1 chromatography is a separation method in which the analyte is contained in a mobile phase and...

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

Chapter One

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

Chromatography is a separation method in which the analyte is

contained in a mobile phase and pumped through a stationary phase.

Sample components interact differently with these two phases and elute

from the column at different retention times tR. Since the first description

of chromatography by Russian botanical scientist Mikhail Semenovich

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.

Chromatographic separations can be described quantitatively with a number

of parameters including the capacity factor k , the selectivity factor α, the

plate number N or height equivalent of a theoretical plate H and the

resolution RS. The optimum flow rate of a chromatographic separation can

be determined with the van Deemter equation. In bioanalytical chemistry,

chromatography is mainly employed for the isolation and purification of

proteins. Reversed phase chromatography can separate biomolecules

according to their interaction with the hydrophobic stationary phase and

the hydrophilic moblile phase. This separation method can be coupled to

an ESI mass spectrometer. Ion exchange chromatography separates

molecules depending on their net charge. Affinity chromatography makes

use of molecular recognition between biomolecules; and size exclusion

chromatography allows for the separation of molecules depending on

their size.

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.


Chromatography is a technique for separating a sample into various

fractions. The heart of any chromatography is the stationary phase (liquid or

solid). The stationary phase is attached to a support, a solid inert material.

The sample (gas, liquid or solid which may or may not dissolved in solvents)

is carried across the stationary phase by a mobile phase (gas or liquid). The

sample components undergo a series of exchanges (partitions) between the

two phases due to the differences in their chemical and physical properties.

These differences govern the rate of movement (called migration) of the

individual components.

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.


5-Purpose of Chromatography

Analytical - determine chemical composition of a sample

Preparative - purify and collect one or more components of a sample

Other classification of Chromatographic Methods

Chromatography is classified according to three ways:


1. According to the physical state of the mobile phase:

Liquid chromatography: This subdivided according to the stationary

phase into liquid-liquid or liquid-solid chromatography.

Gas chromatography: This subdivided according to the stationary

phase into Gas-liquid or Gas-solid chromatography.

2. According to the method of contact between the mobile phase and

stationary phase:

Column chromatography: the stationary phase is placed in a column

through which the mobile phase moves under the influence of gravity or

pressure. The stationary phase is either a solid or a thin, liquid film

coating on a solid particulate packing material or the column’s walls.

Planar chromatography: the stationary phase coats a flat glass, metal, or

plastic plate and is placed in a reservoir containing the mobile phase

which moves by capillary action carrying with it the sample components

3. According to the chemical or physical mechanism responsible for

separating the sample’s constituents.(attractive forces)

Adsorption chromatography: for polar non-ionic compounds

Ion Exchange chromatography: for ionic compounds

Anion: analyte is anion; bonded phase has positive charge

Cation: analyte is cation; bonded phase has negative charge

Partition chromatography: based on the relative solubility of analyte in

mobile and stationary phases

Normal: analyte is non-polar organic; stationary phase MORE polar

than the mobile phase


Reverse: analyte is polar organic; stationary phase LESS polar than

the mobile phase

Size Exclusion chromatography: stationary phase is a porous matrix.

6-The Principle of Chromatography

Chromatography is a separation method where the analyte is contained

within a liquid or gaseous mobile phase, which is pumped through a

stationary phase.

Usually, one phase is hydrophilic and the other lipophilic. The components of

the analyte interact differently with these two phases. Depending on their

polarity, they spend more or less time interacting with the stationary phase

and are thus retarded to a greater or lesser extend. This leads to the

separation of the different components present in the sample. Each sample

component elutes from the stationary phase at a specific time, its retention

time tR (Fig. 1.1). As the components pass through the detector, their signal

is recorded and plotted in the form of a chromatogram.

Chromatographic methods can be classified into

Gas chromatography (GC) and liquid chromatography (LC) depending on

the nature of the mobile phase involved.

Gas chromatography can be applied only to gaseous or volatile substances

that are heat-stable. The mobile phase, an inert carrier gas such as

nitrogen, hydrogen or helium, is pumped through a heated


Figure1-1

column. This column can be packed with a silicon oxide based material or

is coated with a polymeric wax. The sample is vaporised, pumped through

the column and the analytes are detected in the gas stream as they exit the

column. Analyte detection can be achieved by either flame ionisation or

thermal conductivity. GC is not commonly used for the analysis of

biomolecules since large molecular weight compounds such as peptides and

proteins are thermally destroyed before evaporation. Smaller molecules such

as amino acids, fatty acids, peptides and certain carbohydrates can be

analysed if they are modified chemically to increase their volatility. Some cell

cultures produce volatile metabolites such as aldehydes, alcohols or

ketones. These can be analysed readily via GC.

In liquid chromatography, the sample is dissolved and pumped through a

column containing the stationary phase. LC is more versatile than GC as it is

not restricted to volatile and heat-stable samples; the sample only has to

dissolve completely in the mobile phase. Common detection methods are

UV spectroscopy, measurement of refractive index, fluorescence, electrical

conductivity and mass spectrometry.


Modes of operation can be classified as normal and reversed phase

chromatography.

In normal phase chromatography, the stationary phase consists of a

hydrophilic material such as silica particles and the mobile phase is a

hydrophobic organic solvent such as hexane.

In reversed phase chromatography, on the other hand, the stationary

phase is hydrophobic and the mobile phase is a mixture of polar solvents, for

example water and acetonitrile. Biomolecules are generally soluble in polar

solvents; hence, reversed phase chromatography is the method of choice for

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

7-Comparison of classical and bioanalytical chemistry


8-Application of Liquid Chromatography for Bioanalysis

In bioanalytical chemistry, chromatography is mainly used for the

separation, isolation and purification of proteins from complex sample

matrices. In cells, for example, proteins occur alongside numerous

other compounds such as lipids and nucleic acids. In order to be

analysed, these proteins must be separated from all the other cell

components. Then the protein of interest might have to be isolated from

other proteins and purified further. Chromatography is an essential part of

almost any protein purification strategy. A number of different

chromatographic techniques are used for the purification and analysis of

proteins. They can be classified according to the physical principle involved

in the separation process.

