chromatography

Definition and Classification of Chromatography Chromatography course

Dr.Ehab Aboueladab (Assistance. Prof.of Biochemistry, Mansoura University) 1صفحة

Aim Separation Techniques

1-Biological fluids are extremely complex in composition.

2-Chemical analysis would be impossible if it were necessary to

completely isolate each substance prior to its measurement.

3- An optimal method tests for a specific substance in the

presence of all others, requiring no isolation of the substance

under analysis.

4- A test is specific when none of the other substances present

interfere. However, virtually all chemical tests are subject to at

least some interference.

5-This is one of the most important problems in clinical

chemistry. Therefore some type of separation procedure is

required.

7-Separation in clinical chemistry usually is based on

differences in the size, solubility or charge of the substances

involved.



INTRODUCTION

The Russian botanist M. S. Tswett is discovery of

chromatography. He used a column of powdered calcium

carbonate to separate green leaf pigments into a series of

colored bands by allowing a solvent to percolate through the

column bed. Since these experiments by Tswett many scientists

have made substantial contributions to the theory and practice of

chromatography. Not least among these is A. J. P. Martin who

received the Nobel Prize in 1952 for the invention of partition

chromatography (with R. L. K. Synge) and in the same year

with A. T. James he introduced the technique of gas-liquid

chromatography. Chromatography is now an important tool

used in all branches of the chemical and life sciences.

1-Definition of Chromatography

Chromatography is essentially a physical method of

separation in which the components to be separated are

distributed between two phases one of which is stationary

(stationary phase) while the other (the mobile phase) through

it in a definite direction.

2- Classification of chromatographic methods

The common feature of all chromatographic methods is

two phases, one stationary and the other mobile

A classification can be made depending upon whether the

stationary phase is solid or liquid. If it is solid, the method is



termed adsorption chromatography; if it is liquid the method

is partition chromatography.

One of the two phases is a moving phase (the mobile phase),

while the other does not move (the stationary phase). The

mobile phase can be either a gas or a liquid, while the

stationary phase can be either a liquid or solid.

3- Classification scheme

One classification scheme is based on the nature of the two

phases. All techniques which utilize a gas for the mobile phase

come under the heading of gas chromatography (GC). All

techniques that utilize a liquid mobile phase come under the

heading of liquid chromatography (LC). Additionally, we

have gas–liquid chromatography (GLC), gas–solid

chromatography (GSC), liquid–liquid chromatography (LLC),

and liquid–solid chromatography (LSC),

4- Main Type of Chromatography

In general, there are four main types which can be

classified as follows:

4.1-Liquid-Solid chromatography

Classical adsorption chromatography (Tswett column)

Ion-exchange chromatography

4.2. Gas-Solid chromatography

4.3. Liquid-Liquid chromatography

Classical partition chromatography

Paper chromatography



4.4 Gas-Liquid chromatography

5-Separation techniques

Technique Property Description

Precipitation Solubility Some of the substances

precipitate while the

others remain dissolved

Ultra-filtration or

Dialysis

Molecular size Some of the substances

pass through a layer or

sheet of porous material

while the other

substances are retained

Extraction Solubility Some of the substances

dissolve (partition) more

in water. While other

substances dissolve

more organic solvent in

contact with the water

Thin layer

Chromatography

or

Column

Chromatography

Solubility Some of the substances

dissolve (partition) more

in the immobile file of

water on a solid

supporting medium (or

stick more to the

exposed areas of the

solid supporting

medium) while the other

substances dissolve

more in the surrounding

film of flowing organic

solvent



Gas liquid

Chromatography

Solubility Some of the substances

dissolve more in the

immobile film of wax or

oil-like material on a

solid supporting

medium. While the

others dissolve more in

surrounding stream of

flowing gas.

Gel filtration

Chromatography

Molecular Size Some of the substances

diffuse into the pores in

a porous, solid material

while others remain

outside in the

surrounding stream of

flowing water

Ion-exchange

Chromatography

Electrical charge Some of the substances

are bound by immobile

charges on the solid

supporting medium

while others are not

bound

Electrophoresis

Chromatography

Electrical charge The substances with

more charge move faster

and, therefore, further.

Substances with

opposite charges move

in opposite directions.



6-Adsorption chromatography

Adsorption column chromatography is the oldest form of

chromatography. Whether two or more substances of a

mixture can be separated by adsorption chromatography

depends on a number of factors. Most important is the strength

with which each component of mixture is adsorbed and its

solubility in the solvent used for elution. The degree to which

a particular substance is adsorbed depends on the type of bonds

which can be formed between the solute molecules and the

surface of the adsorbent.

Chromatography

Adsorption Chromatography

Solid stationary phase Partition Chromatography

Liquid Stationary Phase

Liquid

mobile phase Gas mobile

phase

Gas mobile

phase Liquid

mobile phase

Adsorption Chromatography Chromatography course


All chromatographic separations are carried out using a

mobile and a stationary phase, the primary classification of

chromatography is based on the physical nature of the mobile

phase. The mobile phase can be a gas or a liquid which gives

rise to the two basic forms of chromatography, namely, gas

chromatography (GC) and liquid chromatography (LC). The

stationary phase can also take two forms, solid and liquid,

which provides two subgroups of GC and LC, namely; gas–

solid chromatography (GSC) and gas–liquid chromatography

(GLC), together with liquid solid chromatography (LSC) and

liquid chromatography (LLC). The different forms of

chromatography are summarized in Table.1

Most thin layer chromatography techniques are considered

liquid-solid systems although the solute normally interacts with

a liquid-like surface coating on the adsorbent or support or, in

some cases an actual liquid coating.



Table 1: The Classification of Chromatography

1-ADSORPTION CHROMATOGRAPHY

In adsorption chromatography the compounds to be

separated are adsorbed onto the surface of a solid material. The

compounds are desorbed from the solid adsorbent by eluting

solvent.

2-Separation of the compounds depends on

1-The relative balance between the affinities of the compounds

for the adsorbent and their solubility in the solvent.

2-The chemical nature of the substances.

3-The nature of the solvent.

4-The nature of the adsorbent.



Solid adsorbents commonly used are alumina, silica gel,

charcoal (active carbon), cellulose, starch, calcium phosphate

gels, calcium hydroxylapatite, and sucrose.

Solvents commonly used are hexane, benzene, petroleum

ether, diethyl ether, chloroform, methylene chloride, various

alcohols (ethyl, propyl, n-buryl and t-butyl alcohols), and

various aqueous buffers and salts, some in combination with

organic solvents

Adsorption chromatography is a column that is packed with the

adsorbents. The adsorbent is prepared and poured into the

column with an inert support at the bottom. Suitable supports

include plastic discs, or sheets of nylon or Teflon fabrics.

The adsorbent bed must be homogeneous and free of bubbles,

cracks, or spaces between the adsorbents and the walls of the

column.

The choice of the eluting solvent, although very important,

depends on the nature of the substances to be separated and

the adsorbent, and hence affords considerable latitude. The

process of eluting the sample components from the adsorbent by

the solvent is termed development. As illustrated in Figure 1,

the compounds in the mixture that are more soluble in the

solvent and have less affinity for the adsorbent move more

quickly down the column.

If the substances are colored, as they were in Tswett's

experiment, they are readily visible as they separate, However,



many substances are not colored, and in these instances, as the

development proceeds, fractions are collected at the bottom of

the column, and the different fractions are analyzed for

compounds of the types that are being separated, For example, if

proteins are being separated,

the fractions would be analyzed for protein by measurement of

the UV absorbance at 280 nm. If carbohydrates or nucleic

acids are being separated analytical measurements for

carbohydrates or nucleic acids. The collection of fractions by an

automatic fraction collector,

Figure 1: Collection of fractions from a column by an automatic fraction



a device that accumulates from an elution column the same

predetermined volume in each of a series of tubes that

automatically change position when the proper volume has been

collected This may be accomplished in various ways. For

example, set volume, with a timer, or by counting drops with a

drop counter. The latter is frequently used and is usually the

most reliable and flexible. The fraction collector may be

equipped with a detection cell that automatically measures some

parameter of the solution going into the tubes and may

correlated with fraction number and automatically recorded. The

detection cell is frequently a small spectrophotometer that can

measure absorbances at a fixed wavelength or at variable

wavelengths. Other detecting cell use index of refraction,

optical rotation, and other properties.



Figure 2: Adsorption chrornatography

A = adsorbent, S=Sample, ES = eluting solvent

(A) Application of sample to the of the column.

(B) Adsorption of sample onto adsorbent.

(C)Addition of elution solvent.

(D) and (E) Partial fraction of sample components.

(F) Complete fractionation of sample.

(G) and (H) Separation of all three components at various stages on the adsorbents.

(I) Elution of the first component from the column.



The substances adsorbed on the column support can be

eluted in three ways

(a) in the simplest method, a single solvent continuously

flows through the column until the compounds have

been separated and eluted from the column

(b) Stepwise elution, in which two or more different

solvents of fixed volume are added in sequence to

elute the desired compounds.

(c) Gradient elution, in which the composition of the

solvent is continuously changing. The latter method is

used to effect separations that are difficult because of a

tendency of component to be eluted in broad. Trailing

bands when a single solvent is used. Gradient elution

frequently provides a means of sharpening the bands,

a simple linear gradient has two solvents, A and B, in

which A is the starting solvent and B is the final

solvent. Solvent B is allowed to flow into solvent A as

solvent A flows into the column. The composition of

solvent A is, thus, constantly changing as solvent B is

constantly being added to A (Fig. 3).



Figure 3: Gradient elution. Flow of solvent B into solvent A With

mixing, continuously changing the composition of solvent A as it flows

into column

Figure 4: Elution of chromatography column with a gradient of

increasing salt concentration.



Gradients other than linear gradients (e.g., exponential,

concave. or convex) may be obtained by introducing a third

vessel and varying the composition of the solvents in the

vessels. These eluting methods are also used with other

column chromatographic methods.

3-Activation of adsorbent

Many adsorbents such as alumina, silica gel, and active

carbon and Mg silicate can obtain commercially, but they

require activation before use. Activation is achieved by heating

and there is usually an optimum temperature for activation, for

e.g. alumina is about 400oC. For reduced activity by the

controlled addition of water, and the subsequent activity is

related to the amount of water added. Brookman and Schodder

established five grades of alumina Grade I is the most active

and the is simply alumina heated at about 350 0

C for several

hours. Grade II has about 2-3% water, Grade III 5-7%, Grade

IV 9-11 %, Grade V film. (Least active) about 15%.

4-Retention

The retention is a measure of the speed at which a substance

moves in a chromatographic system. In continuous development

systems like HPLC or GC, where the compounds are eluted with

the eluent, the retention is usually measured as the retention

time Rt or tR, the time between injection and detection. In

interrupted development systems like TLC the retention is

measured as the retention factor Rf, the run length of the



compound divided by the run length of the eluent

front:

The retention of a compound often differs considerably between

experiments and laboratories due to variations of the eluent, the

stationary phase, temperature, and the setup. It is therefore

important to compare the retention of the test compound to that

of one or more standard compounds under absolutely identical

Conditions.

