Ion Exchange Chromatography of Amino Acids Effect of Resin Particle Size on Column Performance PAUL
B. HAMILTON
Alfred 1. du Ponf Institbte o f the Nemours Foundation, Wilmington, Del.
F A back-washing technique was devised for grading spherical or irregularly shaped particles of sulfonated polystyrene cationic resins, approximately 8% cross-linked, into 20-, 2 5 , 30-,etc., micron diameter classes, each having 60 to 75% of the particles within st 3 microns of the average diameter of the class, and a diameter range of approximately 57 microns. Martin’s (or accidental) diameter, applicable to spheres and irregular particles alike, is proposed as a designation of particle size in favor of the less precise sieve number. Column performance in the separation of amino acids b y ion exchange chromatography a t the rapid flow rate of 39.3 ml. per sq. cm. of resin bed per hour was optimum with spherical particles of 25-micron or irregular particles of 30-micron diameter. A range of 20 to 40 microns of pulverized resin was equally serviceable, if size distribution was uniform throughout the range.
T
of resolution of amino acids obtained on columns of sulfonated polystyrene resins is a function of many factors, including the pH, ionic strength, and flow rate of the developing buffers, the temperature of operation, and the degree of cross linking and particle size of the resin (6, 7 ) . The sharp peaks, ivhich are an essential attribute of systems exhibiting high resolving power, require rapid attainment of equilibrium between stationary and flowing phase, and it has been recognized since the early studies of &layer and Tompkins ( 3 ) that resins of small particle size permit higher rates of flow through the column because equilibrium is attained more rapidly. I n attempting to define precisely the optimum conditions for the operation of an ion exchange column, it is advantageous to be able to secure samples of resin of accurately known particle size, possessing a narrow range of particle size distribution. The need for such resins has been rendered more acute by the recent development of automatic recording apparatus for use in the chromatography of amino acids (9), and the elaboration in this laboratory of an improved system of analysis with controls for automatically changing temperature and HE DEGREE
914
ANALYTICAL CHEMISTRY
buffer solutions in the conventional fraction collector technique. Both methods permit rapid separations and use rapid flow rates through columns of very finely divided resins. Details for obtaining resin particles of a serviceable size range by eliminating the fine and coarse material from commercial preparations have been described by Moore and Stein (6, 7 ) . The sodium form of the sulfonated, fully hydrated resin mas sieved through a standard 200-mesh screen, mith a jet of water, and the particles that passed the 200-mesh screen were reserved. The through-200-mesh resin was then suspended in a large volume of water and after settling, colloidal material and fines were decanted with the supernatant. This procedure i.j someir hat laborious and lacks precision, as the distribution of particle sizes within the 37- to 74-micron range is not revealed. The present investigation was initiated by the observation that the resolution of amino acids was better and the peaks were sharper in chromatograms obtained from the first of two consecutively poured columns. This suggested that a higher proportion of finer particles was present in the first column and that particle size might be responsible for this effect. To study this effect in greater detail, a method of classifying tlie mixed population of the commercial cationic exchange resins in current use in this laboratory was devised, which provided a means of obtaining several classes of particles easily and rapidly. Direct measurement showed that GO to 75% of the diameters in each class were within =t3 microns of the average diameter of the class. The method of classifying spheres of irregular particles of pulverized resins, and the correlation of particle size with column performance in the chromatographic separation of amino acids, are described in the present paper. Particle size can be defined in a manner applicable t o spheres and irregular phrticles alike. The accidental or Martin’s diameter proposed by Martin, Blythe, and Tongue (2) fulfills this condition and entails the simplest measurement. It is defined as the
