Anal. Chem. 1987, 59, 79-05 (30) Ishii, D.; Takeuchi, T. J. Chromatogr. Libr. 1985, 30, 3. (31) Manz, A.; Frobe, 2.; Simon, W. J. Chromatogr. Libr. 1985, 30, 297. (32) Balchunas, A. T.; Capacci, M. J.; Sepaniak, M. J.; Maskarinec. M. P. J . Chromatogr. Sci. 1985, 2 3 , 381. (33) Niessen, W. M. A.; Poppe, H. J. Chromatogr. 1985, 323, 37. (34) Van Wet, H. P. M.; Poppe, H. J. Chromatogr. 199$, 346, 149. (35) Niessen, W. M. A.; Van Vliet, H. P. M.; Poppe, H. Chromatographia 1985, 2 0 , 357. (36) St. Chire, R. L.; Jorgenson, J. W. J. Chromatogr. Sci. 1985, 2 3 , 186. (37) White, J. G.; St. Clalre, R. L.; Jorgenson, J. W. Anal. Chem. 1988, 5 8 , 293. (38) Farbrot, A.; Folestad, S.; Larsson, M. HRC CC, J . High Resolut. Chromatogr. Chromatogr. 1988, 9 , 117. (39) Foley. J. P.; Dorsey, J. G. Chromatographia 1984, 18, 503. (40) Zarrin, F.; Dovichi, N. J. Anal. Chem. 1985, 5 7 , 2690. (41) Eisenman, G. Glass Electrodes for Hydrogen and Other Cations; Marcel Dekker: New York, 1967.
79
(42) Durst, R. A. Ion-Selective Electrodes; Special Publication No. 314, National Bureau of Standards: Washington DC, 1969. (43) Henderson, P. 2.Phys. Chem. 1907, 5 9 , 118. (44) Henderson, P. 2.Phys. Chem. 1908, 6 3 , 325. (45) Milazzo, G. Eiektrochemie I , Grundlagen und Anwendungen ; Birkhauser: Basel, 1980. (46) Lanter, F.; Erne, D.; Ammann, D.; Simon, W. Anal. Chem. 1980, 5 2 , 2400. (47) Kucera, P. J. Chromatogr. 1980, 798, 93. (48) &ais, K.; KrejG, M. J. Chromatogr. 1982, 235,21.
RECEIVED for review, June 2, 1986. Accepted September 15, 1986. This work was partly supported by the Schweizerischer Nationalfonds zur Forderung der wissenschaftlichen Forschung.
Transitory Mobile Phase Environments for Rapid Selectivity Changes in Liquid Chromatography: Application to Organic Dyestuffs Jennifer C. Gluckman, Karel Slais,’ Udo A. Th. Brinkman, and Roland W. Frei* Department of Analytical Chemistry, Free University, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
Translent moblle phase environments created by lnjectlng solvent plugs of varying composJtlon are shown to selectlvety elute compounds according to their chemlcal properties. Wlth several organlc dyestuffs as model solutes, weakly acidic, strongly acldlc, and neutral substances were sequentlaily eluted in a serles of subchromatograms based on pH, lon-palr competitlon, and hydrophobic Interaction mechanlsms, respectlveiy. Elution wlth both lsocratlc plugs and wlth pluggenerated gradlents Is described. The use of transparent columns allowed solute bands to be vlewed durlng the separation process, and the potential for on-cdumn detection and laser scannlng Is dlscussed.
