Jonathan S. NimitzUniversity 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 desiredproduct 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 suchsmall 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 solventor eluant), and a compound distributes itself between the two phases according to itsaffinity for each. The higher the affinity for the mobile phase, the larger the fraction oftime or compound spends there and the faster it moves down the column. •

A chromatography column consists of a tube packed with a solid adsorbent suchas 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 orso for more difficult ones. A small plug of cotton or glass wool is placed at the bottomof the column to prevent the solids from trickling out, then a layer of sand to provide aflat base for the packing material. If the sand were not present, the adsorbent would beuneven at the bottom and would not provide as sharp a separation. Another layer ofsand at the top of the column keeps the top of the adsorbent fiat and protects the columnfrom 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 bepacked by filling it with solvent, then slowly pouring a stream of solid adsorbent at thetop. The latter method (adding dry powder to a solvent-filled column) is generally lessmessy 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 letthe solvent level drop below the top of the adsorbent. If it does, channels and bubbleswill 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 aPasteur pipette in a thin, even layer on the top. This layer is run down through the sandonto 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 a1 : I (by volume) mixture of petroleum ether and ethyl acetate. Collect the green bandof chlorophyll in a tared beaker. On a steam bath in the fume hood carefully evaporatethe 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'soption you may take ultraviolet-visible spectra of the two compounds. Compare thespectra 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.

Layer of sand

Layer of glass wool

Column Chromatography 73

Elution solvent

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solvated adsorbent

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Figure 5-1 A Packed Chromatography Col-umn

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

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Figure 5-2 Types of Solvent Reservoirs

74 Section 5

If the desired components are not visible on the column, the standard procedureis to collect numbered fractions of solvent coming off the column, then to examine themby 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 reducedpressure) to yield the pure compound.

The correct choice of solvent is of course critical in achieving good separation ina reasonable time. The more polar the solvent, the faster it will move a compound downthe column. The approximate order of solvents in terms of eluting power from leastpolar to most polar is listed in Table 5-1; this is the same list that was discussed withrespect to TLC in Section 4.

TABLE 5-1 SOLVENTS LISTED IN APPROXIMATE ORDEROF ELUTING POWER

Least polar

Increasingpower ofelution

Petroleum etherHexaneCarbon tetrachlorideTolueueDicblorornetilaneChloroformDiethy I etherEthyl acetateAcetoneEthanolMethanolWater

Most polar

Often a mixture of solvents is used, such as 5% chloroform in petroleum ether, toobtain the desired polarity. If the solvent is too polar, the compounds move down thecolumn too quickly (at the solvent front) and are not separated. They come off thecolumn still mixed together. If the solvent chosen is too nonpolar, the compounds willmove down the column very slowly if at all. The separation may be very tedious andrequire enormous volumes of solvents.

If the separation has not been carried out before an educated guess must be madeas to the choice of an appropriate solvent. One solution to this problem is to use gradientelution; starting with a nonpolar solvent and gradually changing the solvent to increasing

TABLE 5-2 STRENGTH OF ADSORPTION OF VARIOUSFUNCTIONAL GROUPS ON ALUMINA

Order of elution

HydrocarbonsOlefinsEthersHalocarbonsAromaticsKetonesAldehydesEstersAlcoholsAminesAcids, strong bases

Most weakly absorbed(will elute with nonpolar solvent)

Most strongly absorbed(need a polar solvent to elute)

Column Chromatography 75

polarity. This procedure allows nonpolar compounds to be eluted first, then graduallystrips off the more polar ones from the column.

How strongly a compound adsorbs onto a column depends on its polarity, andtherefore on its functional groups. Adsorption also depends on the nature of the packingmaterial (silica, alumina, etc.). The approximate order of strength of adsorption forvarious functional groups on alumina is shown in Table 5-2. This order means that, forexample, in a mixture containing a carboxylic acid, an ether, and a halide, the halidewould 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 columnsand may be difficult to elute.

Experiment 5.1 is the separation of a mixture of syn and anti isomers of azoben-zene.

00N=N

syn -Azobenzene anti-Azobenzene

Both of these compounds are orange-colored and easy to see on a column. Thesyn isomer has the polar nitrogen-containing portion of the molecule more exposed thanthe anti isomer, so the syn-azobenzene will be more strongly adsorbed on the aluminaand 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 magnesiumion, and a long hydrophobic side chain, and catalyzes the photosynthesis of glucosefrom carbon dioxide. Without chlorophyll, probably none of us would be here!

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CH2 - CH2 - C - 0 - Phytyl

Chlorophyll a

76 Section 5

H3C

CH3 CH3 CH3 H3C

~ ~ ~ ~ ~ ~ ~ ~ ~CH3CH3 CH3 CH3 CH3

p-Carotene

f3-Carotene is the orange pigment found in carrots that functions as a precursor tovitamin 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 isomerizationof II-cis-retinal to the more stable all-trans-retinal accompanied by the sending of avisual signal as a nerve impulse.

