Topics in..
.
Chemical Instrumentation feature Edited by 5. Z. LEWIN,
New
York University,
New
York 3,
N.Y.
These articles, most of which are to be cont7ibuted by guest authors, are intended to serve the readers of this JOURNAL by calling attentima to new developments i n the theory, design, or availability of chemical laboratory instrumentation, or by presenting useful insights and explanations of topics that are of practical importance to those who use, or teach the use of, modern instrumentation and instrumental techniques.
XXIX. Gel Permeation ChromatographyJack Cazes, Mobil Chemkal Cornpony, Metuchen, N.I. Gel permeation chromatography (GPC) is the newest analytical tool available for the fractionat,ion and characterization of polymers. I t is rapidly becoming indispensable in the field of polymer science both for the determination of molecular weight distribution of polymers and for the frxt.ianstion of medium and high molecular weight materials for further characterization by other physical and chemical methods. The basic principle of gel permeation chromatography can, at fint glance, be likened to that of a. nonionic molerulsr sieve having pores ranging in size from 10 to 107 angstroms. The separation of molecules according to size is envisioned to take place within the voids which are nresent in the articles of a ngirl, rn,.+lwknl poly;t?n,nr grl. Thr struerlire of ? u ~ h a gel is at~.+lwgc.u,to tile structure of an ion exchange resin but without its ionic sites. The gel, composed of rigid, crosdinked heads of tapprox~~
~~
~
Figure 1 . Phofomicrogroph of p0iyltyrene beodr.
crora-linked
imately 200 to 300 mesh size, is prepared by suspension copolymerization of styrene with a difunctional monomer (erosti linking agent) such as divinylbenzene in the presence of a diluent. I t is possible to prepare a gel having a given maximum pore size and a given pore sine distribution by proper choice of cross-linking agent and type and cancentrstion of diluent. Figure 1 is a photomicrograph of the spherical beads that are produced. Historicdly, gel permeation chromatography began with observations made during exchange studies; same non-ianic substances were fractionated by passage through an ion exchange column. S e p aration seemed to he according to molecular size. For example, i t wa? noted that ethylene glycol, glycerine, and sugar were separated on a cationic polystyrene resin by elrhon with water; the sugar eluted first,, followed by the glycerine and then the ethylene glycol. I t is not certain whether snch separations were due to a true "molecular sieving" or a t least partly t o some small adsorptive effect. I t was not until the group headed by J. C. Moore of the Dow Chemical Company first r e ported their work in 1963 that gel perme ation chromatography was really horn. Moore, in 1964, described the procedure for reproducihly preparing gels of given maximum pore sizes and illustrated t,he utility of the separation technique for the determination of molecular weight distributions of polymeric substances. I t wm Moore who named this technique "gel permeation chromatography." The separt~tion of molecular species according to size is believed to occur as a result of differences in the extent to which different species permeate the gel particles. Molecules whose size is too great to allow them to enter the gel pores paw through the column solely by way of the interstitial volume, i.e., the space occupied by the solvent outside the porous heads. Smaller molecules permeate the gel t o a
Jock Cam. is presently with the Analytical and Phy-icol Chemistry Deportment of the Mobil Chemical Company. He hor also been ~sfociotedwith both the Orgenic Chemicals and Polymer Departmenh of Mobil during the four yeorr he has been with the compony. Dr. Cores received a B.S. in chemistry (1 9551 a t The City College of New York, and an M S . in orgonic chemistry (19621 ond Ph.D. in organic chemistry 119631 at New York University. Research inkrests hove included organic ryntheris, study of organic free rodicol reac. tion mechanirm,, organic chemicol and polymer process research, and chemicoi instrumentation. Present position is concerned primarily with reparation methods research and opplicotionr. Dr. Cozer has taught ot Queens College 1N.Y.C.I and is presently on the rtoff of University College, Rvtgers University [New Brunrwick, N.J.I.
I
I
.LaRGE
SMALL MOLECULES MOLECULES.
Figure 2. Separation of molecules according to sire by gel permeation chromatography.
(Conlinni~don page A568)
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A567
Chemical Instrumentation greater or lesser degree, depending upon their size and upon the distribution of pore sizes available in the gel particles. Consequently, the largest molecules emerge from bhe column first,, followed by smaller molecules which must follow a more circuitous and tortuous path as they travel bhrough the column. This order of elution-large molecules first, small molecules last-while not surprising here, does stand out in contrast to that which is generally observed in other chromatographic techniques. An idealized view of the over-all process is illustrated schemat,icdly in Figure 2.
