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Water: Chemistry, Analytics, Microbiology
Laboratory practice: Thin Layer Chromatography (TLC). Objectives:
• • • • To familiarize with the TLC technique and its application. To use the TLC technique to separate the components of a mixture of dyes. To use the TLC technique to separate and identify amino acids. To recognize, improve and optimize basic parameters as sample concentration, spotting size, polarity and resolution.
In order to run the TLC practice, the students must pass a pre-lab test. Therefore, it is necessary to review the principles and fundaments about TLC; as: equipment, stationary phase, mobile phase, polarity effect, spotting technique, developing procedures, visualization techniques, reproducibility, etc. The students will work in groups of two members. Each group must bring for the practice a fruit (apple, orange, lemon, peach, etc.). The experiment must start by working with the amino acids due to the long time they need to run. Once the amino acids have been placed inside the developing chamber it is possible to continue with the other parts of the experiment. Record any important observations you make during the course of the analysis such as: spot shape, spot size, spot color, developing time, tailing, difficulties, etc. The evaluation of the whole practice will consist on: pre-lab test, laboratory performance and report.
1. Separation and identification of amino acids: For the experiment, you will use: Unknown amino acids mixture (0.25 % in (HCl 0.5 M in ethanol)). Fruit juice. Several amino acids standards: glycine, proline, alanine, glutamic acid, aspartic acid, phenylalanine, hydroxyproline, tyrosine, tryptophan, valine (0.25 % in (HCl 0.5 M in ethanol)).
After visualization with UV light.. add each solvent system to a depth of about 10 mm. 100 ml neutral solvent system. 2. You should see the solution rising in the capillary. the unknown sample and the fruit juice2. make a mark every 1. Insert the end of a capillary tube into the standard amino acid solution. Cover the internal walls of the chambers with filter paper and wet it with the solvent system used in that chamber. but if a compound shows own fluorescence the spot will be also fluorescent 7 This procedure must be done inside the extractor vapour hood and in the specific place designated for that. and allow all solvent to evaporate. 2 If the fruit is solid. Once the spots have dried3. therefore. for example apple. mark the solvent front with a pencil. Liquid should flow from the capillary to the plate to form a spot. mark every TLC plate with a pencil line about 1. Close the lid. Your goal is to identify which are the components of your unknown sample and which are the free amino acids on the fruit. 4 It is very important that the start line and spots be above the level of solvent. and spot the plate in the next pencil mark. Withdraw the capillary when the liquid has risen to a height of 1-2 cm. on this line.2 3 Pre-coated TLC silica-gel plates 20 X 20 cm 3 Pre-coated TLC cellulose plates 20 X 20 cm 120 ml acid solvent system. Spot the sample a second time to ensure that the material has been placed on the TLC plate. lower the plate carefully into the chamber4. and heat it inside the oven at 110 °C for 5 minutes.10. Repeat the spotting procedure with all the standards.30].3% in (acetic acid 3% in acetone). When you draw the line and also when you spot the plate avoid to push the pencil and capillary strongly on the adsorbent material.5 cm from the bottom1.30]. Next. For visualizing the spots.n-propanol-ammonium hydroxide 34% [70. Into respective developing chambers. Ninhydrine 0. compounds will appear as dark spots. With another capillary tube repeat the process to draw up your second standard amino acid solution..10]. 3 You can use a hairdryer to accelerate the drying. Separation of dyes: For the experiment.n-propanol-water [70. Hold the capillary vertically and briefly touch the filled end to the pencil mark line. watch the plate using the UV light chamber6 (254 nm and 366 nm) and mark them with a pencil. This happens rapidly. you will use: 1 Mixture of lipophilic dyes (0. When the solvent has advanced at least 15 cm (≈ 4 hours)5 remove the plate from the chamber. Circle the new spots with a pencil and calculate the Rf values. let it dry. spray the plate with ninhydrin reagent7.01 % in toluene) Over this line you must spot the sample and standards. you just need to scrape the fruit with a spoon to obtain juice. . These marks will be used to apply your standards and sample. lift the capillary before the spot gets bigger than 2 mm in diameter. so.5 cm from left to right. Softly. Close the lid and monitor the movement of solvent up the plate.. 5 At this time you must continue other parts of the experiment. 6 The silica plates are coated with fluorescent indicator.n-butanol-acetic acid-water [40. . 100 ml basic solvent system.
