BIOCHEMISTRY_PROTEINS, ENZYMES, CHROMATOGRAPHY, Study notes of Biochemistry

Download BIOCHEMISTRY_PROTEINS, ENZYMES, CHROMATOGRAPHY and more Study notes Biochemistry in PDF only on Docsity! 1 PROTEINS AND ENZYMES Proteins  are large, complex molecules that play many critical roles in the body. They do most of the work in cells and are required for the structure, function, and regulation of the body's tissues and organs  Amino acids are the building blocks of proteins. Laboratory Techniques Isoelectric Focusing of Casein  Casein, like proteins, are made up of many hundreds of individual amino acids. Each may have a positive or a negative charge, depending on the pH of the system. At some pH value, all the positive charges and all the negative charges on the [casein] protein will be in balance, so that the net charge on the protein will be zero.  Milk is a mixture of many types of proteins, most of them present in very small amounts. Milk proteins are classified into three main groups of proteins based on their widely different behaviors and forms of existence. They are caseins (80%), whey proteins and minor proteins.  Casein is a heterogeneous mixture of phosphorous containing proteins in milk. Casein is present in milk as calcium salt and calcium caseinate. It is a mixture of alpha, beta, and kappa caseins to form a cluster called micelle. These micelles are responsible for the white opaque appearance of milk.  That pH value is known as the isoelectric point (IEP) of the protein and is generally the pH at which the protein is least soluble. For casein, the IEP is approximately 4.6 and it is the pH value at which acid casein is precipitated. In milk, which has a pH of about 6.6, the casein micelles have a net negative charge and are quite stable. During the addition of acid to milk, the negative charges on the outer surface of the micelle are neutralized (the phosphate groups are protonated), and the neutral protein precipitates. Materials 1. Raw milk - 100ml 2. 0.2N HCl - 50ml 3. Diethyl ether - 50ml 4. 50% Ethanol - 50ml 5. Filter paper strip 6. Beaker 7. Glass stirring rod 8. Centrifugation Device *Alternative - use non-fat milk if centrifuge machine is not applicable* Procedure  Step 1: Measure 100ml of milk in a measuring cylinder and transfer 25ml of milk to four Oakridge centrifuge tubes each.  Step 2: Centrifuge the milk in a centrifuge at 4000rpm at room temperature (25- 30o C) for 20 minutes. This is done to remove the fats and lipids from the mixture.  Step 3: After centrifugation, carefully remove the fats and lipids from the surface of the milk with a spatula.  Step 4: Then transfer the milk from all the tubes into a beaker and add equal volume of distilled water and stir well. Now check the pH.  Step 5: Start adding 0.2N HCl drop by drop into the milk mixture and stir well.  Step 6: Note the PH at which precipitation (white curdy substances) appears. The pH should be 4.6.  Step 7: Take the curdy precipitate and allow it to sediment.  Step 8: Now decant the supernatant using a filter paper and funnel and wash the precipitate with distilled water to remove the salts, then wash with diethyl ether and ethanol.  Step 9: Dry the precipitate and take the weight of the casein and record it. Isolation of Gluten from Flour Gluten is a combination of the natural proteins, glutenin and gliadin, found in the flour. Gluten molecules are activated when flour is moistened and then mixed. When this happens, the glutens literally stretch out as the proteins form longer and longer chains. Isolation of proteins is the process of separating a type of protein from a complex mixture. As for gluten, it is separated through its difference in solubility. Insoluble proteins are easily isolated, removing other substances that are soluble by washing. Materials  Flour (Types of Flour - have different gluten content)  Tap water  Cheesecloth (alternative - clean cloth)  Bowl Methods  Gluten can be readily prepared by gently washing dough under a stream of running water. This removes the bulk of the soluble and particulate matter to leave a proteinaceous mass that retains its cohesiveness on stretching. Gluten comprises some 75% protein on a dry weight basis, with most of the remainder being starch and lipids.  Through a process called centrifugation the major constituents of the flour are separated. The starch and other constituents dissolve, but the gluten, which is not water soluble, does not. Once starch and gluten are separated by centrifugation, the gluten is washed thoroughly and dried. Procedures  Select the flour.  Put the flour in the cheesecloth then make sure there will be a bowl underneath it.  Gently wash the flour under the stream of running water.  Knead to develop the proteins into gluten.  Rinse it until the dough become elastic. Biuret Test  Biuret is a chemical made by heating urea, which causes two urea molecules to condense into one. BIOCHEMISTRY LABORATORY PROTEINS, ENZYMES, AND CHROMATOGRAPHY (3) JAILOUISE A. PEREZ BACHELOR OF SCIENCE IN NURSING 2 Biuret is named for the peptide bonds in the reagent that produces a positive response in the test. For substances (proteins and peptides) with two or more peptide (CO-NH) bonds, it is used as a generic test.  