King Abdulaziz University Faculty of Sciences

Department of Biochemistry Girls Section

Analytical Biochemistry Lab BIOC 343

Edited and Organized by a Collaborative Work with Lecturers Mona Badahdah, Wedam Alghazzawi, and Sherin Bakhash

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Lab # Experiment Page

#

1

Separation methods

Adsorption chromatography (LSC).

EXP. 1: Part I

Separation of amino acids by two-dimensional chromatography by Thin Layer

Chromatography.

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EXP. 1: Part II

Separation of amino acids by two-dimensional chromatography by Thin Layer

Chromatography.

Adsorption chromatography (LSC).

EXP. 2: Separation and identification of carotenoids pigments in tomatoes and carrots by

column chromatography.

Part I: Dehydration and Extraction

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14

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EXP. 2: Separation and identification of carotenoids pigments in tomatoes and carrots by

column chromatography.

Part II: Column chromatography.

Part III: TLC analysis.

Part IV: UV/Vis spectroscopy

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21

4 Measurement of color.

EXP. 3: Determination of wavelength of maximum absorbance of carotenoids.

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26

5 Gel-filtration chromatography.

EXP. 4: Separation of a mixture of blue dextran and cobalt chloride molecules. 30

6 Titration cutve

EXP. 5: Determination of the isoelectric point of glycine 34

7

Dialysis

EXP. 6: A- Separation of a mixture of starch and maltose

EXP. 6: B- Separation of a mixture of Starch and Glucose

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40

8 Centrifugation

EXP. 7: Effect of salt on extractability of proteins. 45

9 Chemical solutions.

EXP. 8: Preparation of buffer and liquid solutions 48

10 EXP. 9: Electrophoresis

Serum protein electrophoresis 55

11 Enzyme linked Immuno Sorbent Assay- ELISA

EXP. 10: 58

Table of Contents

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Introduction

Analytical biochemistry involves the use of laboratory techniques to determine the composition

of biological samples. It includes a qualitative analysis or a quantitative analysis. A qualitative

analysis indicates what is present in the sample. Chromatographic and electrophoretic methods

are an example of such analysis. A quantitative analysis indicates the amount of a particular

substances present in the sample such as spectrophotometric and titrimetric methods.

Areas of chemical analysis and questions they answer:

Quantification:

How much of substance X is in the sample?

Detection:

Does the sample contain the sample X?

Identification:

What is the identity of the substance in the sample?

Separation:

How can the species of interest be separated from the sample matrix for better

quantitation and identification?

The separation of molecules from biological materials is an important part of biochemical work

and often involves the isolation of one molecular species from a mixture of compounds with

very similar properties. Because of this, the usual methods of organic chemistry are inadequate,

and to be able to choose a suitable analytical technique it is essential to know something about

the chemical and the physical properties of the test substances. Generally, the use of extreme

pH, temperature, organic solvents, and oxidizing and reducing agents should be avoided when

dealing with molecules separated from living matter. These factors lead to loss of the biological

activity through denaturation.

NB: Being in the laboratory you must:

1. Wear safety glasses and protective gloves.

2. Clean the bench and dispose of the waste chemicals.

3. Submit a lab report the following week to report your findings.

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In this lab manual, the experimental techniques employ mild conditions and utilize differences

in the basic physical properties of the molecules such as their size, shape, mass, charge,

solubility, and adsorption properties. The techniques involve different degrees of interaction

between three components:

1) Solute (molecules to be separated from a mixture).

2) Mobile phase (liquid or solvent) is the phase that moves through or over stationary phase

carrying the sample with it or it is a carrier sample.

3) Stationary phase (solid), a layer or coating on the supporting medium that interacts with

the analytes.

Common separation technique

Technique

Physical

properties of

solute

Solid phase Solvent

Adsorption chromatography

(LSC) Adsorption

Adsorbent usually

inorganic material Non-polar

Partition chromatography

(LLC) Solubility Inert support

Mixture of

polar and non-polar

solvents

Ion exchange chromatography Ionization Matrix containing

ionized groups Aqueous buffer

Gel-filtration chromatography

(SEC) Size and shape Hydrated gel Usually aqueous

Dialysis Size and shape Semi-permeable

membrane Water

Polyacrylamide-gel electrophoresis

(PAGE) Charge and size Inert support with pores Aqueous buffer

The basis of separation methods in biochemistry involving three components

Solute

(Molecules to be separated)

Solid phase

Usually contain a paper,

thin layer, gel, or

membrane.

Solvent

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Introduction

Chromatography literally means color writing. From Greek "Chroma" color and "graphein" to

write. Chromatography is a physical technique for separating a mixture into its individual

components in which the components to be separated are distributed between two phases, a

stationary phase (solid) and a mobile phase (solvent), due to the differences in their chemical

and physical properties. By passing the mixture over the stationary phase the substances stick

on it to varying degrees. Substances that stick tightly to the stationary phase move very slowly,

while those that stick loosely or do not stick at all move rapidly.

Because the components of the mixture are not chemically combined, they can separate on the

basis of their charge, size, shape, and their solubility. Chromatography is used to analyze,

identify, purify and quantify the compounds.

The technique can be divided into planar and columnar techniques. In planar type, the

stationary phase coats a plane surface (two dimensions surface) and placed in reservoir

containing the mobile phase which moves by capillary action. It can be paper chromatography

or thin layer chromatography. In the columnar type, the stationary phase is placed in a column

through which the mobile phase moves under the influence of gravity or pressure. Planar

techniques have the advantages of faster separation, visualization of formed chromatograms, or

spots, and they are less expensive. However, they are not useful for preparative purposes.

Columnar techniques have the advantages of possibility to yield a large amount of compounds.

However they have the disadvantage of being expensive, time consuming.

Chromatography can be an analytical method, in which the investigator determines the

chemical composition of the components in a very small amount of a mixture, but does not

actually isolate them. A common analytical method is silica-gel thin layer chromatography. Or

it can be a preparative method, in which the investigator uses a large quantity of the mixture

to purify and collect one or more component of it for more advanced use. A common

preparative method involving the same phases is silica-gel column chromatography.

• Purify

• Identify

• Quantify

• Analyze Mixture Components

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Types of Chromatography

There are many forms of chromatography, but all forms work on the same principle. All

chromatographic forms have stationary phase (solid or liquid supported on a solid) and mobile

phase (liquid or gas).The mobile phase flows through the stationary phase and carries the

components of the mixture with it.

Classification according to the chemical or physical mechanism:

1. Partition Chromatography (LLC)

2. Adsorption Chromatography (LSC)

3. Gel filtration Chromatography, or SEC Chromatography

4. Ion Exchange Chromatography uses a charged stationary phase to separate charged

compounds including amino acids, peptides, and proteins. The stationary phase is an

ion exchange resin that carries charged functional groups which interact with oppositely

charged groups of the compound to be retained.

Applications of chromatography

• In pharmaceutical industry (penicillin and other antibiotics).

• In separation of proteins into amino acids.

• In crime scene investigation for DNA and RNA sequencing.

• In many scientific studies to identify unknown organic and inorganic compounds.

• In food and vegetables contained tiny amounts of pesticides and herbicides.

Development (movement or migration of the solvent)

A. Ascending chromatography

Solvent running up the paper or TLC by capillary action. It is most employed

and has the advantage that separation can be carried out in two dimensions.

B. Descending chromatography

In descending paper chromatography, the chromatogram is held vertically,

and the spot of the sample is drooped on the top of the chromatogram.

Solvent drips off the bottom of the paper by gravity. Descending

chromatography is faster because gravity helps the solvent flow but it’s

difficult to set the apparatus.

Solvent

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Detection of spots

Most biological compounds are colorless and can be visualized on the "chromatogram" by:

1. Spraying the paper by specific reagents, as Ninhydrin.

2. Dipping in a solution of the reagent in a volatile solvent, as Iodine vapors.

3. Fluorescence compounds can be visualized with ultraviolet light.

4. Radioactive spots can be located with a detector, or the chromatogram can be pressed

against X-ray film.

Retention factor (Rf)

Different compounds should move different distances on the plate. Some compounds in a

mixture travel as the solvent moves; some stay much closer to the base line. The ratio of the

two distances is calculated. This ratio is called the retention fraction Rf. Individual compound

in a mixture can be identified by its Rf value when compared to one or more standards under

absolutely identical conditions to that of the test compound. If one of the Rf values of a mixture

matches the Rf value of the standard, this suggest the presence of the standard compound in the

mixture. When comparing two different compounds run under identical conditions, the

compound with the larger Rf is less polar because it interacts less strongly with the polar

adsorbent on the TLC. Spots with the same Rf values within experimental error and the same

appearance are likely to be the same compound.

Specifically, the retention fraction is defined as the fractional distance the spot moves compared

to the distance travelled by the solvent front. An Rf value will always be in the range 0 to 1; if

the compound moves, it can only move in the direction of the solvent flow, and cannot move

faster than the solvent. The Rf is constant for a particular compound; as long as you keep

everything else constant; the solvent system, the stationary phase, temperature, amount of

material spotted, pH of the solution, the distance travelled by the solute and solvent, and

thickness of the layer. Rf can be estimated by:

Distance from baseline to spot, x_

Distance from baseline to solvent, y

The Rf is calculated as below:

Rf =

=

3.0 cm 4.5 cm

Solvent front

New position

of compound

Origin

Rf = x = 3.0 = 0.67

y 4.5

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Introduction

Partition chromatography (LLC) is the distribution of similar solutes between two immiscible

liquid phases, the stationary phase and the mobile phase due to differences in partition

coefficients during the flow of mobile phase. It is mainly used for separation of molecules of

small molecular weight. Partitioning occurs between the original solvent which is the mobile

phase and the thin layer of water adsorbed on the cellulose filaments of a prepared filter paper.

Both stationary phase and mobile phase are liquid in nature. Polar molecules get partitioned

into polar phase and vice-verse. It is frequently referred to as liquid-liquid chromatography

(LLC). Paper chromatography and gas-liquid chromatography (GLC) come under this

chromatograph type. Paper chromatography is an analytical technique for separating and

identifying both colored (e.g. pigments) and colorless (e.g. amino acids) mixtures.

Cellulose (non polar) in the form of paper sheets makes an ideal

support medium where water is absorbed to the cellulose fibers

and forms the stationary hydrophilic phase. You can think of

paper as being cellulose fibers with a very thin layer of water

molecules bound to the surface. The stationary phase is more

polar than the mobile organic phase. This method has been

largely replaced by thin layer chromatography, but is still a

powerful teaching tool.

Cellulose is a polymer of the simple

sugar, glucose has - OH groups

Partition

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Introduction

Adsorption chromatography (LSC) is oldest form of chromatography. Tswett developed the

technique around 1900 and demonstrated its use by separating plant pigments.

It utilizes a mobile liquid or gaseous phase that is adsorbed onto the surface of a stationary solid

phase. When a mixture of components undergoes a chromatography process, the components

move through the system at different rates. The difference in rates between the components is

due to the difference in degree of adsorption of the compounds onto the surface of the stationary

phase (adsorbent).The equilibrium between the mobile and stationary phase account for the

separation of different solutes.

