Chromatography

Definition and Classification of Chromatography

Chromatography course

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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬1

Definition and Classification of Chromatography

Chromatography course

INTRODUCTION The Russian botanist M. S. Tswett is discovery of chromatography. He used a column of powdered calcium carbonate to separate green leaf pigments into a series of colored bands by allowing a solvent to percolate through the column bed. Since these experiments by Tswett many scientists have made substantial contributions to the theory and practice of chromatography. Not least among these is A. J. P. Martin who received the Nobel Prize in 1952 for the invention of partition chromatography (with R. L. K. Synge) and in the same year with A. T. James he introduced the technique of gas-liquid chromatography. Chromatography is now an important tool used in all branches of the chemical and life sciences.

1-Definition of Chromatography Chromatography is essentially a physical method of separation in which the components to be separated are distributed between two phases one of which is stationary (stationary phase) while the other (the mobile phase) through it in a definite direction.

2- Classification of chromatographic methods The common feature of all chromatographic methods is two phases, one stationary and the other mobile A classification can be made depending upon whether the stationary phase is solid or liquid. If it is solid, the method is

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬2

Definition and Classification of Chromatography

Chromatography course

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 Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬3

Definition and Classification of Chromatography

Chromatography course

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

Molecular size

Some of the substances pass through a layer or

Dialysis

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

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬4

Definition and Classification of Chromatography

Gas liquid

Solubility

Chromatography course

Some of the substances dissolve more in the

Chromatography

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

Molecular Size

Some of the substances diffuse into the pores in

Chromatography

a porous, solid material while others remain outside in the surrounding stream of flowing water

Ion-exchange

Electrical charge

Some of the substances are bound by immobile

Chromatography

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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬5

Definition and Classification of Chromatography

Chromatography course

6-Adsorption chromatography Adsorption column chromatography is the oldest form of chromatography. Whether two or more substances of a mixture can be separated by adsorption chromatography depends on a number of factors. Most important is the strength with which each component of mixture is adsorbed and its solubility in the solvent used for elution. The degree to which a particular substance is adsorbed depends on the type of bonds which can be formed between the solute molecules and the surface of the adsorbent.

Chromatography

Adsorption Chromatography Solid stationary phase

Liquid mobile phase

Gas mobile phase

Dr.Ehab Aboueladab

Partition Chromatography Liquid Stationary Phase

Liquid mobile phase

Gas mobile phase

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬6

Adsorption Chromatography

Chromatography course

All chromatographic separations are carried out using a mobile and a stationary phase, the primary classification of chromatography is based on the physical nature of the mobile phase. The mobile phase can be a gas or a liquid which gives rise to the two basic forms of chromatography, namely, gas chromatography (GC) and liquid chromatography (LC). The stationary phase can also take two forms, solid and liquid, which provides two subgroups of GC and LC, namely; gas– solid chromatography (GSC) and gas–liquid chromatography (GLC), together with liquid solid chromatography (LSC) and liquid chromatography (LLC). The different forms of chromatography are summarized in Table.1 Most thin layer chromatography techniques are considered liquid-solid systems although the solute normally interacts with a liquid-like surface coating on the adsorbent or support or, in some cases an actual liquid coating.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬7

Adsorption Chromatography

Chromatography course

Table 1: The Classification of Chromatography

1-ADSORPTION CHROMATOGRAPHY In adsorption chromatography the compounds to be separated are adsorbed onto the surface of a solid material. The compounds are desorbed from the solid adsorbent by eluting solvent. 2-Separation of the compounds depends on 1-The relative balance between the affinities of the compounds for the adsorbent and their solubility in the solvent. 2-The chemical nature of the substances. 3-The nature of the solvent. 4-The nature of the adsorbent.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬8

Adsorption Chromatography

Chromatography course

Solid adsorbents commonly used are alumina, silica gel, charcoal (active carbon), cellulose, starch, calcium phosphate gels, calcium hydroxylapatite, and sucrose. Solvents commonly used are hexane, benzene, petroleum ether, diethyl ether, chloroform, methylene chloride, various alcohols (ethyl, propyl, n-buryl and t-butyl alcohols), and various aqueous buffers and salts, some in combination with organic solvents Adsorption chromatography is a column that is packed with the adsorbents. The adsorbent is prepared and poured into the column with an inert support at the bottom. Suitable supports include plastic discs, or sheets of nylon or Teflon fabrics. The adsorbent bed must be homogeneous and free of bubbles, cracks, or spaces between the adsorbents and the walls of the column. The choice of the eluting solvent, although very important, depends on the nature of the substances to be separated and the adsorbent, and hence affords considerable latitude. The process of eluting the sample components from the adsorbent by the solvent is termed development. As illustrated in Figure 1, the compounds in the mixture that are more soluble in the solvent and have less affinity for the adsorbent move more quickly down the column. If the substances are colored, as they were in Tswett's experiment, they are readily visible as they separate, However, Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬9

Adsorption Chromatography

Chromatography course

many substances are not colored, and in these instances, as the development proceeds, fractions are collected at the bottom of the column, and the different fractions are analyzed for compounds of the types that are being separated, For example, if proteins are being separated, the fractions would be analyzed for protein by measurement of the UV absorbance at 280 nm. If carbohydrates or nucleic acids are being separated analytical measurements for carbohydrates or nucleic acids. The collection of fractions by an automatic fraction collector,

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

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬10

Adsorption Chromatography

Chromatography course

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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬11

Adsorption Chromatography

Chromatography course

Figure 2: Adsorption chrornatography A = adsorbent, S=Sample, ES = eluting solvent (A) Application of sample to the of the column. (B) Adsorption of sample onto adsorbent. (C)Addition of elution solvent. (D) and (E) Partial fraction of sample components. (F) Complete fractionation of sample. (G) and (H) Separation of all three components at various stages on the adsorbents. (I) Elution of the first component from the column.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬12

Adsorption Chromatography

Chromatography course

The substances adsorbed on the column support can be eluted in three ways (a)

in the simplest method, a single solvent continuously flows through the column until the compounds have been separated and eluted from the column

(b)

Stepwise elution, in which two or more different solvents of fixed volume are added in sequence to elute the desired compounds.

(c)

Gradient elution, in which the composition of the solvent is continuously changing. The latter method is used to effect separations that are difficult because of a tendency of component to be eluted in broad. Trailing bands when a single solvent is used. Gradient elution frequently provides a means of sharpening the bands, a simple linear gradient has two solvents, A and B, in which A is the starting solvent and B is the final solvent. Solvent B is allowed to flow into solvent A as solvent A flows into the column. The composition of solvent A is, thus, constantly changing as solvent B is constantly being added to A (Fig. 3).

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬13

Adsorption Chromatography

Chromatography course

Figure 3: Gradient elution. Flow of solvent B into solvent A With mixing, continuously changing the composition of solvent A as it flows into column

Figure 4: Elution of chromatography column with a gradient of increasing salt concentration.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬14

Adsorption Chromatography

Chromatography course

Gradients other than linear gradients (e.g., exponential, concave. or convex) may be obtained by introducing a third vessel and varying the composition of the solvents in the vessels. These eluting methods are also used with other column chromatographic methods. 3-Activation of adsorbent Many adsorbents such as alumina, silica gel, and active carbon and Mg silicate can obtain commercially, but they require activation before use. Activation is achieved by heating and there is usually an optimum temperature for activation, for e.g. alumina is about 400oC. For reduced activity by the controlled addition of water, and the subsequent activity is related to the amount of water added. Brookman and Schodder established five grades of alumina Grade I is the most active 0

and the is simply alumina heated at about 350 C for several hours. Grade II has about 2-3% water, Grade III 5-7%, Grade IV 9-11 %, Grade V film. (Least active) about 15%. 4-Retention The retention is a measure of the speed at which a substance moves in a chromatographic system. In continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the retention is usually measured as the retention time Rt or tR, the time between injection and detection. In interrupted development systems like TLC the retention is measured as the retention factor Rf, the run length of the Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬15

Adsorption Chromatography

compound

divided

Chromatography course

by

the

run

length

of

the

eluent

front:

The retention of a compound often differs considerably between experiments and laboratories due to variations of the eluent, the stationary phase, temperature, and the setup. It is therefore important to compare the retention of the test compound to that of one or more standard compounds under absolutely identical Conditions. 5-Plate theory The plate theory of chromatography was developed by Martin

and

Synge.