distance betiyeen opposite sides of a particle, measured crosswise of the field and on a line which bisects the projected area of the particle. The arithmetical average of these measured distances of a field of particles is the average accidental diameter. Measurement and computation of Martin’s diameter can be readily made from enlarged photographs of a microscope field of resin particles taken under medium magnification. According to the thorough discussion by Chamot and Mason ( I ) , 3lartin’s diameter gives results in satisfactory agreement with those from the three actual dimensions of the individual particles. Throughout the present study, Martin’s diameter, in microns, is used to specify particle size, and the less precise, more cumbersome designation of mesh number is avoided. RESINS INVESTIGATED
The only resin in bead form investigated was some Dowex 50-X8, hydrogeli form, purchased in 1953, which contained a high percentage of spheres with diameters in the 20- to 74-micron range. The Dowex 50-X4, Domex 50-XS, Dowex 5OTY-X8, and Dowex 50-X12 (The Dow Chemical Co., hlidland, Mich.) from current commercial production contained such small percentages of spheres below 74 microns that they were not studied further. The resin powders investigated were prepared from spheres which were fragmented in a ball mill, shearing plate, or other type of pulverizer. They comprised irregular particles, a high proportion of which were in the 20- to 50-micron range. One of the pulverized resins was Amberlite CG-120, Type 111, sodium form (Rohm & Haas Co., Philadelphia, Pa., available from Fisher Scientific Co., Fairlawn, N. J.), which is the resin used for the chromatography of amino acids in the most recent procedures evolved by Moore, Spackman, and Stein (4, 8, 9 ) , and also by this laboratory. The other resins were Permutit Q-ZP, sodium form (The Permutit Co., Birmingham, N. J.), and Dowex 50W-X8, hydrogen form (Bio-Rad Laboratories, Berkeley, Calif.). All are sulfonated polystyrene polymers, approximately 8 to 8.5% cross-linked, of medium porosity, with an exchange capacity of 5 meq. per gram of dry resin. The wet density of the hydrogen form of Dowex 50-X8 and
I
I
I
6 0 - 2-LITER FUNNCL
I
I
I
I
1
I
I
Input flow rate (ml./minute) = IIR'(rne.x) x V(5takes) (37
1
D -V (CALCULATED ) AMBERLITE C G - ! 2 0 TYPE A---A OOWEX 5 0 - X E ( B E A D S ) x x x x DOWEX 50W-X8 e--*
50 -
B-B
W
.--.
6 LITER FUNNEL D-V (CALCULATED) AMBERLITE CG-I20 TYPE I II A--A DOWEX 50-X8(BEADS)
-
*---e
Y
I
0
50
I
I
I
I
I
I
I
I
100 150 200 250 300 350 400 450 FLOW RATE INPUT, ML.PER MINUTE
-
I
500
Figure 1. Experimentally determined relationship between flow rate into 2and 6-liter separatory funnels and diameter of particles removed
Ainberlite CG 120 is 1.21; the wet density of the sodium form is 1.27. 60 a* cm./minute (Stokes' Ian-) (1) PRELIMINARY PREPARATION OF RESINS
All resins were processed in the form in which they were received from the manufacturers-Le,, Dowex resins in the hydrogen form, Amberlite resin in the sodium form. Transfer of the dry resin from shipping container to glass vessel, and the initial hydration, were carried out under adequate ventilation in a fume hood to eliminate inhalation of particles. Depending on the size of back-washing chamber to be used subsequently, approximately 350 ml. of settled resin volume for a 2-liter vessel, or 1000 ml. for a 6-liter vessel, was suspended in 12 liters of water and stirred violently with a powerful mixer for 20 minutes to break up clumps and to become hydrated. After settling for 4 hours, the supernatant containing much colloidal material and fines was removed with an aspirator pump and discarded. The resin was then resuspended in water, so that 1 liter of slurry contained approximately 175 ml. of settled resin volume. Pulverized resins were violently agitated for 3 hours to break up partially fragmented pieces to minimize later shedding of very fine particles. Excessive colloidal material, which was present in some powders, was further removed, through repetition once or twice more of the suspension, settling, and removal of the supernatant. This was especially necessary with some lots of the Amberlite resin, which generated a voluminous intractable froth when the dry powder was first hydrated. THEORETICAL CONSIDERATIONS
A sphere falling freely through a liquid reaches a uniform linear downward velocity defined by the relationship :
