10-critical micelar concentration

8/6/2019 10-Critical Micelar Concentration

1/25

1

THIN LAYER

CHROMATOGRAPHY

VISUALISATION

REAGENTS

ELBERTUS KRUISWIJK


2/25

2

Forword

The idea for this book was born in 2004 when I was coming to the end of writing my named

organic reactions book. This book is a collection of reagents that I have collected of the last 5

years working as a chemist. It is definitely not complete and does not come near the classic and

very good book by Egon Stahl.

I hope that organic chemists find this book useful. The layout is as follows. The first 20pages deal with the background of chromatography.

There are more than 250 different dips mentioned in this book in alphabetical order. If you

use the electronic version you can search with the search option in Adobe acrobat. Otherwise the

index is a possible starting point. In the index the reagents are written in small letters, and the

compounds you are trying to detect in capital letters. At each page the preparation of the spray or

dip solution is described in detail. Under the heading treatment, you can find how to use the

reagent and what colour of spots you can expect. The necessary chemicals are given andreferenced to the 2005 2006 Aldrich catalogue, in some cases to other catalogues. I have

referenced to the Aldrich catalogue, because in most organic labs this is the most commonly used

catalogue. I like to make clear that I have not been sponsored by Aldrich. Supplier codes and CAS

number are also provided. References are given to the Merck index or Beilstein and selective

journal publications. Under the heading comments some additional information has been given if

necessary and there is some room left under notes to add your own comments. Structures are

provided where necessary.This book can be used by anyone who is active in practical organic chemistry.

None of the mentioned reagents have been specially tested during the preparation of this

book nor by me nor by the proof readers. If there are any comments about the entries, please

contact me at [email protected] and please do contact me.

Of course, I am indebted to the following group of people who were willing to volunteer to

proof read this book. In random order many thanks to Jelle Brinksma, Kiadis, Groningen, The

Netherlands, thank you Jelle you are always very supportive and Richard Tucker, West Monmouth

School, Pontypool. Thank you all for your time and fruitful discussions.

Bert Kruiswijk

Aberaman, 27-08-2005.
mailto:[email protected]:[email protected]


3/25

3


4/25

4

CHROMATOGRAPHY

The term chromatography is derived from the Greek word chromagraphein , i.e. chroma =

colour, graphein = write. Chromatography was invented by the Russian botanic Mikhail Tswett

who using this method proofed the existence of different kinds of chlorophyll. He used a leaf

extract on a column filled with calcium carbonate. Coloured bands were made visible after eluting

the column with solvent. It took until 1931 when Kuhn, Winterstein and Lederer used this methodagain for the separation of carotenoids. In 1941 Martin and Synge developed partition

chromatography and were awarded the Novel prize in 1952. However, already in 1948 A. Tiselius

received the same prize in recognition of his contribution to electrophoresis and adsorption

chromatography.

There are several techniques to separate substances, all of the techniques depend upon

the difference in distribution of the various compounds in the applied mixture between the mobile

phase and the stationary phase. This book will only consider thin layer chromatography (analysis),normally abbreviated as TLC. If the reader is interested in the mathematical background of

chromatography it is recommended to consult specialist handbooks.

THE STATIONARY PHASE

Many different materials are capable of retaining both solvents and solutes. The two most

commonly used as stationary phase (adsorbent) are silica gel (SiO 2) and alumina (Al 2O 3). Both

compounds are supplied highly purified and finely powdered. They are readily dispersed into the

atmosphere and inhaled. Therefore they should always be handled in the fume hood and if this is

not possible the use of a facial dust mask is recommended. The active centres on the surface of

the adsorbents do not possess the same adsorption power. This is a result of the special

orientation, the chemical character and the conformation of the adsorption places. In the

adsorption processes of silica gel and alumina are not only electrostatic interactions important but

also hydrogen bonding plays an important role. Both silica gel and alumina can be purchased in

large quantities, figure 1 .


5/25

5

Figure 1

Silica gel

Silica gel has a good linear capacity and hardly shows any catalytic character that can lead

to the decomposition of certain compounds. It has a large specific surface area (300 800 m 2/g)

and a large pore volume (> 0.7 ml/g). The pore diameter and specific surface can be changed

during the preparation of the silica gel. For spherical particles the ratio between surface and

volume equals 6/d. If the diameter (d) decreases, the specific surface area increases. Vicinal and

geminal hydroxyl groups are responsible for the adsorption process, figure 2 .

