Chemical Instrumentation 5.
L LEWIN, N e w
York University, Washington Square, New York 3, N. Y.
feature
low solute concentrations, the proportion of the component that is extracted into the second or stationary phase (phase 1 in the diagram) is greater t,han a t higher concentrations. Since the rate of travel of the component down the column, under the influence of the flowing phase (phase 2 in the diagram), is slower, the greater the proportion extracted, the component will move down slowly where C O ~ ~ . ~ > J ~ O ~ its concentration in the leading edge of the zone is low, and more rapidly where it is high. Hence, the portion length of s packed column causes the s ~ p - of the sane having a, high concentration xratian between elution peaks to increaw, will tend to overtake and swallow-up but the consequent increased retention the low concentration rrgion of the front, volume makes t,he penks less easy t o with the result that the leading edge discern. Increasing the crass-sectional ! d l tend to he sharp and to correspond diameter of the column permits larger t o a high conccntrstion. The tailing samples to be chromatographed, but the a t the back edge of the zone is due to the tendwcy for the fronts to boeome irregular fact that the amount of component end to angle away from the horizontal desorbed by fresh solvent moving down also increases because of the difficulty of maintaining a h i ~ hdegree of uniformity in a large bed of very small particles. When effluent is collected, the more irregular and non-horizontal the fronts are, the more diffuse and indistinct does the semration of eom~onents mnem to be, even though the actual sepsrstion of these components on the adsorbent ilself may be good. This fiituation is illustrated by the diagramsgiven in Figure 16. The degree of sharpness of fronts, as well a s of the leading edges of elution peaks, is determined by the physical chemical nature of the adsorption or distribution equilibrium (cf. Figures 2 and 3 ) . In most cmes, 8. ~hromatogmm Figure 16. lllvrtration of the fact that the more d l be better, the closer to equilibrium irregular 01 non-horizontal the fronts ore wilhin it is possible to maintain conditions a column, lhe less well-resolved doer the elution throughout the length of the column. chromotogrom become. For example, consider a convex distrihution curve, such as 111 in Figure 2. At is always a fraction of what had been adsorbed just before, so that the net amount adsorbed a t a given place decreases as a geometrical ( i . , exponentid) function of the volume of the eluant. The various possible shapes of the partition curves (Figures 2 and 3) show that many types of chromatographic behavior are possible, ranging from self-sharpening fronts to fronts that become progressively less sharp with distance of travel. The aohievement of equilibrium conditions in the column is facilitated by the use of an adsorbent of small particle z size and high porosity. However, eolumn packing8 of fine particles have a high resistance to fluid flow, and if the eluant takes too long to pass through the column, much af the sharpness of separation can he lost as a result of the tendency for ML. 8. diffusion to wipe out concentration differences. Consequently, i t is common Figure 15. Graphs of concentrotion versus practice in column ehromatogmphy with volume of emvent for very well resolved certain adsorbents to use fine particles chromotogroms of o three-component sample. A. ~ m t a development. i B. ~ h t i o ndevelop. (e.g., No. 325 mesh) and to apply a presment. sure of several atmospheres to the top
T h i s series of articles presents a swuev 4f the basic principles, characteristics, and limitatio& of those instruments Gh&h find im&wtak applications i n chemical work. The emphasis i s on commercially available equipment, and approzimate prices are quoted to show the ordm of rnkgnitzde of East of the uarious types of design and construction.
14. Chromatographic Equipment In column chromatography, the diameter and length of the column container, as well as the particle size and packing of the adsorbent bed, have controlling effects on the sharpness of the separations obtained. Ideally, in an elution ehromatogram i t would be most desirable if the several components could be resolved completely from each other, with welldefined boundaries for each zone; in s. frontal chromatogram one might hope for sharp, almost discontinuous tctnsitions from on? front to another. These conditions, which are shown schematically in Figure 14, a m of course not observed
A
FRONTAL
E. E L U T I O N
Figure 14. Schemolic representation of hypothetically perfect frontal and elution chromatograms, neglecting the nabre of the physical phenomena giving rise to the reparations.
