SYNTHESIS OF PROTEIN BY GREEN METHOD

The main problems and their solutions which came during the project are as follows:-

Problem :- How to do work ?

Solution :- Follow the following steps :1) Collect the particular waste or less usable part or row material from environment. 2) Extract the protein from that material 3) Identify the extracted proteinious part
A) Isolate by a) Paper Chromatography

b) Gel filtration Method. B) Detection of that part confirm by spectral methode C) Conformation by laboratrical tests also.

The material collection from environment 1)The material collected should be waste or less usable. 2)The material should be cheap. We collect the animals part which are waste and those may be 1) 2) 3) Vein --------> Goat ---------> [A] Lung -------> Goat --------> [B] Skin --------> Hen ---------> [C]

[A] -----> It is a part of Goat which is not so much usable and not have so much food value or any commertial importance. So we can use this type of material. [B] -----> It is a part of Goat which is protein rich side but but have food value i.e we can say that it is not a waste material. [C] -----> It is a part of Hen which can be found in a large quantity as a waste material from chicken butcher shop.

Test to check the presence of protein

First problem arises :1)Extraction of protein from the material

For extraction of material there are three steps which are as follows :-

i) ii)

Dissolve the whole part into the suitable solvent In order to detect the presence of protein content in the material sufficient quantity is required.

iii) Isolate the particular protein part only from whole solution.

We have collected some reference to detect the perfectly suitable solvent for the material we collected.

But the collected material contains the animal tissue not the vegetable tissue. Reference on the collection of tissue protein from the cells. *---------*

To dissolve the material we used two solvents. One is selected from above reference and another on the basis of collected information are :-

Sr.N o. 1.

NaOH It is strong base

KOH It is less stronger than NaOH It is used in bath soaps as less harmful to skin.

2.

It is used to dissolve the vegetable cell those are having cell wall.

From above information and below results. ---------ml 10% NaOH solution + ----------g of material [A] After 3 days (Approx.)

Shows complete dissolution of clear solution. -------ml 10 % solution of KOH + -------g of material [B] After 3 days (Approx.)

Shows complete dissolution

Make different concentrations both solutions and observed the time required for it.

Sr.N o.

NaOH

Time Required

Observations

1.

5% conc. 2 ml/ 0.02g

4 Days (Approx. ) 4 Days (Approx. ) 2 Days (Approx. )

Some part is insoluble and solution is not clear

2.

7% conc. 2 ml/ 0.02g

Completely soluble but the colour of solution becomes some reddish in colour

3.

10% conc. 2 ml/ 0.02g

Almost dissolved but the solution is not clear

4.

12% conc. 2 ml/ 0.02g

2 Days (Approx. ) 5 to 7 hours (Approx. ) 2 hours and 20 Minutes (Approx. )

Completely soluble and solution is but the colour of solution becomes some reddish in colour

5.

15% conc. 2 ml/ 0.02g

Completely not soluble.

6.

20% conc. 2 ml/ 0.02g

Completely soluble but the colour of solution becomes some reddish in colour

7.

30% conc. 2 ml/ 0.02g

10 Completely Soluble and Minutes clear solution is (Approx. obtained )

Sr. No. 1.

KOH

Time Required

Observations

5% conc. 2 ml/ 0.02g

4 Days (Approx. ) 4 Days (Approx. ) 24 Hours (Approx. )

Some part is insoluble

2.

7% conc. 2 ml/ 0.02g

Completely soluble and clear solution is obtained

3.

10% conc. 2 ml/ 0.02g

Completely dissolved

4.

12% conc. 2 ml/ 0.02g

12 Hours (Approx. )

Completely soluble and solution is transparent

5.

15% conc. 2 ml/ 0.02g

5 to 7 hours (Approx. )

Completely soluble.

6.

20% conc. 2 ml/ 0.02g

10 to 20 Completely dissolved Minutes (Approx. ) 10 Completely Soluble but Minutes there may be possibility (Approx. of degradation. )

7.