The

plate

theory

describes

the

chromatography system, the mobile and stationary phases, as being in equilibrium. The partition coefficient K is based on this equilibrium, and is defined by the following equation:

K is assumed to be independent of concentration, and can change if experimental conditions are changed, for example temperature is increased or decreased. As K increases, it takes longer for solutes to separate. For a column of fixed length and flow, the retention time (tR) and retention volume (Vr) can be measured and used to calculate K Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬16

Adsorption Chromatography

Chromatography course

6- Column chromatography 1. Small plug of wool (or cotton) 2. Sand to cover "dead volume" 3. Silica gel, length = 5.5 - 6 inch (Note 1inch=2.54cm). 4. Tap column on bech (carefully) to remove air bubbles inside the column 5. Add solvent system 6. Add sand on top of silica 7. The top of the silica gel should not be allowed to run dry. 8. Sample is diluted (20-25% solution) 9. The sample is applied by pipette 10. Solvent used to pack the column is reused 11. Walls of column are washed with a few milliliters of eluant 12. Column is filled with eluant 13. Flow controller is secured to column and adjusted 2.0 in / min.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬17

Adsorption Chromatography

Chromatography course

Figure 5

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬18

Adsorption Chromatography

Chromatography course

Column as that illustrated in Fig.5 may be used:  Typical chromatographic column.  Mixture sorbed on top of column.  Partial separation  Complete separation Table 2: Common adsorbents and the type of compounds Solid

Suitable for separation of

Alumina

Steriods, vitamins, ester, and alkaloids

Silica gel

Steriods, amino acids, alkaloids

Carbon

Peptides, carbohydrates, amino acid

Magnesium

Porphyrins

carbonate Magnesium

Steriods, ester, glycerides, alkaloids

silicate Magnesia

Similar to alumina.

Ca(OH)2

Carotenoids.

CaCO3

Carotenoids and xanthophylls.

Ca Phosphate

Enzymes, protein, and polynucleotide

Starch

Enzymes.

Sugar

Chlorophyll.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬19

Thin layer chromatography

Chromatography course

Thin layer chromatography This technique is particularly useful for the separation of very small amounts of material. The general principle involved is similar to that involved in column chromatography, i.e. it is primarily adsorption chromatography, although other partition effects may also be involved. A glass sheet is covered by a uniform thin layer of an adsorbent. Adsorbents used in TLC, differ from column adsorbents. It contain a binding agent such as calcium sulphate, which facilitates the adsorbent sticking to the glass plate. The plates are prepared by spreading slurry of adsorbent in water over them, starting at one end, and moving progressively to the other and then drying them in an oven at 100120°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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬20

Thin layer chromatography

Chromatography course

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

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬21

Thin layer chromatography

Chromatography course

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)

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬22

Thin layer chromatography

Chromatography course

Destructive techniques Staining Solutions immerse the plate as completely as possible in the stain and remove it quickly. Heat carefully with a heating

Stains Anisaldehyde PMA Vanillin Ceric sulfate DNP Permangante

Use/Comments Good general reagent, gives a range of colors Good general reagent, gives blue/green spots Good general reagent, gives a range of colors Fairly general reagent, gives a range of colors Mainly for aldehydes and ketones, gives orange spots 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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬23

Thin layer chromatography

Chromatography course

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 2,4,6trimethylpyridine 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)

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬24

Thin layer chromatography

Chromatography course

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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬25

Thin layer chromatography

Chromatography course

Examples of some TLC separation systems Compounds Amino acids

Adsorbent Silica Gel G

Mono and di saccharides

Kieselguhr G (sodium acetate) Kieselguhr G (sodium phosphate pH5) Silica Gel G

Neutral lipids Cholesterol Esters Carotenoids

Silica Gel G

Phospholipids

Silica Gel G

Kieselguhr G

Solvent system (v/v) 96% Ethanol/water (70/30) Butan-1-ol/acetic acids/ water (80/20/20) Ethyl acetate/propan-1-ol (65/35). Butan-1-ol / acetone/phosphate buffer pH5 (40/50/10) Petroleum ether/diethyl ether/acetone (90/10/1) Carbon tetrachloride/ chloroform (95/5) Petroleum ether/propan-1ol (99/1) 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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬26

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 multipleascent technique. For descending chromatography, papers 22 cm wide and 56 cm long can be used. To facilitate the flow of solvent from the paper, the bottom of the paper is serrated with a pair of pinking shears. Three pencil lines are drawn 25 mm apart at the top of the sheet, and small aliquot of the sample (10-50 ml) is placed at a marked spot on the third line. The spot is kept as small as possible by adding the aliquot in small increments. With drying in between. This may be expedited with a hair dryer. Several samples, including standards, are placed 15-25 mm apart.

The paper is then folded along the other two lines and placed in the solvent trough of the descending tank (Fig. 1). Which has been equilibrated with solvent beforehand to ensure a saturated atmosphere. The paper is irrigated with solvent until the solvent reaches the bottom or for a longer period, allowing the solvent to flow off the end of the paper, if necessary. The chromatogram is then removed dried and developed to reveal the locations of the compounds. (Part II gives methods of locating carbohydrates, amino acids. proteins. nucleotides and nucleic acids and lipids.) In ascending chromatography, a paper approximately 25 cm x 25 cm is used. A pencil line 20-25 mm from the bottom is drawn across the paper

Fig. 1 Steps in descending paper chromatography

and aliquots (10-50l) of the samples and standards are spotted approximately 15-25 mm apart along the line. The spots are dried and the paper is rolled into a cylinder and stapled so that the ends of the paper are not touching (Fig. 2). Solvent is poured into the bottom of a chromatographic chamber, and the cylinder is placed inside. The chamber is closed and solvent is allowed to flow up

Fig. 2 Steps in ascending paper chromatography the paper by capillary action. The chamber may be a simple wide-mouth, screw top, gallon jar or a cylinder with a ground-glass edge and a glass plate top. As with descending chromatography, the chamber should be equilibrated

with

solvent

beforehand.

Contrary

to

a

popular

misconception, if the chamber has been sealed and is airtight, the paper docs not have to be removed as soon as the solvent reaches the top. When

multiple ascents are performed, the paper is removed, thoroughly dried, and returned to the chamber for another ascent of solvent. The resolved compounds on a paper chromatogram may be detected by their color if they are colored, by their fluorescence if they are fluorescent, by a color that is produced from a chemical reaction on the paper after spraying or dipping the chromatogram with various reagents, or by autoradiography if the compounds are radioactive. Identification of compounds on a chromatogram is usually based on a comparison with authentic compounds (standards). A quantitative comparison may be made by measuring the Rf , which is the ratio of the distance the compound migrates to the distance the solvent migrates. A better comparison is the ratio of the distance a particular compound migrates to the distance a particular standard migrates. For example, in the separation of carbohydrates, the standard might be glucose and the ratio would be RGlc or for amino acids, the standard might be glycine and the ratio would be RGly A useful modification is two-dimensional paper chromatography, in which the sample is spotted in the lower left-hand corner and irrigated in the first dimension with solvent A. The chromatogram is removed from the solvent dried, turned 90, and irrigated in the second dimension with solvent B, giving a two-

Fig. 3 Two-dimensional paper or thin-layer chromatography dimensional separation (Fig. 3). An application of this procedure has been developed for the study of enzyme specificity in which a solution of the enzyme is sprayed onto the chromatogram between the first irrigation and the second to see what products are formed by the action of the enzyme on the compounds separated in the first dimension. Paper chromatography has been used to establish the structural homology of a series of oligomers obtained by enzymic synthesis, by acid or enzymic hydrolysis, or by isolation from a natural source. The R F of each separated homologue is determined and a French-Wild plot is made by plotting log [RF / (1-RF)] against the number of monomers in the oligomer. If the isolated compounds fall on a straight line of this plot, they belong to a homologous series, differing from each other by one monomer residue

(Fig. 4). Compounds separated by paper chromatography may be quantitatively determined. Aliquots (50-200,l) of the solution containing the substances to be separated and quantitatively determined are streaked along the separation line. Aliquots of the solution (5-10,l) are also spotted on the two outside edges of the streak and are used as location standards. The chromatogram is irrigated in the usual way, and vertical sections of the location standards are cut out and developed to reveal the positions of the compounds. After drying, these standards are placed alongside the streaked sections and the undeveloped compounds are located; horizontal strips containing the individual compounds are cut out and

Fig. 4. French-Wild plots (log RF / 1-RF), versus number of monomer units per molecule) correlating paper chromatographic mobility with the number of homologous monomer residues in oligosaccharide molecules.

Fig. 5. Elution of compounds from paper chromatograms for preparative chromatography or quantitative determination

eluted with water. To accomplish the elution, tabs of chromatographic paper are stapled to the narrow ends of each strip. As shown in Figure 5, one end is fitted with two pieces of glass (cut microscope slides), which arc held together with rubber bands, and the bottom end is cut tapered, like a pipet tip. This assembly is played so that one end lies in a chromatographic trough containing water, and the elution of the strip occurs by capillary flow of the water down the paper strip into a baker. Usually less than 1 mL of water is sufficient to effect quantitative elution, the samples are quantitatively diluted to a specific volume, and a chemical analysis is performed for the specific compound separated. This technique also may be used as a preparative procedure to obtain small quantities of pure compound from a mixture of compounds. In an alternate quantitative procedure, the compounds in the sample are radioactively labeled and separated in the usual way, and an autoradiogram is prepared. The labeled compounds are located on the chromatogram by comparing their positions on the autoradiogram. The radioactive compounds are cut out and placed into a liquid scintillation cocktail, and the radioactivity is determined by heterogeneous liquid scintillation counting

Paper Chromatography What is Chromatography? Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/or quantify the mixture or components.

