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  • 1. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Osama Barayan - 1091105869 Faculty of Information Science & Technology HCB 1019 – BIOCHEMISTRY 1 LECTURE: AMELIA BINTI KASSIM Date: 27–6-2012 PRACTICAL 2 Separation and Identification Techniques using Paper Chromatography Page 1 of 6
  • 2. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Introduction: Chromatography is a process which can be used to isolate the various components of a mixture. There are a number of different types of chromatography in use, including gas, liquid, paper, and gel permeation chromatography, and this process can get quite involved, especially with complex mixtures. It is also an extremely useful addition to a variety of fields, including pure and applied sciences, forensics, and athletics, among many others. The process relies on the fact that different molecules will behave in different ways when they are dissolved in a solvent and moved across an absorbent medium. In a very simple example, one could take ink and make a mark on a piece of paper. The paper could be dipped into water, and the capillary action of the water would pull the ink through the paper. As the ink moved, its ingredients would separate out, revealing a distinctive pattern which could be used to determine the components of the ink. Chromatography, a group of methods for separating very small quantities of complex mixtures, with very high resolution, is one of the most important techniques in environmental analysis. The ability of the modern analytical chemist to detect specific compounds at ng/g or lower levels in such complex matrices as natural waters or animal tissues is due in large part to the development of chromatographic methods. The science of chromatography began early in the twentieth century, with the Russian botanist Mikhail Tswett, who used a column packed with calcium carbonate to separate plant pigments. The method was developed rapidly in the years after World War II, and began to be applied to environmental problems with the invention of the electron capture detector (ECD) in 1960 by James Lovelock. This detector, with its specificity and very high sensitivity toward halogenated organic compounds, was just what was needed to determine traces of pesticides in soils, food and water and halocarbon gases in the atmosphere. This happened at exactly the time when the effect of anthropogenic chemicals on many environmental systems was becoming an issue of public concern. Within a year, it was being applied to pesticide analysis. The pernicious effects of long lived, bioaccumulating pesticides, such as DDT, would have been very difficult to detect without the use of the ECD. The effect of this information on public policy has been far- reaching. The basis of all types of chromatography is the partition of the sample compounds between a stationary phase and a mobile phase which flows over or through the stationary phase. Different combinations of gaseous or liquid phases give rise to the types of chromatography used in analysis, namely gas chromatography (GC), liquid chromatography (LC), thin layer chromatography (TLC), and supercritical fluid chromatography (SFC). Chromatography has increased the utility of several types of spectroscopy, by delivering separate components of a complex sample, one at a time, to the spectrometer. This combination of the separating power of chromatography with the identification and quantitation of spectroscopy has been most important in environmental analysis. It has enabled analysts to cope with tremendously complex and extremely dilute samples Page 2 of 6
  • 3. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Materials, reagents and equipments: Trypsin Light Green Congo Red Orange G Buffer pH7 Buffer pH3 Aspartic Acid Valine Solvent A (80% Methanol) Solvent B (0.05% Ninhydrin in 80% Methanol) Beakers Chromatography tank containing glass bead Sample applicators Chromatography paper strips Methods: Experiment I Separation of dyes Three dyes are supplied: Orange G, Congo Red and Light Green 1. Place 1-2 drops of Orange G in one beaker, Congo Red in another beaker and similarly for Light Green in the third. 2. Get a TLC sheet (avoid touching the coated surface since fingerprints can leave significant quantities of protein and will affect the experiment). Draw a light pencil line about 3/4cm or 0.75cm from the bottom on both side of the sheet and fold it into half. 3. Gently spot a tip or small drop of each color onto the pencil mark with applicator (the spot should be as small as possible) side by side for 10 times and allow the sheet to dry. 4. At another side of the sheet, place three larger spots side by side for about 20 times of the drop. (Figure 1) 5. Fold the paper in half and stand for about 5 mins to allow the spots to dry. 6. Add sufficient amount of solvent A to the bottom of the beaker until the solvent just cover the glass bead. 7. Place the paper strip in the beaker, sample side down, cover it with lid. Allow the solvent to travel up the sheet in 5 to 10 mins or until the solvent has reach almost the line of folded paper. 8. Remove the TLC sheet carefully by forceps and allow it to dry. Note the positions of the solvent immediately that define how far the solvent has traveled and let it dry. Allow the chromatogram to dry completely and note the position of each spot later. 9. Take another paper strip and spot the amount of dyes which you now think will give good spots. Mix the three dyes together in one beaker and spot this mixture at the other end, but use three times the amount that necessary for any dye, since they are now diluted with each other. Allow the spots to dry and develop the chromatogram as before. Page 3 of 6
  • 4. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 10. Calculate Rf values for each amino acid according to the following equation: Rf = Distance traveled by spot Distance traveled by solvent Record Rf values for each sample you tested in the table shown below. 11. Elaborate on your observations for both sides of the TLC sheets. 12. Keep the chromatograms for reference and label fully. 3 large spots 3 small spots about 3/4cm Figure 1 Experiment 2 Separation of amino acids 1. Place 1-2 drops of amino acid Valine into a beaker and Aspartic acid in another beaker. 2. Get a TLC sheet (avoid touching the coated surface since fingerprints can leave significant quantities of protein and will affect the experiment). Draw a light pencil line about 3/4cm or 0.75cm from the bottom on both side of the sheet. 3. Gently spot a tip or small drop of each amino acid onto the pencil mark with applicator (the spot should be as small as possible) side by side for 10 times and allow the sheet to dry. 4. At another side of the sheet, place two larger spots side by side for about 20 times of the drop. (as in Figure 1) 5. Fold the paper in half and stand for about 5 mins to allow the spots to dry. 6. Develop this in solvent B. Add sufficient amount of solvent B to the bottom of the beaker until the solvent just cover the glass bead. 7. Place the paper strip in the beaker, sample side down, cover it with lid. Allow the solvent to travel up the sheet in 5 to 10 mins or until the solvent has reach the line of the folded paper. 8. Remove the TLC sheet carefully by forceps and allow it to dry. Note the positions of the solvent immediately that define how far the solvent has traveled and let it dry. Note the position of each spot later. Allow the chromatogram to dry completely. Page 4 of 6 . . . . . .