Typical examples include reversed phase chromatography, ion

exchange chromatography, affinity chromatography and size exclusion

chromatography (SEC) (Table 1.1). These are outlined in more detail in the

following sections.

Separation methods for proteins9- Basic Chromatographic Theory


The optimisation of chromatography is aimed towards completely

separating all of the components of a sample in the shortest possible time.

This can, for example be achieved by modifying the composition of the

mobile phase, choosing a different stationary phase or by changing the flow

rate. A typical chromatogram is depicted in (Fig. 1.2).

Fig. 1.2. Definition of retention time, tR, and peak width, w.

The sample is injected into the chromatographic column at t = 0 s.

Substances that are not retarded by the stationary phase leave the column

at zero retention time, t0, corresponding to the flow rate of the mobile

phase. Compounds A and B are retarded by the stationary phase and leave

the column at their retention times tR (A) and tR(B), respectively. The peak

width, w, is defined as the intersection of the tangents on each side of the

peak with the baseline. These basic parameters, retention time and peak

width, can be used to derive a number of other parameters that express the

quality of the achieved chromatographic separation. In the following

paragraphs, a brief summary of the most important parameters of

chromatographic theory are discussed.

The capacity factor k' (equation1. 1) describes the velocity of the analyte

relative to the velocity of the mobile phase. Each compound spends a


different amount of time interacting with the mobile and stationary phase.

The average velocity of a sample compound is dependent on how much time

it spends in the mobile phase. If k' is much smaller than 1, then the analyte

moves too quickly and the elution time is so short that an exact

determination of tR is difficult. If the sample moves too slowly, the separation

time is very high. A good value for k’ would be between about 1 and 5. The

selectivity factor α (equation 1.2) describes the relative velocities of the

analytes with respect to each other. The selectivity describes how well a

chromatographic method can distinguish between two analytes.

capacity factor

(equation 1.1)

selectivity factor

(equation 1.2)

The efficiency of a chromatographic separation is dependent on band

broadening. If band broadening is large, peaks can overlap and resolution is

lost.

Band broadening for a column of length L is quantitatively expressed in the

concept of height equivalent of a theoretical plate, H, or simply plate

numbers, N (equations 1.3 and 1.4). The larger the number of plates N

and the smaller H is, the better the chromatographic efficiency.


(equations 1.3)

(equations 1.4)

The parameters that influence band broadening can be approximated by the

van Deemter equation (equation 1.5) which is valid for gas and liquid

chromatography as well as capillary electrophoresis

(equations 1.5)

In this simplified equation, the height of theoretical plates, H, is given as a

sum of three terms. The first term, A, describes the influence of the column

packing on band broadening. This so-called Eddy diffusion is constant for

a given column and independent of the flow rate. The second term, B/u,

describes the diffusion in or opposed to the direction of flow. This

longitudinal diffusion is inversely proportional to the flowrate u. The third

term, C·u, describes the resistance to mass transfer between the stationary

and mobile phase which is directly proportional to the flow rate. By plotting H

as a function of u, the optimum flow rate for a chromatographic separation

can be determined (Fig. 1.3).


Fig. 1.3. A van Deemter plot for the determination of the optimum flow rate.

The ultimate goal of a separation is to achieve a high resolution, Rs,

(equations 1.6 and 1.7). If Rs = 1.5, then peaks of identical area overlap by

only 0.3 %, an Rs = 1 equals a peak overlap of 4 %. Peak resolution can be

optimized by increasing the selectivity and minimizing band broadening.

Resolution

(equations 1.6)

valid for α < 1.2

(equations 1.7)


As can be seen from equation 1.7, the capacity factor k' has a great

influence on the resolution. Usually the components in the sample have a

wide variety of k values. If conditions are optimised such that the first

compounds to elute have k' values between the optimum of 1 and 5, then the

other compounds with higher k values elute much later and show excessive

band broadening. If, on the other hand, conditions are optimised for the later

eluting compounds, then the resolution will be poor for the compounds that

elute first. This general elution problem can be overcome by decreasing k'

during the separation. In LC, the composition of the mobile phase can be

changed during the separation. This is called a gradient elution as

opposed to an isocratic elution, where the composition of the mobile phase

remains unchanged during the separation process. In GC, a temperature

gradient can be applied during separation rather than operating under

isothermic conditions. Generally, the first step in trying to achieve a good

separation of the sample mixture is to choose a stationary phase with which

the analyte can interact. Then, the composition and gradient of the mobile

phase can be chosen to optimise the capacity factor and resolution.

Chromatographic theory as outlined in the above paragraphs can be

applied to the analysis of smaller molecules such as amino acids,

peptides and short biopolymers. Care has to be taken for larger

biomolecules such as high molecular weight proteins. These often show

different behavior and the theory can only be applied to a limited extent.


10-Reversed Phase Liquid Chromatography

Normal phase chromatography was developed many years before reversed

phase chromatography was investigated. Initially, stationary phases were

made of polar materials such as paper, cellulose or silica gel and the

mobile phase consisted of non-polar solvents such as hexane or

chloroform. Only at a later stage were these phase polarities reversed. Polar

solvents such as water and acetonitrile were

Fig. 1.4. Surface groups used for stationary phases in reversed phase

chromatography range from ethyl silane (C2) to n-octadecyl silane (C18).

used in combination with non-polar stationary phases. These were obtained

by etherification of the polar hydroxyl groups of the silica gel with long alkyl

chains. Reversed phase chromatography is the method of choice for the

separation of smaller biomolecules such as peptides, amino acids,

carbohydrates and steroids, which are soluble in water/acetonitrile

mixtures. The separation of proteins can be problematic as organic solvents

such as acetonitrile can decrease the protein’s solubility and cause

denaturisation.

