Jonathan S.

Nimitz
University of New Mexico

PRENTICE HALL, Upper Saddle River, New Jersey 07458

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Overview

Frequently when isolating a natural product or after carrying out a reaction the investi-
gator wishes to isolate one or more components of a mixture in pure form. If the desired
product is easily purified by distillation or recrystallization, these are usually the meth-
ods of choice. However, many compounds decompose on heating or are present in such
small quantities that distillation or recrystallization are impractical. In these cases col-
umn chromatography can often be used to carry out the desired separation.
Column chromatography operates on the same principles as gas or thin-layer chro-
matography: There is a stationary phase (the packing) and a mobile phase (the solvent
or eluant), and a compound distributes itself between the two phases according to its
affinity for each. The higher the affinity for the mobile phase, the larger the fraction of
time or compound spends there and the faster it moves down the column. •
A chromatography column consists of a tube packed with a solid adsorbent such
as alumina (AI203), silica (Si02), or Florisil (magnesium silicate, Mg2Si04). Usually,
an adsorbent-compound weight ratio of 25: 1 is used for easy separations and 100: I or
so for more difficult ones. A small plug of cotton or glass wool is placed at the bottom
of the column to prevent the solids from trickling out, then a layer of sand to provide a
flat base for the packing material. If the sand were not present, the adsorbent would be
uneven at the bottom and would not provide as sharp a separation. Another layer of
sand at the top of the column keeps the top of the adsorbent fiat and protects the column
from disruption as new solvent is poured in at the top. A column may be packed wet,
with the adsorbent poured in as a slurry in the desired solvent. A column can also be
packed by filling it with solvent, then slowly pouring a stream of solid adsorbent at the
top. The latter method (adding dry powder to a solvent-filled column) is generally less
messy for an inexperienced chromatographer. An illustration of a properly packed col-
umn appears in Figure 5-1, accompanied by drawings of some types of solvent reser-
voirs in Figure 5-2. It is important to keep the column wet at all times and not to let
the solvent level drop below the top of the adsorbent. If it does, channels and bubbles
will form in the packing and the efficiency of separation will be decreased.
After the column is packed the mixture to be separated is placed dropwise with a
Pasteur pipette in a thin, even layer on the top. This layer is run down through the sand
onto the column by letting a few drops out of the stopcock at the bottom. Solvent is

72
 Column Chromatography 81

of f)-carotene as it emerges from the bottom of the column, then begin eluting with a
1 : I (by volume) mixture of petroleum ether and ethyl acetate. Collect the green band
of chlorophyll in a tared beaker. On a steam bath in the fume hood carefully evaporate
the fractions containing the two products and record their masses on an analytical bal-
ance. Scrape up and examine the green film of chlorophyll formed. At the instructor's
option you may take ultraviolet-visible spectra of the two compounds. Compare the
spectra obtained with the literature spectra shown in Figure 5-3.

700 600 500 400 nm

Figure 5-3 Visible absorption spectra of chlorophyll and f3-carotene in petroleum ether.

Reference:

McKONE, A. T., "The Rapid Isolation of Carotenoids from Foods," Journal of Chemical Edu-
calion (1979) 56, 676.
 Column Chromatography 73

Elution solvent

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Column of
solvated adsorbent

'. :';n-1
Layer of sand

Layer of glass wool Stopcock

Figure 5-1 A Packed Chromatography Col-

umn

added at the top and the stopcock is opened to allow the solvent to move down the
column. The components of the mixture at the top of the columnm separate into bands
and move down the column at different speeds. Sometimes (as in this experiment) the
compounds are visible and it is easy to tell where they are on the column. In other cases
it may be necessary to use an ultraviolet absorption detector (which follows absorbance
of the eluant and indicates when organic compounds are coming off the column) or to
check the eluant by TLC.