Solvation and chemical interactions within the mobile phase play an important role in liquid chromatographic separations. Compound retention, a function of the dynamic equilibrium of individual solute molecules between the stationary and mobile phases, may be significantly affected by changing the chemical properties of either phase. For a given analytical column, however, altering the mobile phase composition is the simplest and most direct means of modifying the separation. Solvent strength and polarity are among the two most widely varied mobile phase parameters (1-3). However, additional factors, such as changing pH (4,5),ionic strength (4,5), and/or the presence of competing (5, 6 ) , complex-forming (6),or ion-pairing (5-7) additives in the eluent, may alter compound retention by several orders of magnitude. The importance of mobile-phase interactions in liquid chromatography (LC) combined with the fact that the eluent is, by definition, a constantly flowing and, therefore, potentially changing medium has recently led to the examination of transitory mobile phase changes as they affect compound separation. Berry (8-10) has generated both cation- and anion-pairing chromatographic modes on a temporary basis using a single, reverse-phase, gradient elution system. By O n leave from the Institute o f Analytical Chemistry, Czechoslovak Academy of Sciences, 611 42 Brno, Czechoslovakia. 0003-2700/87/0359-0079$01.50/0
”injection-loading” two sample volumes of 1 M ion-pairing agent onto the column prior to sample introduction and subsequently eluting the agent with a full mobile phase gradient, he was able to examine cation-pairing, reverse-phase, and anion-pairing separation consecutively. Although injection-loading is not a new concept (11-15), it has been used primarily to shorten column equilibration times (11-14) or to place large amounts of sample onto the column for trace analysis (15). In addition to the more traditional injection loading, Berry has also extended this concept to include transient alterations in the mobile phase created by injecting plugs of weak eluent 10-25 times the volume of the sample (10). Carefully timing these injections during a chromatographic run allowed (10) two overlapping peaks to be resolved when the low solvent strength segment reached them. Temporary mobile phase environments have also been created through sample-injection-generatedion-pair gradients (16). Simultaneousintroduction of both solute and counterion into a chromatographic column effectively created a counterion concentration gradient in the mobile phase (16),focusing the solute a t the column head and subsequently eluting it at a characteristic counterion concentration (16). Alternatively, peak compression may be achieved by creating narrow mobile phase zones whose composition varies from that of the bulk eluent. For example, in ion-pairing systems injecting the sample in a solution of an organic anion can create a depletion zone of one of the mobile phase components (17 ) . It has been shown (17 ) that the peak for an analyte which coelutes with this region will be extremely narrow. Unfortunately this focusing effect can be applied to only one specific compound within the sample, making the effective volume of the “alternative mobile phase” equal only to this narrow band and, consequently,reducing the peak capacity (18) of the alternate separation system to unity. Unlike previous work, the present study used chemical interactions and solvation phenomena withii transitory mobile phase environmenb to achieve the selective elution of various compound types contained within a single sample. With the same bulk eluent and without column switching, several 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
“subchromatograms”,each having a substantial and variable peak capacity, can be obtained within the context of a single chromatographic run. In the present paper, pH, ion-pair competition, and solvent strength have been utilized to selectively elute weak and strong acids and neutral compounds contained in a single sample. Although the model system consisted of only these three chemical groups, the technique is general and may be readily extended to a large variety of compound types. Several of the 256 organic color additives permitted for use in cosmetic products by the European Economic Community (EEC) (19)were selected as model solutes in this initial study. The analysis of these dyestuffs, which represent a wide range of compound classes, is a difficult analytical problem important in the establishment of appropriate safety regulations and in the future monitoring of their use. In particular, most synthetic dyes contain a variety of compounds due to the impurity of the reactants used in their synthesis and to the formation of many side products (20). Indeed, some of the dyes are purposefully mixtures, all components of which have not been adequately characterized, despite the large effort that has been directed toward their analysis (20-25). The successive, selective elution of specific compound types from a single sample as demonstrated in the present study can provide readily available additional information on the nature and chemical properties of many dye components. In addition, the use of glass analytical columns allows the separation to be visually monitored, simplifying optimization of the plug composition and providing immediate information on the number and nature of sample components. Previously employed to follow the retention of various proteins in reverse-phase LC (26),the transparent glass columns used in the reported work permitted the equilibration of the various dyes between the stationary and the mobile phases to be followed in some detail, yielding a great deal of chemical information. In addition, there is the potential for direct on-column detection (i.e., using laser scanning) along the entire column. In this way, the separation may be instantaneously monitored at any point during the chromatogram (see also ref 36) and a t any place along the column, after which time a subsequent plug can be injected and the analysis continued.