SiO

OH

Si OH SiHO OH

Vicinal Geminal

Figure 2


6/25

6

Furthermore, these hydroxyls can form hydrogen bonds in different manners, figure 3 .

Si OHO

Si OH

Free

Si OO

Si OH

H

Vicinal OH

Si O

O

Si O

H

H

OH

H

Hydrogen bonded with water

Figure 3

Alumina

On the surface of aluminium oxide are acidic (Al 3+) and basic (O 2-) groups present, as

result acidic compounds will be strongly adsorbed. The specific surface are is smaller than for

silica gel (100 200 m 2/g) and the pore volume is also smaller (0.2 0.3 ml/g). Alumina can

however catalytically decompose acidic compounds, furthermore chemisorption can take place.

Like silica gel alumina may be regarded as a typical polar sorbent and the order of separation of

compound classes in alumina and silica gel is generally similar. Carbon-carbon double bonds

contribute somewhat more to compound adsorption energy on alumina as compared to silica gel,

and hence compounds differing only in relative unsaturation (e.g. aromatic hydrocarbons) are

generally better separated on alumina than on other polar adsorbents such as silica gel. Active

aluminas are markedly sensitive to the differing shapes of various aromatic hydrocarbons and

some of their derivatives permitting an excellent separation of many aromatic isomers. The

preferential adsorption of acidic substances on alumina offers many useful separation possibilities

in the case of weak acids and this effect can be further enhanced by the use of basic solvents.

Stronger acids however tend to chemisorb on alumina, requiring the use of acid treated aluminas.

Acidic and neutral aluminas are useful for the separation of base sensitive materials. It should be

noted that separation order maybe considered as distinct adsorbent subtypes.

Activity

The activity is governed by the amount and the adsorption power of the active centres on

the surface of the adsorbent. First of all the activity is dependent on the nature of the adsorbent.

For a certain adsorbent the activity is not constant. The amount of water plays an important role.

On the surface of polar adsorbent is normally a variable amount of water molecules bound. These

water molecules form hydrogen bonds with the hydroxyl groups of the adsorbent, as result thatadsorption places are being blocked. Because the active centres differ in adsorption power, the

amount of water has not only influence on the amount of possible adsorption places but also on


7/25

7

the activity. The most active places are occupied first. The adsorption power of the remaining

places will decrease and the retention value increases. The remaining places form a more

homogenous group, which is important for the quality of the separation. The more homogeneous

the adsorption power of the active sites, the larger the linear capacity. This is the reason why it is

not always good to use an adsorbent with the highest activity.

Activity grade

There are five activity grades depending on the amount of water present. In activity grade I

all the water has been removed. The higher the number of the activity grade the higher the

amount of water and the lower the activity is, table 1 . The adsorbed water can be removed by

heating. Up to 150 oC only the adsorbed water will be removed. Other reactions take place above

200 oC. The geminal hydroxyl groups split off water with as result that the amount of adsorption

places decreases. The vicinal groups split off water above 500o

C and the specific surface areadecreases. Above 1200 oC all remaining water is removed and silica gel will have a hydrophobic

character. The activity grade can be checked by using an elution solvent saturated with a known

percentage of water.

Table 1: Activity grades.

Activity grade Al 2O3 amount of water (%) SiO 2 amount of water (%)

I - -

II 3 10

III 6 12

IV 10 15

V 15 20

ELUTION SOLVENTS

In most cases the structure of the component is constant. This means that the composition

of the elution solvent is the most important factor in adsorption chromatography. It is the elution

solvent that interacts with the component and with the adsorbent. Small changes in the

composition of the elution solvent can have major effects in the separation.

The elution solvent passes the component during chromatography. If this reaches new

adsorption places, it means that there will be an equilibrium between the elution solvent and the

adsorbent. This equilibrium can be written down as equation 1 :


8/25

8

[C] + [EA] [CA] + [E] eq. 1

= concentration of free component= occupied places elution solvent= occupied places component

= concentration elution solvent molecules

[C][EA][CA]

[E]

The side of the equilibrium depends on the difference in affinity strength of the elution

solvent and the component towards the adsorption places. If the affinity of the elution solvent for

the adsorbent is larger than the affinity of the component the equilibrium will lay on the left. In that

case the fraction of the component in the mobile phase is larger and the component will move

down the column or up the plate. This mainly depends on the polarity of the elution solvent.