The stationary phase usually consists of porous silica particles with non-

polar surface groups (Fig. 1.4), obtained from etherification of the initial


hydroxyl groups of the silica particle with silanes containing non-polar

hydrocarbon chains. Any chain length from ethyl silane (C2) to n-octadecyl

silane (ODS) (C18) is used, although octyl silane (C8) and ODS are the most

commonly employed chain lengths. For analytical separations, the particle

size is typically 5 μm or smaller. In preparative liquid chromatography, where

the goal is to isolate a compound of interest for further analysis or

investigation, larger particles with a higher capacity and larger column

diameters are used. The pore size of the silica particles is usually about 10

nm, resulting in a very large surface area, as much as 100 to 400 m2/g.

This gives the analytes ample opportunity to interact with the stationary

phase whilst flowing through the separation column.

The mobile phase is based on a polar solvent system consisting of an

aqueous buffer and acetonitrile or methanol. Gradient elution is often

employed to increase resolution and shorten separation times. This is

achieved by increasing the organic solvent and thus decreasing the mobile

phase polarity and the retention of less polar analytes during the separation

process. Solvents can be classified according to their elution strength and

polarity (Fig. 1.5).

Buffer systems based on ammonium acetate, phosphate or hydrogen

carbonate are usually added at concentrations of about 20 mM to adjust the

pH of the mobile phase to values between 2 and 8. Ion pairing reagents can

be used at low concentrations, typically 0.1%, to increase the hydrophobicity

of charged analytes. They


Fig. 1.5. Solvents ordered according to polarity and elution speed of the

analytes.

Fig. 1.6. Instrumental setup of an HPLC gradient system.

form ion-pair complexes with the analyte. Anionic ion pairing reagents such

as trifluoroacetic acid (TFA) bind to positively charged analytes,

whereas cationic ion pairing reagents such as tetra alkyl ammonium salts

can be used to bind to negatively charged analytes. These complexes are

retarded more by the stationary phase and are thus easier to separate than

the largely unretained charged analytes alone.

In modern chromatography, the separation columns are tightly packed with

small particles of about 1–5μm in diameter. To achieve ambient flow rates in

these columns, high pressures of up to 300–400 bars must be generated. A

typical instrumental setup for this high pressure or high performance liquid

chromatography (HPLC) is shown in Fig. 1.6.


Computer controlled pumps move the mobile phase through the system.

Aqueous solvent A and organic solvent B are mixed to the desired

composition. In the case of gradient elution, the composition is gradually

altered during the separation.

Sample volumes are injected with either a manual loop and valve system or

automatically via an auto sampler. Depending on the column dimensions

sample volumes can be as low as several nL and as high as a mL. Often the

column is situated inside an oven which is thermostatically regulated to

maintain a constant temperature. After eluting from the column, the analytes

pass through the detector.

UV detection using a fixed wavelength could be performed at λ = 210 nm

for peptides and λ = 254 nm or λ = 280 nm for proteins. More expensive

instruments have diode array detectors (DAD) which can take several

whole spectra per second and allow for more unambiguous identification.

High sensitivity can be achieved via fluorescence detection of derivatised

amino acids and peptides. A more recent development is to couple

liquid chromatography systems to an electrospray ionisation mass

spectrometer, ESI-MS.

Mass spectrometry allows universal detection at very high sensitivity and

also gives structural information about the analyte. However, not all buffers

commonly employed for liquid chromatography are compatible with mass

spectrometers.

In recent years, there has been a trend to develop ever smaller liquid

chromatography systems. LC systems on micro and even nanoscales have

been demonstrated. Shorter and smaller columns with smaller particles offer

faster analysis times, decreased solvent consumption and require less


sample. The differences between preparative, analytical, micro and nano

LC are summarized in Table 1.2.

Table 1.2. Differences between preparative, analytical, micro and nano

liquid chromatography.


Chapter Two

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.

1-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, 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 2-1, 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 2-1: 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 2-2: Adsorption chrornatography

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

(A) Application of sample to 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

In the simplest method, a single solvent continuously flows through

the column until the compounds have been separated and eluted from

the column

Stepwise elution, in which two or more different solvents of fixed

volume are added in sequence to elute the desired compounds.

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.

2-3).

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.

2-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

Figure 2-3: Gradient elution. Flow of solvent B into solvent A With mixing,

continuously changing the composition of solvent A as it flows into column


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%.

3-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.

4-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

5- 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 2-5


Table 2-1: 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.


Chapter Three

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. 3-1). This 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. 3-1 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. 3-2). 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.3-2 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 does 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. 3-3 Two-dimensional paper or thin-layer chromatography


dimensional separation (Fig. 3-3). 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.

Fig.3-4. 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 3.4, 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

In paper chromatography, the mobile phase (solvent) carries the

components of the sample on the stationary phase (filter paper) separating

them according to the differences in the migration rate (depends on the

molecular weight , polarity and adsorption ability)

Components

For one-dimensional paper chromatography, either ascending or

descending development can be carried out in simple units. Descending

development is more often used because it is faster and more suitable for

long paper sheets.

The stationary phase (filter paper)


The mobile phase (solvent may be in a reservoir)

Procedures

1. Make the initial line on the paper.

2. Apply the solvent alone on the initial line.

3. Wait till the solvent migration is stopped, then make the final line.

4. Spot the sample, and then apply the solvent either in ascending or

descending or concentric manner.

5. In case of colored sample: Calculate the rate of flow (Rf) directly then

compare it with stander in order to know the unknown sample (qualitatively).