EXPERIMENTAL SECTION Apparatus. A schematic diagram of the HPLC system is shown in Figure 1. A Kontron (Zurich, Switzerland) Model 414
pump was used without pulse dampening to deliver a mobile phase flow of 0.4 mL/min. Two Valco (Houston,TX) six-port injection valves were used for sample and plug introduction. The sample valve, which was returned to the “load” position during plug injection, contained a 30-kL loop, while that used for plug introduction contained a loop of 0.5 mL (1.0 mm id.), 1.4 mL (1.0 mm i.d.), 3.0 mL (2.0 mm i.d,), or 5.6 mL (2.0 mm id.). The plug-introductionloops were coiled as tightly as possible (- 1-cm coil diameter) to facilitate mixing within the loop. For gradient generation, an additional 1-mL mixing coil with either a 1 or 2 mm i.d. (1-cm coil diameter) was added after valve C to generate a more gently sloping plug profile, as previously described (27) and illustrated in Figure 1B. In addition, a segment of small inner diameter tubing (0.25 mm i.d. X 75 cm length, 1-cm coil diameter) was placed after the mixer. All tubing used was made from stainless steel. The analytical column was a 10 cm X 3 mm i.d. Chromspher 5 ym C-18 (Chrompack, Middelburg, The Netherlands) glass column housed in a stainless steel cylinder from which a window was cut to allow easy on-column viewing of the solute bands. A Perkin-Elmer (Norwalk, CT) Model LC-55 variable wavelength UV-visible detector monitored the absorbance at 500 nm for dye samples and at 280 nm for acetone injections. Chromatograms were recorded with a Kipp and Zonen (Delft, The Netherlands) Model BD-8 multirange recorder. Chemicals and Reagents. HPLC grade methanol, acetone, acetic acid, nitric acid. and sodium acetate were obtained from
I’
plug introduction column
waste recorder
a
1
31UQ introduction
1
recorder
1
Figure 1. Schematic diagram of the analytical system for (A) isocratic transient mobile phases and (B) gradient generation.
J. T. Baker (Deventer,The Netherlands). Tetrabutylammonium hydroxide (TBAOH, 40% in water) was purchased from Janssen Chimica (Beerse,Belgium) and octanesulfonic acid (sodium salt) was obtained from Aldrich Europe (Beerse, Belgium). The cosmetic dye standards used as model compounds are listed in Table I along with several of their designations, their structures, and their compound-type assignments. Acid green 25, pigment red 3, and solvent red 23 were purchased from Aldrich. Solvent yellow 94 was obtained from Thomas Scientific (Swedesboro, NJ) and the remaining dyes came from D. F. Anstead, Ltd. (Essex, U.K.). All chemicals were used without further purification. Water was purified in-house with a Milli-Q system (Waters Millipore, Milford, MA). Methods. The mobile phase delivered by the pump (system mobile phase) was consistently 0.03 M TBAOH, 45/1/0.5/53.5 (v/v) CH30H/CH3COOH/HN03/H20(pH 3.0). The dye samples were dissolved in the mobile phase when possible. However, the neutral dyes required that 10% (v/v) tetrahydrofuran be added to the mobile phase for dissolution. The red and yellow dyes were injected at a concentration of approximately 0.1 mg/mL. This concentrationwas doubled for the blue and green dyes due to their relatively poorer absorption at 500 nm. Exact sample concentrations were not considered critical in this qualitative study, provided no overloading was observed.
RESULTS AND DISCUSSION There are two primary attractions to using transient, plug-generated mobile phases for selective elution in LC. First, it is a universal technique which can be applied to samples containing compounds with different chemical properties. Second, it allows diverse separation systems to be used with minimal column reequilibration. Because of the numerous parameters involved in establishing such a system, a series of choices was necessary to select an illustrative model. To begin, it was desired to combine a transparent analytical column with easily visible model solutes so that the behavior of the various compound types in different mobile phase environments could be followed directly on the column. In addition, this combination would provide the opportunity for future work with on-column detection and peak-shape monitoring. The cosmetic colorants were chosen as commercially important visible substances having a wide range of chemical properties. The fundamental chromatographic system was selected next, since it would form the basis for all chemical interactions. Although the dyes have been successfully analyzed by both
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
81
Table I. Model Cosmetic Dyes compound number
C.I. registry C.I. designationa number
F.D.A. designationb
structure
compound type
source
I
solvent yellow 94
45350:l
D+C yellow no. 8
weak acid
Thomas Scientific
I1
(Fluorescein)c acid green 25
61570
D+C green no. 5
strong acid Aldrich NnO3S
I11
acid yellow 36
13065
Ext. D+C yellow no. 1
strong acid D.F. Anstead, Ltd. NaOSS,
strong acid D. F. Anstead, Ltd.