d i e r e G is the gravitational constant, q the viscosity of water, d2 the density of n-ater, and dl the density and a the radius in centimeters of the sphere. For convenience in present calculations, the computed velocity was converted to centimeters per minute by the factor 60. As a corollary, a sphere, when placed in a fluid column within which a vertical gradient of linear velocity has been established, maximum a t the bottom and minimum a t the top, would rise (with diminishing velocity) to the = V@takes). Such level where a gradient of linear velocity n-ould be approximated in a V-shaped vessel, placed vertically, if fluid were to flow a t a constant rate up into the vessel. The minimum linear velocity of flo\v would occur a t the widest part and may be computed with sufficient accuracy, as the volume input per minute to the vessel divided by the volume of a layer 1 cm. in depth located a t the maximum internal diameter: r ( " P . "!In)
=
ml./minute input IrR2(ln&x)
cm./minute
(2)
\There R(m,,xlis the internal radius in centimeters, a t the widest part of the vessel. It folloivs that those particles with v i ( S t o k e s ) greater than or equal to V(up,mzn) will remain below or a t the level of the maximum diameter. Parmln) ticles with V-(Stokea) less than vCUp, mould be carried up and out the top of the vessel. Input flow rate may be calculated which, for any particular sized particle located a t the widest diameter, V(up)= v ( S t o k e s ) , by Equation 3:
Linear flow a t R(max, is a limiting rate n-hich governs the size of particle liberated from the vessel. Thus, a graded stratification of particles can be achieved in a relatively short Vshaped column; a considerably longer tube would be needed for comparable stratification in a vessel with parallel walls. For practical purposes these conditions are realized when water f l o w at a constant rate up into an ordinary separatory funnel of the Squibb type. K i t h resin in the funnel there appears t o be sufficient turbulence generated a t the point of entry-i.e., immediately above the stopcock-to reduce axial streaming, so that for practical purposes, approximately uniform velocity is obtained throughout the cross-sectional area a t any diameter level. To eompute V(stokes) from Equation 1 for particles of diameter 20, 25, 30, etc., microns, the following values n ere used: G = 980 cm./sec.?, 7 = 0.00894 poise a t 25' C., d2 = 1.00 a t 25' C., and dl = 1.21, the wet density of the hydrogen form of the resin. Substituting the computed values of v ( B t o k e s ) and the appropriate value of R(m,v-i.e., 7.4 or 11.5 cm.-for the 2- or Miter funnel, respectively, Equation 3, input flow rates were calculated. In Figure 1 the dashed curve, D-V, illustrates the relationship between particle size and the calculated input flow rates. APPARATUS FOR FRACTIONATION AND RESULTS
Depending on the amount of resin processed, either a 2-liter (Corning 6400) or 6-liter (Scientific Glass Apparatus Co., 5-1875) pear-shaped separatory funnel of the Squibb type was filled with well-mixed slurry (175 ml. of settled resin volume per liter) and clamped vertically. A source of water a t constant temperature was joined by rubber tubing through a flowmeter (Emil Greiner and Co., New York, Predictability flowmeter G9145B with calibration chart), to the lower stem of the funnel. I n the present instance a 100-gallon reservoir provided a continuous supply of distilled water a t an adequate pressure head and a t the ambient temperature (25' C.). Cold tap water, warmed to 25' C. by passing through a heat-exchange coil immersed in a warm bath, could probably be used if the water were not too hard; deionized water is preferable. The top of the funnel was closed with a rubber stopper provided with a right-angled glass tube, and a short length of rubber tubing discharged the outflow into suitable collection vessels. Flow was regulated by the funnel stopcock and the rate determined by means of the flowmeter and its accompanying calibration chart. AlternaVOL. 30, NO. 5, MAY 1958
915
Table I. Analysis of Dowex 50-X8 Hydrogen Form Spherical Particles with a 2-Liter Separatory Funnel (Squibb Type) at 2 5 " C.
Fraction So.
Flow Rate, hll./Min,
Martin's Av. Settled Volume Diameter, Ill. 3% of total Microns
Range of Diameter, hlicrons
So. of Particles
within
zb3 hlicrons of
hv. Diameter, 7, / "
30 65 125 240 390 540
1 2 3 4
5 6
Residue
7
17.1 62.4 100.0 92.0 53.4 22.6 77.2
4.1 14 7 23.6 21.7 12.6 5.3 18.2
14.5 21 28 39 50 63
18-27 23-36 33-45 44-58 56-71
73 74 72 64 i9
Table II. Analysis of Amberlite CG-120 Type 111, Sodium Form, Pulverized Irregular Particles with a 6-Liter Separatory Funnel (Squibb Type) a t 2 5 " C.
Fraction NO.