In adsorption chromatography there are two types of forces one is force attracting solutes to

adsorbent and second is force tending to remove solutes from adsorbent to move with the

mobile phase. During separation of components, when we use a polar stationary phase, non-

polar compounds will be less strongly attracted to stationary solid phase and will spend more

time in the moving phase and will move faster and will appear closer to the top. They will have

relatively high Rf values. Polar compounds will be more strongly attracted by hydrogen

bonding or dipole-dipole attractions to the stationary solid phase and will spend less time in the

moving phase and they aren't going to travel very fast up the stationary phase and appear near

to the bottom. They will have relatively low Rf values. Column chromatography and thin layer

chromatography (TLC) come under this chromatograph type.

Solvent front

Compound B

Compound A

More polar

Less polar

Origin

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Experiment 1

The separation of amino acids by two-dimensional chromatography

Thin Layer Chromatography (TLC)

Introduction

Thin layer chromatography (TLC) is one of the simplest, fastest, easiest and least expensive of

sevsral chromatographic techinque used in quantitative and qualitative analysis to separate

organic compounds and to test the purity of compounds.

Many compounds with varying functional groups may be used as the stationary phase and

several types of interactions can aid in developing the desired separation (i.e. Van der Waals

forces, electrostatic interactions, hydrogen bonding, dipole–dipole etc.).

The stationary phase consisting of very thin layer of adsorbent material mainly silica gel or

aluminum oxide, forming a thin layer (0.1 – 0.3 mm) on a flat, inert carrier sheet (glass, plastic

or aluminum plate). The surface of alumina is more polar than that of silica gel. The mobile

phase is various organic solvent or mixture of solvents that is less polar than silica gel. TLC has

advantages of choice between different stationary phases, otherwise it is

similar to paper chromatography.

Common uses of Thin-Layer Chromatography: 1. To determine the number of components in a mixture.

2. To determine the identity of two substances.

3. To monitor the progress of a reaction.

4. To determine the effectiveness of a purification.

5. To determine the appropriate conditions for a column chromatographic separation.

6. To monitor column chromatography.

Principle

Two-dimensional TLC chromatography

(Two-way TLC chromatography) involves

using two solvents and rotating the paper

90° in between. This useful for separating

complex mixtures of similar compounds

for example amino acids.

Ninhydrin, which is originally yellow,

will react with a free α-amino group. This

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group is present in all amino acids, proteins, peptones or peptides in which ninhydrin react with

such compounds in the pH range of 4-8 to give blue to violet to red color. The blue color changes

to a purple when viewed in artificial light. Other compounds also react if present, and these

include primary and secondary aliphatic amines and some non-aromatic heterocyclic nitrogen

compounds. The imino acids, proline and hydroxyl proline, also react with ninhydrin but they

give a yellow color. Ninhydrin can be used qualitatively (e.g. for chromatographic

visualisation) or quantitatively (e.g. for peptide sequencing). The test is sensitive enough that

ninhydrin can be used for the visualization of fingerprints.

Materials and chemicals:

• Chromatography glass jar with lid

• Chromatography paper or filter paper: Whatman No.1,

(10 cm x 10 cm)

• Capillary tubes or Pasteur pipettes

• Hairdryer or drying oven at 105°C

• Solvent 1: ethanol: water (7:3 v/v)

• Solvent 2: n-butanol: acetic acid: (8:2:2 v/v).

The solvent should be made up fresh on the day.

• Ninhydrin reagent (Dissolve 0.2 gm in 100 ml of aceton just before use).

• Mixture of amino acids (Alanine, histidine, leucine, proline, argnine).Prepare small

volumes of 10 gm/liter solutions in 1 molar HCl (IRRITANT), a drop of acid are needed

to bring the compound into solution.

Part I

Procedure

1. Select five amino acids whose Rf values differ widely.

2. Obtain paper or TLC plate (10X10) and place it onto a paper towel. Be

careful to touch the paper or TLC sheet only on the edges, without

touching your fingers to the surface.

3. Using a pencil, draw a faint line (on the powdery side of the TLC), about

1 cm from the bottom and about 1 cm from top of both paper and TLC

(Solvent front). The line should be parallel to the bottom edge. Do not

allow the pencil to dig into the coating on the plate at all. Then faintly draw one small hash

mark, about 2 cm towards the left corner.

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4. Spot 10-20 ul of a single spot of amino acids mixture onto the paper or

the plate (up to a total of 3 times). To do this, dip one end of the capillary

tube into the top of the solution so that only the clear portion of the

liquid enters the capillary tube. Then lightly and quickly touch the

capillary tube to the surface of the paper or the TLC plate. Try to keep

the spot as small possible, preferably around 1mm, Dry the spot in a

current of air.

5. Use forceps to place the paper or TLC into the jar. Make sure that when the TLC plate is

placed in the solvent bath, the solvent does not immerse the spot of the sample mixture. The

solvent is below the spot, but then moves up across the spot, carrying the components of

the sample mixture up the plate at different rates. If the spot is immersed in the solvent at

the beginning, it will simply dissolve in the solvent and not rise up the plate.

6. Position the paper or the plate so that it is not touching the sides of the jar, and make sure it

does not curve or buckle in the jar. Covering the jar makes the atmosphere in the jar

saturated with solvent vapor. Saturating the atmosphere in the jar with vapor stops the

solvent from evaporating as it rises up the paper or the TLC plate.

7. As the solvent slowly travels up the paper sheet or TLC plate, the different components of

the mixture travel at different rates. As the solvent migrate up the paper or the TLC plate,

do not disturb the jar.

8. Watch the paper sheet or the TLC plate carefully. Once the solvent migrates almost to the

top of the plate use forceps to remove it and let the solvent evaporate from it in the hood.

Part II

Procedure

9. Arrange the frame so that the second edge to which the spot was adjacent now dips in the

second solvent. Cover the jar with the lid. Once the solvent migrates almost to 1the top of

the plate use forceps to remove the plate and immediately mark the solvent front with a

pencil, before the solvent evaporates.

10. Dry the paper or the TLC plate by hairdryer. Rapidly spray the paper or the TLC plate

through the Ninhydrin reagent and allow the acetone to evaporate. Develop the colors by

heating at 105° C for 2-3 minute or using a hairdryer. The outcome of chromatography

experiment is a chromatogram.

11. Carefully circle the spots with a pencil and mark the center of each spot. Place your name

on the front or back of your TLC plate.

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Experiment 2

Separation and identification of carotenoids pigments in tomatoes

and carrots by column chromatography

Introduction

Carotenoids are organic, fat-soluble pigments that are naturally occurring in plants and some

other photosynthetic organisms. These compounds are responsible for the red, yellow, and

orange color of fruits and vegetables. There are over 600 different

carotenoids known. Two classes of carotenoids are known xanthophylls

and carotenes. Since animals are incapable of synthesizing carotenoids,

they must obtain them through their diet. The two commonest

carotenoids are lycopene, a red pigment found in tomato and beta-carotene,

a yellow pigment found in carrots. Other pigments also occur in the leaves of plants but they

are not obvious because their colors masked by the chlorophylls in live, healthy leaves. These

pigments become visible in the fall when the leaf dies and the chlorophyll rapidly decompose.

Carotenes act as a precursor of vitamin A. Beta-carotene cleaves to form two molecules of

vitamin A when it is ingested. Vitamin A plays an important role in vision. Beta-carotene is a

powerful antioxidant, and has been shown to help guard against cancer and heart disease.

Lycopene is also an antioxidant stop free radical production, and it may lower the risk of

prostate cancer. The best food source of lycopene is processed tomato products such as ketchup,

tomato paste, and tomato juice.

Principle

This experiment performed to try and tentatively identify and isolate lycopene and beta-

carotene from two foods rich in them, tomato paste and carrots using refluxing, column

chromatography and thin layer chromatography. Analyze the fractions by thin-layer

chromatography to determine if the fraction contains more than one component. Since both

lycopene and β-carotene are colored pigments that absorb light in the UV and visible range,

they can be identified from their maximum wavelength.

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PART I: Dehydration and Extraction

In this part you will extract pigments from tomato paste and carrots, using ethanol and

chloroform as organic solvents. The experiment should be performed under a portable fume

cupboard giving all-round visibility.

Materials and chemicals

• Condenser

• Hot plate

• Beaker

• Filter paper

• Separation funnel

• Glass funnel

• Round-bottom flask

• Two Conical flask

• Tomato paste or carrot (natural or baby food)

• Ethanol (95 %)

• Chloroform

• Saturated sodium chloride solution

• Anhydrous sodium sulfate

Procedure

1. In a small beaker place 3 gm of tomato paste, 9 gm carrots, and 30

ml of 95 % absolute ethanol. Then stir the suspension for at least

five minutes with a glass rod. Ethanol is added to remove water from

the sample, since chloroform immiscible with water and will not

effectively extract the carotenoids until the water is removed.

2. Filter to separate ethanol from the plant sample through a small

funnel. Press out all the liquid of the semi-solid residue collected in

the funnel by pressing it gently with the flat side of a spatula and discard the solvent. All

glassware from this point must be dry.

3. Return the solid residue, with or without any adhering filter paper, to a

round-bottom flask, then add 15 ml of chloroform. The flask is fitted

with a condenser and refluxed at 40°C for 4 minutes. This will extract

the β-carotene and lycopene from the plant. The yellow or red extract is

separated from the solid residue by decantation, and then the solid

residue is returned to the flask.

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4. Repeat the extraction procedure two more times with 15 ml chloroform each time. Combine

the organic solvent extracts in a flask and discard the plants.

5. Pour the combined organic solvent extracts into a separation funnel; add

20 ml of saturated sodium chloride solution (aid in layer separation).

Invert the funnel and shake vigorously for 2 minutes to extract carotenoids.

Release the pressure by opening the lid occasionally.

6. Replace the funnel in the stand and remove the lid. Allow the layers to

separate and then slowly drain the colored lower layer (Chloroform is more dense than water)

through a funnel which has a cotton with 4 spatula tips of anhydrous sodium sulfate on top

of it. This removes any water from the colored extract.

7. Evaporate the colored extract on a hot plate under the hood until

about 1 ml of solvent remains in beaker. Do not allow the solvent to

completely evaporate.

8. Warp the flask with foil to avoid light oxidation, label it with your name

and place it in a beaker. Store it in the freezer until the next lab.

PART II: Column chromatography

In this part you will use the preparative method of column chromatography to separate two

major fractions of pigments, the carotenoids from extract. Since the different components are

colored differently, the separation is easily followed visually. Many factors can affect the

separation. These include: adsorbent, polarity of the column or solvents, size of the column,

and the rate of flow. The carotenoids separating according to the differences in polarity.

Principle

Column chromatography (CC) is one of the most useful methods in biochemical work in

separation and purification of both solids and liquids. This separation

method involves the same principles as TLC, but can be applied to

separate larger quantities than TLC. The technique of TLC was useful

in determining the type and number of ingredients in the mixture, but

it was not helpful for collecting the separated components. We could

only separate and visualize the spots. If we needed to collect the

separated materials, column chromatography could be used. Column

chromatography allows us to separate and collect the compounds

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individually. This type of chromatography has the same method of separation as the thin layer

chromatography but instead of a thin layer, the stationary phase was packed on a vertical

column.The stationary phase or adsorbent is first selected; the most common adsorbents are

silica gel (SiO2) and alumina (Al2O3). Both silica and alumina are polar adsorbents but retain

different molecules. Alumina is basic and retains acidic compounds while silica, less polar than

alumina, is acidic and retains basic compounds.