890).020).890) and ethyl acetate (ε=6.5 X 9 cm. Therefore. xylene (ε=2. methanol (ε=32.270). and allow all solvent to evaporate. When solvent has advanced to the top pencil line. Mark your TLC plates with a narrow pencil line 1. ethyl acetate (ε=6. Turn the plate 90 degrees and run it in the second direction with the solvent system toluene-ethyl acetate [9:1]. By using one-dimensional TLC it is not possible to separate all the components of the dye mixture. spot the dyes mixture.5 cm from the left edge.5 cm from the bottom and 1. Mark your 10 X 10 cm TLC plate with a narrow pencil line 1. Developing solvents: cyclohexane (ε=1. you will use: Mixture of lipophilic dyes (0. 1 Pre-coated TLC silica-gel plate 10 X 10 cm. methylene chloride (ε=9.080).630). preserving the shape and taking note of the color of each spot.01 % in toluene) 11 Pre-coated TLC silica-gel plates 1. Polarity effect: For the experiment. Run the plate in the first direction with xylene and let it dry.5 X 9 cm Developing solvents: cyclohexane (ε=1.700). remove the plate from the chamber.5 cm from the bottom and 1 cm from the top. Use a pencil to outline each observed spot on the plate. ethanol (ε=24. 3. acetone (ε=20. Spot the dyes mixture in the center of the bottom line. toluene (ε=2. In the intersection of these lines. Use a pencil to outline each observed spot on the plate.3 10 Pre-coated TLC silica-gel plates 1.330).300). Using tweezers.5 X 9 cm 11 Pre-coated TLC aluminium oxide plates 1.37) Into respective developing chambers add 3 ml of each solvent and close the lid.020) in the following proportions: Cyclohexane (%) 100 90 80 70 60 50 40 30 20 10 0 ethyl acetate (%) 0 10 20 30 40 50 60 70 80 90 100 . lower the plate into the developing chamber. diethyl ether (ε=4.379). it is necessary to run a bi-dimensional TLC with two different solvent systems. Close the lid and monitor the movement of solvent up the plate. The solvents will not run at the same speed. water (ε=80.
4 0. plot the Rf values you got in this practice for each stationary phase.3 0.6 0.5 0. compare the Rf values obtained for all stationary phases and mobile phases with the Rf values reported in the bibliography.1 0 hyp phe pro gly ala glu asp amino acids acid medium neutral medium basic medium val trp tyr 3. neutral or basic) affect the elution of amino acids? What stationary phase – solvent system do you recommend to separate amino acids? What stationary phase is more polar: cellulose or silica gel? What amino acids are more polar? Is the chemical structure of the amino acids related with the polarity? TLC of aminoacids Stationary phase:_________ 0.2 0.9 retardation factor (Rf) 0.8 0. Conclusions and recommendations. calculate in each plate the retardation factor (Rf) for the first and for the last eluted spot. Results and discussions. Answer to the questionnaire.4 Run each set of plates with the different solvent systems. For all amino acid standards. as shown in the example. 1. Once developed. and explain: How does the solvent medium (acid. 2. What are the components in your unknown amino acid sample and in the fruit? Explain how you made the interpretation of your results. Questionnaire. Report: The corresponding report must include: Observations.7 0. . For the amino acids.
4 0.607 6.194 5. 6.542 3. the Rf values obtained using aluminium oxide are smaller? Polarity effect Stationary phase:______________ 1 0.5 0.2 0.716 3.7 0.3 0.129 3. at the same polarity. What would happen if the same solvent is used in the first and in the second development of a bidimensional TLC?. With the Rf values obtained in the polarity effect part.955 4.5 4. polarity (X axis) for the two different stationary phases.9 0.368 4.020 have almost the same Rf value? What spot belongs to the more polar dye component? Why.020 are completely separated? Why the two spots at polarity = 6. plot a graph (as indicated below) between Rf values (Y axis) vs. Why is it necessary to run a bi-dimensional TLC with different solvents?. And if you use aluminium oxide? 7.1 0 1. What would you expect to happen to the dyes mixture if you use cellulose instead of silica gel as stationary phase.890 and shorter than 6.303 2. What differences can you note between running the dyes with one and two dimensional TLC? 5.890 have a Rf = 0? Why the two spots at polarity larger than 1. and explain: Why the two spots at polarity = 1.6 0.8 0.020 polarity first spot last spot The polarity value is calculated using the following equation: retardation factor (Rf) polarity = where: ∈c C c + ∈a C a 100 ∈ c= dielectric constant of cyclohexane Cc= proportion (%) of cyclohexane ∈ a= dielectric constant of ethyl acetate Ca= proportion (%) of ethyl acetate .781 5.890 2.