A Biuret test is a chemical test that determines whether or not a sample contains a peptide link. It is based on the biuret reaction, in which an alkaline copper sulfate- treated peptide structure with at least two peptide linkages creates a violet color. The colored coordination complex is generated by the Cu2+ ion and the peptide bond's carbonyl oxygen (>C=O) and amide nitrogen (=NH).  The solution changes color from blue to purple once this complex has formed. The more purple the tint, the more peptide-copper complexes are present. The color intensity is proportional to the number of peptide bonds present in the responding protein molecule, as well as the number of protein molecules present in the reaction system.  A solution of sodium hydroxide (NaOH) or potassium hydroxide (KOH), hydrated copper (II) sulfate, and potassium sodium tartrate is used to make the Biuret reagent. The alkaline medium is made out of sodium hydroxide and potassium hydroxide, with potassium sodium tartrate added to chelate and thereby stabilize the cupric ions in the solution, or to keep their solubility in alkaline solution. Biuret Test Principle  The copper (II) present in the reaction binds itself to the nitrogen atoms that are present in the protein peptides. Since this test is not greatly disturbed by the presence of amino acids in the sample, it can be used to gauge the concentration of proteins in whole tissue samples. However, the samples of proteins that are purified via ammonium sulfate ((NH4)2SO4) precipitation are not ideal for this test since buffers like ammonia interfere with it.  Now, four nitrogen atoms donate lone pairs to form coordinate covalent bonds with the cupric ion, resulting in the formation of a chelate complex. This chelate complex has the ability to absorb light with a wavelength of 540nm, which imparts a purple colour to it. Therefore, the formation of a purple coloured complex indicates the presence of proteins in the analyte. Procedure  Step 1: Take 3 clean and dry test tubes.  Step 2: Add 1-2 ml of the test solution, egg albumin, and deionized water in the respective test tubes.  Step 3: Add 1-2 ml of Biuret reagent to all the test tubes.  Step 4: Shake well and allow the mixtures to stand for 5 minutes.  Step 5: Observe for any color change. Observation and Interpretation  No color change, i.e., the solution remains blue Proteins are absent (Negative Biuret Test)  The solution turns from blue to deep purple Proteins are present (Positive Biuret Test) Application 1. It can be used to detect the amount of protein in the urine. 2. Biuret reaction with protein is applicable to the quantitative determination of total protein by spectrophotometric analysis. Ninhydrin Test  The ninhydrin test is a chemical test which is utilized and performed to detect the presence of ammonia and whether a given analyte contains amines or α- amino acids. Ninhydrin is most commonly used as a forensic chemical to detect ―fingerprints‖.  In order to execute this test, ninhydrin is being added to the test solution of the analyte. Ninhydrin reacts with the α-amino group of primary amino acids producing ‗Ruhemann‘s purple‘. The chromophore formed is the same for all primary amino acids. The intensity of the colour formed depends on the number and chemical nature of the amino groups being analysed. The optimum pH for the overall reaction is 5.5. Ruhemann‘s purple has a spectral maximum at 570 nm  An amino group that belongs to a free amino acid goes through a chemical reation with ninhydrin, which acts as an oxidizing agent. The amino acid goes through oxidative deamination that results to the freedom of CO2, NH3, and an aldehyde alongside hydrindantin (which is a reduced type of ninhydrin) when it is presented to the ninhydrin, Materials 1. Test tubes 2. Test tube stand 5  Sakaguchi reagent: 1% 1-naphthol in alcohol with a few drops of a 10% sodium hypobromite solution in bromine water.  40 percent sodium hydroxide a case study (0.1 percent of arginine or 0.1 percent of creatine) Required Materials  Tubes for testing  Stand for test tubes  Pipettes Procedure  Step 1: In a test tube, 3 ml of the test solution is added, followed by 1 ml of 40 percent NaOH, which is properly mixed.  Step 2: Then, in the same test tube, two drops of 1- naphthol are added and thoroughly mixed.  Step 3: 4-5 drops of sodium hypobromite (10%) or bromine water are now added.  Step 4: Color development in the test tube is monitored. Results  POSITIVE: The creation of red color indicates a positive result on the Sakaguchi's test. This suggests that an arginine or guanidinium molecule is present.  NEGATIVE: The absence of red color indicates a negative result in Sakaguchi's test. This suggests that arginine or a guanidinium molecule is missing. Application  Sakaguchi's Assay is a biochemical test for detecting arginine in proteins, either free or mixed.  The test is qualitative, but with the addition of urea, which stabilizes the colored result, it can be made quantitative. Limitations  Because Sakaguchi's reaction is sluggish, quantitative examination of the colored result is not possible with this test.  