Lab E - Extraction of Bixin Part 1

What is bixin?

A natural product that is isolated from the seeds of Bixa Orellana, commonly known as the annatto tree

T or F: Bixin, lycopene, and carotene belong to a group of highly colored organic molecules called carotenoids which are characterized by 11 double bonds in conjugation.

True

What are the three compounds which annatto extract contains?

Bixin, methylbixin, and norbixin

What is the difference in functional groups between bixin, norbixin, and methylbixin?

Bixin - carboxylic group on one side, methyl ester on the other

Methylbixin - both groups are methyl esters

Norbixin - both groups are carboxylic acids

Arrange bixin, norbixin, and methyl bixin in order of increasing polarity

Methyl bixin < Bixin < Norbixin

The frequency of light is ____ related to the wavelength

inverseley

What is the equation for color in organic compounds?

E = hv = hc/lambda

v = frequency

c = speed of light

lambda = wavelength of light

A compound that absorbs blue light will appear ___ and a compound that absorbs green light will appear ___

orange, red

The UV region of the magnetic spectrum, compared to visible and infrared light, has ____ frequency and ____ wavelength. It also has ____ energy

increasing, decreasing; increasing

What happens during absorption at UV and visible wavelengths?

promotion of electron from HOMO to LUMO, increase in energy

Extended conjugation reduces _____ gap and ____ wavelength

HOMO to LUMO; increased (allows them to absorb at longer wavelengths)

As the number of conjugated pi bonds increases, the energy difference between the ground and the excited state ____, which allows absorption to occur at ___.

In other words, more conjugation = absorption at ____ wavelengths

decreases, longer

longer

Red light at one end of the EM spectrum has ____ wavelength and ___ frequency; violet light at the other end of the spectrum has ____ wavelength and ____ frequency

high, low

low, high

Observed color is _____ to wavelength of absorbed color

complementary **know color wheel

Give a general overview of lab E

- Add 1 g of annatto seeds and 5 mL of 10% EtOH in DCM to the original beaker

- Stir for 5 min, then vacuum filter via buchner

- Filtrate transferred to 50 mL RB, solids transferred back to original filter

- Add an additional 5 mL of 10% EtOH in DCM and stir for 5 min, then vacuum filter via buchner again

- Solids transferred back to original beaker, filtrate transferred tot he 50 mL RB flask

- Repeat the 5mL addition of 10% EtOH in DCM a total of 4x to total 20 mL of solvent used

- Run 3 TLC plates to determine which solvent to use for column chromatography; save all these 3 TLC plates for final lab

- Place a dash on faintly yellow colored places and a circle around any location that is darker yellow/orange

- Filtrate in 50 mL RB is then rotovap'd and save the annatto extract for column chromatography

What is the purpose of using 3 different solvents to develop TLC plates of annatto extract?

To determine the best separation of the multiple components

What are the physical properties of n-hexane, et2o, tBME, water, DCM

densities:

0.66

0.71

0.74

1.00

1.33

bps:

69

35

55

100

40

What observations will you have for each of the solvents in TLC for lab E?

DCM - no separation

10% etoh in DCM - everything runs to the top

3% etoh/DCM - intermediate

How do you increase Rf values? Why does this work?

increase solvent polarity because the polar groups will compete with the compound for the polar OHs on the SiO2 plate

How do you decrease Rf values?

decrease solvent polarity

How does rotary evaporation work?

It applies vacuum to reduce the boiling point of the solvent to prevent toxic vapors from escaping (e..g: DCM)

Which solvent do you end up using in lab E and F?

3% etoh in dcm

What is each lane on your 3 TLC plates?

lane 1 - annatto extract

lane 2 - more concentrated spot of same extract

lane 3 - bixin standard

What are some important points to keep in mind during lab E's procedure?

- do not we the filter paper w/ water (solubility)

- change the filter paper after each extraction

- do not leave the vacuum on for too long when working with DCM

What are the waste disposal procedures for lab E?

annatto seeds go in biohazard

buchner funnel filter paper goes in biohazard

vacuum filter flask, buchner funnel, and 60 mL beaker are rinsed with solvent and emptied into CHO halogenated red can

used TLC capillary tubes go into red sharps container

Why do you do 4 extractions with 5mL instead of 1 with 20 mL?

To improve the % recovery of bixin