30% conc. 2 ml/ 0.02g

Conclusion:-From the above data it is concluded that KOH of range 10%,12%,15% and 20% solution is more preferable as a solvent. But, 10 12 15 20 % % % % Solution Solution Solution Solution Required --------> 24 Hours --- (P) Required --------> 12 Hours --- (Q) Required --------> 5 to 7 Hours --- (R) Required --------> 20 Minutes --- (S)

observations :(S) May be more quicker than all other but there is chances of Degradation.

(R) Also avoidable because of chances of denaturation of chain.

(Q) Provides results as required and less chances of degradation but required time is more.

But, (P) require 24 hours is more beneficial which helps to form more clear solution. So, (P) is preferred.

Now the problem is over come.

Use of 10 % KOH solution as solvent to dissolve the material which we used is confirmed.

Collection of Data for protein tests or to separate the particular protein form whole solution.
Rererences:When the protein is filtered through filter paper containing specific size of holes through which only protein can pass through it. It is obtained from the submitted reference paper. In reference paper there is use of whatmann filter paper no. 42. Which allow protein to pass through it. But we had used 3 types of Whatmann filter papers which are as follows:Whatmann Filter paper No. 42 Whatmann Filter Whatmann Filter paper No. 01 paper No. 41 Shows filtration specifically of protein part and remaining part is separated

Does not provide Protein part do any type of not pass filtration through it because of very small size of pores on the filter paper

Result:-It is concluded that Whatmann filter paper No. 42 can be used conveniently to filter protein.

We had solved the problem of separation but now we have to confirm that the filtered part is particularly protein or not. So, to detect that we had to perform the test for the presence of protein. There are various tests for protein detection but, we will use such tests which are easily available and can be performed systematically. Those tests are as follow:*__________________________* TEST OF BIURET:( For testing amino linkage) :- Take all the filtrate which is obtained after the process of filtration. In this filtrate add (NH4)2 SO4 (Ammonium sulphate). This solution is added in (1:1) and (1:3) ratio. Saturated solution of ammonium sulphate is prepared and added 3 ml into the 1 ml of above filtrate in test tube A. Now in test tube B take 2 ml of ammonium sulphate solution and add it to 2 ml of above filtrate. Keep both the test tubes in a test tube stand and observe. The 3:1 ratio shows the formation of precipitate within few minutes. 1)Sat. Ammonium sulphate(3 ml) & 1 ml filtrate Precipitate is obtained in 2 to 3 minutes.(a) 2)Sat. Ammonium sulphate (3 ml) &1 ml filtrate Precipitate is obtained in 19 to 20 hours.(b)

(a) is more preferable than (b) so procedure for

(a) should be followed

Now collect the precipitate which is formed above and use it for the detection test of protein or we have to use this ppt. to check the presence of amino acid chain. So, in order to confirm the presence of protein we have to follow the following tests. Test 10 % copper sulphate solution + 1 ml precipitated solution, boil the solution for 5 minutes. Observation The colour of CuSO4 is converted to very dark navy blue colour.

Result:- From the observation of above test i.e. dark navy blue colour itself indicates the presence of peptide linkage of amino acids.

Upto here we detected the results for the given sample contain protein or not. So all the 3 samples or waste materials are used to detect the presence of protein in all the three samples. So, to detect that the above procedure should be repeated for [B] and [C].

2)0.02 g of material [B] is dissolved in 2 ml of 10% KOH solution and it is labeled as (p).

3)0.02 g of material [C] is dissolved in 2 ml of 10% KOH solution and it is labeled as (q).

2) (p) filter by Whatmann filter paper no.41

Collect the filtrate (2 ml) + 2 ml Sat.(NH4)2SO4 Solution (1:1 Ratio)

(a)Precipitate occurs with in 10 to 20 minutes + 10% CuSO4, Boil

Dark blue colour is observed

3)(q) filter by Whatmann filter paper no.41

Collect the filtrate (2 ml) + 2 ml Sat.(NH4)2SO4 Solution (1:3 Ratio)

(b) Precipitate occurs with in few seconds + 10% CuSO4, Boil

Dark blue colour is observed

NOTE:i) The point should be noted that the ammonium sulphate which is required should be in less ratio. ii) Both the samples gives Biuret test which shows that both the samples consist of protein.