5-Plate theory

The plate theory of chromatography was developed by

Martin and Synge. The plate theory describes the

chromatography system, the mobile and stationary phases, as

being in equilibrium. The partition coefficient K is based on

this equilibrium, and is defined by the following equation:

K is assumed to be independent of concentration, and can

change if experimental conditions are changed, for example

temperature is increased or decreased. As K increases, it takes

longer for solutes to separate. For a column of fixed length and

flow, the retention time (tR) and retention volume (Vr) can be

measured and used to calculate K



6- Column chromatography

1. Small plug of wool (or cotton)

2. Sand to cover "dead volume"

3. Silica gel, length = 5.5 - 6 inch (Note 1inch=2.54cm).

4. Tap column on bech (carefully) to remove air bubbles inside

the column

5. Add solvent system

6. Add sand on top of silica

7. The top of the silica gel should not be allowed to run dry.

8. Sample is diluted (20-25% solution)

9. The sample is applied by pipette

10. Solvent used to pack the column is reused

11. Walls of column are washed with a few milliliters of eluant

12. Column is filled with eluant

13. Flow controller is secured to column and adjusted 2.0 in /

min.



Figure 5



Column as that illustrated in Fig.5 may be used:

Typical chromatographic column.

Mixture sorbed on top of column.

Partial separation

Complete separation

Table 2: Common adsorbents and the type of compounds

Solid Suitable for separation of

Alumina Steriods, vitamins, ester, and alkaloids

Silica gel Steriods, amino acids, alkaloids

Carbon Peptides, carbohydrates, amino acid

Magnesium

carbonate

Porphyrins

Magnesium

silicate

Steriods, ester, glycerides, alkaloids

Magnesia Similar to alumina.

Ca(OH)2 Carotenoids.

CaCO3 Carotenoids and xanthophylls.

Ca Phosphate Enzymes, protein, and polynucleotide

Starch Enzymes.

Sugar Chlorophyll.

Thin layer chromatography Chromatography course


Thin layer chromatography

This technique is particularly useful for the separation of very

small amounts of material. The general principle involved is similar to

that involved in column chromatography, i.e. it is primarily adsorption

chromatography, although other partition effects may also be involved. A

glass sheet is covered by a uniform thin layer of an adsorbent.

Adsorbents used in TLC, differ from column adsorbents. It contain a

binding agent such as calcium sulphate, which facilitates the adsorbent

sticking to the glass plate. The plates are prepared by spreading slurry of

adsorbent in water over them, starting at one end, and moving

progressively to the other and then drying them in an oven at 100-

120°C. Drying serves to remove the water and to leave a coating of

adsorbent on the plate. Equipment is available which will ensure the

production of an even coating of adsorbent over a series of glass plates.

The normal thickness of slurry layer used is 0.25 mm for qualitative

analysis, but layers up to 5-10 mm thick may be made for preparative

work.



The sample is applied to the plate by micropipette or syringes, as spot

2.5 cm from one end and at least an equal distance from the edge. The

solvent is removed from the sample by the use of an air blower. All spots

should be placed on equal distance from the end of the plate.

Separation takes place in glass tank which contains the developing

solvent (mobile phase) to a depth of 1.5 cm , this is allowed to stand for

at least 1 hour with a glass plate over the top of the tank to ensure that

the atmosphere within the tank becomes saturated with solvent vapor.

Then, the thin layer plate is placed vertically in the tank so that, it stands

in the solvent with the end bearing the sample in the solvent.

The cover plate is replaced and separation of the compounds then occurs

as the solvent travels up the plate. After the solvent had reached the

wanted level, the run is stopped. The chromatographic separation is

completed the spots of the component substances can be detected by

different methods:

1-Many commercially available TLC adsorbents contain a fluorescent

dye, the plate is examined under UV light, the separated components

show up as blue, green, black area.

2. Spraying the plate with 50% sulphuric acid and heating so,

the compounds become charred and show spots



3. Spraying the plates with specific color reagents will stain up certain

compounds e.g. ninhydrin for amino acid (aa) , aniline for aldoses.

Solvents Universal TLC System:

petroleum ether - ethyl acetate

Very polar solvent additives:

methanol > ethanol > isopropanol

Moderately polar additives:

acetonitrile > ethyl acetate > chloroform, dichloromethane >

diethyl ether > toluene

Non-polar solvents:

cyclohexane, petroleum ether, hexane, pentane

TLC Visualization (Detecting the spots)

Non-destructive techniques:

1. Ultraviolet lamp. Shows any UV-active spots

2. Plate can be stained with iodine.

Bottle containing silica and a few crystals of iodine

(especially good for unsaturated compounds)



Destructive techniques

Staining Solutions immerse the plate as completely as possible in the

stain and remove it quickly. Heat carefully with a heating

Stains Use/Comments

Anisaldehyde Good general reagent, gives a range of colors

PMA Good general reagent, gives blue/green spots

Vanillin Good general reagent, gives a range of colors

Ceric sulfate Fairly general reagent, gives a range of colors

DNP Mainly for aldehydes and ketones, gives

orange spots

Permangante Mainly for unsaturated compounds and alcohols,

gives yellow spots

Thin-Layer Chromatography of Amino acids

Amino acids may be separated by two-dimensional TLC using either

silica gel or cellulose as the separating medium. Two different solvents

are used for each type of TLC plate and a different type of separation is

achieved for each type. The amino acids are visualized with two types of

ninhydrin spray for the silica gel and the cellulose gel media.

Ninhydrin Sprays for amino acid detection

For silica gel TLC: The plate is sprayed with a solution of 300 mg of

ninhydrin + 3 ml of glacial acetic acid + 100 ml of butyl alcohol and

heated for 10 minutes at 110°C.



For cellulose TLC:

The plate is sprayed with a solution of 500 mg of ninhydrin + 350

ml of absolute ethanol + 100 ml of glacial acetic acid + 15 ml of 2,4,6-

trimethylpyridine and heated for 10 minutes at 110°C.

Two-dimensional TLC separation of amino acids.

On silica gel G with

solvent I, chlorolorm-17% methanol (v/v)-ammonia (2:2:1, v/v/v) and

solvent II, phenol-water (75:25, v/v).

on cellulose MN 300 with

solvent III, 1-butanol-acetone-diethylamine-water (10:10:2:5,v/v/v/v,

pH 12.0) and

solvent IV, 2-propanol-formic acid (99%)-water (40:2:10, v/v/v, pH 2.5)



Thin-Layer Chromatography of Carbohydrates

Carbohydrates may be separated on commercial silica gel plates

using a variety of solvents to achieve specific separations. The results of

the separation depend on the particular plate used. Whatman K5 silica gel

and Merck silica gel 60 plates give good results.

Solvent for TLC separations of carbohydrates

Solvent: Acetonitrile-water (35:15, v/v) with four ascents (45 minutes

each for a 20-cm plate) will separate mono-, di , and trisaccharides

The visualization of carbohydrates on thin layer silica gel plates is

obtained by spraying with sulfuric acid-methanol (1: 3, v/v) followed by

heating for 10 minutes at 110-120°C. Most carbohydrates give black to

brown spots on a white background.



Examples of some TLC separation systems

Compounds Adsorbent Solvent system (v/v)

Amino acids Silica Gel G 96% Ethanol/water (70/30)

Butan-1-ol/acetic acids/

water (80/20/20)

Mono and di

saccharides

Kieselguhr G (sodium

acetate)

Kieselguhr G

(sodium phosphate pH5)

Ethyl acetate/propan-1-ol

(65/35). Butan-1-ol /

acetone/phosphate buffer

pH5 (40/50/10)

Neutral lipids Silica Gel G Petroleum ether/diethyl

ether/acetone (90/10/1)

Cholesterol

Esters

Silica Gel G Carbon tetrachloride/

chloroform (95/5)

Carotenoids Kieselguhr G Petroleum ether/propan-1-

ol (99/1)

Phospholipids Silica Gel G Chloroform/methanol/water

(65/25/4)

Advantages of TLC.

The speed at which separation is achieved. With a volatile solvent

as the mobile phase the time involved may be as low as 30 minutes, but

even with non-volatile solvents the time involved is rarely longer than

90 minutes.

PAPER CHROMATOGRAPHY

Paper chromatography is a type of liquid-liquid partition

chromatography that may be performed by ascending or descending

solvent flow. Each mode has its advantages and disadvantages.

Ascending chromatography involves relatively simple and inexpensive

equipment compared with descending chromatography and usually gives

more uniform migration with less diffusion of the sample "spots."

Descending chromatography, on the other hand is usually faster because

gravity aids the solvent flow and with substances of relatively low

mobility. The solvent can run off the paper. Giving a longer path for

migration. To resolve compounds with low mobility. Ascending

chromatography may be performed more than once utilizing a multiple-

ascent technique.

For descending chromatography, papers 22 cm wide and 56 cm

long can be used. To facilitate the flow of solvent from the paper, the

bottom of the paper is serrated with a pair of pinking shears. Three pencil

lines are drawn 25 mm apart at the top of the sheet, and small aliquot of

the sample (10-50 ml) is placed at a marked spot on the third line. The

spot is kept as small as possible by adding the aliquot in small

increments. With drying in between. This may be expedited with a hair

dryer. Several samples, including standards, are placed 15-25 mm apart.

The paper is then folded along the other two lines and placed in the

solvent trough of the descending tank (Fig. 1). Which has been

equilibrated with solvent beforehand to ensure a saturated atmosphere.

The paper is irrigated with solvent until the solvent reaches the bottom or

for a longer period, allowing the solvent to flow off the end of the paper,

if necessary. The chromatogram is then removed dried and developed to

reveal the locations of the compounds. (Part II gives methods of locating

carbohydrates, amino acids. proteins. nucleotides and nucleic acids and

lipids.)

In ascending chromatography, a paper approximately 25 cm x 25

cm is used. A pencil line 20-25 mm from the bottom is drawn across the

paper

Fig. 1 Steps in descending paper chromatography

and aliquots (10-50l) of the samples and standards are spotted

approximately 15-25 mm apart along the line. The spots are dried and the

paper is rolled into a cylinder and stapled so that the ends of the paper are

not touching (Fig. 2). Solvent is poured into the bottom of a

chromatographic chamber, and the cylinder is placed inside. The chamber

is closed and solvent is allowed to flow up

Fig. 2 Steps in ascending paper chromatography

the paper by capillary action. The chamber may be a simple wide-mouth,

screw top, gallon jar or a cylinder with a ground-glass edge and a glass

plate top. As with descending chromatography, the chamber should be

equilibrated with solvent beforehand. Contrary to a popular

misconception, if the chamber has been sealed and is airtight, the paper

docs not have to be removed as soon as the solvent reaches the top. When

multiple ascents are performed, the paper is removed, thoroughly dried,

and returned to the chamber for another ascent of solvent.

The resolved compounds on a paper chromatogram may be detected by

their color if they are colored, by their fluorescence if they are

fluorescent, by a color that is produced from a chemical reaction on the

paper after spraying or dipping the chromatogram with various reagents,

or by autoradiography if the compounds are radioactive. Identification of

compounds on a chromatogram is usually based on a comparison with

authentic compounds (standards). A quantitative comparison may be

made by measuring the Rf

, which is the ratio of the distance the

compound migrates to the distance the solvent migrates. A better

comparison is the ratio of the distance a particular compound migrates to

the distance a particular standard migrates. For example, in the separation

of carbohydrates, the standard might be glucose and the ratio would be

RGlc or for amino acids, the standard might be glycine and the ratio would

be RGly

A useful modification is two-dimensional paper chromatography,

in which the sample is spotted in the lower left-hand corner and irrigated

in the first dimension with solvent A. The chromatogram is removed from

the solvent dried, turned 90, and irrigated in the second dimension with

solvent B, giving a two-

Fig. 3 Two-dimensional paper or thin-layer chromatography

dimensional separation (Fig. 3). An application of this procedure has

been developed for the study of enzyme specificity in which a solution of

the enzyme is sprayed onto the chromatogram between the first irrigation

and the second to see what products are formed by the action of the

enzyme on the compounds separated in the first dimension.