Elution strength

The elution strength of a solvent is measured against n-pentane. The elution strength of n-

pentane is set at zero. Although in practice the elution strength can be measured experimentally, it

is possible to calculate it from the adsorption energy, equation 2 . The higher the adsorption

energy is, the larger the elution strength will be.

= _ EAe

o

o

EAe

= elution strength= adsorption energy elution solvent= necessary space at the surface

eq. 2

Eluotropic series Solvents can be ranked in order according to their elution strength, table 2 . What is

frequently forgotten is that the order in the eluotropic series is not the same for different

adsorbents. The eluotropic series holds for straight phase and reversed phase (in opposite

direction of course), but is only valid for pure distilled solvents. Impurities can have major

influences on the series. As expected the order of the eluotropic series runs in the order of polarity

and relative permittivity.


9/25

9

Table 2: Eluotropic series. o elution strength, r relative permittivity, speed coefficient over a

distance of 75 mm.

Solvent o SiO 2 o Al2O3 r

Least polar n-Pentane 0,00 0,00 1.8

n-Hexane 0.03 0.01 1.9 10.6

Cyclohexane 0.03 0.04 2.0 6.3

Carbon

tetrachloride

0.11 0.18 2.2 6.7

Di-isopropyl ether 0.21 0.28 3.9

Benzene 0.25 0.32 2.3

Chloroform 0.26 0.40 4.8 10.5

Dichloromethane 0.32 0.42 8.9

Diethyl ether 0.38 0.38 4.3

Ethyl acetate 0.38 0.58 6.1 10.9

Acetone 0.47 0.56 21.4 14.7

Dioxane 0.49 0.56 2.2

Acetonitrile 0.50 0.65 37.5 14.0

Pyridine - 0.71 12.4

n-Propanol 0.63 0.82 21.8

Ethanol 0.69 0.88 25.8

Methanol 0.73 0.95 33.6 6.5

Most polar Water - - 80.5

Speed coefficient

For TLC the speed at which the elution solvent is running is very important. is the speed

coefficient, a higher value indicates a shorter analysis time, eq. 3. The speed the elution solvent is

running at is not constant, it decreases with the height of the solvent. It is therefore important to

quote over a certain distance. In table 2 some values are given for Merck kieselgel 60 plates.

= ______ (xf + x s )

2

tf + t seq. 3

xf xs

tf ts

= distance between baseline and front= distance between solvent surface and baseline

= running time between baseline and front= running time between solvent surface and baseline


10/25

10

POLARITY OF COMPONENTS

The choice of elution solvent depends mainly on the polarity of the component. Polarity is

not a simple property, but rather a composite of different physical properties. The greatest

contributor to the polarity of a molecule will be its ability to form hydrogen bonds. This requires an

X-H bond, where X is an electronegative element (commonly N, O, or S). Common examples of

compounds like this are alcohols and water (-OH) and amines (-NH). Another factor is the dipolemoment of a compound. This is a physical constant that can be found in a reference book or

calculated with reasonable precision; the dipole moment represents the overall imbalance of

electron density in a molecule. Compounds with many polarized bonds (where the elements have

differing electronegativities, like C-O, C-N, C=O, etc.) tend to have large dipole moments. A quick

survey of the number of heteroatoms in a compound will offer a rough estimate of dipole. Alkanes

have essentially zero dipole moment, whereas compounds like acetonitrile (CH 3CN) and acetone

(CH 3C=OCH 3) have appreciable dipoles. Since dipole moment is a vector sum of individual bonddipoles, symmetrical molecules will frequently have zero dipole moment. The dielectric constant of

a material also influences polarity; it is a number whose value represents the ability of a substance

to isolate ions from one another. Once again, this value can be found in reference books, and

ranges from 1.9 for n-hexane to about 80 for water. The dielectric constant is not as useful of a

measure of polarity, however, because there is not a good way to get a qualitative feel for

dielectric constant values without going into the lab.

