research summaries for teachers
analytical L.
8. ROGERS
Pvrdue University
Lafayefle, Indiana. Dcspite the fact that separations, including variorrs forms of chromatography, have been used by chemists for many years, the next five years appear destined t o produce some revolutionary advances in analytical (and preparative) chromatography. The advances will stem in large measure from studies in gas chromatography which have made possible the rapid evaluation of fundamental variables. As a result, liquid chromatography may prove t o be competitive (in time) with gas chromatography as it is present,ly practiced, and chromatographic isolations of enantiomers and isotopically substituted species may become a rapid quantitative procedure that will be relatively easy to do. When a chemist performs an analysis, he is applying informat,ion theory (1,2) whether or not he is aware of it. I n analysis, just as in other forms of communication, one of the chief goals is to "get the message across" with a prescribed accuracy and reliability in a minimum length of time. The analytical chemist in industry, who is faced with the need for cont,rolling a continuous process, is perhaps the one most keenly aware of the variety of ways to minimize the required time. However, many of the guiding principles for optimizing chromatographic separations are appropriate for inclusim in an undergraduate analytical course so that they can be understood and applied more widely by all chemists. II Started in 1948
A chemist's intuition tells him that if he has two or more pairs of substances, the easiest pair to separate by particular technique will be the one for which the quotient for the two distribution ratios will be largest. For example, substances having-distribution ratios of 20 and 2, respect,ivelg, should he easier to separate than those having ratios of 20 and 5. But what about the Roger's eolnmn initiates the EDITOR'S NOTE: PI.O~BSSOI. ieatwe "llesesreh Summaries for Teachers" pwmised in t,he July 1!167 editorial. I n this and in each of the next five ismes, t,his column will be written by a distingnished chemist vepresenliug m e of the six sob-disciplines of chernistry, i.e., anslytical, biochemistry, iuorganic, organic, phy.iical, theoretical. During the second six months a second set of research summaries will appear. Next mont,h Pmfeswr Jweph IIirsehfelder will present, a, topic i n theoretical chemistry.
chemistry latter pair compared t o a third pair with values of 2 and 0.5 where both have the same quotient? I n 191S, Bush and Densen (3) made an astute observation relating t o pairs of substances being separated by liquidliquid extraction in a pseudo-countercurrent process analogous to chromatography. They suggested that, for a given amount of work (e.g., number of transfers) the resolution was maximum for a given value of the quotient of the distribution ratios when the product of the distribution ratios was unity. Because a distribution ratio, K, is a function not only of the concentration of the substance in each phase hut also the volumes of the phases (eqn. (I)), Bush and Densen showed how to calculate
the volume ratio that would permit two compounds to be separated using a minimum number of transfers. A similar result has been derived for columnar chromatographic processes in which substances are eluted from a column using an operation called "single withdrawal" (4). I n l%O, Purnell and Quinn (5) showed that the minimum number of theoretical plates was required for a chromatographic separation when the larger of the two distribution ratios was hetween 2 and 3. To grasp what this means, in gas chromatography the "air peak" gives a good estimate of B,, the "interstitial volume" in the column; the chromatographic peak for a substance is found a t (K 1) Vi. Hence, the second of two peaks should appear a t only 3 4 times the air peak. If a sample contains several components, the most difficult pair to resolve should be treated in that way. How can the values for the distribution ratios be changed? I n gas-liquid and liquid-liquid chromatography, one of the easiest wajTs is by changing the amount (percentage by weight) of liquid held by the inert support (6). (Use of a Golay [open-tube] columu (7) represents a useful hut extreme way for decreasing the liquid-to-gas ratio by a factor of 30LLi00.) I n liquid-solid chromatography, the concentmtion of reagent can be changed. A computer search (8) for optimum conditions can he made when detailed information is available (9). If a sample contains a large number of substances, representing a wide range of distribution ratios, it is advantageous t o consider the use of pmgmmmed changes in temperature ( l o ) ,flow (11, 12), or concen-
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tration of a reagent (IS, 14) in order to minimize the time required. However, one should not overlook the fact that there is an interplay, for example, between flow rate of the carrier and rate of temperature change which can, in turn, be optimized. Finally, i t is worth noting that the counterpart to prescribed resolution in minimum time is maximum resolution in a fixed time (16, 16). I n gas chromatography, lighter liquid loadings or deactivated absorbents can usually be used a t lower column temperatures to effect sizable gains in resolution. Basic Studies of Column Performance