6. In case of the colorless sample: use UV-lamb to detect the spot

position then determine the (Rf). Rf depends on the temp., solvent, type of

paper Rf = distance of sample migration / distance of solvent migration

Applications

1. Separation of amino acids

2. Separation of the plant pigments

Advantages

1. Simple

2. Cheap

Disadvantages

1. Time consuming.

2. Need high quantity of sample.

3. With weak solvent power.

4. limited use

5. Difficulty detection of spots

6. Difficulty isolation of separated substances.


Chapter Four

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 contains 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.

Summary for TLC

Principle

As in paper chromatography

Components

glass or plastic plate: as a support to the stationary phase

stationary phase (silica gel, alumina or agar)

mobile phase solvent system

Procedures

(a) Preparing the plate

1- Prepare a glass plate. for example (20X20)Cm

2- Dissolve suitable amount of the silica gel in water path.

3- Spread it on glass plate homogeneously. Then wait till solidification.

(b) Running the sample


1- Make the initial and final line as in PC

2- Spot the sample on the initial line and then apply the solvent either in

ascending or descending or concentric manner

3- For example: add the plate on a tank containing the solvent

(ascending) where the solvent move by the capillary action carrying

with it the components of the sample.

4- The plate is removed from the tank and dried. (Additional separation

can be achieved in two dimensional TLC)

5- According to the sample type (if colored, if colorless, if florescent),

identification (qualitative) occurs either by Rf, UV, spraying colored

reagent or autoradiography.

6- Compare it with stander in order to know the unknown sample

components.

7- We can separate the sample by cutting the silica layer by spatula, then

dissolve it with the same solvent then filter for further purification.

Applications

1- Environmental application from water analysis (especially pesticides) to

plant residues

2- Pharmaceutical applications from stability and impurity studies to drug

monitoring in biological fluid

3- Biomedical compounds (organic acids, lipids, carbohydrates and

steroids)

4- Food analysis from carcinogens, drug residues , preservatives and

flavors

Disadvantages

1- Time consuming


2- Limited to non-volatile compounds

3- Less accurate and less sensitive

Advantages

1- Need small quantity of sample.

2- With greater solvent power.

3- easy detection of spots

4- easy isolation of separated substances


The procedure of two-dimensional thin-layer chromatography


Developing solvent mixtures that have been recommended for two dimensional TLC separation of

underivatised amino-acids

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.


Chapter Five

Gel filtration

Biomolecules are purified using chromatography techniques that separate

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

5.1. and Table 5.1.

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 5.1.


Fig. 5.1 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. 5. 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. 5.3. 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

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

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


volume, Vt, of buffer has passed through the column, Fig. 5.4.

Fig. 5.4.Theoretical chromatogram of a high resolution fractionation (UV

absorbance).


Separation examples

Fig. 5.5 Cytochrome C, Aprotinin, Gastrin I, Substance P,

(Gly)6, (Gly)3 and Gly

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.5.7.


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

industrial scale


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.

Determination molecular weight

Ve – V0

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

Vt – V0

Where Ve = elution volume for the protein

Vo = column void volume

Vt = total bed volume

On semi logarithmic 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.5.2).


Product Fractionation

range, Mr

(globular

proteins)

pH stability Bed

volume

ml/g

dry

Sephad

ex

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

1×103–5×103 Long term: 2–13

Short term: 2–13

4-6 170–520 μm

Sephadex G-

25 Medium

1×103–5×103 Long term: 2–13

Short term: 2–13

4-6 85–260 μm

Sephadex G-

25 Fine

1×103–5×103 Long term: 2–13

Short term: 2–13

4-6 35–140 μm

Sephadex G-

25 Superfine

1×103–5×103 Long term: 2–13

Short term: 2–13

4-6 17–70 μm

Sephadex G-

50 Fine

1×103–3×104 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 5.10. 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. 5.10. 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 = Gel Permeation Chromatography =

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.5.11 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 5.12. Cross-linked dextrans

are commercially produced by Pharmacia 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.5.12. Structure of epichlorohydrin cross linked Dextran


Table 5.1: 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. 5.13).

Fig.5.13. 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 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.5. 14).

D-galactose (-1->4) 3, 6-Anhydro-L-galactose Fig.5.14. 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

5.1 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. 5.15).

Fig.5.15. 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.


Summary Gel filtration chromatography

(Size-exclusion chromatography)

Principle

This technique separate proteins according to their size and shape, as they

pass through a stationary phase (cross-linked polymer =sephadex) by the

help of mobile phase (without binding). Larger proteins or molecules, which

can not penetrate the sephadex pores, move around the sephadex in space

between them faster than the smaller molecules which may penetrate the

sephadex pores taking long time to elute from the column.

Components

1. Column: as a support to the stationary phase

2. Stationary phase (pours matrix in the form of spherical particles,

stable, inert e.g. sephadex or agarose)

3. Mobile phase (buffer system)

Procedures

1. (Loading step): spherical particles of the sephadex are packed into the

column

2. (Sampling step): sample is applied to the column

3. Buffer (mobile phase) and sample move through the column. The

sample components diffuse in and out of the pores of the matrix (sephadex)

according to their size.

4. Larger proteins or molecules move faster than the smaller molecules

and leave the column first

5. Separation completed as the entire buffer volume is passed.

Applications

1. Separation of neutral proteins and larger molecules including polymers


and biomolecules according to size.

