Anal. Chem. 1991,63,2183-2188
E. S.; Bobbit, D. R. Anal. Chem. 1984, 56, 1457- 1460. Renn, C. N.; Synovec, R. E. Anal. Chem. 1989, 61, 1387-1393. Synovec. R. E.; Johnson, E. L.; Bahowick, T. J.; Sulya, A. W. Anal. Chem. 1990 62, 1597-1603. Okada, T.; Kuwamoto, T. Anal. Chem. 1985, 57, 829-833. Wilson, S. A.; Yeung,
Haddad, P. R.; Jackson, P. E. Ion Chromatography: Principles and Applications; Elsevier: New York, 1990; pp 84-94. Strong, D. L.; Dasgupta, P. K.; Friedman, K.; Stilllan, J. R. Anal. Chem. 1991. 63, 480-486. Strong, D. L.; Joung, C. U.; Dasgupta, P. K. J . Chromatogr. 1991, 546, 159-173. Okdada, T.; Dasgupta, P. K. Anal. Chem. 1989, 61, 548-554. Tanaka, K.; Fritz, J. S. Anal. Chem. 1987, 59, 708-712. Gupta, S.; Dasgupta, P. K. J . Chromatogr. Sci. 1988, 26, 34-38. Matsui, K.; Kikuchi, Y.; Hiyama, T.; Tobita, E.; Kondo. K.; Akimoto, A.; Seita, T.; Watanabe, H. US. Patent 4,567,206, Jan 28, 1986.
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(17) Dasgupta, P. K. In Ion Chromatography; Tatter, J. G., Ed.; MarcelDekker: New York, 1987; pp 191-367. (18) Curtis. M. A.; Shahwan, G. J. LC-GC 1988, 6, 158-164. (19) Smaii, H. Ion Chromatography; Plenum: New York, 1990; p 170.
RECEIVED for review May 13, 1991. Accepted July 12, 1991. This research was supported by Dionex Corp. and by the Office of Basic Energy Sciences, U S . Department of Energy, through Grant DE-FG05-84ER13281. However, this report has not been subjected to review by the DOE and no endorsement should be inferred.
Effect of Displacer Impurities on Chromatographic Profiles Obtained in Displacement Chromatography Jie Zhu, Anita M. Katti,' and Georges Guiochon* Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-1501, and Division of Analytical Chemistry, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831
A malor problem encountered durlng the development of a method In displacement chromatography has been Identified and k discussed. Experimental resutts are presented by using the model system of phenol as the solute and 4-methylcatechol a8 the dlsplacer. Elution results showed that the dlsplacer contained several lmpurltles elutlng prior to the major component. Analysis of the displacement profiles showed that several of these lmpurltles plerced through the sample train elutlng at the front of the solute band. Thls phenomenon 1s studied In detall, and theoretical and experlmental rerutto are presented. The results show that for the production of hlgh-purtty products, displacement chromatography mwt be carrled out wlth a dkplacer free of early elullng Impurltles.
t
Column
INTRODUCTION Among the important practical problems encountered during the development of a method for displacement chromatographic separations, one problem in particular has never been discussed in detail. This is the behavior of these impurities in the displacer solution which are less retained than the displacer. This behavior will determine the purity requirements for the displacer. It is important to understand how these impurities may interfere with the purification process in order to select a displacer and develop a method that allows the products to be isolated at the purity required. The present work attempts to shed light on this issue. Displacement chromatography is an important separation mode for the isolation of solutes (I) at the semipreparative and process scale. For this mode of chromatography,a general theory has been developed to predict band shapes, assuming competitive Langmuir equilibrium isotherms and no bandbroadening processes in the column (2,3). Specific studies
*Tow h o m correspondence should be sent a t t h e U n i v e r s i t y of Tennessee. Present address: E i c h r o m Industries, 8205 S. Cass Ave, Suite 107, Darien, IL 60559. 0003-2700/91/0363-2183$02.50/0
Fraction Collector
Flgurr 1. Schematics of the instrument set up: pump A, displacer solution: pump 6, carrier (water).