the new solvent is allowed to move a longer distance than the previous one. the solvent is allowed to run just a few distance on the plate. and draw at least 3 pictures to explain this powerful technique. You are trying to determine a TLC solvent system which will separate the compounds X. In every consecutively development. 9. with different solvents at each time (of course. an so on. In order to get an idea of the small amount of sample used usually in TLC. between each development the solvent on the plate must be complete evaporated). explain how the solvent polarity must be changed to obtain the desired separation. a TLC plate is developed several times.. You ran the compounds on a silica gel TLC plate using hexane/ethyl acetate 95:5 as the eluting solvent and obtained the chromatogram below. Based on this information. Y. in the same direction (one dimensional TLC). Take as reference the dyes mixture used in this practice (0. At the beginning.01%). 10. In this technique.6 8. and Z. calculate approximately the amount of sample (in µg) in every spot. How could you change the solvent system to give better separation of these three compounds? . An interesting TLC technique is the one known as “High Performance Thin Layer Chromatography (HPTLC)”.
C = corrosive. T+ = very toxic. Hazard signs. F = flammable. 5: Substances and products with a low hazard potential. Xi = irritating.1: Extremely strong toxins..O = oxidizing (fire-promoting). 2: Water polluting substance.nwg: Non-water polluting substance. 4: Substances and products that must be considered harmful. teratogenic). 5S: Approved for selfhandling. F+ = extremely flammable. 10 . RA: Radioactive substances.. 1*: Very strong toxins (carcinogenic. 3: Highly water polluting substance. 1: Slightly water polluting substance. F: Not subject to toxicity classification. Xn = harmful. 9 8 Water pollution risks (Wassergefährdungsklassen. BT: Narcotic substances.. mutagenic. N = dangerous for the environment. 3: Strong toxins. T = toxic.7 Safety Data Sheet of chemical reagents to be used in the practice Condensed formula C2H5NO2 C5H9NO2 C3H7NO2 C5H9NO4 C4H7NO4 C9H11NO2 C5H9NO3 C9H11NO3 C11H12N2O2 C5H11NO2 HCl C4H10O C2H4O2 C3H8O NH4OH C9H6O4 C6H12 C7H8 C8H11 CH2Cl2 C4H10O C4H8O2 C3H6O C2H5O CH4O Reagent Glycine Proline Alanine Glutamic acid Aspartic acid Phenylalanine Hydroxyproline Tyrosine Tryptophan Valine Hydrochloric acid 1-Butanol Acetic acid 1-Propanol Ammonium hydroxide Ninhydrin Cyclohexane Toluene Xylene Methylene chloride Diethyl ether Ethyl acetate Acetone Ethanol Methanol Lipophilic dyes Poisson WGK9 Class8 T+ F 4 F F 2 4 3 4 2 3 4 4 4 4 4 5 5 F 3 1 2 nwg nwg 1 nwg nwg nwg nwg nwg nwg 1 1 1 1 2 3 1 2 2 2 1 1 1 1 1 T Hazard signs10 Xn Xi C O F N X X X X X X X X X X X X X X X X X X X X X X X X X X Poisson class. WGK). 2: Very strong toxins.
8 List of materials to be used in the practice # 1 1 3 12 1 15 4 4 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 Material Oven UV-Lamp Development chamber for 20x20 cm plates Development chamber for 10x3 cm plates Development chamber for 10X10 cm plates Capillary tubes TLC 20x20 cm Si-gel plate TLC 20x20 cm cellulose plate TLC 20x20 cm aluminium oxide plate Measurement cylinder 10 ml Measurement cylinder 25 ml Measurement cylinder 100 ml Pipette 10 ml Beaker 100 ml Beaker 600 ml Ruler Pencil Tweezers Scissors Sample and standard vials Hair dryer Waste container TLC plate holder 20x20 cm TLC guide spotting plate Reagent spray Remarks .
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