Lab D: Identification of an Unknown using Thin Layer Chromatography

Question 1

Chromatography “Color Writing” Definition

Answer 1

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

Question 2

What does chromatography “color writing” help with? (4)

Answer 2

• 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)

Question 3

What are 2 benefits of chromatography “color writing”?

Answer 3

• no restriction on sample type: organic, inorganic, biological, or medical
• high sensitivity: detection of μg amounts or less (10-6 g)

Question 4

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

Answer 4

1. stays as it
2. fine solid with lots of SURFACE AREA
3. retains sample by SURFACE INTERACTION (based on polarity of sample)
4. an obstacle course
5. thin layer (250um) of silica gel (SiO2)

Question 5

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

Answer 5

1. flows around stationary phase
2. fluid (liquid or gas)
3. helps move sample along
4. “motivating force”
5. a mixture of hexane (9mL) and ethyl acetate (1mL) <– for our experiment

Question 6

what are 4 types of chromatographic methods?

Answer 6

1. TLC (thin layer chromatography)
2. LC (liquid chromatography): SP (silica gel or Alumina) + MP (a liquid)
3. 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
4. GC (gas chromatography): MP (a gas)

Question 7

what are 2 examples of adsorbents for the stationary phase?

Answer 7

1. silica gel (SiO2) - most commonly used; inexpensive stationary phase
2. aluminum oxide (aka alumina): Al2O3

Question 8

what are the results of interaction with ONLY stationary phase?

Answer 8

• 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

Question 9

describe the relationship between silica gel’s (SiO2) chemical makeup and its polarity

Answer 9

• 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

Question 10

what is the composition of a TLC plate?

Answer 10

250μm silica gel layer impregnated with a fluorescent Indicator, on a plastic backing

Question 11

what is the function of CaSO4 in TLC plates?

Answer 11

CaSO4 is added to help silica gel bind to a TLC plate
- Compounds which fluoresce (ZnS which adsorbs at 254 nm)

Question 12

what were the 3 compounds used in this lab?

Answer 12

1. fluorene
2. 9 - hydroxyfluorene
3. phenyl benzoate

Question 13

what is the retention factor (Rf) equation?

Answer 13

AKA ratio of the front or ratio of distances

Rf = distance travelled by COMPOUND (or SOLUTE) / distance traveled by SOLVENT (or ELUENT)

Question 14

describe the relation between polarity of compounds and their Rf values and the distance traveled on the TLC plate

Answer 14

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

Question 15

describe the 4 steps f thin layer chromatography

Answer 15

1. Application of Sample: Use a different TLC Capillary tube (open on both ends) to spot the standard solution of each solute.
2. Development of Sample: This is when the separation
   actually occurs inside the TLC Development Chamber
3. Visualization of Sample: The TLC plate will be viewed
   under UV light
4. Interpretation of Results: Comparison of Retention factors

Question 16

describe how to prepare and spot a TLC plate

Answer 16

1. ALWAYS SPOT on the ROUGH SIDE of the TLC plate
   (smooth side is plastic backing)
2. Draw the baseline lightly with pencil 1.5 cm from the bottom of the plate
3. Gently and lightly draw 4 “x” marks on the baseline
4. 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

Question 17

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?

Answer 17

solvent front approx 1cm from top
baseline 1.5cm from bottom

Question 18

what is the ELUENT? and what is it for this experiment?

Answer 18

eluting solvent (the solvent used to run the TLC plate/what the plate is put in)

9mL hexanes: 1mL ethyl acetate

Question 19

draw the schematic of the developing chamber to run a TLC plate

Question 20

the baseline of the TLC plate is ABOVE or BELOW the solvent level in the beaker?

Answer 20

baseline ABOVE solvent level

Question 21

describe how to visualize the spots on the TLC plates

Answer 21

1. mark solvent front and allow solvents to evaporate from surface of TLC plate
2. view results under UV light/chamber
3. use a pencil to mark the observed bright PINK spots

Question 22

what is a safety precaution when using the UV chamber?

