Lab J: Preparation of Bixin (TLC) Flashcards
TLC is a technique that can ___ and ___
TLC can be used to
1. monitor reactions
2. evaluate the purity of samples
what is bixin?
bixin is a natural product that is isolated from the seeds of Bixa Orellana (aka annatto tree)
- bixin extract is a carotenoid and is orange-red in color
carotenoids definition
naturally occurring pigments
the annatto extract contains ___ (3 things)
bixin
norbixin
methyl bixin
compare the structural and polarity differences between bixin, norbixin, and methyl bixin
bixin: has a COOH group on one side + methyl ester group on the other side
methyl bixin: both groups are methyl esters
norbixin: both groups are COOHs (carboxylic acids)
–> these differences in functional groups results in differences in polarities which allows us to separate them by column chromatography
–> highest polarity
1. norbixin
2. bixin
3. methyl bixin
(COOH is more polar than an ester group due to the presence of hydrogen bonding)
what are the 3 different TLC solvents used in this lab?
- dichloromethane (DCM)
- 3% ethanol in DCM
- 10% ethanol in DCM
–> trying to determine the optimal solvent system that you will use for column chromatography
how do you determine which is the optimal solvent system to use for column chromatography using TLC?
looking for a solvent that…
1. gives you good separation of the multiple components
2. the bixin moves sufficiently off the baseline so that you can isolate it in column chromatography later on
what are 2 examples of carotenoids besides bixin that are mentioned?
- beta-carotene - orange color
- lycopene - red color
what do beta-carotene, lycopene, bixin and its derivatives all have in common?
they all have 11 double bonds in conjugation
relation between the TLC Rf value of bixin and the speed at which the compound will move off the column during column chromatography
if the Rf value of bixin is too HIGH –> compound will move off the column very QUICKLY which will not allow separation to occur
if the Rf value of bixin is too LOW –> compound will move off the column too SLOWLY (difficult to get the compound to elute off the column)
what is the optimal distance that bixin should move off the baseline in TLC such that it will have optimal separation in column chromatography?
In the optimal separation, the bixin should move off the baseline (Rf
between 0.25 to 0.5) and show significant distance from all other compounds
what are 2 safety hazards/classifications of DCM (dichloromethane)
carcinogenic compound (causes cancer)
flammable (boiling point of 39.6C)
–> need to keep DCM covered at all times and keep fume hoods closed when you aren’t using them
what is a safety precaution for this lab?
DOUBLE GLOVING! - always when using DCM
what is a real world application of bixin
bixin is a primary ingredient used in the food coloring industry
what should you keep in mind while doing the vacuum filtrations in regards to the filter paper?
- use a NEW filter paper each time
- do NOT wet the filter paper with water
why do we not wet the filter paper for vacuum filtration with water?
after doing the vacuum filtration, you will end up with a phase separation (organic phase with the 10% DCM and the aqueous phase with water)
vacuum filtration is filtering what out?
the annatto seeds are left on the filter paper and the annatto extract is being collected in the filtrate flask underneath the vacuum filtration
why do we repeat vacuum filtration 4 times?
because doing several smaller extractions compared to doing one larger extraction gives you better yield for the desired product (which in this case is the annatto extract)
all 3 TLC plates have the same 3 lanes which are…
lane 1 (from the left): 1 spot of annatto extract
lane 2: 2 spots of annatto extract
lane 3: bixin standard (from TA) - the reference
what is the final color of the annatto extract (after rotary evaporation?
deep red, brick color
–> coated around the walls of the RB flask
describe what the 3 developed TLC plates look like afterwards
DCM (the most nonpolar - relatively)
- only methyl bixin moves up the TLC plate (methyl bixin is the most nonpolar –> has no affinity to the silica gel –> travels highest)
- no separation of bixin at all
10% EtOH in DCM (most polar solvent)
- bixin does travel up the plate, but gets too close to the solvent front (so slightly better than pure DCM but still not clearly separated)
- methyl bixin is even further up from bixin –> no clear separation between the two
3% EtOH in DCM (middle one): there are 3
- bixin is the middle spot and is clearly separated from both methyl bixin and norbixin
- bixin travelled approx. to the middle of the TLC plate
what is the chosen solvent for column chromatography?
3% EtOH in DCM
- since bixin shows clear separation from both norbixin (at the bottom) and methyl bixin (at the top of the TLC plate)
compare the differences in solvent polarity
least polar
DCM
3% EtOH in DCM
10% EtOH in DCM
most polar solvent
–> as you increase the polarity of solvent, the spotted compounds traveled LARGER distances up the TLC plate –> HIGHER Rf values
compare how norbixin, bixin, and methyl bixin should travel up the TLC plates
norbixin (most polar) –> travels the least up the plate (essentially does not move at all)
bixin (intermediate polarity) –> the middle spot
methyl bixin (most nonpolar) –> travels the most up the plate (since it has low affinity to the polar stationary phase of silica gel)
describe the relationship between the polarity of the solvent and how far the compounds typically travel
as you increase the polarity of solvent, the spotted compounds typically travel LARGER distances up the TLC plate –> HIGHER Rf values
what are 3 main procedures of this lab?