Table I component
k'
a, m g / m g
b, m L / m g
phenol 4-methylcatechol
9.3 12.7
18.5 25.5
0.7 0.7
have also been presented in order to understand the effect of various parameters on the band shape, such as the sample size, the displacer concentration, and the column length (4-6). Mass-transfer effects in displacement chromatography have also been presented (7,8).Through systematic experimental work, theoretical predictions of the effect of sample size and the displacer concentration have been confirmed by measuring independently the isotherm and determining the individual 0 1991 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 19, OCTOBER 1, 1991
0
2
4
6
0
Concertration
n
0
1
2
1
Mobile Phose
component band profiles (9,101.Additionally, operation of displacement separations as a continuous process has been proven feasible (11). Recently, the application of displacement chromatography for the isolation of peptides has been presented (12). Due to the need for high-purity products for the various industries, displacement chromatography has been compared and contrasted with other operating modes such as overloaded elution (13)and gradient chromatography (14). Displacement chromatography also has a unique property to separate and, at the same time, to enrich considerably impurities present in the sample (15-17). Two-component frontal analysis shows that the less retained component exhibits a slight concentration enrichment before the front of the later eluting component appears (18). However, the importance of impurities in the displacer during displacement development is not well understood. In this paper the effect of the relative retention of the less retained impurities in the displacer, of their concentration in the displacer solution, and of the sample size are studied through theoretical calculations and experimental findings.
4
(~g/rl)
Figure 2. Measured isotherms of the sample (solid line, phenol) and
of the displacer (dotted line, 4-methylcatechol). Column: length, cm, 4.6 mm i.d., phase ratio, 0.501.
15
EXPERIMENTAL SECTION Equipment. Displacement chromatography was performed by pumping the carrier and the displacer solutions, respectively,
k' I=3.5
r
35
25
25
3
3.5
Re:ention Volume (FI) 0
"I
k ' I=8.6
25
3
35
Retention Volume
(ml)
Flgure 3. Calculated displacement chromatogram showing the effect of the retention factor of the impurity. Conditions: same column as In Figure 2; flow rate, 0.2 mL/min; carrier, water; feed, phenol (15 mg/mL in 1 mL of carrier); loading factor, 68%; displacer concentration, 25 mg/mL in carrier; impurity concentratlon, 2 mg/mL. Key: profiles of sample (dashed line), displacer (solid line), and impurity (dasheddotted line). Retentlon factor of impurity (k'& (a) 1.0; (b) 3.5; (c) 5, loading factor = 68%; (d) 8.6.
ANALYTICAL CHEMISTRY, VOL. 63, NO.
19, OCTOBER 1, 1991
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of the loading factor of the sample. Experimental conditions are the same as for Figure 3c, except loading factor, 20%.
were purchased from Burdick and Jackson (Muskegon, MI). All chemicals were used without further purification. Experimental Procedures. Isotherm Measurement. The Langmuir isotherm parameters a and b for each component were measured by the RTM method (20), by measuring the retention time of a very small pulse (near infinite dilution or under linear elution conditions) and by recording several overloaded elution profiles. Good agreement was observed between the reduced experimental profiles and the Langmuir profile. For this system the Langmuir model accounts well for the adsorption data in the concentration range studied. Displacement Development. The most common way of performing a displacement experiment is to employ two six-port valves to inject the sample and to switch from carrier to displacer. Instead, in these experiments a single 10-port valve was used to perform at the same time the injection and the switching (Figure 1). The displacement experiment begins by equilibrating the column with the carrier (water) pushed through pump B. During equilibration, the loop is filled with the sample (phenol) and the displacer solution is pumped with pump A into waste (outlet A) through the valve. As soon as the column is equilibrated,the valve is switched to let the displacer solution push the feed through the loop into the column. Fractions (40 pL) are collected continuously. Upon elution of the displacer front, the column is washed extensively with methanol and reequilibrated with the carrier water. Analysis of the Fractions. Without further sample workup, aliquots of the fractions are reanalyzed by using the Gilson 2321401 processor as an autosampler. The band profiles of the individual components in the displacement train were determined by quantitative analysis of the collected fractions.
