A Study Of The Absorption Rates Of Protein Biology Essay

Published:

This essay has been submitted by a student. This is not an example of the work written by our professional essay writers.

Proteins are defined as the large molecule that is composed of one or more chains of amino acids in specific manner. Protein's study is very vast and divided into many areas such as food processing and manufacturing, biotechnology, biology and mostly important in the medicine. In the vast studies of proteins "Adsorption study of protein" is very popular and complicated. (1) Adsorption is a surface phenomenon means it is related with interaction of any surface like - solid - solid interaction, solid - liquid interaction and liquid - liquid interaction etc.

As we mentioned earlier, Adsorption of protein is very complicated study because here we going to study the adsorption of protein on to the solid surface in the water and protein shows folding and unfolding nature in the water. Adsorption phenomenon based on a principle that is whenever an interface formed (1)

between two different phases it has a high free energy than bulk phase. So the interface becomes thermodynamically stabilized by adsorbing other substance different than the solvent molecule. But in specific, the adsorption of protein on to the solid surface is depend on the structure and change in conformation of protein, hydrophobic and hydrophilic interactions, and the charges on their surface. (1)

The proteins are large amphiphatic molecules this characteristic make them intrinsically surface active molecule and it causes a surface interaction. The main causes for the surface interaction are conformational entropies and restricted nobilities, intermolecular forces, Lewis acid - base forces, columbic forces, hydrophobic interactions, van der Waals forces.(3,4) additionally intramolecular forces within the protein molecules that might cause alteration of protein confirmation. The determination of extent of conformational changes is very difficult and even more its prediction. So for determination of protein adsorption kinetic study of the adsorption process is required. (5)

So, the objective of this project is to study the protein adsorption on to the solid surfaces like Microcrystalline Cellulose and Silica at different pH conditions. It includes a ultra violate - vis spectrometry study to measure concentration adsorbed and in solution. This study is under taken with pH 4, 5, 6 & 7.

The protein for study used is Bovine Serum Albumin (BSA) and the adsorption BSA on to the MCC and Silica was studied as the function of pH.

Significance of protein adsorption on solid surfaces

Protein adsorption on solid interfaces and protein adsorption on liquid/ solid interfaces are process of significance to our daily lives and other fields like food processing, medicine, and biotechnology etc. (39, 40)

Combination of some natural polymers and some protein can be essential and promising for medicinal applications. (39)

Due to protein adsorption on solid surface is so common,

It has much significance in various fields - (41)

Medical Significance

Biocompatibility of medical and dental implants

Lysine-laden surfaces that can adsorb proteins to dissolve blood clots

Diagnostic devices and biosensors (41,43)

Drug delivery schemes for pharmaceuticals

Biomedical research and genetic engineering

Industrial Significance

Food and beverages processing equipments

Production equipment for protein purification and separation

Optical switching via protein adsorbed to an optical surface

Emerging significances include

Biomimetics, e.g., self healing surfaces mimic protein adsorption such as natural "anti freeze proteins". (42)

Materials engineering, e.g., antibodies binding to specific crystal surfaces to control crystal growth. (42)

Table 1: Conditions at which adsorption is predicted are marked "Yes "and absence of adsorption are marked "No ". (2)

Structural

stability

Electrical   charge

Sorbent surface

Hydrophobic

Electrical charge

Hydrophilic

Electrical charge

+

-

+

-

Protein

Stable

+

Yes

Yes

No

Yes

-

Yes

Yes

Yes

No

Unstable

+

Yes

Yes

Yes

Yes

-

Yes

Yes

Yes

Yes

Hydrophobic dehydration dominates adsorption

Electrical interactions or structural changes in proteins dominate adsorption

Bovine Serum Albumin ( BSA )

1.2.1 Physiochemical properties

Bovine serum albumin is a protein having low content of tryptophan and methionine and high content of cystine and charged amino acids, aspartic and glumatic acids, lysine and arginine. The glycine and iso leucine content of BSA are lower than other protein. (6) The BSA molecule is composed of three homologous domains (I, II, III) and which are divide into nine loops ( L1-L9) by 17 disulphide bond. (7)

The multi domain structure of BSA is responsible for the anomalous behaviour of the protein under denaturing conditions. The domains of BSA are dissimilar in hydrophobicity, net charge and ligand binding sites and each has distinct function apart from the others. There is some evidence that between 400 and 500 C, BSA partially unfolds, exposing apolar residues to the molecular surface, facilitating reversible protein - protein interaction.(8) BSA has some physiochemical properties are as follows