2. The determination of formula weights.

Disadvantages

1. Limited applications

2. Low purification

Advantages

1. Provides a rapid means for separating larger molecules

2. Use only one buffer (coast effective)

3. Do not need elution step because there are no bonds formed.

Note: Gel Filtration

• Separation based on size

• Molecular sieve chromatography

• Size exclusion chromatography

• Media composed of crosslinked polymers

• Pore size of matrix determines degree of interaction

• Larger molecules are excluded and migrate faster

• Smaller molecules are included and are retained longer

• Dextran (=Sephadex®)

• Agarose (=Sepharose®)

• Polyacrylamide choose matrix with desired characteristics

• Size range

• does not interact with solute

• include 0.15-1 M NaCl in buffer

• Load sample in smallest possible volume

• elute in one column volume


Practical Considerations

Sephadex

Code Range (kDa)

G-25 1-5, G-50 2-30, G-100 4-150, G-150 5-300, G-200 5-600

Applications:

• Purification

• Desalting

• Size determination

Calculating Size


Chapter Six

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 6.1. 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 6.2. 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 6.1. 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

ter) 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 column 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

Summary Ion-exchange chromatography

Principle

Ion exchange chromatography separates molecules (proteins) according to

their differences between the overall charges. The proteins to be separated

must have a charge opposite to that of stationary phase in order to bind. Ion

exchange has two types according to the stationary phase charge:

1. Cation-exchanger: in which the stationary phase is charged

negatively in order to binds with positive molecules (cations)

2. Anion-exchanger: in which the stationary phase is charged positively

in order to binds with negative molecules (anions)


A-Cation-exchange chromatography

Cation-exchange chromatography can be classified as: either strong or

weak. A strong cation exchanger contains strong acid which stable along

pH1-14. Whereas, weak cation exchanger contains weak acid which loss its

charge as the pH decrease below 4-5

The sample must be charged positive in order to bind with the negative

matrix (strong or weak acid). H+

B-Anion-exchange chromatography

Anion-exchange chromatography can be classified as: either strong or

weak. A strong anion exchanger contains strong base which stable along

pH1-14. Whereas, weak anion exchanger contains weak base which loss its

charge as the pH increase over 9

The sample must be charged negative in order to bind with the positive

matrix (strong or weak base).OH-

Components

1. The column containing the stationary phase (anion or cation

exchanger) on suitable matrix

2. Washing and eluting buffer

3. pump to withdrew the buffer

4. Detector


Procedures

Before carry out the process, you must answer two important questions:

a) What is the sample charge? If +Ve, use cation exchanger. if –Ve, use

anion exchanger

b) What is the suspected strength of the charge? If weak +Ve, use weak

cation exchanger, if strong +Ve, use strong cation exchanger, if weak –Ve,

use weak anion exchanger, if strong –Ve use strong anion exchanger.

e.g. the sample is weak negative proteins. So we will use anion exchanger

contain weak base.

1. (Loading step): the column is packed with the matrix that charged with

weak positive charge by adding weak base e.g. DEAE- cellulose (stationary

phase)

2. (Sampling step): apply the sample in the column: the negatively

charged proteins bind to positively charged matrix whereas; the positively

charged proteins flow down to the exterior. Some negative charged

contaminants can bind to matrix.

3. (Washing step): apply washing buffer (Tris-HCL) to remove the

contaminants remaining the target proteins.

4. (Elution step): now, we need to separate the target proteins from the

matrix, so we apply an eluting buffer that has the same charge of protein in


order to substitutes it (ion exchange). Separation can be done also by ion

exclusion and ion pairing.

5. (Gradient step): make gradient elution with different buffer till you

obtain 100% correct proteins. i.e. repeat washing and eluting steps with

different buffer

6. (Detection step): after separation carry out detection by electrophoresis

Applications

1. Separation and detection of ions and ionized species.

2. Separation and purification of components from mixture

3. Identification of ionic impurities

Disadvantages

1. Analytes can be misidentified

2. Analytes are performed sequentially

3. Analysis consume eluent

Advantages

1. Selective to charge

2. Separation and detection of ions and ionized species

Relation between pH, Pi & ion exchange

pH: is quantitative description of the acidity of an aqueous solution.

pH= -Log [H]+ =Log 1/[H]+


Isoelectric point (Pi): is the point at which the protein charge reach zero.

If pH > PI use anion exchange

If pH < PI use cation exchange

pH = pK + Log conjugated base / conjugated acid

Protein

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

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

Anion exchange resin Cation exchange resin

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

Isoelectric point (pI)

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


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


Chapter Seven

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 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.

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.

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.


Chapter Eight

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. 8.1. 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.


Chapter Nine

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 9.1) Effective Range of Separation of DNA by PAGE

Acrylamide

(% 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 9.1 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 9.2.


The polyacrylamide slab is prepared between two glass plates that are

separated by spacers Figure 9.3.

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 9.4


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 9.5

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 9.2


Figure 9.6

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 9.7

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.

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 pH

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, and 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.


The polymerization reaction of acrylamide and methylenebisacrylamide


Chapter Ten

Question

Consider the following TLC plate when answering questions (1) and (2). The

plate was developed in hexanes.

1) The Rf of Compound C is: a) 1.0 b) 0.8 c) 0.5 d) 3.5

2) A fresh TLC plate is spotted with compounds A, B, and C, but this time the

solvent is isopropanol. How will this affect the Rf of compound A?

a) The Rf of A in the new solvent will be the same as in hexanes

b) The Rf of A in the new solvent will be greater than that in hexanes

c) The Rf of A in the new solvent will be less than that in hexanes

3) The TLC plate is prepared by drawing a light line 1 cm from the bottom of

the plate to mark where the pain relievers should be spotted. This line is

drawn with: a) a pencil b) a black pen c) a blue pen d) a red pen

4)Which of the following methods can be used to visualize the spots on a

developed TLC plate?

a) spraying the plate with a reagent b) iodine (I2) crystals in a jar

c) visualization by the color of the compound d) all of the above

5) The three compounds below are run on a silica gel TLC plate developed

in hexanes-ethyl acetate (70:30).


a) The carboxylic acid will run the fastest and the alcohol the slowest

b) The aromatic will run the fastest and the carboxylic acid the slowest

c) The alcohol will run the fastest and the aromatic the slowest

6) Which of the following solvent mixtures is more polar?

a) ethyl acetate/hexanes 50:50 b) ethyl acetate/hexanes 80:20

c) ethyl acetate/hexanes 20:80 d) all of the above

7) If two substances are run on the same TLC plate have the same Rf value,

they are:

a) Likely, but not necessarily, the same compound

b) Definitely the same compound

c) Likely, but not necessarily, different compounds

d) Definitely different compounds

8-Amino acid analyzers are instruments that automatically separate amino

acids by cation exchange chromatography. Predict the order of elution (first

to last) for each of the following sets of amino acids at pH = 4.