Similarly, the hypochlorite may damage some of the guanidinium groups in the solution, causing problems with testing findings. Lead Acetate Test Lead Acetate test (or Lead Sulfide test) is a biochemical test for the detection of amino acids like cysteine and cystine. The test is a specific test for the detection of amino acids containing sulfur, S-H group in cysteine, and S-S group in cystine. The test is mainly called lead acetate test as the reagent for the test is lead acetate. Even though the test is specific for the detection of sulfur-containing amino acids, methionine doesn‘t give a positive result in this test. Reaction Involved The sulfur-containing amino acid such cysteine and cysteine (sulfhydryl/thiol group) reacts with lead acetate under alkaline conditions to form a brown precipitate. These sulfur- containing amino acids are degraded in strongly alkaline media to release sulfide ion (S2-) in the form of H2S (hydrogen sulfide). The sulfide ions can react with lead (II) acetate to form a brownish-black precipitate. Materials 1. Test tube 2. Test tube stand 3. Pipettes Reagents 1. 2% lead acetate solution 2. 40% NaOH 3. Sample Methods  In a test tube, 2 ml of the amino acid solution is taken. To this, 2 ml of NaOH is added, and a few drops of lead acetate.  The solution is boiled for a minute. Once the test tube cools down, observe the solution.  The test tube is then observed for the formation of a precipitate Results 6  POSITIVE: There would be a formation of black precipitate.  NEGATIVE: There would be no formation of black precipitate. Bradford Assay Background  In 1976, an American scientist named Marion M. Bradford developed the highly known and widely used Bradford protein assay. What Bradford created is a procedure for determining the concentration of protein in a solution in a fast, easy, and accurate way. It also determines the protein content of cell fractions and asses protein concentrations for gel electrophoresis.  Bradford assay‘s principle results in the color change from brown to blue by the binding of protein molecules to Coomassie dye under acidic conditions. This assay method contributes to formation of the protein-dye complex and strictly measures the presence of the basic amino acid residues, arginine lysine, and histidine. Also, in the Bradford assay, samples that are out of range can be retested within minutes. Use The Bradford assay is used to calculate the concentration of the total amount of protein in a given sample. It utilizes standards to both assess the amount of protein in samples and to subtract any background due to interfering substances that can shift the ratios between the three forms of the dye. Samples that have protein concentrations higher than the concentrations in the linear range must therefore be diluted and re-assayed to obtain a more accurate estimation of the protein concentration. Materials and Methods 1. Summarized Procedures a. Dilute protein sample with buffer (or protein standards) a. To fit in the standard curve (linear curve) b. Linear curve – forms line using different data sets, data is in real world set ups and maintain pattern- we attempt the pattern to represent plots or trend, real world set ups may give us scattered lines b. Add Bradford reagent c. Incubate for 5 minutes d. Read absorbance using spectrophotometer a. Spectrophotometer- equipment; light will pass on the sample and will be measured through its color of light. The color of light in the electromagnetic spectrum has a specific wavelength. The result of the sample when mix to Bradford reagent is blue, we use color wheel to determine its contrary color which is orange. The wavelength of orange color is around 600-650 nm. Bradford assay can be measured in 595 nm. b. Refer to the established standard curve equation to estimate protein concentration of sample. 2. Full materials & procedures (long version) a. Kindly refer to this link https://www.youtube.com/watch?v=vfY3mVOlGBU Conclusion  The Bradford assay is based on the dye name Coomassie brilliant blue G-250 that binds to the side chain of basic amino acid. The sulfonic acid groups of the dye interact with the positive amino group of the proteins found in the basic amino residues present in lysine, arginine, and histidine; this interaction makes the brown color of Coomassie blue G-250 into a blue color.  To determine the amount of concentration of a protein, first is we need to examine and measure a solution without any protein content, next is we need a reference solution where a known protein concentration is measured into 3 different concentration sample of an increasing optical density such as 2 and 4 microgram so that we can determine an unknown protein sample by the use of spectrophotometer and with extrapolation, we can calculate the protein concentration of the unknown protein sample. ENZYME Enzyme  Proteins that operate as biological catalysts are known as enzymes. Catalysts help to speed up chemical reactions. Substrates are the molecules on which enzymes can function, and the enzyme changes the substrates into various molecules called products.  Enzymes are essential in different functions like respiration, digesting food, muscle, and nerve function. But for this type of experiment, we are going to focus on the food digestion. Salivary Amylase Enzyme Assay Salivary Amylase  Food are made up of a large molecules like lipids, carbohydrates, and proteins that are too big to move to our blood, in regard with this we need to digest it into smaller molecules. And this is where physically processes like chewing and chemically processes by mean of special proteins called enzymes takes place.  