(Final) Isolation of Chlorophyll And Carotenoid Pigments From Spinach

In this video, we will demonstrate how to isolate Pigments From Spinach. You will need Frozen Spinach, Magnesium Sulfate,
Sand, and a Mortar and Pestle. Weight about 3g of Frozen Spinach, and 3g
of Magnesium Sulfate. Add the Spinach, Magnesium Sulfate, and a
pinch of Sand to the mortar. Crush the mixture for about 5 minutes using
the pestle. Make sure to wear gloves and work inside the
fume hood. You will also need 5mL of Acetone, 4mL of
Petroleum Ether, and 1mL of water. Next, weight around 3g of the spinach mixture,
and carefully add it to a test tube. You can use a spatula or make a funnel from
a weighting paper. Add 5mL of Acetone to the test tube, and stopper
with a stopper. Shake for 5 minutes. Be sure to hold your thumb over the stopper
so it can’t pop out Remove the stopper occasionally to remove
the pressure inside the test tube. Use the pipet to transfer as much of the liquid
as possible into a clean small test tube. The pigment will be in the acetone. Add 4mL of the Petroleum Ether to the new
test tube. Stopper and shake for a minute. Then add 1mL of water, stopper, and shake
for another minute. The stopper tends to pop out at this step,
so hold it tightly with your thumb while shaking. Occasionally remove the stopper to release
pressure. Next, centrifuge for about 1 minute to separate
the layer. Remember to balance the centrifuge with anothertest tube of equal mass or volume on the opposite of your test tube. After the centrifuge stop completely, carefully
remove your test tube. You could see the layer. The petroleum and the pigment are in the top
layers. In Part 2 we will demonstrate how to set up
a drying column. You will need Sand, a Pipet, Sodium Sulfate,
Cotton and a Ring Stand. Place a small piece of cotton at the end of
the pipet. Use a paper clip to push it to the bottom. Next, add about a quarter inch of sand. Make sure to keep the sand level. Followed by 1 inch of Sodium Sulfate. Put a small test tube under the drying column. Carefully pipet the green petroleum ether
layer (top layer) into the top of the drying column and let it run through the Sodium
Sulfate into the collection tube. Cover the collection tube with parafilm and
leave it in the drawer over the weekend to dry Part 3 we will be separating the pigments
using column chromatography. You will need sand, about 5 mL each of ethanol,
petroleum ether, 70/30 petroleum ether and about 3 mL Alumina as well as the dried pigment
from part 2. You will need to rehydrate your pigment using
about 0.5mL of Petroleum Ether. Prepare the column using a funnel, a chromatography
column, and stopcock from the microscale kit. Transfer the alumina into a flask. Add Petroleum Ether and mix. In order to successfully prepare your alumina
in the column, pour it into your column while mixing so the solid will not settle to the
bottom of the flask. Lightly tap the column with a pencil to help
the alumina settle and avoid crack. If the collection is slow, you can pipet the excess
Petroleum Ether from the column, which can be reuse to mix the alumina in the flask. Be careful not to pipet the settle alumina. Repeat the process until there is about 1
inch of alumina. Add a quarter inch of sand into the column,
as in part 2, make sure the sand is level. Once the remaining Petroleum Ether is nearly
absorbed into the sand, add half of the green pigment into the column. Make sure the column does not get dry. Once the green solution has seeped into the
alumina, add more Petroleum Ether. If the yellow band begin to separate from
the green band, continue to add Petroleum Ether until the yellow band get near the bottom
of the column, and then collect in test tube 2 If the yellow band does not separate from
the green band, then change to a more polar solvent, such as the 70:30 Petroleum Ether,
until the yellow band passes through the column. After you have collected the yellow pigment,
switch to test tube 3 to collect the intermediate between the green and yellow pigment until
you can see the green band move to the bottom of your test tube. Here you can see our green band is moving
down. Once the green is almost at the bottom, switch
to test tube 4 to collect the green pigment. If the green band does not move, change to
a more polar solvent, Ethanol. We add the Ethanol to move the green band
down. Here we can see the green is moving to the
bottom of the column. We switch the collection tube 4 to collect
the green pigment. Here you can see our 4 fractions. Test tube 1 has the Petroleum Ether, which
is colorless. Test tube 2 has our yellow pigment. The intermediate is test tube 3. You can see it is between the yellow and green
color. And test tube 4 showing the green pigment. Test tube 2 and 4 will be use to the following
TLC lab, which is not covered in this video.

1 Comment

  1. arthaye August 10, 2019 at 10:53 pm

    thanx a lot

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