Answer 22

do not look directly into the UV viewing chamber

• the wavelength is 254nm in the UV lamp

Question 23

iodine visualization

Answer 23

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

Question 24

describe how to interpret TLC plate results

Answer 24

1. 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
2. Circle gently with a pencil all observed spots (when
   viewed under UV light)
3. determine RETENTION FACTORS (Rf) for each spot detected
4. compare the Rf values of the 3 standards and the unknown to identify your unknown

Question 25

do identical Rf values prove that two compounds are the same?

Answer 25

NO Identical Rf values suggest, but do not prove that compounds are the same

Question 26

describe chromatography and how are they separated

Answer 26

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

Question 27

what 2 things does the distance that a compound travels up the TLC plate depend on?

Answer 27

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

Question 28

what is a pitfall to using Rf values in identifying compounds?

Answer 28

sometimes 2 different compounds can have the same Rf value

Question 29

relation between polarity of SOLVENT and distance a compound will travel on TLC plate

Answer 29

general rule: a more POLAR solvent will move a compound FARTHER UP the TLC plate

Question 30

why is it possible for us to use UV light to see the spots on the TLC plate?

Answer 30

because all 3 compounds used in this lab contains a BENZENE RING which absorbs UV light --> PINK spot under UV light

Question 31

what are the safety hazards for hexanes and ethyl acetate?

Answer 31

both flammable and can cause irritation to the skin or eyes

Question 32

since the ratio for the solvent is 9 : 1 for hexanes : ethyl acetate, is the solvent more polar or nonpolar?

Answer 32

NONPOLAR - hexane is nonpolar; ethyl acetate is polar

Question 33

what could happen if the baseline of the TLC plate is NOT above the solvent level?

Answer 33

the samples you spotted may be dissolved into the solvent

Question 34

where do you dispose the TLC solvent mixture and the scrap TLC plates?

Answer 34

TLC solvent mixture --> C, H, O nonhalogenated waste container scrap TLC plates --> biohazard waste boxes acetone waste --> waste acetone rinsings

Question 35

describe the relation between polarity of the solvent and distance travelled by compounds on TLC plate

Answer 35

the MORE POLAR the solvent, the FARTHER it will move the compounds up a TLC plate --> all Rf values for the compounds will increase

Question 36

elution

Answer 36

the process of the compounds moving up the TLC plate

Question 37

what draws up the developing solutions in the TLC capillary tubes?

Answer 37

capillary action

Question 38

On TLC plate, what is the order of the 3 compounds from the BOTTOM of the plate? (phenyl benzoate, 9-hydroxyfluorene, fluorene)

Answer 38

most bottom (aka most polar) 1. 9-hydrozyfluorene 2. phenyl benzoate 3. fluorene

Question 39

why are we able to see the 3 compounds/spots on the TLC plate through the UV chamber?

Answer 39

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

Question 40

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?

Answer 40

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

Question 41

for nonpolar compounds, what types of forces bind them to the silica gel?

Answer 41

only van der waals forces bind nonpolar compounds to the silica gel

Question 42

for polar compounds, what types of forces bind them to the silica gel?

Answer 42

stronger dipole-dipole forces and/or hydrogen bonding

Question 43

why do you want to keep the spots very small and spread apart from each other?

Answer 43

spots become larger by diffusion during development so they need to be far apart to keep clean separation and no overlapping of spots

Question 44

why do you need to cover the beaker/developing chamber?

Answer 44

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

Question 45

what is the importance of solvent vapors in the developing chamber?

Answer 45

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

Question 46

what happens if the baseline of the TLC plate is BELOW the solvent level?

Answer 46

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

Question 47

what is filter paper used for in TLC?

Answer 47

Filter paper is used to saturate the chamber with solvent vapors by wicking solvent into the upper region of the chamber

Question 48

what is a solvent stall and the results of it?

Answer 48

solvent stall: when the solvent front does not move as expected up the TLC plate --> results in inaccurate or manipulated Rf measurements

Question 49

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?

Answer 49

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