Ion Exchan e Chromatography of Amino Study of Effects of High Pressures and Fast Flow Rates PAUL B. HAMILTON Alfred 1. du Pont institute of the Nemours Foundation, Wilmington
b Glass columns and pump connections to them were designed to tolerate fluid pressures in excess of 600 p.s.i. Linear Aow rates up to 0.1 7 cm. sec.-I through the columns packed with 2.4, 4.6, or 8.0 X cm. diameter resin particles were used. Pressure as a function of particle diameter, column length, flow rate, and temperature were studied. The effect of flow rate and particle diameter on the resolution of some amino acids is indicated. Multiple section columns were made and tested.
I
THE ION EXCHANGE chroniatography of amino acids, flow rates through columns used for analytical work have ranged from 0.00174 to 0.0131 cm. sec.-l a t pressures up to 65 p.s.i. (5, 9, IO). More recently, flow rates of 0.022 em. sec.-l a t pressures of approximately 200 p.s.i. have been reported (6). This paper reports flow rates up to 0.17 cm. sec.-l with pressures as high as 600 p s i . The columns ranged from 0.218 t o 0.636 cm. in diameter, 67, 108, and 156 em. in length. They were packed with spherical particles of Dowex 50-X8. Glass columns suitable for operation a t elevated pressures and pump connections to them are described and the usefulness of the latter in making multisectional columns is indicated. The algebraic expression for resolution which was suggested by Hamilton, Bogue, and Anderson ( 7 ) is used to discuss the effect of flow rate and particle diameter on the resolutioii of some amino acids.
N
99,
Del.
was provided with a 60-second timer (Cycle-Flex Reset Timer Series KO. HP2, Eagle Signal Corp., Moline, Ill.). The time interval per fraction was checked by a stop watch. Temperature control, pump calibration, column conditioning, developing buffer solutions, and amino acid test solutions were as described previously (6, 7 ) . Standard procedures for determining amino acid concentrations in the effluent fractions and for plotting the chromatograms were used (6-8). The volume of test solution and the concentration of amino acids (feed pulse volume and concentration) met Glueckauf's criterion (4, for starting bands small enough t o be without effect on the width of the elution peaks, as discussed by Hamilton, Bogue, and Anderson (7). The columns were of borosilicate glass, standard capillary for the small columns, precision bore tubing (Fischer & Porter Co., Hatboro, Pa.) for the larger ones. A nominal 1/4-inch pipe flange was tooled onto each end of the column. Supporting filters were cut
from porous Teflon sponge inch thick, grade 5-55, Liquid Nitrogen Processing Co., Chester, Pa.) with a cork borer. The Teflon filters fitted snugly but needed only gentle pressure for their insertion. The columns were provided with jackets. Connections to the pump system and to the fraction collector are shown in Figure 1: the couplings were stainless steel (Fischer & Porter Co., Hatboro, Pa.) ; the Tcflon capillary was gage 22, extra heavy walled (Pennsylvania Fluorocarbon Go., Philadelphia, Pa.). The flare on the end of the polyethylene tubing was made by careful warming a t the edge of a gas flame. Details for the construction of a capillary column are shown in Figure 2. Multisection columns were made similar to that shown in Figure 3. Gage 22 Teflon capillary, extra heavy walled, was used to join the sections. The resin packing was prepared from commercial Dowex 50-X8 or Dowex 5OW-X8 spherical beads, minus 400 mesh. The resin was classified hydraulically (5) to obtain a cut of the
TOP
7 -To
/FEMALE
PUMP COUPLING, S/ST'L.
