SUBJECT: Chromatography of spinach pigments
DATE: 2/96; 2/97

Dear Biolabbers,
In lab this week, the students are separating the pigments found in spinach
leaves. The leaves are ground in acetone, the slurry is filtered through
cheesecloth and then distributed to students in amber glass bottles. The
chromatograms are run using a 9:1 mixture of petroleum ether and acetone as
the chromatography solvent. Students get the four bands they have
predicted they should see: chlorophylls a and b, xanthophyll and
beta-carotene. However, at the solvent front, a fifth band is always
found. This band is bright yellow. Does anyone out there know what it is?
Sincerely,
Michael Dini
Texas Tech Univ.
y3mld@ttacs.ttu.edu


You don't describe the type of TLC plates. However, you may be
fractionating the xanthophylls. you may be seeing lutein fractionate
from violaxanthin + neoxanthin. Just a guess.

John Markwell Phone: 402-472-2924
Dept. Biochemistry FAX: 402-472-7842
University of Nebraska Internet: markwell@unl.edu
Lincoln, NE 68588-0664


WARNING... BE CAREFUL GRINDING IN ACETONE... at a previous job,
a colleag put his acetone/spinach slurry in a BLENDER, it exploded,
and badly burned one student!!

From: YOUR FRIENDLY NEIGHBORHOOD BIOLOGIST <MOSSRE@wofford.edu>


the band is an additional carotenoid pigment. There are any number of
carotenoids - in fact since this is "at the solvent front" it is likely
a mixture. Consult

Dave McNeely, Biology, University of Texas at Brownsville, 80 Fort Brown,
Brownsville, TX 78520; mcneely@utb.edu


Michael Dini's question and John Markwell's reply raised a question for me. We
do a chromatographic separation that sounds very much like Michael's and I
thought the pigment order was
chlorophyll b
chlorophyll a
xanthophyll 2
xanthophyll 1
(gray, "ides")
and carotene at the solvent front.

Would someone please clarify this for me.

thanks,
Christine Case
case@smcccd.cc.ca.us


If run on a silica gel plate, I would expect the following pigments:
carotenoids at solvent front, followed by a dull grey band of chlorophyll
decomposition products (depending on how old the spinach prep is), followed
by chlorophyll a then b then three xanthophylls (one fairly bright with the
others fairly dull).

Roger Christianson 503-488-0223 (home)
Department of Biology 503-552-6747 (office)
Southern Oregon State College 503-552-6415 (fax)
1250 Siskiyou Boulevard rchristi@wpo.sosc.osshe.edu
Ashland, OR 97520


Sorry Biolabbers,
I should have specified that we were doing paper chromatography to separate
the spinach pigments. Thanks all, for the quick responses!
Yours,
Michael Dini
Texas Tech Univ.
y3mld@ttacs.ttu.edu


We run the same basic lab everyone else is describing. We
use a paper matrix instead of silica and use ethanol extracts
instead of acetone (much easier to work with!). On our
chromatograms the carotene elutes with the solvent front
followed by xanthophyll, which is not resolved into
multiple bands. I base my identification of the bands on the
orangish tinge to the carotene (the xanthophyll is lemon-
yellow) and comparisons to controls- you can get carotene
from extracting a package of frozen squash and xanthophyll
from corn (with the corn its a good idea to boil it in water
first to remove some of the starch). These extracts are a
little faint for reliable chromatography, but they're excellent
if you want an absorption spectrum from a
spectrophotometer.

John Dickerman
Northern Illinois University
DeKalb, IL 60115
T80JWD1@WPO.CSO.NIU.EDU


We also do a separation of spinach in petroleum ether/ acetone on paper.
The separation is based on the polarity of the pigments - least polar
should migrate farthest. So at/ near the solvent front is carotene
followed by xanthophyll, chl a and chl b. This order is the same as most
other replies to the list. Every once in a while we get odd results.
Check all the components of your set up for contamination. In another
chromatography lab, a faint blue line always appeared that we never did
nail down. Alconox residue was suspected. What are the dtudents using to
mark the origin line? Pen will chromatograph or maybe an organic in the
pencil. How clean are your chromoatography jars? Could the spinach have
been sprayed with something? Some foods are color enhanced for your
shopping pleasure. If all else fails, maybe you could elute the band and
give it your biochem lab as an unknown to id.