There are different types of food molecules we need to digest and those are the lipids, carbohydrates, and proteins. For this type of digestion, we are going to focus on carbohydrates.  Carbohydrates types of food can be found on rice and pasta. The simplest carbohydrates are sugars which can be joined into a big chain called starch. These complex carbohydrates can be digested by these carbohydrates‘ enzyme called amylase. Amylase is a special type of carbohydrates that breaks down starch into smaller molecules which can be broken down further into glucose which is small enough to be moved into the blood. 7  Amylase can be found on saliva, where carbohydrates will first start to be broken down chemically. Effects of Different Factors on Enzyme Activity 1. pH a. pH affects the enzymes activity by affecting the structure of the enzymes itself. b. As enzyme changes in pH, the structures of the enzyme changes by means that the substrate and the enzymes cannot bind completely because of there charges. Many amino acids in an enzyme molecule carry a charge and these charges contributes to the folding of the enzyme molecule, its shape, and shape of the active site. c. Thus changing the pH will affect the charges on the amino acid molecules, which can affect the shape of the active sites. d. There are some instances where pH can work on different enzymes with their own optimum pH. Here for example the optimum pH of the salivary amylase is at 6.8 pH level starting from 4.5, the optimum pH is the highest point of activity of the enzymes. Here we can see as the pH decreases from the optimum pH, the rate of enzyme activity decreases, and as pH increases from optimum pH, rate of enzyme activity decreases. Enzyme Optimum pH Salivary Amylase 4.5 – 6.8 Stomach Protease (pepsin) 1.5 – 2.0 Pancreatic Protease (trypsin) 3.5 – 8.0 e. The symmetrical shape of this enzyme activity shows that below the optimum pH, the enzymes start to denature. And above the optimum pH, the enzymes start to denature as well. Denature of denaturation means that the enzymes active site loses it specific 3D shape like on the temperature the enzymes break down, the enzymes cannot also bind to it substrate. 2. Temperature a. As the temperature increases, the rate of enzyme activity also increases which gives the enzyme a more chance to bind with its substrate. b. Optimum temperature - the highest rate of enzyme activity (for human the optimum temperature is at 37 degrees Celsius). Beyond the optimum temperature the rate of enzyme activity decreases. Because as the temperature goes higher the chemical bond (intra and intermolecular bonds) that hold the enzymes molecules begin to break down or denature because the enzyme molecules gain even more kinetic energy. (To be more specific the active site of the enzyme can be destroyed and therefor the substrate can no longer bind to the active site). c. At low temperature enzyme activity is almost zero as well, because the kinetic energy of enzyme and substrate moves slower and the chances for them to bind as enzyme substrate complex are low, which therefore conclude having a low temperature gives a low rate of activity for enzyme. 3. Substrate Concentration a. As the temperature increases, the rate of an enzyme-catalyzed reaction increases as the temperature increases. At low temperatures, the rate increases again because the enzyme becomes denatured and can no longer function. b. Increasing the temperature (which is a measure of the thermal energy in the system), therefore, increases the frequency of the collisions and thus increases the rate at which the product is formed. c. As you increase the concentration of the substrate, the enzyme activity increases as well as the rate of the reaction. In addition, as 10 2. Immobilization of antibody a. Add diluted antibody to each well of a 96-well ELISA plate. Seal the plate to prevent evaporation, and allow it to incubate at 4°C for 15-18 hours to immobilize the antibody. 3. Washing a. Remove the diluted antibody, and wash 3 times with washing solution. 4. Blocking buffer a. Add blocking buffer to each well, and allow it to incubate at 37°C for 1 hour to reduce non- specific binding of the target protein to the well 5. Washing a. Remove the blocking buffer, and wash 3 times with a washing solution. 6. Addition of Samples\ a. Dilute the samples with sample dilution buffer, and add 100 µL of each sample to each well. For the calibration curve, prepare a dilution series of the standard on the same plate. Allow it to incubate at 37°C for 1 hour. 7. Washing a. Remove the samples, and wash 5 times with washing solution. 8. Addition of Detection Antibody a. Dilute the detection antibody in sample dilution buffer, and add 100 µL to each well. b. Allow it to incubate at 37°C for 1 hour. 9. Washing a. After reaction, remove the detection antibody, and wash 5 times with washing solution. 