Table 2: Some physiochemical characteristics of BSA. (9)

Parameter

Value

Molecular weight

From composition

From physical data

Molecular dimensions ( equilateral triangle)

Side

Depth

Isoelectric point

Conformational Stability

66,267

66,700

8.0 nm

3.0 nm

4.7 - 5.1

Low

Fig. 1: Location of disulfide bonds, loops and domains in BSA structure. (10)

Bovine serum albumin has another characteristic to undergo reversible conformational isomerisation with changes in pH. The isomeric forms are Expanded (E), Fast (F), Normal (N), Basic (B), and Aged (A). The changes in forms cause due to the folding unfolding of their domain. The isomeric forms at different pH values are shown in the figure below. (11)

Fig. 2: Isomeric form of BSA changes with different pH range. (7)

Bovine serum albumin is not uniformly charged within the primary structure. Unlike the asymmetric charge distribution on the primary structure, the distribution on the tertiary structure seems fairly uniform.(6,7)

Fig.3: Space filling model of BSA molecule with basic residues coloured in blue, acidic residue in red and neutral one in yellow. (7

Besides above mentioned physiochemical properties BSA has some functional properties also.

1.2.2 Functional properties

Foaming

Foaming is defined as formation and stabilization of gas bubbles in a liquid. Proteins diffuse to the air - water interface and reduce surface tension. Proteins unfold at the interface and its association produce an intermolecular cohesive film with some degree of elasticity. (12)

Gelation

When BSA is heated it forms a soluble aggregate through disulphide and noncovalent bond.(13) Polymerized molecules forms soluble aggregate during the early stages and subsequent polymerization leads to formation of a rigid gel network.(14,15)

Ligand - binding

BSA has a very good property to bind reversibly an incredible variety of ligands. BSA has a high affinity for fatty acids, haematin, bilirubin a broad affinity for small negatively charged aromatic compounds.(16)

Microcrystalline Cellulose (MCC)

Microcrystalline cellulose is a naturally occurring substance, it is very stable, safe and physiologically inert. MCC is basically cellulose derived from the wood pulp. MCC can only derived from special grade of alpha cellulose. MCC is ideal excipient. Naturally occurring polymer composed of glucose units by a beta 1-4 beta glycosidic bond.(17) The first self suspending range of MCC was developed in 1964 then launched in USA later.

1.3.1 Physiochemical and functional properties of MCC

Purified, partially depolymerised cellulose prepared by treating alpha cellulose, obtained as a pulp from fibrous plant material, with mineral acids. The degree of polymerisation is typically less than 400. Mostly the average particle size is below than 150 μm. MCC is fine, white or almost white, odourless, free flowing crystalline powder. It is used as emulsifier, stabilizer, anticaking, dispersing agent.(18)

Chemical formula - (C6H10O5) n

Molecular structure

Fig. 4: Molecular structure of microcrystalline cellulose.(19)

Microcrystalline cellulose is insoluble in water, ethanol, and dilute mineral acids. Slightly soluble in sodium hydroxide solution. MCC has specialised rheological characters that shows a unique functional property when it fully dispersed in water using sufficint shear MCC particles form a microscopic three dimensional network of crystal. The main cause that facilitate the formation of this structure is the soluble copolymers by acting as water swelling capillaries between the microcrystals, forcing them apart during the hydration. (20, 21)

Rheological consideration (22,23,24)

Recommended application areas

Food

Pharmaceutical

Recommended solvent systems

Water

Mixtures of water with water miscible organic solvents

Table.3: Solvent systems and solubility of Microcrystalline Cellulose.(25,26)

Solvent system

Solubility

Water

Insoluble, Dispersible

Dilute Alkali

Partially soluble, Swells

Dilute Acid

Insoluble, Resistant

Organic Solvent

Insoluble, Inert

Oil

Insoluble, Inert

Ionic Charge

Powdered grades : Non-ionic

Colloidal grades : Anionic (because of presence of the protective colloid)

Compatibility/ Stability Characteristics

Excellent Thermal stability

Readily flocculated by electrolytes, cationic polymers and surfactant

Table.4: Other physiochemical properties of Microcrystalline Cellulose. (25, 26)