Paper chromatography has been used to establish the structural

homology of a series of oligomers obtained by enzymic synthesis, by acid

or enzymic hydrolysis, or by isolation from a natural source. The RF of

each separated homologue is determined and a French-Wild plot is made

by plotting log [RF / (1-RF)]

against the number of monomers in the oligomer. If the isolated

compounds fall on a straight line of this plot, they belong to a

homologous series, differing from each other by one monomer residue

(Fig. 4). Compounds separated by paper chromatography may be

quantitatively determined. Aliquots (50-200,l) of the solution

containing the substances to be separated and quantitatively determined

are streaked along the separation line. Aliquots of the solution (5-10,l)

are also spotted on the two outside edges of the streak and are used as

location standards. The chromatogram is irrigated in the usual way, and

vertical sections of the location standards are cut out and developed to

reveal the positions of the compounds. After drying, these standards are

placed alongside the streaked sections and the undeveloped compounds

are located; horizontal strips containing the individual compounds are cut

out and

Fig. 4. French-Wild plots (log RF / 1-RF), versus number of monomer units per

molecule) correlating paper chromatographic mobility with the number of

homologous monomer residues in oligosaccharide molecules.

Fig. 5. Elution of compounds from paper chromatograms for preparative

chromatography or quantitative determination

eluted with water. To accomplish the elution, tabs of chromatographic

paper are stapled to the narrow ends of each strip. As shown in Figure 5,

one end is fitted with two pieces of glass (cut microscope slides), which

arc held together with rubber bands, and the bottom end is cut tapered,

like a pipet tip. This assembly is played so that one end lies in a

chromatographic trough containing water, and the elution of the strip

occurs by capillary flow of the water down the paper strip into a baker.

Usually less than 1 mL of water is sufficient to effect quantitative

elution, the samples are quantitatively diluted to a specific volume, and a

chemical analysis is performed for the specific compound separated. This

technique also may be used as a preparative procedure to obtain small

quantities of pure compound from a mixture of compounds.

In an alternate quantitative procedure, the compounds in the sample are

radioactively labeled and separated in the usual way, and an

autoradiogram is prepared. The labeled compounds are located on the

chromatogram by comparing their positions on the autoradiogram. The

radioactive compounds are cut out and placed into a liquid scintillation

cocktail, and the radioactivity is determined by heterogeneous liquid

scintillation counting

Paper Chromatography

What is Chromatography?

Chromatography is a technique for separating mixtures into their

components in order to analyze, identify, purify, and/or quantify the

mixture or components.

Uses for Chromatography

Chromatography is used by scientists to:

Analyze – examine a mixture, its components, and their relations

to one another

Identify – determine the identity of a mixture or components

based on known components

Purify – separate components in order to isolate one of interest for

further study

Quantify – determine the amount of the a mixture and/or the

components present in the sample

Separate • Analyze

• Identify

• Purify

• Quantify

Components Mixture

Real-life examples of uses for chromatography:

• Pharmaceutical Company – determine amount of each chemical

found in new product

• Hospital – detect blood or alcohol levels in a patient’s blood

stream

• Law Enforcement – to compare a sample found at a crime scene

to samples from suspects

• Environmental Agency – determine the level of pollutants in the

water supply

• Manufacturing Plant – to purify a chemical needed to make a

product

Definition of Chromatography

Detailed Definition:

Chromatography is a laboratory technique that separates

components within a mixture by using the differential affinities of the

components for a mobile medium and for a stationary adsorbing medium

through which they pass.

Terminology:

• Differential – showing a difference, distinctive

• Affinity – natural attraction or force between things

• Mobile Medium – gas or liquid that carries the components

(mobile phase)

• Stationary Medium – the part of the apparatus that does not

move with the sample (stationary phase)

Simplified Definition:

Chromatography separates the components of a mixture by

their distinctive attraction to the mobile phase and the stationary phase.

Explanation:

• Compound is placed on stationary phase

• Mobile phase passes through the stationary phase

• Mobile phase solubilizes the components

• Mobile phase carries the individual components a certain

distance through the stationary phase, depending on their

attraction to both of the phases

Illustration of Chromatography

Components Affinity to Stationary Phase Affinity to Mobile Phase

Blue ---------------- Insoluble in Mobile Phase

Black

Red

Yellow

Mixture Components

Separation

Stationary Phase

Mobile Phase

Principles of Paper Chromatography

Capillary Action – the movement of liquid within the spaces of a

porous material due to the forces of adhesion, cohesion, and surface

tension. The liquid is able to move up the filter paper because its

attraction to itself is stronger than the force of gravity.

Solubility – the degree to which a material (solute) dissolves into a

solvent. Solutes dissolve into solvents that have similar properties.

(Like dissolves like) This allows different solutes to be separated by

different combinations of solvents.

Separation of components depends on both their solubility in

the mobile phase and their differential affinity to the mobile phase

and the stationary phase.

Paper Chromatography Experiment

What Color is that Sharpie?

Overview of the Experiment

Purpose:

To introduce students to the principles and terminology of

chromatography and demonstrate separation of the dyes in Sharpie Pens

with paper chromatography.

Time Required:

Prep. time: 10 minutes

Experiment time: 45 minutes

Costs:

Less than $10

Materials List

• 6 beakers or jars

• 6 covers or lids

• Distilled H2O

• Isopropanol

• Graduated cylinder

• 6 strips of filter paper

• Different colors of Sharpie pens

• Pencil

• Ruler

• Scissors

• Tape

Preparing the Isopropanol Solutions

Prepare 15 ml of the following isopropanol solutions in appropriately

labeled beakers:

- 0%, 5%, 10%, 20%, 50%, and 100%

Preparing the Chromatography Strips

Cut 6 strips of filter paper

Draw a line 1 cm above the bottom edge of the strip with the pencil

Label each strip with its corresponding solution

Place a spot from each pen on your starting line

Developing the Chromatograms

Place the strips in the beakers

Make sure the solution does not come above your start line

Keep the beakers covered

Let strips develop until the ascending solution front is about 2 cm

from the top of the strip

Remove the strips and let them dry


Observing the Chromatograms

Concentration of Isopropanol

0

% 20

% 50

% 70

% 100

%

Alternative Experiments

Protein purification Chromatography


1. Ammonium Sulfate Fraction of Protein Mixtures

Increasing the salt concentration to a very high level will

cause proteins to precipitate from solution without denaturation if

done in a gentle manner. First, we want to understand why the

protein precipitates. A protein in a buffer solution is very highly

hydrated, in other words, the ionic groups on the surface of the

protein attract and bind many water molecules very tightly:

This graphic illustrates how proteins in solution are hydrated

by water molecules. When a lot of salt, such as ammonium sulfate,

is added to the protein solution, the salt ions attract the water

molecules away from the protein. This is partly since the salt ions

have a much greater charge density than the proteins. So as the salt

is added and these small ions bind water molecules, the protein

molecules are forced to interact with themselves and begin to



aggregate:

So when enough salt has been added, the proteins will be

begin to precipitate. If this is carried out at a cold temperature like

in ice, the proteins will precipitate without denaturation. Thus, the

proteins can be collected by centrifugation and then redissolved in

solution using a buffer with low salt content.

This process is called "Salting Out" and works best with divalent

anions like sulfate, especially ammonium sulfate which is highly

soluble at ice temperatures.

Salting out or ammonium sulfate precipitation is useful for

concentrating dilute solutions of proteins. It is also useful for

fractionating a mixture of proteins. Since large proteins tend to

precipitate first, smaller ones will stay in solution. Thus, by

analyzing various salt fractions, one can find conditions where the



protein you are studying precipitates and many of the other

proteins in the mixture stay in solution. The end result is that you

are also able to increase the purity of your protein of interest while

you concentrate it using an ammonium sulfate fractionation

procedure.

2. Dialysis of Proteins

After a protein has been ammonium sulfate precipitate and

taken back up in buffer at a much greater protein concentration

than before precipitation, the solution will contain a lot of residual

ammonium sulfate which was bound to the protein. One way to

remove this excess salt is to dialyze the protein against a buffer

low in salt concentration.

This graphic illustrates the dialysis process. First, the



concentrated protein solution is placed in dialysis bag with small

holes which allow water and salt to pass out of the bag while

protein is retained. Next the dialysis bag is placed in a large

volume of buffer and stirred for many hours (16 to 24 hours),

which allow the solution inside the bag to equilibrate with the

solution outside the bag with respect to salt concentration. When

this process of equilibration is repeated several times (replacing the

external solution with low salt solution each time), the protein

solution in the bag will reach a low salt concentration:

The graphic illustrates the complete dialysis process, except

for it suggests you do this with distilled water. Really you want to

do this process with buffer to prevent the protein from denaturing

due to the fact that distilled or deionized water is too low in salt

and may have an undesirable pH for your protein, which may



cause it to denature.

In fact, dialysis is a good way to exchange the buffer the protein is

in at the same time you get rid of excess salt. For example, the

GOT after ammonium sulfate precipitation contains a mixture of

buffers as well as excess salt. So we use the buffer we want for the

next step in the purification, which is ion-exchange

chromatography, as the external solution during dialysis. After the

dialysis process, the protein solution is dialyzed against the starting

buffer for the ion-exchange chromatography step, not only will the

salt be removed but the protein will now be in the buffer needed

for the next step and ready to go. Sometimes, proteins will

precipitate during the dialysis process and you will need to

centrifuge the solution after dialysis to remove any particles which

would interfere with the next step – such as ion-exchange

chromatography where particles would clog the column and

prevent the chromatography step from working. In addition, you

may lose enzyme activity during dialysis. So it is a good idea to

keep some of your protein solution as a sample before it is put in

the dialysis bag so that it can be assayed for enzyme activity

before and after dialysis.

3. Alternative Methods for Desalting and Concentration of Proteins

There are several ways to get rid of excess salt in a protein

solution. One rapid method is to use a small gel filtration column



which contains a gel with very small pores which will only allow

water and salt inside the gel particles and will exclude the protein.

This method works very well and can be done at 4°C so that little

or no enzyme activity is lost during processing. A small amount of

dilution of the protein solution will take place during processing,

but it is possible by this method to exchange the buffer and prepare

the protein solution.

Another way to both concentrate a protein and exchange the

buffer, which completely avoids precipitation, is called

ultrafiltration:

Ultrafiltration is done a device which can withstand high

pressure. First, the ultrafiltration device is fitted with an ultrafilter

membrane of the desired molecular weight cut off such that you

protein of interest will be retain in the cell. Next, the pressure cell



is filled with the protein solution and nitrogen gas at about 50 psi is

applied while the cell is stirred gently at 4°C. After about 1 hour,

the solution will be decreased in volume usually without loss of

activity. To exchange the buffer the cell is filled with the desired

buffer and the concentration process are repeated.

Desalting

Before an ion-exchange chromatographic step or after an

ammonium sulfate fractionation, it is usually necessary to remove the salt

from the solution of protein. Desalting is accomplished in one of two

ways: dialysis or gel filtration.

Dialysis

Dialysis is performed by filling a section of dialysis tubing (a semi

permeable membrane) having a sufficiently small molecular weight "Cut-

off", with the protein solution, and placing the filled tubing in a large

volume of buffer. The decrease in salt concentration can be calculated

easily from the ratio of the volumes inside and outside of the bag.

