Lab D: Identification of an Unknown using Thin Layer Chromatography Flashcards
Chromatography “Color Writing” Definition
Simple and economical method for separation of components in a mixture
identification of unknowns by comparison with known reference samples
- routinely used as a qualitative analytical tool
What does chromatography “color writing” help with? (4)
- Helps follow the progress of a chemical reaction.
- Helps determine the effectiveness of a purification (aka how pure a compound is)
- Helps determine how many components are in a mixture of compounds
- Helps determine the conditions for macroscopic separations (Column Chromatography)
What are 2 benefits of chromatography “color writing”?
- no restriction on sample type: organic, inorganic, biological, or medical
- high sensitivity: detection of μg amounts or less (10-6 g)
Stationary Phase:
1. What does it do?
2. What is it? (physical form)
3. How does it effect sample movement?
4. Analogous to…
5. specifically for TLC
- stays as it
- fine solid with lots of SURFACE AREA
- retains sample by SURFACE INTERACTION (based on polarity of sample)
- an obstacle course
- thin layer (250um) of silica gel (SiO2)
Mobile Phase:
1. What does it do?
2. What is it? (physical form)
3. How does it effect sample movement?
4. Analogous to…
5. specifically for TLC
- flows around stationary phase
- fluid (liquid or gas)
- helps move sample along
- “motivating force”
- a mixture of hexane (9mL) and ethyl acetate (1mL) <– for our experiment
what are 4 types of chromatographic methods?
- TLC (thin layer chromatography)
- LC (liquid chromatography): SP (silica gel or Alumina) + MP (a liquid)
- HPLC (high performance liquid chromatography): utilizes high pressure exerted by mechanical pumps to force the mobile phase through a very small diameter column packing that contains the stationary phase
- GC (gas chromatography): MP (a gas)
what are 2 examples of adsorbents for the stationary phase?
- silica gel (SiO2) - most commonly used; inexpensive stationary phase
- aluminum oxide (aka alumina): Al2O3
what are the results of interaction with ONLY stationary phase?
- HIGHLY POLAR molecules will interact strongly with the polar Si-OH bonds in the silica gel adsorbent –-> resulting in SLOW movement up the TLC plate
- WEAKLY POLAR molecules are held less tightly than the polar species on the silica gel adsorbent –-> resulting in QUICKER movement up the TLC plate
describe the relationship between silica gel’s (SiO2) chemical makeup and its polarity
- extended covalent network of tetrahedral Si atoms bridged by “O” atoms –> terminating in very POLAR SILANOL (Si-OH) groups –> creating very polar surface
- the presence of these OH groups results in the surface of silica gel to be highly polar
what is the composition of a TLC plate?
250μm silica gel layer impregnated with a fluorescent Indicator, on a plastic backing
what is the function of CaSO4 in TLC plates?
CaSO4 is added to help silica gel bind to a TLC plate
- Compounds which fluoresce (ZnS which adsorbs at 254 nm)
what were the 3 compounds used in this lab?
- fluorene
- 9 - hydroxyfluorene
- phenyl benzoate
what is the retention factor (Rf) equation?
AKA ratio of the front or ratio of distances
Rf = distance travelled by COMPOUND (or SOLUTE) / distance traveled by SOLVENT (or ELUENT)
describe the relation between polarity of compounds and their Rf values and the distance traveled on the TLC plate
more polar compounds = small Rf values = shorter distance traveled on the TLC plate
less polar compounds = large Rf values = longer the distance traveled on the TLC plate
describe the 4 steps f thin layer chromatography
- Application of Sample: Use a different TLC Capillary tube (open on both ends) to spot the standard solution of each solute.
- Development of Sample: This is when the separation
actually occurs inside the TLC Development Chamber - Visualization of Sample: The TLC plate will be viewed
under UV light - Interpretation of Results: Comparison of Retention factors
describe how to prepare and spot a TLC plate
- ALWAYS SPOT on the ROUGH SIDE of the TLC plate
(smooth side is plastic backing) - Draw the baseline lightly with pencil 1.5 cm from the bottom of the plate
- Gently and lightly draw 4 “x” marks on the baseline
- Use capillary tubes that are labelled as TLC capillary tube (and not MP capillary tubes) to draw up the solution from the vial onto the TLC plate
- (Be sure to spot lightly to avoid making a break in the silica gel coating on the plate that will hamper the separation
how far is the solvent front from the top of the TLC plate?
how far is the baseline from the bottom of the TLC plate?
solvent front approx 1cm from top
baseline 1.5cm from bottom
what is the ELUENT? and what is it for this experiment?
eluting solvent (the solvent used to run the TLC plate/what the plate is put in)
9mL hexanes: 1mL ethyl acetate
draw the schematic of the developing chamber to run a TLC plate
the baseline of the TLC plate is ABOVE or BELOW the solvent level in the beaker?
baseline ABOVE solvent level
describe how to visualize the spots on the TLC plates
- mark solvent front and allow solvents to evaporate from surface of TLC plate
- view results under UV light/chamber
- use a pencil to mark the observed bright PINK spots
what is a safety precaution when using the UV chamber?
do not look directly into the UV viewing chamber
- the wavelength is 254nm in the UV lamp
iodine visualization
Colorless Organic Compounds (alkanes, alcohols, ethers) can also be visualized by absorption of Iodine Vapor
- Yellowish-brown colored spots form from the reaction of substances with Iodine vapor
describe how to interpret TLC plate results
- Mark the solvent front (eyeball it to be approx. 1 cm from the top of the TLC plate), as soon as you remove the TLC plate from the beaker
- Circle gently with a pencil all observed spots (when
viewed under UV light) - determine RETENTION FACTORS (Rf) for each spot detected
- compare the Rf values of the 3 standards and the unknown to identify your unknown
do identical Rf values prove that two compounds are the same?
