Lab.3. Colloidal solutions - umlub.pl · Lab.2. Colloidal solutions and gel electrophoresis 2 Colloid systems (or simply colloids) are a specific form of disperse systems. They include

Lab.2. Colloidal solutions and gel electrophoresis

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Key words:

(Colloidal solutions) disperse systems, lyophilic and lyophobic colloids, properties of

colloids, coagulation, sedimentation processes, factors influence on coagulation, Hardy-

Schultz rule, (Gel electrophoresis) Separation methods, properties of compounds (structure,

charge), migration in electric field, electrophoretic mobility, μE, capillary electrophoresis

Literature:

P. Monk; Physical Chemistry, Understanding our chemical world, Wiley 2004, pp. 504-523.

D.A. Skoog, F.J. Holler, T.A. Nieman: Principles of Instrumental Analysis; 996 -1020

J. Crowe. T. Bradshaw, P. Monk, Chemistry for the Biosciences. The essential concepts;

Oxford University Press, 2006; Chapter 17, pp. 515 - 549.

F. Rouessac, A. Rouessac, Chemical Analysis, Modern Instrumentation Methods and

Techniques, 6th ed., 2004, Chapter 8.

Theoretical background

System in which one substance is distributed throughout another in the form of finely divided

particles is called disperse system. They usually consist of two phases: the dispersed phase,

consisting of the suspended solid particles and the dispersive medium surrounding them.

Compare three solutions: true solution (e.g. table salt or sugar in water), colloid solutions

(particles of proteins 100 – 1000 times greater than water molecule) and sand in water.

a) b) c)

Na+ AgCl H2O Cl- • • • •

H2O H2O SAND •••

Solution NaCl in water Colloid system Sand in water

Fig. 1. a) Diameter of ions ~ diameter of water molecules; b) diameter of colloid particles are

100 – 1000 times greater than diameter of water molecules; c) diameter of sand particles 106

times higher than diameter of water molecules.


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Colloid systems (or simply colloids) are a specific form of disperse systems. They include

systems with a comparatively high degree of dispersion, in which the particle size range from

0.10 to 1000 nm (1 nm = 1∙10-9 m). The particles of colloid systems cannot be seen through

an ordinary microscope.

There is an extremely great variety of colloid systems. They are widespread in nature, and

find application in industrial processes. Many natural substances such as milk, blood, egg

albumin, numerous vegetable and animal tissues are colloid systems. Other disperse systems

are clouds, fog and volcanic dust.

Lyophilic and lyophobic colloids

A lyophilic (solvent attracting) colloid is one whose particles have a strong attraction for the

molecules of the dispersion medium (usually water, in this case hydrophilic colloids), binding

large numbers of them into so called solvent shells, on the contrary, in a lyophobic (solvent

repelling) colloid the particles do not interact so strongly with the molecules of the

surrounding medium.

Colloid systems differ widely with respect to stability, some can be preserved unchanged for

long time – others are comparatively unstable. There are two kinds of processes that lead to

the disintegration of colloid systems: sedimentation and coagulation processes. In

sedimentation – the particles of the dispersed phase settle out on the bottom of beaker.

In coagulation processes the particles of the dispersed phase adhere to one another increasing

in size. If the particles are sufficiently large, they settle under the force of gravity.

The following conditions are necessary for the stability of lyophobic and lyophilic colloids:

• the particles must be very small,

• must carry electric charges of like sign,

• must form solvate shells.

The first condition prevents sedimentation, the second and third hinder coagulation.

Let us consider small colloid particle called a micelle. Suppose we have solution of AgCl in

water. The nucleus of each particle of this solution is composed of great number of molecules

of the AgCl (solid). Let m be the average number of molecules in one such nucleus. The n Cl-

and n – x Ag+ ions adsorbed on the nucleus collectively form a particle, while x Ag+ ions are

concentrated in the solution immediately surrounding it.

The structure of a micelle of this solution can be represented by the following formula:


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[(m AgCl), n Cl-, n – x Ag+] x- + x Ag+

micelle

The structure of a micelle corresponding to this model is depicted in Fig. 2.

Ag+

Ag+ Ag+

Ag+

Ag+

Ag+

Ag+

Fig. 2. The silver chloride adsorbed chloride ions and became negatively charged.

It is well seen that the micelle has negative electric charge. The system, however, is neutral,

because the electric charge on the micelles of AgCl is compensated by the opposite charge on

the ions in the surrounding medium.

Micelles of the hydrophilic colloids, e.g. proteins strongly interact with molecules of water

(surrounding medium), which create so-called solvent shell. This solvent shell protects the

protein molecules against the coagulation.