Dialysis requires a few hours, after which the bag may be

transferred to fresh buffer if the reduction in salt concentration effected

by one cycle is deemed to be insufficient. In dialysis, all small molecules,

including salt ions, metal ions and cofactors, pass through the membrane,

which retains only macromolecules. Neither tightly bound metal ions and

cofactors, nor counterions to the macromolecule are effectively removed.

Since the initial solution in the bag is of much greater osmotic

strength than the surrounding buffer, the bag generally increases in

volume. The volume of the contents of the bag must be measured after

dialysis if either total protein or total enzyme units are to be calculated.

Ion Exchange Chromatography

Since proteins have different net charge and charge distribution,

ion exchange chromatography can be an effective purification tool. For

bench-top preparations, usually gravity-flow columns are employed, but

HPLC and automated HPLC-like systems have grown in popularity. For

gravity flow or for use with low pressure peristaltic pumps, ion exchange

media are usually carbohydrate based. Charged groups are attached to

solid supports (“inert phase”) such as Sepharose, Sephadex and cellulose.

Since these carbohydrates are compressible, they are not used in higher-

pressure systems, and more rigid inert phases such as TSK (a polyether-

coated gel) are used. For higher pressures, reinforced Polysaccharides,

and organically coated silica (e.g., TSK) are used. The resins, especially

poly (styrenediviny1benzene) described by HIRS for use with enzymes

were used by MOORE and STEIN in their famous amino acid analyzer.

They are commonly employed for ion exchange chromatography of small

molecules, but have given way to the ion exchange polysaccharides for

preparative applications in enzymology. The charged groups used with

the solid supports depend to some extent on the chemistry of the support

material itself, but are remarkably similar. Groups containing charged

nitrogen atoms are almost universally used for anion exchange media.

These include, from strong to weak, quaternary amino methyl or ethyl

(QAE), tertiary amino (diethylaminoethyl, DEAE, or diethylaminomethyl)

and secondary plus tertiary nitrogens (polyethylenimine, PEI). The

quaternary amino compounds are positively charged at any pH, but the

others must be used at a pH below the pK, of the protonated form (- 10,

for DEAE). The conjugate base of the strongly acidic sulfonic acid (i.e.,

alkyl or aryl sulfonate) and the weakly acidic carboxylic acids (e.g.,

carboxymethyl, CM) are the most common charged groups employed in

cation exchangers. The carboxymethyl packing must be used at a pH

above their pK4. Methods for determining the optimal pH for separation

of proteins depends, of course, on the proteins. Since most proteins are

acidic, they are negatively charged at pH 7-8. They therefore adsorb to a

positively charged stationary phase to which they act as counterions,

providing that other anions are not available to play the role of

counterion. The cationic stationary phase is known as an anion

exchanger because it functions by exchanging one anionic counterion for

another. Anionic proteins may bind more tightly to anion- exchange

stationary phases than simple salts because they possess more negative

charges than a simple anion. However, it is not the total charge on a

protein, but the charge density that determines the affinity. More

precisely, it is the charge distribution. Since a protein may interact with a

stationary phase on one side at a time, proteins with densely charged

patches may be bound more tightly. At pH values below the isoelectric

point of a protein, the net charge is positive, so negatively charged

stationary phases (cation exchange phases) are used. If a protein has an

isoelectric point near neutrality, either a cation exchange or an anion

exchange system can be used, depending on the pH employed. The

important considerations in choosing an optimal pH for separation of

enzymes by ion exchange chromatography have been reviewed. Protein

solutions are generally desalted, then loaded onto a column packed with a

stationary phase having the appropriate charge. Loading can often be

done as rapidly as the columns will flow without undue pressure; proteins

that adsorb are retained at the top of the column. As long as the capacity

of the column is not exceeded, liters of a (desalted, buffered) crude

extract can be loaded onto a column of modest size, so that a pre-

chromatography concentration step is not needed. After loading, the

column is washed with the loading buffer to remove unabsorbed and

weakly adsorbed proteins. The adsorbed proteins are then eluted by

washing the column with buffers of increasing salt concentration (e.g.,

NaCl), which corresponds to increasing solvent strength. This method of

elution using a series of isocratic (constant strength) elutions of

progressively increasing strength is known as batch elution. The ion

having a charge of the same sign as the protein can act as a displacing ion

by competing for charged sites on the stationary phase. At some

concentration, the eluting ion competes effectively with the protein,

which accordingly, spends a larger fraction of its time in the mobile

phase, leading to elution. This concentration would be ideal to purify the

protein of interest providing that more loosely bound proteins were

removed first, because it affords the maximum discrimination among

the charge densities of the proteins still on the column. However, the

protein might elute as a broad, dilute band. A simple and common

solution to elution is to employ a linear concentration gradient of salt,

Such a gradient can cover a range from 0 to 1 M NaCl over the volume of

a few hundred ml to a few liters, depending on the dimensions of the

column and the steepness of the gradient desired.

A major advantage of gradient elution is that proteins having a

wide range of affinities for the column can be eluted in a single run. The

information obtained from a gradient elution may be used to determine an

optimum salt concentration to be used in isocratic elution, but the

procedure is not straightforward. The theory of gradient elution is messy,

even in the simplest case. One egregious misstatement appears in

numerous papers on enzyme purification “the enzyme elutes at such and

such a concentration of sodium chloride”. Because the gradient travels

much more rapidly in the column than the protein (the protein is retained

to some extent), the concentration of sodium chloride in which the

enzyme actually appears at the bottom of the column is much higher than

the concentration at which it began to elute appreciably. Thus, the

concentration in which it appears to elute (concentration of sodium

chloride in the fraction in which the activity appears) is much too strong

for use as an isocratic eluent. In addition, the concentration in which the

enzyme appears varies with the dimensions of the column; longer

columns cause the enzyme to appear to elute in a higher salt

concentration, simply because the gradient progresses as the enzyme

moves down the column. To exercise maximum control over the system,

it is useful to separate the effects of pH from those of ionic strength

during ion exchange chromatography. One of the ions involved in the

buffering system bears the same charge as the protein and can therefore

act as a displacing ion. The concentration of this ion should not change

with pH, so it should not be the one involved in the equilibrium with

solvent protons. Buffering ions selected for use in ion exchange

chromatography should have the same charge as the column, i.e., cations

for an anion exchange column, anions for cation exchange. Hence,

phosphate buffers are used for cation exchange chromatography, and

Tris (for instance) buffers are used for anion exchange. It is necessary

for the column to be completely equilibrated with the starting solvent.

Equilibration can be checked by measurement of both pH and ionic

strength (e.g., by conductivity) prior to loading the column. Elution from

an ion-exchange column could also be accomplished using a change in

pH. Stepwise pH changes are sometimes employed, but do not generally

produce high resolution of complex mixtures. Reproducible continuous

pH gradients are difficult to obtain because so many of the components in

the system engage in acid-base equilibria. A workable system along these

lines has been devised using a proprietary mixed-bed packing and a

multi-component buffer system to elute proteins at their isoelectric pH.

The process is called chromatofocusing because of a loose analogy to

isoelectric focusing gel electrophoresis.

Gel filtration

Biomolecules are purified using chromatography techniques that separate

them according to differences in their specific properties, as shown in

Figure 1. and Table 1.

Property Technique

Size Gel filtration (GF), also called size

exclusion

Charge Ion exchange chromatography

(IEX)

Hydrophobicity Hydrophobic interaction

chromatography (HIC)

Reversed phase chromatography

(RPC)

Biorecognition (ligand specificity) Affinity chromatography (AC)

Table 1.

Fig. 1. Separation principles in chromatography purification.

Gel filtration has played a key role in the purification of enzymes,

polysaccharides, nucleic acids, proteins and other biological

macromolecules. Gel filtration is the simplest and mildest of all the

chromatography techniques and separates molecules on the basis of

differences in size. The technique can be applied in two distinct ways:

1. Group separations:

The components of a sample are separated into two major groups

according to size range. A group separation can be used to remove high

or low molecular weight contaminants (such as phenol red from culture

fluids) or to desalt and exchange buffers.

2. High resolution fractionation of biomolecules:

The components of a sample are separated according to differences

in their molecular size. High resolution fractionation can be used to

isolate one or more components, to separate monomers from aggregates,

to determine molecular weight or to perform a molecular weight

distribution analysis.

Gel filtration can also be used to facilitate the refolding of denatured

proteins by careful control of changing buffer conditions.

Gel filtration is a robust technique that is well suited to handling

biomolecules that are sensitive to changes in pH, concentration of metal

ions or co-factors and harsh environmental conditions. Separations can

be performed in the presence of essential ions or cofactors, detergents,

urea, guanidine hydrochloride, at high or low ionic strength, at 37 °C

or in the cold room according to the requirements of the experiment

Gel filtration in practice

Gel filtration separates molecules according to differences in size

as they pass through a gel filtration medium packed in a column. Unlike

ion exchange or affinity chromatography, molecules do not bind to the

chromatography medium so buffer composition does not directly affect

resolution (the degree of separation between peaks).

Separation by gel filtration

Gel filtration medium is packed into a column to form a packed bed. The

medium is a porous matrix in the form of spherical particles that have

been chosen for their chemical and physical stability, and inertness (lack

of reactivity and adsorptive properties). The packed bed is equilibrated

with buffer which fills the pores of the matrix and the space in between

the particles. The liquid inside the pores is sometimes referred to as the

stationary phase and this liquid is in equilibrium with the liquid outside

the particles, referred to as the mobile phase as shown in Figure 2.

Gel filtration is used in group separation mode to remove small

molecules from a group of larger molecules and as a fast, simple solution

for buffer exchange. Small molecules such as excess salt (desalting) or

free labels are easily separated. Samples can be prepared for storage or

for other chromatography techniques and assays. Gel filtration in group

separation mode

is often used in protein purification schemes for desalting and

buffer exchange

.

Fig. 2. Common terms in gel filtration

Sephadex G-10, G-25 and G-50 are used for group separations.

Large sample volumes up to 30% of the total column volume (packed

bed) can be applied at high flow rates using broad, short columns. Figure

3 shows the elution profile (chromatogram) of a typical group separation.

Large molecules are eluted in or just after the void volume, Vo as they

pass through the column at the same speed as the flow of buffer. For a

well packed column the void volume is equivalent to approximately 30%

of the total column volume. Small molecules such as salts that have full

access to the pores move down the column, but do not separate from each

other. These molecules usually elute just before one total column volume,

Vt, of buffer has passed through the column. In this case the proteins are

detected by monitoring their UV absorbance, usually at A280 nm, and

the salts are detected by monitoring the conductivity of the buffer.

Fig. 3. Typical chromatogram of a group separation. The UV (protein)

and conductivity (salt) traces enable pooling of the desalted fractions and

facilitate optimization of the separation.

The theoretical elution profile (chromatogram) of a high

resolution fractionation. Molecules that do not enter the matrix are eluted

in the void volume, Vo as they pass directly through the column at the

same speed as the flow of buffer. For a well packed column the void

volume is equivalent to approximately 30% of the total column volume

(packed bed). Molecules with partial access to the pores of the matrix

elute from

Sample: (His)6 protein eluted from HiTrap™

Chelating HP with

sodium phosphate 20 mM,

sodium chloride 0.5 M,

imidazole 0.5 M, pH 7.

Column: HiTrap Desalting 5 ml

Buffer: Sodium phosphate 20 mM,

Sodium chloride 0.15 M, pH 7.0

Void volume :Vo,

Total column volume :Vt

the column in order of decreasing size. Small molecules such as salts that

have full access to the pores move down the column, but do not separate

from each other. These molecules usually elute just before one total

column volume, Vt, of buffer has passed through the column, Fig. 4.