in practice; illustrations of the characteristic appearance of very good frontal and elution ohromatagrams are given in Figure 15. It will be noted that the transitinn between successive fronts becomes less distinct, and the tailing or diffuseness of an elution peak becomes greater, RS the retention volume increilaes. The retenlion volume of a component
~ ~ t ~ ~ S ~ , " : " ~ZY;,"~",~ , " , ~ ~ ~Eb6:j, " that component to the bottom (i% to cause it to "break through'' and become rvidrnt in the efflumt). Incrensing the
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Chemical Instrumentation of the ~rruving liquid phase t o whivve a. reasonable rate of flow. Thus, x glass column for this type of work should be made of thick-mlled tubing capable of withstanding 3-4 ntmasphcrrs (rf. Figure 10). The advant,ages of the use of oolnnnm packed with srlsorhent stem principally from the flcribilitv these over with respect t o t h r composition of the twophase system (i.e., choice of ~dsorhrnts and elnent,s), and the pos~ibilityof nd, justing t,he operating conditions (e.g., column rlimensiuns, Bow r a t r ) t o the characteristics of the sample bring malyeed. A packed column is most appropriate if semimicro or micro scale samples art. t o I E worked up. The disadvnntages of parked columns include the ncemsitp of cither extruding :tnd carving up thc adsorbent bed in order t o sopsratc t,hr zunrs of components, or of eollpcting, evaporating, or i,therwisc aualyzing large volumes (or a largr nunnher oismall volumes) of effluent. Column chromatography is often very timoconsuming and t d i o u s . Thc locntion of the various (,lot\-colored) romponmts within thr c o l ~ t ~ nis n generally not w e c~ptil,lrt , trst, ~ and i t is not pussible to run n stanclnnl s m ~ p l oside-by-sidc v i t h an unknorrn O I La givrn column. h 4s aconsequwre of the mmigulative inconv~nienccof r o h ~ m nchromntogrxl~hie praerdores, mr~rheffort hes b ~ m dcvotcd to the development of alternative, more attradive techniques. The most surwssful of thpse terhniques is p n p o rhromnlography, in r h i r h a strip or &rt
Figure 17. The Gordon-Mirco multiple tube equipment of Microchemical Specidtie5 Co. for p o p e , strip chrom.tography.
of palxr, composed of cellulose or glass fibers, nnd, if desired, impregnnted with ion exchange resins, serves ss the solid phase. The paper may function as s surface-active phase (adsorplion chromatography), or merely as a suppwt,ing substrate t o immobilize a film of n liquid (Continued on page A 5 i l )
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Chemical instrumentation (parlition chrmnatography), or it may combine these functions a8 well as some degree of ion exchange activity. The two principal means employed for creating the Bow of the developing liquid over the solid or supported-liquid ~ h i t s eare the ascending and the descending techniques. In both cases, the motive force is supplied by capillary action; in the former, this is opposed by the force of gravity, in the latter, gravity aids the capillmy foroe.
Ascending Paper Chromatogmphs Far ascending paper chromatography, it is only necessary to mount the paper strip or sheet so that i t hangs vertically, with ite bottom dipping into a reservoir of the developing liquid. In order t o prevent irregular and irreproducihle Bow patterns and temperature inhomogeneities, the paper must be contained in a sealed volume which is saturated with the vapor of the developing Liquid, so that there is no tendency for vapor t o distill from the wetted portions of the paper. If the wet paper touches its container a t any
Kurtz-Mirornon frame of Kensco for ~scendingpaper strip mounting.
Figure 18.
point except where it is in contact with the pool of liquid on the bottom, siphoning of liquid will occur, and the chromsttographio development will be vitiated. .4 number of containers and mounting (Conlinued on page A572)
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Chemical Instrumentation accessories are commercially availsble far ascending work, ranging from simple, stoppered tubes for single strips, to large battery jars capable of holding a hundred or more strips for simultaneous devclopmcnt. A convenient sin-tube unit for multiple
Tube dhromatography Labbarstory, onodbnmsiannl nsccnding, W8) is shown in Figure 17. Ench tube consists of a Pyrex cylinder open a t both ends, fitted with polyethylene stoppers. A solvent cup with sloping sides, so that the paper strip can dip into the liquid without touching the sides, sits on the lower stopper, and s. clip holder for the psper strip is ~nountedin the upper stopper. Special cups are also mailable for attschment to the upper stopper so bhnt these units can be used for descending ehromatogrzphy (6 cups, $20). Polyethylene or Teflon clamps are employed for mounting the paper strips on a glass frame in the apparatus made by Kensington Scientific Corp. (Iiensco), Berkeley 10, California, fonnerly California Laboratory Equipment Co. (Calab), ss illustrated in Figure 18. The funnel attached to the verbiesl tube is used far adding solvmt to the bottom of the container. S t n n d d units are svailable consisting of n glass tank with closefitting cover, a rack for holding 20 frames each of which carries up t o 7 one-inch wide paper strips, and 280 polyethylene clamps (No. l W i ,$159). A cont,ainer for ascending chromatography with square paper sheets, so that
Figure 19. Kenrco stainless steel tank ascending paper chrom~togrophy.