A fine powder of the adsorbent in the column serves as the stationary phase, and mixtures of

substances in appropriate solvents are applied to the top of the column and passed through the

column, more or less like filtration. Non-polar compounds are going to come off the column

first. Polar compounds are going to move slowly and are going to come off column last because

they will be attracted to the polar column packing by hydrogen bonding or dipole-dipole

attractions.

The solvent (eluent), instead of rising by capillary action up a TLC, flows down through the

column filled with the adsorbent by gravity action or by the application of air pressure.

The column is a piece of cylindrical glass (polyethylene) tubing with a stopcock attached at the

bottom of the column to control the flow of the solvent through the column, or a piece of flexible

tubing is often attached at the bottom of the column, and a screw clamp would be placed on the

flexible tubing at the bottom to stop or regulate the flow of the solvent.

Column chromatography can be small or big, according to the amount of material which needs

to be loaded onto the column. It is often convenient to use burette and Pasteur pipette as a

chromatography column.

Materials and chemicals • Column chromatography

• Beakers

• Glass rod

• Conical flask

• Glass funnel

• Pasteur pipette

• 2 Small vials

• Crude carotenoids (dried pigment)

• Petroleum ether (60-80°C)

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Procedure

A. Preparation of the column

1. Wear gloves and goggles at all times.

2. Place a very small glass wool plug in the bottom of the column with a long glass rod, until

all entrapped air is forced out. The plug is just to keep the solid alumina from entering the

stopcock. The plug must not interfere with the flow of the solvent through the stopcock.

3. Clamp the column to a ring stand in vertical position.

4. Close the stopcock, and fill the column with 6 ml of the eluent.

5. Place a small beaker under the stopcock of the column to catch any solvent that may

accidentally drip out of the tip of your column.

B. Preparation of the slurry

These include dry packing and the slurry method. The slurry method normally achieves the best

packing results. Prepare slurry (mixture of a solvent and an un-dissolved solid) by pouring dry

alumina slowly into a beaker of double volume of petroleum ether, a little at a time while

swirling to form thick but flowing slurry. The addition of adsorbent to solvent should be

followed strictly because the adsorbent will solvate and liberate heat. If the solvent is added to

the adsorbent, it may boil away almost as far as it is added due to heat evolved, especially if

ether or another low boiling solvent is used.

C. Packing of column

1. Quickly and carefully pour the slurry (10-12 cm) into the top of a

clean and dry column with a glass funnel. Stir by glass rod and pour

immediately to maximize the amount of alumina that goes into the

column instead of remaining behind in the beaker. Gently tape the

column constantly by tubing or rubber stopper on the side during the

pouring of slurry to help the alumina settle uniformly, compact, and

to remove air bubbles inside the column.

2. Place a beaker under the column outlet, open the screw clamp, and

allow the solvent to drain into it. Add more solvent as necessary. The

solvent collected prior to the sample can be re-used.

3. Use a plastic pipette to rinse any alumina that is sticking to the sides

of the column. Allow the alumina to settle while eluent continues to

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drip into the beaker. Once the alumina has settled, carefully add about 1cm of sand to the top

of the column to prevent it from being disturbed when fresh solvent is added.

4. When finish packing, drain the excess solvent until it just reaches the top level of alumina

(about 0.5 ml). Close the screw clamp. Your column is now “packed”.

5. Never let the solvent drop below the top of the adsorbent to prevent the column from going

dry out as the column progresses. Cracks will form within the alumina column if it dries, and

compounds can fall down the cracks instead of partitioning between mobile and stationary

phases. The success of separation will be dependent on how well you pack the column.

D. Adding the sample (pigment)

5. The sample is first dissolved in a very small amount of the solvent. It is important to use a

minimum amount (1-1.5 ml) of solvent to dissolve the pigments. A thin horizontal band of

sample is best for an optimal separation. If the extract has dried out, re-dissolve it in 1ml of the

solvent.

6. Place a beaker under the column then open the clamp at the tip of the column allowing the

solvent to drain just to the top of the column 1 mm above the surface of the column.

7. Add about 1 ml (and save the remaining for the thin layer chromatography later) of the crude

plant pigment extract drop wise to the top of the sand using a long Pasteur pipette being careful

not to disturb the top of the column. Allow the sample to adsorb onto the top of the alumina

before adding more eluting solvent.

E. Sample collection

8. Carefully add solvent to the top of the column. To avoid disturbing the top of the column,

it’s a good idea to carefully pipette an inch or two of solvent onto the column instead of pouring

solvent directly onto the column. If the sample is colored, and the fresh layer of solvent acquires

this color, add small amount of eluting solvent until the solvent above the top of the column is

colorless.

9. Fill the column to the top with solvent. Continue adding solvent at the top.

6. The components of the sample (pigment) begin to move down the column and separate

according to their polarity. The yellow beta-carotene (less polar) moves rapidly through the

column, while the red lycopene (polar) moves slowly.

5. Collect solvent in a beaker until you see the band of the yellow β-carotene is just above the

stopcock of the column. Change the collecting beaker and begin collecting the yellow fraction

in the first vial.

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6. Switch collection vial and allow the solvent to run down to just the top

of the column and then add second solvent mixture of 1:5 petroleum

ether/chloroform to elute the orange/red lycopene band. Increasingly

more polar solvents will be used to elute the various components from

the column.

7. Switch beaker again when there appears to be a color change and begin

to collect the orange band in the second vial when it reaches the middle to the lower half of the

column to minimizes contamination of the lycopene with β-carotene.

8. The process is discontinued when the band(s) desired is (are) off the column. Save the

collected fractions for TLC analysis and UV-Vis spectrum. If the compounds to be separated

from column chromatography are colorless, small equal size fractions of the eluent are collected

sequentially in labeled vials and the composition of each fraction is analyzed by thin layer

chromatography.

Petrolum ether

Alumina

Stopcock

Tomato sample

Glass wool

Clamp

20

PART III: TLC analysis of sample fractions

In this part you will use the analytical method of thin layer chromatography to estimate the

number of pigments in the extract and the purity of the separation.

Applying the sample

Procedure

1. Using capillary tubes, spot the solutions to be analyzed on the silica gel side of the TLC plate

along the pencil line. One spot should be from the yellow fraction β-carotene from the column.

One spot should be from the red fraction lycopene from the column. One spot should be from

the colored crude extract that was not ever put on the column. The spots should be very small

in diameter, but clearly visible. If they are not visible, place enough of each sample on the plate.

3. Follow the steps as in experiment 1 page 13.

4. Immediately circle the visible spots with pencil since the colors may fade over time.

5. Estimate the presence of β-carotene and lycopene in the sample through the calculation of

the Rf values. β-carotene carried the highest Rf values. Lycopene had the lowest values.

PART IV: UV/Vis spectroscopy

In this part you will explain the colors of pigment fractions by measuring the absorption of

electromagnetic spectrum of each, using UV-visible spectrophotometer. Also you will correlate

the results of the two types of chromatography with your spectral data.

21

Introduction

Most biological compounds in water are colorless and undetectable to the human eye. To test

for their presence we must find a way to “see” them. We can study colorless compounds in two

different ways. One is that they can often react with other substances to form colored derivatives

that we can see and measure. Another is that colorless compounds usually absorb light in the

region of the spectrum that is not visible to the naked eye. Absorption of this light can be

measured, even if we cannot observe it unaided.

Colormetry is "the measurement of color" and a colorimetric method is "any technique used to

evaluate an unknown color in reference to known colors". Two commonly used types of color

measurement equipment are colorimeters and spectrophotometers. A colorimeter or

spectrophotometer can be used to measure the amount of colored light absorbed by a test sample

that is itself colored or can be reacted to produce a color by measuring the amount of light of a

given wavelength that is transmitted by a sample in reference to a colorless sample (blank). The

most important idea in measurement of color is that the intensity of the color from the reaction

is directly proportional to the concentration of the substance being tested and the absorbance of

the substance is proportional to its concentration.

Light and Electromagnetic spectrum (EMS)

The full range of wavelengths of light is called the electromagnetic spectrum. The

electromagnetic spectrum includes gamma rays, X-rays, ultraviolet, visible, infrared,

microwaves, and radio waves. The only difference between these different types of radiation is

their wavelength or frequency.

Sunlight is a portion of the electromagnetic spectrum given off by the sun, particularly infrared,

visible, and ultraviolet.

22

Visible light

Or white light is most of the light that the human eye can only see ranging from 400nm–700nm.

It is a very small part of the electromagnetic spectrum. It is primarily made up of seven different

colors (or wavelength) of rainbow: red, orange, yellow, green, blue, indigo, and violet. It is

most important energy source which travels through space as wave. It is the only energy we can

see it in the form of color.

White light decomposed into its component colors by a prism or grating produces the color

spectrum. Each color of these has its own wavelength (each light color is defined by its

wavelength). The reddish color is the longest wavelength (lower energy). The greenish is the

mid-size, while the violet has the shortest wavelength (higher energy). Humans are especially

sensitive to wavelength around 655 nm (red).

Invisible light

Infrared light (IR) is long wavelength (700nm-900nnm) before red in the visible spectrum.

Infrared can be felt, but it cannot be seen by the naked eye but we can feel it as heat. Everything

emits infrared light as heat. Because of this, movement can be detected in the dark with infrared

detectors.

Ultraviolet light (UV) is short wavelength of light (200nm-400nm) beyond violet in the visible

spectrum. Most are reflected by the ozone layer, but some do get into our atmosphere. These

rays damage unprotected skin and have been known to cause skin cancer.

Color

Color is the visual effect that is caused by the spectral composition of the light emitted,

transmitted, or reflected by objects. A color solution appears colored because molecules in the

solution absorb light of particular colors (wavelengths) and transmits light of the wavelengths

that it does not absorb. The particular wavelengths of light that a given solution absorbs

determine the color we perceive. The perceived color (not absorbed) has a complementary

relationship with the color of the light absorbed. The wavelengths of light that are not absorbed

are transmitted or reflected completely to our eyes. If something absorbs all the colors, it

23

appears black, if it reflects everything, it appears white. Many substances appear to be colorless

to the eye since they absorb only UV and IR. A color wheel can be used to predict the

appearance of the sample. A color wheel, illustrates the approximate complementary

relationship between the wavelengths of light absorbed and the wavelengths transmitted or

reflected. A color has the greatest absorbance in its complementary color range. In general, if

one color of light is absorbed, the sample will appear to be the color on the wheel opposite to

the one absorbed.

For example, Tomatoes appear to be Red because contain a carotenoid known as "Lycopene".

Lycopene absorbs most colors of the visible light that are not red and reflects mainly red back

to the human eye, thus tomato appears to be Red. We see the complementary color (the opposite

color on the wheel) to the color absorbed.

Wavelength

Wavelength is the distance between two peaks of the light wave which determines its color. It

is commonly designated by the Greek letter Lambda (λ). Common units of measurement are

angstroms and nanometers (nm). One nanometer equals one billionth of a meter, or microns.

The choice of the correct wavelength for testing is important. It is interesting to note that the

wavelength that gives the most sensitivity for a test factor is the complementary color of the

test sample. Different wavelengths of light appear to our eyes as different colors, as shown

beside.