Cl- Cl- Cl-

Cl- Cl-

Cl- Cl-

Cl- Cl-

Cl-

m AgCl

Ag+

Ag+

Ag+

Ag+

Ag+


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Fig. 3. Micelle of protein with water shell.

Colloidal systems exhibit several properties. They are optical (Tyndall effect), kinetical or

mechanical (Brownian movement, sedimentation) and electrical (zeta potential,

electrophoresis).

Electrophoresis (from the Greek words electron = electron; and phoresis = carrying) was

introduced by Arnie Tiselius, to separate human serum into albumin, α – globulin, β –

globulin and γ – globulin.

Electrophoresis is a separation method in which charged particles (ions), migrate under the

influence of an electric field at various rates, depending upon their charge to mass ratios. Such

situation is demonstrated in Fig. 1.

P R O T E I N

H H O

H H O

H H O

H H O

H H O

H H O

O H H

O H H

O H H

O H H

O H H

H H O

H O

H O H

O H H

O H H

+

+

+ +

+ +

+ + +

+

+ +

H


5

V start line

((9

Gel + buffer = stationary phase

Gel + buffer = stationary phase

Fig. 1. Migration of the charged particles: red spots (+) and white spots (―), under the

influence of an electric field.

One of the key elements in a gel electrophoresis system is the gel itself. Polyacrylamide and

agarose gel are the principal medium for electrophoresis. The gel matrix acts as a medium

reducing diffusion, so that separated sample components remain positioned in sharp zones. In

addition, the gel acts as a molecular sieve, separating molecules according to their size.

Second key element is buffer. The electrical current in an electrophoresis cell is carried

largely by the ions supplied by buffer, which also maintain proper pH and provide a medium

for heat dissipation. Third key is voltage.

In an electric field, E, an ion with charge q, moves toward opposite electrode due to

Coulombic force. Electrophoretic mobility, μE, is a function of viscosity, η, of the buffer and

radius, r, of an ion (charged particle). The electrophoretic mobility is described by equation:

μE = q / 6 Π η r (1)

It is well seen (from the equation 1), that in the buffer of constant viscosity, small, highly

charged species have high mobilities whereas large, minimally charged species have low

mobilities.

In the main, the relative mobility of individual molecules depends on several factors.

1. Molecular characteristics

Cathode (-) Anode (+)


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The size and shape of molecules influences their migration rate. As a molecule increases in

size, its migration rate declines because of frictional forces in the electrophoretic buffer

medium. Globular molecule exhibits a different (higher) migration rate from that of fibrous

one. A molecule's charge depends on its dissociation at the pH of the buffer solution used. As

a molecule's charge increases, its migration rate also increases. Note that depending on the

nature of the net charge, the charged particles will migrate to the cathode (red spots) or to the

anode (white spots). Electrophoresis of positively charged particles (cations) is called

cataphoresis, while electrophoresis of negatively charged particles (anions) is called

anaphoresis. The order of molecules migration is presented in Fig. 2.

Fig. 2. The order of molecules migration in gel electrophoresis.

2. Buffer properties

The ions of the buffer used in the electrophoretic run conduct the current applied between the

cathode and anode. The molecules in the sample interact with the ions in the buffer depending

on the pH of the buffer. This interaction affects the migration rate of the molecules. Buffers

with high ionic strengths cause an increase in current, which produces a greater amount of

heat and reduction in the molecule migration rate. Buffers with very low ionic strength lead to

problems in molecule diffusion; this impairs the separation of electrophoretic bands.

3. Electrical field characteristics

The migration of molecule depends on the characteristics of the electric field that is applied to

the sample dispersed in the buffer. For a given voltage, the current that passes through the

electrophoretic medium depends on the potential gradient and the medium's resistance. As the

potential gradient increases, molecule migration speeds up. As the length of the

electrophoretic medium that molecules traverse increases, molecules encounter greater

resistance. This hampers their migration.

++ --

catode anode

start


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4. Temperature effects

The time taken for separation of sample components is proportional to the voltage applied to

the medium. An increase in voltage increases the migration velocity. However, high voltages

generate an excessive amount of heat. Increased temperature alters the viscosity and electrical

conductivity of the electrophoretic medium. This affects the migration rate as well.

Temperature changes may also cause a change in the conformation of molecules, which

reduces the migration velocity and efficiency of electrophoretic separation.

There is also fully automated capillary electrophoresis (CE), shown in Fig. 3, besides of lab

gel electrophoresis,

Fig. 3. Schematic of capillary electrophoresis instrumentation.