- vacuum filtration x4 (extraction of annatto extract)
- TLC with 3 different developing solvents (DCM, 3% EtOH in DCM, 10% EtOH in DCM –> 3% EtOH in DCM was the chosen one for column chromatography)
- rotary evaporation (to isolate the annatto extract from the 10% DCM solvent used in vacuum filtration)
why was a rotary evaporation used?
rotary evaporation was used to remove the initial 10% DCM solvent that we used for vacuum filtration (extraction) of the annatto extract and to leave behind only the annatto extract in the RB flask
–> aka isolate the annatto extract by removing the solvent it was initially in (10% DCM)
what is the color of the annatto extract in 10% DCM solvent when doing the TLC plates
dark orange/red-ish
waste disposal:
TLC capillary tubes –>
TLC developing solvents –>
acetone rinsings waste –>
TLC capillary tubes –> red SHARPS container
TLC developing solvents –> “CHO halogenated and acetone rinsings” container
acetone rinsings waste –> “CHO halogenated and acetone rinsings” container
what is the energy equation ?
E = hv = hc/lambda
v = frequency of light (Hz, s^-1)
c = speed of light = 3x10^8 m/s
lamdba = wavelength of light
h = plank’s constant
what is the order of the visible light spectrum on the electromagnetic spectrum from lowest frequency/longest wavelength to highest frequency/shortest wavelength?
(700nm)
red
orange
yellow green
blue
violet
(400nm)
what is the relationship between wavelength and frequency and energy?
wavelength and frequency are inversely proportional to each other
frequency and energy are directly proportional to each other
what is the order of the different waves from order of longest wavelength/lowest frequency to shortest wavelength/highest frequency?
(longest wavelength)
radio, TV waves
microwaves
infrared
visible
ultraviolet
x-rays
gamma rays
(shortest wavelength)
what does the absorption at UV and visible wavelengths indicate?
the promotion of electrons from HOMO to LUMO (excited state)
what is the relation between the number of conjugated pi-bonds (double bonds) and absorption?
the number of conjugated pi-bonds increase –> the energy difference between HOMO and LUMO (aka the ground state and the excited state) DECREASE –> frequency gets lowered –> they absorb at LONGER wavelengths
–> increase in conjugation = increase in wavelength of absorption
at what wavelengths does an alkene with 2 double bonds, 3, and 6 absorb light at?
2 double bonds (conjugated) - 217nm
3 double bonds - 268nm
6 double bonds - 364nm
what is the relation between observed color and the color of the wavelength absorbed?
the observed color that we see is COMPLEMENTARY to the color of the wavelength that was absorbed
ie. if the color absorbed was 430nm (violet), then the color we see is yellow
describe the color wheel and the complementary colors
orange - blue
yellow - violet
green - red
what is the stationary phase and mobile phase for this lab’s specific TLC?
stationary phase: thin layer of SILICA GEL SiO2 (250um)
mobile phase:
1. DCM
2. 3% EtOH in DCM
3. 10% EtOH in DCM
what are the 4 types of chromatographic methods?
- TLC - thin layer chromatography
- LC - liquid chromatography
- HPLC - high performance liquid chromatography
- GC - gas chromatography
what causes silica gel to be so polar?
SiO2 structure is an extended covalent network of tetrahedral Si atoms connected by O atoms and the surface terminates in Si-OH groups –> very polar surface
what is the function of CaSO4 and ZnS in silica gel?
CaSO4 - helps the silica gel bind to the TLC plate
ZnS - the fluorescent marker/indicator impregnated in the silica gel
what are 2 absorbents commonly used for stationary phase of TLC?
- silica gel SiO2
- aluminum oxide (aka alumina) Al2O3
what do you need to keep in mind when doing the vacuum filtration (extraction 4x) on the annatto seeds
- do not wet the filter paper with water
- change the filter paper between each vacuum filtration
- when working with DCM, do not leave the vacuum on for too long
total volume of 10% EtOH in DCM used for extraction: 20mL (5mL each time)
what to do if your gloves become damaged, grossly soiled or contaminated? (3 steps)
- remove the gloves
- wash hands for 20 seconds with soap and warm water
- replace with new pairs of gloves
waste disposal procedure
annatto seeds (waste) –>
buchner funnel filter paper –>
vacuum filer flask, buchnel funnel and 50mnL beaker –>
TLC capillary tubes –>
annatto seeds (waste) –> biohazard waste container
buchner funnel filter paper –> biohazard box
vacuum filter flask, buchnel funnel and 50mnL beaker –> rinse with acetone and pour into “CHO halogenated and acetone rinsings” waste container
TLC capillary tubes –> red SHARPS container