NO
Identical Rf values suggest, but do not prove
that compounds are the same
describe chromatography and how are they separated
a set of techniques used to separate organic compounds by using a solvent (aka mobile phase) to move the compounds over a solid (aka stationary phase)
the compounds are separated based on how strongly the compounds bind to the stationary phase via NONCOVALENT interactions vs how well the compounds DISSOLVE in the solvent
what 2 things does the distance that a compound travels up the TLC plate depend on?
the distance that the compound travels up the plate depends on
1. how strongly it binds to the silica gel VERSUS
2. how well it dissolves in the solvent
what is a pitfall to using Rf values in identifying compounds?
sometimes 2 different compounds can have the same Rf value
relation between polarity of SOLVENT and distance a compound will travel on TLC plate
general rule: a more POLAR solvent will move a compound FARTHER UP the TLC plate
why is it possible for us to use UV light to see the spots on the TLC plate?
because all 3 compounds used in this lab contains a BENZENE RING which absorbs UV light –> PINK spot under UV light
what are the safety hazards for hexanes and ethyl acetate?
both flammable and can cause irritation to the skin or eyes
since the ratio for the solvent is 9 : 1 for hexanes : ethyl acetate, is the solvent more polar or nonpolar?
NONPOLAR
- hexane is nonpolar; ethyl acetate is polar
what could happen if the baseline of the TLC plate is NOT above the solvent level?
the samples you spotted may be dissolved into the solvent
where do you dispose the TLC solvent mixture and the scrap TLC plates?
TLC solvent mixture –> C, H, O nonhalogenated waste container
scrap TLC plates –> biohazard waste boxes
acetone waste –> waste acetone rinsings
describe the relation between polarity of the solvent and distance travelled by compounds on TLC plate
the MORE POLAR the solvent, the FARTHER it will move the compounds up a TLC plate –> all Rf values for the compounds will increase
elution
the process of the compounds moving up the TLC plate
what draws up the developing solutions in the TLC capillary tubes?
capillary action
On TLC plate, what is the order of the 3 compounds from the BOTTOM of the plate? (phenyl benzoate, 9-hydroxyfluorene, fluorene)
most bottom (aka most polar)
1. 9-hydrozyfluorene
2. phenyl benzoate
3. fluorene
why are we able to see the 3 compounds/spots on the TLC plate through the UV chamber?
all three compounds used for this TLC experiment include a BENZENE ring, which ABSORBS UV radiation –> thus we can see it as PINK spots under UV light
If you spotted a compound too heavily on a TLC plate, how would the resulting spot for that compound appear, after the TLC plate has developed?
The resulting spot for that compound will be very large and will probably overlap with the other spots since spots become larger by DIFFUSION during development
for nonpolar compounds, what types of forces bind them to the silica gel?
only van der waals forces bind nonpolar compounds to the silica gel
for polar compounds, what types of forces bind them to the silica gel?
stronger dipole-dipole forces and/or hydrogen bonding
why do you want to keep the spots very small and spread apart from each other?
spots become larger by diffusion during development so they need to be far apart to keep clean separation and no overlapping of spots
why do you need to cover the beaker/developing chamber?
to prevent the eluent from evaporating and impurities (dust and airborne particles) from contaminating the chamber, and to saturate the developing chamber with solvent vapors
what is the importance of solvent vapors in the developing chamber?
prevents solvent evaporation from TLC plate to ensure consistend migration of the mobile phase
prevents uneven solvent fronts: without sufficient vapor, the solvent can evaporate unevenly as it moves up the plate, which can lead to streaking and distorted spots
helps maintain optimal solvent polarity, which leads to clearer spots and differences between compounds
what happens if the baseline of the TLC plate is BELOW the solvent level?
the spots will leach into the solvent and could dissolve directly into the solvent instead of traveling up the plate via capillary action –> loss of sample and poor separation of spots
what is filter paper used for in TLC?
Filter paper is used to saturate the chamber with solvent vapors by wicking solvent into the upper region of the chamber
what is a solvent stall and the results of it?
solvent stall: when the solvent front does not move as expected up the TLC plate
–> results in inaccurate or manipulated Rf measurements
what would happen if you change the solvent from 100% ethyl acetate to 1:1 ethyl acetate: hexane?
what would happen if you change the solvent from 100% hexanes to 1:1 ethyl acetate: hexane?
100% ethyl acetate: solvent is too polar and there is too high affinity for the mobile phase –> the two compounds are overlapping and at the top of the TLC plate
100% hexanes: solvent is too nonpolar and there is too low affinity for the mobile phase –> the two compounds are overlapping and at the bottom of the TLC plate
–> when it is a 1:1 ratio for the solvent, the polarity gives good separation of the two compounds (neither one is too up top or too at the bottom)
–> the more polar the solvent, the higher the compounds travel up the TLC plate