using two separate Gilson (Middleton,WI) Model 302 pumps into two distinct porta of a 10-port Valco (Houston,TX) pneumatically actuated valve fitted with a 1-mL loop, a column, and various waste streams. Figure 1illustrates schematically the configuration of the valve. The column effluent was monitored by using a Spectroflow 757 variable-wavelength UV detector (Applied Biosystems, Ramsey, NJ) at 290 nm. Fractions were collected and reanalyzed with a Model 232-401 automatic sample processor (Gilson, Middleton, WI) (19). Columns and Chemicals. A Vydac (Hesperia, CA), 5 pm, Protein & Peptide C18 column, 0.46 X 25 cm, was used for the displacement experiments. A column, 0.46 X 5 cm, packed inhouse with Vydac 10 pm material was used for the off-line fraction analysis. CMethylcatechol was purchased from Aldrich (Milwaukee,WI). Phenol was purchased from two manufacturers, Aldrich and Mallinckrodt (St. Louis, MO). HPLC grade water and methanol
RESULTS AND DISCUSSION Isotherm Parameters. The Langmuir isotherm parameters and the capacity factor of each component are listed in Table I. The isotherms of phenol and 4-methylcatechol are shown in Figure 2. These isotherms are well accounted for by the Langmuir model. Thus, for the simplets displacement experiment of one solute, these compounds can be used, phenol as the solute and 4-methylcatechol as the displacer. Theoretical Study. The theoretical predictions of band profiles in displacement chromatography were obtained by numerical calculation of the solution of the system of partial differential equations with the appropriate boundary conditions and by assuming competitive Langmuir isotherms (4, 9 , 1 3 , 16). In this theoretical study, we attempted to model the experimental system that includes one solute, representing phenol, a solution containing the displacer, representing the
3.3
3.8
Retention Volume (ml) Flguro 4. Calculated displacement chromatogram showing the effect
~ ~ ' 0 . q5 / m l
C'I = 9
c2=2.0 q / m l
r 5
3
5.5
Retention Volume (mi)
15
3
3.5
Retention Volume (ml)
Figure 5. Calculated displacement chromatogram showing the effect of the concentration of the impurity. Experimental conditions are the same as for Figure 3, except retention factor and impurity concentration: (a) k' = 9 and concentration of impurity = 0.5 mg/mL (curve 1) and 2 mglmL (curve 2); (b) k' = 5.0 and concentration of impurity = 0.025 mg/mL (curve 1) and 0.25 mg/mL (curve 2); (c)k' = 1.O and concentration of impurity = 0.025 mg/mL (curve 1) and 0.25 mg/mL (curve 2).
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 19, OCTOBER 1, 1991
k P I =2 . 5 0
I
I1
OJ
............
0
..0
? 0?
.p..
0n
*
9 L“
1 ; :-;:; 1 7
, ‘7
k t I =11.0
..................
19
2‘
Retention Time (mi;)
,
#’..
?
::.,
0
17.5
18
, .-.-,-.-
18.5
19
19.5
Retention Volumn (ml) Flgure 8. Calculated dlsplacement chromatogram for a binary mixture with an impurity in the displacer showing the influence of the impurity retention: (chromatogram 1)k’of impurity, 2.5;(chromatogram 2) k’ of impurity, 7.0; (chromatogram 3)k’of impurity 9.03;(chromatogram 4) k’of impurity, 1 1 .O. Experimental conditions are the same as for Figure 3, except column length (100 cm), sample composition (1:l binary mixture), sample Concentration (10mg/mL of each component), and retention factor (k’, = 7.5,k’, = 9.0,k’, = 12.7). The impurity concentration is 0.2 mg/mL in the displacer solution (0.8% of displacer), in all cases.
4-methylcatechol, and one impurity of the displacer. This theoretical study involves observing the band profiles as the concentration and the retention factor of the impurity in the displacer are varied systematically. This approach permits an understanding of the influence of these parameters on the quality of the purification achieved by displacement chromatography. It also suggests the conditions under which experiments should be carried out. The effects of the capacity factor of the displacer impurity; of the sample amount and of the concentration of the impurity in the displacer are illustrated in Figures 3-5; respectively. For the sake of clarity in Figures 3 and 4,the concentration of the impurity in the displacer is rather large, 8% (Wimpuiity/Wdisplecer).