Mechanism of the Separation The general picture of what occurs in and near the gel particles during the s e p aration process is not thoroughly understood. It is not known whether the process involves a true, reversible, dynamic equilibrium, or near equilihriom, or possibly, one which is diffusion-controlled. I t is of interest t o examine some experimental observations in order to understand the situation that exish The resolut,ionachieved with a given gel permeation column is often improved as the eluent flow rate is decreased. For all practical purposes, resolution may be continuously improved by a continued decrease in flow rate, a.lmost down to a flow rate of zero. This is a common observation in liquid-solid chromatography. However, in gel permeation chromatography the improved resolution manifests itself as a decrease in peak width rather than as an increase in the differences between the elution volumes of the individual components. Elution volume, for a given component, remains almost constant.. If the mechanism of gel permeation chramatography involved a diffusion-controlled process, one might expect elution volume to increase with decreased flow rate. One must keep in mind, however the possihi1it.y that changes in flow rat,e might not affect diffusion within or near the gel pores; changes in flow rate might influence the diffusion of molecules through the interstitial volume alone. Another interesting ohservation can he made. The chromatogram obtained for a mixture is usually the graphical sum of the chromatograms obtained for the individual components. In other words, each molecule "does not know" that other molecules are nearby, provided that the column hss not been overloaded. As with other chromatographic techniques, this is a necessary condition if the technique is to he reproducible and generally useful. Resolution is improved by an increase in temperature. Elution volume dDes change with temperature. The improved resolution is commonly attributed to a decrease in the viscosity of the solvent as the temperature is increased. This results in greater freedom of movement of the molecules in solution, allowing them to enter snd leave the gel's pores with greater ease. This idea is further supported by the observation that two different solvents (Continued on page A670)
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Journal of Chemical Education
Chemical instrumentation having the same viscosity give almosL identical resolution for a given sample. Diffusion control of the separation process is certainly suggested. To date, no systematic st,udy aimed a t the elucidation of the mechanism of the gel permeation chromatographic process has been reported. Perhaps this lack of basic data is the result of fascination with
ready yielded rapid answers to many questions involving the molecular weight distributions of polymers that were heretofore left unanswered because of the extreme difficulty involved in earlier methods. There is no doubt,, however, that some workers will turn to more basic problems involving the gel permeation chromatographic technique itself. Instrumentation The instrumentation reqoired far gel permeation chromatography ran he ai simple as a glaw tube packed with the rigid, crosslinked polystyrene beads mspended in a soitahle solvent if only coarse fractionation is required. However, in order to achieve precision approaching the ela4cal molecular weight methods, several parameters, such as flow rate and temperatore must be closely controlled. The complexity of the equipment is determined by several factors, including the degree of reproducibility that is required, the sample size, and the exbent of aotomation and convenience of operation that is desired. U a d y , however, the final decision a5 to the extent of refinement of t,he instrumentation is a compromise between the factors mentioned above and the cost of the eqnipment needed to achieve the end result,. An attempt will be made here to examine the role of each functional unit in the idealised gel permeation ehromntograph and to criticdiy discuss the advantages and disadvantages of the design features involved in its construction. The instrumentation needed to carry out gel permeation chromstogrrvphie separations can be conveniently discussed under the following functions1 headings:
I. Solvent handling 2. Sample introduction
3. Fractionation 4. Detection, estimation, and collection The degree of sophistication of the instrumentation used to carry out these fun* tions is limited only by the ingenuity and budget of the designer, and ult,imately, the user of the equipment. Solvent Handling
The solvent handling system must store the solvent, remove gases that may he dissolved in the solvent, and carry the solvent, a t a constant rate of flow, pmt the sampling device and to the head of the column. Constancy of flow rate is of prime importance since resolution can be (Continued o n page A678)
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Chemical Instrumentation adversely affected by variations in flow rate. Changes in resolution resulting from an erratic flow rate lead to poor reproducibility, and chromatograms recorded under such conditions do not lend themselves to eompilrison. Furthermore, the use of gel permeation chromatography as a meana of determining molecular weight distribution involves calibration of the equipment with standards of known molecular weight. Such calibration requires a, stable and reproducible solvent flow rate. A non-pulsing liquid pump would appear ~
~i~~~~ 3.