But, 1) (p) gives precipitate in very large quantity. 2) (q) gives precipitate in very very less quantity.

From the above collected data it is prove that [A] is the better waste material because is of less use it gives sufficient quantity of precipitate of peptide linkage.

Conclusion:Here it is confirmed that [A] material should proceed for next step. Upto this step the perfect selection of the material is decided. Now this material is collected in large quantity and store is next problem before us.

Now the next problem arises in front of us.

When the material is collected in the form of precipitate, that collected material should be preserved in the proper way. From Reference we follow the two methods those methods are as follows:-

Method I:Keep this material at room temperature The material provides the results upto two days but after that results are in disturbed condition called (R)

Method II :Keep this material at 00C in Refrigerator. The material provides the results upto one month called (S) So,The (R) is to be avoided and we should follow (S) method.

NOTE:-

While following the (S) method one precaution should be taken that we should not defreeze the material after its preservation, otherwise the degradation of material may take place. So, to avoid degradation of material we should avoid the defreezing of the material.

For good results we should always use the fresh precipitate but we can store the material in as it is form but, dont use the precipitate which is freezed. So, when we need to proceed the detection of present type and no. of protein in that freshly prepared precipitate should be used.

The next problem which arises in front of us is the isolation and the conformation of the type of the protein content into that precipitate.

isolation

Defination:- To separate the no. of protein content as quantity and quality both of them. the chart which is given below gives us the detail of R.F. values of the particular amino acid. For isolation we follow the three methods which are as follows:1)Chromatography 2)gel filtration 3)spectroscopically prof

Collected information is as follow SDS-PAGE (PolyAcrylamide Gel Electrophoresis)

The purpose of this method is to separate proteins according to their size, and no other physical feature. In order to understand how this works, we have to understand the two halves of the name: SDS and PAGE. SDS Since we are trying to separate many different protein molecules of a variety of shapes and sizes,

we first want to get them to be linear so that the proteins no longer have any secondary, tertiary or quaternary structure (i.e. we want them to have the same linear shape). Consider two proteins that are each 500 amino acids long but one is shaped like a closed umbrella while the other one looks like an open umbrella. If you tried to run down the street with both of these molecules under your arms, which one would be more likely to slow you down, even though they weigh exactly the same? This analogy helps point out that not only the mass but also the shape of an object will determine how well it can move through and environment. So we need a way to convert all proteins to the same shape - we use SDS.

Figure 1. This cartoon depicts what happens to a protein (pink line) when it is incubated with the denaturing detergent SDS. The top portion of the figure shows a protein with negative and positive charges due to the charged R-groups of the particular amino acids in the protein. The large H represents hydrophobic domains where nonpolar R-groups have collected in an attept to get away from the polar water that surrounds the protein. The bottom portion shows that SDS can break up hydrophobic areas and coat proteins with many negative charges which overwhelms any positive charge in the protein due to positively charged R-groups. The resulting protein has been denatured by SDS (reduced to its primary structure) and as a result has been lenearized.

SDS (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it. Therefore, if a cell is incubated with SDS, the membranes will be dissolved and the proteins will be soluablized by the detergent, plus all the proteins will be covered with many negative charges. So a protein that started out like the one shown in the top part of figure 1 will be

converted into the one shown in the bottom part of figure 1. The end result has two important features: 1) all proteins contain only primary structure and 2) all proteins have a large negative charge which means they will all migrate towards the positve pole when placed in an electric field. Now we are ready to focus on the second half PAGE PAGE If the proteins are denatured and put into an electric field, they will all move towards the positive pole at the same rate, with no separation by size. So we need to put the proteins into an environment that will allow different sized proteins to move at different rates. The environment of choice is polyacrylamide, which is a polymer of acrylamide monomers. When this polymer is formed, it turns into a gel and we will use electricity to pull the proteins through the gel so the entire process is called polyacrylamide gel electrophoresis (PAGE). A polyacrylamide gel is not solid but is made of a laberynth of tunnels through a meshwork of fibers (figure 2).

Figure 2. This cartoon shows a slab of polyacrylamide (dark gray) with tunnels (different sized red rings with shading to depict depth) exposed on the edge. Notice that there are many different sizes of tunnels scattered randomly throughout the gel.