(a) Gly, Asp, His

(b) Arg, Glu, Ala

(c) Phe, His, Glu

9-Predict the relative order of paper chromatography Rf values for the amino

acids in the following mixture: Ser, Lys, Leu, Val, and Ala. Assume that the

developing solvent is n-butanol, water, and acetic acid.


10-In what order would the following proteins be eluted from a DEAE-

Cellulose ion exchanger by an increasing salt gradient. The pH1 is listed

for each protein.

Egg albumin, 4.6 , Pepsin, 1.0 , Serum albumin, 4.9

Cytochrome c, 10.6, Myoglobin, 6.8, Hemoglobin, 6.8

11-Draw the elution curve (Abs 280 nm vs. fraction number) obtained by

passing a mixture of the following proteins through a column of Sephadex G-

100. The molecular weight is given for each protein.

Myoglobin, 16,900 Myosin, 524,000

Catalase, 222,000 Serum albumin, 68,500

Cytochrome c, 13,370 Chymotrypsinogen, 23,240

1-Answer: (c) Rf is the distance the spot travels divided by how far the

solvent travels. In this case, the answer is 5 cm/10 cm, or 0.5.

2-Answer: (b) isopropanol is more polar and a stronger eluter than is

hexanes, and thus will carry compound A and also compounds B and C

farther along the plate than will hexanes. (Remember: all compounds travel

faster when a polar solvent is used.)

3- Answer: (a) Always use a pencil to mark a TLC plate. If a pen is used, the

pigments in the ink will move up the plate as the plate is developed; pencil

lines are not so affected

4- Answer: (d) All of the above. In the TLC experiment, the compounds will

be visualized by their color

5-Answer: (c) The eluting order of these three classes of compounds

fastest (highest Rf) to slowest (lowest Rf) is: (less polar) aromatic >


alcohol > carboxylic acid (most polar). This is because carboxylic

acid are the most polar and aromatics the least polar of the three

types of compounds. (Remember: all compounds travel faster when a

polar solvent is used.)

6- Answer: (b) Ethyl acetate (an ester) is more polar than hexanes (a

hydrocarbon solvent = non plolar). In (b), the solvent has a greater

percentage of this more polar solvent than in (a).

7- Answer: (a) Compounds which have the same Rf value when run on the

same TLC plate are likely the same compound. This is especially true when

the TLC plate is run of a reaction mixture: in such a case the chemist usually

has a pretty good idea of what compounds might be present, and thus

chooses and runs appropriate standards. In this situation, if an "unknown"

has the same Rf as a standard, it's a pretty good indication that the

compound is the same as the standard. However, if the entire organic

compounds is considered, many different compounds will have the same Rf

in a particular TLC system, and thus Rf values as a means of compound

identification is limited.

8. (a) Asp, Gly, His

(b) Glu, Ala, Arg

(c) Glu, Phe, His

9. Ser, Lys, Ala, Val, Leu

10. Cyt c, myoglobin = hemoglobin, serum albumin, egg albumin, pepsin

11. Myosin, catalase, serum albumin, chymotrypsinogen, myoglobin,

Cytochrome c


12-Name the techniques described for separating cellular proteins: a) Taking advantage of unique structural or functional properties of a protein, this technique specifically removes the protein of interest from a solution. b) Proteins leave the mobile phase, associating with a negatively charged immobile structure, such as bead or resin. c) Proteins are separated on the basis of their ability to migrate in an electric field, an indicator of relative size. d) Proteins are chromatographically separated on the basis of size. Answer a- Affinity chromatography b- Cation exchange chromatography c- Electrophoresis d- Gel filtration/ size-exclusion chromatography ========================================================= 13-Amino acid analyzers are instruments that automatically separate amino acids by cation-exchange chromatography. Predict the order of elution (first to last) for each of the following sets of amino acids at pH = 4. (a) Gly, Asp, His (b) Arg, Glu, Ala (c) Phe, His, Glu 14-Predict the relative order of paper chromatography Rf values for the Amino acids in the following mixture: Ser, Lys, Leu, Val, and Ala. Assume that the developing solvent is n-butanol, water, and acetic acid. 15-In what order would the following proteins be eluted from a DEAE- cellulose ion exchanger by an increasing salt gradient. The pH1 is listed for each protein. Egg albumin, 4.6 , Pepsin, 1.0 , Serum albumin, 4.9 Cytochrome c, 10.6, Myoglobin, 6.8, Hemoglobin, 6.8 16-Draw the elution curve (Abs 280 nm vs. fraction number) obtained by passing a mixture of the following proteins through a column of Sephadex G-100. The molecular weight is given for each protein.


Myoglobin, 16,900 Myosin, 524,000 Catalase, 222,000 Serum albumin, 68,500 Cytochrome c, 13,370 Chymotrypsinogen, 23,240 Answer 13. (a) Asp, Gly, His (b) Glu, Ala, Arg (c) Glu, Phe, His 14. Ser, Lys, Ala, Val, Leu 15. Cyt c, myoglobin = hemoglobin, serum albumin, egg albumin, pepsin 16. Myosin, catalase, serum albumin, chymotrypsinogen, myoglobin, Cytochrome c ====================================================== 17-What physical characteristics of a biomolecule influence its rate of movement in an electrophoresis matrix? 18. Draw a slab gel to show the results of nondenatunng electrophoresis of the following mixture of proteins. The molecular weight is given for each. Lysozyme A3,930) Egg white albumin D5,000) Chymotrypsin B1,600) Serum albumin F5,400) 19. Each of the proteins listed below is treated with sodium dodecyl sulfate and separated by electrophoresis on a polyacrylamide slab gel. Draw pictures of the final results. (a) Myoglobm (b) Hemoglobin (two a subunits, molecular weight = 15,500; two /3 subumts, molecular weight = 16,000) 20. Explain the purpose of each of the chemical reagents that are used for PAGE. (a) acrylamide (d) sodium dodecyl sulfate (b) N, /V'-methylene-bis-acrylamide (e) Coomassie Blue dye (c) TEMED (f) bromophenol blue 21. What is the main advantage of slab gels over column gels for PAGE? 22. Is it possible to use polyacrylamide as a matrix for electrophoresis of


