Faculty of Natural Sciences Instrumental Analytical Chemistry

Campus Duisburg

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.

Organization:
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.

Procedure:
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)).

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.- n-butanol-acetic acid-water [40,10,10]. 100 ml neutral solvent system.- n-propanol-water [70,30]. 100 ml basic solvent system.- n-propanol-ammonium hydroxide 34% [70,30]. Ninhydrine 0.3% in (acetic acid 3% in acetone).

Into respective developing chambers, add each solvent system to a depth of about 10 mm. Cover the internal walls of the chambers with filter paper and wet it with the solvent system used in that chamber. Close the lid. Softly, mark every TLC plate with a pencil line about 1.5 cm from the bottom1. Next, on this line, make a mark every 1.5 cm from left to right. These marks will be used to apply your standards and sample. Insert the end of a capillary tube into the standard amino acid solution. You should see the solution rising in the capillary. Withdraw the capillary when the liquid has risen to a height of 1-2 cm. Hold the capillary vertically and briefly touch the filled end to the pencil mark line. Liquid should flow from the capillary to the plate to form a spot. This happens rapidly, therefore, lift the capillary before the spot gets bigger than 2 mm in diameter. Spot the sample a second time to ensure that the material has been placed on the TLC plate. With another capillary tube repeat the process to draw up your second standard amino acid solution, and spot the plate in the next pencil mark. Repeat the spotting procedure with all the standards, the unknown sample and the fruit juice2. Once the spots have dried3, lower the plate carefully into the chamber4. Close the lid and monitor the movement of solvent up the plate. When the solvent has advanced at least 15 cm ( 4 hours)5 remove the plate from the chamber, mark the solvent front with a pencil, and allow all solvent to evaporate. For visualizing the spots, watch the plate using the UV light chamber6 (254 nm and 366 nm) and mark them with a pencil. After visualization with UV light, spray the plate with ninhydrin reagent7, let it dry, and heat it inside the oven at 110 C for 5 minutes. Circle the new spots with a pencil and calculate the Rf values. Your goal is to identify which are the components of your unknown sample and which are the free amino acids on the fruit. 2. Separation of dyes: For the experiment, you will use: 1

Mixture of lipophilic dyes (0.01 % in toluene)

Over this line you must spot the sample and standards. When you draw the line and also when you spot the plate avoid to push the pencil and capillary strongly on the adsorbent material. 2 If the fruit is solid, for example apple, you just need to scrape the fruit with a spoon to obtain juice. 3 You can use a hairdryer to accelerate the drying. 4 It is very important that the start line and spots be above the level of solvent. 5 At this time you must continue other parts of the experiment. . 6 The silica plates are coated with fluorescent indicator, so, compounds will appear as dark spots, 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.

3 10 Pre-coated TLC silica-gel plates 1.5 X 9 cm. 1 Pre-coated TLC silica-gel plate 10 X 10 cm. Developing solvents: cyclohexane (=1.890), xylene (=2.270), toluene (=2.379), diethyl ether (=4.330), ethyl acetate (=6.020), methylene chloride (=9.080), acetone (=20.700), ethanol (=24.300), methanol (=32.630), water (=80.37)

Into respective developing chambers add 3 ml of each solvent and close the lid. Mark your TLC plates with a narrow pencil line 1.5 cm from the bottom and 1 cm from the top. Spot the dyes mixture in the center of the bottom line. Using tweezers, lower the plate into the developing chamber. Close the lid and monitor the movement of solvent up the plate. The solvents will not run at the same speed. When solvent has advanced to the top pencil line, remove the plate from the chamber, and allow all solvent to evaporate. Use a pencil to outline each observed spot on the plate, preserving the shape and taking note of the color of each spot. By using one-dimensional TLC it is not possible to separate all the components of the dye mixture. Therefore, it is necessary to run a bi-dimensional TLC with two different solvent systems. Mark your 10 X 10 cm TLC plate with a narrow pencil line 1.5 cm from the bottom and 1.5 cm from the left edge. In the intersection of these lines, spot the dyes mixture. Run the plate in the first direction with xylene and let it dry. Turn the plate 90 degrees and run it in the second direction with the solvent system toluene-ethyl acetate [9:1]. Use a pencil to outline each observed spot on the plate. 3. Polarity effect: For the experiment, you will use: Mixture of lipophilic dyes (0.01 % in toluene) 11 Pre-coated TLC silica-gel plates 1.5 X 9 cm 11 Pre-coated TLC aluminium oxide plates 1.5 X 9 cm Developing solvents: cyclohexane (=1.890) and ethyl acetate (=6.020) 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