In Figure 3, the capacity factor of the impurity, as measured under analytical conditions, increases from 1 (Figure 3a) to 3.5 (Figure 3b), 5.0 (Figure 3c), and 8.6 (Figure 3d), holding all other parameters constant. The capacity factor of the solute and the displacer are 9.3 and 12.7, respectively. In all four cases the impurity is less retained than the sample component. The general trend observed in Figure 3 is that the smaller the capacity factor of the impurity in the displacer, the more severe the perturbation of the train front. In the specific situation where the impurity is weakly retained (Figure 3a), a high concentration spike of the impurity appears at the front of the solute band. During elution of the solute band, the profile of the impurity exhibits a plateau having a concentration slightly higher than its concentration in the displacer solution. This is called the intermediate
Flgure 7 Experimental chromatograms wkh phenol as sample. Curve 1 is from one manufacturer, curve 2 from the other. (a, top) Frontal analysis of the pure phenol. Experimental conditions: Concentration of phenol, 25 mg/mL in carrier; flow rate, 0.2 mL/min; UV detection 290 nm. (b, bottom) Displacement chromatograms of the two phenol samples. Experimental conditions: feed, 1 mL of 25 mg/mL solution of phenol in carrier; concentration of displacer, 25 mg/mL; flow rate, 0.2 mL/min; UV detection 290 nm; same column as for Figure 2. I
plateau and is due to the competitive behavior between the impurity and the phenol. During elution of the displacer, the profile of the impurity in the displacer also exhibits a plateau having a concentration that is the same as its concentration in the displacer solution. As the retention factor of the impurity increases (Figure 3b,c), the concentration of the spike in the front of the sample band decreases to zero and the concentration of the intermediate plateau increases. When the retention factor of the impurity is of the same order as
4NALYTICAL CHEMISTRY, VOL. 63, NO. 19, OCTOBER 1, 1991
9 v
17
19
21
23
Retentien Time (min)
d
19
21
2
Retention T i n e ( m i d Figure 8. Reconstructed displacement chromatogram from off-line fraction analysis. The experimental conditions are the same as for Figure 7b. (a, top) Profiles of the Impurities. (b, bottom) Profile of the sample (phenol, 0)band and the dlsplacer front (0).
the sample component, the impurity does not reach the sample band front and there is no intermediate plateau where the sample band elutes. However, a broad peak appears at the interface between the sample rear boundary and the displacer front (Figure 3d). In Figure 4, the loading factor of the sample has been reduced from 68% (as in Figure 3c) to 2070,providing a sample band that is approximately 3 times narrower. The displacer impurity pierces more easily this narrower sample band and a spike appears a t the front of the sample band in Figure 4, while in Figure 3c the same impurity penetrates into the sample band but does not pass it. The spike is nearly twice
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as high in Figure 4 as it was in Figure 3a, with a less retained impurity. Thus, the lower the loading factor of the sample, the more serious the perturbation to the band front. Figure 5 presents several displacement chromatograms where both the concentration and the capacity factor of the impurity in the displacer are varied simultaneously. These figures show that the concentration of the impurity in the displacer has no effect on the ability of the impurity to pierce through the sample band. It is also observed that the higher the concentration of the impurity in the displacer, the more it contaminates the solute band. These figures also suggest that there is a system of dimensionless variables where, for example, all the chromatograms in Figure 5a would coincide. Since the two-component system is the simplest realistic example of practical utility, it is important to illustrate this phenomenon with a sample containing two components and a displacer with one impurity. The isotherm parameters used in this study for the two solutes and the displacer are given in the caption of Figure 6. This figure illustrates how the impurity in the displacer distributes itself as a function of its capacity factor. The concentration of the impurity in the displacer was uniformly 0.8%. When the capacity factor of the impurity in the displacer is below that of the earliest eluting solute, a spike appears in front of the earliest eluting solute band (chromatogram 1 in Figure 6). As the capacity factor of the impurity increases, it moves more slowly in the column, thus giving a broad band. When the capacity factor of the impurity in the displacer is 7.0, a broad band appears under the first component band (Figure 6b). When the capacity factor