Fig. 4.Theoretical chromatogram of a high resolution fractionation (UV

absorbance).

Separation examples

Fig. 5. Cytochrome C, Aprotinin, Gastrin I, Substance P,

(Gly)6, (Gly)3 and Gly

Comparison of the selectivity of Superdex 75 prep grade and Superdex

200 prep grade for model proteins Figure.6

Superdex 75 prep grade (a)

gives excellent resolution of the three proteins in the Mr range 17 000 to

67 000 while the two largest proteins elute together in the void volume.

Superdex 200 prep grade (b) resolves the two largest proteins completely. The three smaller proteins

are not resolved quite as well as the larger ones or as in (a). The void

volume (Vo) peak at 28 minutes in (b) is caused by protein aggregates.

Fig. 6. Columns : a) HiLoad 16/60 Superdex 75 prep grade

b) HiLoad 16/60 Superdex 200 prep grade

Sample : 1. Myoglobin 1.5 mg/ml, Mr 17 000

2. Ovalbumin 4 mg/ml, Mr 43 000

3. Albumin 5 mg/ml, Mr 67 000

4. IgG 0.2 mg/ml, Mr 158 000

5. Ferritin 0.24 mg/ml, Mr 440 000

Sample volume : 0.5 ml

Buffer : 0.05 M phosphate buffer,

0.15 M NaCl,

0.01% sodium azide, pH 7.0

Flow : 1.5 ml/min (45 cm/h)

Media Selection Chromatography media for gel filtration are made from porous

matrices chosen for their inertness and chemical and physical stability.

The size of the pores within a particle and the particle size distribution

are carefully controlled to produce a variety of media with different

selectivities. Today's gel filtration media cover a molecular weight

range from 100 to 80 000 000, from peptides to very large proteins and

protein complexes. Figure.7

Superdex is the first choice for high resolution, short run times and high recovery.

Sephacryl is suitable for fast, high recovery separations at laboratory and industrial

scale

Superose offers a broad fractionation range, but is not suitable for large scale or

industrial scale separations.

Sephadex is ideal for rapid group separations such as desalting and buffer exchange.

Sephadex is used at laboratory and production scale, before, between or after other

chromatography purification steps.

The selectivity of a gel filtration medium depends solely on its pore

size distribution and is described by a selectivity curve. Gel filtration

media are supplied with information on their selectivity, as shown for

Superdex in Figure 8. The curve has been obtained by plotting a

partition coefficient Kav against the log of the molecular weight for a

set of standard proteins

Fig. 8. Selectivity curves for Superdex

Fig. 9. Defining fractionation range and exclusion limit from a selectivity curve.

Selectivity curves are usually quite linear over the range Kav = 0.1

to Kav = 0.7 and it is this part of the curve that is used to determine the

fractionation range of a gel filtration medium Figure 9.

Determination molecular weight

Ve – V0

Kav =--------------

Vt – V0

where Ve = elution volume for the protein

Vo = column void volume

Vt = total bed volume

On semilogarithmic graph paper, plot the Kav value for each protein

standard (on the linear scale) against the corresponding molecular

weight (on the logarithmic scale). Draw the straight line which best fits

the points on the graph. Then, Calculate the corresponding Kav for the

component of interest and determine its molecular weight from the

calibration curve.

Sephadex: Rapid group separation of high and low molecular weight

substances, such as desalting, buffer exchange and sample clean up

Sephadex is prepared by cross-linking dextran with epichlorohydrin.

Variations in the degree of cross linking create the different Sephadex

media and influence their degree of swelling and their selectivity for

specific molecular sizes (Table. 2 ).

Product Fractionation

range, Mr

(globular

proteins)

pH stability Bed volume

ml/g dry

Sephadex

Particle size,

wet

Sephadex G-10 <7×102

Long term: 2–13

Short term: 2–13 2-3 55–165 μm

Sephadex G-25

Coarse

1×103–5×10

3 Long term: 2–13

Short term: 2–13 4-6 170–520 μm

Sephadex G-25

Medium

1×103–5×10

3 Long term: 2–13

Short term: 2–13 4-6 85–260 μm

Sephadex G-25

Fine

1×103–5×10

3 Long term: 2–13

Short term: 2–13 4-6 35–140 μm

Sephadex G-25

Superfine

1×103–5×10

3 Long term: 2–13

Short term: 2–13 4-6 17–70 μm

Sephadex G-50

Fine

1×103–3×10

4 Long term: 2–10

Short term: 2–13 9-11 40–160 μm

• Sephadex G-10 is well suited for the separation of biomolecules such

as peptides (Mr >700) from smaller molecules (Mr <100).

• Sephadex G-50 is suitable for the separation of molecules Mr >30000

from molecules Mr<1500 such as labeled protein or DNA from

unconjugated dyes. The medium is often used to remove small

nucleotides from longer chain nucleic acids.

• Sephadex G-25 is recommended for the majority of group separations

involving globular proteins. These media are excellent for removing

salt and other small contaminants away from molecules that are greater

than Mr 5000. Using different particle sizes enables columns to be packed

according to application requirements

Sephadex is prepared by cross-linking dextran with

epichlorohydrin, illustrated in Figure 10 The different types of

Sephadex vary in their degree of cross-linking and hence in their degree

of swelling and selectivity for specific molecular sizes, as shown

Fig. 10. Partial structure of Sephadex.

Why use different techniques at each stage

In order to final removal of trace contaminants. Adjustment of pH,

salts or additives for storage. Then, end product of required high level

purity Therefore, The technique chosen must discriminate between the

target protein and any remaining contaminants

Gel Filtration (or)

Gel Permeation Chromatography (or)

Size Exclusion Chromatography

Size exclusion chromatography(SEC), also called gel permeation

Chromatography (GPC) or gel filtration chromatography(GFC) is a

technique for separates molecules according to their molecular size. Gel

particles form the stationary phase of this type of chromatography; the

mobile phase is the solution of molecules to be separated and the eluting

solvent, which most frequently is water or a dilute buffer. The sample is

applied to the gel, if the molecules are too large for the pores; they never

enter the gel and move outside the gel bed with the eluting solvent. Thus,

the very large molecules in a mixture move the fastest through the gel bed

and the smaller molecules, which can enter the gel pores, are retarded and

move more slowly through the gel bed. In gel chromatography, molecules

are, therefore, eluted in order of decreasing molecular size

Fig.1 Gel permeation chromatography. Open circles represent porous gel molecules:

large solid Circles represent molecules too large to enter the gel through the pores,

and smaller solid circles represent molecules capable of entering the gel pores

Three types of polymers are principally used-dextran,

polyacrylamide, and agarose

Dextran is a polysaccharide composed of (-1--->6)-linked glucose

residues with (-1,3) branch linkages. It is synthesized from sucrose by

an enzyme produced by the bacterium Leuconostoc mesenteroides B-

512F. The dextran is cross-linked to various extents by reaction with

epichlorohydrin to give gel beads with different pore sizes Fig.2. Cross-

linked dextrans are commercially produced by Pharrnacia Fine

Chemicals, lnc., (Uppsala, Sweden), and sold under the trade name

Sephadex. Sephadex gels in the so-called G-series, where the G-

numbers refer to the amount of water gained when the beads are

swelled in water (Table 1) have different degrees of cross-linking, hence

different pore sizes. This gives gels that have capabilities of separating

different ranges of molecular weights and have different molecular

exclusion limits. The exclusion limit is the molecular weight of the

smallest peptide or globular protein that will not enter the gel pore.

Sephadex G-10, the highest cross-linked dextran, has a water regain of

about 1mL/g of dry gel and Sephadex G-200, the lowest cross-linked

dextran, has a water regain of about 20 mL/g of dry gel. In the swelling

process, the gels become filled with water.

Fig.2. Structure of epichlorohydrin cross linked Dextran

Table 1: Properties of gels used in gel permeation (filtration) chromatography

Gel

Water

regain

(mL/g)

Exclusion

limit

Maximum

hydrostatic

pressure cm

H2O

Maximum

flow rate

(ml,min)

Sephadex G-10 1.0 700 200 100

Sephadex G-15 1.5 1500 200 100

Sephadex G-25 2.5 5000 200 50

Sephadex G-50 5.0 30000 200 25

Sephadex G-75 7.5 70000 160 6.4

Sephadex G-100 10.0 150000 96 4.2

Sephadex G-150 15.0 300000 36 1.9

Sephadex G-200 20.0 600000 16 1.0

Sepharose 6B NA 4 x 106 200 1.2

Sepharose CL 6B NA 4 x 106 >200 2.5


Sepharose CL 4B NA 20 x 106 120 2.17


Sepharose CL 2B NA 40 x 106 50 1.25

Bio-Gel P-2 1.5 1800 >100 110

Bio-Gel P-4 2.4 4000 >100 95

Bio-Gel P-6 3.7 6000 >100 75

Bio-Gel P-10 4.5 20000 >100 75

Bio-Gel P-30 5.7 40000 >100 65

Bio-Gel P-60 7.2 60000 100 30

Bio-Gel P-100 7.5 100000 100 30

Bio-Gel P-150 9.2 150000 100 25

Bio-Gel P-200 14.7 200000 75 11

Bio-Gel P-300 18.0 400000 60 6

Bio-Gel A-0.5m NA 500000 >100 3

Bio-Gel A-1.5m NA 1.5 x 106 >100 2.5

Bio-Gel A-5m NA 5 x 106 >100 1.5

Bio-Gel A-15m NA 15 x 106 90 1.5

Bio-Gel A-50m NA 50 x 106 50 1.0

Bio-Gel A-150m NA 150 x 106 30 0.5

Bio-Gel is a trade name of Bio-Rad Laboratories

Sephadex and Sepharose are trade name of Pharmacia Fine Chemical

Polyacrylamide gels are long polymers of acrylamide cross-linked with

N.N'methylene-bisacrylamide (Fig. 3).

Fig.3. Structure of cross-linked polyacrylamide

The gels are commercially produced by BioRad Laboratories, Richmond.

California, as the Bio-Gel P series. Like the Sephadex G series. the Bio-

Gels differ in degree of cross-linking and in pore size; the Bio-Gels,

however. have a wider range of pore sizes than is available in the

Sephadex G series See Table. 1 for the exclusion limits and properties of

the different Bio-Gels.

Agarose is a gel material with pore sizes larger than cross-linked dextran

or polyacrylamide. Agarose is the neutral polysaccharide fraction of agar.

It is composed of a linear polymer of D-galactopyranose linked ( 1->4)

3,6 anhydro-L-galactopyranose, which is linked (1-> 3) (Fig. 4).

D-galactose (-1->4) 3,6-Anhydro-L-galactose

Fig.4. Structure of the repeating unit of agarose, D-galactopyranose linked (-1->4)

to 3,6-anhydro-L-galactopyranose, which is linked (-1-3) to the next D-

galactopyranose residue

When the polysaccharide is dissolved in boiling water and cooled, it

forms a gel by forming inter-and intramolecular hydrogen bonds. The

pore sizes are controlled by the concentration of the agarose. High

molecular weight materials such as protein aggregates, chromosomal

DNA, ribosomes, viruses, and cells have been fractionated on agarose

gels. Bio-Rad markets the agarose Bio-Gel A series with different

molecular exclusion limits, and Pharmacia markets agarose as Sepharose

and Sepharose CL. The latter is Sepharose cross-linked by reacting with

alkaline 2,3-dibromopropanol to give an agarose gel with increased

thermal and chemical stability. Table 1 gives the properties of the

different Sephadex, Bio-Gel, and Sepharose gels.