“Briefly, the ends of a narrow – bore, fused silica are placed in buffer reservoirs. The content

of the reservoirs is identical to that within the capillary. The reservoirs also contain the

electrodes used to make electrical contact between the high voltage power supply and

capillary. Sample is loaded onto capillary by replacing one of the reservoirs (usually at the

anode) with a sample reservoir and applying electric field. After replacing the buffer

reservoir, the electric field is applied, and separation performed. Optical detection of mixture

component can be made at the opposite end, directly through capillary wall.” (D. Heiger,

High performance capillary electrophoresis, Agilent Technologies, 2000)


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1. Some information from organic chemistry

As you know in inorganic chemistry we have: acidic, basic and neutral compounds.

Acidic: HCl; HNO3; H2SO4; H3PO4

Basic: NaOH; Ca(OH)2; Fe(OH)3

Neutral: NaCl; K2SO4; CO2; N2

In organic chemistry we have the same situation. We instantly experience a sour, when

consuming anything containing vinegar. The component within the vinegar causing the

sensation is ethanoic acid also called acetic acid. This acid can dissociate into ions:

H O H O

H - C - C – O – H → H – C – C – O- + H+

H H

or simply:

CH3 – COOH → CH3 – COO- + H+

An acid is a substance capable of donating proton, and in case of organic acid, such as acetic

acid, proton can be release only from – COOH group, (but not from -CH3 group). We say that

- COOH group is the acidic group.

There are many organic acids like:

CH3COOH ethanoic acid

CH3CH2COOH propanoic acid

CH3CH2CH2COOH butanoic acid

- COOH benzoic acid

In organic chemistry we also have basis, mainly amines. Amines are derivatives of ammonia

(NH3), where one or more hydrogen atoms have been replaced by an alkyl group (CH3- ; CH3-

CH2-), e.g.:

CH3 – NH2 methyl amine CH3 – CH2 – NH2 ethyl amine.

Amino group can be also introduced into acetic acid, to get amino acid:

CH2 – COOH + NH3 → CH2 – COOH + HCl

Cl H – N:

H

amino acid (glycine)


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Aqueous ammonia is more properly called ammonium hydroxide, because NH3 dissolved in

water can abstract a proton from water molecule and generate the hydroxide ion, OH-,

according to the reaction:

H H

H – N: + H2O → H – N: H+ + OH-

H H

As a result of this reaction, we obtained ammonium ion NH4+ and OH- ion. In the same way,

amino group in the amino acid can attach hydrogen ion and become positively charged ion.

Proteins consists of many (more than 100 molecules) of amino acids. They possess both,

acidic group (-COOH) and basic group (-NH2), which can carry positive, negative or zero net

charge, depending on the pH of local environment.

Conclusion: many important biological molecules such as amino acids, peptides, proteins,

nucleotides, and nucleic acids, possess ionizable groups (-COOH, -NH2, phosphates), and at

any given pH, exist in solution as electrically charged species either as cations (+) or anions

(-).

EXPERIMENTAL PART

PART I. Establishing the coagulative force of various electrolytes

Procedure:

Put the baker with 300 cm3 distilled water on hot plate and keep until the water begin to boil.

Keeping the water boiling, drop in by pipette 30 cm3 of 2 % solution FeCl3. After leaving

obtained sol to cool down.

To ten tubes add 10 cm3 of sol. To other five tubes add 1, 3, 5, 7, 9 cm3 solution of KCl (c = 4

M L-1) and complete volume to 10 cm3 with distilled water. Take five tubes with solution of

electrolyte and mix with five solution of sol.

Shake vigorously the solution in tubes. After, observe the tubes, looking perpendicularly to

the surface of liquid. In the tubes where the process of coagulation took place, the solution is

turbid. Notice the number of tube where coagulation took place using asterisks (Table 1).


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Table 1. Estimation of coagulation force of KCl solution

Number of tubes 1 2 3 4 5

Volume of KCl (cm3) 1 3 5 7 9

Volume of water (cm3) 9 7 5 3 1

Volume of solution (cm3) 10 10 10 10 10

Fill empty spaces with sign X for the test tubes

in which coagulation happened

On the base results from the first experiment estimate the power of KCl as coagulate agent.

100020

=ca

mk ( mM L-1)

mk – coagulate force, mM L-1

a – volume of electrolyte cause coagulation of 20 cm3 of sol,

c – concentration of electrolyte M L-1.

A coagulation force of KCl is equal......................................

The same procedure repeats with solution of K2SO4. The volume of added to the tubes are: 1,

3, 5, 7, 9 cm3 of (c = 0.001 M L-1). Notice the number of tube where coagulation took place

using asterisks (Table 2).

Table 2. Estimation of coagulation force of K2SO4 solution.