Figure 3. This is a top view of two selected tunnels (only two are shown for clarity of the diagram). These tunnels extend all the way through the gel, but they meander through the geland do not go in straight lines. Notice the difference in diameter of the two tunnels.

Now we are ready to apply the mixture of denatured proteins to the gel and turn on the current (figure 4). If all the proteins enter the gel at the same time and have the same force pulling them towards the other end, which ones will be able to move through the gel faster? Think of the gel as a tiny forest with many branches and twigs throughout the forest but they form tunnels of different sizes. If we let children and

adults run through this forest at the same time, who will be able to get through faster? The children of course. Why? Because of their small size, they are more easily able to move through the forest. Likewise, small molecules can maneuver through the polyacrylamide forest faster than big molecules.

Figure 4. Cartoon showing a mixture of denatured proteins (pink lines of differen lengths) begins their journey through a polyacrylamide gel (gray slab with tunnels). An electric filed is established with the positive pole (red plus) at the far end and the negative pole (black minus) at the closer end. Since all the proteins have strong negative charges, they

will all move in the direction the arrow is pointing (run to red).

You have to remember that when we work with proteins, we work with many copies of each kind of protein. As a result, the collection of proteins of any given size tend to move through the gel at the same rate, even if they do not take exactly the same tunnels to get through. Back to our analogy of the Forrest... If we were in a hot air balloon above the Forrest and watched 100 children, 100 teenagers, and 100 large adults running through the Forrest, we would see collection (or band) of children moving quickly, a band of teenagers moving slower, and a third band made of adults plodding their way through the Forrest. Likewise, proteins tend to move through a gel in bunches, or bands, since there are so many copies of each protein and they are al the same size. When running an SDS-PAGE, we never let the proteins electrophoresis (run) so long that they actually reach the other side of the gel. We turn off the current and then stain the proteins (normally they are colorless and thus invisible) and see how far they moved through the gel. Figure shows a cartoon

gel and figre 6 shows a one real. Notice that the actual bands are equal in size, but the proteins within each band are of different sizes.

Figure 5. This shows a top view of an SDS PAGE after the current has been on for a while (positive pole at the bottom) and then turned off. The gel (gray box) has five numbered lanes where five different samples of proteins (many copies of each kind of protein) were applied to the gel. (Lane 1, molecular weight standards of known sizes; Lane 2, a mixture of three proteins of different sizes with a being the biggest and c being the smallest protein;

Lane 3, protein a by itself; Lane 4, protein b by itself; Lane 5 protein c by itself.) Notice that each group of the three proteins migrated the same distance in the gel whether they were with other proteins (lane 2) or not (lanes 3-5). The molecular weight standards are used to measure the relative sizes of the unknow proteins (a, b, and c).

Figure 6. This photo shows a variety of different proteins being separated on a gel. This particular image is showing a serial dilution of the same protein sample to indicate how little protein is needed (16 picograms = 16 . 10 -12 grams) in order to be detected. This image was taken from a home page operated by

Hitachi Software (http://www.hitachisoft.com/hitsoft/gs/fmbio/feb.htm)

There is a caveot to this method that you must always keep in mind. SDS-PAGE separates proteins based on their primary structure of size but not amino acid sequence. Therefore, if we had many copies of two different proteins that were both 500 amino acids long, they would travel together through the gel in a mixed band. As a result, we would not be able to use SDS-PAGE to separate these two proteins from each other. SDS-GEL FILTRATION to done of this presager we first step followed is the purification of over protin sample & then collection there is the ammonium salfat provid ppt consist the protine is proved but in that ther is a so much impurities allso present in that to clear that follow the following procejur AIM:- Purification of our sample. Chemicals:- egg cover ,Hcl solution,ammonium buffer silan solution,magnetic stirrer etc. procejur:-take a egg break that very finly without disterbing the whole cover just hole at one side & put

the all inner liquid out reject that part & then the calcium cover of the egg is dissolve in the HCL then kept out the remaining bag which is semipermeable wash that by water add ppt of ammonium sulfate ,hang the bag & deep in to a bekear containing solution of ammonium buffer silane. Arrange the beaker on the magnetic stirrer up to whole night then the solution is kept inner the bag is now pur & separated from all types of salts use this solution for gel filtration SDS.