Composition

Microcrystalline Cellulose

Molecular weight

Equilibrium moisture,%

(58% R.H., 720 F)

Organic extractable, %

Ash, %

Calcium, ppm

Iron, ppm

Copper, ppm

Manganese, ppm

Absolute density

Particle size (Average)

30,000 - 50,000

5

< 0.04

< 0.06

< 60

< 20

< 4

0 - 2

1.55

10 - 150 μm

Silica

Many studies have been dedicated to the determination of adsorption properties and surface chemistry of silica. Silica is one of the most important adsorbents used in chromatography and chemical technology, immobilization of enzymes, as a catalyst support, as well as filler for the polymer.(27) The study of surface chemistry of silica is important but the main centre of attraction is intermolecular interaction at adsorption on silica as well as the mechanism of adsorption on silica. (27)

Silica - Water chemistry

Silica particles in aqueous solution posses a surface charge due to preferential dissolution of surface species and interfacial ion exchange. The magnitude and sign of the surface charge is dependant on the concentration of the potential determining ions and is a function of pH and ionic strength of the solution. When silica is contacted with water the surface of silica is hydrolysed and gives rise silanol at the surface. (28)

The silanol surface is acidic in nature and silica possesses a negative charge at neutral pH. And it possesses zero charge at about pH 2. The surface properties of silica particles are determined by the number and type of silanol groups on the surface and the degree of hydration of the surface. The rate of silica hydrolysis is a function of the solution pH, ionic strength, and temperature. (28)

Fig. 5: Types of silanol groups and siloxane bridges on the surface of amorphous silica, and internal OH groups. (32)

1.4.2 Silica structure and properties

Silica is giant atomic structure in which each silicon atom is bonded to four oxygen, and each oxygen atom to two silicon atoms in such a way that each silicon atom is centre of a regular tetrahedron of oxygen atom.

It is a solid of high Melting point (17000 C), Density 2 and 3 g/cm3 and refractive index in the range 1.5 - 1.6. The electronic configuration of the silicon atom suggests the idea of the formation of bonds based on tetrahedrally oriented hybrid sp3 orbitals. (33)

Fig. 7: Amorphous silica surface. The yellow represent silicon. The re and pink spheres represent bridge oxygen and non bridge oxygen atoms respectively.(34)

Basic structure of silica

Fig. 8: Tetrahedral structure of silica. (34)

Silica occurs in 13 different structural modifications. They all have different structures but their basic properties are same. The chemical bonds in silica are covalent and as above said about the orbitals in which 2 electrons are shared between the atoms. The basic building block in almost all modifications of silica is the SiO4 unit in which a central silicon atom is surrounded by four oxygen atoms. (35)

Langmuir Adsorption Isotherm

The Langmuir isotherm is that which fits in the equation developed by the Langmuir. This isotherm has a specific shape that rapidly adsorptions at low pressure of gas / vapour and then reaches a plateau well below P0, after any further increases in pressure do not cause an increase in adsorption. More and more of the available adsorption sites become occupied, further increases in pressure result in comparatively little increase in the amount adsorbed. At a certain pressure all the adsorption sites are occupied and further adsorption stops and it giving a plateau region.

The Langmuir isotherm can only occur in situations where the entire surface is covered with equally accessible, identical adsorption sites, and the presence of an adsorbed molecule on one site does not hinder adsorption to a neighbouring site. (36) Adsorption can be monolayer or multilayer.

In Monolayer adsorption all the adsorbed molecules are in contact with the surface layer of the adsorbent. In multilayer adsorption the adsorption space accommodates more than one layer of molecules and not all adsorbed molecules are in contact with the surface layer of the adsorbent. (38)

Based on Langmuir's theory, the derived equation of Langmuir isotherm is ;( 37)

Θ = KP/1+KP

Where Θ is the number of sites of the surface which are covered with gaseous molecule, P is pressure and K is equilibrium constant

At lower pressure,

Θ = KP

At higher pressure,

Θ = KP/KP = 1

Fig. 9: Standard Adsorption Isotherm (36)

1.6 UV Spectrometry analysis

This technique is well known for its simplicity, versatility, speed, accuracy and cost effectiveness. The wavelength range of UV radiations start at the blue end of visible light (about 4000 A°) ends at 2000 A°.(30) Ultra violet absorption spectra arise from transition of electrons or electrons within a molecule or an ion from a lower to higher electronic energy level and the ultraviolet emission spectra arise from the reverse type of transition.(29,30)

Fig. 13: UV spectrometry analyses (31)

In the diagram shown above hÏ… is the energy of incident light po is the energy of transmitted light.