24

Experiment 3

Determination of wavelength of maximum absorbance of carotenoids

Principle

The purpose of this experiment is to find the wavelength of maximum absorbance (Lambda

max, λmax) for any colored solution. It is the wavelength of light at which absorbance are

greatest. There may be more than one λmax for a compound. The value of λmax is characteristic

of sample and may be used as an aid in its identification. It is the preferred wavelength to use

for running a calibration curve. The absorbance over the full range of wavelengths constitutes

the absorption spectrum, a plot of absorbance (on the y-axis) as a function of wavelength (on

the x-axis). It is characterized by the wavelength λmax (the highest portion of the curve). The

shape of the spectrum and the wavelength of maximum absorbance are characteristic of the

chemical compound. The absorbance of compounds is also directly related to the concentration

of the sample. You will use a spectrophotometer to do UV-Vis spectroscopy on the extract

(from tomatoes & carrots) and the fractions that you collected during the column

chromatography.

Lycopene and β-carotene pigments may be identified from their absorption spectra. They

absorb light in the 400-500 nm region of the visible spectrum. The process involves recording

the absorbance over the range of 400 nm to 500 nm, usually in intervals of 10 nm.

Make sure a blank is run first. Record the spectrum of your sample from 400-500 nm. Determine

the λmax of any peaks. There are actually several absorption peaks in the electronic spectrum of

lycopene, but the peak of maximum intensity corresponds to a wavelength near 473 nm. β-

carotene behaves similarly on irradiation, but the peak of maximum absorption is found at

shorter wavelengths, around 448 nm.

Material and chemicals

• Spectrophotometer

• Two Cuvettes

• Dropper

• Lycopene

• β – carotene

• Extract of the pigment

25

Procedure

1. Obtain two cuvettes that have been cleaned inside and outside. One will serve as blank (or

reference) and should be filled with petroleum ether (or whatever solvent is used for the

colored compound). The blank must be used to adjust the spectrophotometer every time the

wavelength is changed. If you are working with a single wavelength, the blank should be

placed in the spectrophotometer periodically to check for drift.

You will need a petroleum ether solution of carotene into the glass cuvettes. Do not use an

ordinary test tube for your measurement. Care should be taken not to drop the cuvettes.

Note: It is important to use matched cuvettes, to check this, place at least 2.0 ml of water (or

whatever liquid in the reference cuvette) to the other cuvette (called the sample cuvette) and

read the absorbance. If it is identical to that of the first cuvette (within 0.003 absorbance

units). If not, get two that read the same.

2. Rinse the sample cuvette with a small amount of colored solution, and then fill it 3/4 full.

3. Turn on the spectrophotometer and warm up 15 min. Make sure to set the correct wavelength.

Without a tube in the sample compartment set the mode to transmittance, rotate the front

left knob until the digital display reads 0.00%T.

4. Wipe the blank cuvette with a Kim wipe. Insert the blank into the sample compartment

making sure the line of the cuvette matches the mark on the sample holder, and then close

the lid. Push the mode button changing the readout to absorbance. Rotate the right knob

until the digital display reads 0.00.

5. Remove the blank, wipe the sample cuvette, and insert the colored solution.

6. Record the absorbance at 10 nm intervals from 400 nm to 700 nm. Remember to zero the

spectrophotometer with the blank each time you change the wavelength. The absorbencies

over the full range of wavelengths constitute the absorption spectrum.

7. All of your data must be taken on the same instrument, at the same time. If you need to

change instrument, you must start over and take all of the data for the spectrum again.

8. Record the data in the table in your result sheet and answer the questions.

9. Graph the data to generate absorption spectrum. From this absorption curve you will

determine a λmax for the carotene and from the color wheel you will to obtained their color.

26

Introduction

Size Exclusion Chromatography (SEC), also called gel filtration Chromatography (GFC) or Gel

Permeation Chromatography (GPC) is a form of column chromatography in which molecules

are separated on the basis of their molecular mass as they pass through a column packed with

the stationary phase which is a gel (cross-linked polymer,sephadex) by collecting fractions as

the mobile phase is eluted through the column, with the largest molecules eluting first and the

smallest last.

Sephadex is packed into a column to form a packed bed. The packed bed is equilibrated with

solvent which fills the pores of the matrix and the space between the particles. The liquid inside

the pores sometimes referred to as stationary phase and this liquid is in equilibrium with the

liquid outside the particles, referred to as mobile phase.

The sample is applied in a narrow band at the top of the column and then it is washed through

the column by the mobile phase. Large molecules which cannot enter the sephadex pores are

excluded from the beads and are restricted to move around the beads in space between them.

They elute from the column faster than the smaller molecules after an amount of the mobile

phase equal to (Vo) has passed through the column. A smaller molecule that have full access to

the pores move down the column and elute by (Vt ) of solvent have passed through the column.

Intermediate size molecule that are small enough to pass through some of the pores of the

beads, however, elute from the column in order of decreasing size by various volumes (Ve),

depending on how small they are and what fraction of the pores of the beads are accessible to

them. Small molecule which may penetrate the beads pores taking long time to elute from the

column.

27

Gel filtration works opposite to filtration. It is reliable and simple, little equipment is required,

the procedures are straight forward and good separation and yields are usually obtained.

It is possible to calculate a partition coefficient Kd. For a solute Kd is the extent to which the

molecules can penetrate the pores in stationary phase, which values ranging between 0 and 1.

Kd = Ve – Vo

Vi

If the Kd is greater than 1, then adsorption of the compound on the gel has occurred.

However, because it is difficult to measure Vi precisely, the equation may be modified to

determine available part of the resin (Kav):

Kav = Ve – Vo

Vt – Vo

Where Vt is the total volume of the column (Vt = πr².h).

In order to separate easily two solutes, their values for (Kav) must be significantly different from

each other.

Gel filtration can also be used to determine the molecular weight of a molecule. A calibration

curve has been obtained by plotting a partition coefficient Kav for each standard against the log

of the molecular weight for a set of standard molecules. A linear curve is obtained. Calculate

the corresponding Kav for the unknown and determined its molecular weight from the

calibration curve.

28

Experiment 4

Separation a mixture of blue dextran and cobalt chloride molecules

Principle

A mixture of Blue dextran and Cobalt chloride molecules is applied to a column which contains

sephadex G-25. Blue dextran larger than the largest pores of the gel cannot enter the pores and

therefore passes rapidly down the column between the beads or is “completely excluded” from

the beads, whereas cobalt chloride can access most or all pores. Thus, blue dextran move fast

and elute first, cobalt chloride elute later. The path of cobalt chloride through the gel bed is

much longer than that of blue dextran because they move in and out of the pores of the gel (they

are included).

Both molecules, Blue dextran & CoCl2.6H2O, are colored so the progress of the filtration can

be followed by observing the separation of the colored bands. The eluent is collected in constant

volumes, known as fractions. The collected fractions

analyzed by measuring the extinction of each fraction at 625

nm, λmax of blue dextran and 510 nm, λmax of CoCl2.6H2O.

Sephadex G-25 (the trade name) is one of eight different G-

types ranging from G-10 for small molecules to G-100 for

large molecules. It is most commonly used for gel-filtration

column. It is cross-linked dextran polymer of glucose

residues, semi-permeable, inert (lack of reactivity and adsorptive properties), porous beads with

well-defined range of pore sizes. Degree of cross linking is controlled carefully to yield

different pore sizes. The different types of sephadex vary in their degree of cross-linking and

hence in their degree of swollen and selectivity for specific molecular size. Sephadex G-25

excludes all molecules with a molecular weight greater than 5000, thereby eluting them first. It

is bought as dry beads, the beads swell when water is added.

Blue dextran is a high molecular weight glucose polymer (2,000,000 Daltons) larger than the

exclusion limit of the gel cannot get into the beads because of its large size and therefore

completely excluded from the gel (Kd=0), and eluted when a volume of solvent equal to the

void volume Vo (i.e. volume of liquid between the beads required to exclude "Blue Dextran").

Blue dextran is often used as a marker to determining void volume.

Cobalt chloride, with a low molecular weight (237.93 g) is freely accessible to the gel particles

(Kd=1), and elute at a volume equal to the total volume of solvent Vt, or the sum of Vo and Vi.

29

The elution volume is the volume of liquid required to elute the molecule from the column. The

elution volume of a molecule is, of course, dependent on its size.

Materials and chemicals

• Column chromatography

• Spectrophotometer

• Fifteen test tubes

• Glass rod

• Glass funnel

• Pasteur pipette

• Beaker

• Stand with burette clamp

• Sephadex G25 (excludes all molecules with a molecular weight greater than 5000, thereby

eluting them first).

• Blue dextran in saline

• Cobalt chloride in saline

• Sodium chloride (0.9 %), (Saline)

Procedure

1. Number fifteen small test tubes 1-15. Arrange them in order on a rack.

2. Prepare the gel bead column:

a. Clamp the column vertically.

b. Column (About 12 cm in hight) is filled with semisolid (Swollen) gel beads of Sephadex

G-25 [Note: the Sephadex gel has already been soaking in the elution buffer for 3 to 4

hours]. Be gentle; do not allow gaps or bubbles to form. It may help the beads to settle

by flow a small amount of saline through the column between additions of beads.

c. Equilibrate the column with saline by passing about 10 ml of saline through the column

beads after it has completely settled.

d. Avoid stirring up the top of the column bead when adding saline or samples, as this will

give poor resolutions of the samples.

3. Carefully add the sample mixture containing blue dextran and CoCl2.6H2O to the top of the

column using a pasture pipette. Again, do not stir up the top of the gel. Allow saline solution

to drain down so there is only about 1 mm of buffer above the gel before layering the sample

mixture carefully on top of the gel. Turn off the stopcock to allow the mixture to enter the

gel beads. As soon as it does, continue to add saline solution to the top, filling the space at

the top of the column.

4. Collect the fractions, 3 ml per tube, beginning with tube number “1” Early fractions contain

large molecules while later fractions contain smaller ones. Check to see if there is any color

in each fraction. Be sure to collect all fractions.

30

5. Measure the absorbance spectrum of samples in order to identify each molecule (Blue

dextran at 625 nm, CoCl2.6H2O at 510 nm).

6. Clean up the column as follows: Gently invert and shake the beads into the recycle beaker

for further use. Be patient. Some beads will come out the end. Then you can add some buffer

solution and shake. The rest of the beads will eventually come out. Try slipping a pipette

bulb over the thin end of the tube and onto the body. Try squeezing the bulb to back flush

the beads out. Clean and return your pipette.

7. Wash your test tubes. Line the tubes back up for the rest separation.

Separation a mixture of blue dextran and cobalt chloride through a gel filtration column.

31

Introduction

Titration curve (pH curve) is obtained when pH of given volume of a sample varies after

successive addition of acid or alkali. The curve is usually plotted of pH against the volume of

titrant added. Titration curves (acid-base titration) are very useful for identification of amino

acids.

Using a titration curve, the pI can be empirically determined as the inflection point between the

pK's of the anionic and cationic forms. The pK's value for each dissociable group of an amino

acid can be determined from such a titration curve by extrapolating the midpoint of each

buffering region (the middle flat part of the curve) where the pH changes only slightly upon

addition of either acid or base. The diagram shows that there is a point in the curve where the

amino acid behaves as a "neutral" salt. At this pH, the amino acid is predominantly zwitterions

with a net charge of zero. This point of the titration curve is the isoelectric point, pI and can be

approximated as halfway between the two points of strongest buffering capacity.

Mathematically, the pI can be determined by taking the average of the pK's for the anionic and

cationic forms.

pI = 1/2 ( pKa + pKb).

Where Ka and Kb are the dissociation constants of the carboxyl and amino groups respectively.