1 21' 3a 45
5= 6a
Flow Rate, Ml./?rIin.
Martin's Diameter, Alicrons
Range of Diameter, Microns
No. of Particles within f 3 Microns of Av. Diameter, yo
47 72 165 240 345 488
12.5 17 22 30 34 42
9-2 1 11-23 14-30 20-39 24-43 36-53
86 mi l
69 57 60 53
0 Settled resin volumes of the fractions were approximately equal to each other; sum of fractions 2 t o 6 constituted approximately 55% of total resin volume,
tively, flow rate was determined by timing the collection of a measured volume of outflow; precise adjustment to a predetermined rate was possible but difficult. The outflow was collected in four precipitating jars (&liter capacity each) connected by siphons, or in a single vessel from which water was removed by suction through a tubular fritted filter (Corning 35000) or on a funnel with fritted disk (Corning 36060) which ivas connected to an aspirator pump. Fractions of 20 microns or less were collected in jars; the remaining fractions by either of the tn-o latter procedures. For an experimental deterinination of the relationship between particle diameter and flow rate, a schedule of values of the latter for the 2- and 6-liter funnels were read from the D-I' curves corresponding to 15-, 20-, 25-, etc.. micron particles. Fractionation was conimenced a t a rate which washed out colloidal material and particles of less than 15-micron diameter. The outflow from this wash was opaque with fine particles usually for 20 hours or more, clearing after 30 to 36 hours; it was run to waste. The rate was then increased by stepwise increments in accordance Ivith the above schedules and the corresponding fractions were collected. After each increment in rate, the outflow soon became opaque n ith particles, which reached a maximum and then diminished slowly. The end point of the fraction was assumed to have been reached when the outflo\r again contained very few particles. To determine particle size and distribution within the fractions, photographic analysis was carried out as follon-s. Each fraction was stirred vigorously and a drop of suspension, on a glass 916
*
ANALYTICAL CHEMISTRY
slide under a cover slip, was photographed. Enlarged prints TT-ere made to give an over-all magnification of approximately X 7 0 0 . A substage micrometer was photographed and enlarged to the same degree. The position of the accidental diameter for each particle in the field mas determined by inspection and the distance measured with a millimeter rule. The average, the range, and the frequency distribution of the diameters ivere computed. Measurement of the enlarged micrometer scale, also with the rule, shoned that 1 mm. on the photograph was equiralent to 1.4 microns and the measurements (in millimeters) were accordingly converted to microns by multiplying by 1.4. Typical analyses are shown in Tables I and 11. The diameter range of the pulverized resin fractions was a little greater than those obtained for the spherical resin fractions; a higher percentage of the latter was within 1.3 microns of the average. The results of analysis of both spherical and pulverized resins using 2- and 6-liter funnels are summarized in Figure 1. -4s the computed D-T' curre is used only as a guide for selecting a schedule of flow rates within the range appropriate for fractionating the resins under investigation, it is immaterial whether the curve is calculated for the wet density of the hydrogen or the sodium form. I n the present instance the D-V curve was computed for the hydrogen form. Once the diametervolume input curve for a particular resin is established experimentally, input flow rates are read from the experimental curve to provide a schedule suitable for isolating any particular
size of particle of the resin. Figure 1 s h o w that the flow rate for any specified diameter of spherical particle (Dowex 5O-X8, beads) was approximately 20% greater than that indicated by the D-1' curve for the 2-liter funnel; approximately 1.5% greater for the 6liter funnel. This result is to be anticipated where both the computed and experimentally determined curves are for the hydrogen form of the resin spheres, as input flow rate should be higher to provide a linear flow rate at R(msr, such that - V(Etakes) is a positive quantit5- for any particular sized particle. The irregular particles of the Amberlite (sodium form) resin were isolated at flow rates numerically less than the D-l' rate of corresponding diameters. I n the absence of further experimental evidence, it is tentatively suggested that the greater surface area and irregular shape of the pulverized resin particles affect the diameter-volume input relationship more profoundly than the change in density from the hydrogen to the sodium form. Study of this observation seemed t o add little to the practical aims of the present investigation and it was not pursued further, The particles of pulverized Dowex 50K (hydrogen form) gave a curve similar to the pulverized Amberlite, but the present work offers no worth-while explanation of the differences shown in Figure 1, between these two resins. A total volume of approximately 40 liters of mater was necessary to isolate each fraction from the 2-liter funnel, approximately 60 liters from the &liter funnel. CONDITIONING OF RESINS FOR CHROMATOGRAPHY