1.3.1 Beer- Lambert law (29, 30)

The Beer- Lambert law states that the concentration of a substance in a solution is directly proportional to the absorbance 'A' of the solution.

Absorbance A = (constant) x (concentration) x (cell length).

The law is only true for the monochromatic light that is light of single wavelength and provided that the physical or chemical state of the substance does not change with concentration. When a monochromatic light passes through a homogeneous solution in a cell, the intensity of the emitted radiation depends upon the thickness (I) and the concentration (c) of the solution.

     

Fig. 14: Schematic representation of the transmittance of light (29)

Where, I is the intensity of incident radiation and Io is the intensity of the transmitted radiation. This is sometime expressed in percentage and called %transmittance.

Mathematically, absorbance is related to percentage transmittance T by the expression:

A= log10 (Io/I) = log10 (100/T) =kcL

Where, L= length of the radiation

C =concentration of absorbing molecules in the path

K =extinction coefficient.

Experimental       Procedure…

Experimental procedure

The whole experiment has been carried out at the school of science laboratory of The University of Greenwich, Medway. The instruments and chemicals used in this experiment were supplied by school of science.

Work plan

The experiment was carried out at 4 different pH values of solvent media and at room temperature. Experiment done at 7 and 6 pH is presented here. On the other side, another part of experiment that is at 4 and 5 pH is explained by my colleagues.

The experiment is based on following two processes

Adsorption of Bovine serum albumin on Microcrystalline cellulose

Adsorption of Bovine serum albumin on Silica.

Reproducibility is very much essential in pharmaceutical field; hence all the experiment has carried out twice by keeping same experimental condition.

Experimental method divided in three groups. Each group worked in following 5 sections.

For all three groups experimental condition was same i.e. pH, amount of adsorbent and volume of the medium. Two different solvent systems were used during experiment.

Distilled water pH 7, pH 6 where required.

Isotonic solution (saline solution, 0.9% w/v NaCl) pH 7, pH 6.

Material and instruments

Instruments

- UV/Visible spectroscopy

- pH meter

- Rotator

- Centrifuge

- Micropipette

- Micro filters and syringes

Chemicals

- Bovine serum albumin (BSA)

- Silica

- Microcrystalline cellulose

- Sodium Chloride (NaCl)

- Diluted hydrochloric acid

- Diluted Sodium hydroxide

Method

Section 1

In case of section 1 we studied adsorption of BSA on microcrystalline cellulose at pH 7 by using distilled water as solvent system. Some experimental procedures are described as follows.

- Preparation of stock solution

Two sets of 100 ml distilled water were measured and pH of that water was adjusted by using diluted hydrochloric acid and diluted sodium hydroxide. pH meter was used to adjust the pH of solvent system. 7 pH (+,-, 0.1 unit) was kept constant throughout the experiment. Then accurately 180 mg of bovine serum albumin was weighted and dissolved in one set of 100ml of distilled water. BSA has pH around 5.2-7 which again changes pH of distilled water. Hence pH of BSA solution again adjusted to 7(+, -, 0.1 unit). Then this solution has been used as stock solution for further procedure.

- Preparation of sample solution of BSA

11 x 10 ml centrifuge tubes were taken and labelled as 0.5 ml, 1 ml, 2ml, 3ml, 4ml ...10ml. Then by using micropipette accurate amount of BSA solution was transferred into respected labelled centrifuged tube. Final volume has made up to 10 ml by using distilled water (pH 7).

- Initial absorbance of the sample solution (before adsorption)

All the sample solutions were filtered through 0.45 micron syringe filter into UV cuvettes. Base line correction was done by using pH 7 distilled water at 280 nm wavelength. Then initial absorbances of all samples were taken at 280 nm wavelength by using UV spectroscopy. All the sample solutions from cuvettes were transferred into respected tube. All the observations were recorded on lab note book for future work.

- Addition of Microcrystalline cellulose and adsorption of BSA

11 x 500 mg of microcrystalline cellulose weighted individually and added in to each centrifuge tube. Tubes were allowed to rotate for 24 hours at 20 rpm on rotator. Adsorptions of BSA on microcrystalline cellulose were initiated from this stage.