Figure 5-2 Types of Solvent Reservoirs

74 Section 5

If the desired components are not visible on the column, the standard procedure
is to collect numbered fractions of solvent coming off the column, then to examine them
by TLC or other methods to determine which fractionfs) contain the desired compound.
The desired fractions are then combined and the solvent is removed (often under reduced
pressure) to yield the pure compound.
The correct choice of solvent is of course critical in achieving good separation in
a reasonable time. The more polar the solvent, the faster it will move a compound down
the column. The approximate order of solvents in terms of eluting power from least
polar to most polar is listed in Table 5-1; this is the same list that was discussed with
respect to TLC in Section 4.

TABLE 5-1 SOLVENTS LISTED IN APPROXIMATE ORDER

OF ELUTING POWER

Least polar
Petroleum ether
Hexane
Increasing Carbon tetrachloride
power of Tolueue
elution Dicblorornetilane
Chloroform
Diethy I ether
Ethyl acetate
Acetone
Ethanol
Methanol
Water
Most polar

Often a mixture of solvents is used, such as 5% chloroform in petroleum ether, to

obtain the desired polarity. If the solvent is too polar, the compounds move down the
column too quickly (at the solvent front) and are not separated. They come off the
column still mixed together. If the solvent chosen is too nonpolar, the compounds will
move down the column very slowly if at all. The separation may be very tedious and
require enormous volumes of solvents.
If the separation has not been carried out before an educated guess must be made
as to the choice of an appropriate solvent. One solution to this problem is to use gradient
elution; starting with a nonpolar solvent and gradually changing the solvent to increasing

TABLE 5-2 STRENGTH OF ADSORPTION OF VARIOUS

FUNCTIONAL GROUPS ON ALUMINA

Most weakly absorbed Hydrocarbons

(will elute with nonpolar solvent) Olefins
Ethers
Halocarbons
Aromatics
Order of elution Ketones
Aldehydes
Esters
Alcohols
Amines
Most strongly absorbed Acids, strong bases
(need a polar solvent to elute)
 Column Chromatography 75

polarity. This procedure allows nonpolar compounds to be eluted first, then gradually
strips off the more polar ones from the column.
How strongly a compound adsorbs onto a column depends on its polarity, and
therefore on its functional groups. Adsorption also depends on the nature of the packing
material (silica, alumina, etc.). The approximate order of strength of adsorption for
various functional groups on alumina is shown in Table 5-2. This order means that, for
example, in a mixture containing a carboxylic acid, an ether, and a halide, the halide
would elute first on an alumina column, followed by the ether, followed by the carbox-
ylic acid. Very polar compounds such as carboxylic acids adsorb strongly to columns
and may be difficult to elute.
Experiment 5.1 is the separation of a mixture of syn and anti isomers of azoben-
zene.

00 N=N
syn -Azobenzene anti-Azobenzene

Both of these compounds are orange-colored and easy to see on a column. The
syn isomer has the polar nitrogen-containing portion of the molecule more exposed than
the anti isomer, so the syn-azobenzene will be more strongly adsorbed on the alumina
and will move down the column much more slowly than the anti isomer.
Experiment 5.2 is the isolation of chlorophyll and !3-carotene from spinach. Chlo-
rophyll is a green plant pigment that contains a porphyrin ring system, a magnesium
ion, and a long hydrophobic side chain, and catalyzes the photosynthesis of glucose
from carbon dioxide. Without chlorophyll, probably none of us would be here!

o
N II
CH2 - CH2 - C - 0 - Phytyl
'\ N---Mg---N
! Y'
I
N

Chlorophyll a
76 Section 5

H3C
CH3 CH3 CH3 H3C

~ ~ ~ ~ ~ ~ ~ ~ ~
CH3
CH3 CH3 CH3 CH3
p-Carotene

f3-Carotene is the orange pigment found in carrots that functions as a precursor to

vitamin A. In the liver f3-carotene is cleaved to form vitamin A, which is further COn-
verted to ll-cis-retinal within the eye.

Vitamin A

l1-cis-Retinal

The mechanism of vision involves the enzyme-catalyzed photochemical isomerization

of II-cis-retinal to the more stable all-trans-retinal accompanied by the sending of a
visual signal as a nerve impulse.