Volume of K2SO4 (cm3) 1 3 5 7 9

Volume of water (cm3) 9 7 5 3 1


Fill empty spaces with sign X for

the test tubes in which coagulation

happened

The coagulative force is estimated from equation:

100020

=ca

mk ( mM L-1)

where:


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a – volume of electrolyte cause coagulation of 20 cm3 of sol,


A coagulation force of K2SO4 is equal......................................

K3Fe (CN)6 as the coagulate agent.

This same procedure repeats with solution of K3 Fe (CN)6. The volume of added to the tubes

are: 0,50; 0.75; 1.00; 1.25; 1.50 cm3 of (c = 0.001 M L-1). Notice the number of tube where

coagulation took place using asterisks in Table 2.

Table 2. Estimation of coagulation force of K3 Fe (CN)6


Volume of K3 Fe (CN)6 (cm3) 0.50 0.75 1.00 1.25 1.50

Volume of water (cm3) 9.50 9.25 9.00 8.75 8.50


Fill empty spaces with sign X for the test

tubes in which coagulation happened

The coagulative force is estimated from equation:

100020

=ca

mk ( mM L-1)

where:


a – volume of electrolyte cause coagulation of 20 cm3 of sol, use the volume of K2SO4 from

the first tube in which the coagulation happened


Coagulation forces of K3 Fe(CN)6 is equal......................................

Answer the questions:

1. What kind of ions causes coagulation process?

2. What is the coagulative force of investigated ions (the same or different for each ion)?


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PART II. Gel electrophoresis

Preparation of the agarose gel

Step 1

Weigh 1.11 g of the agarose pour into a beaker (250 mL) and add 110 mL of the

buffer solution of pH 3.6.

Step 2

Similarly prepare next two agarose gels using buffers of pH 6.0 and pH 10.

Step 3

Each beaker with the agarose puts on the hot plate and warm up to boiling. Do not

allow to boil. Take off the beaker from the hot plate and a little cool down.

Step 4

Before pouring agarose, stick adhesive tape on both sides of segment (Fig. 4).

Fig. 4. Electrophoresis chamber scheme.

Step 5

Place the plastic comb into the middle of the tray.

Step 6

Pour agarose into the gel tray, into the special segments, to obtain 5-6 min. layer of the

agarose. Note that there are three segments for each agarose gel of pH.6; pH 6 and pH 10. Let

the gel harden without disturbing it for about 10 minutes.

Step 7

Carefully remove adhesive tape from the gel.

Step 8

Pour the appropriate buffer into special box with two electrodes. Add about 380 mL of buffer

to completely fill the box and to cover the top gel surface with about 1 mm of buffer.


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Remember: we use buffer of the same pH in “the electrode box” and the agarose gel.

Step 9

Carefully remove the comb from the gel by pulling straight out of the solidified gel.

Step 10

Load on the gel, using syringe (10 μl) of 5 dyes and one mixture of dyes. Use a new tip (or

clean!) for each dye and be careful not to puncture the bottom of the well.

Step 11

Carefully place the lid on the gel box.

Step 12

Plug in the power and turn on the machine.

Step 13

Let the gel run undisturbed for about 60 minutes.

Step 14

After 60 minutes, turn off power supply, discontent the patch cords from the power supply

and unplug the unit.

Step 15

Take off the lid and measure migration distances for each dye and dyes in mixture. Result

record in Table 1.

Make sure that the power supply in unplugged and switched off before proceeding.

Table 1. The migration distances of selected dyes during electrophoresis.

Polarization voltage: ........….. V, time in min.: ……………

Solutes

pH = 3.6 pH = 6.0 pH = 10.0

distance

migration distance

migration distance

migration

toward

anode

[mm]

toward

cathode

[mm]

toward

anode

[mm]

toward

cathode

[mm]

toward

anode

[mm]

toward

cathode

[mm]

Malachite green

Rhodamine 6G

PAR 4-(2-irydyloazo) resorcinol

Red Kongo

Azorubine

Mixture dyes


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Based on data from Table 1 make the graph MD (migration distance) vs. pH.

Answer the following questions:

1. Which substances were in mixture?

2. What effects we can observe during the electrophoresis process?

3. Which substances undergo toward anode and why?

4. Which substances undergo toward cathode and why?

Mig

ration

tow

ard cath

od

e

[mm

]

Mig

ration

tow

ard an

od

e [m

m]

Buffer pH

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Lab.3. Colloidal solutions - umlub.pl · Lab.2. Colloidal solutions and gel electrophoresis 2 Colloid systems (or simply colloids) are a specific form of disperse systems. They include