Campus Duisburg

Faculty of Natural SciencesInstrumental Analytical Chemistry

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 toreview 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 continuewith 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 andreport.

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 inethanol)).

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- 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 theinternal walls of the chambers with filter paper and wet it with the solvent system used in thatchamber. 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 andsample.

Insert the end of a capillary tube into the standard amino acid solution. You should see the solutionrising 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 shouldflow from the capillary to the plate to form a spot. This happens rapidly, therefore, lift the capillarybefore the spot gets bigger than 2 mm in diameter. Spot the sample a second time to ensure that thematerial has been placed on the TLC plate. With another capillary tube repeat the process to drawup your second standard amino acid solution, and spot the plate in the next pencil mark. Repeat thespotting 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 monitorthe 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 toevaporate.

For visualizing the spots, watch the plate using the UV light chamber6 (254 nm and 366 nm) andmark 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 andcalculate the Rf values.

Your goal is to identify which are the components of your unknown sample and which are the freeamino acids on the fruit.

2. Separation of dyes:

For the experiment, you will use:

- Mixture of lipophilic dyes (0.01 % in toluene) 1 Over this line you must spot the sample and standards. When you draw the line and also when you spot the plate avoidto 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 compoundshows own fluorescence the spot will be also fluorescent7 This procedure must be done inside the extractor vapour hood and in the specific place designated for that.

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- 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 (ε=1.890), xylene (ε=2.270), toluene (ε=2.379), diethyl

ether (ε=4.330), ethyl acetate (ε=6.020), methylene chloride (ε=9.080), acetone (ε=20.700),ethanol (ε=24.300), methanol (ε=32.630), water (ε=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 themovement of solvent up the plate. The solvents will not run at the same speed. When solvent hasadvanced to the top pencil line, remove the plate from the chamber, and allow all solvent toevaporate.

Use a pencil to outline each observed spot on the plate, preserving the shape and taking note of thecolor 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 cmfrom 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 inthe second direction with the solvent system toluene-ethyl acetate [9:1]. Use a pencil to outline eachobserved 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 (ε=1.890) and ethyl acetate (ε=6.020) in the following

proportions:

Cyclohexane (%) ethyl acetate (%)100 090 1080 2070 3060 4050 5040 6030 7020 8010 900 100

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Run each set of plates with the different solvent systems. Once developed, calculate in each platethe 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 howyou made the interpretation of your results.

2. For all amino acid standards, plot the Rf values you got in this practice for each stationaryphase, 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:_________

00,10,20,30,40,50,60,70,80,9

ala

asp

glu

gly

hyp

phe

pro trp tyr

val

amino acids

reta

rdat

ion

fact

or (R

f)

acid medium neutral medium basic medium

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

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4. What differences can you note between running the dyes with one and two dimensionalTLC?

5. Why is it necessary to run a bi-dimensional TLC with different solvents?. What wouldhappen if the same solvent is used in the first and in the second development of a bi-dimensional TLC?.

6. What would you expect to happen to the dyes mixture if you use cellulose instead of silicagel 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; andexplain:

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 completelyseparated?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 effectStationary phase:______________

00,10,20,30,40,50,60,70,80,9

1

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

polarity

reta

rdat

ion

fact

or (R

f)

first spot last spot

The polarity value is calculated using the following equation:

100aacc CCpolarity ∈+∈

=

where:

∈c= dielectric constant of cyclohexaneCc= proportion (%) of cyclohexane∈a= dielectric constant of ethyl acetateCa= proportion (%) of ethyl acetate

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8. An interesting TLC technique is the one known as “High Performance Thin LayerChromatography (HPTLC)”. In this technique, a TLC plate is developed several times, inthe 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 thebeginning, the solvent is allowed to run just a few distance on the plate. In everyconsecutively development, the new solvent is allowed to move a longer distance than theprevious one, an so on.Based on this information, explain how the solvent polarity must be changed to obtain thedesired 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, calculateapproximately the amount of sample (in µg) in every spot.. Take as reference the dyesmixture 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 asthe eluting solvent and obtained the chromatogram below. How could you change thesolvent system to give better separation of these three compounds?

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Safety Data Sheet of chemical reagents to be used in the practice

Reagent Condensedformula

PoissonClass8

WGK9 Hazard signs10

T+ T Xn Xi C O F NGlycine C2H5NO2 F 2Proline C5H9NO2 4 nwgAlanine C3H7NO2 F nwgGlutamic acid C5H9NO4 F 1Aspartic acid C4H7NO4 - nwgPhenylalanine C9H11NO2 - nwgHydroxyproline C5H9NO3 - nwgTyrosine C9H11NO3 - nwgTryptophan C11H12N2O2 - nwgValine C5H11NO2 - nwgHydrochloric acid HCl 2 1 X1-Butanol C4H10O 4 1 XAcetic acid C2H4O2 3 1 X1-Propanol C3H8O 4 1 X XAmmonium hydroxide NH4OH 2 2 X XNinhydrin C9H6O4 3 3 XCyclohexane C6H12 4 1 X X XToluene C7H8 4 2 X XXylene C8H11 4 2 X XMethylene chloride CH2Cl2 4 2 XDiethyl ether C4H10O 4 1 XEthyl acetate C4H8O2 5 1 X XAcetone C3H6O 5 1 X XEthanol C2H5O F 1 XMethanol CH4O 3 1 X XLipophilic dyes 1 X X

8 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 beconsidered harmful; 5: Substances and products with a low hazard potential; 5S: Approved for self-handling; F: Not subject to toxicity classification; BT: Narcotic substances; RA: Radioactivesubstances.

9 Water pollution risks (Wassergefährdungsklassen, WGK).- nwg: Non-water pollutingsubstance; 1: Slightly water polluting substance; 2: Water polluting substance; 3: Highly waterpolluting substance.

10 Hazard signs.- O = oxidizing (fire-promoting); C = corrosive; F = flammable; F+ = extremelyflammable; T = toxic; T+ = very toxic; Xn = harmful; Xi = irritating; N = dangerous for theenvironment.

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List of materials to be used in the practice

# Material Remarks1 Oven1 UV-Lamp3 Development chamber for 20x20 cm plates12 Development chamber for 10x3 cm plates1 Development chamber for 10X10 cm plates15 Capillary tubes4 TLC 20x20 cm Si-gel plate4 TLC 20x20 cm cellulose plate1 TLC 20x20 cm aluminium oxide plate1 Measurement cylinder 10 ml1 Measurement cylinder 25 ml1 Measurement cylinder 100 ml1 Pipette 10 ml3 Beaker 100 ml1 Beaker 600 ml1 Ruler1 Pencil1 Tweezers1 Scissors

Sample and standard vials1 Hair dryer1 Waste container1 TLC plate holder 20x20 cm1 TLC guide spotting plate1 Reagent spray