A572 / Journal of Chemical Education
~
use of surge tonk to damp out pumping
pable of pumping liquids st a conshot flow rate of a few milliliters a minute against a hack pressure of from 50 to 200 psig are just not readily available. The usual approach to s. problem of this type is to use a pulsing, variable stroke, liquid pump in conjlmdion with a. pulse huffering system; t,he simplest buffer is a surge tank or. as it is sometimes called, a. ballast tank. The surge tank is placed in the solvent st,ream immediately after the pump,. Its volume is chosen so that pulsabons produced hy the pump are damped out,. Solvent flows from the pump into the surge tank and then to the column. This is shown schematically in Figure 3. Solvent must he freed of any dissolved eases mior t o enterine a heated column. t
(Conlint~edon page A574)
Chemical Instrumentation possihle farmstion of gas huhhles within the column. Gaseous spaces in the column packing lead to what is generally called channeling with a resultsnt nonhomogeneous pat,tern of solvent flow through the column. The solvent is forced to flow around the gas packets a t uneven rates and broadened and irregularly shaped chromatographic peaks are observed along with greatly diminished resolut,ion. Tho observed chromat,ographic curves do not reflect the true distribution of the sample being fractionahed. Degassing can he most convenient,ly a* complished by passing the solvent through s. vented heater maintained a t a temperature just below the boiling point of the solvent. Most of the dissolved gases me ihen boiled out of solution and vented to the atmosphere. The hest location for the degasser is probably just ahead of the solvent pump where t,he solvent is s t atmospheric pressure and venting presents no significant problems. Placement of t,he degnsser in s. pressurised part of the system would neces~it,ate heating the solvent t,o a temperature higher than its normal boiling point in order t,o remove dissolved gases. Furthermore, it would be necessary to provide for removal of gases under pressure to a point where they could not redissolve; venting of gases under pressure without loss of solvent would he inconvenient a t best,.
Somple Infrodudion The sample is generally prepared as a to solution in the fixme solvent that is used as the eluent. The sampling system must be capable of introducing the sample solution into the flowing solvent stream a t apoint as close to the head of the column a3 possible. Ideally, the sample should he placed ont,o the column in as short a. time as possible so as to produce a narrow band or "slug" of sample. One way of accomplishing this is to inject the sample solution directly into the solvent in or just ahead of the eolumn with s hypodermic syringe and septum arrange ment of the type generally used in gas chromatographs. Figure 4 illust.rstes such an arrangement. A possible disadvantage of this method of sample introduction is that the syringe is subjected to whatever pressure is prwent in the system s t the time of injeobion. Another more advantageous approach involves the use of a four-port valve arranged as shown in Figure 5. A cslihrated sampleloop is filled with thesample,
8-
SYRINGE
T O COLUMN
Figure 4.
Septurnfsyringe
injection.
(Conlinued on page A678)
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Fractionation
Chemical Instrumentatien
The heart of any column chromatographic instrument is the column, which consists of a shell (usually a length of tubing) filled with the column packing. It is in the column packing that the various species present in the sample are separated from each other. Consequently, one cannot be overly meticulous when preparing s. gel permestion column. The tubing can be made of any now reactive, insoluble material that will contain the packing and with?t,and the operating temperature and pressure. Some suitable, readily svdilahle materials are glass, copper, aluminum, and stainless st,eel. At present it is just about unitnimonsly agreed that the calumn should have a uniform circular cross-section throughant its length, and lhat i t should not be coiled. With such a. column, one is generally able to maintain b homoge-
a t at,mospheric pressure, from a syringe while the valve is in the "filling" position. In this way it is possihle to completely flush out the sample loop with sample and, if desired, heat, the sample in the loop prior to actually passing it into the column. The valve is then rotated to the "emptying" position, placing the sample loop directly into the solvent stream. The amount of sample actually entering the column is determined by the concentration of the sample solution, the size of the sample loop, t,he time that the loop is in the solvent stream, and the flow rate of the solvent. A list of manuiacturers of mdtiport valves is given further on in this paper. Such valves generally cost from $100 to $500 depending upon the material of construction.