Nucleic acids? What are the limitations, if any? 23. Explain the purposes of protein and nucleic acid "blotting." 24. Can polyacrylamide gels be used for the analysis of plasmid DNA with greater than 3000 base pairs? Why or why not? 25. Describe the toxic characteristics of acrylamide and outline precautions necessary for its use. Answer 17. Charge, size 18. From top to bottom: serum albumin, egg white albumin, chymotrypsin, lysozyme 19. (a) Monomer for polymeric gel matrix (b) Monomer for adding cross-linking to gel matrix (c) Catalyst for polymerization process. (d) Detergent that denatures proteins for electrophoresis (e) Dye used to stain proteins after gel electrophoresis (f) Molecule used as a "tracking dye" during electrophoresis 20. The gel matrix in slab gels is more uniform than column gels, which are made individually. 21. Polyacrylamide gels may be used for nucleic acids up to 2000 base pairs. ======================================================== (1) Why is it important to avoid air bubbles in the column during chromatography?

(a) the air in the bubbles might react with the compounds being separated (b) bubbles are toxic and harmful to your health (c) bubbles cause the samples to travel unevenly down the column and thus the components might not separate (d) bubbles block the flow from the bottom of the column

(2) Spinach is green, however the pigments it contains are green, orange, yellow, and grey. Which of the following statements is true?

(a) Chlorophyll is grey and carotene is yellow-orange. (b) Chlorophyll is green and carotene is yellow-orange. (c) Chlorophyll is grey and carotene is green.

(3) A column chromatography procedure for the separation of a polar and a


non-polar compound calls for sequential elution with the following two solvents:

methylene chloride hexanes Which solvent should be used first? (a) methylene chloride (b) hexanes (c) it does not matter which solvent is used first

(4) What happens if you load the spinach pigment mixture onto the column in too much methylene chloride?

(a) the spinach pigments will not separate into individual components because they will travel rapidly down the column (b) the spinach pigments will evaporate (c) too much methylene chloride in the loading solvent will not affect the separation

(5) Of the following compounds, which will be the first to elute from an alumina chromatography column? Last?

(a) a will elute first and b will elute last (b) d will elute first and c will elute last (c) b will elute first and a will elute last (d) b will elute first and d will elute last

(6) If several compounds are present in a sample which is developed on a TLC plate, a column of spots is seen on the developed plate, with: (a) more polar compounds toward the top of the plate and less polar toward the bottom (b) more polar compounds toward the bottom of the plate and less polar toward the top (c) lower boiling compounds toward the bottom of the plate and higher boiling toward the top (d) lower boiling compounds toward the top of the plate and higher boiling toward the bottom 7- TLC is generally used as a qualitative analytical technique for: (a) determining the number of components in a mixture


(b) checking the purity of a compound (c) following the course of a reaction (d) all of the above 8- In general, a compound will move further on a TLC plate with a: (a) non-polar solvent (b) polar solvent 9- Which of the following solvents is more polar? (a) ethyl acetate (b) hexanes 10- The melting point of a pure organic compound: (a) is broad, having a range of 3 degrees or more (b) is sharp, having a range of 1 degree or less (c) has a range of 5.5 degrees (d) varies according to atmospheric pressure 11- Calculate the Rf value for the following compound: spot, 3.0 cm; solvent front, 10.0 cm (a) 3 (b) 10 (c) 0.3 (d) 0 12- A beaker will be used as a "developing jar" in this experiment. When the TLC plate is set in this beaker, the solvent in the beaker must be: (a) above the pencil line used to guide the spotting of samples (b) deep enough to cover the entire TLC plate (c) deep enough to come about halfway up the TLC plate (d) below the pencil line used to guide the spotting of samples 13- Which of the following are flammable? (a) hexanes (b) ethyl acetate (c) ethanol (d) all of the above


No Answer Explanation

1 C The absorbent-packing must homogeneous and even throughout

the length of the column, otherwise the samples will not run at a

steady rate and evenly down the column. Bubbles, dry areas, and

unevenly packed areas channel the solvent unevenly down the

column; if this happens, the compounds do not move down the

column in discreet bands, but instead in streaks, and you will not

be able to collect fractions of pure samples. Also note that the

adsorbent, silica gel or alumina, is delicate and will not work

properly if it is dry.

2 b Carotene is yellow-orange, as it states in the introduction to this

experiment in the Lab Manual. The spinach pigments are air-,

heat-, and light-sensitive. If you do not protect the dried column

fractions from air, light, and heat, they can undergo oxidation,

hydrolysis, and other reactions, leading to more spots than you

see on the column and/or more spots than in the original

mixture.

3 b You must always begin with the least polar solvent, in this case,

hexanes. If you start with the most polar solvent, all of the

compounds will travel down the column very rapidly and

probably will not separate. (Remember: all compounds travel

faster when a polar solvent is used.) By using the less polar

solvent first, only the least polar compounds will travel rapidly

down the column. Once the faster-moving (less-polar)

compound(s) are off the column, you can switch to a polar

solvent to speed the elution of the slower-moving compound(s).

