Structural characterization begins with a purity check!

• Elemental Analysis (EA)

• Thin Layer Chromatography (TLC)

• High Performance Liquid Chromatography (HPLC)Course: 59-320 Analytical Chemistry, R. Letcher

• Gas Chromatography (GC) – only for volatile compounds Course: 59-320 Analytical Chemistry, R. Letcher

• Other chromatographic or electrophoretic methods

History of TLC and EA

Mikhail Tswett (1872-1919) developed chromatography in 1903Though he received some awards by the time he died in 1919 his chromatography seemed to have made little impact. However, in the 1930s, particularly in Eastern Europe, it was rediscovered and then spread worldwide. Physical chemical studies on chlorophyll adsorptionsBerichte der Deutschen botanischen Gesellschaft 24, 316-23 (1906) [as translated and excerpted in Henry M. Leicester, Source Book in chemistry 1900-1950 (Cambridge, MA: Harvard, 1968)]

TLC as we know it today was first described by Ismailov and Shraiber in 1938.“Analysis of Drop-chromatography and its Application in Pharmacy” Farmatzija (Moscow) 1938, 1. TLC became a widely applied analytical method in the 1950s.

The description of the first three elements Ca, Sr, and Ba by Doebereiner in 1817 might also be seen as the discovery of EA. When did Mendelejeff first announced his idea of the “periodic properties of elements”?

Pour solvent into the beaker to a depth of just less than 0.5 cm.

To aid in the saturation of the TLC chamber with solvent vapours, line part of the inside of the beaker with filter paper.Cover the beaker with a watch glass, swirl it gently, and allow it to stand while you prepare your TLC plate.

1. Prepare the developing containerThe developing container for TLC can be a specially designed chamber, a jar with a lid, or a beaker with a watch glass on the top.

Instruction for TLC separations(http://orgchem.colorado.edu/hndbksupport/TLC/TLCprocedure.html)

Thin Layer Chromatography (TLC)

2. Prepare the TLC plateTLC plates are purchased as large sheets and each sheet is cut horizontally into plates which are about 5 cm tall by various widths; the more samples you plan to run on a plate, the wider it needs to be.

Shown are a box of TLC plates, a large un-cut TLC sheet (5 x 20 cm), and small TLC plates which have been cut to a convenient size.Handle the plates carefully so that you do not disturb the coating of adsorbent or get them dirty.

Measure 0.5 cm from the bottom of the plate. Take care not to press so hard with the pencil that you disturb the adsorbent.

Using a pencil, draw a line across the plate at the 0.5 cm mark. This is the origin: the line on which you will "spot" the plate.

Under the line, mark lightly the name of the samples you will spot on the plate, or mark numbers for time points. Leave enough space between the samples so that they do not run together, about 4 samples on a 5 cm wide plate is advised. Use a pencil and do not press down so hard that you disturb the surface of the plate.

3. Spot and run the TLC plate.The sample to be analyzed is added to the plate in a process called "spotting".If the sample is not already in solution, dissolve about 1 mg in a few drops of a volatile solvent such as hexanes, ethyl acetate, or methylene chloride. As a rule of thumb, a concentration of "1%" or "1 gram in 100 mL" usually works well for TLC analysis. If the sample is too concentrated, it will run as a smear or streak; if it is not concentrated enough, you will see nothing on the plate. The "rule of thumb" above is usually a good estimate, however, well-sized and easy to read spots are sometimes obtained by trial and error only.

The solution is applied to the TLC plate with a 1µL microcap. Microcaps give consistently sized spots and are convenient. They are a bit expensive if you use a new one for each spot. Environmentally and financially, it is much better to reuse them by keeping used microcaps in a small beaker containing a small volume of solvent. They can be rinsed by consecutively dropping one end onto paper to empty them and then again into the beaker with solvent. Directions for rinsing and reusing microcaps are given below.Microcaps come in plastic vials inside red-and-white boxes. If you are opening a new vial, you will need to take off the silver cap, remove the white Styrofoam plug, and put the silver cap back on. A small hole in the silver cap allows you to shake out one microcap at a time.

Take a microcap and dip it into the solution of the sample to be spotted. Then, touch the end of the microcap gently to the adsorbent on the origin in the place which you have marked for the sample. Let all of the contents of the microcap run onto the plate. Be careful not to disturb the coating of adsorbent.