• Analyze • Identify • Purify • Quantify

Separate

Mixture

Components Uses for Chromatography

Chromatography is used by scientists to: 

Analyze – examine a mixture, its components, and their relations to one another



Identify – determine the identity of a mixture or components based on known components



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



Quantify – determine the amount of the a mixture and/or the components present in the sample

Real-life examples of uses for chromatography: •

Pharmaceutical Company – determine amount of each chemical found in new product

•

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

•

Law Enforcement – to compare a sample found at a crime scene to samples from suspects

•

Environmental Agency – determine the level of pollutants in the water supply

•

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

Definition of Chromatography Detailed Definition: Chromatography is a laboratory technique that separates components within a mixture by using the differential affinities of the components for a mobile medium and for a stationary adsorbing medium through which they pass.

Terminology: • Differential – showing a difference, distinctive • Affinity – natural attraction or force between things • Mobile Medium – gas or liquid that carries the components (mobile phase) • Stationary Medium – the part of the apparatus that does not move with the sample (stationary phase) Simplified Definition: Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase.

Explanation: • Compound is placed on stationary phase • Mobile phase passes through the stationary phase • Mobile phase solubilizes the components • Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases

Illustration of Chromatography Stationary Phase

Separation

Mobile Phase

Mixture

Components

Components

Affinity to Stationary Phase

Affinity to Mobile Phase

Blue

----------------

Insoluble in Mobile Phase

Black





Red





Yellow



        

Principles of Paper Chromatography 

Capillary Action – the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity.



Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on both their solubility in

the mobile phase and their differential affinity to the mobile phase and the stationary phase.

Paper Chromatography Experiment What Color is that Sharpie?

Overview of the Experiment Purpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography. Time Required: Prep. time: 10 minutes Experiment time: 45 minutes Costs: Less than $10

Materials List • 6 beakers or jars • 6 covers or lids • Distilled H2O • Isopropanol • Graduated cylinder • 6 strips of filter paper • Different colors of Sharpie pens • Pencil • Ruler • Scissors • Tape

Preparing the Isopropanol Solutions Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100%

Preparing the Chromatography Strips  Cut 6 strips of filter paper  Draw a line 1 cm above the bottom edge of the strip with the pencil  Label each strip with its corresponding solution  Place a spot from each pen on your starting line

Developing the Chromatograms  Place the strips in the beakers  Make sure the solution does not come above your start line  Keep the beakers covered  Let strips develop until the ascending solution front is about 2 cm from the top of the strip  Remove the strips and let them dry

Developing the Chromatograms

Developing the Chromatograms

Observing the Chromatograms

0

%

20

50

70

100

%

%

%

%

Concentration of Isopropanol

Alternative Experiments

Protein purification

Chromatography

1. Ammonium Sulfate Fraction of Protein Mixtures Increasing the salt concentration to a very high level will cause proteins to precipitate from solution without denaturation if done in a gentle manner. First, we want to understand why the protein precipitates. A protein in a buffer solution is very highly hydrated, in other words, the ionic groups on the surface of the protein attract and bind many water molecules very tightly:

This graphic illustrates how proteins in solution are hydrated by water molecules. When a lot of salt, such as ammonium sulfate, is added to the protein solution, the salt ions attract the water molecules away from the protein. This is partly since the salt ions have a much greater charge density than the proteins. So as the salt is added and these small ions bind water molecules, the protein molecules are forced to interact with themselves and begin to Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬1

Protein purification

Chromatography

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 Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬2

Protein purification

Chromatography

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 Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬3

Protein purification

Chromatography

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 Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬4

Protein purification

Chromatography

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 Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬5

Protein purification

Chromatography

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 Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬6

Protein purification

Chromatography

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.

Dr.Ehab Aboueladab

(Assistance. Prof.of Biochemistry, Mansoura University)

‫صفحة‬7

Desalting Before an ion-exchange chromatographic step or after an ammonium sulfate fractionation, it is usually necessary to remove the salt from the solution of protein. Desalting is accomplished in one of two ways: dialysis or gel filtration.

Dialysis

Dialysis is performed by filling a section of dialysis tubing (a semi permeable membrane) having a sufficiently small molecular weight "Cutoff", with the protein solution, and placing the filled tubing in a large volume of buffer. The decrease in salt concentration can be calculated easily from the ratio of the volumes inside and outside of the bag.

Dialysis requires a few hours, after which the bag may be transferred to fresh buffer if the reduction in salt concentration effected by one cycle is deemed to be insufficient. In dialysis, all small molecules, including salt ions, metal ions and cofactors, pass through the membrane, which retains only macromolecules. Neither tightly bound metal ions and cofactors, nor counterions to the macromolecule are effectively removed. Since the initial solution in the bag is of much greater osmotic strength than the surrounding buffer, the bag generally increases in volume. The volume of the contents of the bag must be measured after dialysis if either total protein or total enzyme units are to be calculated.

Ion Exchange Chromatography Since proteins have different net charge and charge distribution, ion exchange chromatography can be an effective purification tool. For bench-top preparations, usually gravity-flow columns are employed, but HPLC and automated HPLC-like systems have grown in popularity. For gravity flow or for use with low pressure peristaltic pumps, ion exchange media are usually carbohydrate based. Charged groups are attached to solid supports (“inert phase”) such as Sepharose, Sephadex and cellulose. Since these carbohydrates are compressible, they are not used in higherpressure systems, and more rigid inert phases such as TSK (a polyethercoated gel) are used. For higher pressures, reinforced Polysaccharides,

and organically coated silica (e.g., TSK) are used. The resins, especially poly (styrenediviny1benzene) described by HIRS for use with enzymes were used by MOORE and STEIN in their famous amino acid analyzer. They are commonly employed for ion exchange chromatography of small molecules, but have given way to the ion exchange polysaccharides for preparative applications in enzymology. The charged groups used with the solid supports depend to some extent on the chemistry of the support material itself, but are remarkably similar. Groups containing charged nitrogen atoms are almost universally used for anion exchange media. These include, from strong to weak, quaternary amino methyl or ethyl (QAE), tertiary amino (diethylaminoethyl, DEAE, or diethylaminomethyl) and secondary plus tertiary nitrogens (polyethylenimine, PEI). The quaternary amino compounds are positively charged at any pH, but the others must be used at a pH below the pK, of the protonated form (- 10, for DEAE). The conjugate base of the strongly acidic sulfonic acid (i.e., alkyl or aryl sulfonate) and the weakly acidic carboxylic acids (e.g., carboxymethyl, CM) are the most common charged groups employed in cation exchangers. The carboxymethyl packing must be used at a pH above their pK4. Methods for determining the optimal pH for separation of proteins depends, of course, on the proteins. Since most proteins are acidic, they are negatively charged at pH 7-8. They therefore adsorb to a positively charged stationary phase to which they act as counterions,

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

cationic stationary phase is known as an anion

exchanger because it functions by exchanging one anionic counterion for another. Anionic proteins may bind more tightly to anion- exchange stationary phases than simple salts because they possess more negative charges than a simple anion. However, it is not the total charge on a protein, but the charge density that determines the affinity. More precisely, it is the charge distribution. Since a protein may interact with a stationary phase on one side at a time, proteins with densely charged patches may be bound more tightly. At pH values below the isoelectric point of a protein, the net charge is positive, so negatively charged stationary phases (cation exchange phases) are used. If a protein has an isoelectric point near neutrality, either a cation exchange or an anion exchange system can be used, depending on the pH employed. The important considerations in choosing an optimal pH for separation of enzymes by ion exchange chromatography have been reviewed. Protein solutions are generally desalted, then loaded onto a column packed with a stationary phase having the appropriate charge. Loading can often be done as rapidly as the columns will flow without undue pressure; proteins that adsorb are retained at the top of the column. As long as the capacity of the column is not exceeded, liters of a (desalted, buffered) crude extract can be loaded onto a column of modest size, so that a pre-

chromatography concentration step is not needed. After loading, the column is washed with the loading buffer to remove unabsorbed and weakly adsorbed proteins. The adsorbed proteins are then eluted by washing the column with buffers of increasing salt concentration (e.g., NaCl), which corresponds to increasing solvent strength. This method of elution using a series of isocratic (constant strength) elutions of progressively increasing strength is known as batch elution. The ion having a charge of the same sign as the protein can act as a displacing ion by competing for charged sites on the stationary phase. At some concentration, the eluting ion competes effectively with the protein, which accordingly, spends a larger fraction of its time in the mobile phase, leading to elution. This concentration would be ideal to purify the protein of interest providing that more loosely bound proteins were removed first, because it affords the maximum discrimination among the charge densities of the proteins still on the column. However, the protein might elute as a broad, dilute band. A simple and common solution to elution is to employ a linear concentration gradient of salt, Such a gradient can cover a range from 0 to 1 M NaCl over the volume of a few hundred ml to a few liters, depending on the dimensions of the column and the steepness of the gradient desired.