In 1933, van Deemter, et al., (17) proposed an equatiou for relating column efficiency, as measured by the height equivalent to a theoretical plate (HETP) to the linear flow rate of the moving phase. That paper stimulated many studies which have led to several important extensions and major refinements of their equation. For example, there is evidence that the efficiency levels off at very high flow rates rather than continuously decreasing (18-20). Another interesting result has been the finding by Sternberg and Poulson (21) that the efficiency for a small-diameter packed column goes through a decided maximum when the ratio of the column diameter to particle diameter is between 2 and 3. Other studies have shown that highly efficient columns can take many forms (22) including one produced by packing a thick-walled glass capillary and pulling it out to about ten times its original length ($8). The role nlaved bv the worositv of the ~ a c k i-n ais also " being actively studied. It is known that molecules, particularly polymers and other large molecules, can he fractionated on the basis of size, e.g., by the relative abilities of molecules to diffuse into a porous solid. "Gel filtration" is the term that has been applied by biochemists to their separations carried out in aqueous media ($4, 25) ; "gel permeation" is the corresponding term that has been applied to fractionations of polymers in nonaqueous systems (26). The latter area is an especially active one a t the present time. The fact that large molecules are less able than small molecules to diffuse into a porous gel means that they are eluted sooner. Such behavior is opposite to that for adsorption in which larger molecules (in a homologous series) are generally retained longer. Clearly, because of the interplay between the two mechanisms, a separation based upon adsorption using a porous adsorbent like silica will have optimum conditions which will depend upon the particular mixture of adsorbates to be separated. As a result, the critical importance of porosity characteristics of adsorbents is receiving increased attention (21, 22). Iciselev and his co-workers (27, 28) have made extensive studies of the relationships between porosity, adsorption behavior, and chromatographic properties.
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High-pressure Chromatography
The recent trend toward the study of decidedly higher inlet pressures (>lo00 atm) has two goals: making very fast analyses, and making a new type of separation. While greater speeds are to be welcomed in gas chromatography (29), they are expected to have their greatest impact on the various forms of liquid 8
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chromatography. It appears very likely that liquid columnar chromatography can be made closely competitive in speed with thin-layer chromatography (SO) and with gas chromatography as it is ordinarily performed today (51). Furthermore, instead of the low efficiencies one might have anticipated at high flow rates, markedly higher efficiencies have been predicted on the basis of "complete and rapid" turbulence in the moving phase (32, 33). High-pressure chromatography is also opening up a new area of separations: gas chromatography at supercritical pressures (54, 35). By operating a column at a high outlet pressure as well aa a high inlet pressure, and by using as a mobile phase, a polar and/or polarizable substance, e.g., CC12F2,above its critical pressure, the moving phase acts like a low-viscosity liquid. Relatively nonvolatile compounds, such as porphyrins and polyalcohols, which are presently handled better by liquid chromatography, can be chromatographed quickly under such conditions. Optimized High-pressure Analyses of the Future
It is quite evident that many separations which are currently viewed as very difficult or "impossible" will soon be feasible using high-pressure chromatography under carefully optimized conditions. Separations of enantiomers (56, $7) as well as diastereoisomers (88, 39) will be more widely applied. Similarly, separations of mixtures of isotopically substituted organic compounds (40, 41) and studies of isotope effects using doublelabeling techniques (@) should become more nearly routine for the chemist. Clearly, the science of chromatography has just started to develop. It offers a challenging area for basic studies that will allow it to fulfill its bright promise for the future. Literature Cited ( 1 ) GOLDGTINE, H. H., Seiaee, 133, 1395 (1961). ( 2 ) Snaw, R. R., Sciaee, 140, 606 (1963). (3) BUSH, M. T., AND DENSEN,P. M., Anal. Chem., 20, 121 (1948). (4) ROGERS,L. B., "Principles of Separations" in "Treatise on Analytical Chemistry," (Editors: KOLTAOFF, I. M., AND ELVING, P. J.), Interscience Publishers, a division of John Wiley & Sons, Inc., New York, Part I, Vol. 2, p. 917 ff. (5) PURNELL, J. H. AND QUINN,C. P., "An Approach to Higher Speeds in Gas-Liquid Chromatography" in "GasChromatogrsphy," (Editor: SCOTT,R. P. W.), Butterworth, Washington, 1960, pp. 184-198. J. P., RESCHKE,R. F., FRED( 6 ) HISHT.A,C., MESSERLEY, EmCKs, D. I