Sample preparation and electrophoresis Equipment and reagents Gel electrophoresis apparatus; e.g., the BioRad Mini-PROTEAN 3. Electrophoresis power supply capable of delivering at least 300 V and 200 mA; e.g., the Bio-Rad PowerPac 300 or PowerPac 1000. Electrode buffer. See Table 2. Consult the apparatus instructions for the volume required.

Method 1) Assemble the electrophoresis apparatus and insert the gels according to the manufacturers instructions. With some electrophoresis cells, it is sometimes easier to load the wells before the gel assembly is placed in the buffer tank. 2) Remove the combs from the gels and rinse the wells with electrode buffer. 3) Fill the upper reservoir of the electrophoresis cell so that buffer fills the wells. 4) Mix one volume of protein sample with at least two volumes of the appropriate sample buffer Prepare protein standards or markers in the same manner as the samples. Sample volumes depend on the types of proteins, the type of detection, and the size of the sample wells. Volumes loaded into the wells are often of the order of tens of microliters. 5)Heat samples for SDS-PAGE (Laemmli or Tricine) at 95C for 2 min in a temperature block or water bath. (Make sure that 2-mercaptoethanol is included in the sample buffer for SDS-PAGE. Do NOT heat samples for non-denaturing conditions (McLellan or Ornstein-

Davis systems). 6) Load the samples carefully in the wells of the gels with a microsyringe or micropipette; e.g., Bio-Rad Prot/Elec pipette tips. The glycerol in the sample buffers provides density for layering the samples under the buffer in the wells. 7) Place the gel assembly in the lower-electrode buffer tank, if sample loading was done outside the tank. Add electrode buffer to the electrode chambers as necessary. It is often unnecessary to immerse the gels completely in buffer. 8) Put the lid on the tank and connect the leads to the power supply. For most gel types, the anode (+) (red lead) is at the bottom of the tank and the cathode (-) (black lead) is at the top. However, it is necessary to reverse the leads with CTAB gels and with the high pH McLellan gels. For safety, power supplies for electrophoresis should have with isolated grounds. 9) Turn on the power supply and set it for the electrical conditions recommended by the manufacturer of the electrophoresis cell; e.g., 200 V constant voltage for Laemmli gels run in the Bio-Rad Mini-Protean 3.

10)Allow the run to proceed until the blue tracking dye from the sample buffer reaches the end of the gel (about 40 min for Laemmli gels in the Mini-PROTEAN3). It is not necessary to remove the gel immediately at the end of a run. Gels can be left for extended periods at least overnight without deterioration of the band patterns. Molecular mass estimation SDS-PAGE has become the most popular method of gel electrophoresis because it can be used to estimate molecular masses (1, 3, 8, 34, 44). To a first approximation, migration rates of SDS polypeptides are inversely proportional to the logarithms of their molecular masses. The larger the polypeptide, the slower it migrates in a gel. Molecular masses are determined in SDS-PAGE by comparing the mobilities of test proteins to the mobilities of known protein markers. At one time, when samples for SDS-PAGE were run in individual tubes, it was necessary to normalize to a common parameter so that the different tube gels could be compared. This was because tube gels

differ in length. The normalizing parameter that is still used is the relative mobility, Rf, defined as the mobility of a protein divided by the mobility of the ion front. In practice, when all gels are run for the same length of time, Rf is calculated as the quotient of the distance traveled by a protein from the top of the resolving gel divided by the distance migrated by the ion front. The distance to the ion front is usually taken as the distance to the tracking dye (measured or marked in some way before staining). With slab gels, this normalization is less important provided that a lane of standards is run in the same gel as the samples whose masses are to be determined. It is sufficient to compare migration distances of samples and standards. Plots of the logarithm of protein molecular mass (log Mr) versus the migration distances fit reasonably straight lines. In each gel, a lane of standard proteins of known molecular masses is run in parallel with the test proteins. After staining the gel to make the protein bands visible (see the Application Focus about the Detection of Proteins in Gels on this website), the migration distances are measured from the top of the resolving gel. The gel is calibrated with a plot of log Mr vs. migration distances for the