Ext. D+C violet no. 2
IV
acid violet 43
60730
V
pigment red 3
12120
neutral
Aldrich
VI
solvent violet 3
60725
neutral
D.F. Anstead, Ltd.
VI1
solvent red 23
26100
neutral
Aldrich
D+C red no. 17
"C.I. indicates the Color Index (30). bFood and Drug Administration (U.S.A.). cCommon name. normal- (25, 28) and reverse-phase (20-25) liquid chromatography, a (2-18 reverse-phase system was used for the present work in order to minimize complicating water saturation effects present with silica surfaces and to reduce the time required for stationary phase equilibrium. The six model dyes (Table I) were selected on the basis of their availability, their range of chemical properties, their easy visibility, and their ability to absorb light at a common wavelength of 500 nm. The system mobile phase (i.e., that delivered by the pump) was designed to retain all model compounds (k'> 501, while remaining miscible with the elution plugs and providing a rapid reequilibration of the stationary phase. Thus, a moderate strength eluent with a rather high (0.03 M) concentration of a short-chain ion-pairing agent (TBA') and a pH greater than the pK, of the strongly acidic model dyes (group 2) but less than the pK, of the weakly acidic compounds (group 1) was chosen. Eluent Plugs. Since all three model compound groups (weak acids, strong acids, and neutrals) were retained at the top of the column in the system mobile phase, the introduction order of the elution plugs was critical in order to achieve sequential elution. After each plug was tested (described below) with each compound type, it was evident that a strong transient mobile phase eluted all solutes, while a plug containing an organic anion to compete with the dye molecules and, thus, break their TBA ion pairs eluted only the acidic dyes. A slightly basic solution, on the other hand, selectively
eluted the previously un-ionized weak acids alone. Therefore, the appropriate elution order was determined to be weakly acidic (pK, > 4) solutes followed by strongly acidic (pK, < 2.5) compounds and finally all remaining neutral substances. The plug volume was the next consideration. The column void volume was determined to be 0.3 mL by using acetone as a nonretained solute. Thus, plugs generating transient mobile phases with constant compositions equal to and greater than the column volume were examined. The profiles of plugs having 0.5, 1.4,3.0, and 5.6 mL total volumes are presented in Figure 2. All four volumes successfully eluted any single compound. However, the peak capacity of the temporary column/mobile phase system is limited by the mobile phase volume. On the other hand, the duration of the transient system is lengthened by the plug volume (cf., e.g., Figure 2, part A vs. part D). Thus, a compromise volume yielding adequate peak capacity without undue length is ideal. For the isocratic portion of this study, a plug volume of 1.4 mL was selected. Weak Acids. All acids that are less than 10% ionized at pH 3 were considered weak acids for the purpose of this study. From among the cosmetic dyes this includes all compounds having only carboxylic acid groups. Due to the presence of many interfering strongly acidic and/or neutral impurities in the available dye standards, a single weak acid (fluorescein, C.I. no. 45350), pK, = 4.5 (29) was chosen for the purpose of illustration (Table I). It should be noted, however, that all
82
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
Table 11. Elution Plugs" and Target Compounds
dyes
plug composition 46:54 (v/v) CH30H:H20 0.1 M NaOAc pH 8.4
i
i
il
I
weak acid
retention time
weak acid
4.5
45:55 (v/v) CH3OH:HZO I1 0.3 M C,S03-(Nasalt) I11 pH3 (adjusted with CH3COzH) IV
strong acid
9010 (v/v) CH30H:pH 3 H20 (adjusted with CH3C02H)
neutral
V
2.4
3.0 4.7 4.8
100% CHBOH
VI neutral 3.9 "Listed in order of introduction, system mobile phase was 45:1:0.5:53.5 (v/v) CH3OH:CH&!OzH:HNO,:H,0, 0.03 M TBAOH (PH 3).