Measuring absorbance after adsorption

After 24 hour sample has dislodge from rotator and allow to centrifuge for 15 min and at 6000rpm. All the solid microcrystalline cellulose has settled down and clear supernatant solution has taken out carefully without contamination of MCC in supernatant solution. This supernatant solution was filtered through 0.45 micron syringe filter. Base line correction done by using pH 4 distilled water. Absorbance of the entire sample has taken at 280 nm wave length by using UV spectroscopy. All the solution from cuvette has transferred in to respected tube and allows rotating for 48 hr and 72 hr. same procedure carried out for 48 hr and 72 hr. Data obtained from UV absorbance was recorded into lab book for future work.

NOTE: Above explained procedure followed for pH 6 also.

Section 2

In section 2 we studied adsorption of BSA on silica surface at pH 6 by using distilled water as solvent system. Some experimental procedures are described as follows.

- Preparation of stock solution

In this case the experimental procedure was same as previous section 1 experiment. (Refer ----)

- Preparation of sample solution of BSA

In this case the experimental procedure was same as previous section 1 experiment. (Refer ----)

- Initial absorbance of the sample solution (before adsorption)

In this case the experimental procedure was same as previous section 1 experiment. (Refer ----)

- Addition of silica and adsorption of BSA

11 x 500 mg of 0.5 micron silica weighted individually and added in to each centrifuge tube. Tubes were allowed to rotate for 24 hours at 20 rpm

on rotator. Adsorptions of BSA on silica were initiated from this stage.

- Measuring absorbance after adsorption

After 24 hour sample has dislodge from rotator and allow to centrifuge for 15 min and at 6000rpm. All the solid silica has settled down and clear supernatant solution has taken out carefully without contamination of silica in supernatant solution. This supernatant solution was filtered through 0.45 micron syringe filter. Base line correction done by using pH 6 distilled water. Absorbance of the entire sample has taken at 280 nm wave length by using UV spectroscopy. All the solution from cuvette has transferred in to respected tube and allows rotating for 48 hr and 72 hr. same procedure carried out for 48 hr and 72 hr. Data obtained from UV absorbance was recorded into lab book for future work.

Section 3

Here in section 3 we studied adsorption of BSA on microcrystalline cellulose at pH 6 by using 0.9%w/v NaCl solution as solvent system. Some experimental procedures are described as follows.

- Preparation of stock solution

Two set of 90 mg of sodium chloride was weighted accurately and one set dissolved in 100 ml distilled water. pH of that water was adjusted by using diluted hydrochloric acid and diluted sodium hydroxide. pH meter was used to adjust the pH of solvent system. 6 pH (+,-, 0.1 unit) was kept constant throughout the experiment. Then accurately 180 mg of bovine serum albumin was weighted and dissolved in 100ml of saline solution. BSA has pH around 5.2-7 which again changes pH of saline water. Hence pH of BSA solution again adjusted to 6 (+, -, 0.1 unit). Then this solution has been used as stock solution for further procedure. Remaining set of NaCl dissolved in 100 ml distilled water.

- Preparation of sample solution of BSA

The experimental procedure was same as previous section 1 experiment. Saline water as solvent system instead of distilled water. (Refer ----)

- Initial absorbance of the sample solution (before adsorption)

The experimental procedure was same as previous section 1 experiment. Base line correction was done by using saline water. (Refer ----)

- Addition of microcrystalline cellulose and adsorption of BSA

11 x 500 mg of microcrystalline cellulose weighted individually and added in to each centrifuge tube. Tubes were allowed to rotate for 24 hours at 20 rpm on rotator. Adsorptions of BSA on microcrystalline cellulose were initiated from this stage.

- Measuring absorbance after adsorption

After 24 hour sample has dislodge from rotator and allow to centrifuge for 15 min and at 6000rpm. All the solid microcrystalline cellulose has settled down and clear supernatant solution has taken out carefully without contamination of microcrystalline cellulose in supernatant solution. This supernatant solution was filtered through 0.45 micron syringe filter. Base line correction was done by using pH 6 saline water. Absorbance of the entire sample has taken at 280 nm wave length by using UV spectroscopy. All the solution from cuvette has transferred in to respected tube and allows rotating for 48 hr and 72 hr. same procedure carried out for 48 hr and 72 hr. Data obtained from UV absorbance was recorded into lab book for future work.