Functional groups in order of increasing polarity :

Hydrocarbons < ethers < tertiary amine < nitro < dialkyl amines < ketone < aldehyde < primary

amine < alcohol < phenol < alkanoic acid < sulfonic acid

In general :

Hydrocarbons < weak Lewis base < strong Lewis base < weak Lewis acid < strong Lewis acid

Estimating Dipole Moments

The dipole moment of a compound is the vector sum of all the dipoles of all the bonds in

the molecule (It is also related to the first derivative of the molecule's energy with respect to an

applied electrical field, but that is way beyond the context of this book.). It is a physical property

that can be measured in the lab or calculated with accuracy. These individual bond dipoles are

related to the polarisation of the bonds. Bonds between the same kinds of atoms share electron

density equally and thus have zero dipole. Bonds between different atoms will have polarized


11/25

11

bonds and thus dipoles. The magnitude of these dipoles is related to the difference in

electronegativity between the atoms being bonded, and its direction is always pointing toward the

electron-richer atom. We are normally only interested in molecules with significant dipole

moments. A consequence of this is that we can neglect C-C and C-H bonds when examining a

molecule; these bonds have very small dipoles (0 in the case of C-C) and thus contribute very little

to the net dipole of the molecule. We thus only need to focus on bonds involving heteroatoms.There are certainly many polarized bonds with appreciable dipoles. However, it is sufficient just to

look at the groups as a whole; we are trying to do a simple assessment, after all. To get a

qualitative feel for the dipole of the molecule, we can simply say that the dipole due to the nitro

group is opposing that of the carboxamide, so they tend to cancel each other out and the result is

a small dipole. This would be useful in comparing it to the m-nitrobenzamide, for instance. This

simple, qualitative analysis of dipole moment will hopefully bring you a long way toward

understanding the polarity of molecules. Just remember, dipoles that point in opposing directions tend to cancel each other out while dipoles pointing in similar directions tend to reinforce each

other .

THIN LAYER CHROMATOGRAPHY

Thin Layer Chromatography is a technique that is used all the time in the organic laboratory

because it is quick and inexpensive. It is used everyday in the research lab for everything from

monitoring reactions to assessing compound purity. In TLC, the stationary phase is a thin coating

of silica (SiO 2) or sometimes alumina (Al 2O 3) on a plate (commonly glass, plastic, or aluminium),

figure 4 .

Figure 4

Silica and alumina are the common choices for the stationary phase because they are very

inexpensive. They are also both very polar. The 20 x 20 cm TLC plates can be cut in smaller


12/25

12

pieces using a guillotine or a paper cutter, figure 5 . There are still people who prefer to make their

own chromaplates. The preparation of these plates is described in Vogels Textbook of Practical

Organic Chemistry, page 200, 5 th edition. Ready made 5 x 10 cm plates are also available, figure

6.

Figure 5

Figure 6

Although there is little choice in the stationary phase, a mobile phase can be selected to

suite the needs of a given separation. In general in TLC separations, the mobile phase isrelatively non-polar. Thus, polar compounds will be strongly retained on the plate and non-polar

ones will move with the non-polar mobile phase. Solvents are chosen with the opposite polarity to


13/25

13

the stationary phase so that relative retentions can be easily predicted. Consider a TLC

separation with a polar stationary phase and a polar mobile phase (like water, for instance). Now,

the stationary and mobile phases are competing for the compound, and it is very difficult to predict

where the spots will end up. The utility of these chromatography techniques lies in our ability to

predict their results; thus, they are not useful if the results are convoluted.

Applying sample

Because thin layer chromatography is an extremely sensitive procedure it is important to

bring a small quantity of the sample on to the TLC plate. In order to do this a micro pipette has to

be used. Some people use commercially available spotters, or use Pasteur pipettes but it is better

to use a melting point tube open at both sides, figure 7 .

Figure 7

To prepare these pipettes, the middle of the tube has to be heated on a Bunsen burner

(colourless flame) until the tube softens and the flame turns yellow. Quick pull the tube apart after

it is removed from the flame. Let the tube cool down and carefully break the tube in the middle of

the thin section, figure 8.

Figure 8

Before the sample is brought on to the TLC plate, the baseline has to be drawn with a

pencil on to the bottom of the plate approximately 1 cm from the end, figure 9 .


14/25

14

1 cm

Figure 9

Now dissolve a small quantity of the sample in the least polar solvent in which it is soluble.

Mark two or three spots with pencil on the baseline and apply the spot on to the plate. The spot of

the baseline has to be kept as small as possible. A second spot can be applied containing 2 or 3

times as much compound. The easiest way of doing this is, is to respot. Again the spot has to be

kept as small as possible. If there is room enough on the plate a third spot with even more

compound can be applied, figure 10 .