A major advantage of gradient elution is that proteins having a wide range of affinities for the column can be eluted in a single run. The information obtained from a gradient elution may be used to determine an optimum salt concentration to be used in isocratic elution, but the procedure is not straightforward. The theory of gradient elution is messy, even in the simplest case. One egregious misstatement appears in numerous papers on enzyme purification “the enzyme elutes at such and such a concentration of sodium chloride”. Because the gradient travels much more rapidly in the column than the protein (the protein is retained to some extent), the concentration of sodium chloride in which the enzyme actually appears at the bottom of the column is much higher than the concentration at which it began to elute appreciably. Thus, the concentration in which it appears to elute (concentration of sodium chloride in the fraction in which the activity appears) is much too strong for use as an isocratic eluent. In addition, the concentration in which the enzyme appears varies with the dimensions of the column; longer columns cause the enzyme to appear to elute in a higher salt concentration, simply because the gradient progresses as the enzyme moves down the column. To exercise maximum control over the system, it is useful to separate the effects of pH from those of ionic strength during ion exchange chromatography. One of the ions involved in the buffering system bears the same charge as the protein and can therefore

act as a displacing ion. The concentration of this ion should not change with pH, so it should not be the one involved in the equilibrium with solvent protons. Buffering ions selected for use in ion exchange chromatography should have the same charge as the column, i.e., cations for an anion exchange column, anions for cation exchange. Hence, phosphate buffers are used for cation exchange chromatography, and Tris (for instance) buffers are used for anion exchange. It is necessary for the column to be completely equilibrated with the starting solvent. Equilibration can be checked by measurement of both pH and ionic strength (e.g., by conductivity) prior to loading the column. Elution from an ion-exchange column could also be accomplished using a change in pH. Stepwise pH changes are sometimes employed, but do not generally produce high resolution of complex mixtures. Reproducible continuous pH gradients are difficult to obtain because so many of the components in the system engage in acid-base equilibria. A workable system along these lines has been devised using a proprietary mixed-bed packing and a multi-component buffer system to elute proteins at their isoelectric pH. The process is called chromatofocusing because of a loose analogy to isoelectric focusing gel electrophoresis.

Gel filtration Biomolecules are purified using chromatography techniques that separate them according to differences in their specific properties, as shown in Figure 1. and Table 1.

Property Size Charge Hydrophobicity

Biorecognition (ligand specificity) Table 1.

Technique Gel filtration (GF), also called size exclusion Ion exchange chromatography (IEX) Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC) Affinity chromatography (AC)

Fig. 1. Separation principles in chromatography purification. Gel filtration has played a key role in the purification of enzymes, polysaccharides,

nucleic

acids,

proteins

and

other

biological

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

chromatography techniques and separates molecules on the basis of differences in size. The technique can be applied in two distinct ways: 1. Group separations: The components of a sample are separated into two major groups according to size range. A group separation can be used to remove high or low molecular weight contaminants (such as phenol red from culture fluids) or to desalt and exchange buffers. 2. High resolution fractionation of biomolecules: The components of a sample are separated according to differences in their molecular size. High resolution fractionation can be used to isolate one or more components, to separate monomers from aggregates, to determine molecular weight or to perform a molecular weight distribution analysis. Gel filtration can also be used to facilitate the refolding of denatured proteins by careful control of changing buffer conditions. Gel filtration is a robust technique that is well suited to handling biomolecules that are sensitive to changes in pH, concentration of metal ions or co-factors and harsh environmental conditions. Separations can be performed in the presence of essential ions or cofactors, detergents, urea, guanidine hydrochloride, at high or low ionic strength, at 37 °C or in the cold room according to the requirements of the experiment

Gel filtration in practice Gel filtration separates molecules according to differences in size as they pass through a gel filtration medium packed in a column. Unlike ion exchange or affinity chromatography, molecules do not bind to the chromatography medium so buffer composition does not directly affect resolution (the degree of separation between peaks).

Separation by gel filtration Gel filtration medium is packed into a column to form a packed bed. The medium is a porous matrix in the form of spherical particles that have been chosen for their chemical and physical stability, and inertness (lack of reactivity and adsorptive properties). The packed bed is equilibrated with buffer which fills the pores of the matrix and the space in between the particles. The liquid inside the pores is sometimes referred to as the stationary phase and this liquid is in equilibrium with the liquid outside the particles, referred to as the mobile phase as shown in Figure 2. Gel filtration is used in group separation mode to remove small molecules from a group of larger molecules and as a fast, simple solution for buffer exchange. Small molecules such as excess salt (desalting) or free labels are easily separated. Samples can be prepared for storage or for other chromatography techniques and assays. Gel filtration in group separation mode

is often used in protein purification schemes for desalting and buffer exchange

.

Fig. 2. Common terms in gel filtration Sephadex G-10, G-25 and G-50 are used for group separations. Large sample volumes up to 30% of the total column volume (packed bed) can be applied at high flow rates using broad, short columns. Figure 3 shows the elution profile (chromatogram) of a typical group separation. Large molecules are eluted in or just after the void volume, Vo as they pass through the column at the same speed as the flow of buffer. For a well packed column the void volume is equivalent to approximately 30% of the total column volume. Small molecules such as salts that have full

access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt, of buffer has passed through the column. In this case the proteins are detected by monitoring their UV absorbance, usually at A280 nm, and the salts are detected by monitoring the conductivity of the buffer.

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

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

and conductivity (salt) traces enable pooling of the desalted fractions and facilitate optimization of the separation. The theoretical elution profile (chromatogram) of a high resolution fractionation. Molecules that do not enter the matrix are eluted in the void volume, Vo as they pass directly through the column at the same speed as the flow of buffer. For a well packed column the void volume is equivalent to approximately 30% of the total column volume (packed bed). Molecules with partial access to the pores of the matrix elute from

the column in order of decreasing size. Small molecules such as salts that have full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt, of buffer has passed through the column, Fig. 4.

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

absorbance). Separation examples

Fig. 5.

Cytochrome C, Aprotinin, Gastrin I, Substance P, (Gly)6, (Gly)3 and Gly

Comparison of the selectivity of Superdex 75 prep grade and Superdex 200 prep grade for model proteins Figure.6 Superdex 75 prep grade (a) gives excellent resolution of the three proteins in the Mr range 17 000 to 67 000 while the two largest proteins elute together in the void volume. Superdex 200 prep grade (b) resolves the two largest proteins completely. The three smaller proteins are not resolved quite as well as the larger ones or as in (a). The void volume (Vo) peak at 28 minutes in (b) is caused by protein aggregates.

Fig. 6. Columns

:

a) HiLoad 16/60 Superdex 75 prep grade b) HiLoad 16/60 Superdex 200 prep grade

Sample

:

1. Myoglobin 1.5 mg/ml, Mr 17 000 2. Ovalbumin 4 mg/ml, Mr 43 000 3. Albumin 5 mg/ml, Mr 67 000 4. IgG 0.2 mg/ml, Mr 158 000 5. Ferritin 0.24 mg/ml, Mr 440 000

Sample volume :

0.5 ml

Buffer

:

0.05 M phosphate buffer, 0.15 M NaCl, 0.01% sodium azide, pH 7.0

Flow

:

1.5 ml/min (45 cm/h)

Media Selection Chromatography media for gel filtration are made from porous matrices chosen for their inertness and chemical and physical stability. The size of the pores within a particle and the particle size distribution are carefully controlled to produce a variety of media with different selectivities. Today's gel filtration media cover a molecular weight range from 100 to 80 000 000, from peptides to very large proteins and protein complexes. Figure.7

Superdex is the first choice for high resolution, short run times and high recovery. Sephacryl is suitable for fast, high recovery separations at laboratory and industrial scale Superose offers a broad fractionation range, but is not suitable for large scale or industrial scale separations. Sephadex is ideal for rapid group separations such as desalting and buffer exchange. Sephadex is used at laboratory and production scale, before, between or after other chromatography purification steps.

The selectivity of a gel filtration medium depends solely on its pore size distribution and is described by a selectivity curve. Gel filtration media are supplied with information on their selectivity, as shown for Superdex in Figure 8. The curve has been obtained by plotting a partition coefficient Kav against the log of the molecular weight for a set of standard proteins

Fig. 8. Selectivity curves for Superdex

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

Selectivity curves are usually quite linear over the range Kav = 0.1 to Kav = 0.7 and it is this part of the curve that is used to determine the fractionation range of a gel filtration medium Figure 9.

Determination molecular weight

Ve – V0 Kav =-------------Vt – V0 where Ve = elution volume for the protein Vo = column void volume Vt = total bed volume On semilogarithmic graph paper, plot the Kav value for each protein standard (on the linear scale) against the corresponding molecular weight (on the logarithmic scale). Draw the straight line which best fits the points on the graph. Then, Calculate the corresponding Kav for the component of interest and determine its molecular weight from the calibration curve.

Sephadex: Rapid group separation of high and low molecular weight substances, such as desalting, buffer exchange and sample clean up Sephadex is prepared by cross-linking dextran with epichlorohydrin. Variations in the degree of cross linking create the different Sephadex

media and influence their degree of swelling and their selectivity for specific molecular sizes (Table. 2 ). Product

Sephadex G-10 Sephadex G-25 Coarse Sephadex G-25 Medium Sephadex G-25 Fine Sephadex G-25 Superfine Sephadex G-50 Fine

Fractionation range, Mr (globular proteins) <7×102

pH stability

Bed volume ml/g dry Sephadex

Particle size, wet

Long term: 2–13 Short term: 2–13 Long term: 2–13 Short term: 2–13

2-3

55–165 μm

4-6

170–520 μm

1×103–5×103

Long term: 2–13 Short term: 2–13

4-6

85–260 μm

1×103–5×103

Long term: 2–13 Short term: 2–13

4-6

35–140 μm

1×103–5×103

Long term: 2–13 Short term: 2–13

4-6

17–70 μm

1×103–3×104

Long term: 2–10 Short term: 2–13

9-11

40–160 μm

1×103–5×103

• Sephadex G-10 is well suited for the separation of biomolecules such as peptides (Mr >700) from smaller molecules (Mr <100). • Sephadex G-50 is suitable for the separation of molecules Mr >30000 from molecules Mr<1500 such as labeled protein or DNA from unconjugated dyes. The medium is often used to remove small nucleotides from longer chain nucleic acids. • Sephadex G-25 is recommended for the majority of group separations involving globular proteins. These media are excellent for removing salt and other small contaminants away from molecules that are greater than Mr 5000. Using different particle sizes enables columns to be packed according to application requirements

Sephadex

is

prepared

by

cross-linking

dextran

with

epichlorohydrin, illustrated in Figure 10 The different types of Sephadex vary in their degree of cross-linking and hence in their degree of swelling and selectivity for specific molecular sizes, as shown

Fig. 10. Partial structure of Sephadex.