Section 4

Here in section 4 we studied adsorption of BSA on silica at pH 6 by using 0.9%w/v NaCl solution as solvent system. Some experimental procedures are described as follows.

- Preparation of stock solution

The experimental procedure was same as previous section 1 experiment. Saline water as solvent system instead of distilled water. (Refer ----)

- Preparation of sample solution of BSA

The experimental procedure was same as previous section 3 experiment. Saline water as solvent system instead of distilled water. (Refer ----)

- Initial absorbance of the sample solution (before adsorption)

The experimental procedure was same as previous section 1 experiment. Base line correction was done by using saline water. (Refer ----)

- Addition of 0.5 micron silica and adsorption of BSA

11 x 500 mg of silica weighted individually and added in to each centrifuge tube. Tubes were allowed to rotate for 24 hours at 20 rpm on rotator. Adsorptions of BSA on silica were initiated from this stage.

- Measuring absorbance after adsorption

The experimental procedure was same as previous section 3 experiment. Silica was used as adsorbent instead of MCC. (Refer ----)

Section 5

In section 5 we tried to study adsorption of BSA on MCC at different ratio. Using distilled water as solvent system.

- Preparation of stock solution

Three sets of BSA: MCC (500mg) were prepared having ratio 3:1, 2:1, and 1:1 respectively. Distilled water was measured and pH of that water was adjusted by using diluted hydrochloric acid and diluted sodium hydroxide. pH meter was used to adjust the pH of solvent system. 6 pH (+,-, 0.1 unit) was kept constant throughout the experiment. Then this solution has been used as stock solution for further procedure.

- Preparation of sample solution of BSA

3 x 10 ml centrifuge tubes were taken and labelled as 3:1, 2:1, and 1:1.

- Initial absorbance of the sample solution (before adsorption)

All the sample solutions were filtered through 0.45 micron syringe filter into UV cuvettes. Base line correction was done by using pH 6 distilled water at 280 nm wavelength. Then initial absorbances of all samples were taken at 280 nm wavelength by using UV spectroscopy. All the sample solutions from cuvettes were transferred into respected tube. All the observations were recorded on lab note book for future work.

- Addition of Microcrystalline cellulose and adsorption of BSA

3 x 500 mg of microcrystalline cellulose weighted individually and added in to each centrifuge tube. Tubes were allowed to rotate for 24 hours at 20 rpm on rotator. Adsorptions of BSA on microcrystalline cellulose were initiated from this stage.

- Measuring absorbance after adsorption

After 24 hour sample has dislodge from rotator and allow to centrifuge for 15 min and at 6000rpm. All the solid microcrystalline cellulose has settled down and clear supernatant solution has taken out carefully without contamination of MCC in supernatant solution. This supernatant solution was filtered through 0.45 micron syringe filter. Base line correction done by using pH 6 distilled water. Absorbance of the entire sample has taken at 280 nm wave length by using UV spectroscopy. All the solution from cuvette has transferred in to respected tube and allows rotating for 48 hr and 72 hr. same procedure carried out for 48 hr and 72 hr. Data obtained from UV absorbance was recorded into lab book for future work.

Result

And

Discussion….

Result and discussion:­

After performing the planned work we got some data basically got the absorbances of each dilution. These absorbances are then used to calculate the concentration and adsorbed amount. This data is plotted in the graph as below.

Section 1

1. (Distilled water at pH 7 + BSA + MCC) graph plotted for 24, 48 hrs.

Graph 1: Initial concentration of BSA with Adsorbed amount of BSA mg at pH 7

Above graph plotted between initial concentrations and adsorbed amount of BSA in mg. the solution condition maintained at pH 7. The graph shows both 24 and 48 hrs observation. From the graph it is found that there is no adsorption of BSA on MCC at pH 7. The reason for it is the "surface charge" on the both surfaces. BSA has all positive charge from pH 2.5 to 4.8 and MCC molecules are negatively charged at all pH range 3.5 to 9.0. So due to repulsive electrostatic forces there is no adsorption at pH 7.

But, as the graph showing the adsorbed amount of BSA is in negative because MCC has swelling property in neutral, acidic and alkaline media. So at pH 7 the MCC particle swells and there will be an increased specific surface area. This swelling is more supported by the shear that is by centrifugation process. The more we centrifuge more the swelled particles release micro fibrils that can be easily analyse with UV spectroscopy at the same frequency that is used for BSA.