standards. The migration distances of the test proteins are compared with those of the standards. Interpolation of the migration distances of test proteins into the standard curve gives the approximate molecular masses of the test proteins. Pore-gradient SDS-PAGE gels can also be used to estimate molecular masses. In this case, log Mr is proportional to log (%T). With linear gradients, %T is proportional to distance migrated, so that the data can be plotted as log Mr vs. log (migration distance). Standard curves are actually sigmoid in shape The apparently linearity of a standard curve may not cover the full range of molecular masses for a given protein mixture in a particular gel. However, log Mr is sufficiently slow, in a mathematical sense, to allow fairly accurate molecular mass estimates to be made by interpolation, and even extrapolation, over relatively wide ranges. The approximate useful ranges of single- percentage SDS-PAGE gels for molecular mass estimations is as follows: 40,000 to 200,000 Da, 7.5%T; 30,000 to 100,000, 10%T; 15,000 to 90,000 Da, 12%T; 10,000 to 70,000 Da, 15%T. Mixtures of standard proteins with known molecular masses are available commercially for calibrating electrophoresis gels.

2)Chromatography of amino acids Amino acids have no color. Therefore all of these procedures need to be carried out "blind", and the results will be seen when a revealing agent (ninhydrin) is sprayed on the resulting chromatogram. You are provided with a number of solutions of amino acids, and solution X (a mixture of 2 amino acids).Do not use the same pipette for more than one liquid. Chromatography paper must not be touched with the hands (at the bottom, at least). Use plastic/rubber gloves, and work on a clean surface (e.g. inside page of pad of paper). Cut a suitable length of chromatography paper (slightly longer than the glass chamber) and mark it with a pencil line about 1.5 cm from the bottom. Again using a pencil, put 3 marks on the line forming crosses, the outer ones labelled with (3 letter codes for) the amino acids you are going to use, and X in the middle. Put your name at the top.

Using 3 different pipettes, place a drop of each amino acid, and the mixture X, at the appropriate positions on the line. If you have time, gently warm the paper and repeat the spotting process (on the same positions) to raise the concentration of the amino acids, but ensure that the paper is completely dry before proceeding. Partially fold the paper in half length ways, to remove its tendency to curl up. In a fume cupboard or a secluded (well ventilated?) area of the lab : Using a funnel, pour a small amount of chromatography solvent (butanol/ethanoic acid) into the glass chamber (to about 1 cm depth). Place on the lid to allow the atmosphere to become saturated with vapor. Leave the chamber on the bench in its final position, so that it does not splash up; bring the paper to meet it. Line up the paper with the (outside of the) chamber, and either fold over the top or cut it so that it will fit. The solvent should touch the lower part of the paper but not cover the drops on the line.

Attach the paper to the lid, and then place the lid on. The paper should not touch the sides of the chamber. The solvent should gradually rise up the paper, passing the line and heading upwards. After about 3 hours, the liquid should have risen about three-quarters of the height of the paper. If the solvent nears the top of the paper, proceed immediately to the next stage. Remove the paper from the apparatus, and use a pencil to mark the position of the solvent front. Up to 5 pieces of chromatography paper can be placed across a clean A4 sheet of paper, stapled at the top of the chromatograms. Place the chromatograms into an oven at about 45 C to dry. Either in a fume cupboard or outside on a still day, spray ninhydrin evenly over the paper. Care: ninhydrin is dissolved in an inflammable solvent.

Return the chromatograms to the oven to develop the color. Spots should be visible as purplish smears on the paper. The image of the spots may be enhanced if the sheet is photocopied on a darker than normal setting.

Calculation of Rf values Measure the distance from the start line to the solvent front and to the front of each spot.