SAMPLE s*soLvENT
SAMPLE
ms
SOLVENT
TO COLUMN
TO COLUMN
\j
\j
WASTE
WASTE
FILLING LOOP Figure
I
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Use of multi-port valve for somple introduction.
Journal of Chemical Education
EMPTYING LOOP
neous solvent flaw pallern. .4 nsrrowly coiled column W U produce severely broadened overlapping peaks as a. result of the fact that the solvent flowing along the outer circumference of the coil must travel a longer distance than the solvent dong the inner circumference. No doubt there artre some investigators who will attempt to use column made with tubing of circular crosssections, but having a conical shape, in attempts to compensate far the decreasing pressure differences measnred between the exit of the column and points successively further away from the hesd of the column. Others will probably attempt to use square, triangular, or annular tubing, or tubing with other cross-sections as has been tried in gas chromatography. While such a p proaehes ofttimes produce novel effects, no dist,inct general advantage is realized. A word might be said about the use of annular columns since they might offer some advantages in cases where increased sample capacity is desired or where temperature programming is to be used. By using an annular column, the total cross-sectional area of the pscking can be greatly increased without significantly increasing the thickness of tho packing. I t is possible that annular columns having a. large outside diameter with only a, narrow annulus of packing could present less difficulty in the establishment of homogeneous flow then colnmns having a solid circular cross-section. It is not likely that temperature programming will be used with gel perme*
(Continz~erlon page A679)
Chemical instrumentation tion chromatography, at least a t present, since it would not be expected to produce the drsmatically improved resolution that its m e provides in gas chromatography. However, if temperature programming should be desirable, annular colnmns would offer a solution to the heat transfer problem that is involved in uniformly changing the temperature of the column packing in a reproducible manner. As wit,h any chromatographic column the process of packing a GPC column should be carried out wit,h great care. Uniformity of packing density together with complete absence of voids are absolutely essential. The cross-linked polystyrene gel particles must he kept completely immersed in solvent while being transferred to the column. If the gel particles are allowed to dry, leaving the pores filled with air, i t is virtually impossible to restore the gel to its original state of efficiency. One is generally forced to prepare a f r a h hatch of gel. The efficiency of a gel permeation oolumn is commonly expressed in terms of the numher of "t,heoretieal plates" i t contains. The theoretieal plate concept is borrowed from that area. of chemical engineering involving fractional disbillation. A t h e e retical plate, in the ease of distillation, refers to a discreet distillation stage constituting s, simple distillation in which complete equilibrium is established between the liquid and vapor pheses. I n t,he case of gel permeation chromatography, where the two phases involved are in constant motion, i.e., the solvent in the interstitial volume and the solvent within the gel pore, equilibrium is probably never achieved and the true significance of the theoretical plate is lost. It must be realired, moreover, that the calculated theoretical plate in a chromatographic column represent3 smaller separating ability than the theoretieal plate in a distillation column by, perhaps, s. factor of t,wenty-five or fifty. Nevertheless, i t is still convenient to odcleulste the numher of theoretical plates for s chromatographic column as a means of making comparisons hetween the efficiencies of different chromatographic cnlnmns. The number of theoretieal plates in a colnmn is not only a function of the column and its operating parameters (woh as temperature, pressure, solvent, and flow-rate), but also depends upon the sample species being separated. For emmple, if t,he number of theoretical plates in a eolomn is determined under a given set of condit,ions wibh substance A as t,he sample, t,his number will probably be different from that which is determined with substance B, having the same or different elution volume, 35 the sample A. Therefore, if one is to examine a. sample containing several qxcies, then the plate wunt determined using a single p u e subst,ance is, a t best, only an approxim~t,ion of the true plate count for each species present in the sample. The procedure for determining the plate wunt is as follows.
(Continued a page A680)
Chemical Instrumentation 1. A chromatogram is n m with s, single pure sitbstmce as the sample. o-Dichlarobenzene, tetrahydrofurnn, and 1,2,4,-trichlnrobenzene are the snbstancps that are commonly used for this pnrpose in GPC. 2. A briangle is eonstn~ctedon the chromatographic peak by drawing a line across the bsse of the peak and two lines tangent to the sides of the peak a t the points of maximnm slope. This is illustrsted in Figttrc 6.
Figure
6. Calculation of
column plate count.