Column Chromatography 77

APPLICATION: HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

Column chromatography often proves time consum-ing, since low flow rates of solvents are required forequilibration and efficient separation. The need forfaster and higher-resolution column chromatographyhas been addressed by a method called high-pressure(or high-performance) liquid chromatography(HPLC). In HPLC, the liquid phase enters a columnpacked 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 coatedwith a thin layer of porous material (called pellicularbeads) with or without a very thin layer of a liquid,may serve as the stationary phase. These small par-

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ticles provide a larger surface area and the particlespack together more tightly, allowing shorter columnlengths than in standard column chromatography.This tight packing restricts the flow of the solvent,though, and pressure must be applied to push themobile phase through the column. A pump suppliesa constant pressure of up to 10 ,000 psi (for a small,2- to 3-mm diameter column) to force the solventthrough. For chemical inertness and strength, thetubing and column used are constructed of thickpolypropylene, Teflon, or stainless steel. A sche-matic diagram and photograph of an HPLC systemare shown here.

Column

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Schematic of a Liquid Chromatograph

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Waters Delta-Prep 3000 HPLC System (Courtesy of Waters Chro-matography Division of Millipore Corporation)

The liquid emerges from the column at atmo-spheric pressure. In analytical HPLC this eluant isthen passed through a detector to measure com-pounds coming off the column. Common detectorsmeasure ultraviolet light absorption, fluorescence, orrefractive index. The signal from the detector ispassed to a chart recorder, which provides a chro-matogram similar to those obtained in gas chroma-tography (see figure).

In preparative HPLC, separate fractions arecollected 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 onerun.

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78 Section 5

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Separation of Free Amino Acids by HPLC

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HPLC bas the advantage over normal columnchromatography of much faster separations andgreater resolving power. In addition, it can be usedon high-molecular-weight (nonvolatile) compoundsnot amenable to gas chromatography. Since the com-pounds do not undergo heating in HPLC and are onthe column for only a short time, danger of decom-position is diminished. HPLC has been praised forgreatly simplifying the work involved in analyzingand synthesizing complex natural products such asvitamin B 12 and periplanone B, the sex attractant ofthe American cockroach.

Periplanone B (sex attractantof 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 SEPARATIONOF 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 breathingfinely 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 thestopcock. Push a small plug of glass wool to the bottom using a long glass rod. Obtainabout 100 mL of petroleum ether (bp 60-80). Now place a funnel on top of the columnand add about 30 mL of the petroleum ether. Pour a linle sand through the top to settlein a layer about I cm thick on top of the glass wool. The exact thickness is not criticalas long as the top is level. Tap the burette lightly with a pencil or plastic pen to ensureeven packing. Weigh 30 g of alumina and gradually pour it into the top through thefunnel while continuing intermittent tapping. If necessary. excess solvent may be runout into a beaker at the bottom to avoid spilling solvent out the top of the column. Ifwet alumina accumulates at the top and blocks the flow, push it down with a stirringrod. When finished packing the alumina, pour another l-cm layer of sand on top. Thispacking procedure leaves about 15 ern of the column empty at the top for the solventhead. Place a beaker or flask underneath the burette and open the stopcock to drain thesolvent to a point just below the top of the sand. The column is now ready for thesample.

Separation of syn and ant; isomers of azobenzene. Record the meltingrange of a mixture of practical grade syn- and anti-azobenzene. Weigh 100 mg of thismixture. 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 thestopcock 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 carefullyadd petroleum ether almost to the top of the burette.

Place a beaker labeled forerun below the burette tip and open the stopcock tobegin solvent flow down the column. Monitor the solvent level on the top carefully andkeep adding more petroleum ether before the level drops to the sand. If by accident thesolvent level falls below the level of the sand, simply refill the solvent on top andproceed. There may be bubbles in the packing, resulting in slower solvent flow andpoorer separation, but the anri-azobenzene can still be collected.

Observe the orange band of anri-azobenzene moving down the column. The synisomer remains in a band near the top of the column. When the anri-azobenzene beginsto reach the bottom of the burette change to another collection beaker (labeled anti) andcollect the band. When it has been collected shut off the column, add a boiling chip tothe beaker, and evaporate the solvent on a steam bath in the hood. Record the mass andmelting point of the material obtained (lit. mp for anti-azobenzene, 68°). Dispose of theforerun in a properly marked waste bottle in the fume hood.

EXPERIMENT 5.2 ISOLATION OF CHLOROPHYLLAND i3-CAROTENE FROM SPINACHBY 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 thelab, the instructor will take a JO-oz (280-g) package of frozen spinach (which has beenthawed), 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 chlorophylland l3-carotene in the spinach. As an alternative, strained spinach baby food (8 g perperson) 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 insteadof a filter paper. Place a piece of filter paper on the top and squeeze to remove theethanol. Drain excess ethanol from the top as well.

Place the remaining residue on a clean paper towel, remove the glass wool andfilter paper, and press to dry futher. Place the resulting pellet in a lOO-mL beaker andadd 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 cleanbeaker and concentrate it on a steam bath in the fume hood to a volume of 1-2 mL. Becareful 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 columnslightly faster than the green chlorophyll. The quantity of l3-carotene is not great so theband may be faint. Observe and record the shapes of the bands. Collect the yellow band