Each fraction was conditioned by washing under gravity on a funnel with fritted disk (medium porosity) with 6 S hydrochloric acid until the effluent was no longer colored yellowish green. About 9 liters of acid per 500 ml. of resin was needed. The colored substances removed were presumably largely copper or iron, which are known to be introduced as contaminants from the machinery during pulverization. Residual acid was washed out with water and the resin converted to the sodium form by 9 liters of 2N sodium hydroxide run through under gravity. The alkaline solution contained 0.1% Sequestrene (Geigy Chemical Co., 89 Barclay St., New York 8, N.Y.) to ensure removal of traces of those metals, if any were present, whose chlorides were insoluble in the hydrochloric acid. The first half of the alkaline effluent was slightly colored and somewhat opalescent; the last half became progressively clearer. At this stage, some of the pulverized fractions showed more fine particles, apparently generated as a result of the cycling washes. As it was observed that excessive fines pro-
duced higher column operating pressures without contributing to column performance. the above fractions were suspended in 10 liters of 0 . 2 5 sodium hydroxide, and allon-ed to settle for 2 hours. The fines in suspension were removed n-ith the supernatant, which \vas discarded. Occasionally, repetition of this procedure \vas necessary. \Then once clean. tlie fractions, on being suspended in 10 liters of n-ater, exhibited few fines in the supernatant after 60 minutes' scttling. and none after 4 hours, nor did they show appreciable prodnction of more fines thereafter.
Table 111.
Conditions for Isolating 20to 40-Micron Particles from Pulverized Resin Powders
Fraction No. 1 2
3 1
2 3
Martin's Flow Rate, Diameter, ProMl./hIin. hIicrons cedure 2-Liter Separatory Funnel 50 20 Discard 250 20-40 Collect Residue Discard 6-Liter Separatory Funnel 120 20 Discard 480 20-40 Collect ... Residue Discard
AMINO ACID CHROMATOGRAPHY
Chromatographic columns, 0.9 X 100 cm., were poured in 20-cni. sections n-ith suspensions of 1 ml. of resin per 2 voliuiies of 0.2.1- sodium hydroxide! according to the detailed directions of IvIoore, Spacknian, and S k i n
(e).
The bottom of the column was plugged while the initial GO ml. of slurry was poured in. after which the plug was removed. Each section was settled under air pressure of 7 pounds per square inch until well packed. Residual supernatant solution was removed by suction and the nest section was poured directly on the moist surface of the esposed resin bed. The colu11111s 17-ere jacketed and operated a t 50" C.,and solutions were pumped through a t 2,5 ml. per hour, using a llinipunip (225 ml. per hour capacity. Milton Roy Co., Philadelphia, Pa.). This corresponds to a flow rate of 39.3 nil. per sq. em. of resin bed per hour. Tlie columns were conditioned before analysis with 100 ml. of 0.2N sodium hydroxide, followed by 0.1X citrate buffer (0.2S with respect t,o sodium), p H 2.90, until the p H (glass electrode) of the effluent was the same. Two cubic centimeters of a test solution of 29 amino acids dissolved in 0 . 1 S hydrochloric a d n-as placed on the column and forced in under air pressure, 10 pounds per square inch, followed by a n-ash of 1 nil. of 0.LY hydrochloric acid. Development of the column was begun Tvitli 0.1X citrate (0.2A7sodium), p H 3.10 or 3.00, as noted below. At nil. 400 the buffer was changed bo 0.lX citrate (0.25 sodium), p H 4.15, and again a t ml. 600 to 0.4M citrate (0.8S sodium), pH 5.00. Brij-35 (Atlas Powder Co., Wilmington, Del.) was present in 1% concentration in all conditioning solutions and buffers. The preparation of buffer solutions and general column operation have lieen described by Moore and Stein (6). The p H of buffers, their sequence, and rate of flow were those investigated in this laboratory. Column effluent was collected in 2-ml. fractions and analyzed in accordance with the principles described by Moore and Steiii (5, 6). RESULTS
OF CHROMATOGRAPHY