Why use different techniques at each stage In order to final removal of trace contaminants. Adjustment of pH, salts or additives for storage. Then, end product of required high level purity Therefore, The technique chosen must discriminate between the target protein and any remaining contaminants

Gel Filtration (or) Gel Permeation Chromatography (or) Size Exclusion Chromatography Size exclusion chromatography(SEC), also called gel permeation Chromatography (GPC) or gel filtration chromatography(GFC) is a technique for separates molecules according to their molecular size. Gel particles form the stationary phase of this type of chromatography; the mobile phase is the solution of molecules to be separated and the eluting solvent, which most frequently is water or a dilute buffer. The sample is applied to the gel, if the molecules are too large for the pores; they never enter the gel and move outside the gel bed with the eluting solvent. Thus, the very large molecules in a mixture move the fastest through the gel bed and the smaller molecules, which can enter the gel pores, are retarded and move more slowly through the gel bed. In gel chromatography, molecules are, therefore, eluted in order of decreasing molecular size

Fig.1 Gel permeation chromatography. Open circles represent porous gel molecules: large solid Circles represent molecules too large to enter the gel through the pores, and smaller solid circles represent molecules capable of entering the gel pores

Three types of polymers are principally used-dextran, polyacrylamide, and agarose Dextran is a polysaccharide composed of (-1--->6)-linked glucose residues with (-1,3) branch linkages. It is synthesized from sucrose by an enzyme produced by the bacterium Leuconostoc mesenteroides B512F. The dextran is cross-linked to various extents by reaction with epichlorohydrin to give gel beads with different pore sizes Fig.2. Crosslinked dextrans are commercially produced by Pharrnacia Fine Chemicals, lnc., (Uppsala, Sweden), and sold under the trade name Sephadex. Sephadex gels in the so-called G-series, where the Gnumbers refer to the amount of water gained when the beads are swelled in water (Table 1) have different degrees of cross-linking, hence different pore sizes. This gives gels that have capabilities of separating different ranges of molecular weights and have different molecular

exclusion limits. The exclusion limit is the molecular weight of the smallest peptide or globular protein that will not enter the gel pore. Sephadex G-10, the highest cross-linked dextran, has a water regain of about 1mL/g of dry gel and Sephadex G-200, the lowest cross-linked dextran, has a water regain of about 20 mL/g of dry gel. In the swelling process, the gels become filled with water.

Fig.2. Structure of epichlorohydrin cross linked Dextran

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

Water regain (mL/g)

Exclusion limit

Maximum Maximum Gel hydrostatic flow rate pressure cm (ml,min) H2O 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 6 Sepharose 6B NA 4 x 10 200 1.2 6 Sepharose CL 6B NA 4 x 10 2.5 >200 6 Sepharose 4B NA 20 x 10 80 0.96 6 Sepharose CL 4B NA 20 x 10 120 2.17 6 Sepharose 2B NA 40 x 10 40 0.83 6 Sepharose CL 2B NA 40 x 10 50 1.25 Bio-Gel P-2 1.5 1800 110 >100 Bio-Gel P-4 2.4 4000 95 >100 Bio-Gel P-6 3.7 6000 75 >100 Bio-Gel P-10 4.5 20000 75 >100 Bio-Gel P-30 5.7 40000 65 >100 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 3 >100 6 Bio-Gel A-1.5m NA 1.5 x 10 2.5 >100 6 Bio-Gel A-5m NA 5 x 10 1.5 >100 6 Bio-Gel A-15m NA 15 x 10 90 1.5 6 Bio-Gel A-50m NA 50 x 10 50 1.0 6 Bio-Gel A-150m NA 150 x 10 30 0.5 Bio-Gel is a trade name of Bio-Rad Laboratories Sephadex and Sepharose are trade name of Pharmacia Fine Chemical

Polyacrylamide gels are long polymers of acrylamide cross-linked with N.N'methylene-bisacrylamide (Fig. 3).

Fig.3. Structure of cross-linked polyacrylamide

The gels are commercially produced by BioRad Laboratories, Richmond. California, as the Bio-Gel P series. Like the Sephadex G series. the BioGels differ in degree of cross-linking and in pore size; the Bio-Gels, however. have a wider range of pore sizes than is available in the Sephadex G series See Table. 1 for the exclusion limits and properties of the different Bio-Gels. Agarose is a gel material with pore sizes larger than cross-linked dextran or polyacrylamide. Agarose is the neutral polysaccharide fraction of agar. It is composed of a linear polymer of D-galactopyranose linked ( 1->4) 3,6 anhydro-L-galactopyranose, which is linked (1-> 3) (Fig. 4).

D-galactose (-1->4) 3,6-Anhydro-L-galactose Fig.4. Structure of the repeating unit of agarose, D-galactopyranose linked (-1->4) to 3,6-anhydro-L-galactopyranose, which is linked (-1-3) to the next Dgalactopyranose residue

When the polysaccharide is dissolved in boiling water and cooled, it forms a gel by forming inter-and intramolecular hydrogen bonds. The pore sizes are controlled by the concentration of the agarose. High molecular weight materials such as protein aggregates, chromosomal DNA, ribosomes, viruses, and cells have been fractionated on agarose gels. Bio-Rad markets the agarose Bio-Gel A series with different molecular exclusion limits, and Pharmacia markets agarose as Sepharose and Sepharose CL. The latter is Sepharose cross-linked by reacting with alkaline 2,3-dibromopropanol to give an agarose gel with increased thermal and chemical stability. Table 1 gives the properties of the different Sephadex, Bio-Gel, and Sepharose gels. The separations that may be achieved by gel permeation chromatography are based on differences in the molecular sizes of the molecules. The method is used for both preparative and analytical purposes. The latter has been especially useful in determining the molecular weights of proteins. The proteins are chromatographed on a gel column and the

elution volume of the protein determined. Proteins with known molecular weights are also chromatographed and the elution volumes determined. Then, from a plot of log molecular weight versus elution volume, the molecular weight of an unknown protein may be determined (Fig. 5).

Fig.5. Molecular weight determination of proteins by gel permeation chromatography using Sephadex G-100 as the gel bed: log molecular weight is plotted versus elution volume.

Gel chromatography provides a rapid and mild method of removing salts and other small molecules from high molecular weight biomolecules. The sample containing the biomolecules and the salt is passed over a gel column whose exclusion limit is below the molecular weight of the biomolecules. The biomolecules which do not enter the gel emerge in the void volume of the column, while the salts enter the gel and are retarded, and therefore are removed from the biomolecules.

Ion-exchange chromatography Ion-exchange chromatography is a variation of adsorption chromatography in which the solid adsorbent has charged groups chemically linked to an inert solid. Ions are electrostatically bound to the charged groups; these ions may be exchanged for ions in an aqueous solution. Ion exchangers are most frequently used in columns to separate molecules according to charge. Because charged molecules bind to ion exchangers reversibly. Molecules can be bound or eluted by changing the ionic strength or pH of the eluting solvent. Two types of ion exchanger are available: those with chemically bound negative charges are called cation exchangers and those with chemically bound positive charges are called anion exchangers. The charges on the exchangers are balanced by counterions such as chloride ions for the anion exchangers and metal ions for the cation exchangers. Sometimes buffer ions are the counterions. The molecules in solution which are to be adsorbed on the exchangers also have net charges which are balanced by counterions. As an example of an ion-exchange process, let us say that the molecules to he adsorbed from solution have a negative charge (X-), which is counterbalanced by sodium ions (Na +). Such negatively charged molecules can be chromatographed on an anion

exchanger (A+), which has chloride ions as the counterion to give A+Cl-. When (Na+ X-) molecules in solution interact with the ion exchanger, the X- displaces the chloride ion from the exchanger and becomes electrostatically bound to give A+X-, simultaneously releasing sodium ions. This process of ion exchange is illustrated in Figure 1. A similar but opposite process would take place for positively charged molecules (Y+ Cl-) which would be chromatographed on cation exchangers (C-Na+). Thus the cation exchangers will bind positively charged molecules from solution and the anion exchangers will bind negatively charged molecules from solution. One of the inert materials used in this type of chromatography is cross-linked polystyrene, to which the charged groups are chemically bound. In the separation of biologically important macromolecules, such as enzymes and proteins.