for
two-dimensional separations can be carried out by employing two runs with different solvents, the psper sheet being rotated by (10' between runs, is illustrated in Figure 19 (IZensco No. 1013 Stainless Steel Tank. $41.20). The tank dimensions are chosen t,o provide for the mounting of two 8 X &inch wide sheets, with a small (Continxed on page A574)
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Chemical Instrumentation free tank volume, so that vapor equilibration is rapidlyaehieved. The paper sheetis held a t the top hy means of stainless steel spring clips to stainless steel or glass rods. The tank is covered with a flat plate glass cover to permit observation while preventing loss of vapor. In same cases, a specimen migrates up the paper very slowly, and i t may be desired t o continue the ascending chromatogram far long periods. For thia purpose, a cover glass with nmrow slits may he employed to seal the tank; the paper sheets are cut long enough to permit them to stick out of these slits. When the mobile phase reaches the top of the tank, it evaporates from the paper, and fresh liquid continues to mount up the paper to replace that which is lost by evaporation. A large tank constructed entirely of */.-inch thick polyethylene, with a plate glass viewing top, and capable of mounting eight 17 X 17-inch sheets for aseending chromatography is available from Pslo Laboratory Supplies, Inc., New York 7, N. Y. (Chromatography Bench Unit, Model 7700, 8154). A novel annroaeh to the desien of a
fornia, shown in diagrammatic form in Figure 20. A paper strip, one inch wide and up to 17 inches long is placed an
E
A
B
C D
Figure 20. The Chromatobox, manufodured by Research Specidties Co. A. Teflon band. B. Paper strip. C. Glass plater D. G1.s~ rod. E. Holding ring. F. Solvent chamber.
a Teflon strip, and the pair is rolled up into a coil. The Teflon has a n embossed surface of many small raised dots, as shown in Figure 21, so that there is relatively Mtle actual contact with the paper strip; in addition, the non-wettability of Teflon by most solvents helps t o insure that the sample and solvent will travel along the paper, and will not leak off onto the plastic. The coil assembly is placed in a molded plastic box, with the free (wick) end of the paper anohored between glass plates and dipping into B 8mdl d v e n t compartment. Because of the smell size of this unit, saturation of the enclosed atmosphere and development of the chromatogram require only 6-8 ml of solvent (Model A-801,510).
Descending Paper Chrornotogmphs The design of chromatographe for the descending technique diPiers from that (Continued on page A576)
Chemicul Instrumentation -
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employed far ascending work only in the requirement for a, reservoir of solvent t o be laced a t the top ol t h paper ~ sheet, rather than a t the bottom. The typical
Component ports of the ChromatoBox and lid. B. Holding ring. C. ~ l o . 3plater. D. Rod. E. TeRan Bond.
~ i g v r e21.
box
A.
construction of such a r e r e ~ o i ris shou-n in Figure 22. Thc paper is held in a solvent trough by the w i g h t of a glass anchor rod. Precautions must he taken to prevent liquid from siphoning out of the trough by climbing along the paper to the rim of the trough and t h m running down its unrlerside. Thi. ir ;\voided by
Figure 22. Detoii of the design of a typical solvent reservoir and paper wpport for dercending paper chromatography.
making the paper rise up some distance above the trough before it changes dirpetion and falls t o the floor of the container; the anti-siphon rods provide the required support to hold the paper away from the trough. Figure 23 shows a hydro~netrr iar armaratus made by Kensington Scientific c&. for descending or a s k l i n g paper sheet or strip chromatography (KO. 1015, 1 , I t consists oi s. st,ainlcss steel holder for the paper and solvent, and s Pyrex hydrometer jar, 4';s X 24 inches,
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Chemical Instrumentation v i t h a ground flat top and a stoppered late glass cover. For descending work, solvent is placed io the upper trough; for ascending work the solvent is introduced through the hollow center rod into a trough a t tho base. After insertion of the paper inside thewire struts, theapplication of the sample, rhrornittographb~g, drying! and staining em all be carried out w t h a u t touching or handling the paper.
Figwe 23. Kenrco hydrometer jor opparotur for ascending or descending paper chromatogRemovable upper trough. D. Holraphy. A low center rod. E. Center receptacle for solvent which feeds paper roll dipped over center rod to aid in vapor ratvrotion of enclosed rpace. F. Bare trough. H. Wire rtrutr.
