200323226 1 Chromatography Metods Practically

8/11/2019 200323226 1 Chromatography Metods Practically

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1Chromatography CourseDr 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 impossibleif it were necessary to completelyisolate 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 chargeof 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 ofcolored 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


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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 allbranches 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 , 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 p hase chromatography can separate biomolecules

according to their interaction with the hydro phob ic stat ionary phase and

the hydro phi l ic mobl i le phase. This separation method can be coupled to

an ESI mass spectrometer. Ion exchange chromatography separates

molecules depending on their net charge. Aff in i ty chrom atographymakes

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 mobilephase)through it in a definite direction.


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chromatography (GSC), liquidliquid chromatography (LLC), and liquidsolid

chromatography (LSC),

4- Main Type of Chromatography

In general, there are four main types which can be classified asfollows:

4.1-Liquid-Solid chromatography

Classical adsorption chromatography(Tswett column)

Ion-exchange chromatography

4.2. Gas-Solidchromatography

4.3. Liquid-Liquidchromatography

Classicalpartition chromatography

Paper chromatography

4.4Gas-Liquidchromatography

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 retainedExtraction Solubility Some of the substances

dissolve (partition) more in


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water. While other

substances dissolve more

organic solvent in contact

with the waterThin 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


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porous, solid material while

others remain outside in the

surrounding stream of

flowing waterIon-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 bondswhich can be formed between the solute molecules and the surface of the

adsorbent.


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5-Purpose of Chromatography

Analytical- determine chemical composition of a samplePreparative- purify and collect one or more components of a sample

Other classification of Chromatographic Methods

Chromatography is classified according to three ways:


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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 columns 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 samples 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


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Reverse: analyte is polar organic; stationary phase LESS polarthan

the mobile phase

Size Exclusion chromatography: stationary phase is a porous matrix.

6-The Principle of ChromatographyChromatography 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 chromatographycan 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


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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 throughthe 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.


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Modes of operation can be classified as normal and reversed phase

chromatography.

In normal phase chromatography, the stationary p hase consists of a

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


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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 flowrate. 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 retent ion t im e, t0, corresponding to the flow rate of the mobile

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


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(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 constantfor

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, Cu, 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 ratefor a chromatographic separation

can be determined (Fig. 1.3).


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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 = 1equals a peak overlap of 4 %. Peak resolution can be

optimized by increasing the selectivity and minimizing band broadening.

Resolution

(equations 1.6)

valid for < 1.2

(equations 1.7)


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

other compounds with higher k valueselute 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 elutionproblem 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 elut ion as

opposed to an isocratic elution, where the composition of the mobi le 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.


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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 particlesize is typically 5 m 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 mMto 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


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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 15m in diameter. To achieve ambient flow rates in

these columns, high pressures of up to 300400 bars must be generated. A

typical instrumental setup for this high pressure or high performance liquid

chromatography (HPLC) is shown in Fig. 1.6.


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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 = 210 nm

for peptides and = 254 nm or = 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 developm ent is to couple

liquid chromatography systems to an elect rospray ion isation m ass

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 liquidchromatography 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


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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.


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Chapter Two

ADSORPTION CHROMATOGRAPHYIn 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 relativebalance between the aff in i ties of the comp ound s

for the adsorbent and their solubi l i ty in the solvent.

2-The chemical nature of the subs tances.

3-The nature of the solvent.

4-The nature of the adsorbent.

Solid adsorbents commonly used are alumina, si l ica gel , charcoal

(act ive carbo n), cel lulose, starch, and suc ros e.

Solvents commonly usedare hexane, benzene, petroleum ether, diethyl

ether, chlo rofo rm, methylene chlo r ide, var ious alco hols (ethyl , pro py l ,

n-bury l and t-buty l alcohols), and various aqueous b uf fers and sal ts,

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 plast ic disc s, or sheetsof ny lon

or Tef lon fabr ics.The adsorbent bed must be homogeneous and f ree of bubb les, cracks,

or sp aces between the adsorbents and the wal ls of the column .


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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 adrop 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.


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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 threeways

In the simp lest method, a single solvent continuously flows through

the column until the compounds have been separated and eluted from

the column

Stepwise elution, in which twoor mo re di f ferent solvents of fixed

volume are added in sequence to elute the desired compounds.

Gradient elut ion, 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


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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., exponent ial , 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 alumin a, si l ica gel , and act ive carbon and

Mg si l icatecan 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

activityby the controlled addi t ion of w ater, and the subsequent activity is

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

continuously changing the composition of solvent Aas it flows into column


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related to the amount of water added. Brookman and Schodder

established five grades of aluminaGrade I is the most active and the is

simply alumina heated at about 3500

C for several hours. Grade IIhas about

2-3% water, Grade III5-7%, Grade IV9-11 %, Grade Vfilm. (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 anddetection. 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:


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K is assumed to be independent of concentration, and can change ifexperimental conditions are changed, for example temperature is increased

or decreased. As Kincreases, 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. Sandto 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 sandon 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.