Figure 1. The process of anion-exchange chromatography

Cellulose and cross-linked dextran (Sephadex) are used as the solid supports and charged groups such as diethylaminoethyl (DEAE)

or carboxymethyl (CM) are chemically linked to them to give anion and cation and the exchangers respectively. The preparation and commercial availability of these materials beginning in the 1960 provided the biochemist with powerful tools for separation of proteins and nucleic acid Figure 2 presents partial structures of DEAE-cellulose and CM –cellulose

Figure 2. Partial structures of diethylaminoethyl-cellulose and carboxymethylcellulose. The DEAE and CM groups are shown attached to the C6-hydroxyl group of glucose. The DEAE and CM groups are also found attached to the hydroxyl groups of C2 and C3. The total degree of substitution of the DEAE and CM groups must be less than one group per five glucose residues to maintain a water-insoluble product. Table 1. Pretreatment steps for DEAE-cellulose and CM -cellulose ion exchangers

Cellulose

First treatment

DEAE CM

0.5 M HCl 0.5 M NaOH

Intermediate pH 4 8

Second treatment 0.5 M NaOH 0.5 M HCl

The dry ion-exchange celluloses are pretreated with acid and base to swell the exchangers so that they become fully accessible to the charged macromolecules in solution. The weighed exchanger is suspended in 15 volumes (w/v) of the "first treatment," acid or alkali depending on the exchanger (Table. 1), and is allowed to stand at least 30 minutes but not more than 2 hours. The supernatant is decanted and the exchanger is washed until the effluent is at the "intermediate pH" The exchanger is stirred into 15 volumes of the "second treatment" and allowed to stand for

an additional 30 minutes. The second treatment is repeated and the exchanger is washed with distilled water until the effluent is close to neutral pH. The treated exchanger is placed into the acid component of the buffer (the pH should be less than 4.5) and degassed under vacuum 10 cm Hg pressure) with stirring, until bubbling stops The exchanger is then titrated with the basic component of the buffer to the desired pH, filtered, and suspended in fresh buffer to complete the pretreatment. The exchanger is allowed to settle and the "fines" (fragments < 10 m in diameter) above the settled exchanger are removed by decantation. Buffer is added to the exchanger so that the final volume of the slurry is l50% of the settled wet volume of the exchanger. The column is then packed with the slurry of the exchanger, the sample is applied, and elution is performed as described for adsorption chromatography. Three general methods are used for eluting molecules from the exchanger: (a) Changing the pH of the buffer to a value at which binding is weakened (i.e., the pH is lowered for an anion exchanger and raised for a cation exchanger), (b) Increasing the ionic strength by increasing the concentration of salt in the elution solvent, thereby weakening the electrostatic interactions between the adsorbed molecule and the exchanger, and

(c) Performing affinity elution. In affinity elution the adsorbed molecule is usually a macromolecule that is desorbed from the affinity ligand by adding a molecule that is charged and of opposite signs to the net charge on the macromolecule and has a specific affinity for the macromolecule. Thus, the reduction of the net charge on the macromolecule weakens its electrostatic interaction with the exchanger sufficiently to permit the elution of the macromolecule from the affinity ligand. The stages of anion exchange chromatography.

An example of the use of ions exchange resins Is the purification of cytochrome C: Cytochrome C has an isoelectric point (pI) of 10.05; that is at pH 10.05 the number of positive charges will equal the number of negative charges. A cloumn containing a cation exchanger buffered, at pH 8.5,

is prepared. This column has a full negative charge. Cytochrome C at pH 8.5 has a full positive charge. An Impure solution of cytochrome C at pH 8.5 placed on the column, and water is passed through the column (the pI of proteins is usually 7.0 or less) but cytochrome C is held firmly by electrostatic attraction to the resin heads. If the

eluting

solvent pH

is

raised

to

about

10,

the

cytochrome C will now has a net zero charge and will pass rapidly through as a pure component.

Gel Filtration

Ion exchange chromatography

Affinity chromatography

Histadine ,Aspartic,glycine,tyrosine In anion exchange chromatography,which seprate first and why

AFFINITY CHROMATOGRAPHY Affinity chromatography is a specialized type of adsorption chromatography in which a specific type of molecule is covalently linked to an inert solid support. This specific molecule called a ligand, has a high binding affinity for one of the compounds in a mixture of substances. The process uses the unique biological property of the substance to bind to the ligand specifically and reversibly and provides a high degree of selectivity in the isolation and purification of biological molecules

Fig. 1. The steps of affinity chromatography

A solution containing the substance to be purified. Usually a macromolecule such as a protein (enzyme, antibody, hormone. etc.). Polysaccharide or nucleic acid is passed through a column containing an insoluble inert polymer to which the ligand has been covalently attached. The ligand may be specific competitive inhibitors, substrate analogues, product analogues, coenzymes and so on. Molecules in the mixture not having affinity for the ligand pass through the column. Wide molecules that have specific affinity for the ligand are bound and retained on the column. The specifically adsorbed molecules) can be eluted by changing the ionic strength the pH or by the addition of a competing ligand. In one chromatographic step. The method is capable of isolating a single substance in a pure form. It has thus become a powerful tool in the isolation and purification of enzymes, antibodies, antigens, nucleic acids. Polysaccharides, coenzyme or vitamin binding proteins, repressor proteins, transport proteins, drug or hormone receptor structures and other biochemical materials.

The Inert Support and the Ligand The inert solid supports are the same materials discussed in the preceding sections: cross-linked dextran cross-linked polyacrylamide, agarose and cellulose. The macromolecules to be separated should not be retarded by a gel filtration process but should be retarded only by the

specific interaction with the ligand. The ligand must be a molecule that display, special and unique affinity for the macromolecule to be purified it also must have a chemical group that can be modified for covalent linkage to the solid support without destroying or seriously decreasing its interaction with the macromolecule to be purified. Also for successful affinity chromatography, the chemical groups of the ligand that arc critical for the binding of the macromolecule to be purified must be sufficiently distant from the solid support to minimize steric interference with the binding process. This steric problem has been solved by adding a long, hydrocarbon chain spacer arm to the solid support and coupling the ligand to the end of the arm. Alternatively the hydrocarbon arm may be attached to the ligand and the arm attached to the solid support.

Attachment of the Ligand to the Solid Support The polysaccharide solid supports-cross-linked dextran, agarose, and cellulose can be activated by reaction with alkaline cyanogen bromide. The products that arc formed upon coupling of the activated polysaccharides with amino compounds are derivatives of amino carbonic acid. The reactions are the following:

If the ligand contains an amino group, it can be coupled directly to the activated polysaccharide. A spacer arm can be introduced by sequential reaction with a diaminoalkane and glutaraldehyde. The amino group on the ligand can then be coupled to the free aldehyde group.

If the ligand contains an aldehyde group instead of an amino group, it can be coupled directly to the free amino group of the diaminoalkane. Ligands may be coupled to polyacrylamide by displacing the amide group

of the polyacrylamide by heating with a diaminoalkane (c), followed by reaction with glutaraldehyde (d).

The Schiff base that results from the reaction of glutaraldehyde with an amino group may be stabilized by reduction with sodium cyanoborohydride without affecting the aldehyde group. The ligand can then be coupled to the aldehyde group.

Another method of activating polyacrylamide is to form the hydrazide derivative by reaction with hydrazine hydrate. When an amino, aldehyde, or hydrazide group is incorporated onto the solid support, the support becomes activated so that ligands may be attached through amino, carboxyl, phenolic, or imidazole groups.

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

(Equation 1.0) where E = the electric field in volts/cm q = the net charge on the molecule f = frictional coefficient, which depends on the mass and shape of the molecule V = the velocity of the molecule The charged particle moves at a velocity that depends directly on the electrical field (E) and charge (q) but inversely on a counteracting force generated by the viscous drag (f ) The applied voltage represented by E in Equation 1.0 is usually held constant during electrophoresis, although some experiments are run under conditions of constant current (where the voltage changes with resistance) or constant power (the product of voltage and current). Under constant-voltage conditions, Equation 1.0 shows that the movement of a charged molecule depends only on the ratio q/f. For molecules of similar conformation (for example, a collection of linear DNA fragments or spherical proteins), varies with size but not shape; therefore, the only remaining variables in Equation 1.0 are the charge (q) and mass dependence of (f ) meaning that under

such conditions molecules migrate in an electric field at a rate proportional to their charge-to-mass ratio. The movement of a charged particle in an electric field is often defined in terms of mobility, , the velocity per unit of electric field (Equation 2.0).

(Equation 2.0) This equation can be modified using Equation 1.0.

(Equation 3.0) In theory, if the net charge, (q), on a molecule is known, it should be possible to measure (f) and obtain information about the hydrodynamic size and shape of that molecule by investigating its mobility in an electric field. Attempts to define (f) by electrophoresis have not been successful, primarily because Equation 3.0 does not adequately describe the electrophoretic process. Important factors that are not accounted for in the equation are interaction of migrating molecules with the support medium and shielding of the molecules by buffer ions. This means that electrophoresis is not useful for describing specific details about the shape of a molecule. Instead, it has been applied to the analysis of purity and size of macromolecules. Each molecule in a mixture is expected to have a unique charge and size, and its mobility in an electric field will therefore be unique. This expectation forms the basis for analysis and

separation by all electrophoretic methods The technique is especially useful for the analysis of ammo acids, peptides, proteins, nucleotides, nucleic acids, and other charged molecules. Method of Electrophoresis All types of electrophoresis are based on the principles just outlined. The major difference between methods is the type of support medium, which can be either cellulose or thin gels. Cellulose is used as a support medium for low-molecular-weight biochemical such as ammo acids and carbohydrates, and polyacrylamide and agarose gels are widely used as support media for larger molecules. Geometries (vertical and horizontal), buffers, and electrophoretic conditions for these two types of gels provide several different experimental arrangements, as described below.