Carefully place the TLC plate into the chamber so that the solvent front runs horizontally. Remove the plate just before the solvent reaches the top and mark the solvent front with a pencil. Let the solvent fully evaporate and mark (circle) visible spots with a pencil. The ratio between the distances of the sample and the solvent front from the base line is call the Rf-value.

4. Visualize the spotsMost samples are not coloured and need to be visualized with a UV lamp (254 nm). Hold a UV lamp over the plate and mark any spots which you see lightly with a pencil.Beware! UV light is damaging both to your eyes and to your skin! Make sure you are wearing your goggles and do not look directly into the lamp. Protect your skin by wearing gloves.Spots that are neither visible nor absorbing at 254 nm might become visible by exposure to iodine (iodine chamber) or treatment with spray reagents.A versatile method is first spraying the plate with a 3% w/v solution of Vanillin powder in

ethanol and then with a 3% v/v solution of conc. H2SO4 in ethanol (do not saturate the plate).

Gentle heating with an air blower brings out initial spots with specific colours and subsequent

hard heating might bring out more spots.

If the TLC plate runs samples which are too concentrated, the spots will be streaked and/or run together. If this happens, you will have to start over with a more dilute sample to spot and run on a TLC plate.

Predicting column resolution using TLC

∆CV (column volume) not ∆Rf is a reliable predictor of column behavior. ∆CV can be calculated using the relationship of CV = 1/Rf

Rf .90 .80 .07 .06 .05 .04 .03 .02 .01

CV 1.10 1.25 1.40 1.65 2.00 2.50 3.33 5.00 10.0

Generally, TLC conditions should be chosen that will provide Rf values in the range of 0.15 - 0.35, which for column chromatography would result in CV’s of 2.8 - 6.7.

Choosing the right solvent and the right stationary phase

A solvent must dissolve the sample well. Streaking spots appear if the solubility of the annalite is not high enough.

Polar solvents for non-polar stationary phases and vice versa.

Three popular stationary phases are silica, aluminum oxide, and RP-18

What solvent would you choose for each of these stationary phases?

Elemental Analysis (EA)

Elemental analysis on carbon, hydrogen, and nitrogen is the oldest

investigation performed to characterize and/or prove the elemental composition of

an organic or inorganic sample. In fact, it used to be the only available routine

method before modern spectroscopy was established in the 50s. The majority of

organic compounds only contain the elements C, H, N, and O and the latter is

seldom determined separately.

Our departmental analyser gives weight percentages of C, H, N done by

combustion in O2 and gas chromatographic analysis of CO2, H2O and NO2.

As an option S can be measured as SO2 and oxygen as CO.

Other elements such as Cl, Br, and I must be determined by other means.

Perkin Elmer 2400 Series II CHNS/O Analyzer

Measuring PrincipleThe sample under test is weighed in using a tin capsule. The required amount is 2 to 3 mg of organic material and can hardly exceed 10 mg, if inorganic matter with little carbon content is investigated. After folding the capsule (looking rather like wrapped tin foil) the sample is placed in the autosampler.The tin capsule enclosing the sample falls into the reactor chamber where excess oxygen is introduced before. At about 990 °C the material is "mineralized". Formation of carbonmonoxide is probable at this temperature even under these conditions of excess oxygen.The complete oxidation is reached at a tungsten trioxide catalyst which is passed by the

gaseous reaction products. The resulting mixture should thus consist of CO2, H2O und NOx. But

also some excess O2 passes the catalyst.

The product gas mixture flows through a silica tube packed with copper granules. In this zone, held at about 500 °C, remaining oxygen is bound and nitric/nitrous oxides are reduced. The

leaving gas stream includes the analytically important species CO2, H2O und N2. Eventually

included SO2 or hydrohalogenides are absorbed at appropriate traps.

High purity helium (Quality 5.0) is used as carrier gas. Finally the gas mixture is brought to a defined pressure/volume state and is passed to a gas chromatographic system. Separation of the species is done by so called zone chromatography. In this technique a staircase type signal is obtained and the step height is proportional to the substance amount in the mixture.Blank values are taken from empty tin capsulesCalibration is done by elemental analysis of standard substances supplied by the instrument's manufacturer for this purpose.

