TLC

Sketch of apparatus
Fig 8.9 and Fig 1 from Handout

Uses for this TLC experiment (know more from book)
a way to tell if compound is pure
a way to see what compounds are in a sample by comparison
monitor progress of a reaction
determine identity of two substances
determine the appropriate conditions for a column chromatography separation
to monitor column chromatography

analytic technique vs. purifying technique
Melting Point and TLC are analytic techniques
Recrystallization and Distillation are purifying techniques

TLC is a commonly used tool to
observe separation of biomolecules in biochemistry
identifying illicit drugs or dyes in fibers in forensic chemistry
analyzing components of medicinal plants

General Chromatography Technique
based on components of a mixture being differentiated by exposure by two competing phases

Tools for the Experiment
1. TLC plates
2. Capillary tubes
3. Developing chamber
4. Standards
5. Reference solution
6. Unknown
7. Solvents

TLC plates
plastic plates covered with an adsorbent material (stationary phase)
The material we will be using is silica but alumina is another common adsorbent

For TLC, a binder like calcium sulfate added to
hold adsorbents on the plate

You must know what happens if you silica for very nonpolar compounds and if you use alumina for very polar compounds
Silica: Samples travel all the way too the top of the plate or will travel with the solvent front

Capillary tubes (open at both ends)
called spotters

Developing chamber
a bottle with a top with filter paper inside

Standards
Acetaminophen, Aspirin, Caffeine (solids)

Reference solution
a mixture of acetaminophen, aspirin, and caffeine

Unknowns
anacin, excedrin, tylenol

Solvents
methylene chloride for dissolving unknowns
– eluent (mobile phase)
ethyl acetate

What are typical characteristics for solvents used as eluents? Why?
having low boiling points that allow them to be easily evaporated and low viscosities that allow them to migrate rapidly

You want to know if you want high/low bp
May have to choose something based on bp

Solvent rises up the TLC plate by capillary action (rising up; opposite gravity) which is the ability of a
liquid filling the spaces b/w the solid particles in opposition to the gravitational force

May be given list of eluents and must choose based on characterization i.e must be solube

Extra Considerations for TLC eluent
Mixture to be separated must be soluble in eluent
– if not the spotted sample will stay on the spotting line
Intermolecular attractions between eluent and compounds determine
– solubility of compounds in mobile phase
– like dissolves like

Using eluent with too high a polarity with respect to samples
all spots will move to top of TLC plate ; more polar an eluent the faster a compound moves

Using eluent with too low a polarity with respect to samples
all spots will stay towards the bottom of the TLC plate;

What do polar compounds require
Require polar eluents to overcome attractions to the adsorbent

If you use a mixture of solvent, the pair of solvents MUST be
miscible

Stages of Experiment
Spotting
Developing
Visualizing

Spotting Stage (Preparation)
Draw a line using a pencil 1 cm from the bottom of the TLC plate (spotting line)
Draw a line 5 mm from the top of the plate (solvent front).
Make sure that the lines are drawn lightly. Do not flake off adsorbent.

Why shouldn’t you flake off adsorbent?
If flaked a gap might form that may stop the flow of the eluent or cause eluent to run crookedly

Why is pencil always used to mark a plate and why is ink never used to mark the plate?
It will chromatograph like any other organic compound and give flawed results; Because the graphite (carbon) is inert

Spotting
Use Capillary tubes to take sample
Then lightly touch it to the TLC plate to apply a spot of sample onto the spotting line (1 cm line).
Be careful again not to flake off adsorbent (spotter has sharp edges)
Label spots with a pencil

To avoid streaking spots
Try to make spots small and compact
– by touching lightly on the TLC plate
Also allow the solvent to dry completely between application to the same spot. Otherwise the spot will become too large
Sample should not be too concentrated

What problem could streaking spots lead to?
Streaking may lead to the overlap of two or more compounds, thus skewing results

If a sample contains many components then spots may just run together and
appear as a streak

Additional points: Don’t put spots too
close together on spotting line (1 cm between each spot)
– they may bleed into each other
close to the edge
– leads to inaccurate RFs ( not enough solvent or adsorbent surrounding spot – unequal forces for each spot)