PLEXIGLAS WASHER
b Figure 1. nections
-TEFLON --RUBBER -TEFLON '----O-RING,
!!--
Column con-
F L A R E 1
Top, pump connection to glass column suitable for fluid pressures up to ca. 700 p.5.i. Bottom, connection of effluent capillary which leads to fraction c oII e ct o r
COLLAR STOPPER WASHER NEOPRENE
GASKET, NEOPRENE-ASBESTOS
GASKET, NEOPRENE- ASBES1-05
APPARATUS AND MATERIALS
TEFLON, POROUS
The apparatus employed was that previously described (6) but the laboratory-made gage was replaced by a 600pound gage with chemical diaphragm (E. S. Gauge No. 1901-T, 0-600 p.s.i. with AB2 diaphragm seal and Type 25B, Porosity E, pressure snubber) and the polyethylene fittings were replaced by Hastelloy C (Swagelok, Crawford Fitting Co., Cleveland, Ohio). The column effluent mas collected on a time flow fraction collector. For fraction times of less than 1 minute the collector
FLARE-
TEFLON 0 - R I NG. NEOPREN E ~ L T E F L O WASHER N @-RUBBER
0-TEFLON
STOPPER
COLLAR A PLE x I G L.AS WASH E R -FEMALE COUPLING, S/ST'L. POLYETHYLENE TUBING CAPILLARY, GAUGE 22
0
BOTTOM
/
VOL. 32, NO. 13, DECEMBER 1960
e
177
p-
Ill/
\!\ I\
b
SPECIAL HEAYY WALL 1.D-028 0.D 068"
= 020"
Figure 2. Details of connection of effluent capillary to columns less than 6.36 mm. (0.25 inch), internal diameter P u m p connection is similar
mean particle diameter desired with a range of approximately i15 microns of the mean. Each cut was then passed through the classification procedure a second time to obtain a cut of narrow particle size range. The particle sizes were 2.4 X cm. with 80y0 within 1 0 . 2 x 10-8 cm. of the mean, 4.6 X cm. with 75% within 0.1 X 10+ cm. of the mean, and 8.0 X cm. with 83% within 1 0 . 5 X 10+ cm. of the mean. The capacities were 5.22, 5.27, and 5.28 meq. per dry gram, and the wet resin densities were 1.275, 1.264, and 1.275 grams. per cc., respectively. The densities correspond to cross linkings of 8.3, 7.7, and 8.3%, respectively. The resin was slurried in 0.214' sodium hydroxide and the columns were packed in either single or multiple sec'cions. T o pack a capillary, a reservoir was temporarily clamped to the top of the column and the column and reservoir were filled with 0.2N sodium hydroxide. Enough resin was then poured,. as a slurry, into the top of the reservoir.
0.414-, and 0.636-em. diameter columns, 108 cm. long were essentially the same as those that have been previously published ;Figure 4, reference (S)]. The volume of the fractions collected was reduced in proportion to the cross-sectional area of the column, a 1ml. fraction volume being taken as standard for the 0.636-cm. column. The loading of each solute was scaled down similarly. With the 0.218-cm. capillary column satisfactory peaks were obtained when the column loading was 0.1 pmole of each amino acid. Sectional columns of 3, 4, or 6 sections of 0.4- or 0.636-em. diameter were made and tested. Three-section columns were the moat practical. They were the easiest to assemble, were low enough for bench operdion, and gave chromatograms that were the same as those obtained with a single column of the same length of resin bed. With foursection columns, slight widening of the base of the peak was observed, but the loss of resolution was not significant. Widening of the peaks and some loss of resolution was observed with the six-section column. Resolution. I n a detailed theoretical analysis of the ion exchange process, Hamilton, Bogue, and Anderson (7) defined resolution by the ratio
where tia, f i b are the elution volumes of two peaks whose separation is of interest, and ua, Ob, are the respective dispersions of the peak. For complete resolution of a and b, RE 2 2. A similar definition has been proposed in the field of gas chromatography (8). Both U and u are experimentally deternlinable quantities. ii is obtained by inspection of the chromatogram and FUM? CONNEC,TION
RESULTS
Hydraulic Characteristics. Typical results with 0.636-cm. diameter columns are shown. T h e pressure drop was proportional t o column length (Figure 4) and inversely proportional to the square of t h e particle diameter (Figure 5 ) . The linear flow rate (volumetric input divided b y cross-sectional area) was proportional t o the pressure (Figure 6). T h e d a t a of Figures 4, 5 , and 6 were obtained a t 5 4 O C. The effect of temperature on pressure is shown in Figure 7. The hydraulic characteristics of Dowex 50X8 and Dowex 5OW-X8 were indistinguishable. Column Dimensions. T h e sequence a n d position of solutes in chromatograms obtained with 0.218-, 0.286-,
1780
e
ANALYTICAL CHEMISTRY
L.-
Figure column
3. Schematic of three-section
Sections are joined by Teflon capillary and details of connections are shown in Figures l and 2
630-
I
I
0
-L__i
75 100 125 150 2 COLUMh -ENG'q-Z-cr
Figure 4. Pressure at top of column as function of column length