10. Addition of enzyme-linked secondary antibody 11. Washing 12. Addition of substrate solution 11 13. Addition of stop solution 14. Measuring using plate reader 4 Types of Elisa 1. Direct Elisa - An antigen or sample is directly immobilized on the plate in a direct ELISA, and a conjugated detection antibody binds to the target protein. After that, the substrate is added, resulting in a signal proportional to the amount of analyte in the sample. Direct ELISAs are less specific than sandwich ELISAs since just one antibody is utilized. a. Advantages i. Quick because only one antibody and fewer steps are used. ii. Cross-reactivity of secondary antibody is eliminated. iii. Fast and simple protocol b. Disadvantages i. Cell Smear: Adhere non-adherent cells on coverslip with chemical bond ii. Immunoreactivity of the primary antibody might be adversely affected by labeling with enzymes or tags. iii. Labeling primary antibodies for each specific ELISA system is time- consuming and expensive. iv. No flexibility in choice of primary antibody label from one experiment to another. v. Minimal signal amplification. 2. Indirect Elisa - An indirect ELISA is similar to a direct ELISA in that it uses a plate to immobilize an antigen, but it also contains an amplification detection step. First, a primary detection antibody that is unconjugated is introduced and binds to the specific antigen. After that, a conjugated secondary antibody directed against the main antibody's host species is added. The amount of antigen bound in the well is thus proportional to the signal produced by the substrate. a. Advantages i. A wide variety of labeled secondary antibodies are available commercially. ii. Versatile because many primary antibodies can be made in one species and the same labeled secondary antibody can be used for detection. iii. Maximum immunoreactivity of the primary antibody is retained because it is not labeled. iv. Sensitivity is increased because each primary antibody contains several epitopes that can be bound by the labeled secondary antibody, allowing for signal amplification. b. Disadvantages i. Cell Smear: Adhere non-adherent cells on coverslip with chemical bond ii. Cross-reactivity might occur with the secondary antibody, resulting in nonspecific signals. iii. An extra incubation step is required in the procedure. 3. Sandwich Elisa - The most frequent type of ELISA is a sandwich ELISA. Matching antibody pairs are two specific antibodies that are used to sandwich the antigen. A microplate is covered with a capture antibody, the sample is added, and the protein of interest binds and immobilizes on the plate. The next step is to add a conjugated-detection antibody, which binds to an extra epitope on the target protein. Substrate is added, and a signal proportional to the amount of analyte in the sample is produced. Sandwich ELISAs are very selective since they require two antibodies to bind to the protein of interest. a. Advantages i. High specificity: the antigen/analyte is specifically captured and detected ii. Suitable for complex (or crude/impure) samples: the antigen does not require purification prior to measurement iii. Flexibility and sensitivity: both direct or indirect detection methods can be used b. Disadvantages i. Longer protocol ii. Challenging to develop 4. Competitive Elisa - When the protein of interest is too tiny to sandwich with two antibodies successfully, 12 competitive ELISAs are routinely utilized. A capture antibody is put on a microplate, similar to a sandwich ELISA. A conjugated antigen is utilized instead of a conjugated detection antibody to complete the binding with the antigen present in the sample. The less conjugated antigen in the sample, the less it will bind to the capture antibody. When the substrate is introduced, the resulting signal is inversely proportional to the amount of protein in the sample. a. Advantages i. Ability to quantitate small molecules b. Disadvantages i. Less specific since you are only using 1 antibody ii. Requires a conjugated antigen Conclusion ELISA or Enzyme-Linked Immunosorbent Assay, also known as Enzyme Immunoassay (EIA), is a biochemical technique used to detect and quantify compounds like antigens, antibodies, and hormones. The amount of compound in the sample corresponds to the amount of substrate converted to colored product by enzyme-linked antibodies. It is useful to diagnose HIV, rotavirus, syphilis, Zika virus, and many others. There are four types of ELISA, namely, Direct ELISA, Indirect ELISA, Sandwich ELISA and Competitive ELISA. The major difference between Direct and Indirect ELISA is that only one antibody is used in Direct ELISA, while Indirect ELISA requires two antibodies. In both Direct and Indirect ELISA, it is the antigen that is immobilized to the plate, meanwhile in Sandwich ELISA, it is the antibody that is immobilized to the plate, and this antibody is called a capture antibody. Meanwhile, Competitive ELISA is a type of ELISA wherein antigens compete with each other for the binding with antibodies. The lower the intensity of color in Competitive ELISA means there is a higher amount of targeted antigen. The steps of this assay could be summarized into 6 steps, which are: 1. Antibody coating of wells 2. Protein capture 3. Antibody detection 4. Addition of enzyme linked secondary antibody 5. Addition of substrate solution 6. Analysis Summary To conclude, the three experiments exhibit the activities of proteins. Bradford Assay is focused on measuring the number of proteins then Salivary Amylase Enzyme Assay explains that proteins are controlled by other factors such as pH level, temperature, substrate concentration, and inhibition, while Elisa detects a specific protein. CHROMATOGRAPHY Chromatography  Greek chroma meaning ‗color‘; graphein meaning ‗writing‘  Physical process where the solutes of a sample mixture are separated as a result of their differential distribution between stationary and mobile phase  Mikhail Tsvet- Russian-Italian botanist; Father of Chromatography; Credited for the development of Chromatography; Produced a colorful separation of plant pigments through a column of calcium carbonate Principles of Chromatography  Usually based on the principle of partition of solute between two phases  Mobile Phase/Eluent- refers to the mixture of substances to be separated dissolved in a liquid or a gas; Stationary Phase- porous solid matrix through which the sample contained in a mobile phase percolate  Column o usually contains the stationary phase o either packed or open tubular o Packed Columns (filled with particles of the stationary phase) o the stationary phase is coated on the inside of the column in open tubular columns  Analyte- substance to be separated during chromatography  Eluate- solvent leaving column; Eluite- sample leaving column  Chromatogram o graphical representation of detector response, concentration of analyte in the effluent, other quantity used as a measure of effluent concentration o the retention time or volume is when a solute exits the injector and passes through the column and the detector o data represented by the chromatogram are used to help identify and quantify solute/solutes o eluting solutes are displayed graphically as a series of peaks, usually referred to as Chromatographic Peaks (width, height, area)  Molecules more soluble in mobile phase moves fast; Molecules less soluble in mobile phase, takes longer time to move 15  Step 3: SPOT THE TLC PLATE- Spot the plate by using a capillary tube. After that press 3 times firmly to the plate in order to deposited the solution.  Step 4: DEVELOP THE PLATE- Get the TLC developing chamber and put the TLC plate inside. Cover it for a while then after a few minutes the solvent will go up to the TLC plates. In this phase where the mobile phase which is the solvent that we put in the TLC developing chamber moves forward to the stationary phase which is the TLC plate. It does it by the capillary action, the two components will move up the plate at different rates. Two components will interact of hydroxyl group in the silica. First polar compounds that has strongly absorbed to the stationary phase that cause slow movement while the Second is non-polar compound that move quickly cause of weak interaction in the silica.  Step 5: VISUALIZE THE SPOTS- Once the solvent slightly move up, remove it before the solvent hits the top of the plate. Then mark the solvent line with a pencil. Let the TLC plate dry. When the spots are not visible use UV light and trace all the spots results in the experiments. Analysis  Sample #1: In solvent 1 where the 75% of a polar solvent use it will move up quickly while the less polar in the solvent two move slowly to the solvent point. It is also show that the red spot on the stationary phase move slowly due to the strongly absorbed on the silica gel and the blue spot which is the less polar molecule has weakly absorbed on the silica gel that cause blue spots move rapidly than to the red spots.  Sample #2: If the both sample solvent has same polar, it will still the same results that shown on the TLC plate.  Sample #3: In the solvent #1 where 100% of non-polar used, there no move and it will stay on the starting line. While the solvent #2 used 100% polar both of the two spots moves fast to the solvent line point. There will be no sticky to the polar silica gel. Layer VS Paper Chromatography  Time saving- Thin layer chromatography experiment takes 3-4 hours while the paper chromatography takes 13-16 hours  Rigid support- In paper chromatography use support of cellulose paper which is extremely flexible. On the other hand TLC is use support of on rigid plastic, mental or glass. In terms of fast development TLC has more advantage than to Paper chromatography due to the type of support that used in experiment. With the use of proper rigidity, diffusion will be going less and the formation of well-defined spots.  Heating- Thin layer chromatography can be heated if it is necessary for developing spots. While the sheets of paper are not capable of that procedure.  Choice of support- TLC has various of support substance choices such as adsobernt, liquid coated and fluorescence induces.However the paper chromatography was limited in the choice of different kind of paper. Application  Phytohemistry - used for plant chemistry, biochemistry and molecular biology o Biological activities of plant compounds. It can be used on thin layer chromatography to recognize the variation of plant compounds such as anti-oxidative, antibacterial, or antifungal. In the process to test the antioxidants on the TLC plates can be sprayed with 2,2- diphenhyl- picrylhydrazyl (DPPH) and methanol. It will turns out this compound have free radical like deep purple color. After the reaction with the antioxidants, it turns out to yellow. By examine the presence of anti-bacterial or anti-fungal compounds, TLC plate can be incubated with microorganism. It can monitor the inhibited growth of microorganism.  Food and Cosmetic Industry - to distinguish all the measurements of colors, ingredients, preservatives, and sweetening agents in food and cosmetic products.  