3. The distancefrom t,hepointaf sample iniection to the aoex of the neak snd
where
P = plates per foot f = no. of ieet,of column z = ml elution volume from injection to peak apex d = width of base in ml. Detection, Esfimotion, and Collection Detection and estimation of sample components eluting from the column can be carried out either by continuously monitoring the effluent stream or by collecting fractions of the eli~ate and estimating the sample components present in each fraction individually. The advantages of continuous monitoring include convenience of operation and the ability to couple such a system to other automatic electronic devices for recording and integration of the ohromatographic curve. An advantage inherent in the collection of fractions is the ability to characterize the various species present in each fraction by independent instrumental and chemical methods. Of course, the mast desirable approach would be to continuously monitor the column effluent and collect fractions a t some paint beyond the conbinuous monitor. The usual sample size in analytical scale gel permeation chromatography is only a few milligrams; the sample emerges from the column over a. twenty-five to one hundred milliliter elution volume. This means that each milliliter of eluate contains less than one milligram of sample. Here i t becomes necessary to use a very sensitive detection metbod having a high signal-to-noise ratio. Advantage can generally be taken of one or more of the physical properties of the substances being
(Cnlinued on p a p A683)
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Chemical Instrumentation examined. For example, one might measure such properties as refractive index, dielectric constant, UV absorption, or fluorescence. With samples that are labeled with radioactive isotopes, one can continuously monitor the radioactivity of the column effluent. I n cases where fractions are collected, one can make use of both chemical and physical properbies of the sample for characterization of the fractions. Colored substances can be estimated calorimebricdly, while colorless suhstanoes may be converted to colored or fluorescent ones by addition of rtppsopriate reagents. A dramatic increase in detectian sensitivity can be realised, very often, by using a differential device. For example, one can readily measure a, difference in refractive index with much greater sensitivity than one is able to measure a. single value of refractive index. With this in mind, one can design the chromatographic system such that it has a dual strezm arrangement (analogous to "double beam" in speetrophotometty), one stream carrying only solvent and a second stream carrying solvent p h a sample. In such a system, one can then rnonit,or the difference in a physical property between the two streams. This technique lends i& self well to the application of servomechanics1 control, recording, and computatiou. The choice of a particular
A582 / Journal of Chemical Education
detection device for the construction of a I~horatorygel permeation chromatograph often is governed by the avaihhility, in the laboratory, ..of an instrument that is c a ~ a b l e of measuring a given physical property. A detailed discussion of s.11 of the possible detection devices, however, is beyond the scope of this paper. However, there is one detection device that ought to he discussed here because of it3 broad applicability and ease of operation. The automatic recording differential refractometer is capable of precisely measuring the difference in refractive index between two flowing liquid streams. The usefulness of this type of detector in GPC stems from the fact that it is applicable to a wide variety of organic compounds. I t does not depend upon the presence of a. given functional group or other structural feature of the molecules being detected. Theonly requirement is that there be a difference in refractive index between the solvent and the sample being examined. A commercial unit (manufactured by Waters Associates, Framingham, Mass., $5000) employs a dual barrier layer photocell detection system and is claimed to be capable of detecting a refractive index difference of 1 X lo-' refractive index units. Another (manufsctured by Phoenix Precision Instrument Ca., Philadelphia, Pa., 84840) employs a monochromatic source, chopper, and single photomultiplier, a combination which is claimed to result in good long-term stability. This unit e m detect a difference of 5 X 10-7 refractive index units.
Collection devices most often involve a mechanism for sensing the flow of liquid from the column by counting drops or by measuring a. volume through the use of a calibrated syphon. Somebimes a timing device is employed, although this is not as desirable as drop-counting or volumetric syphoning. Since, in gel permeation chromatography, a given species will emerge from the column at. a given elution volume, its retention time will vary with variations in flow-rate. Timing devices do not take into account or correct far variations in flaw-rate, while volumetric syphoning and drop counting do. A detailed discussion of commercially available fraction cutting and collection devices is the subject of an earlier paper in this series [Lewin, S. Z., J. CHEM.EDUC.,38, A515, A567, A713, A789 (1961)). A list of manofaet.urers of these devices is aiven a t the end of the present paper.
The conelusion of Ule article, "Gel Permeation Chromtographu" bg Jack Cazes will be published in the August 1966 issue.