  • 5. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 9. After development, take the paper strip from the tank and heat it, either in an oven at 70 – 110 ºC for 2 to 3 mins or over a hot plate. Amino acids will give purple or yellow colors when they react with ninhydrin and the spots of Valine and Aspartic acid will show up dense colorations of the paper. Note the positions of the spots and note any trailing of the spots; extensive trailing means that the chromatogram is overloaded that means there is too much application of the sample. 10. Take another paper strip and spot the amount of amino acids which you now think will give good spots. Mix the two amino acids together and spot onto another paper strip but use two times the amount that necessary for any dye. Develop again in solvent B, heat and note the separation achieved. 11. Determine the Rf values for these two amino acids on all the chromatograms. 12. Elaborate on your observations on both sides of the TLC sheets. Keep the chromatograms for reference and label fully. Results: Experiment 1 Distance from origin to the centre of x10 spot x20 Rf value Solvent front 4.0 cm 8.2 cm - Orange G 7.7 cm 8.2 cm Congo Red 5.1 cm 4 cm Light Green 3.5 cm 8.2 cm Experiment 2 Distance from origin to the centre of x10 spot x20 Rf value Solvent front 8.2 cm 8.2 cm - Valine 5.7 cm 7.0 cm Aspartic acid 8.1 cm 8.10 cm The pic shows when adding 20 drop it become very obvious Page 5 of 6
  • 6. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Questions: 1. Why do different compounds travel different distances on the piece of paper? Two reasons: 1. They have different solubilities in the solvent that's being used. 2. They bind to the paper to differing degrees. This is more true with silica platesrather than paper, but it is a possibility. 2. In this experiment, name the stationary phase and the mobile phase. With paper chromatography the stationary phase is the paper while the mobile phase is the solvent in which the sample is dissolved. 3. How is an Rf value useful? What will the range of a Rf value? You are given Rf values for particle A and particle B, 0.2 and 0.8, what is your conclusion on the mobility of these particles? RF value is the distance travelled by a component of a mixture relative to the solvent. That is, it is the distance travelled by the component divided by the distance travelled by the solvent. RF value for any component has a fixed value less than unity. By calculating RF value for a component and comparing it with known values, a given component can be identified. The lower the Rf value the less soluble the pigment is in the solvent. Rf = Distance traveled by spot Distance traveled by solvent Rf = 0.2 = 0.25 0.8 4. Base on your experiment results, which has the highest mobility and which particle is the least mobile? The highest mobility with 20 times of the drop and the lowest mobility with 10 times of the drop. 5. What is chromatography used for? Chromatography is a process which can be used to isolate the various components of a mixture. There are a number of different types of chromatography in use, including gas, liquid, paper, and gel permeation chromatography, and this process can get quite involved, especially with complex mixtures. It is also an extremely useful addition to a variety of fields, including pure and applied sciences, forensics, and athletics, among many others. 6. List the major sources of errors you can observe in this experiment. A major source of error in this method is the reproducibility of the injection, especially if manual injections are being made using a syringe. Since most chromatographic samples are only a microliter or less, accurate measurement is difficult. Automatic injectors and sampling valves can reduce this error to a few percent. Page 6 of 6
  • 7. HBC 1019 Biochemistry 1 Trimester 1, 2012/2013 Questions: 1. Why do different compounds travel different distances on the piece of paper? Two reasons: 1. They have different solubilities in the solvent that's being used. 2. They bind to the paper to differing degrees. This is more true with silica platesrather than paper, but it is a possibility. 2. In this experiment, name the stationary phase and the mobile phase. With paper chromatography the stationary phase is the paper while the mobile phase is the solvent in which the sample is dissolved. 3. How is an Rf value useful? What will the range of a Rf value? You are given Rf values for particle A and particle B, 0.2 and 0.8, what is your conclusion on the mobility of these particles? RF value is the distance travelled by a component of a mixture relative to the solvent. That is, it is the distance travelled by the component divided by the distance travelled by the solvent. RF value for any component has a fixed value less than unity. By calculating RF value for a component and comparing it with known values, a given component can be identified. The lower the Rf value the less soluble the pigment is in the solvent. Rf = Distance traveled by spot Distance traveled by solvent Rf = 0.2 = 0.25 0.8 4. Base on your experiment results, which has the highest mobility and which particle is the least mobile? The highest mobility with 20 times of the drop and the lowest mobility with 10 times of the drop. 5. What is chromatography used for? Chromatography is a process which can be used to isolate the various components of a mixture. There are a number of different types of chromatography in use, including gas, liquid, paper, and gel permeation chromatography, and this process can get quite involved, especially with complex mixtures. It is also an extremely useful addition to a variety of fields, including pure and applied sciences, forensics, and athletics, among many others. 6. List the major sources of errors you can observe in this experiment. A major source of error in this method is the reproducibility of the injection, especially if manual injections are being made using a syringe. Since most chromatographic samples are only a microliter or less, accurate measurement is difficult. Automatic injectors and sampling valves can reduce this error to a few percent. Page 6 of 6