Polyacrylamide Gel Electrophoresis (PAGE) Gels formed by polymerization of acrylamide have several positive features in electrophoresis: A) High resolving power for small and moderately sized proteins and nucleic acids (up to approximately 1 X 106 daltons), B) Acceptance of relatively large sample sizes, C) Minimal interactions of the migrating molecules with the matrix, and

D) Physical stability of the matrix that gels can be prepared with different pore sizes by changing the concentration of cross-linking agents. Electrophoresis through polyacrylamide gels leads to enhanced resolution of sample components because the separation is based on both molecular sieving and electrophoretic mobility The order of molecular movement in gel filtration and PAGE is very different, however in gel filtration, large molecules migrate through the matrix faster than small molecules The opposite is the case for gel electrophoresis, where there is no void volume in the matrix, only a continuous network of pores throughout the gel. The electrophoresis gel is comparable to a single bead in gel filtration. Therefore, large molecules do not move easily through the medium, and the rate of movement is small molecules followed by large molecules. Polyacrylamide gels are prepared by the free radical polymerization of acrylamide and the cross-linking agent N,N'- methylene-bisacrylamide. Chemical polymerization is controlled by an initiator-catalyst system, ammonium persulfate-N,N,Nَ,Nَ tetramethylethylenediamine (TEMED). Photochemical polymerization may be initiated by riboflavin in the presence of ultraviolet (UV) radiation. A standard gel for protein separation is 7.5% polyacrylamide. It can be used over the molecular size range of 10,000 to 1,000,000 daltons; however, the best resolution is obtained in the range of 30,000 to 300,000 daltons. The resolving power

and molecular size range of a gel depend on the concentrations of acrylamide and bis-acrylamide Lower concentrations give gels with larger pores, allowing analysis of higher-molecular-weight biomolecules In contrast, higher concentrations of acrylamide give gels with smaller pores, allowing analysis of lower-molecular-weight biomolecules (Table 1.0) Effective Range of Separation of DNA by PAGE Acylamide

Range of Separation

Bromophenol

Xylene Cyanol

(% W/V)

(bp)

Blue

35

1000-2000

100

450

50

80-500

65

250

80

60-400

50

150

120

40-200

20

75

200

5-100

10

50

Polyacrylamide electrophoresis can be done using either of two arrangements, column or slab. Figure 1.0 shows the typical arrangement for a column gel. Glass tubes (10 cm X 6 mm l.d.) are filled with a mixture of acrylamide, N,N'-methylene-bis-acrylamide, buffer, and free radical initiator catalyst. Polymerization occurs in 30 to 40 minutes. The gel column is inserted between two separate buffer reservoirs. The upper

reservoir usually contains the cathode and the lower the anode. Gel electrophoresis is usually carried out at basic pH, where most biological polymers are anionic; hence, they move down toward the anode. The sample to be analyzed is layered on top of the gel and voltage is applied to the system. A "tracking dye" is also applied, which moves more rapidly through the gel than the sample components. When the dye band has moved to the opposite end of the column, the voltage is turned off and the gel is removed from the column and stained with a dye. Chambers or column gel electrophoresis is commercially available or can be constructed from inexpensive materials.

Slab gels are now more widely used than column gels. A slab gel on which several samples may be analyzed is more convenient to make and use than several individual column gels. Slab gels also offer the advantage that all samples are analyzed m a matrix environment that is identical in composition. A typical vertical slab gel apparatus is shown in Figure 2.0.

The polyacrylamide slab is prepared between two glass plates that are separated by spacers Figure 3.0.

The spacers allow a uniform slab thickness of 0.5 to 2.0 mm, which is appropriate for analytical procedures. Slab gels are usually 8 X 10 cm or 10 X 10 cm, but for nucleotide sequencing, slab gels as large as 20 X 40 cm are often required. A plastic "comb" inserted into the top of the slab gel during polymerization forms indentations in the gel that serve as sample wells. Up to 20 sample wells may be formed. After polymerization, the comb is carefully removed and the wells are rinsed thoroughly with buffer to remove salts and any unpolymerized acrylamide. The gel plate is clamped into place between two buffer reservoirs, a sample is loaded into each well, and voltage is applied. For visualization, the slab is removed and stained with an appropriate dye. Perhaps

the

most

difficult

and

inconvenient

aspect

of

polyacrylamide gel electrophoresis is the preparation of gels. The monomer, acrylamide, is a neurotoxin and a cancer suspect agent; hence, special handling is required. Other necessary reagents including catalysts and initiators also require special handling and are unstable- In addition, it is difficult to make gels that have reproducible thicknesses and compositions. Many researchers are now turning to the use of precast polyacrylamide gels. Several manufacturers now offer gels precast in glass or plastic cassettes. Gels for all experimental operations are available including single percentage (between 3 and 27%) or gradient gel concentrations and a variety or sample well configurations and buffer

chemistries. Several modifications of PAGE have greatly increased its versatility and usefulness as an analytical tool. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophorosis (SDS-PAGE) The electrophoretic techniques previously discussed are not applicable to the measurement of the molecular weights of biological molecules because mobility is influenced by both charge and size. If protein samples are treated so that they have a uniform charge, electrophoretic mobility then depends primarily on size (see Equation 2.0). The molecular weights of proteins may be estimated if they are subjected to electrophoresis in the presence of a detergent, sodium dodecyl sulfate (SDS), and a disulfide bond reducing agent, mercaptoethanol. This method is often called "denaturing electrophoresis." When protein molecules are treated with SDS, the detergent disrupts the secondary, tertiary, and quaternary structure to produce linear polypeptide chains coated with negatively charged SDS molecules. The presence of mercaptoethanol assists in protein denaturation by reducing all disulfide bonds. The detergent binds to hydrophobic regions of the denatured protein chain in a constant ratio of about 14 g of SDS per gram of protein. The bound detergent molecules carrying negative charges mask the native charge of the protein In essence, polypeptide chains of a constant charge/mass ratio and uniform shape are produced The electrophoretic

mobility of the SDS-protein complexes is influenced primarily by molecular size the larger molecules are retarded by the molecular sieving effect of the gel, and the smaller molecules have greater mobility Empirical measurements have shown a linear relationship between the log molecular weight and the electrophoretic mobility Figure 4.0

In practice, a protein of unknown molecular weight and subunit structure is treated with 1% SDS and 0.1 M mercaptoethanol in electrophoresis buffer. A standard mixture of proteins with known molecular weights must also be subjected to electrophoresis under the same conditions. Two sets on standards are commercially available, one for low-molecular-weight proteins (molecular weight range 14,000 to

100,000) and one for high-molecular weight proteins D5,000 to 200,000) Figure 5.0

a stained gel after electrophoresis of a standard protein mixture After electrophoresis and dye staining, mobilities are measured and molecular weights determined graphically SDS-PAGE is valuable for estimating the molecular weight of protein subunits This modification of gel electrophoresis finds its greatest use in characterizing the sizes and different types of subunits in oligomeric proteins. SDS-PAGE is limited to a molecular weight range of 10,000 to 200,000. Gels of less than 2.5% acrylamide must be used for determining molecular weights above 200,000, but these gels do not set well and are very fragile because of minimal cross-linking. A modification using gels of agarose-acrylamide mixtures allows the measurement of molecular weights above 200,000.