Pharmaceutical Industry o It can control and monitor all the substances in the testing of pharmaceutical formulations. o One of the example is identifying drugs in body fluids. It can be used in Thin layer chromatography of forensic studies, such as urine and blood test that can identify the presence of the drugs on the body.  Biochemical Analysis - In this Thin layer chromatography examperiment which often used this to identify, differentiate and characters the compound and metabolites in the following: o Blood o Body fluids o Urine o Serum 16 Column Chromatography  Column chromatography is a method in chemistry that is used to isolate a single chemical component from a mixture that has been dissolved in a fluid.  It separates substances via differential adsorption of compounds to the adsorbent as the compounds pass along the column at various speeds, allowing them to be separated into fractions.  This procedure can be employed on a small or big scale to purify materials for use in future investigations. This technique is an example of the type of chromatography used by researchers at Cambridge University. Ion Exchange Chromatography  Ion exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their affinity to ion exchangers.  It offers a huge sample-handling capacity, a broader application (particularly to proteins and enzymes), a low cost, great resolving ability, and eases scale-up and automation, making it one of the most flexible and commonly used liquid chromatography methods. In this process two types of exchangers i.e., cationic and anionic exchangers can be used. o Cationic exchangers possess negatively charged group, and these will attract positively charged cations. These exchangers are also called ―Acidic ion exchange‖ materials, because their negative charges result from the ionization of acidic group. o Anionic exchangers have positively charged groups that will attract negatively charged anions. These are also called ―Basic ion exchange‖ materials. Materials Pump, injector, column, suppressor, detector, and recorder or data system are examples of typical IC equipment. 1. Pump - The IC pump is regarded as one of the most critical components in the system, since it must deliver a consistent flow of eluent through the IC injector, column, and detector. 2. Injector - A sample introduction can be done in a variety of ways. The most basic approach is to utilize an injection valve. Liquid samples can be injected directly, whereas solid samples merely need to be dissolved in a suitable solvent. Injectors should be able to inject liquid samples ranging in volume from 0.1 to 100 ml with good repeatability and under high pressure. 3. Columns - The column material may be stainless steel, titanium, glass, or an inert plastic such as PEEK (Polyether ether ketone), depending on its eventual usage and region of application. Depending on whether it is to be used for standard analytical purposes, microanalysis, high speed analyses, or preparative work, the column's width can range from around 2mm to 5 cm and its length can range from 3 cm to 50 cm. The guard column is located ahead of the dividing column. This acts as a protective element, extending the separation column's life and effectiveness. They are reliable columns that filter or eliminate particles that block the separation column. 4. Suppressor - The suppressor suppresses the background conductivity of the chemicals used to elute samples from the ion-exchange column, improving the conductivity measurement of the ions under test. IC suppressors are membrane-based devices that are meant to convert ionic eluent to water in order to increase sensitivity. 5. Detectors - Electrical conductivity detectors are widely used. 6. System of data - A pre-programmed computer integrator may suffice in routine analysis when no automation is required. A more sophisticated device, such as a data station or minicomputer, is required for greater control levels. Mechanism of Separation  The stationary phase in this method is an insoluble porous resinous substance containing fixed charge- carrying groups. These groups are loosely complexed with counter-ions of opposite charge.  The reversible exchange of these ions occurs when a liquid mobile phase containing ionised or partly ionised molecules of the same charge as the counter-ions passes through the system.  The pace of migration and hence the degree of separation between the different solute species is determined by the degree of affinity between the stationary phase and feed ions.  The most common form of stationary phase is a synthetic copolymer of styrene and divinyl benzene (DVB), which is manufactured as micrometer-sized beads. Controlling the amount of DVB applied carefully determines the degree of cross-linking and, as a result, the porosity of the resinous structure.  Big pores in low-cross-linking resins allow large ions to diffuse into the resin beads and permit fast ion exchange. Highly cross-linked resins feature holes that are comparable in size to tiny ions.  The selection of a certain resin will be heavily influenced by the application. Chemical modifications of the resin can be used to introduce cation (+) or anion (-) exchange characteristics.  