Brenda Simmers
University of Toledo
bsimmer@uoft02.utoledo.edu

Plant pigment chromatography

DATE: 2/97

 

 

Biolab experts,

 

I need some help trouble-shooting our chromatography lab. All we get are

smears - no separation. Someone suggested keeping the TLC strips dry - we

disiccated them for 2 weeks and that didn't solve the problem.

 

We use commercially prepared solvent & pigments. I tried spinach leaves

extracted with acetone - still no separation.

 

If you have a good, simple protocol that works, please share it with me.

 

Thanks

 

-Frank

-------------------------------------------------------------------------------

Frank R. Hensley, Ph.D. Biology 111/112 Web page:

Dept. of Biology UNC-Greensboro http://www.uncg.edu/~frhensle

FHensley@uncg.edu

910-334-5391 x23

 

 

Frank,

 

The acetone extract doesn't work (no surprise to you, huh). All the pigments

aren't soluble in acetone and it doesn't evaporate quickly. We always get great

results making our own solvent and extract.

 

General notes: 1. paper and TLC both work

2. make small spots and let each spot dry before adding the next

spot

3. spot >5 times

 

Chromatography solvent

9 Petroleum ether: 1 acetone

 

Concentrated chlorophyll extract

Add 200 ml pet ether to 200 ml acetone extract of pigments in a

separatory

funnel. Add four drops of water and hsake. Discard aqueous acetone layer

(bottom). The top will be a wet ether solution of pigment. Dry this layer by

adding 1 tsp anhydrous sodium sulfate and agitating vigorously. Decant into a

small wide-mouthed jars for student use. Should be prepared the same day the

extract is to be used.

 

Christine Case

Skyline College

case@mscccd.cc.ca.us

 

 

Biolabbers:

 

In our Intro Bio lab we do a TLC using plastic-backed TLC strips. They

come in pre-cut 5cm X 20 cm strips from VWR Scientific.

 

We extract spinach pigments from spinach leaves by grinding them with

acetone and a little sand. The key to getting a good extract seems to be

using dark green leaves and not including the stems or veins. After the

solution is a very dark green, 5-10 minutes of grinding with a mortar and

pestle, we filter it using regular coarse filter paper.

 

The students then apply many drops (20-25) with a capillary tube to the

TLC strip. We use a 2 isooctane : 1 acetone : 2 ethyle ether solvent

system. We prepare the solvent ourselves and replace it in the

chromatography jars (tall thin jars, also available from VWR) between each

lab because the ether evaporates off quite rapidly.

 

The separation is usually very good. We have found that the smearing is

usually due to the ether evaporating off and changing the solvent sytem.

The colors are quite clear but fade after a few minutes. So I usually

have the students circle the pigment spots with a pencil immediately and

hold the TLC strip up to the window, assuming the lab is during the day

and there is some light out there, to see and identify the colors. The

problem we have is that the Rf values we usually measure don't match the

Rf table we have, which means that one of these years I will probably have

to give in and run a series of standards.

 

Sorry if this is wordy, contact me if you have any questions.

 

Good-luck,

 

Rosemary

 

**********************************************************

Rosemary E. Boone Department of Biological Sciences

rmboone+@pitt.edu University of Pittsburgh

(412) 624-9325 G2 Clapp Hall

Fax: (412) 624-4759 Pittsburgh, PA 15260

 

 

 

>The acetone extract doesn't work (no surprise to you, huh). All the pigments

>aren't soluble in acetone and it doesn't evaporate quickly.

 

We've mostly used our own acetone extract, prepared by drying spinach

leaves, crumbling, then adding acetone and filtering. Carotenoids,

xanthophyll, and chlorophylls a and b are generally reasonably well

separated, though the chlorophylls run very close together.

 

Last fall I also got excellent separation of the chlorophylls (though not

much carotene) using a technique from an ABT article for which I don't have

the reference at the moment: lay a leaf on the chromatography paper and rub

across it to crush tissue onto the paper. I used the bottom edge of a film

canister. Quick and undeniably dirty, but very direct in its impact! My

students were raising beans for a growth project, and I used leaves from my

own crop of bean plants for the photosynthesis lab. I haven't tried eluting

pigments from these chromatograms for spectrophotometry, as I have for

spinach extract, but it would probably be easier since the chlorophylls

separate more cleanly.