32

Experiment 5

Determination of the isoelectric point of glycine

Principle

Amino acids are the building blocks of proteins. All of the amino acids found in proteins are

classified as ampholytes substances because they contain a carboxyl group (-COOH) that acts

as an acid and an amino group (-NH2) that acts as a base, i.e. when they are titrated with acid,

it act as a base, and with base, it act as an acid.

Amino acid as bases:

In strongly acidic conditions a positive ion forms:

An amino acid behaves as a base and the (-COO-) ion gains a proton.

Amino acids as acids:

In strongly alkaline conditions a negative ion forms:

An amino acid behaves as an acid and the (-NH+) ion loses a proton.

At neutral pH amino acids such as glycine are almost always present as Zwitterions; the

dominate forms of amino acids. Also known as dipolar ion. It is a neutral molecule has both

positive (the protonated amine group) and a negative (the deprotonated carboxyl group)

electrical charge. The net charge of this molecule equals zero. The pH at which the amino acid

exists as Zwitterion is called isoelectric point pI (isoelectric pH, IEP). The isoelectronic point is

the pH at which the amino acid has no net charge. Each amino acid has a different pI.

33

If acid is added to a solution containing the zwitterion, the carboxylic group captures a hydrogen

(H+) ion, and the amino acid becomes positively charged. If base is added, removal of the H+ ion

from the amino group of the zwitterion produces a negatively charged amino acid.

(Acidic medium) (Basic medium)

Hydrogen acceptor Zwitterion Hydrogen donor

Positive ion (cation) (neutral) Negative ion (anion)

At a pH below the pI proteins carry a net positive charge. Above the pI they carry a net negative

charge. At pI the solubility is often minimal and the protein will accumulate (precipitate) and it

will not migrate in an electrical field at all. the electrolytic mobility is minimum. This is also

true for individual amino acids.

Due to a preponderance of weakly acid residues in almost all proteins, they are nearly all

negatively charged at neutral pH. We can use pI in separation method as ion exchange

chromatography (choose stationary phase) and protein electrophoresis (choose buffer pH).

In this experiment, you will identify an unknown amino acid through acid-base titration

(titration curve).

As an example for glycine, a simple amino acid has two

dissociation steps corresponding to loss of H+ from the

acidic carboxyl group at low pH followed by l oss of H+

from the more basic amino group at high pH. Comparisons

between experimental and literature pKa values can allow

the identification of a specific amino acid.

Materials and Chemicals

• pH meter and standard buffer solutions

• Magnetic stirrer and bar

• Graduated burette and holder

• Beaker (400 ml)

• Graduated cylinder (20 ml)

• Distilled water

• NaOH solution (0.5 N)

34

• H2SO4 solution (0.25 N)

• Amino acid (Glycine)

Procedure

1. Use the electronic magnetic stirrer device. Place a magnetic

bar into 400 ml beaker, and turn on the stirrer so that the bar

turns at a moderate speed. Turn off the stirrer.

2. Place approximately 20 ml of your selected amino acid

solution into the beaker. Assemble the titration stand,

graduated burette, and pH meter so the electrode is

submerged into the amino acid solution and the burette is positioned to drop solution into

the beaker. Be sure the electrode is clear of the magnetic stir bar.

3. Record the initial pH of the amino acid solution and the initial reading of your burette.

4. Turn on the stirrer, check that the magnetic bar does not collide with the electrodes.

5. Fill the titration burette with about 10 ml of 0.5 N NaOH solution. It is recommended that

you allow the first portion of NaOH solution to run through the burette to clear the bottom

constriction from residual distilled water (Discard this volume).

6. Record the starting volume reading on the burette, and then carefully titrate the amino acid

solution by drop wise addition of the 0.5 N NaOH.

7. Record the volume of added NaOH and pH of the solution throughout the titration until a pH

12 is reached. You do not have to take a reading for each pH unit shown, but try not to skip

more than two units. Turn the stirrer off each time before reading the pH.

8. Repeat the titration (Step 5 to 7) with 0.25 N of H2SO4 until a pH 1.5 is reached.

Note: Use care in adding the H2SO4 and NaOH, as the pH may change slowly or rapidly

depending on the pH and buffering range of the ionizable group(s). In the beginning, the pH

will change very rapidly with each addition of titrant. As you get closer to the pKa of the

ionizable group (near the equivalence point) a single drop may change the pH much more

slowly. When this phenomenon occurs, it is strongly recommended that you proceed drop

by drop.

35

Introduction

The movement of molecules through a cell membrane is termed osmosis or diffusion. Diffusion

is the movement of molecules (solute or particles). Osmosis is the movement of water

molecules. Such movement is principally possible because nutritive molecules are smaller than

membrane micro pores. If the molecules are too large, no molecular transfer, or diffusion occur.

Through dialysis, molecules will separate according to their size by use a semi permeable

membrane that transmit selectively taking into consideration the concentration differences of

the two solutions on either side of the membrane. This porous membrane selectively allows

smaller solutes to diffuse from a high concentration solution to a low concentration solution

across the semi-permeable membrane until equilibrium is reached while the larger molecules

are retaining. This process is affected by the variables of temperature, viscosity, and pressure

gradient across the membrane. Also it is affected by the solvent, the nature of the membrane

and the size of pores or molecular weight cut-off (MWCO). Only the molecules or ions smaller

than MWCO will move out of the membrane. The rate of dialysis is greatest in distilled water.

Synthetic and natural membranes are commonly used. In the following experiment, cellophane

dialysis tubing serves as an excellent representation of the cell membrane. Cellophane is a thin

transparent sheet made of regenerated cellulose (manufactured by the conversion of

natural cellulose to a soluble cellulosic derivative). It is the most commonly used as dialysis

material. The regenerated cellulose is derived from cotton (cotton fibers are composed of

mostly α-cellulose). It has a symmetric pore structure. The membrane contains traces of

sulpher compounds, metal ions, and some enzymes.

Dialysis is best carried out with freshly prepared tubing since, once it is wet, it becomes very

susceptible to attack by micro-organisms. If it has to be stored, then the tubing is best kept with

a trace of benzoic acid in the solution.

36

Experiment 6

A- Separation of a mixture of starch and maltose

Principle

Starch consists of two large molecules:

amylose of molecular weight 50,000

and amylopectin of molecular weight

1,000,000, neither of which will pass

through a dialysis membrane. Salivary

amylase converts the starch to maltose,

molecular weight 360, which diffuses

out the membrane. A solution of our

sample (starch and salivary amylase) is

placed into a dialysis bag such as cellophane and the bag is sealed by knotting it at both ends.

The sealed bag is then immersed in a relatively large volume of the solvent (distilled water).

Maltose that is smaller than the pores of membrane (MWCO) allow moving freely across out

of the membrane into the solvent. Starch is greater than the pore diameter and will retain inside

the dialysis bag.

Materials and chemicals

• Test tube rack to hold 16 small test tubes

• Two beakers

• Magnetic stirrer and bar

• Cellophane tubing

• Dropper, Pipette (2 ml)

• Hot plate

• Salivary amylase (1 ml saliva diluted to 5 ml with distilled water)

• Iodine solution (5 mmol/L in 30 g/L Potassium iodide)

• Soluble starch (20 g/L)

• Sodium chloride solution (1g/L) buffered with 0.02 mol/L Sodium phosphate pH 6.8).

• Fehling’s solution.

Preparing of the dialysis tubing

1. A suitable size and length of dialysis tubing is selected (about 8 cm). Always handle the bag

with gloves.

2. Boil the bag for 30 min in alkaline EDTA (Na2Co3, 10 g/L: EDTA, 1mmol/L) to avoid the

loss of activity of molecules dialyzed and to make the dialysis sac soft. After boiling the

tubing is washed with distilled water.

37

3. Soak the bag in water. Never let the dialysis bag dry out once it has been wetted.

Procedure

1. Label sixteen small test tubes 1-16. Number test tubes 1 S through 8 S - and 1 M through 8

M (S is for starch an M is for maltose). Arrange them in order on a rack.

2. In test tubes 1 M through 8 M place one ml of Fehling’s solution (Test for maltose).

3. Place one ml of iodine solution into each test tube numbered 1 S through 8 S (Test for starch).

4. Seal one end of the tubing with a string. Carefully fill the sac with the reaction mixture by

pipetting 2.5 ml starch, 0.5 ml phosphate buffer and then 0.5 ml salivary amylase.

5. At 0 min, quickly, pipette 1.5 ml of the reaction mixture (From inside the bag). Add 0.5 ml

to tube # 1 S and 1 ml to tube # 1 M. Heat test tube # 1 M in a boiling water bath for 5min.

Observe result for starch identification (Blue color). Observe result for maltose identification

(Red ppt).

6. Tie the second end of the tubing. Immerse the tubing in the 400 ml beaker containing 100

ml of the distilled water, the solvent. Place the beaker on a stir plate and stir in the cold for

at least 90 min (it takes about 2 hours for dialysis to be completed). Stirring help in the entry

of the water inside the sac and help in getting out of small molecules out of the sac.

7. Pipette 1.5 ml from the solvent (outside) every 15 min interval during the 90 min. Examine

each fraction (7 fractions) for the presence of starch or maltose as in step 5.

8. At the end of the experiment, cut the dialysis sac, carefully remove the dialyzed from the sac

with a pipette and squeeze out as much as possible of whatever is left into the test tubes # 8S

(0.5 ml) and # 8 M (1ml). Examine the dialyzed (Inside the sac) for the presence of starch or

maltose as in step 5.

Incomplete separation Incomplete hydrolysis Complete hydrolysis

38

Experiment 6

B-Separation of a mixture of starch and glucose

Materials and chemicals

• Glucose Solution

• Starch Solution

• Iodine Solution

• Glucose Test Strips

• Dialysis Tubing

• String & Scissors

• 400 ml Beaker

• 10 ml Graduated Cylinder

Procedure

1. Fill the 400 ml beaker ¾ full with tap water. Add 10 drops of Iodine solution and stir well

(what the color of the water). Iodine will turn blue-black in the presence of starch.

2. Test the solution in the beaker for the presence of glucose by dipping a glucose test strip

into it. After 30 seconds, compare the color on the strip to the color chart on the side of the

bottle.

3. Remove the tubing from the water and rub one of the ends between your thumb and pointer

finger to open. Once open, submerge it in water again for about thirty seconds.

4. Seal one end of the tubing with a string. Carefully pour into the tubing 5 ml of starch

solution and 5 ml of glucose solution as well, using the 10 ml graduated cylinder. Tie the

second end of the tubing.

5. Immerse the tubing in the 400 ml beaker of the solvent and allow it to remain undisturbed

for 15-20 minutes. Remove the tubing from the beaker.

6. Retest the solution in the beaker and in the bag with a new glucose test strip.

7. Test the solution in the beaker and in the bag for the presence of starch by notice any

change in the color of both solutions.

8. Record your observations in the data table for Initial and final status information.

39

Sedimentation

The motion of molecules in solutions or particles in solutions in response to an external force

as gravity, centrifugal force or electric force. The particles sediment at different rates depending

on:

• Properties of molecules (size, shape, density)

• Properties of solvent or gradient material (density, viscosity, temperature)

• Interaction between the solute molecules and the solvent gradient material.

Centrifugation

Centrifugation is one of the most important and widely applied techniques that used to separate

immiscible liquids and solids from liquids by artificially controlling the gravity of a solution.