Figure 10


15/25

15

The development of the TLC plate

After the TLC plate is spotted it has to be transferred with tweezers in to the development

tank. This tank holds the solvent system. The baseline is not allowed to sink in to the solvent

system otherwise the compound will diffuse in to the solvent system.

Although a large variety of development tanks are commercially available ( figure 11 and

figure 12 ), the cheapest way is to use a beaker with a watch glass as lid. Jam jars can also beused if they have a flat and straight bottom. To saturate the inside atmosphere of the tank, the

tank can be lined with filter paper.

Figure 11

The solvent front should rise in a straight horizontal line until it reaches the top of the plate

approximately 1 cm from the end. The plate is removed with tweezers and the solvent front is

marked with pencil before all the solvent is evaporated. Because flammable and toxic solvents are

used, it is recommended to allow the solvent to evaporate in the fume cupboard.

Figure 12


16/25

16

The visualisation of the TLC plate

There are numerous reagents available to visualise TLC plates. It is the goal of this book to

give an overview of all the available visualisation agents. Even when the material being analysed

is coloured it is necessary to treat the TLC plate to visualise any no-coloured spots that may be

present in the sample. The two most useful means of analysis are ultraviolet-light and iodine

vapour. The TLC plate can be dipped into a stock solution of the reagent ( figure 13 ) or the platecan be sprayed with a diffuser, figure 14 .

Figure 13

Figure 14


17/25

17

Ultraviolet light

Never look directly at the ultraviolet light source and wear gloves because ultraviolet light

can damage your eye sight and skin. Ultraviolet light of two different wavelengths is normally

used. Both silica and alumina TLC plates can be supplied with the fluorescent compound zinc

sulphide. The box of plates possesses the markings 254 or F 254 . Under ultraviolet light of 254 nm

the zinc sulphide in the adsorbent will fluoresce green, figure 15 . The TLC plate has to bethoroughly dried because some solvents can mask any products present. The compounds present

in the sample will show up as dark spots on the green background. The spots can be marked with

a soft pencil.

Ultraviolet light of 356 nm is used to visualise aromatics and molecules with extended

conjugated -electrons. The compounds present in the sample will show up as purple spots.

Figure 15

Iodine vapour The easiest way to make an iodine tank is to use a jar with a plastic coated lid in to which a

few crystals of iodine are placed. The iodine crystals can be mixed with a small amount of sand

covering the bottom. The developing plate has to be observed at intervals. Normally, within

minutes yellow to dark brown spots show up on a light brown background. However, compounds

like alcohols, acids and halides can give a negative stain (white spot on a light brown background)

initially. Sometimes it can take several hours before spots show up. A major drawback is that

aluminium TLC plates are attacked by the iodine to give a dark brown mess.


18/25

18

The retention factor R f

In TLC, the most important influence on the retention of compounds will be their polarity

relative to the stationary and mobile phases. The more polar a compound is, the more strongly it

will be retained by the stationary phase. Conversely, the less polar a compound is, the more likely

it will move with the mobile phase. In order to get a quantitative assessment of the behaviour of a

compound in TLC, we use R f , which is a relative scale of the distance the compound moved. R f isdefined as the distance the compound moved (from where it was spotted on the plate) divided by

the distance the solvent moved (from where the compound was spotted). Thus, the scale is

linear, with R f = 0 corresponding to a spot that does not move, and R f = 1 corresponding to a spot

that moved with the solvent all the way down the plate. If a compound moved 3/4 of the way

down the plate, it would have an R f of 0.75, and so on. R f is dimensionless and must be between

0 and 1, so if your numbers come out otherwise, you have made a mistake in your calculation,

figure 16 .

Solvent front

Baseline

a b

R f = =ab

Distance moved by product spot

Distance moved by solvent front

Figure 16

The distance the solvent travelled is measure from where the compound was spotted on

the plate to the solvent front recorded when the plate was finished eluting. The distance that the

compound travelled is measured from where it was spotted to where it ended up on the plate.

In TLC, spots with the same R f are probably the same compound. Thus, running a spot of

a known material and an unknown next to each other allows us to determine whether they have

the same identity or not. If an authentic sample is available, the known sample and the unknown

sample have to be double spotted on to the TLC plate, either side of the mixed spot. Any

difference in R f value between the two samples will show up for the mixed spot. The mixed spot


19/25

19

will look like a figure of eight, figure 17 . Even when the mixed spot does not show up as a

figure of eight the double spotting should be repeated using a different solvent system.