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Figure 2-5


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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.


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Chapter Three

PAPER CHROMATOGRAPHY

Paper chromatography is a type of liquid-liquid partitionchromatography that may be performed by ascending or descending

solvent f low. Each mode has its advantages and disadvantages.

Ascending c hromatography involves relatively simple and inexpensive

equipment compared with descending chromatography and usually gives

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

Descending chrom atography, 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 thesolvent 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


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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


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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 sprayingor 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


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(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. Forexample, in the separation of carbohydrates, the standard might be glucose

and the ratio would be RGlcor 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


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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 andthe 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


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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. Descendingdevelopment is more often used because it is faster and more suitable for

long paper sheets.

The stationary phase (filter paper)


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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

Appl icat ions

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 use5. Difficulty detection of spots

6. Difficulty isolation of separated substances.


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Chapter Four

Thin layer chromatography

This technique is particularly useful for the separation of very smallamounts of material. The general pr inc ip le 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, di f fer f rom colum n adsorb ents. 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 dry ing them in an oven at 100-120C. Dryingserves 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.


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The sample is applied to the plate by micropipette or syringes, as spot2.5

cm from one endand 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

(mo bi le 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 vert ical lyin 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 s topp ed. 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 blu e, gr een, blackarea.

2. Spraying the plate with 50% sulphur ic acid and heat ing so, the

compounds become charred and show spots

3. Spraying the plates with speci f ic color reagents will stain up certain

compounds e.g. ninh yd r in for am ino acid (aa) , ani l ine for aldoses.

SolventsUniversal TLC System:

Petroleum ether - ethyl acetate

Very polar solvent addi t ives:


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Methanol > ethanol > isopropanol

Moderately po lar addi t ives:

Acetonitrile > ethyl acetate > chloroform, dichloromethane > diethyl ether >toluene

Non-polar so lvents:

Cyclohexane, petroleum ether, hexane, pentane

TLC Visualization (Detecting the spots)

Non-dest ruct ive techniqu es:

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)

Destruct ive techniq ues

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/greenspots

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


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Thin-Layer Chromatography of Amino acidsAmino acids may be separated by two-dimensional TLC using either

si l ica gel or cel lulos eas 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 sprayfor the silica gel and the cellulose gel media.

Ninhydr in Sprays for amino acid detect ion

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 110C.

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 110C.

Two-dimensional TLC separation of amino acids.

On s i l ica gel Gwith

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

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


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on cel lulose MN 300with

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


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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 si l ica gel plates is

obtained by spraying with sul fu r ic acid -methanol (1: 3, v/v) followed by

heat ing for 10 m in utes at 110-120C. 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)


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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 speedat which separation is achieved. With a volatile solventas

the mobi le phasethe time involved may be as low as 30 minu tes, but even

with non-volatile solvents the time involved is rarely longer than 90

minutes.

Summ ary 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


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2- Limited to non-volatile compounds

3- Less accurate and less sensitive

Advantages1- Need small quantity of sample.

2- With greater solvent power.

3- easy detection of spots

4- easy isolation of separated substances


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The procedure of two-dimensional thin-layer chromatography


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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


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phase. Therefore, compounds soluble to organic component move faster than

compounds soluble to aqueous component. -Thus, molecules are separated

according to their polarities.


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Chapter FiveGel 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.


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Fig. 5.1Separation principles in chromatography purification.

Gel filtration has played a key role in the puri f icat ion of enzymes,

polysacchar ides, nucle ic acids, prote ins and other bio logical

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 orlow molecular weight contaminants(such as phenol red from culture fluids)

or to desalt and exchange buffers.


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2. High resolution fractionation of biomolecules:

The components of a sample are separated according to di f ferences

in their mo lecular size.High resolution fractionation can be used to isolateone or more components, to separate monomers from aggregates, to

determine m olecular w eightor 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 beperformed

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

medium is a porous matr ixin the form of sphericalparticles that have been

chosen for their chemical and physical stabi l i ty, and inertness (lack of


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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

stat ionary phaseand this liquid is in equilibrium with the liquid outside theparticles, referred to as the mobi le phaseas shown in Figure 2.

Gel filtration is used in group separation mode to remove small

molecules from a group of larger moleculesand 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 pur i f icat ion schemes for

desalting and buffer exchange


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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

mo lecules are eluted in or just af ter the void volum e, Voas they pass

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

packed column the void v olum e is equivalent to approximately 30% of

the tota l colum n volum e. Small molecules such as salts that have fullaccess 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


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detectedby monitoring theirUV absorbance, usually atA280 nm, and the

sal ts are detectedby monitoring the cond uct iv i ty of the buf fer.

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, Voas they pass directly through the column at the same speed as

the flow of buffer. For a well packed column the vo id vo lumeis 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.


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

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

absorbance).


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Separation examples

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

(Gly)6, (Gly)3and Gly

Media Selection

Chromatography media for gel filtration are made from p orous

matr iceschosen 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'sgel filtration media cover a molecular weightrange from 100 to80 000 000, from peptides to very large proteins and protein complexes.