When it is not balanced and it goes up crooked how do you know results are accurate?
you don’t

Note: the sample needs to be concentrated enough-otherwise
no spots will be visible during visualization stage

Developing Stage
Place TLC plate in a developing chamber that has 4mm high level of ethyl acetate. Make sure solvent level is below spotting line so that solvent does not dissolve samples on the spotting line

Why is it suggested that one uses tweezers to place TLC plate in developing chamber? !!!
Don’t want surface of hand touching the top of TLC plate (we will handle by holding by sides)
gloves still have chemicals/oils you don’t want to get on TLC plates

The RF for a compound is a constant from one experiment to the next providing the following are constant:
– solvent system
– adsorbent (silica or aluminum)
– thickness of adsorbent (difficult to control)
– amount of material spotted (difficult to control)
– temperature (difficult to control)
Keeping the last three constant is difficult so relative RFs are used

What can you do if two different compounds have the same RF in one eluent?!!!!
changing the stationary or mobile phase will usually effect their separation

Two samples of the same compounds will
always have the same relative RF in every eluent

You should know the relative polarity of functional groups
alkane, alkyl halides, alkenes, dienes, aromatic hydrocarbons, ethers, esters, ketones, aldehydes, amines, alcohols, phenols, carboxylic acids, sulfonic acids.

Column chromatography (purifying technique)
used for separation and purification of both solids and liquids when carrying out microscale experiments

What is the difference of how column chromatography works vs. thin-layer chromatography? Where do you put solvent?
column: mixture loaded on top (solvent poured on top) TLC: mixture loaded on bottom : solvent is reservoir on the bottom

Separation begins with a relatively low polarity solvent. Why?
to allow compounds to adsorb to stationary phases
then polarity of solvent is slowly increased to desorb compounds and allow compounds to move with the mobile phase

What may occur if polarity is changed too suddenly?
cause heat evolution as the alumina or silica gel adsorbs the new solvent

Stages
Packing column
Adding sample
Eluting column

What is the problem of collecting very large fractions?
column chromatography is a length process; may get more than one compound in a particular fraction

Isolating Separated Compounds; What is TLC’s role in this stage
Spotting some of the starting material or the product on the TLC plate as a standard will help in the identification

Limitation for TLC and column chromatography-
cannot be used on volatile compounds having boiling points below 150 degrees C

Experiment- 1st TLC plate
spot 3 standards
-develop with ethyl acetate
-visualize using UV
– trace spots with pencil and take measurements

Experiment- 2nd TLC plate
spot unknown (note #) and reference solution
– develop using ethyl acetate
– visualize using UV
– trace spots with pencil and take measurements

From 1st TLC plate, you should be able to identify the spots of the reference solution on the 2nd plate
From reference solution you should be able to identify your unknown using the information on your handout

Why should you make sure the bottom of the TLC plate is level with bottom of developing chamber
if not then solvent will rise up unevenly on TLC plate

What effect might moving or jostling the development chamber during the developing stage have on how it rises?evenly? unevenly?!!!
the eluent will rise up unevenly

Make sure top is placed back on so
solvent does not evaporate

Evaporation of solvent during this stage causes the !!!
development of the TLC plates to be messy and ineffective !!!

Before developing a new TLC plate
add solvent to correct level

Make sure solvent does not rise up past solvent front (5 mm line) Why?
if it does, spots in visualization stage will be difficult to evaluate; RF would be too high

Make sure filter paper is in the dev. chamber
to saturate dev. chamber w/ solvent vapors

Avoid leaning plate against the filter paper because!!
Eluent on the filter paper can absorbed by adsorbent and interfere with ascending eluent.
Put filter paper on one side plate on another

Where should the solvent come from?
bottom

This extra eluent may prevent the compounds travelling from
moving up the plate in a straight line

Leaning
have wetness come from sides since filter paper is wet

Pay close attention to apparatus: identify and can be drawn wrong so you should known what goes where

Visualizing stage
Dry the TLC plate
Since the solvents and solutions you are using are colorless, you need to use visualization technique

Two common ways to visualize plates
1. UV light-used for conjugated compounds (don’t shine on eyes; shine on plate)
– Shine on TLC spots and you will see purple spots, take pencil and trace the spots. Any place where there isn’t spots it would appear florescent green.
2. I2 vapor – for most compounds (usually with double bonds)

Other methods
stains, charring are associated with the presence of a particular functional group.