The separations that may be achieved by gel permeation chromatography

are based on differences in the molecular sizes of the molecules. The

method is used for both preparative and analytical purposes. The latter

has been especially useful in determining the molecular weights of

proteins. The proteins are chromatographed on a gel column and the

elution volume of the protein determined. Proteins with known molecular

weights are also chromatographed and the elution volumes determined.

Then, from a plot of log molecular weight versus elution volume, the

molecular weight of an unknown protein may be determined (Fig. 5).

Fig.5. Molecular weight determination of proteins by gel permeation chromatography

using Sephadex G-100 as the gel bed: log molecular weight is plotted versus elution

volume.

Gel chromatography provides a rapid and mild method of removing salts

and other small molecules from high molecular weight biomolecules. The

sample containing the biomolecules and the salt is passed over a gel

column whose exclusion limit is below the molecular weight of the

biomolecules. The biomolecules which do not enter the gel emerge in the

void volume of the column, while the salts enter the gel and are retarded,

and therefore are removed from the biomolecules.

Ion-exchange chromatography

Ion-exchange chromatography is a variation of adsorption

chromatography in which the solid adsorbent has charged groups

chemically linked to an inert solid. Ions are electrostatically bound to the

charged groups; these ions may be exchanged for ions in an aqueous

solution. Ion exchangers are most frequently used in columns to separate

molecules according to charge. Because charged molecules bind to ion

exchangers reversibly. Molecules can be bound or eluted by changing the

ionic strength or pH of the eluting solvent.

Two types of ion exchanger are available: those with chemically

bound negative charges are called cation exchangers and those with

chemically bound positive charges are called anion exchangers. The

charges on the exchangers are balanced by counterions such as chloride

ions for the anion exchangers and metal ions for the cation exchangers.

Sometimes buffer ions are the counterions. The molecules in solution

which are to be adsorbed on the exchangers also have net charges which

are balanced by counterions. As an example of an ion-exchange process,

let us say that the molecules to he adsorbed from solution have a negative

charge (X-), which is counterbalanced by sodium ions (Na

+). Such

negatively charged molecules can be chromatographed on an anion

exchanger (A+), which has chloride ions as the counterion to give A

+Cl

-.

When (Na+ X

-) molecules in solution interact with the ion exchanger, the

X- displaces the chloride ion from the exchanger and becomes

electrostatically bound to give A+X

-, simultaneously releasing sodium

ions. This process of ion exchange is illustrated in Figure 1. A similar

but opposite process would take place for positively charged molecules

(Y+ Cl

-) which would be chromatographed on cation exchangers (C

-Na

+).

Thus the cation exchangers will bind positively charged molecules from

solution and the anion exchangers will bind negatively charged molecules

from solution.

One of the inert materials used in this type of chromatography is

cross-linked polystyrene, to which the charged groups are chemically

bound. In the separation of biologically important macromolecules,

such as enzymes and proteins.

Figure 1. The process of anion-exchange chromatography

Cellulose and cross-linked dextran (Sephadex) are used as the

solid supports and charged groups such as diethylaminoethyl (DEAE)

or carboxymethyl (CM) are chemically linked to them to give anion

and cation and the exchangers respectively. The preparation and

commercial availability of these materials beginning in the 1960 provided

the biochemist with powerful tools for separation of proteins and

nucleic acid Figure 2 presents partial structures of DEAE-cellulose and

CM –cellulose

Figure 2. Partial structures of diethylaminoethyl-cellulose and carboxymethyl-

cellulose. The DEAE and CM groups are shown attached to the C6-hydroxyl group

of glucose. The DEAE and CM groups are also found attached to the hydroxyl groups

of C2 and C3. The total degree of substitution of the DEAE and CM groups must be

less than one group per five glucose residues to maintain a water-insoluble product.

Table 1. Pretreatment steps for DEAE-cellulose and CM -cellulose ion

exchangers

Cellulose First treatment Intermediate

pH

Second

treatment

DEAE 0.5 M HCl 4 0.5 M NaOH

CM 0.5 M NaOH 8 0.5 M HCl

The dry ion-exchange celluloses are pretreated with acid and base to

swell the exchangers so that they become fully accessible to the charged

macromolecules in solution. The weighed exchanger is suspended in 15

volumes (w/v) of the "first treatment," acid or alkali depending on the

exchanger (Table. 1), and is allowed to stand at least 30 minutes but not

more than 2 hours. The supernatant is decanted and the exchanger is

washed until the effluent is at the "intermediate pH" The exchanger is

stirred into 15 volumes of the "second treatment" and allowed to stand for

an additional 30 minutes. The second treatment is repeated and the

exchanger is washed with distilled water until the effluent is close to

neutral pH. The treated exchanger is placed into the acid component of

the buffer (the pH should be less than 4.5) and degassed under vacuum

10 cm Hg pressure) with stirring, until bubbling stops The exchanger is

then titrated with the basic component of the buffer to the desired pH,

filtered, and suspended in fresh buffer to complete the pretreatment. The

exchanger is allowed to settle and the "fines" (fragments < 10 m in

diameter) above the settled exchanger are removed by decantation.

Buffer is added to the exchanger so that the final volume of the slurry is

l50% of the settled wet volume of the exchanger. The column is then

packed with the slurry of the exchanger, the sample is applied, and

elution is performed as described for adsorption chromatography.

Three general methods are used for eluting molecules from the

exchanger:

(a) Changing the pH of the buffer to a value at which binding is

weakened (i.e., the pH is lowered for an anion exchanger and raised for a

cation exchanger),

(b) Increasing the ionic strength by increasing the concentration of salt

in the elution solvent, thereby weakening the electrostatic interactions

between the adsorbed molecule and the exchanger, and

(c) Performing affinity elution. In affinity elution the adsorbed molecule

is usually a macromolecule that is desorbed from the affinity ligand by

adding a molecule that is charged and of opposite signs to the net

charge on the macromolecule and has a specific affinity for the

macromolecule. Thus, the reduction of the net charge on the

macromolecule weakens its electrostatic interaction with the exchanger

sufficiently to permit the elution of the macromolecule from the affinity

ligand.

The stages of anion exchange chromatography.

An example of the use of ions exchange resins

Is the purification of cytochrome C:

Cytochrome C has an isoelectric point (pI) of 10.05; that is at pH 10.05

the number of positive charges will equal the number of negative

charges. A cloumn containing a cation exchanger buffered, at pH 8.5,

is prepared. This column has a full negative charge. Cytochrome C at

pH 8.5 has a full positive charge. An Impure solution of cytochrome

C at pH 8.5 placed on the column, and water is passed through

the column (the pI of proteins is usually 7.0 or less) but

cytochrome C is held firmly by electrostatic attraction to the resin

heads. If the eluting solvent pH is raised to about 10, the

cytochrome C will now has a net zero charge and will pass rapidly

through as a pure component.

Gel Filtration

Ion exchange chromatography

Affinity chromatography

Histadine ,Aspartic,glycine,tyrosine

In anion exchange chromatography,which seprate first and why

AFFINITY CHROMATOGRAPHY

Affinity chromatography is a specialized type of adsorption

chromatography in which a specific type of molecule is covalently linked

to an inert solid support. This specific molecule called a ligand, has a

high binding affinity for one of the compounds in a mixture of

substances. The process uses the unique biological property of the

substance to bind to the ligand specifically and reversibly and provides a

high degree of selectivity in the isolation and purification of biological

molecules

Fig. 1. The steps of affinity chromatography

A solution containing the substance to be purified. Usually a

macromolecule such as a protein (enzyme, antibody, hormone. etc.).

Polysaccharide or nucleic acid is passed through a column containing an

insoluble inert polymer to which the ligand has been covalently attached.

The ligand may be specific competitive inhibitors, substrate analogues,

product analogues, coenzymes and so on. Molecules in the mixture not

having affinity for the ligand pass through the column. Wide molecules

that have specific affinity for the ligand are bound and retained on the

column. The specifically adsorbed molecules) can be eluted by changing

the ionic strength the pH or by the addition of a competing ligand. In one

chromatographic step. The method is capable of isolating a single

substance in a pure form. It has thus become a powerful tool in the

isolation and purification of enzymes, antibodies, antigens, nucleic acids.

Polysaccharides, coenzyme or vitamin binding proteins, repressor

proteins, transport proteins, drug or hormone receptor structures and

other biochemical materials.

The Inert Support and the Ligand

The inert solid supports are the same materials discussed in the

preceding sections: cross-linked dextran cross-linked polyacrylamide,

agarose and cellulose. The macromolecules to be separated should not be

retarded by a gel filtration process but should be retarded only by the

specific interaction with the ligand. The ligand must be a molecule that

display, special and unique affinity for the macromolecule to be purified

it also must have a chemical group that can be modified for covalent

linkage to the solid support without destroying or seriously decreasing its

interaction with the macromolecule to be purified. Also for successful

affinity chromatography, the chemical groups of the ligand that arc

critical for the binding of the macromolecule to be purified must be

sufficiently distant from the solid support to minimize steric interference

with the binding process. This steric problem has been solved by adding a

long, hydrocarbon chain spacer arm to the solid support and coupling the

ligand to the end of the arm. Alternatively the hydrocarbon arm may be

attached to the ligand and the arm attached to the solid support.

Attachment of the Ligand to the Solid Support

The polysaccharide solid supports-cross-linked dextran, agarose,

and cellulose can be activated by reaction with alkaline cyanogen

bromide. The products that arc formed upon coupling of the activated

polysaccharides with amino compounds are derivatives of amino carbonic

acid. The reactions are the following:

If the ligand contains an amino group, it can be coupled directly to

the activated polysaccharide. A spacer arm can be introduced by

sequential reaction with a diaminoalkane and glutaraldehyde. The amino

group on the ligand can then be coupled to the free aldehyde group.

If the ligand contains an aldehyde group instead of an amino group,

it can be coupled directly to the free amino group of the diaminoalkane.

Ligands may be coupled to polyacrylamide by displacing the amide group

of the polyacrylamide by heating with a diaminoalkane (c), followed by

reaction with glutaraldehyde (d).

The Schiff base that results from the reaction of glutaraldehyde

with an amino group may be stabilized by reduction with sodium

cyanoborohydride without affecting the aldehyde group. The ligand can

then be coupled to the aldehyde group.

Another method of activating polyacrylamide is to form the hydrazide

derivative by reaction with hydrazine hydrate. When an amino, aldehyde,

or hydrazide group is incorporated onto the solid support, the support

becomes activated so that ligands may be attached through amino,

carboxyl, phenolic, or imidazole groups.

Gel electrophoresis The movement of a charged presented by Equation 1.0 subjected to an

electric field:

(Equation 1.0)

where

E = the electric field in volts/cm

q = the net charge on the molecule

f = frictional coefficient, which depends on the mass and shape of the

molecule

V = the velocity of the molecule

The charged particle moves at a velocity that depends directly on the

electrical field (E) and charge (q) but inversely on a counteracting force

generated by the viscous drag (f ) The applied voltage represented by E in

Equation 1.0 is usually held constant during electrophoresis, although

some experiments are run under conditions of constant current (where the

voltage changes with resistance) or constant power (the product of

voltage and current). Under constant-voltage conditions, Equation 1.0

shows that the movement of a charged molecule depends only on the

ratio q/f. For molecules of similar conformation (for example, a

collection of linear DNA fragments or spherical proteins), varies with

size but not shape; therefore, the only remaining variables in Equation

1.0 are the charge (q) and mass dependence of (f ) meaning that under

such conditions molecules migrate in an electric field at a rate

proportional to their charge-to-mass ratio. The movement of a charged

particle in an electric field is often defined in terms of mobility, , the

velocity per unit of electric field (Equation 2.0).