4 Run each set of plates with the different solvent systems. Once developed, calculate in each plate the retardation factor (Rf) for the first and for the last eluted spot.

Report:
The corresponding report must include: Observations. Results and discussions. Conclusions and recommendations. Answer to the questionnaire.

Questionnaire.
1. What are the components in your unknown amino acid sample and in the fruit? Explain how you made the interpretation of your results. 2. For all amino acid standards, plot the Rf values you got in this practice for each stationary phase, as shown in the example, and explain: How does the solvent medium (acid, 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:_________

0,9 retardation factor (Rf) 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 hyp phe pro gly ala glu asp amino acids acid medium neutral medium basic medium val trp tyr

3. For the amino acids, compare the Rf values obtained for all stationary phases and mobile phases with the Rf values reported in the bibliography.

5 4. What differences can you note between running the dyes with one and two dimensional TLC? 5. Why is it necessary to run a bi-dimensional TLC with different solvents?. What would happen if the same solvent is used in the first and in the second development of a bidimensional TLC?. 6. What would you expect to happen to the dyes mixture if you use cellulose instead of silica gel as stationary phase. And if you use aluminium oxide? 7. With the Rf values obtained in the polarity effect part, plot a graph (as indicated below) between Rf values (Y axis) vs. polarity (X axis) for the two different stationary phases; and explain: Why the two spots at polarity = 1,890 have a Rf = 0? Why the two spots at polarity larger than 1,890 and shorter than 6,020 are completely separated? Why the two spots at polarity = 6,020 have almost the same Rf value? What spot belongs to the more polar dye component? Why, at the same polarity, the Rf values obtained using aluminium oxide are smaller?

Polarity effect Stationary phase:______________

1 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0 1,890 2,303 2,716 3,129 3,542 3,955 4,368 4,781 5,194 5,607 6,020 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

6 8. An interesting TLC technique is the one known as High Performance Thin Layer Chromatography (HPTLC). In this technique, a TLC plate is developed several times, in the same direction (one dimensional TLC), with different solvents at each time (of course, between each development the solvent on the plate must be complete evaporated). At the beginning, the solvent is allowed to run just a few distance on the plate. In every consecutively development, the new solvent is allowed to move a longer distance than the previous one, an so on. Based on this information, explain how the solvent polarity must be changed to obtain the desired separation; and draw at least 3 pictures to explain this powerful technique. 9. In order to get an idea of the small amount of sample used usually in TLC, calculate approximately the amount of sample (in g) in every spot.. Take as reference the dyes mixture used in this practice (0.01%). 10. You are trying to determine a TLC solvent system which will separate the compounds X, Y, and Z. 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. How could you change the solvent system to give better separation of these three compounds?

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.- 1: Extremely strong toxins; 1*: Very strong toxins (carcinogenic, mutagenic, teratogenic); 2: Very strong toxins; 3: Strong toxins; 4: Substances and products that must be considered harmful; 5: Substances and products with a low hazard potential; 5S: Approved for selfhandling; F: Not subject to toxicity classification; BT: Narcotic substances; RA: Radioactive substances.
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Water pollution risks (Wassergefhrdungsklassen, WGK).- nwg: Non-water polluting substance; 1: Slightly water polluting substance; 2: Water polluting substance; 3: Highly water polluting substance. Hazard signs.- O = oxidizing (fire-promoting); C = corrosive; F = flammable; F+ = extremely flammable; T = toxic; T+ = very toxic; Xn = harmful; Xi = irritating; N = dangerous for the environment.
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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