Due to gravity many particles in solution will settle at the bottom of a container. Other particles

extremely small in size (very light) will not separate unless subjected to high centrifugal force

(measured as Xg gravity) provided by a centrifuge. By putting the solution in a centrifuge and

rotating particles at a high rate of speed. This creates a centrifugal force and very light particles

will fall out and form a pellet at the bottom of the tube.

Relative centrifugal force

Relative centrifugal force (RCF) is the measurement of the force applied to a sample within a

centrifuge. This can be calculated using the following calculation.

g = RCF = 0.00001118 r (rpm)2, where:

g = Relative Centrifuge Force

r = rotational radius (centimeters, cm)

rpm = rotating speed (revolutions per minute, r/min)

An RCF of 500Xg indicates that the centrifugal force applied is 500 times greater than earth

gravitational force.

Common example of the use of centrifugal force

1. In a washing machine, it is centrifugal force generated in the spin cycle.

2. To separate cream from whole milk.

3. To separate uranium 235, to produce nuclear energy.

40

Centrifuge

It is an apparatus that rotates at high speed and by centrifugal force

separates substances of different densities. The materials with the highest

density travel towards the bottom of the centrifuge tube at a higher rate

of speed than they would under the force of normal gravity.

Centrifuge can be considered devices for increasing the effects of the

earth's gravitational pull to quicken the precipitate of substances to the

bottom. The material is placed in a centrifuge tube which is then placed

in rotor. It is separates into pellet (precipitate) and supernatant or

supernatant (The clarified liquid above the pellet). The pellet (denser

materials) is settled at bottom of the centrifuge tube. The supernatant

(liquid) is then either quickly decanted from the tube without disturbing

the precipitate, or withdrawn with a Pasteur pipette.

Types of centrifuges

The major distinguishing features between centrifuge types are speed and capacity.

According to speed there are:

• Low-speed centrifuge (maximum speed 5000 rpm)

• High-speed centrifuge (speed up to about 25,000 rpm.

• Ultracentrifuge (speed up to 70,000 rpm). Speeds up to 100,000 rpm are available on

typical modern versions. At such speeds, the centrifuge chamber must be evacuated and

refrigerated to counter the heat generated as a result of friction.

There are different types of laboratory centrifuges:

• Micro centrifuges (devices for small tubes, micro tubes, from 0.2 ml to 2.0 ml). They

meet all modern requirements of various types of labs for routine sample preparations.

• Clinical centrifuges (moderate-speed devices used for clinical applications like blood

collection tubes)

• Multipurpose high-speed centrifuges

Types of rotors

There are three types of rotors, the rotating unit of the centrifuge, fixed angle, swinging-

bucket, and vertical rotors. Fixed-angle and swinging-bucket rotors are the most common

styles for bench top, low speed, and high-speed floor-model centrifuge applications. Vertical

rotors are used primarily in ultracentrifugation.

Supernatant

41

Experiment 7

Effect of salt on extractability of proteins

Principle

The purpose of this lab is to know how proteins can be separated from a tissue homogenate into

different fractions by centrifugation. The process of centrifugation is based on the fact that

proteins have differences in size, shape and density. At each step of centrifugation, more dense

particles are separated from less dense particles. In biological systems proteins exist in a variety

of forms; some are in free soluble form and some are bound to nucleic acids, lipid or sugar.

Each type has to be extracted differently.

You will also need to measure the concentration of protein in each fraction by taking a sample

of the original homogenate and the supernatants. The procedure you will use to determine

protein concentration is the Biuret method. It permits the determination of protein

concentration by measuring the intensity of the purple color at 540 nm.

Materials and chemicals

• Centrifuge

• Balance

• Spectrophotometer

• Water bath (37o C)

• Mortar and pestle

• Filter paper & Muslin

• Measuring cylinder, Glass funnel & Beaker

• Centrifuge tube, test tube & Cuvette

• Dropper & Pipette

• Cold distilled water

• Liver tissue

• Biuret reagent

• NaCl )0.2 M(, NaCl (1.0 M), &NaCl + triton X (1.0 M)

42

Procedure

A. Preparation of homogenate

Prepare a homogenate of a biological tissue (liver) as follows:

1. Weigh 5 gm of liver. Dry it by folding between folds

of filter paper sheet and drop it in mortar.

2. Using a scissors, mince it into small pieces. Add 10

ml of cold distilled water and grind well by pestle

until a uniform paste is obtained.

3. Filter the homogenate through 2 or 3 layers of muslin

into the measuring cylinder to remove any lumps that

may be present. Rinse the mortar with 5 ml of cold

distilled water and combine it in the measuring

cylinder with the original homogenate.

4. Complete the volume to 20 ml with cold distilled

water (Label it as F0).

5. Pipette 0.1 ml of (F0) into test tube for Biuret assay.

B. Before carrying the procedure

• Remember to take a 0.1 ml samples of each fraction (0 to 4 ).

• Do all the following procedure in the cold, keeping all solutions on ice.

• If a refrigerated centrifuge is not available the centrifuge holders need to precooled to keep

them at or near the operating temperature.

• Carry out the procedure with the homogenate into two centrifuge tubes according to the

diagram in the next page.

43

Procedure

Biuret assay

44

Chemical solutions

Introduction

Chemical solutions is a homogeneous mixtures in which one substance (the solute) is dissolved

in another substance (the solvent), but not chemically combined. A solution may exist in

any phase. The three types of solution are:

1. Gaseous Solution includes gases or vapors dissolved in one another. Two or more gases

can form a solution. Air is an example of a gaseous solution.

2. Liquid Solution contains a liquid solvent in which gas, liquid, or solid is dissolved.

Water is the most common liquid solution. Many things can be dissolved in it. Table

salt is an example of a solid dissolved in a liquid.

In this lab you will deal with liquid solution and you will learn how to prepare it.

3. Solid Solution is a mixture of solids spread equally throughout one another. Metal is

an example of a solid solution at room temperature.

Preparing of a solution is something that is very critical to many applications in science,

medicine, cooking and other fields. Lab experiments often require preparation of chemical

solutions in their procedure. That is, two or more substances are mixed together in known

quantities. This may involve weighing a precise amount of dry material or measuring a precise

amount of liquid. Preparing solutions accurately will improve an experiment's safety and

chances for success. When you are making a solution of given compound, you need to know

what concentration you want, and what volume you want. These are important because they

relate to how much of your compound you need. You will also need to find the molecular

weight of the compound. We look at preparation of these chemical solutions by weight

(w/v) [Mass of solute (g)/Volume of solution (ml)] and by volume (v/v) [Volume of solute

(ml)/Volume of solution (ml)].

Particularly in chemistry, solutions are made using molar concentration or molarity. Molarity

(M ( is one of the most common ways to express the concentration of the solution because they

directly relate the moles of the solute to the volume of solution. A mole of a compound is its

molecular weight in grams. A one molar (1M) solution has one mole of a solute dissolved in

1000 ml of solution (1L), or MW of a solute dissolved in 1000 ml of solution (1L).

45

For example, the molecular weight of NaCl is 58.54. Therefore:

1M solution of NaCl has 58.54 g NaCl dissolved in dH2O with a final volume of 1 liter.

0.1 M solution of NaCl has 5.85 g in 1 liter or 0.585 g in 100 ml or 0.058 g in 10 ml.

0.2 M solution of NaCl has 11.71 g in 1 liter or 1.17 g in 100 ml or 0.117 g in 10 ml.

Concentration in percent means "in a hundred" and by convention, a 10% w/v solution contains

10 grams of a solute in a total volume of 100 ml of the solution.

Meniscus

The meniscus is the upward or downward curve at the upper surface of a liquid in a container,

caused by surface tension. A meniscus may be either concave or convex, depending on the

liquid and the surface. A concave meniscus curves downward; if you are looking down from

the top, it curves away from you. In contrast, a convex meniscus curves upward; if you are

looking down into the container, the meniscus curves toward you.

Most liquids have concave menisci because the molecules of those liquids are more strongly

attracted to the walls of their container than to each other. The liquid climb the walls of the

container instead of lying flat, causing the downward curve. This occurs between water and

glass. Conversely, some liquids, like mercury, have a convex meniscus because the molecules

of the liquid are more strongly attracted to each other than the walls of the container. So instead

of climbing the walls, they pile on top of each other in the middle, pulling away from the sides

of the container.

Meniscus

Incorrect reading

(Too high)

Correct reading

(at eye level)

Incorrect reading

(Too low)

46

Experiment 8

A- Preparation of liquid solutions

1. Preparation of solutions from solid materials

To prepare a solution of a specific concentration by dissolving a solid in a liquid,

you must know the concentration and total volume of the solution you want to make (probably

given to you) so that the specific amount of solute is measured.

In this lab, you will prepare the following solutions:

0.1 M Na2HPO4 and 0.1 M NaH2PO4

Perform the following calculations to determine how many grams of your samples you need to

weigh out to prepare 100.0 ml, of a 0.1 M Na2HPO4 or 0.1 M NaH2PO4.

Calculation

Molarity (M) = No. of moles of solute

Volume of solution (L)

Molarity (M) = Weight (gm) X 1000

Volume (ml) X Molecular weight

Weight (gm) = Molarity (M) X Volume (ml) X Molecular weight

1000

Determine MW using a periodic table by adding the atomic mass of each atom in the

chemical formula.

Procedure

1. Calculate the weight of chemical needed to make 100 ml of solution using the above formula.

2. Take a 100 ml beaker and place it on the balance. Tare the balance (set it to zero). Weigh

out the required amount in gram of Na2HPO4 or NaH2PO4.

3. Add approx. 1/2 the volume of dH2O to the beaker and stir with a glass rod until the solid

is completely dissolved or stirring with magnetic stir.

4. Transfer the solution to 100 ml volumetric flask. Rinse out the beaker and the glass rod with

dH2O. Do this at least twice. Add water to just below the line on the volumetric flask.

5. Add the final drops with a dropper to ensure that the bottom of the meniscus is at the fill

mark. Put the lid on the flask and invert to mix the solution.

6. Label your solution with your name, the concentration, the date, and the contents, e.g.

0.1 M Na2HPO4.

47

2. Preparation of solutions from liquid materials

To prepare a solution of a specific concentration from a liquid, you must know the concentration

and total volume of the solution you want to make (probably given to you) so that the specific

volume of liquid is measured.

In this lab, you will prepare the following solutions

0.2 N HCl

0.05 N H2SO4

Perform the following calculations to determine the specific volume of your assigned samples

to prepare 25 ml of a 0.2 N HCl or 25 ml of a 0.05 N H2SO4.

Note: When preparing a strong acid, always add acid to water (same for a strong base).

Calculation

Normality (liquid) =Percentage x Specific gravity x 1000

Equivalent weight

Equivalent weight = Molecular weight

No. of H+ or OH-

Volume = Normality x Volume x Equivalent weight

Specific gravity x Percentage x 1000

Procedure

1. Calculate the volume of chemical needed to make 25 ml of solution using the above

formula.

2. Add approximately ¾ of the water from the wash bottle to a 25 ml volumetric flask.

3. Measure the specific volume of HCl or H2SO4 with a pipette then transfer it to the flask.

4. Put the lid on the flask and invert several times to mix the solution.

5. Add more water, if necessary, but do not go over the fill mark. Add the final drops with a

dropper to ensure that the bottom of the meniscus is at the fill mark. Put the lid on the flask

an again invert to mix the solution.

6. Label your solution with your name, the concentration, the date, and the contents, e.g. 0.1N

HCl.