APPLICATION: HIGH-PERFORMANCE
Column chromatography often proves time consum-
ing, since low flow rates of solvents are required for
equilibration and efficient separation. The need for
faster and higher-resolution column chromatography
has been addressed by a method called high-pressure
(or high-performance) liquid chromatography
(HPLC). In HPLC, the liquid phase enters a column
packed with very fine particles (10 to 50 um in di-
ameter) of a solid adsorbent. Several materials, in-
cluding silica, alumina, or small glass spheres coated
with a thin layer of porous material (called pellicular
beads) with or without a very thin layer of a liquid,
may serve as the stationary phase. These small par-
Injection valve
Pump
Proportioning valves
Solvent containers
WWW
ABC
Schematic of a Liquid Chromatograph
Waters Delta-Prep 3000 HPLC System (Courtesy of Waters Chro-
matography Division of Millipore Corporation)
Column Chromatography 77
LIQUID CHROMATOGRAPHY (HPLC)
ticles provide a larger surface area and the particles
pack together more tightly, allowing shorter column
lengths than in standard column chromatography.
This tight packing restricts the flow of the solvent,
though, and pressure must be applied to push the
mobile phase through the column. A pump supplies
a constant pressure of up to 10 ,000 psi (for a small,
2- to 3-mm diameter column) to force the solvent
through. For chemical inertness and strength, the
tubing and column used are constructed of thick
polypropylene, Teflon, or stainless steel. A sche-
matic diagram and photograph of an HPLC system
are shown here.
Column
Detector
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ww Waste bottle.
The liquid emerges from the column at atmo-
spheric pressure. In analytical HPLC this eluant is
then passed through a detector to measure com-
pounds coming off the column. Common detectors
measure ultraviolet light absorption, fluorescence, or
refractive index. The signal from the detector is
passed to a chart recorder, which provides a chro-
matogram similar to those obtained in gas chroma-
tography (see figure).
In preparative HPLC, separate fractions are
collected and evaporated to yield the purified com-
ponents of the original mixture. By using large col-
umns with diameters of 3-4 ern, it is sometimes
. possible to separate up to 15-20 g of material in one
run.
78 Section 5

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Separation of Free Amino Acids by HPLC

a carbon-cobalt HPLC bas the advantage over normal column

o bond chromatography of much faster separations and
greater resolving power. In addition, it can be used
on high-molecular-weight (nonvolatile) compounds
not amenable to gas chromatography. Since the com-
pounds do not undergo heating in HPLC and are on
the column for only a short time, danger of decom-
position is diminished. HPLC has been praised for
greatly simplifying the work involved in analyzing
and synthesizing complex natural products such as
vitamin B 12 and periplanone B, the sex attractant of
o the American cockroach.

Periplanone B (sex attractant

of the American cockroach)
 ColumnChromatography 79

Questions

1. How would you suggest eluting the syn-azobenzene?

2. If grease were present initially on the stopcock. where would you find it after performing the
chromatography?
3. What is the purpose of column chromatography?
4. What are the advantages of this method over other purification methods? The disadvantages?
5. Arrange the following solvents in order of increasing polarity: dichloromethane, methanol.
hexane.
6. Arrange the following compounds in expected order of elution: benzoic acid. I-heptanol,
l-bromobutane.
7. What will happen if the sand layers are uneven or there are bubbles in the packing?

EXPERIMENT 5.1 MICROSCALE SEPARATION

OF AZOBENZENES
Estimated Time:
2.5 hours

Special Hazards

Petroleum ether is highly flammable; keep it away from all flames or heat sources.
Azobenzene is a cancer suspect agent; avoid skin contact or ingestion. Avoid breathing
finely powdered inert materials such as alumina or silica; they can cause lung damage.