of the impurity in the displacer is 9.03, a broad band appears under the second component band. Lastly, when the capacity factor of the impurity in the displacer is 11.0, a broad peak appears between the second component band and the displacer front. Experimental Study. We show in Figures 7 and 8 experimental results that have been obtained while developing a displacement method for phenol and alkyl phenols. Methylcatechol oxidizes spontaneously in contact with air, giving compounds that absorb strongly in the UV and visible ranges; slowly, the solution turns black. Although care was taken to use freshly prepared solutions, the elution and displacement results showed that impurities were always present in the displacer solutions. Several different methylphenols were investigated as displacers. Figure 7a illustrates the detector response upon performing frontal analysis of phenol. The two experiments were performed several weeks apart with phenol samples from different manufacturers. The retention times of the front differ by about 2%, which corresponds to the long-term reproducibility of the flow rate setting. An impurity less retained than the main component gives a spike a t the front of the sample component, phenol. This chromatogram shows that the two samples have slightly different purities. However, the concentration of each of these impurities is low because we observe a spike with a pointed tip. If the amount were large, we would observe, instead of a spike, a plateau or rectangular band. This is the shape observed in the frontal analysis of binary mixtures (1421). The fact that the concentration is small, but the peak is relatively tall, indicates that the absorbance at the detector wavelength is high compared to the case with the phenol sample. Figure 7b illustrates the displacement chromatograms of a l-mL injection volume of each of these two phenol samples. The two spikes are much higher than those in Figure 7a, and they have nearly the same height. This proves that most of the impurities in the chromatograms of Figure 7b come in a large part from the displacer. In Figure 8, we show the reconstructed displacement chromatogram obtained from the analysis of the fractions
Anal. Chem. 1991, 63, 2188-2193
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collected during an experiment similar to the one shown in Figure 7b. The profiles in Figure 8a are those of the displacer impurities, while the profile in Figure 8b represents the band of the sample and the displacer. For these two figures, a considerably expanded time scale has been used, for the sake of clarity. As the impurities have not been identified, their concentrations are given in units of peak area in the analytical chromatograms. The concentration profiles of two early eluting components (symbols A and 0 in Figure 8a) are qualitatively similar to those in Figure 3a. The third profile (symbol +) appears to be qualitatively similar to Figure 3b, and the last eluting impurity (symbol 0)is qualitatively similar to Figure 3c. Therefore, the profiles of the impurities agree well with theoretical predictions. These results demonstrate the potentially dramatic influence of the displacer impurities on the band profiles of the sample components, the strong need to use a pure displacer or to purify it carefully before starting a displacement experiment, and the definite requirement to make sure that any remaining impurities are harmless to the performance of the products.
CONCLUSION As long as the displacer contains impurities that are less retained than the displacer, one band at least, the last one in the isotachic train, will be contaminated by this impurity. The smaller the retention of a displacer impurity, the larger the number of bands which it will contaminate. The higher the impurity concentration, the higher is its concentration in the solute band. In principle, the effect of an impurity could be reduced to some extent by increasing the sample loading. Accordingly, the displacer should be a high-purity chemical and strict specifications regarding its content in weakly retained components must be fulfilled. Alternately, the displacer should be chosen so that it and its impurities can be removed readily from the purified product and that their concentrations can be assayed easily. Only those impurities whose presence in the purified products is inconsequential can be tolerated. In some cases, these restrictions may render more difficult and costly the development of a separation method by displacement chromatography.
ACKNOWLEDGMENT We deeply thank VYDAC for the C18column used for the displacement chromatography and the adsorbent used in the column for the off-line fraction analysis. We acknowledge the help provided by Yong-Shen Zhang in drawing the figures. Registry No. Phenol, 108-95-2.