I
15
10
5
0
-tirne(min)
Figure 2. Elution profiles for isocratic plugs of differing volume: (A) 0.51 mL, (6)1.4 mL, (C) 3.0 mL, and (D)5.6 mL; column, 100 X 3.0 mm i.d. 5 pm Chromspher C-18; system mobile phase, 0.03 M TBAOH in 45/1/0.5/53.5 (v/v) MeOH/CH,CO2H/HNO3/H,O(pH 3); flow rate, 0.4 mL/min; plug composition, 0.02% (v/v) acetone in mobile phase; detection by UV absorbance at 280 nm.
weak acids tested (and their various impurities) were eluted according to the mechanisms described below. The influence of ionic equilibria on the distribution of carboxylic acids between the stationary and mobile phases has been described in the literature for reverse-phase and reverse-phase ion-pair LC (30). Accordingly, this retention time dependence was utilized as the fundamental principle for the selective elution of the weak acids. Since these compounds are primarily neutral a t pH 3, a slightly basic plug was chosen to ensure their complete ionization. The plug composition was optimized to achieve Gaussian peak shapes and relatively rapid elution based mainly on hydrophobic interactions between the solute molecules and the stationary phase. The optimum composition was determined to be at a pH of 8.4 with 46% methanol and 0.1 M NaOAc in water (see Table I1 and Figure 3). Forty-six percent methanol was used in the plug so as to make its organic strength equal to that of the system mobile phase, in which the acetic acid acted largely as an organic additive. Also, since the retention time of dissociated acids is known (30)to decrease with increasing mobile phase ionic strength, adding 0.1 M NaOAc helped to ensure the rapid elution of the weak acids. Strong Acids. Dyes with pK, values less than approximately 2.5 were considered to be strong acids. Among the model compounds, those with sulfonic acid groups fell into this category. Acid yellow (C.I. no. 13065), acid violet ((2.1. no. 60730), and acid green (C.I. no. 61570) were selected as
-
10 5 0 ttme(min) Figure 3. Chromatogram illustrating the selective elution of six cosmetic colorants: I, solvent yellow 94; 11, acid green 25; 111, acid yellow 36; IV, acid violet 43; V, pigment red 3; VI, solvent violet 3; plug A, 46/54 (v/v) MeOH/H,O, 0.1 N NaOAc (pH 8.4); plug B, 45/55 (v/v) MeOH/H,O, 0.3 N octylsulfonate (Na sait) (pH 3);plug C, 90/10 (v/v) MeOH/H,O @H 3); plug D, 100% MeOH (acidicpH values adjusted with CH3C03H). Plug volume was 1.4 mL; detection was by UV absorbance at 500 nm. All other conditions are as in Figure 2.
25
20
15
model solutes (Table I). Again, all sulfonic acids examined behaved in a similar manner. As indicated in Table 11,the optimum plug composition was found to be 0.3 M C8S03- (sodium salt) in 45/1/0.5/53.5 CH30H/CH3COOH/HN03/H20 at pH 3. The transient mobile phase composition was kept identical with that of the system mobile phase with the exception of the added organic anion so as to minimize the variety of chemical interactions occurring on the column. In order to facilitate column reequilibration to the system mobile phase, octylsdfonate was selected as the anion because its chain length provided a compromise between easy removal from the column and adequate ion-pairing properties. A concentration of 0.3 M was selected as the lowest one resulting in a reasonable movement of the dye. The elution mechanism for these compounds was based on establishing a competitive equilibrium between the TBA+ ion-pairing agent and a high concentration of an alternative sulfonic acid which would pair with it (31-33). As a plug containing an excess of this acid entered the column, the large number of alternative sulfonic acid molecules shifted the
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1. JANUARY 1987
I 15
83
I
3o
t~
Fbun 5. Graphical representab of peak p n l o n on wlumn as a function of separation time for ttm strong acid dyes: 0. compound 11; A. wmpound Ill: 0.compound I V .