48

Buffer solution and pH

Introduction

pH (Potential of hydrogen) describes the amount of H+ cations in a

solution. It measures the acidity and alkalinity of the solution.

Concentration of these ions can change in very wide range (a scale

from 0 to 14). Aqueous solutions at 25ºC with a pH less than 7.00

are acidic and pH greater than 7.00 is alkaline. In neutral solution

such as pure water, the pH is equal 7.00.

The formal definition of pH is the negative logarithm of the

hydrogen ion concentration,

pH = -log[H+].

Thus, it is evident that the pH is inversely proportional to the acidity.

Henderson-Hasselbalch equation can be used for pH calculation of a solution containing pair

of acid and conjugate base.

pH = pKa + log ([A-]/[HA])

[A-] = molar concentration of a conjugate base (M)

[HA] = molar concentration of a weak acid (M)

pKa = the effective ionization constant of the acid Measurement of pH

1. pH indicators

Acid - base titration with a dye; for example, phenolphthalein change

the color when a specific pH is reached. It is changing to pink color in

alkaline medium and it changes to colorless in acidic medium.

2. pH paper strips.

There are different types of pH papers. Universal strips are often used to

check pH from the 1-14 range and for checking pH in narrow range also.

The litmus stripes have two versions: blue ones and red ones. It used to

check the solution acid or base. If the blue strip immersed in acid, the color

will change to red color and will not change the color if immersed in base

solution, and the red strip do the opposite.

49

pH meter

The most common and reliable method for measuring pH which measure the

electro-chemical potential between a known liquid inside the glass electrode

and an unknown liquid outside. The pH meter consists of two electrodes, a

reference electrode and a glass electrode. Both electrodes are suitably

combined in one electrode.

Buffer

In many biological analyses it is necessary to maintain the pH relatively constant regardless of

small amounts of acids or bases added and require buffer systems to resist the change in pH.

Buffer is an aqueous solution consisting of a mixture of a weak acid, HA, and its conjugate

base, A- or a weak base, B, and its conjugate acid, BH+. Its pH will not change significantly

when a small amount of acid or base is added to it. Buffer solutions are maintain pH at nearly

constant value in a wide variety of chemical applications. It will act as neutralizing agents that

contain both positive and negative ions of a weak acid.

Weak acid reacted with any added OH- to give water (HA + OH- H2O). Conjugate base

of weak acid reacted with any added H+ to give weak acid again (A- + H+ HA).

There are three primary buffer systems that regulate the H+ concentration in the body fluids to

prevent acidosis or alkalosis:

1. Carbonic Acid Buffering System (H2CO3 and HCO3) - Major ECF buffer

2. Phosphate Buffering System - Urinary buffer

3. Protein Buffer System - Major ICF buffer

Without buffers, our blood pH would change too quickly. For example, without buffers,

imagine drinking orange juice (citric acid). The weak acid will lower our blood pH and will

lead into a lot of trouble. The enzymes in our body will not be optimal. Then we would run

into trouble.

These buffers are non-toxic to cells, and are not absorbed through cell membranes. The

concentration, temperature, and ionic composition of the medium have minimum effect on the

buffering capacity. They are resistant to enzymatic and non-enzymatic degradation.

Furthermore, they are transparent to visible and ultraviolet light.

50

3. Preparation of buffer solutions

In this lab you will prepare phosphate buffer. Phosphate buffer is one of the more common

buffers used for many molecular biology procedures and is made up of a mixture of dihydrogen

phosphate (NaH2PO4) and its conjugate base, hydrogen phosphate (Na2HPO4). The dihydrogen

phosphate acts as the weak acid because it donates an H+ to the OH- to form water. The

hydrogen phosphate will accept excess H+ to create dihydrogen phosphate. The buffer is most

commonly prepared at pH 7.

Before making a buffer, you must know the component of the buffer, desired pH, the

concentration and total volume of the buffer you want to make (probably given to you).

Procedure

1. Make up 0.1 M monobasic stock NaH2PO4 and 0.1 M dibasic stock Na2HPO4 as in page 47

2. To make 100 ml of a 0.1 M sodium phosphate buffer, combine the two components of each

stock as described in the table1 below according to the desired pH.

3. Check the pH using a pH meter and adjust the pH as necessary using phosphoric acid or

sodium hydroxide.

4. Once you have reached the desired pH, add d.H2O to bring up to 100 ml.

5. Label the volumetric flask. The label includes your name, chemical name of substance, the

pH, the concentration, and the date, e.g. 0.1 M phosphate buffer pH 6.8.

Preparation of phosphate buffer

Vol. of Na2HPO4

(0.1 M)

Vol. of NaH2PO4

(0.1 M) Calculated pH Measured pH

10 ml 10 ml 6.77

9 ml 11 ml 6.61

8 ml 12 ml 6.52

6 ml 14 ml 6.33

11 ml 9 ml 6.79

12 ml 8 ml 6.88

14 ml 6 ml 7.07

51

4. Dilution

Solutions are often prepared by dilutions of a concentrated solution, usually a laboratory

stock solution. Given the initial concentration of the stock solution,Ci, desired concentration

and volume of the diluted solution, its preparation is straightforward. The following equation

is useful to do calculations involving dilutions

Ci Vi (stock solution) = Cf Vf (new solution)

Ci = initial concentration to be diluted (stock solution)

Vi = initial volume to be diluted (stock solution)

Cf = final concentration to prepare (new solution)

Vf = final volume to prepare (new solution)

The volume to be taken from the initial solution can be calculated as

Vi = CfVf / Ci

To make 200 mL of a 0.1 M HCl (new solution) from a solution of 1.0 M HCl (stock

solution), we need 20 ml of 1.0 M HCl.

Procedure

1. Measure 20 ml of 1.0 M HCl (stock solution) with a pipette then transfer it to a 200 ml

volumetric flask.

2. Add approximately ¾ of the water volume to the volumetric flask.

3. Put the lid on the flask and invert several times to mix the solution.

4. Add more water, if necessary, but do not go over the fill mark.

5. Add the final drops with a teat pipette to ensure that the bottom of the meniscus is at the fill

mark. Put the lid on the flask an again invert to mix the solution.

6. Label your solution with your name, the concentration, the date, and the contents, e.g. 1.0M

HCl.

Note: When diluting a strong acid, always add acid to water (same for a strong base).

52

Principle

Electrophoresis is a technique used for the separation and sometimes purification of

macromolecules especially proteins and DNA, or RNA fragments that differ in size, charge or

conformation. It refers to the movement of charged particles (ions, molecules, macromolecules)

through a porous matrix under the influence of electric field. Particles will move through the

matrix at different rates, according to their charge, toward either the negative pole (cathode) if

positively charged or the positive pole (anode) if negatively charged. Similar charge molecules

will migrate according to their size. Smaller molecules migrate faster through the gel matrix

than the larger molecules.

The pH of an electrophoresis gel is determined by the buffer used for that gel. If the pH of the

buffer is above the pI of the protein being run, the protein will migrate to the positive pole. If

the pH of the buffer is below the pI of the protein being run, the protein will migrate to the

negative pole of the gel. If the protein is run with a buffer pH that is equal to the pI, it will not

migrate at all.

The rate of migration through the electric field depends on the strength of the field, the net

charge, size and shape of the molecules, and the ionic strength, viscosity and temperature of the

buffer.

Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and

nucleic acids, possess ionizable groups and, therefore, at any given pH, exist in solution as

charged species either as cations (+) or anions (-).

Materials and chemicals

a. Supporting media

b. Buffer

c. Power supply: provides the regulated electric current

d. Detection and Quantification

e. Electrophoresis apparatus: tank, electrodes, tray, and combs.

A. Supporting medium:

1. Paper: Whatman filter paper and Cellulose acetate paper

2. Gel: Agarose gel is a linear polysaccharide extracted from seaweed

Starch gel: from the partial hydrolysis of potato starch.

53

Polyacrylamide gel: is a cross-linked polymer, sponge like structure made of acrylamide.

The pH of an electrophoresis gel is determined by the buffer used for that gel. If the pH of the

buffer is above the pI of the protein being run, the protein will migrate to the positive pole. If

the pH of the buffer is below the pI of the protein being run, the protein will migrate to the

negative pole of the gel. If the protein is run with a buffer pH that is equal to the pI, it will not

migrate at all.

B. Buffer composition (ionic strength, pH)

Commonly used buffer

• TBE (Tris-borate-EDTA)

• TAE (Tris-acetate-EDTA)

• Barbital buffer and TE (Tris-EDTA) for protein

Function of buffer:

• Carries the electric current

• Determine the electric charge

• Establish the pH

At low ionic strength the proteins will carry a relatively large proportion of the current and so

will have a relatively fast migration. At high ionic strength, most of the current will be carried

by the buffer ions and so the proteins will migrate relatively slowly. The buffer also will

generate greater heat.

C. Detection and Quantification

After the separation is complete, the molecules can be visualized by treating the gel with a stain

such as ethidium bromide, silver, or coomassie blue, which bind to the molecules but not to the

gel itself.

Bands in different lanes that end up at the same distance from the top contain molecules that

passed through the gel with the same speed, which usually means they are approximately the

same size. There are molecular weight sizes markers can be used to compare it with those of

the unknown to determine their size.

D. Determination of Molecular Weight

A linear relationship exists between the logarithm of the molecular weight of an SDS-denatured

polypeptide, or native nucleic acid, and it’s Rf. The Rf is calculated as the ratio of the distance

migrated by the molecule to that migrated by a marker dye-front. A simple way of determining

relative molecular weight by electrophoresis (MW) is to plot a standard curve of distance

54

migrated vs. log MW for known samples and read the log MW of the unknown sample after

measuring distance migrated on the same gel.

55

Alb

um

in

Alp

ha

1

Alp

ha

2

Be

ta

G

am

ma

Experiment 9

Serum protein electrophoresis

Principle

Serum protein electrophoresis (SPEP) is an easy, inexpensive method of separating proteins

based on their net charge, size, and shape by applying an electric current to them while they are

in a gel, a firm jelly-like substance. Protein electrophoresis is used to identify the presence of

abnormal proteins, to identify the absence of normal proteins, and to determine when different

groups of proteins are present in unusually high or low amounts in blood or other body fluids.

Proteins do many things in the body, including the transport of nutrients, removal of toxins,

control of metabolic processes, and defense against invaders. Protein electrophoresis also can

provide information about the molecular weights and charges of proteins, the subunit structures

of proteins, and the purity of a particular protein preparation. SDS-PAGE, Sodium dodecyle

sulfate-polyacrylamide gel electrophoresis, is the most commonly technique used for proteins.

The 2 major types of protein present in the serum are albumin and the globulin proteins.

Albumin is the major protein component of serum and represents the largest peak that lies

closest to the positive electrode. Globulins have higher molecular weights than albumins but

comprise a much smaller fraction of the total serum protein. Four Globulin categories are

represented: alpha-1, alpha-2, beta, and gamma. In some cases, the beta fraction is further

divided into beta 1 and beta 2, with the gamma fraction being closest to the negative electrode.

(+)

The major components of serum proteins

Materials:

• Barbiturate buffer (0.07mol/liter, pH 8.6)

(-)

56

• Agarose gel (1 % w/v in the barbiturate buffer: heat the agarose at 90oC until the lumps

are dissolved, then cool to 55oC).