Figure 17

Two-dimensional thin layer chromatography

In two-dimensional thin layer chromatography the sample is analysed with two different

solvent systems and is an extremely sensitive method to analyse a sample. It is necessary to use

a square cut TLC plate. At one corner the sample is spotted approximately 1 cm from both edges.

After the initial development, the plate is left to dry and then turned 90 o, so that the eluted

components are at the bottom of the TLC plate. The plate is for the second time developed but

now with a different solvent system. The result is an increase in resolution.

Furthermore, if the TLC plate is left to dry for 15 30 minutes between the two runs and

then run with the same solvent system on visualisation the spots should appear on a diagonal line.

If this not happens than the components of the sample are not stable to the TLC conditions, figure

18 .

Figure 18


20/25

20

Another useful elution technique for the separation of closely running components is known

as multiple elutions and is used only with preparative thin layer chromatography. Unfortunately,

this topic falls outside the scope of this book.

Unusual shaped spots

A perfect developed TLC plate will show a clear round spot. In some instances a longstreak will be observed. This means that the sample is a very complex mixture. However, if the

plate is overloaded this can be the reason for the loss of resolution. Spotting the plate again with a

lower amount of compound will normally solve this problem. Another reason can be the low

solubility of the compound in the eluting system. Changing the eluting solvent solves this problem.

Compounds with strong acidic or alkaline functional groups can stick to the active sites of

the adsorbent. Adding a few drops of formic or acetic acid for acids or triethylamine or ammonia

for alkaline compounds can solve these problems.Careless spotting can damage the adsorbent and results in a U shaped spot. While the use

of polar solvents will result in doubling of the spots.

When the right TLC conditions are found for the optimal separations of the compounds the

column can be prepared for the separation.

TLC Visualisation Reagents

This is a selection of the many available TLC visualization reagents. Below each title is the

type of compounds or structure which can be detected with the specific reagent. When beginning

work with these reagents, acquired any MSDS (material safety data sheets) or look in the Merck

index to see if there are any extra precautions needed in safely using them.

Before spraying, plates should be well dried in the fume hood of residual solvents and

components. Amines and organic acids used in the mobile phases may adversely affect the

visualization reaction being attempted. If heating to remove these components is done,

consideration should be given so that loss of components or their decomposition is avoided (by

lowering the temperature or using a shorter time in the oven).

Always spray any of these reagents onto plates in a well ventilated hood while wearing

safety glasses. Also apply moderate amounts to the plate so it always appears dull and flat (if it

looks wet, you have sprayed too much). You can always overspray to enhance the detection.

When information about the results of using the visualization reagents was available, this

was put under each reagent as Treatment. If not give, the user will have to do a few experiments

to see what the results might be. Always remember to look under normal light and also short and

long wavelength ultraviolet light so as not to miss any possibilities.


21/25

21

Important Notes Please read carefully :

When using the older texts, if they suggest putting the components into benzene, always

substitute toluene since it is much less toxic. Likewise, as mentioned above, if the components are

added to pure water, these solutions cannot penetrate well into the polymer bound layers (hard

layers) now sold. Substitute 5% methanol in water or 5% ethanol in water as the makeup solvent

in these instances.

In past years various visualisation reagents were made up with benzidine . It had been

used for the detection of terpene aldehydes, flavonoids, carbohydrates and phenols. This reagent

is no longer recommended for use since it is now classified as a carcinogen. Other suitable

visualizers for the detection of the above compounds can be found in this and the newer

references listed above.

A few words about the supports the layers might be coated onto and the use of these

visualization reagents (and subsequent heating). Most of these reagents can be used on silica

gel, bonded silica gel, or cellulose plates with glass, plastic, or aluminium supports. The

exceptions are strong acid containing visualizers which cannot be used with aluminium supports

(which obviously would react with the aluminium to dissolve it). Also when heating, plastic

supports are limited to about 110C only. Plastic supported plates when heated at any

temperature should be placed on a metal or glass plate in an oven so they heat evenly. Some

ovens have a metal mesh or grid, which would heat the plastic support unevenly leading to

warping and lifting of the layer. If you have questions, please consult the manufacturer about the

suitability of any procedure you intend on using.

Some TLC plates when manufactured have an inorganic fluorescent indicator added to the

slurry poured to make the final plates. This type of indicator will not dissolve or elute off. They are

activated at 254 nm or 360 nm (see recommendations of the manufacturer for that type of plate).