For each spot, calculate the Rf value (Rf means relative to front): distance moved by spot distance moved by solvent front Compare the values you obtain with reference Rf values. Different solvents and different types or makes of chromatogaphy papers will give slightly different results. One or both of the spots from solution X may be at the same level as another (known) amino acid alongside it. This should assist in identification. Rf value 0.38 0.20 0.5 0.24 0.4 0.13

Amino acid alanine arginine asparagine aspartic acid cysteine glutamine

glutamic acid glycine histidine isoleucine leucine lysine methionine phenylalanine proline not a true amino acid shows up as yellow serine threonine tryptophan tyrosine valine

0.30 0.26 0.11 0.72 0.73 0.14 0.55 0.68 0.43 0.27 0.35 0.66 0.45 0.61

CHROMATOGRAPHY:We found some information on the isolation of amino acids by paper chromatography. So we will use the paper chromatography for particular isolation. Chemical:- n-Butanol, acetic acid, distilled water, actone etc. Apparatus:- capillary tube, glass jar, beaker, watch glass Etc.

Procedure:- We had taken fresh precipitate which is obtained in above procedure called (b) precipitate and taken a filter paper called CHR-1 or Whatmann filter paper No. 1 should be used but the length of the filter paper should be 14 cm and width should be 2 cm and then draw the base line at bottom and draw top line at about 12 cm distance from base line. Now dissolve the precipitate in n-Butanol. The dissolved part should be spotted at the center of base line and care should be taken that the spot should be very fine. So we should use the micro capillary for spotting. Repeat this process of spotting for 3 to 5 times and and it to dry completely.

Now take a solvent into a glass jar and add n-butanol, acetic acid and distilled water in the specified ratio. Now take the paper and dip into the jar and take the precaution that the bottom line should not dip into the solvent. Now cover the jar with the help of watch glass.

Now allow the liquid to flow up to the upper line then remove the paper from the jar and then

heat it at 40-50 0C in heater and then spray a reagent called nin hydrin on it( Prepare this reagent in 0.4% solution of acetone) .Then again allow the paper to dry in heater up to 40-50 0C and observe the paper.

Observation:We had used two types of mobile phases one is as it is shown in reference material and another one is over modification by using lots of type mobile phases. Ist one as it is in reference follows Take n-Butanol= 12 ml. Acetic acid= 3 ml. & Water= 5 ml.

Chromatography of Amino acids:On chromatography paper we had observed four spots separately. So, may be four types of amino acids are present called them as A,B,C & D. Chromatogra phy No. Colour A Violet B Pink Greyish Violet I. II. III. 0.472 0.470 0.469 0.18 0.21 0.30 0.08 0.093 0.130 0.94 0.62 0.78 C D Yellowish Violet

Now the results are as above but there is not confirmly isolation is occur so, we observe the chromatography paper by using different types of solvents.Those are as follows:-

Sr. Observation

n-

Acetic

No . 1. 2. 3. 4. 5. 6. Isolation is not clear Isolation is not clear So much disturbed 4 bands clearly seperated Disturbed Bands So much mixed

Butanol acid (ml) 12 12 12 12 12 12 3 3 3 5 5 5 (ml)

Water (ml) 5 6 7 8 10 15

CONCLUSION:From the above results system No. 4 is used. Repeated three times and results are collected those results are as follows:-

Section II-(12:5:8)(n-Butanol:Acetic acid:Water)

(P) Color Light Violet (10.1)/ (13.3) IV V 0.759 0.803

(Q) Orange (7.7)/ (13.3) 0.578 0.606 Pink

(R)

(S) Violet (4.5)/ (13.3) 0.338 0.314

(5.9)/ (13.3) 0.44 0.44

1] Observation:Four different amino acids may be present because four types of colored bands are observed. Details of those bands are as follows:-

[A] Tryosin(0.45) proline(0.43) Ctstein(0.40)

[B] Lysine(0.14) Histadine(0.11) Arginin(0.2) Glutamine(0.13) Asparatic(0.24) Glycine(0.26) Glutamic Acid(0.3)

[C] Glutamine(0.13) Lysine(0.14) Histadine(0

[D]

Valine(0.61) L-Isoleusin(0.72) Lusin(0.73) Phenylalanine(0.68) Tryptophan(0.66) Metheonine(0.55)

RESULT:From the above data we can conclude that the present precipitate or sample may contain following types of proteins or amino acids:1) 2) 3) 4)