Procedure:

Packing the column. Securely clamp a 50-mL buret vertically and close the
stopcock. Push a small plug of glass wool to the bottom using a long glass rod. Obtain
about 100 mL of petroleum ether (bp 60-80). Now place a funnel on top of the column
and add about 30 mL of the petroleum ether. Pour a linle sand through the top to settle
in a layer about I cm thick on top of the glass wool. The exact thickness is not critical
as long as the top is level. Tap the burette lightly with a pencil or plastic pen to ensure
even packing. Weigh 30 g of alumina and gradually pour it into the top through the
funnel while continuing intermittent tapping. If necessary. excess solvent may be run
out into a beaker at the bottom to avoid spilling solvent out the top of the column. If
wet alumina accumulates at the top and blocks the flow, push it down with a stirring
rod. When finished packing the alumina, pour another l-cm layer of sand on top. This
packing procedure leaves about 15 ern of the column empty at the top for the solvent
head. Place a beaker or flask underneath the burette and open the stopcock to drain the
solvent to a point just below the top of the sand. The column is now ready for the
sample.

Separation of syn and ant; isomers of azobenzene. Record the melting

range of a mixture of practical grade syn- and anti-azobenzene. Weigh 100 mg of this
mixture. Dissolve this sample in I mL of petroleum ether with slight warming. Care-
fully and evenly add this solution to the top of the column using a dropper. Open the
stopcock to run the sample barely below the sand surface. Add about 1 mL of fresh
80 Section 5

petroleum ether to the top and similarly run this down to the sand level. Now carefully
add petroleum ether almost to the top of the burette.
Place a beaker labeled forerun below the burette tip and open the stopcock to
begin solvent flow down the column. Monitor the solvent level on the top carefully and
keep adding more petroleum ether before the level drops to the sand. If by accident the
solvent level falls below the level of the sand, simply refill the solvent on top and
proceed. There may be bubbles in the packing, resulting in slower solvent flow and
poorer separation, but the anri-azobenzene can still be collected.
Observe the orange band of anri-azobenzene moving down the column. The syn
isomer remains in a band near the top of the column. When the anri-azobenzene begins
to reach the bottom of the burette change to another collection beaker (labeled anti) and
collect the band. When it has been collected shut off the column, add a boiling chip to
the beaker, and evaporate the solvent on a steam bath in the hood. Record the mass and
melting point of the material obtained (lit. mp for anti-azobenzene, 68°). Dispose of the
forerun in a properly marked waste bottle in the fume hood.

EXPERIMENT 5.2 ISOLATION OF CHLOROPHYLL

AND i3-CAROTENE FROM SPINACH
BY GRADIENT ELUTION
Estimated Time:
2.5 hours

Special Hazards

See Experiment 5.1. Ethyl acetate is also flammable.

Procedure

Read the procedure for Experiment 5.1 and prepare a column as described. Befo;e the
lab, the instructor will take a JO-oz (280-g) package of frozen spinach (which has been
thawed), place it in a blender with 400 mL of absolute ethanol, and blend it thoroughly.
This process extracts most of the water into the ethanol, while leaving the chlorophyll
and l3-carotene in the spinach. As an alternative, strained spinach baby food (8 g per
person) may be used and stirred thoroughly with JO mL of absolute ethanol.
Take approximately 20 mL of this spinach-ethanol paste (or the baby food-
ethanol mixture) and place it in a funnel containing a small plug of glass wool instead
of a filter paper. Place a piece of filter paper on the top and squeeze to remove the
ethanol. Drain excess ethanol from the top as well.
Place the remaining residue on a clean paper towel, remove the glass wool and
filter paper, and press to dry futher. Place the resulting pellet in a lOO-mL beaker and
add 20 mL of CH2Cl2. Stir thoroughly to extract the pigments into the dichloromethane.
Decant the solution away from the bulk of the spinach residue, then filter into a clean
beaker and concentrate it on a steam bath in the fume hood to a volume of 1-2 mL. Be
careful not to heat to dryness because the large, complex pigment molecules may be-
come oxidized and discolored if overheated.
Place the sample on the column as described in Experiment 5.1. Elute with petro-
leum ether and observe that the yellow band of l3-carotene moves down the column
slightly faster than the green chlorophyll. The quantity of l3-carotene is not great so the
band may be faint. Observe and record the shapes of the bands. Collect the yellow band