In Figure 2. the chromatogram obtained with a column of 55-micron
Dowex 50-X8 spherical particles is compared with that obtained from a column of 25-micron particles of the sanie resin, both columns being operated a t a flow rate of 25 ml. per hour. The superior resolving power of the 25-micron column is clear. In this chromatogram the position of methionine was not established. Whether it was degraded on the column or incorporated with the cystine was not determined. Resolution of a column of 45-micron particles u-as intermediate between these two. Resolution of a column of 35-micron particles n as comparable to that obtained with the 25-micron particles, but the peaks were broader. This sequence of chromatograms provided evidence that as particle size decreased, resolution progressively improved, and that a t the relatively high rate of flow of 25 ml. per hour, optimum resolution was obtained with spherical particles 25 microns in diameter. [Spackman, hloore, and Stein (8)have found that a t lower flow rates of 10 to 15 ml. per hour, comparable resolution may be obtained with coarser particles.] No improvement in either resolution or form of the peaks was observed when 20-micron particles were used, but column operating pressures were increased from 45 to 65 pounds per square inch. A similar series of chromatograms was obtained with columns packed with pulverized Amberlite CG120 Type 111, or n-ith Dowex 50K-Xg. Optimum resolution with the pulverized resins was obtained 1%-ithparticles of 30-micron diameter when a flow rate of 25 ml. per hour \vas used. Cystine and valine were not resolved nhen column development commenced 15-ith pH 3.10 buffer. In later experiments, these two amino acids were resolved with p H 3.00 buffer, as illustrated in Figure 3. I n Figure 3, chromatograms obtained with a column of Amberlite CG-120 Type 111, 30-micron particles, before and after wishing the resin with G S hydrochloric acid and 2N sodium hydroxide are compared. The marked impairment of resolution of the un-
nashed resin could be attributable to the large amount of metallic ions present, perhaps also to other unidentified substances. The necessity for thorough cleaning of the resins is strikingly apparent. After the effect of particle size on resolution had been determined v i t h the narrow range fractions for column flow rates of 25 ml. per hour (Figure 2), a 20- and 40-micron fraction of the Amberlite resin, n hich embraces the n idest useful range of particle diameter for this rate of flow, was run for coniparison. As indicated in Table 11, particle size of this resin was evenly distributed throughout this range. The resolution obtained was similar to that shown in Figure 3,B. Chromatograms obtained n ith 31and 39-micron fractions of Dowes 5OV*-X8 Ivere compared ivith that of a 20- to 45-micron fraction of the sanie resin, all operated a t flow rates of 25 nil. per hour. All three chromatogranis showed equally good resolution of amino acids and the resolution did not differ from that obtained with the hniberlite resin, except in a felv minor respects. A single chromatogram using Perniutit Q-ZP, 20- to 42-micron range, indicated that this resin was probably serviceable, but it was not studied further. The results demonstrate that good chromatograms can be obtained from 0.9 x 100 em. columns a t f l o ~rates of 39.3 ml. per sq. em. per hour with 20- to 45-micron fractions of the pulverized resins, if there is uniform distribution of size throughout the range. If size distribution mere shifted markedly toward the upper limit, some loss of resolving power would occur a t this high rate of flon.. For routine preparative purposes, a more conservative range-e.g., 20 to 40 microns-Ivould ensure columns n ith good resolving power and sharp peaks, even if a disproportionate distribution toward the upper limit of the range were present, and it would render unnecessary a more detailed preliminary particle analysis. Similar considerations suggest that a 20- to 35-micron range of spherical particles should be most serviceable. These wider diameter ranges allowed flow rates of 25 ml. per hour through 0.9 X 100 em. columns, a t moderate operating pressures of 40 to 50 pounds per square inch. They also constituted reasonable proportions of the particular lots of the unclassified resins investigated: 50% of the Amberlite CG-120 Type 111, 75% of the Donex 50K-X8. Table I11 summarizes recommended procedures for isolating 20- to 40micron particles from pulverized resins. u-ith the 2- and 6-liter separator>funnels. Kithin the practical limits desired, these conditions are applicable to either the hydrogen or sodium form. VOL. 30, NO. 5, MAY 1958
917
ARG
0
100