Working RangeMineralization and detection covers every species of the analyte elements in the sample. Beside purely organic samples various metal organic compunds and even inorganic samples as carbides and nitrides have successfully been characterized.The detection limit for carbon and nitrogen at sample amounts of 2 to 3 mg was found to be at about 0,05 w-% (500 ppm) in what case the uncertainty stays at about 0,02 w-%.According to the apparate's supplier the instrument's uncertainty in the medium range stays below 0,3 w-% as required by journals to prove the expected composition. With very carbon rich samples we found this tolerance was slightly exceeded (still within 0,5 w-%).

Problems and InterferencesThe weighing of oily or fluid substances is impossible using the thin walled tin capsules. For this purpose alumina pans with a lid are available. These pans are tightly closed by cold welding to prevent loss of sample by spillage and evaporation. As the blank value for nitrogen is dramatically increased be the enclosed volume of air the determination limit for N in liquid samples is increased to 0,1 to 0,2 w-%.With highly viscous or even glassy materials elemental analysis is even impossible with the above method.

It has long been known that phosphorus can interfere in the mineralization of organic material. In literature the formation of glassy P2O5 xH2O yC has been described. Elemental analysis of phosphorus containing compounds can thus suffer from systematic deviations in the determined carbon content exceeding the tolerance limit of 0,3 w-%. This effect can be controlled by the addition of vanadium pentoxide (V2O5).Fluorine is mineralized to form HF which reacts at the wall of the silica tubes which form the main part of the reaction zone. The gaseous products, such as SiF4 and relatives, can cause systematic errors which rarely become significant with respect to the 0,3 w-% tolerance.The mineralization of metal containing samples can also be affected by interferences. By modification of the method most of these can be compensated for.Percentage Composition

CxHyOz (9.83 mg) + excess O2 x CO2 (23.26 mg) + y/2 H2O (9.52 mg)

millimoles of CO2 = 23.26 mg/ 44.01mg/mmol = 0.5285 mmoles of CO2

mmoles of CO2 = mmoles of C in original sample

(0.5285 mmoles of C)(12.01mg/mmol C) = 6.35 mg of C in original sample

CxHyOz (9.83 mg) + excess O2 x CO2 (23.26 mg) + y/2 H2O (9.52 mg)

mmoles of H2O = 9.52 mg/ 18.02 mg/mmol = 0.528 mmoles of H2O

mmoles of H2O = 1/2 mmoles of H in original sample

(0.528 mmoles of H)(2)(1.008mg/mmol H) = 1.06 mg of H in original sample

Weight Percentage CompositionCxHyOz (9.83 mg) + excess O2 x CO2 (23.26 mg) + y/2 H2O (9.52 mg)%C = 6.35 mg/9.83 mg x 100 = 64.6% %H = 1.06 mg/9.83 mg x 100 = 10.8% %O = 100-(64.6 + 10.8) = 24.6%

Calculation of Empirical Formulaassume for example a 100g sample 64.6% of C: 64.6 g/12.01 g/mol = 5.38 moles of C 10.8% of H: 10.8 g/1.008 g/mol = 10.7 moles of H 24.6% of O: 24.6 g/16.0 g/mol = 1.54 moles of O Thus: C5.38 H10.7 O1.54

converting to simplest ratio:C5.38/1.54; H10.7/1.54; O1.54/1.54 C3.50 H7.00 O1.00 = C7H14O2

What are possible structures?

Problem 1

Which of the three stationary phases (silica, aluminum oxide, or RP-18) and what solvent would you choose for the TLC separation of the following two component mixtures.

How accurate must the EA of your sample be if you want to detect 1 mol%, 10 mol%, or 20 mol% impurity of the phthalodinitrile starting material in the phthalocyanine product, a commercial pigment. 3 mg of sample was weighed in and the resolution limit is set to 0.05 w%.

N

N

NN

N

N

NN

CN

CN

CuCu, heat

OC8H17

OC8H17

OC8H17

OH

+Br

Br COOH

COOH Br

COOH

COOH

+

OC8H17

OC8H17NC

NCOC8H17

OC8H17NC

+1 2

3