During development, samples travel along with solvent up the TLC plat. Each pure compound shows up as
one dev. spot directly above where it was spotted on the spotting line.

Does a single spot on a TLC spot guarantee a single substance. Why or why not?!!!
No ; pure’ must see 1 spot but if its 1 spot it is not necessary pure.

The more polar a compound, the ____ farther it travels up a TLC plate
less

Retardation Factor (RF)
distance sample traveled/distance solvent traveled

The more polar the _________the RF
Smaller

TLC is typically used to determine the correct adsorbent and solvent for column chromatography. Why is that possible?
TLC and column chromatography use some absorbents or TLC and column chromatography both use silica and alumina

TLC can be used to monitor column chromatography. What specifically does the TLC do to achieve this purpose?
Analyze fractions of column chromatography

What advantage is obtained when a TLC is used to determine the number of components in a mixture?
It is sensitive, fast simple, and inexpensive analytical technique

Which use of TLC allows for one to determine the optimum time to halt a reaction?
Monitoring progress of a reaction

You spot a sample that contains a mixture of 3 compounds of varying polarities on a TLC plate and develop it. Assuming that all other factors (adsorbent, technique) were appropriate, what kind of solvent would cause all the spots to congregate in the following manner when visualized under UV light?
spots congregated at top of plate
A solvent with too high a polarity in respect to the mixtures
spots congregated at bottom of plate
a solvent with too low a polarity in respect to the mixtures

You spot two very polar compounds on a TLC plate and develop it in order to distinguish the two compounds. Which adsorbent would most likely produce the result where all spots stay at the bottom of the plate when visualized under UV light if all other factors (solvent, technique) are accounted for?
A solvent that is too low in polarity

During the development stage of the TLC experiment, a student accidentally left the top off the developing chamber and the eluent evaporated. Why is this a problem?
Because the developing chamber wouldn’t be saturated with vapors and the TLC plate would become ineffective and messy

A student develops a TLC plate and realizes during the visualization stage that the eluent had dissolved the samples during the development stage. How could the student have prevented this situation?
By making sure the eluent is below the spotting line

During a TLC experiment, a student used only 0.5 mL of solvent to dissolve a solid unknown instead of the necessary 1.5 mL before spotting the unknown on the TLC plate. What is the likely observation on the developed plate when it is visualized?
There would be too much sample during spotting which would cause poor separation of the compound and it would be smeared

You take a TLC of a compound and you find that the Rf of the compound is much higher than expected. What action would have produced the above result?
solvent rose up past solvent front

Why is it important that solvents for TLC (eluents) have low boiling points?
So they can saturate the developing chamber with their vapors effectively

Why is it important that solvents for TLC (eluents) have low viscosities?
Allows them to migrate rapidly

If 15 grams of an organic solid is dissolved in 80 mL of water, how many grams remain in the water after extraction with 50 mL of a solvent which has a partition (distribution) coefficient with water of 8? Show calculations.

What advantage is obtained when a TLC is used to determine the number of components in a mixture?
Aids in planning further analytical and separation steps

What is the reason a TLC would be taken after one carries out a distillation?
To examine effectiveness of purification

One of the uses of TLC is to monitor the progress of a reaction. What kind of info does TLC provide in this instance?
When to halt rxn and the effect of changing temp, concentrations and solvent w/o having to isolate product

Why can TLC be use to determine the appropriate conditions for a column chromatographic separation?
Adsorbents used for TLC same as adsorbents for column chromatography

One of the uses of TLC is to determine the number of components in a mixtures. What does this help in achieving?
Aids in planning further analytical and separation step

How do you ensure that you make your spots on the spotting line of the TLC plate small and compact?
By touching the capillary tube lightly on the TLC

A student accidentally lets the eluent run all the way to the top of the TLC plate…
The spot in the visualization phase will be difficult to evaluate or the Rf will be too high

A student develops a TLC plate w/o placing filter paper in the developing chamber, why would this omission hinder the rising of the solvent up the TLC plate?
Developing chamber would not be saturated with solvent vapors