4 a Too much of this polar solvent - methylene chloride - has the

same effect on separation as does using the most polar solvent

first. If a mixture to be analyzed by column chromatography will

only dissolve in a polar solvent, use as little of the solvent as

absolutely necessary to load the solvent onto the column

5 d The order of elution of organic classes of compounds is (from

fastest to slowest): ethers (b) > ketones (a) > esters (c) >


carboxylic acids (d).

6 b TLC separates on the basis of strength of the adsorption of

compounds to the TLC plate (the adsorbent). The strength of this

adsorption is greater the more polar a compound, and therefore

a polar compound will not move as far up a plate as would a non-

polar compound. (Note that compounds are spotted near the

bottom of a TLC plate and travel up the plate with the solvent.)

7 d TLC is a quick, powerful tool to check a reaction mixture or

solution of a compound. By determining the number and Rf

values which appear on a developed TLC plate, you will know

how many components are in the mixture, know if a reaction has

proceeded to produce product, or know if a compound is pure.

8 b From the introduction, "generally, the more polar a solvent is,

the more effective it is at eluting both polar and non-polar

compounds.

9 a Ethyl acetate is more polar than hexanes.

10 b Pure compounds have a narrow melting point range, 1 degree or

less if the compound is very pure. A melting point range of 2

degrees or less indicates that a substance is pure enough for

most laboratory purposes.

11 c The Rf is the distance traveled by the compound divided by the

distance traveled by the solvent.

12 d The solvent must not be above the pencil line used to guide the

spotting of the samples. If it is, the samples will dissolve into the

reservoir of solvent instead of traveling up the plate.

13 d hexanes, ethyl acetate, and ethanol are all flammable.

1-Discuss following in detail a)Biospecific chromatography b)Gel Filtration 2-Write briefly on the following a) Ion exchange chromatography: Principles, properties and uses b) Affinity chromatography


3-write about isolation and purification of biological molecules based on ligand specificity 4-Discuss the following material for gel chromatography 5-write on the affinity chromatography 6-write on the following: a) Thin layer chromatography b) Ion exchange chromatography according to:

(1) Type of ion exchanger (2) Preparation of the anion sorbent (3) Methods are used for eluting molecules from the exchanger.

7- Discuss the following: a)Classification of chromatographic methods b)Adsorption chromatography 8- Write briefly on Separation techniques method used in chromatography 9- Write briefly on the following a) Adsorption chromatography b) different ways used in eluted the substances adsorbed on the column 10- Write briefly on Separation mechanisms in adsorption chromatography 11- Write briefly on Common adsorbents and the type of compounds adsorption Chromatography 12- Factor affecting on separation of the compounds in adsorption Chromatography 13- Write briefly on common adsorbents used for TLC 14- Discuss the following:

a) Thin-Layer Chromatography of Amino acids b) Thin-Layer Chromatography of Carbohydrates

15-write briefly on chromatography techniques used to separates molecules on the basis of differences in size 16- Write briefly on determination molecular weight using gel filtration 17- Write briefly on Size Exclusion Chromatography 18- Write briefly on Gel Permeation Chromatography 19- Write briefly on Gel Filtration 20- Write briefly on properties of gels used in gel permeation (filtration) chromatography 21- Compare between three types of polymers are principally used in Gel Filtration


chromatography (dextran, polyacrylamide, and agarose). 22-Definition of Chromatography and Principles of Paper Chromatography.

23-Differentiate between the use of a cation exchange resin and an anion exchange resin in terms of whether the charged sites are positive or negative and whether cations or anions are exchanged. 24-Match each term to one of the statements: a) A chromatography configuration in which the stationary phase is spread across a glass or plastic plate. (b) A chromatography type in which the stationary phase is a liquid. (c) A chromatography type designed to separate dissolved ions. (f) A chromatography configuration that utilizes a fraction collector. (g) The only chromatography type described by the letters GLC. (h) One of two chromatography configurations in which the mobile phase moves by capillary action opposing gravity. 1-Partition chromatography 2-Adsorption chromatography 3-Ion exchange chromatography 4-Size exclusion chromatography 5-Paper chromatography 6-Thin-layer chromatography 7-Electrophoresis ========================================================== 25-Tell what each of the following refer to: GC, LC, GSC, LSC, GLC, LLC, SEC, GPC, DEAE cellulose and CM cellulose 25-Consider a mixture of compound A, a somewhat nonpolar liquid, and compound B, a somewhat polar liquid. Tell which liquid, A or B, would emerge from a chromatography column first under the following conditions and why: (a) A polar liquid mobile phase and a nonpolar liquid stationary phase (b) A nonpolar liquid mobile phase and a polar liquid stationary phase 26-We have studied four chromatography types. One of these is partition chromatography. Answer the following questions concerning partition chromatography yes or no: (a) Can the mobile phase be a solid? (b) Can the mobile phase be a liquid?


(c) Can the mobile phase be a gas? (d) Can the stationary phase be a solid? (e) Can the stationary phase be a liquid? (f) Can the stationary phase be a gas? 27-Answer the following questions either true or false (a) The stationary phase percolates through a bed of finely divided solid particles in adsorption chromatography. (b) The mobile phase can be either a liquid or a gas. (c) The mobile phase is a moving phase. (d) Partition chromatography can only be used when the mobile phase is a liquid. (e) Adsorption includes LSC. (f) In partition chromatography, the mobile phase partitions or distributes itself between the sample solution and the stationary phase. (g) If the stationary phase is a polar liquid substance, nonpolar components will elute first. (h) Size exclusion chromatography separates components on the basis of their charge. (i) Gel permeation chromatography is another name for size exclusion chromatography. (j) Ion exchange chromatography is a technique for separating inorganic ions in a solution. (k) Paper chromatography is a type of LLC. (l) Thin-layer chromatography and open-column chromatography are two completely different configurations of GSC. (m) It is useful to measure Rf values in open-column chromatography. (n) Rf values are used for quantitative analysis. (o) TLC refers to thin-layer chromatography

1 chromatography is a separation method in which the analyte is contained in a mobile phase and...

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