Figure.5.7.


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

industrial scale


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Sephadexis ideal for rapid group separations such as desalting and buffer

exchange.

Sephadexis used at laboratory and production scale, before, between or

after other chromatography purification steps.Determination molecular weight

VeV0

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

VtV0

WhereVe= elution volume for the proteinVo= column void volume

Vt= total bed volume

On semi logarithmic graph paper, plot the Kav value for each protein

standard (on the l inear scale) against the corresponding molecular

weight (on the logar i thmic 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).


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Product Fractionation

range, Mr

(globularproteins)

pH stability Bed

volume

ml/gdry

Sephad

ex

Particle size,

wet

Sephadex G-

10

700)from smaller molecules (Mr 30000 frommolecules Mr


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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 moleculesto 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


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Fig.5.11Gel 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

Dextranis a polysaccharide composed of (-1--->6)-linked glucose residues

with (-1, 3) branch linkages. It is synthesized from sucroseby an enzyme


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produced by the bacter ium Leuconostoc mesenteroides B -512F. The

dextran is cross-linked to various extents by reaction with epichlorohydr in

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 th e

so -cal led G-ser ies, where the G-numbers refer to the amou nt of w ater

gained wh en the beads are sw el led 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.


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Fig.5.12. Structure ofepichlorohydr in c ross l inked Dextran


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Table 5.1: Properties of gels used in gel permeation (filtration)

chromatography

Gel

Waterregain

(mL/g)

Exclusionlimit

Maximumhydrostatic

pressure

cm H2O

Maximumflow 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 10 200 1.2

Sepharose CL 6B NA 4 x 10 >200 2.5


Sepharose CL 4B NA 20 x 10 120 2.17



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Sepharose CL 2B NA 40 x 10 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 10 >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 10 50 1.0

Bio-Gel A-150m NA 150 x 10

6

30 0.5

Bio-Gel is a trade name of Bio-Rad Laboratories, Sephadex and

Sepharose are trade name of Pharmacia Fine Chemical


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Polyacrylamide gels are long polymers of acrylamide cross-linked withN.N'methylene-bisacrylamide (Fig. 5.13).

Fig.5.13. Structureof 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 seriesfor the exclusion limits and properties of the different Bio-Gels.

Agaroseis a gel material with pore sizes larger than cross-linked dextran or

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


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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-galactoseFig.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) tothe 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 serieswith 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 gelwith increased thermal and chemical stability. Table

5.1gives the properties of the different Sephadex, Bio-Gel, and Sepharose


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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.


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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.


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Summary Gel filtration chromatography

(Size-exclusion chromatography)

Principle

This technique separate proteins according to their size and shape, as theypass 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 first5. Separation completed as the entire buffer volume is passed.

Applications

1. Separation of neutral proteins and larger molecules including polymers


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and biomolecules according to size.

2. The determination of formula weights.

Disadvantages1. 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 crosslinkedpolymers

Pore size of matrix determines degree of interaction

Larger moleculesare excluded and migrate faster

Smaller moleculesare 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


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Practical Considerations

Sephadex

Code Range (kDa)

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

Purification

Desalting

Size determination

Calculating Size


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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 negat ive charges are cal led cat ion exchangers and those with

chemically bound posi t ive charges are cal led anion exc hangers. 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


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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

cat ion and the exchangers respect ively. The preparation and commercial

availability of these materials beginning in the 1960 provided the biochemist

with powerful tools for separat ion of p roteins and nucle ic acid Figure 2

presents partial structures of DEAE-cellulose and CMcellulose

Figure 6.2. Partial structures of diethylaminoethyl-cellulose and carboxymethyl-

cellulose.


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The DEAE and CMgroups are shown attached to the C6-hydroxyl group of

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

groups of C2and C3. The total degree of substitution of the DEAE and CM

groupsmust 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


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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 exch anger is sus pended in 15

volumes (w/v) of the "f i rs t treatment," acid o r alkali depending on th e

exchanger (Table. 1),and is al low ed to stand at least 30 m inutes but no t

mo re 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


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is washed with distilled water until the effluent is close to neutral pH. The

treated exchanger is placed into the acid component of the buf fer (the pH

shou ld 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 o f the buf fer 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 st rength by increasing the concentration of salt in

the elution solvent, thereby weakening the electrostatic interactions between

the adsorbed molecule and the exchanger, and


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(c)Performin g aff ini ty elut ion. 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 opp osi te signs to the net chargeon

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:


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Cytoch rom e C has an isoelectr ic p oint (pI) of 10.05; that is at pH

10.05 the number of positive charges will equal the number of negative

charges. A columncontaining a cation exchangerbuffered, at pH 8.5, is

prepared. This columnhas a full negative charge. Cytochrome Cat 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)


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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


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Procedures

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

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


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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

Documents

200323226 1 Chromatography Metods Practically