Why is it important to remove the tlc plate from the developing chamber as soon as the solvent reaches the solvent front?
Rf will be too high or spots at visualization stage will be difficult to evaluate

List 2 precautions you take while spotting
Spots must be small and compact and sample must not be too concentrated

Why should one not stick the capillary tube directly into the reagent bottle
To avoid contamination

What precaution does one have to take when marking a tlc plate?
Not to flake off the absorbent

What might happen if you use silica as your absorbent to distinguish between a set of very polar solutes
Samples will not migrate very far from spotting line

You can use 2 different visualization methods to visualize colorless substances, list which method is used for what kind of colorless compound
Uv- conjugated and iodine for most compounds

What is important that the top of the chromatography jar be kept on while developing a tlc plate?
So solvent does not evaporate

Why is it important not to dissolve the unknowns in more than 1 ml of methylene chloride?
Samples will be too dilute to be spotted

Spotting precautions:
Make sure lines are drawn lightly on plate so as to not flake off adsorbent
If flaked, the solvent will not rise up the plate effectively
Lightly touch capillary tubes to the plate when spotting
Try to avoid spots from being smeary
Try to make spots small and compact
Sample should not be too concentrated

Developing precautions:
Make sure solvent level is below spotting line
So that the solvent does not dissolve the samples on the spotting line
Make sure top is placed back on developing chamber
So that solvent does not evaporate – evaporation of solvent during this stage causes the developing of the plates to be messy and ineffective (before developing a new plate, add solvent to correct level)
Make sure solvent does not rise up past solvent front
If it does, spots in visualization stage will be difficult to evaluate (RF would be to high)
Make sure filter paper is in developing chamber
The reason is to saturate developing chamber with solvent vapors

Visualizing precautions:
Since the solvents and solutions you are using are colorless, you need to use visualization techniques:
UV light – used for conjugated compounds
I2 vapor – for most compounds (brown spot)
During development, samples travel along with solvent up a TLC plate
Each pure compound shows up as one developed spot directly above where it was spotted on the spotting line.
More polar a compound, less is travels up a TLC plate

Why should bores of capillaries be small
To avoid smearing of spots

What are typical characteristics for solvents used as eluents
Low boiling points that allow them to be easily evaporated and low viscosities that allow them to migrate rapidly

Characteristics of solvents that allow them to migrate rapidly
Low boiling point, low viscosity, high fluidity

Adsorbent materials and specific uses (what kinds of adsorbents would be better for what kind of substances)
Alumina- when anhydrous, is more active – it will adsorb substances more strong; adsorbent of choice when the separation involves relatively nonpolar substances such as hydrocarbons, alkyl halides, ethers, aldehydes, and ketones
Silica- less active- used to separate more polar substrates such as alcohols, carboxylic acids, and amines

Table 8.2 (order of solute migration)
Fastest→slowest:
Alkanes > alkyl halides > alkenes > dienes > aromatic hydrocarbons > aromatic halides > ethers > esters > ketones > aldehydes > amines > alcohols > phenols> carboxylic acids > sulfonic acids

Uses of TLC and how it is used – all 6
To determine the number of components in a mixture
To determine the identity of two substances
If two substances spotted on the TLC plate give spots in identical locations, they may be identical. If the spots are not the same, the substances cannot be the same
To monitor the progress of a reaction
By sampling a reaction from time to time, you can watch reactants disappear and reappear using TLC. The optimum time to talk the reaction can be determined, and the effect of changing such variables as temperature, concentrations, and solvents can be followed without having to isolate the product
To determine the effectiveness of a purification
To determine the appropriate conditions for column chromatography
Column chromatography is used to separate and purify larger quantities of material. The correct adsorbent and solvent used to carryout the chromatography can be determined rapidly by TLC (which would be unsatisfactory for the large quantities of material)
To monitor column chromatography
As the column chromatography is carried out and the solvent is collected in small flasks, the fractions can be analyzed by TLC to determine which ones have the desired components of mixture

Effects of using too polar or too nonpolar a solvent in respect to sample
Too polar- all spots will ove to top of TLC plate
Too nonpolar- all spots will stay towards the bottom of the TLC plate