LITERATURE CITED Horvath, Cs. I n The Scknce of Chromatography; Bruner, F., Ed.; Journal of Chromatography Library; Elsevler: Amsterdam, The Netherlands, 1985,p 179;Vol. 32. Mlemerlch, F.; Klein, 0. MultJcomponent Chromato6xephy. A Theory of Interference; Marcel Dekker: New York, 1970. Rhee, H.-K.; Amundson, N. R. AIChE J. 1982. 2 8 , 423. Kattl, A. M.;Guiochon, 0. J. Chromatogr. 1988, 449, 25. Gu, T.; Tsai, G.J.; Tsao, G. T. Biotechnol. Bioeng. 1991, 37, 65. MorbMelli, M.; Stortl, G.; Carra, S.; Niederjaufner, G.; Pontoglio, A. Chem. Eng. Sci. 1985,40. 1155. Subramanian, G.; Phillips, M. W.; Cramer, S. M. J. Chromatogr. 1988, 439, 341. Golshan-Shirazi, S.;Guiochon, G. Anal. Chem. 1989, 61, 1960. Gulochon, G.; Ghcdbane, S.; Golshan-Shirazi, S.; Huang, J. X.; Katti, A.; Lin, B. C.; Ma, 2. Talanta 1989, 36. 19. Frenz, J.; Horvath, Cs. AIChE J. 1985, 31, 400. DeCarll, J. P., 11; Carta, G.; Byers, C. H. AIChE J. 1990, 36, 1220. Cardinal, F.; Ziggiotti, A.; Viscomi, G. C. J. Chromatogr. 1990, 499,
37. Katti, A. M.;Dose, E. V.; Guiochon, G. J. Chromatogr. 1991, 540, 1. Antia, F.; Horvath, Cs. Ber. Bunsen-Ges. Phys. Chem. 1989, 93,
961. Frenz, J.; Bourell, J.; Hancock, W. S. J. Chromatogr. 1990, 512, 299. Ramsey, R. S.;Katti, A. M.; Guiochon, G. Anal. Chem. 1990, 62,
2557. Katti, A. M.;Guiochon, G. C. R . Acad. Sci., Ser. 2 1989, 309 (11),
1557. Jacobson, J.; Frenz, J.; Horvath, Cs. Ind. Eng. Chem. Res. 1987, 26, 43. Katti, A. M.; Guiochon, G. Am. Lab. 1989, 21 (lo),17. Golshan-Shirazi, S.;Guiochon, G. Anal. Chem. 1988, 60, 2364. Zhu, J.; Katti, A. M., Guiochon, G. J. Chromatogr.,in press.
RECEIVEDfor review April 2, 1991. Accepted July 1, 1991. This work was supported in part by Grant CHE-8901382 from the National Science Foundation and by the cooperative agreement between the University of Tennessee and the Oak Ridge National Laboratory. We acknowledge support of our computational effort by the University of Tennessee Computing Center.
CORRESPONDENCE Pulsed Rapid Heating Method for Volatilization of Biological Molecules in Multiphoton Ionization Mass Spectrometry Sir: Multiphoton ionization (MPI) mass spectrometry has been shown to be a very powerful technique for chemical analysis (1). In MPI, molecules are ionized by absorbing two or more photons from a pulsed laser beam and the resulting ions are detected by, usually, a time-of-flight mass spectrometer (TOFMS) (1-4)or a Fourier transform mass spectrometer (FTMS) (5-9). Advantages of using MPI as an ionization source for mass spectrometry include high sensitivity, high selectivity, and the ability of controlling the mass fragmentation patterns. Moreover, supersonic jet spectroscopy (SJS) has been combined with MPI mass spectrometry in a TOFMS to further enhance the sensitivity and selectivity of the MPI 0003-2700/91/0363-2188$02.50/0
technique (1,10,11). By incorporating SJS with MPI mass spectrometry, a two-dimensional detection scheme based on mass spectrum and jet-cooled wavelength spectrum can now be uniquely employed for identification of molecules. However, in the past, both MPI mass spectrometry and SJS were limited to the studies of volatile molecules. In order to extend the techniques for the study of thermally labile biochemicals, a method for the vaporization of these molecules without thermal decomposition must be developed. Thus, pulsed laser desorption (LD) (1, 12, 13) and, more recently, fast atom bombardment (FAB) (14)have been used for the generation of neutrals from nonvolatile and thermally labile 0 1991 American Chemical Society