(Equation 2.0)

This equation can be modified using Equation 1.0.

(Equation 3.0)

In theory, if the net charge, (q), on a molecule is known, it should be

possible to measure (f) and obtain information about the hydrodynamic

size and shape of that molecule by investigating its mobility in an electric

field. Attempts to define (f) by electrophoresis have not been successful,

primarily because Equation 3.0 does not adequately describe the

electrophoretic process. Important factors that are not accounted for in

the equation are interaction of migrating molecules with the support

medium and shielding of the molecules by buffer ions. This means that

electrophoresis is not useful for describing specific details about the

shape of a molecule. Instead, it has been applied to the analysis of purity

and size of macromolecules. Each molecule in a mixture is expected to

have a unique charge and size, and its mobility in an electric field will

therefore be unique. This expectation forms the basis for analysis and

separation by all electrophoretic methods The technique is especially

useful for the analysis of ammo acids, peptides, proteins, nucleotides,

nucleic acids, and other charged molecules.

Method of Electrophoresis

All types of electrophoresis are based on the principles just

outlined. The major difference between methods is the type of support

medium, which can be either cellulose or thin gels. Cellulose is used as a

support medium for low-molecular-weight biochemical such as ammo

acids and carbohydrates, and polyacrylamide and agarose gels are widely

used as support media for larger molecules. Geometries (vertical and

horizontal), buffers, and electrophoretic conditions for these two types of

gels provide several different experimental arrangements, as described

below.

Polyacrylamide Gel Electrophoresis (PAGE)

Gels formed by polymerization of acrylamide have several positive

features in electrophoresis:

A) High resolving power for small and moderately sized proteins

and nucleic acids (up to approximately 1 X 106 daltons),

B) Acceptance of relatively large sample sizes,

C) Minimal interactions of the migrating molecules with the

matrix, and

D) Physical stability of the matrix that gels can be prepared with

different pore sizes by changing the concentration of cross-linking

agents. Electrophoresis through polyacrylamide gels leads to enhanced

resolution of sample components because the separation is based on both

molecular sieving and electrophoretic mobility The order of molecular

movement in gel filtration and PAGE is very different, however in gel

filtration, large molecules migrate through the matrix faster than small

molecules The opposite is the case for gel electrophoresis, where there

is no void volume in the matrix, only a continuous network of pores

throughout the gel. The electrophoresis gel is comparable to a single bead

in gel filtration. Therefore, large molecules do not move easily through

the medium, and the rate of movement is small molecules followed by

large molecules.

Polyacrylamide gels are prepared by the free radical polymerization

of acrylamide and the cross-linking agent N,N'- methylene-bis-

acrylamide. Chemical polymerization is controlled by an initiator-catalyst

system, ammonium persulfate-N,N,N,َN َ tetramethylethylenediamine

(TEMED). Photochemical polymerization may be initiated by riboflavin

in the presence of ultraviolet (UV) radiation. A standard gel for protein

separation is 7.5% polyacrylamide. It can be used over the molecular size

range of 10,000 to 1,000,000 daltons; however, the best resolution is

obtained in the range of 30,000 to 300,000 daltons. The resolving power

and molecular size range of a gel depend on the concentrations of

acrylamide and bis-acrylamide Lower concentrations give gels with

larger pores, allowing analysis of higher-molecular-weight biomolecules

In contrast, higher concentrations of acrylamide give gels with smaller

pores, allowing analysis of lower-molecular-weight biomolecules

(Table 1.0) Effective Range of Separation of DNA by PAGE

Acylamide

(% W/V)

Range of Separation

(bp)

Bromophenol

Blue

Xylene Cyanol

35 1000-2000 100 450

50 80-500 65 250

80 60-400 50 150

120 40-200 20 75

200 5-100 10 50

Polyacrylamide electrophoresis can be done using either of two

arrangements, column or slab. Figure 1.0 shows the typical arrangement

for a column gel. Glass tubes (10 cm X 6 mm l.d.) are filled with a

mixture of acrylamide, N,N'-methylene-bis-acrylamide, buffer, and free

radical initiator catalyst. Polymerization occurs in 30 to 40 minutes. The

gel column is inserted between two separate buffer reservoirs. The upper

reservoir usually contains the cathode and the lower the anode. Gel

electrophoresis is usually carried out at basic pH, where most biological

polymers are anionic; hence, they move down toward the anode. The

sample to be analyzed is layered on top of the gel and voltage is applied

to the system. A "tracking dye" is also applied, which moves more

rapidly through the gel than the sample components. When the dye band

has moved to the opposite end of the column, the voltage is turned off

and the gel is removed from the column and stained with a dye.

Chambers or column gel electrophoresis is commercially available or can

be constructed from inexpensive materials.

Slab gels are now more widely used than column gels. A slab gel on

which several samples may be analyzed is more convenient to make and

use than several individual column gels. Slab gels also offer the

advantage that all samples are analyzed m a matrix environment that is

identical in composition. A typical vertical slab gel apparatus is shown in

Figure 2.0.

The polyacrylamide slab is prepared between two glass plates that are

separated by spacers Figure 3.0.

The spacers allow a uniform slab thickness of 0.5 to 2.0 mm, which is

appropriate for analytical procedures. Slab gels are usually 8 X 10 cm or

10 X 10 cm, but for nucleotide sequencing, slab gels as large as 20 X 40

cm are often required. A plastic "comb" inserted into the top of the slab

gel during polymerization forms indentations in the gel that serve as

sample wells. Up to 20 sample wells may be formed. After

polymerization, the comb is carefully removed and the wells are rinsed

thoroughly with buffer to remove salts and any unpolymerized

acrylamide. The gel plate is clamped into place between two buffer

reservoirs, a sample is loaded into each well, and voltage is applied. For

visualization, the slab is removed and stained with an appropriate dye.

Perhaps the most difficult and inconvenient aspect of

polyacrylamide gel electrophoresis is the preparation of gels. The

monomer, acrylamide, is a neurotoxin and a cancer suspect agent; hence,

special handling is required. Other necessary reagents including catalysts

and initiators also require special handling and are unstable- In addition,

it is difficult to make gels that have reproducible thicknesses and

compositions. Many researchers are now turning to the use of precast

polyacrylamide gels. Several manufacturers now offer gels precast in

glass or plastic cassettes. Gels for all experimental operations are

available including single percentage (between 3 and 27%) or gradient

gel concentrations and a variety or sample well configurations and buffer

chemistries. Several modifications of PAGE have greatly increased its

versatility and usefulness as an analytical tool.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophorosis

(SDS-PAGE)

The electrophoretic techniques previously discussed are not applicable

to the measurement of the molecular weights of biological molecules

because mobility is influenced by both charge and size. If protein samples

are treated so that they have a uniform charge, electrophoretic mobility

then depends primarily on size (see Equation 2.0). The molecular

weights of proteins may be estimated if they are subjected to

electrophoresis in the presence of a detergent, sodium dodecyl sulfate

(SDS), and a disulfide bond reducing agent, mercaptoethanol. This

method is often called "denaturing electrophoresis." When protein

molecules are treated with SDS, the detergent disrupts the secondary,

tertiary, and quaternary structure to produce linear polypeptide chains

coated with negatively charged SDS molecules. The presence of

mercaptoethanol assists in protein denaturation by reducing all disulfide

bonds. The detergent binds to hydrophobic regions of the denatured

protein chain in a constant ratio of about 14 g of SDS per gram of protein.

The bound detergent molecules carrying negative charges mask the native

charge of the protein In essence, polypeptide chains of a constant

charge/mass ratio and uniform shape are produced The electrophoretic

mobility of the SDS-protein complexes is influenced primarily by

molecular size the larger molecules are retarded by the molecular sieving

effect of the gel, and the smaller molecules have greater mobility

Empirical measurements have shown a linear relationship between the

log molecular weight and the electrophoretic mobility Figure 4.0

In practice, a protein of unknown molecular weight and subunit

structure is treated with 1% SDS and 0.1 M mercaptoethanol in

electrophoresis buffer. A standard mixture of proteins with known

molecular weights must also be subjected to electrophoresis under the

same conditions. Two sets on standards are commercially available, one

for low-molecular-weight proteins (molecular weight range 14,000 to

100,000) and one for high-molecular weight proteins D5,000 to 200,000)

Figure 5.0

a stained gel after electrophoresis of a standard protein mixture

After electrophoresis and dye staining, mobilities are measured and

molecular weights determined graphically SDS-PAGE is valuable for

estimating the molecular weight of protein subunits This modification of

gel electrophoresis finds its greatest use in characterizing the sizes and

different types of subunits in oligomeric proteins. SDS-PAGE is limited

to a molecular weight range of 10,000 to 200,000. Gels of less than

2.5% acrylamide must be used for determining molecular weights above

200,000, but these gels do not set well and are very fragile because of

minimal cross-linking. A modification using gels of agarose-acrylamide

mixtures allows the measurement of molecular weights above 200,000.

Agarose Gel Electrophoresis

The electrophoretic techniques discussed up to this point are useful for

analyzing proteins and small fragments of nucleic acids up to 350,000

daltons (500 bp) in molecular size; however, the small pore sizes in the

gel are not appropriate for analysis of large nucleic acid fragments or

intact DNA molecules. The standard method used to characterize RNA

and DNA in the range 200 to 50,000 base pairs 50 kilobases) is

electrophoresis with agarose as the support medium.

Agarose, a product extracted from seaweed, is a linear polymer of

galactopyranose derivatives. Gels are prepared by dissolving agarose in

warm electrophoresis buffer. After cooling the gel mixture to 50°C, the

agarose solution is poured between glass plates as described for

polyacrylamide. Gels with less than 0.5% agarose are rather fragile and

must be used in a horizontal arrangement (Figure 4.8). The sample to be

separated is placed in a sample well made with a comb, and voltage is

applied until separation is complete. Precast agarose gels of all shapes,

sizes, and percent composition are commercially available. Nucleic acids

can be visualized on the slab gel after separation by soaking in a solution

of ethidium bromide, a dye that displays enhanced fluorescence when

intercalated between stacked nucleic acid bases. Ethidium bromide may

be added directly to the agarose solution before gel formation. This

method allows monitoring of nucleic acids during electrophoresis.

Irradiation of ethidium bromide treated gels by UV light results in

orange-red bands where nucleic acids are present. The mobility of nucleic

acids in agarose gels is influenced by the agarose concentration and the

molecular size and molecular conformation of the nucleic acid. Agarose

concentrations of 0.3 to 2.0% are most effective for nucleic acid

separation Table 2.0

Figure 6.0

The separation of DNA fragments on agarose gels. Like

proteins, nucleic acids migrate at a rate that is inversely proportional

to the logarithm of their molecular weights; hence, molecular weights

can be estimated from electrophoresis results using standard nucleic

acids or DNA fragments of known molecular weight. The DNA

conformations most frequently encountered are superhelical circular

(form I), nicked circular (form II), and linear (form III). The small,

compact, supercoiled form I molecules usually have the greatest mobility,

followed by the rodlike, linear form III molecules. The extended, circular

form II molecules migrate more slowly. The relative electrophoretic

mobility of the three forms of DNA, however, depends on experimental

conditions such as agarose concentration and ionic strength.