2. (Distilled water at pH 6 + BSA + MCC) graph plotted for 24, 48, 72 hrs.

Graph 2: Initial concentration of BSA with Adsorbed amount of BSA mg at pH 6

Above graph is same as previous graph drawn between initial concentration and amount adsorbed. The same thing happens here at pH 6. No adsorption of BSA onto the surface of MCC. The same thing is repeated here by MCC characterising swelling behaviour.

Section 2

(Distilled water at pH 6 + BSA + Silica) graph plotted for 24, 48, 72 hrs.

Graph 3: Initial concentration of BSA with Adsorbed amount of BSA mg at pH 6

The above graph is between initial concentration and adsorbed amount of BSA onto silica surface at pH 6. Actually at this pH the silica and BSA both has negative charge on their surface as MCC. But, here BSA shows a nice and higher adsorption on the silica surface as it is an electrostatic interaction. This is can be explained as follows, as previously said both surfaces are charged and these surfaces are surrounded by counter ions in aqueous solution. These counter ions are then diffusely distributed around the surface in the electrical double layer. The electrical double layer of silica and BSA overlap when they approach one another. These overlapping layers will form a redistribution of charges in the interface. This redistribution of charges driven by electrostatic interaction. The charge on the silica and on the protein will be adjusted.

Section 3

(Distilled water at pH 6 + BSA + MCC + 0.9%w/v NaCl) graph plotted for 24, 48, 72 hrs.

Graph 4: Initial concentration of BSA with Adsorbed amount of BSA mg at pH 6

The graph drawn is in between initial concentration and adsorbed amount of BSA at pH 6 but this time the media used was distilled water containing 0.9% Sodium Chloride. As graph the salt couldn't affect the adsorption process till 24 hrs. But, after 48 hrs the MCC surface showing a minor adsorption this is because the distribution of salt ions on the both surface. This distribution of salt ions then causes an electrostatic interaction between which cause adsorption of BSA onto the MCC surface.

Section 4

(Distilled water at pH 6 + BSA + MCC + 0.9%w/v NaCl) graph plotted for 24, 48, 72 hrs.

Graph 5: Initial concentration of BSA with Adsorbed amount of BSA mg at pH 6

The same graph is plotted between initial concentration and adsorbed amount of BSA onto the surface of silica. But here the media used is distilled water at pH 6 containing 0.9% Sodium Chloride. There is sufficient adsorption when only silica is used. But here the media is different and containing 0.9% Sodium Chloride which additionally supports the adsorption process. The distribution of salt ions causes more adsorption. So in this case double layer overlapping and salt ion distribution plays a role to increase the adsorption.

Section 5

(Distilled water at pH 7 + BSA higher conc. + MCC) graph plotted for 24 hrs

Observation and calculation

Sample

Initial conc.

Eq. Conc. mg/ml (C)

Amt. Adsorbed of BSA(X)[(m-c)x10]

X/m

C/X/m

ÆŸ (fractional coverage)

mg/ml

Mg

A1

97.45

85.54

11.91

23.82

3.5911

0.988007

A 2

65.92

56.94

8.98

17.96

3.1703

0.983903

A 3

32.96

29.52

3.44

6.88

4.2906

0.960441

A 1 r

87.07

76.35

10.72

21.44

3.5611

0.98668

A 2 r

52.87

44.7

8.17

16.34

2.7361

0.98196

A 3 r

25.81

23.35

2.46

4.92

4.7493

0.94605

Where,

Initial Conc. = Concentration of solution before addition of MCC.

Eq. Conc. = Concentration of solution after addition of MCC.

And these concentrations are calculated based on Beer-Lambert equation.

A = abc

Where, A= absorbance,

a = molar coefficient for BSA (0.667)

b = path length

c = concentration of BSA

X/m = Mass adsorbed of BSA per unit weight (gm) of MCC

Where ÆŸ = fractional coverage of MCC.

p= equilibrium concentration of BSA.

Slope and intercept obtained from graph ---- of C/X/m against Eq. Conc. of BSA.

Graph 6: Initial concentration of BSA with Adsorbed amount of BSA mg at pH 7

The above graph is plotted between initial concentration and adsorbed amount of BSA onto the surface of MCC at pH 7. The previously mentioned adsorption of BSA on MCC is also at pH 7 but the previous work done with the ratio of BSA conc. and MCC conc. it was 0.036 and there is no adsorption at all that's why in this case the ratio is increased to 1, 2 and 3. That means only BSA conc. has increased and MCC concentration remained same.