is the elution volume (read on the axis) corresponding to C,, of the peak The measurement of dispersion is fully described by Hamilton, Bogue, and Anderson ( 7 ) . Resolution and Flow Rate. To demonstrate the effect of flow rate on resolution, R R was computed for each of t h e pairs of solutes, glycinealanine and aspartic acid-serine, utilizing the experimentally determined values of D and Q of the flow series data of Hamilton, Bogue, and Anderson ( 7 ) . I n Figure 8, RE is plotted as ordinate, U , as abscissa. The aspartic acid-serine and glycinealanine curves (for the 2.4 X l0l8 cm. diameter particles) were extrapolated (dashed lines) by calculating -RE for values of U , above 0.18 cm. sec.-Il using Equation 12 of Hamilton, Bogue, and Anderson (7') which defines RR in terms of column and resin variables. Since Equation 12 neglects possible liquid diffusional effects, it is likely that experiments would shorn that RE = 2 a t flow rates somewhat lower than those indicated, for above flow rates of 0.2 cm. sec.-l with 2.4 X IO+ cm. diameter particles, liquid diffusional effects would become increasingly important. Qualitatively it can be anticipated that the higher the flo~+rate the greater will be the contribution of liquid diffusional effects in diminishing R E . It is apparent, however, that for small particle diameters, relatively high flow rates may be used without sacrificing complete resolution of these particular amino acid pairs. Equation 14 [of reference ( r ) ]which also neglects possible liquid diffusional effects, gives the minimum time necessary for the elution of two completely resolved components-Le., RE = 2. Applying this equation to the separation of glycine and alanine on an 0.636 X 108 cm. column packed with 2.4 X 10-3 cm. diameter particles and operated a t a linear flow rate of 0.4 cm. sec.-l, i t can be predicted that both amino acids would emerge, resohed, in approldmately 20 minutes; the volumetric input to the column would have to be approximately 460 cc. per hour, and
i i
300-
4 Figure 5. Pressure at top of column as function of
250 -
14
particle diameter
.q200W
5
M
150100-
50-
l/dp?cm2
the pressure would be on the order of 1600 p s i . The limitations of increased flow rate are also apparent. For example, in the routine analysis of an amino acid mixture, in which all components are qualitatively known, flow rate may be increased to the point where resolution is just complete-i.e., R = 2-but the limit will be governed by that pair of amino acids whose elution peaks first begin to overlap (providing the presEure a t that flow rate can be tolerated). If unknon n solutes in relatively low concentrations are present, slow flow rates may be desirable to obtain maximum resolution lest the small peaks become overlapped by adjacent larger peaks. Resolution and Particle Size. T h e beneficial effect on RE of reducing d, is also indicated in Figure 8, in which RE for glycine-alanine is shown for 2.4 X 4.6 X low3, and 8.0 X low3cm. particle diameters. Theoretically, the best resolution would be obtained with the smallest possible particle diameters, other variables being constant. Mitigating against too great reduction of particle size is the corresponding increase in pressure, which may rapidly exceed reasonable tolerances. Some reduction in pressure might be achieved by reducing column length without serious reduction of resolution. The more general problem
0
of optimizing all column variables is discussed in reference ( 7 ) . Exemplifying the manipulation of column variables and operating conditions in accord with some of the general principles described, the work of Stewart (11) on the separation of some of the rare earth elements is of qualitative interest. Flow rates up to 0.34 cm. per second through very short narrow columns were examined. Graded particle sizes were used to pack the columns. Pressure v a s applied by the elevation of a confining mercury reservoir; pressure data were not reported. The effect of flow rate and particle size on resolution p a s qualitatively in agreement with the present work. The problem of computing the percentage of cross-contamination of solutes, represented by adjacent overlapping peaks, has been fully dealt with by Glueckauf (5, 4). Glueckauf’s work was also discussed later by Cornish
IO0 200 300 430 500 6GC 700 PRESSLRE- P S I
Figure 6. Pressure at top of column as function of flow rate
Midland, llich., is grateiuliy acknowiedged. LITERATURE CITED
(1) Cornish, F. W., An$ s t 83,634 (1958). (2) Desty, D. H., &a8 Chromatography,” Vol. XI, Komenclsture Recornmendations, Academic Press, New York, 1958. (3) Glueckauf, E., “Ion Exchange and Its Applications,” pp. 34-46, Society of Chemical Industry, 1953, (4) Glueckauf E., Trans. Faraday Sac. 51, 34 (1955).
(5) Hamilton. P. B.. ANAL. CHEN. 39. 914 (1958): (6) Hamilton, P. B., Anderson, R , A., Ibid., 31, 1504 (1959). (7) Hamilton, P. B., Bogue, D. C: Anderson, 1%.A., Ibid., 32, 1782 (1960)‘; (8) Moore, S., Stein, W. H., J . Bial. Chem. 176, 367 (1948). (9) IMd.,192, 663 (1961). (10) Spackman, D. E., Moor2, S,, W i n , W. H., ANAL.CHEM.30, 1191)(?93X). (11) Stewart, D. C . , Wid., 27. 127:) (1955). RECEIVEDfor review July 11, ’960. -4ccepted September 14, 196C. ~
(1). ACKNOWLEDGMENT
A gift of Dowex 50W-X8, minus 400 mesh by The Dow Chemical Co ,
:acio
I
500 .-rn
45 0
7400
W LT
2 350 v, E300 w
250
35
55 65 75 TEMPERATURE-”C
45
85
Figure 7. Pressure at top of column as function of column temperature
0
OC5
010
015
020 025 O?C 035 040 il,CW
SEC-
Figure 8. Resolution of amino acid pairs cs function of linear flow rate through toCumn VOb. 32, F!4. 14, DECEMBER 1960
‘B
‘fr(41