Agarose Gel Electrophoresis The electrophoretic techniques discussed up to this point are useful for analyzing proteins and small fragments of nucleic acids up to 350,000 daltons (500 bp) in molecular size; however, the small pore sizes in the gel are not appropriate for analysis of large nucleic acid fragments or intact DNA molecules. The standard method used to characterize RNA and DNA in the range 200 to 50,000 base pairs 50 kilobases) is electrophoresis with agarose as the support medium. Agarose, a product extracted from seaweed, is a linear polymer of galactopyranose derivatives. Gels are prepared by dissolving agarose in warm electrophoresis buffer. After cooling the gel mixture to 50°C, the agarose solution is poured between glass plates as described for polyacrylamide. Gels with less than 0.5% agarose are rather fragile and must be used in a horizontal arrangement (Figure 4.8). The sample to be separated is placed in a sample well made with a comb, and voltage is applied until separation is complete. Precast agarose gels of all shapes, sizes, and percent composition are commercially available. Nucleic acids can be visualized on the slab gel after separation by soaking in a solution of ethidium bromide, a dye that displays enhanced fluorescence when intercalated between stacked nucleic acid bases. Ethidium bromide may be added directly to the agarose solution before gel formation. This

method allows monitoring of nucleic acids during electrophoresis. Irradiation of ethidium bromide treated gels by UV light results in orange-red bands where nucleic acids are present. The mobility of nucleic acids in agarose gels is influenced by the agarose concentration and the molecular size and molecular conformation of the nucleic acid. Agarose concentrations of 0.3 to 2.0% are most effective for nucleic acid separation Table 2.0

Figure 6.0

The separation of DNA fragments on agarose gels. Like proteins, nucleic acids migrate at a rate that is inversely proportional

to the logarithm of their molecular weights; hence, molecular weights can be estimated from electrophoresis results using standard nucleic acids or DNA fragments of known molecular weight. The DNA conformations most frequently encountered are superhelical circular (form I), nicked circular (form II), and linear (form III). The small, compact, supercoiled form I molecules usually have the greatest mobility, followed by the rodlike, linear form III molecules. The extended, circular form II molecules migrate more slowly. The relative electrophoretic mobility of the three forms of DNA, however, depends on experimental conditions such as agarose concentration and ionic strength. Isoelectric Focusing of Proteins Another important and effective use of electrophoresis for the analysis of Proteins are isoelectric focusing (IEF), which examines electrophoretic mobility as a function of pH. The net charge on a protein is pH dependent. Proteins below their isoelectric pH (pH I or the pH at which they have zero net charge) are positively charged and migrate to a medium of fixed pH toward the negatively charged cathode at a pH above its isoelectric point, a protein is deprotonated and negatively charged and migrates toward the anode If the pH of the electrophoretic medium is identical to the pHI of a protein, the protein has a net charge of zero and does not migrate toward either electrode. Theoretically, it should be possible to separate protein molecules and to estimate the pH:

of a protein by investigating the electrophoretic mobility in a series of separate experiments in which the pH of the medium is changed. The pH at which there is no protein migration should coincide with the pHI of the protein. Because such a repetitive series determine the pHI, IEF has evolved as an alternative method for performing a single electrophoresis run in a medium of gradually changing pH. Figure 7.0

illustrates the construction and operation of an IEF pH gradient. An acid, usually phosphoric, is placed at the cathode; a base, such as triethanolamine, is placed at the anode. Between the electrodes is a medium in which the pH gradually increases from 2 to 10. The pH gradient can be formed before electrophoresis is conducted or formed during the course of electrophoresis. The pH gradient can be either broad (pH 2-10) for separating several proteins of widely ranging pH I values or narrow (pH 7-8) for precise determination of the pHI of a single protein. P

in Figure 7.0 represents different molecules of the same protein in two different regions of the pH gradient. Assuming that the pH in region 1 is less than the pHI of the protein and the pH in region 2 is greater than the pHI of the protein, molecules of P in region 1 will be positively charged and will migrate m an applied electric field toward the cathode. As P migrates, it will encounter an increasing pH, which will influence its net charge. As it migrates up the pH gradient, P will become increasingly deprotonated and its net charge will decrease toward zero. When P reaches a region where it's net charge is zero (region 3), it will stop migrating. The pH in this region of the electrophoretic medium will coincide with the pHI of the protein and can be measured with Illustration of isoelectric a surface microelectrode, or the position of the protein can be compared to that of a calibration set of proteins of bown pHI values. P molecules in region 2 will be negatively charged and will migrate toward the anode. In this case, the net charge on P molecules will gradually decrease to zero as P moves down the pH gradient, and P molecules originally in region 2 will approach region 3 and come to rest. The P molecules move in opposite directions, but the final outcome of IEF is that P molecules located anywhere m the gradient will migrate toward the region corresponding to their isoelectric point and will eventually come to rest in a sharp band; that is, they will "focus" at a point corresponding to their pHI. Since different protein molecules in

mixtures have different pHI values, it is possible to use IEF to separate proteins In addition; the pHI of each protein in the mixture can be determined by measuring the pH of the region where the protein is focused. The pH gradient is prepared in a horizontal glass tube or slab. Special precautions must be taken so that the pH gradient remains stable and is not disrupted by diffusion or convective mixing during the electrophoresis experiment. The most common stabilizing technique is to form the gradient in a polyacrylamide, agarose, or dextran gel. The pH gradient is formed in the gel by electrophoresis of synthetic polyelectrolyte, called ampholytes, which migrate to the region of their pHI values just as proteins do and establish a pH gradient that is stable for the duration of the IEF run. Ampholytes are low-molecular-weight polymers that have a wide range of isoelectric points because of their numerous ammo and carboxyl or sulfonic acid groups. The polymer mixtures are available in specific pH ranges (pH 5-7, 6-8, 3.5-10, etc.). It is critical to select the appropriate pH range for the ampholyte so that the proteins to be studied have pHI values in that range. The best resolution is, of course, achieved with an ampholyte mixture over a small pH range (about two units) encompassing the pHI of the sample proteins. If the pHI values for the proteins under study are unknown, an ampholyte of wide pH range (pH 3-10) should be used first and then a narrower pH range selected for use. The gel medium is prepared as previously described

except that the appropriate ampholyte is mixed prior to polymerization. The gel mixture is poured into the desired form (column tubes, horizontal slabs, etc.) and allowed to set. Immediately after casting of the gel, the pH is constant throughout the medium, but application of voltage will induce migration of ampholyte molecules to form the pH gradient. The standard gel for proteins with molecular sizes up to 100,000 daltons is 7.5% polyacrylamide; however, if larger proteins are of interest, gels with larger pore sizes must be prepared. Such gels can be prepared with a lower concentration of acrylamide (about 2%) and 0.5 to 1% agarose to add strength. Precast gels for isoelectric focusing are also commercially available. The protein sample can be loaded on the gel in either of two ways. A concentrated, salt-free sample can be layered on top of the gel as previously described for ordinary gel electrophoresis. Alternatively, the protein can be added directly to the gel preparation, resulting in an even distribution of protein throughout the medium. The protein molecules move more slowly than the low-molecular-weight ampholyte molecules, so the pH gradient is established before significant migration of the proteins occurs. Very small protein samples can be separated by IER. For analytical purposes, 10 to 50 g is a typical sample size. Larger sample sizes (up to 20 mg) can be used for preparative purposes.

Common abbreviations in chromatography GF:

gel filtration (sometimes referred to as SEC: size exclusion chromatography) IEX: ion exchange chromatography (also seen as IEC) AC: affinity chromatography RPC: reverse phase chromatography HIC: hydrophobic interaction chromatography CIPP: Capture, Intermediate Purification and Polishing MPa: megapascals psi: pounds per square inch SDS: sodium dodecyl sulphate CIP: cleaning in place A280nm, UV absorbance at specified wavelength A214nm: Mr: relative molecular weight N: column efficiency expressed as theoretical plates per meter Ve: elution volume is measured from the chromatogram and relates to the molecular size of the molecule. Vo: void volume is the elution volume of molecules that are excluded from the gel filtration medium because they are larger than the largest pores in the matrix and pass straight through the packed bed Vt: total column volume is equivalent to the volume of the packed bed Rs: resolution, the degree of separation between peaks Kav and partition coefficient and log molecular weight, terms used logMr: when defining the selectivity of a gel filtration medium In product names HMW: high molecular weight LMW: low molecular weight HR: high resolution pg: prep grade precision column PC: SR: solvent resistant

Protein

------------Negative--------------------------o-----------------------Positive----------------------(pH>pI)

(pH=pI)

(pH
anion exchange resin

cation exchange resin

(resin is positive A+)

(resin is negative C-)

At pH > pI, protein net charge is negative At pH < pI, protein net charge is positive At pH = pI, protein net charge is zero Isoelectric point (pI)

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

nonpolar molecules lack charge polar, uncharged molecules carry one or more partial charges

-Organic component of the solvent continues migrating, thus forming the mobile phase. -Therefore, compounds soluble to organic component move faster than compounds soluble to aqueous component. -Thus, molecules are separated according to their polarities.

Differential precipitation Salt (or other solute) is added to a solution that contains a mixture of proteins Ammonium sulfate is most popular for “salting out” -Effective, highly soluble, & does not tend to denature protein Individual proteins precipitate at specific [(NH4)2SO4] Depends on properties of specific solute (salt), not ionic strength per se Precipitated proteins are: - isolated in centrifuge - resuspended in low salt buffer - dialysis or gel filtration can be used to remove residual precipitant (if necessary) Often used in two steps: • Salt out some impurities w/ lower concentration that does not precipitate POI • Use higher concentration for selective precipitation of POI Protein purification Prior requirements for devising a purification method: • Source tissue – Known or likely to contain “high” levels of POI

– Available in suitable (preparative) quantities Possible (& potentially misleading) alternative: clone gene & use expression system • Exogenous system may not recapitulate protein processing/covalent modifications • POI may function as part of a complex (w/ addn’l subunits derived from other genes) • Assay method that is: – Specific for POI & relatively insensitive to other components in extracts – Linear and quantitative: • measured activity should be proportional to amount of POI • Amount of activity is expressed in “units”: – e.g., amount of S � P per time – “katal” (kat) is preferred unit: moles per second – If POI is not enzyme, assay may be based on binding (direct or indirect) – Suitably sensitive – Optimized with respect to pH, ionic strength, temp., substrate or ligand concentration, etc.

The polymerization reaction of acrylamide and methylenebisacrylamide

The three major forms of alanine occurring in titrations between pH 1 and 14

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