Containers for paper chromatography are most generally made of glass or of stainless stocl. The metal const,metions are more compact and rugged, and permit the solvent trough to he mounted on hrackets bolted to the upper walls. However, stainless steel is corroded by halogencontaining substances, such as HC1, and its me should be avoided when solvents containing such substances are to be employed. Glass containers have the advantage of being resistant to all the solvents used in chromntagraphic work, but t h e mounting of a solvent troueh presenta problems. One approach is the use of sn annular ledge near the tap of a glass cylinder as n support for the trough (see Figure ?-I),or sn all-gl.lass support rack reaching from the hottoln to the upper portions of the cylinder.
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Chemical Instrumentation When some stainless steel oarts can be
Figure 24. Ail-glon apparatus for descending paper chrornotography. Solvent trough is mounkd by resting on on onnulor ledge.
Horizontal Paper Chromatographs Several convenient devices have heen proposed for horizontal paper chinmatogrnphy with circular discs. A cornnwreinlly available design that permits the rapid cornparisou of unknowns rind
4; 0 '
Figure 25. Upper solvent troughs may be held in ploce in gloss opporatvs b y means of rtainlelr steel spring bands, or b y o rack rondruction.
standards is the Kawerau apparatus. shown in Figure 26, which is made I,?the Shmdon Scientific Co., Ltd., London: U. S. distributor, Consolidated Laboretories, Inc., Chicago H e i ~ h t s , Illinois (No. 2020, 14inch diameter unit, S28.20). A slotted paper disc is placed with its center resting an the top of a glass capillary tube which dips into a solvent reservoir. This capillary serves as a. wick t o feed solvent t o the center of the paper, from which point i t spreads out radially by (Continued on page A582)
A580 i Journal of Chemical Education
Chemical instrumentation "apillnry nelion. The rxdinl slots dividr the paper into tive pqunl sertors, sn that five samplm m u hc drveloperl simultaneously.
Figure 26. Kowerau opparotm for horizontal paper disc ~ h r o m ~ t o g r o p h y .Upper drawing shows construction details; lower shows o typical chromdogram. A Dish 8 . Glarr support far tube. C. Slotted poper circle. D. Cover. Lower photo: Typical poper disc chromatogram with 5 different specimens.
Centrifugally-Accelerated Chromatographs -\n nsrmdioe chromatoeram mav rr-
f hrranse the flow of dcvrlopw is acrekri~trd by tho pull of gravity. Instruments have lwcn designed in nhirh the forrr of gravits is replaced hy eentrifugi~liorce, and by tllis means rhromatograms vhich wor~ld require a e v ~ r n l h a w s I,? thc descending technique can bc rompletcd in minutes. h srhemetir disgmm of the- Chromatofuge (51205) of Lahlinc, I . Chicago 22, Illinois, is sho~vain Figure 27. A circle of tilter paper, up t o 18 inches in diameter, is fitted ont,o a shaft t h a t is rotated by n variahle-speed motor ( u p t o !a00 rpm). I t is held in place by a Teflon gasket, and is supported on an array of metal pins. Whilc the paper is whirled, solvent is fed onto the nhwt continuously through a rapillary feed tube mounted close to the rotating shaft,. The solvent R o ~ vrate is controlled hy t,he diameter oi the capillary, and a. set of interchange a b k rnpillnry tubrs ia providod t o permit (Continued on page A584)
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Chemical Instrumentation chuicc: of flow rates.
IL
Centrifugal form
w ~ u s e s the solvent to Haw radially oot-
ward, produring ehmmnbographie d ~ ~ e l o p B
\
A
C
'\
D
\\
F M
G '\
E,L
'~$2.'i
n ' b
Figure 27. The construction d e t d r of the Chromotofuge manufactured by Lobline, Inc. A Cover. 8. Collector trough. C. Filter poper circle. D. Support pins. E. Rotating head. F. Solvent feed pipet. G. Hold-down nu^ H. Drive shaft and bearing osrembly. J. 'lr-hp variable speed drive. K. Spring I. Short bore capillary feed closure real. tube. M. Teflon sealing ring.