• Tracking dye (bromophenol blue, 0.1 g/liter)

• Electrophoresis equipment

• Human serum

• Coomassie blue (0.25 % w/v in methanol: acetic acid: water, 5: 1: 5)

• Destaining solution (methanol: acetic acid: water, 5: 1: 5)

Procedure:

1. Prepare 1% agarose solution, by measuring 1g agarose into a conical flask and add 100 ml

barbiturate buffer.

2. Microwave or stir on a hot plate until agarose is dissolved and solution is clear.

3. Allow solution to cool to about 55oC before pouring.

4. Place comb in gel tray about 1 inch from one end of the tray and position the comb

vertically.

5. Pour the gel solution into tray to about half the comb to be filled. Allow the gel to solidify

about 20 minutes at room temperature.

6. Gently remove the comb, place tray in electrophoresis chamber and cover with the buffer

(the same buffer used to prepare the agarose) just until wells are submerged.

7. To prepare samples, add 1 μl of tracking dye for every 5 μl of serum. Mix well and then

load 5 μl of serum sample per well.

8. Run the samples at 90 volts until dye markers have migrated an appropriate distance.

9. Stain the gel in Coomassie blue for 10 minutes. Remove the excess stain with the destaining

solution.

57

Introduction

Enzyme-linked immunosorbent assay (ELISA), also known as an enzyme immunoassay (EIA),

is a biochemical technique used mainly in immunology to identify and quantify an antibody

(gamma globulin proteins; immunoglobulin Ig) or an antigen (foreign objects) in a sample such

as serum or urine depending how the test is designed. It is similar in principle to

radioimmunoassay (RIA), but depends on an enzyme rather than a radiolabel. The molecule as

a hormone or drug is detected by antibodies that have been made against it. An antibody reacts

with the concerned antigen in a highly specific manner to produce an Antigen-Antibody

complex.

ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple

positive or negative result for a sample. In quantitative ELISA, the amount of colored product

is proportional to the amount of enzyme-linked antibody that binds, which is directly related to

the amount of antibody that was present to bind antigen or antigen that was present to bind

antibody. If known amounts of antigen or antibody are added, a

standard curve, which is typically a serial dilution of the target,

can be constructed by plotting each standard optical density vs.

the standard concentrations on graph paper. The concentration of

each unknown antigen or antibody can then be extrapolated and

determined from the standard curve.

ELISA results are reported as a number using a spectrophotometer, spectrofluorometer, or other

optical device. This test is determining the "cut-off" point between a positive and negative

result.

Unknowns that generate a signal that is stronger than the known

sample are called "positive"; those that generate weaker signal are

called "negative."

Most commonly, ELISAs are performed in 96-well (or 384-well)

usually polystyrene microtiter plates, which will passively bind

antibodies and proteins.

96 - well microtiter plate

58

Principle

• Use of enzyme-labelled immunoglobulin to detect antigens or antibodies.

• The enzyme converts a colorless substrate (chromogen) to a color product, indicating

the presence of Ag:Ab binding.

• Optical density measured by microtiter-plate reader

Advantages of ELISA

• Less costly and safest.

• Easy visualization of results with high level of accuracy.

• Specific and highly sensitive assay that can detect protein at the picomolar to nanomolar

range.

• Easily automated for performance of large numbers of tests.

• Require minimal reagents.

• Qualitative detection or Quantitative measurement of either antigen or antibody.

• Wells can be coated with antigens or antibodies.

• Can be done by personnel with only minimal training.

Applications of ELISA

• Measure antibody levels (allergies, vaccines)

• Detect viruses (hepatitis, HIV, venereal diseases)

• Detect hormonal changes (pregnancy)

• Detect circulatory inflammatory markers (cytokines)

Requirements for ELISA test

• Purified antigen (if you want to detect or quantify antibody).

• Purified antibody (if you want to detect or quantify antigen).

• Standard solutions (positive and negative controls).

• Sample to be tested.

• Microtiter plates: plastic trays with small wells in which the assay

is done.

• Wash fluid (buffer).

• Enzyme-labeled antibody and enzyme substrate.

• ELISA reader (spectrophotometer) for quantitative measurements.

59

Enzyme labels

Enzyme labels are used to detect the binding of antigen-antibody complex. It should have high

specific reactivity. The reactivity should be retained after linking of the enzyme to the

antigen/antibody. It should be easily coupled to antigen-antibody complex and must stable.

Enzymes used in labelling should not be normally present in the patient samples. Examples of

enzyme labels are Horseradish peroxidase, Alkaline phosphatase, and Glucose oxidase.

Stages in ELISA

1. The adsorption of either antigen or antibody to the plastic solid phase (micro-titer plate).

2. The addition of the test sample and subsequent reagents.

3. The incubation of reactants (formation of antigen-antibody complex).

4. The separation of bound and free reactants by washing.

5. The binding of enzyme-conjugated antibody or antigen to the target antigen or antibody.

6. The addition of substrate (production of a visible signal).

7. The visual or spectrophotometric reading of the assay.

Types of ELISA assay

Variation of different types of ELISA assay depends upon the labeling and signal detection

methodology. ELISA used for detecting antibody-antigen complex by fixing either antigen or

antibody.

Direct ELISA

For the detection of antigens. It uses the directly labeling antibody itself. It is considered to be

the simplest type of ELISA. A sample containing the target antigen (known) is adsorbed/coated

to the microtiter plastic plates followed by an enzyme-labeled antibody (detecting Ab) directly

adsorbs to the antigen. The color or the signal

produced as a result of addition of substrate is

proportional to antibodies in the sample.

Indirect ELISA

Ag

E

Substrate

Target antigen

Primary antibody conjugate

Direct ELISA

60

For antibody detection. The well coated with antigento be tested.

Indirect ELISA is a two-step ELISA which involves two binding

process of primary antibody and labeled secondary antibody.

The primary antibody is incubated with the antigen followed by

the incubation with the secondary antibody. It differs than direct

ELISA in that the primary antibody is not labeled. The color or

the signal produced as a result of addition of substrate is

proportional to antibodies in the sample.

Sandwich ELISA

To detect the antigen. It measures the amount of antigens

between two layers of antibodies (i.e. capture and detecting

antibody). The plate is coated with capture antibody rather

than antigen. Antigen added binds to the capture antibody.

Then detecting antibody binds to the antigen just like a

sandwich. An enzyme-conjugated secondary antibody

added binds to detecting antibody followed by a substrate to

produce a color. The color or the signal produced as a result

of addition of substrate is proportional to antigen.

Competitive ELISA

It measures the amount of antigen in a sample. In this type of ELISA, an antibody is bound to

the well and antigen is labeled instead of the antibody. The labeled antigen with enzyme

competes with the sample antigen (unlabeled) for the binding to the capture antibody (same

target). The color or the signal produced is inversely proportional to antigens in the sample. For

example, the absence of the antigen in the sample will result in a dark color, whereas the

presence of the antigen will result in a light color or no color as the concentration of the antigen

increases. Absence of signal indicates a positive sample.

References:

1. Plummer D.T., An introduction to Practical biochemistry, Tata McGraw – Hill.

Substrate

Sandwich ELISA

Secondary antibody conjugate

Target antigen

Detecting

antibody

Capture antibody

Ag

E

E

Ag

Substrate

Target

antig

Primary

antibo

Indirect ELISA

Secondary antibody conjugate

E

Ag

E

Ag

E

Ag

E

Ag

Competitive ELISA

Ag

Ag

Ag

E

Target antigen

Antigen conjugate

Capture antibody

Ag

61

2. http://www.columbia.edu/cu/biology/courses/c2005/hand04.html

3. http://www.morrisonlabs.com/ph_study_guide.htm

4. http://www.ph-meter.info/

5. Carlsberg Research Centre history page, http://www.crc.dk/history.shtml

6. Definitions of pH scales, standard reference values, measurement of pH, and related terminology

7. http://hyperphysics.phy-astr.gsu.edu/hbase/chemical/ph.html

8. http://www.virtualsciencefair.org/2005/wali5s0/public_html/pH_scale.htm

9. http://www.gcsechemistry.com/aa29.htm

10. http://www.bbc.co.uk/schools/gcsebitesize/chemistry/chemicalreactions/0acidsbasesrev2.shtml

11. http://www.seafriends.org.nz/dda/ph.htm

12. http://znk.ptfos.hr/dodatno/suradnja/ph%20meter.htm

13. http://www.chem.tamu.edu/class/majors/tutorialnotefiles/buffers.htm

14. Pictures are captured & drawn by KAU/Biochemistry department.

15. http://www.aai.org/committees/education/Curriculum/gel.htm

16. Plummer D.T., An introduction to Practical biochemistry, Tata McGraw – Hill.

17. David J Holme and Hazel Peck (1993) analytical biochemistry, second edition, Longman Singapore,

Singapore, pp. 143-145.

18. http://www.colby.edu/chemistry/CH367/laboratory/

19. http://www.unm.edu/~rrobergs/titration.htm

20. http://www.daviddarling.info/encyclopedia/G/glycine.html

21. http://www.hannainst.co.uk/acatalog/Getting_the_best_from_your_Colorimeter.html

22. http://dl.clackamas.edu/ch105-04/importan.htm

23. http://faculty.uca.edu/~march/bio1/scimethod/spectro.htm

24. http://abacus.bates.edu/~ganderso/biology/resources/spec20.html

25. http://www.biology.lsu.edu/introbio/tutorial/webtutorial/spectrophotometry.php

26. http://services.juniata.edu/ScienceInMotion/chem/labs/spec/maxwave.doc

27. http://www.diycalculator.com/sp-cvision.shtml

28. http://www.wfu.edu/academics/chemistry/courses/CC/index.htm

29. http://www.piercenet.com/browse.cfm?fldID=F88ADEC9-1B43-4585-922E-836FE09D8403

30. http://immunoassays.blogspot.com/

31. http://exploreable.wordpress.com/2011/05/25/elisa-enzyme-linked-immunosorbent-assay/

32. Holme DJ and Peck H, Analytical Biochemistry (2nd edition).

33. cellbiologyolm.stevegallik.org/node/74

34. http://ga.water.usgs.gov/edu/meniscus.html

Useful links:

• Buffer solution

• pH

• Amino acid titration curve and pKa values

• ph-meter

• Buffer solutions

• Solutions

• Phosphate buffer

• Buffer preparation

• Buffered solution

• ph_meter

62

• http://www.moafa.com/c3.htm

• Chromatography-online

• Chromatography menu

• Chromtutorial

• Biochemlab

• See these videos for TLC: Video 1 and Video 2

See this video for the differences between absorption and adsorption

• TLC and Column Chromatography part 1

TLC and Column Chromatography part 2

TLC and Column Chromatography part 3

TLC and Column Chromatography part 4

• The spectrophotometer: A demo and practice experiment

• Lycopene

• Carotenoids

• Column chromatography

• Column Chromatography Procedures

• Separatory Funnel Extraction Procrdure

• Principles of gel filtration chromatography

• http://www.hoeferinc.com/

• http://en.wikipedia.org/wiki/Gel_electrophoresis#cite_note-1

• http://www.mcb.uct.ac.za/sdspage.html

• http://www.spectrapor.com/dialysis/Fund.html

• www.membrane-mfpi.com/labdialy.html

• http://www.membrane-mfpi.com/t_instrs.html

• http://web.siumed.edu/~bbartholomew/course_material/protein_methods.htm

• http://highered.mcgraw-

hill.com/sites/0072556781/student_view0/chapter33/animation_quiz_1.html

• http://www.sumanasinc.com/webcontent/animations/content/ELISA.html