Isoelectric Focusing of Proteins

Another important and effective use of electrophoresis for the analysis of

Proteins are isoelectric focusing (IEF), which examines electrophoretic

mobility as a function of pH. The net charge on a protein is pH

dependent. Proteins below their isoelectric pH (pHI or the pH at which

they have zero net charge) are positively charged and migrate to a

medium of fixed pH toward the negatively charged cathode at a pH

above its isoelectric point, a protein is deprotonated and negatively

charged and migrates toward the anode If the pH of the electrophoretic

medium is identical to the pHI of a protein, the protein has a net charge

of zero and does not migrate toward either electrode. Theoretically, it

should be possible to separate protein molecules and to estimate the pH:

of a protein by investigating the electrophoretic mobility in a series of

separate experiments in which the pH of the medium is changed. The pH

at which there is no protein migration should coincide with the pHI of the

protein. Because such a repetitive series determine the pHI, IEF has

evolved as an alternative method for performing a single electrophoresis

run in a medium of gradually changing pH.

Figure 7.0

illustrates the construction and operation of an IEF pH gradient. An acid,

usually phosphoric, is placed at the cathode; a base, such as

triethanolamine, is placed at the anode. Between the electrodes is a

medium in which the pH gradually increases from 2 to 10. The pH

gradient can be formed before electrophoresis is conducted or formed

during the course of electrophoresis. The pH gradient can be either broad

(pH 2-10) for separating several proteins of widely ranging pHI values or

narrow (pH 7-8) for precise determination of the pHI of a single protein. P

in Figure 7.0 represents different molecules of the same protein in two

different regions of the pH gradient. Assuming that the pH in region 1 is

less than the pHI of the protein and the pH in region 2 is greater than the

pHI of the protein, molecules of P in region 1 will be positively charged

and will migrate m an applied electric field toward the cathode. As P

migrates, it will encounter an increasing pH, which will influence its net

charge. As it migrates up the pH gradient, P will become increasingly

deprotonated and its net charge will decrease toward zero. When P

reaches a region where it's net charge is zero (region 3), it will stop

migrating. The pH in this region of the electrophoretic medium will

coincide with the pHI of the protein and can be measured with

Illustration of isoelectric a surface microelectrode, or the position of the

protein can be compared to that of a calibration set of proteins of bown

pHI values. P molecules in region 2 will be negatively charged and will

migrate toward the anode. In this case, the net charge on P molecules will

gradually decrease to zero as P moves down the pH gradient, and P

molecules originally in region 2 will approach region 3 and come to rest.

The P molecules move in opposite directions, but the final outcome of

IEF is that P molecules located anywhere m the gradient will migrate

toward the region corresponding to their isoelectric point and will

eventually come to rest in a sharp band; that is, they will "focus" at a

point corresponding to their pHI. Since different protein molecules in

mixtures have different pHI values, it is possible to use IEF to separate

proteins In addition; the pHI of each protein in the mixture can be

determined by measuring the pH of the region where the protein is

focused. The pH gradient is prepared in a horizontal glass tube or slab.

Special precautions must be taken so that the pH gradient remains stable

and is not disrupted by diffusion or convective mixing during the

electrophoresis experiment. The most common stabilizing technique is to

form the gradient in a polyacrylamide, agarose, or dextran gel. The pH

gradient is formed in the gel by electrophoresis of synthetic

polyelectrolyte, called ampholytes, which migrate to the region of their

pHI values just as proteins do and establish a pH gradient that is stable for

the duration of the IEF run. Ampholytes are low-molecular-weight

polymers that have a wide range of isoelectric points because of their

numerous ammo and carboxyl or sulfonic acid groups. The polymer

mixtures are available in specific pH ranges (pH 5-7, 6-8, 3.5-10, etc.). It

is critical to select the appropriate pH range for the ampholyte so that the

proteins to be studied have pHI values in that range. The best resolution

is, of course, achieved with an ampholyte mixture over a small pH range

(about two units) encompassing the pHI of the sample proteins. If the pHI

values for the proteins under study are unknown, an ampholyte of wide

pH range (pH 3-10) should be used first and then a narrower pH range

selected for use. The gel medium is prepared as previously described

except that the appropriate ampholyte is mixed prior to polymerization.

The gel mixture is poured into the desired form (column tubes, horizontal

slabs, etc.) and allowed to set. Immediately after casting of the gel, the

pH is constant throughout the medium, but application of voltage will

induce migration of ampholyte molecules to form the pH gradient. The

standard gel for proteins with molecular sizes up to 100,000 daltons is

7.5% polyacrylamide; however, if larger proteins are of interest, gels with

larger pore sizes must be prepared. Such gels can be prepared with a

lower concentration of acrylamide (about 2%) and 0.5 to 1% agarose to

add strength. Precast gels for isoelectric focusing are also commercially

available. The protein sample can be loaded on the gel in either of two

ways. A concentrated, salt-free sample can be layered on top of the gel as

previously described for ordinary gel electrophoresis. Alternatively, the

protein can be added directly to the gel preparation, resulting in an even

distribution of protein throughout the medium. The protein molecules

move more slowly than the low-molecular-weight ampholyte molecules,

so the pH gradient is established before significant migration of the

proteins occurs. Very small protein samples can be separated by IER. For

analytical purposes, 10 to 50 g is a typical sample size. Larger sample

sizes (up to 20 mg) can be used for preparative purposes.

Common abbreviations in chromatography

GF: gel filtration (sometimes referred to as SEC: size exclusion

chromatography)

IEX: ion exchange chromatography (also seen as IEC)

AC: affinity chromatography

RPC: reverse phase chromatography

HIC: hydrophobic interaction chromatography

CIPP: Capture, Intermediate Purification and Polishing

MPa: megapascals

psi: pounds per square inch

SDS: sodium dodecyl sulphate

CIP: cleaning in place

A280nm, A214nm:

UV absorbance at specified wavelength

Mr: relative molecular weight

N: column efficiency expressed as theoretical plates per meter

Ve: elution volume is measured from the chromatogram and

relates to the molecular size of the molecule.

Vo: void volume is the elution volume of molecules that are

excluded from the gel filtration medium because they are

larger than the largest pores in the matrix and pass straight

through the packed bed

Vt: total column volume is equivalent to the volume of the packed

bed

Rs: resolution, the degree of separation between peaks

Kav and

logMr:

partition coefficient and log molecular weight, terms used

when defining the selectivity of a gel filtration medium

In product names

HMW: high molecular weight

LMW: low molecular weight

HR: high resolution

pg: prep grade

PC: precision column

SR: solvent resistant

Protein

------------Negative--------------------------o-----------------------Positive-----------------------

(pH>pI) (pH=pI) (pH<pI)

anion exchange resin cation exchange resin

(resin is positive A+) (resin is negative C-)

At pH > pI, protein net charge is negative At pH < pI, protein net charge is positive At pH = pI, protein net charge is zero

Isoelectric point (pI)

At pH > pI, use an anion exchange resin (positive resin) At pH < pI, use a cation exchange resin(negative resin)

nonpolar molecules lack charge

polar, uncharged molecules carry one or more partial charges

-Organic component of the solvent

continues migrating, thus forming the

mobile phase.

-Therefore, compounds soluble to organic

component move faster than compounds

soluble to aqueous component.

-Thus, molecules are separated according

to their polarities.

Differential precipitation Salt (or other solute) is added to a solution that contains a

mixture of proteins

Ammonium sulfate is most popular for “salting out”

-Effective, highly soluble, & does not tend to denature protein

Individual proteins precipitate at specific [(NH4)2SO4]

Depends on properties of specific solute (salt), not ionic strength

per se

Precipitated proteins are:

- isolated in centrifuge

- resuspended in low salt buffer

- dialysis or gel filtration can be used to remove residual

precipitant (if necessary)

Often used in two steps:

• Salt out some impurities w/ lower concentration that does not

precipitate POI

• Use higher concentration for selective precipitation of POI

Protein purification

Prior requirements for devising a purification method:

• Source tissue

– Known or likely to contain “high” levels of POI

– Available in suitable (preparative) quantities

Possible (& potentially misleading) alternative: clone gene &

use expression

system

• Exogenous system may not recapitulate protein

processing/covalent modifications

• POI may function as part of a complex (w/ addn’l subunits

derived from other

genes)

• Assay method that is:

– Specific for POI & relatively insensitive to other components

in extracts

– Linear and quantitative:

• measured activity should be proportional to amount of POI

• Amount of activity is expressed in “units”:

– e.g., amount of S � P per time

– “katal” (kat) is preferred unit: moles per second

– If POI is not enzyme, assay may be based on binding (direct or

indirect)

– Suitably sensitive

– Optimized with respect to pH, ionic strength, temp., substrate

or ligand

concentration, etc.

The polymerization reaction of acrylamide and methylenebisacrylamide

The three major forms of alanine occurring in titrations between pH 1 and 14

4. F. F. Runge, Farbenchemie, I and II (1834, 1843). 5. F. F. Runge, Ann. Phys. Chem. XVII, 31, 65 (1834); XVIII, 32, 78 (1834). 6. F. F. Runge, Farbenchemie III, 1850. 7. F. Goppelsroeder, Zeit. Anal. Chem. 7, 195 (1868). 8. C. Sch¨onbein, J. Chem. Soc. 33, 304–306 (1878). 9. D. T. Day, Proc. Am. Philos. Soc. 36, 112 (1897). 10. D. T. Day, Congr. Intern. P´etrole Paris 1, 53 (1900). 11. D. T. Day, Science 17, 1007 (1903). 12. M. Twsett, Ber. Deut. Bot. Ges. XXIV 316 (1906). 13. M. Twsett, Ber. Deut. Bot. Ges. XXIV 384 (1906). 14. M. Twsett, Ber. Deut. Bot. Ges. XXV 71–74 (1907). 15. R. Kuhn, A. Wunterstein, and E. Lederer, Hoppe-Seyler’s Z. Physiol. Chem. 197, 141–160 (1931). 16. A. Tiselius, Ark. Kemi. Mineral. Geol. 14B(22) (1940). 17. J. N. Wilson, J. Am. Chem. Soc. 62, 1583–1591 (1940). 18. A. Tiselius, Ark. Kemi. Mineral. Geol. 15B(6) (1941). 19. A. J. P. Martin and R. L. M. Synge, Biochem. J. (Lond.) 35, 1358 (1941). 20. R. Consden, A. H. Gordon, and A. J. P. Martin, Biochem. J. 38, 224–232 (1944). 21. S. Claesson, Arkiv. Kemi. Mineral. Geol. 23A(1) (1946). 22. A. J. P. Martin, Biochem. Soc. Symp. 3, 4–15 (1949). 23. E. Cremer and F. Prior, Z. Elektrochem. 55, 66 (1951); E. Cremer and R. Muller, ZElektrochem. 55, 217 (1951). 24. C. S. G. Phillips, J. Griffiths, and D. H. Jones, Analyst 77, 897 (1952). 25. A. T. James and A. J. P. Martin, Biochem. J. 50, 679–690 (1952). 26. E. Glueckauf, in Ion Exchange and Its Applications, Society of the Chemical Industry, London, 1955, pp.34–36. 27. J. J. van Deemter, F. J. Zuiderweg, and A. Klinkenberg, Chem. Eng. Sci. 5, 271–289 (1956). 28. M. J. E. Golay, in Gas Chromatography, V. J. Coates, H. J. Noebels, and I. S. Fagerson, eds., Academic Press, New York, 1958, pp. 1–13. 29. J. C. Giddings, Dynamics of Chromatography, Part I, Principles and Theory, Marcel Dekker, New York, 1965, pp. 13–26.

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