 

Maren

 

 

Maren H. Brown, Ph.D. brownm@goliath.sunyocc.edu

Professor of Biology office (315) 469-2405

Onondaga Community College fax (315) 469-2593

Syracuse NY 13215

 

 

 

I've always had great luck with the protocol from my old plant phys. lab

book: "Experiments in Plant Physiology," by Witham, Blaydes &

Devlin...although I'm sure there's something more recent out there (my

copy is 1971! But the results are great!) Let me know if you want me to

fax you a copy.

 

***************************************************************

Louise Baxter email: baxterl@cwu.edu

Department of Biological Sciences phone: 509-963-2745

Central Washington University fax: 509-963-2730

Ellensburg, WA 98926

 

 

I've had good luck with a very simple spinach extraction. I take a 10

oz package of frozen spinach, microwave it for 5-10 minutes until

most of the water melts (which I then decant), cover with 1 liter of

95% ethanol, and boil (in a fume hood!) for a few minutes. Filter it

first through cheesecloth and then filter paper. This gives a very

dark extract that separates into all 4 pigments on paper

chromatography. It is stable for at least a week if stored in an

amber bottle. I keep it in the freezer over the weekend when I

prepare it ahead of time, but it doesn't fade too much at room

temperature during the week of the lab.

 

The chromatography system we use is paper, not silica. A simple

solvent of 90% petroleum ether/10% acetone works fine, but we get

better separation of chlorophylls a & b with 10% acetone/ 5%

n-heptane/ 85% mixture of hexane isomers.

 

As I said, the ethanol extract gives all 4 bands, but it is primarily

chlorophyll. As someone else noted, it's a good idea to circle the

lighter bands quickly before they fade. One good side effect of this

is that it can be diluted with more ethanol to give a chlorophyll

solution that is good enough to do an absorption spectrum on a spec

20.

 

The same basic method works for other pigments. For carotene, heat

and remove as much water as you can from frozen squash and then boil

in ethanol until it's orange. Xanthophyll is a bit more complex: Boil

frozen sweet corn in salted water to remove as much starch as

possible. Run through a blender and filter through cheesecloth.

Discard the filtrate and collect the mass of pulverized seed coats

for the hot ethanol extraction. This extract is lemon-yellow when

done. The xanthophyll and carotene extracts aren't concentrated

enough for good chromatography, but are fine for absorption spectra

and a lot easier than trying to elute the bands from the

chromatography strips.

 

John Dickerman

Northern Illinois University

T80JWD1@WPO.CSO.NIU.EDU

 

 

I hesitate to write this because it's SO SIMPLE... but Michael Clark's lab

manual (published by Suspended Animations) has students rolling a

penny over fresh spinich on top of a piece of filter paper, then suspend

the paper in the chromatography solvent as usual. We always get 4

bands with this simple method. The lab book is good for science skills

classes or remedial classes.

Sue Hutchins

Itasca Community College

Grand Rapids, MN

shutchins@it.cc.mn.us

 

 

Hi, how does rolling a penny on top of spinach transfer photosynthetic

pigment to the paper. This sounds great and I would love to try it but Im

missing something.

 

"Thomas Pitzer" <pitzert@fiu.edu>

 

 

 

You have to press hard. It squeezes the juice (including pigments) out of the

spinach leaf and into the paper. As the chromatography solvents move up and

encounter the stain they suspend the pigments and carry them up the paper. We

use a quarter (and, most likely, a larger piece of filter paper).

 

Dave Williams

profdhw@aol.com

Science Division

Anne Arundel Community College

Arnold, MD 21012

 

 

 

Thanks to all who gave me help with chromatography. I determined that the

solvent we had purchased was no good, probably due to differential

evaporation.

 

Several people suggested a 9 pet ether:1 acetone solvent. It works well. I

have also heard that 15:1 is great, and I'll probably try it too.

 

Spinach can be applied directly to silica gel TLC strips, but requires

more care than using chromatography paper. The gel flakes off the plastic

if you press too hard. Many people use a coin to apply the spinach, but

several other tools will work (like rubbing with forceps).

 

Thanks again for all the help!

 

-Frank

 

-------------------------------------------------------------------------------

Frank R. Hensley, Ph.D. Biology 111/112 Web page:

Dept. of Biology UNC-Greensboro http://www.uncg.edu/~frhensle

FHensley@uncg.edu

910-334-5391 x23

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