As the conc. of BSA increased there is well mannered adsorption seen. The graph shows as we increase the conc. of BSA the adsorbed amount also increases and will form a plateau. Higher conc. leads to higher adsorption because of electrostatic repulsive forces between two protein molecules. Due to loads and loads of BSA conc. each molecule repels each other because of same charge on surface. But then these molecules competitively bind on the surface of MCC. Actually the surface of MCC and the surface of BSA have same charges on their surface but for BSA, the surface of MCC is different and mainly adsorption takes place due to the hydrophobic interactions between both surfaces.

Graph 7: Initial concentration of BSA with Fractional coverage area of MCC at pH 7

The above graph plotted between initial concentration of BSA and fractional coverage of the MCC surfaces. The graph clearly shows as the concentration increase fractional coverage area also increases. But the time at which all adsorption sites are blocked or occupied by the BSA molecules the fractional coverage area remains constant and it forms a plateau means no increase in fractional coverage area after that. An option is there for increase in further fractional coverage area is Desorption.

Discussion:

The main objective of this work is to study the adsorption of protein on to solid surfaces at different conditions such as pH, media, ionic strength etc. the above discussed work is at pH 6 and 7. Adsorption of protein on to the surface varies at different pH values. As earlier said this work is carried out with other pH also that is 4 and 5. Basically the adsorption of protein governed by two forces hydrophobic and electrostatic interactions between protein and the solid surface.

The data obtained at pH 6 and 7 is far different than the pH 4 and 5. The pH and ionic strength affects adsorption on both MCC and silica surface. Another key of adsorption is "Iso-electric point" of protein (BSA) which is in the range of 4.7 - 5.1 so, BSA adsorption occurs in between this range. Hence, there is high adsorption of BSA on to MCC at pH 5. Also at pH 4 BSA showing a much higher adsorption than at pH 5. On the other hand at pH 6 and 7 there is no adsorption at all. This adsorption on to the MCC surface is governed by the electrostatic interaction.

The further addition of 0.09% of sodium chloride also affects the adsorption of protein. It sometime supports the adsorption and sometime not. Because it depends on the distribution and interaction of salt ions that is, Na+ and Cl- with the both surface. So depending on the distribution of salt ions attraction and repulsion occurs.

On the other hand adsorption of BSA on silica surface occurs at all pH 4, 5 and 6. Here also hydrophobic and electrostatic interaction supports the adsorption. At pH 4 and 5 adsorption governs by the opposite charges of both surfaces. But at pH 6 the adsorption assisted by the hydrophobic interaction. The further addition of 0.09% of sodium chloride supports the adsorption of BSA on to silica surface at all pH.

Study of adsorption at higher concentrations of BSA also carried out. The study shows higher adsorption of BSA on MCC at all 4, 5 and 6 pH this adsorption particularly assisted by the hydrophobic interactions.

Conclusion . . . . . . . .

Conclusion:

Overall the two factors hydrophobic and electrostatic interactions play an important role in the adsorption of BSA on to the surface of MCC and silica. These forces and the results obtained at different pH leads to following conclusion -

Adsorption of bovine serum albumin to microcrystalline cellulose was determined as function of protein concentration and pH of its aqueous solution. The maximum adsorption value attained at the BSA iso-electric point.

The overall adsorption was governed by hydrophobic and electrostatic interactions it sometimes supports the adsorption and sometimes not depends on the pH and ionic strength of the solution.

Adsorption of BSA on to the surface of silica is very well mannered and the adsorption attained at all 4, 5 and 6 pH. And the adsorption at 6 pH assisted by hydrophobic interaction instead electrostatic.

As the ratios of BSA and MCC increased up to 1, 2 and 3 the adsorption values were also increased and attained at all pH. Where, the adsorption has not seen at pH 6 at lower concentrations of BSA.

References…………..

Writing Services

Essay Writing
Service

Find out how the very best essay writing service can help you accomplish more and achieve higher marks today.

Assignment Writing Service

From complicated assignments to tricky tasks, our experts can tackle virtually any question thrown at them.

Dissertation Writing Service

A dissertation (also known as a thesis or research project) is probably the most important piece of work for any student! From full dissertations to individual chapters, we’re on hand to support you.

Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.