ment of any sperimrns which had prcvioraly been plimd an the paper. Special provision is made for saturating t h r eneloscd atmosphcro with solvent and preventing l a s ~of solvmt vapor during the centrifugation. Reproducibility of migration rvrlocities is aided by thc incorporation of fast starting and braking mechanisms, and the specd of ratstion is shorvn on a eontinuonsrending t b chomctcr. A smaller and sirnplcr vorsion of this type of apparatus is manufactured by Precision Scientific Ca., Chicago 47, Illinois (Hi-Speed Chrometograph, $250). A directdrive ac motor is employed, n i t h a ehoicc of rotation speeds hetween 300 and 1500 rpm. Solvent is fcrl onto t,he papcr through a 0.1-mm orifice in a stainless steel nozzle, and t h r rat,ct.c of flow is controlled by adjusting the air prcssure over the solvont in n res~rvoir t h a t feeds tho nozzle. Paper cirrlcs up t o 121/2 inrhus in diameter can he arcommodaterl. An explosion-proof version of this instrument ( S i 5 ) is available, in which a n air-driven motor is usctl, t o avoid any possibility of n spark from an electric motor igniting solvent vapors which may ho present in achromatography Inhoratory.
Thermally-Accelerated Chromatographs The migration velocity of i n m y constituents undcr the influence of %: moving liquid phasc can he n m r k ~ d l yincrcascd hy raising thc temperature, and efforts have heen mndc t o speed up chromatographic development teehniqucs by operating a t elcvatcd temperatures. This has not paved successful in the stan&wd ascending or descending trehniqurs heeaus,: of the ditfirulty of maintaining uniform and comt,nnt conditions throughout thc system. Howevor, x t ~ r h n i q u e t,aaed upon n. horisontslly supported pnprr shret contwincd in a flat, senled bray nhieh is kept in a thermost,stically-controllrtl (Continued on page A5861
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Chemical lnstrumentution oven has bwn found t o surered iu achieving ntleql!nt,r separations in 2 t o H hours for constitwnts which require $8 t o 96 hours hy the room-temperature ascending technique. The m ~ t h a d of m o u n t i n ~ the paper is i l l u s t m t d in Figure 28. One end of the papcr sheet is hrnt down :and insortcd in x soivrrbt
FILTER PAPER
G L A S S ROD
TROUGH
Figure 28. Arrangement of paper and feed trough in the Speedi-Cell elevated temperature migrotion rhornber.
trough; thr shwt is wpported on a wriw of rrlrtss rods, and thc othor end is wrr;ttrd to prorl!r are the concentrations of compound I a t equilibrium in phase 1 and phase 2 of the solvent system. This fact allows a very mathematical analysis of the quantitative results ohtainerl in counter-currmt extraction, and permits the direct comparison of erperimental a n d theoretical results. Volume
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Number 9, September 1961
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Chemical Instrumentation commercial models of this instrument are available from Precision Scientific Co., Chicago 47, Illiuois (Junior Model, complete with power supply, $510; Senior Model, $1195).
Figure 29. Details of the design d McDonald's lonograph for paper electrophorerir with free, horizontal strips.
Th? pnprr strips are thoroughly wetted by a huKer solntion, and are mounted so that they rest light,l>-on the moisture equilihrirm hridges, and are pulled taut by the spring-loaded end clamps. The ends of the pnpw strips dip into electrode compartments a h i r h are filled with buffer solution and which contain platinum electrodes for application of the electric field. Caw is taken to insure that the paper are p~rfectly level and horizontal, and that the liquid levela in all compartments are exactly equal, so t,hat there will he no tendenev for liquid phase t,o siphon from one point to another. Since the passage of elerbric current through the liquid on the paper generates heat, it is also essential t,hat heat be condncted away rapidly, so t,hat it does not cause liquid t o distill off the paper, thereby creating irregular flow rmttern~and interferine with the electrophoresis. In this instrument, the applied voltage and the consequent currents are m o d w t e ; for runs made a t temperatures below room tempwature, no special cooling is required. At room tempemtme, if the power input t o the paper strip (P = V X I) is 0.25 watts or more, the air in the chamber is replaced by helium, which has a mnrh higher thermal conductivity. Although i t may be true in some cases that t,he electromobility of a component on a wetted paper strip is the same as i t is in the free liquid, this is the exception rather than the rule. I n general, adsorptive, ion exchange, and partition effects are of prime importance, and paper electrophorenis is a form of chromatography (as defined in this discussion) rathm than of electrophoresis. This is t r w , whether or not solvent flow is per-
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(Continued on pagr A5.90)
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Chemical Instrumentation mitted to cantrihute to the migration induced by the electric field I n some paper clectrophorcsis devices, solvent flow is scrupulously %voided,
sin. An -