In industrial operations, ion exchange chromatography is widely used. This technology is used to separate transition metals, remove trace metals from industrial effluents, and purify a variety of organic chemicals and medicines. When compared to other forms of stationary phase, the resin matrix is generally very affordable. Although ion exchange chromatography is the most extensively used large-scale chromatographic method, it is confined to ionisable, water-soluble compounds. 17 Procedure Key steps in the ion exchange chromatography procedure are listed below:  Ion exchange separations are carried out mainly in columns packed with an ion-exchanger.  These ionic exchangers are commercially available. They are made up of styrene and divinyl benzene. Example. DEAE-cellulose is an anionic exchanger, CM-cellulose is a cationic exchanger.  The choice of the exchanger depends upon the charge of particle to be separated. To separate anions ―Anionic exchanger‖ is used, to separate cations ―Cationic exchanger‖ is used.  First the column is filled with ion exchanger then the sample is applied followed by the buffer. The tris- buffer, pyridine buffer, acetate buffer, citrate and phosphate buffers are widely used.  The particles which have high affinity for ion exchanger will come down the column along with buffers.  In next step using corresponding buffer separates the tightly bound particles.  Then these particles are analyzed spectroscopically. Analysis Fluoride, chloride, nitrate, nitrite, and sulfate concentrations may be measured using ion chromatographs. They may also determine the concentrations of important cations such as lithium, sodium, ammonium, potassium, calcium, and magnesium. For water chemistry analysis, ion chromatography is utilized.  The ion exchangers basically contain charged groups covalently linked to the surface of an insoluble matrix.  The charged groups of the matrix can be positively or negatively charged.  When suspended in an aqueous solution, the charged groups of the matrix will be surrounded by ions of the opposite charge.  In this ―ion cloud‖, ions can be reversibly exchanged without changing the nature and the properties of the matrix. Significance/Application Ion exchange chromatography is the most extensively used technology for separating and purifying charged biomolecules such as polypeptides, proteins, polynucleotides, and nucleic acids. The major reasons for its success as a separation method are its wide application, high capacity and simplicity, and excellent resolution. Ion exchange chromatography is widely utilized in a variety of industrial applications, including the following:  Separation and Purification of blood components such as albumin, recombinant growth factors and enzymes.  Biotechnology - Analytical applications such as quality control and process monitoring  Food and clinical research - to study wheat varieties and the correlation of proteinuria with different renal diseases.  Fermentation - Cation exchange resins are used to monitor the fermentation process during ß-galactosidase production. Size Exclusion Chromatography (SEC)  Size-exclusion chromatography (SEC) is a chromatographic technique used for separating substances according to their molecular size, or more correctly, hydrodynamic volume.  It is often described as a chromatographic-based method that developed in 1955 and has been used to fractionate molecules based on their hydrodynamic dimensions.  Further, according to ChemBam, it is a method where separation of different compounds occurs according to their size (hydrodynamic volume) measured by how efficiently they penetrate the pores of the stationary phase.  It is usually applied to large molecules or macromolecular complexes such as proteins and industrial polymers. There are two basic types of size exclusion chromatography.  One is gel permeation chromatography (GPC), which uses a hydrophobic column packing material and a non-aqueous mobile phase (organic solvent) to measure the molecular weight distribution of synthetic polymers.  The other is gel filtration chromatography (GFC), which uses a hydrophilic packing material and an aqueous mobile phase to separate and measure the molecular weight distribution of molecules soluble in water, such as polysaccharides and proteins. Materials As indicated by Mohammad (2020), coming up next are the Instruments that are utilized in doing this investigation: 1. Stationary Phase/Gel - Gels that are usually utilized incorporate cross-linked dextran, agarose, polyacrylamide, poly acryloyl morphine, and Polystyrenes. In addition, there are three sorts of gel and these are the following: a. Soft gel separation of proteins i. E.g. dextran (Sephadex), polyacrylamide gel b. Semi-rigid gels separation of non-polar polymers in non-polar solvents. i. E.g. bio beats c. Highly rigid gels and glasses separation of polar solvents. 2. Mobile Phase/Buffer - the fluid used to disintegrate the biomolecules to make the mobile phase is generally known as a buffer. The combination of biomolecules broken up in the cradle is known as the sample. 3. 3. Sample Preparation - The sample preparation should be ready in weakened fixations (under 2 mg/ml). A decent solvent can break up a sample to any extent in the scope of temperatures. Tests with expansive sub- atomic weight dissemination might require higher focus. It is prescribed to filter the sample solution