When activated the fluorescent indicator will turn a green or white (depending on the

indicator added) and the compounds appear as dark spots or shadows against this background. If

viewing at other than the activation wavelength, the compounds might also have some

fluorescence of their own, so various colours against a dark background would be seen.


22/25

22

For enhancement of fluorescence spots more stable and greater intensity:

1% solution of paraffin oil in hexane can be used. Spray this solution evenly over the TLC

plate.

20% solution of triethanolamine in isopropanol.

10 g triethylamine filled up to 100 ml with dichloromethane.

Useful websites:

AcrosOrganics http://www.acros.be

Debayer http://www.debayer.com

Lancaster http://www.lancastersynthesis.com

Matrixent email: [email protected] , contact Shah Hemal

Sigma-Aldrich http://www.sigmaaldrich.com/Local/SA_Splash.html
http://www.acros.be/mailto:[email protected]://www.sigmaaldrich.com/Local/SA_Splash.htmlhttp://www.sigmaaldrich.com/Local/SA_Splash.htmlmailto:[email protected]://www.acros.be/


23/25

23

GENERAL REAGENTS

Glacial acetic acid, 99.7+%, ACS reagent, Aldrich 242853 [64-19-7]

Acetone, 99.5+%, ACS reagent, Aldrich 179124 [67-64-1]

Acetonitrile, anhydrous, 99.8%, Aldrich 271004 [75-05-8]

28% Ammonia, ACS reagent, Aldrich 320145 [1336-21-6] 1-Butanol, 99.4+%, ACS reagent, Aldrich 360465 [71-36-3]

Tert -Butanol (2-methyl-2-propanol), 99.5+%, HPLC grade, Aldrich 308250 [75-65-0]

Carbon tetrachloride, 99.9+%, HPLC grade, Aldrich 270652 [56-23-5]

Chloroform, 99.9+%, ACS HPLC grade, Aldrich 528722 [67-66-3]

Cyclohexane, anhydrous, 99.8%, Aldrich 227048 [110-82-7]

Diethyl ether, anhydrous, 99.7%, Aldrich 296082 [60-29-7]

Absolute ethanol, denaturated, Aldrich 443611 [64-17-5]Ethyl acetate, anhydrous, 99.8%, Aldrich 270989 [141-78-6]

Hydrochloric acid, 37%, ACS reagent, Aldrich 258148 [7647-01-0]

Isopropanol, anhydrous, 99.5%, Aldrich 278475 [67-63-0]

Methanol, 99.9%, ACS spectrophotometric grade, Aldrich 154903 [67-56-1]

1-Propanol, anhydrous, 99.7%, Aldrich 279544 [71-23-8]

Pyridine, anhydrous, 99.8%, Aldrich 270970 [110-86-1]

Sulphuric acid, 95 98%, ACS reagent, Aldrich 258105 [7664-93-9]


24/25

24

ACETIC ACID

IODINE CONTAINING COMPOUNDS

SPRAY SOLUTION :50% Acetic acid solution is prepared.

TREATMENT :

Dry the plates at 100 oC, after cooling spray with a small amount of reagent and irradiate some

minutes with unfiltered ultraviolet light. The compounds produce violet to brown spots.

CHEMICALS :

See general reagents.

REFERENCES :

Merck , 13 , 56.

COMMENTS :

For further enhancement spray with 10% acetic acid and irradiate again, spots will turn blue.

NOTES :


25/25

ACETIC ANHYDRIDE SULPHURIC ACID (LIEBERMANN BURCHARD REAGENT)

TRITERPENOID GLYCOSIDES, STEROIDS AND GALLIC ACID

SPRAY SOLUTION :This reagent has to be prepared fresh. 5 ml Acetic anhydride is placed in an ice bath, to this is

added 5 ml concentrated sulphuric acid. The mixture is added to 50 ml ice cold absolute ethanol.

For the detection of Gallic acid ethanol is omitted.

TREATMENT :

After the plate has been sprayed, it is heated for 10 minutes at 110 oC. The compounds produce

spots visible under ultraviolet light (360 nm).

CHEMICALS :

Acetic anhydride, 98+%, ACS reagent, Aldrich 242845 [108-24-7]

REFERENCES : Merck , 13 , 57.

COMMENTS :

Ethanol can be replaced by methanol or chloroform.

NOTES :

10-critical micelar concentration

Documents