at any given time. A horizontal uvnn sedion gives their arrival
I
Figure 4. Photographs Illustrating the elution of compounds 11-IV following plug ihleaiar: (A) acid yellow 36 (111) and acid violet 43 (IV) Immediately following 1nje.ction of plug containing 45/55 MeOH/H,O, 0.3 N NaC,SO, (pH 3.0k ( E E ) e l u h of strm acids wimin plug. Note appearance and selectivity changes for acid green 25 (11). Chromatographic conditions are given in Figure 3.
ion-pair equilibrium in their favor, displacing the ionized dye molecules and causing them to elute in an order determined primarily by hydrophobic interections. Thus,the moat drastic differences in elution time and bandwidth were observed for dyes having a greater number of sulfonic acid groups and a smaller number of aromatic rings. Figures 3-5 illustrate this effect nicely. The relative positions of the three sulfonic acid bands are evident in Figure 4 in which the course of their separation is followed from shortly after plug injection (part A) until just prior to the elution of compound I1 (part E). Component 11, having two acidic groups, moves more rapidly than do components III and N, which each contain only one sulfonic acid moiety. A graphical interpretation of the photographs is presented in Figure 5. In fact, two-dimensional information about the behavior of the solutes can be obtained from this plot. A vertical cross section of the graph indicates the in8tantaneous distribution of the components along the column
time a t any particular point on the column. Neutrals. The remaining cosmetic colorants were neutral compounds as exemplified by pigment red 3 (C.I. no. 12120) and solvent violet 3 (C.I. no. 60725),which varied in structure from acid violet 43 only by the absence of a sulfonic acid group (Table I). The neutral dyes differed from the other compound classes in that not all were equally well eluted hy plugs of the same composition. This difference was directly due to the strictly hydrophobic elution mechanism. The same retention rules that govern reverse-phase LC, therefore, dictated the choice of plug composition. As shown in Figure 3 and Table I1 pigment red 3, which contains only three aromatic rings, was nicely eluted in W/10CH,0H/H20, while solvent violet 3, having four rings, was not eluted within this plug. A bamient mobile phase of 100%methanol, which was suffciently strong to elute solvent violet 3, failed to resolve the two injected neutrals. Thus, to separate these two substances well, two plugs were required. If, however, the compounds of interest had already been eluted in a previous plug, the injection of a strong solvent might be desired to remove the neutrals rapidly. Column Regeneration a n d Reproducibility. It is important to fully regenerate the column to its original state in order to ensure g o d reproducibility between chromatographic runs. Although exact regeneration requirements depend on the speeific separation system employed, in the present study only two column volumes of the system mobile phase were needed to obtain reproducible data. The retention time (measured from plug injection) repeatability for the model mixture shown in Figure 3 was 1.9% (SD = 0.2, n = 14) averaged over all six compounds. Gradient Generation. In an extension of the work 4 t h isocratic transient mobile phases, gradient generation within the plug was briefly explored to determine its potential utility. Gradients were created by using the two largest plug-injection loops (i.e., 3 and 5.6 mL) followed by mixing loops of various inner diameters and volumes as shown in Figure 1B. A 75 cm long segment of small (0.25 mm) inner diameter tubing was placed after the mixing loop since poor flow dynamics at thia point resulted in a high degree of peak tailing and splitting without it. A detailed description of this technique for gradient generation is given elsewhere (27). Several gradient profiles obtained by using various combinations of the plug-injection and mixing loops are illustrated in Figure 6. The gradient generated by a 3-mL plug with a 1-mL, 2-mm-i.d. mixing loop (Figure 6B) was best suited to
84
ANALYTICAL CHEMISTRY, VOL. 59, NO. 1, JANUARY 1987
-
time (mid
Figwe 6. Elution profiles of transient mobile phase gradients generated by dmerent plug/mixer combinations: (A) plug = 3.0 mL, mixer = 1.0 mL, 1 mm i.d.; (B) plug = 3.0 mL, mixer = 1.0 ml, 2 mm i.d.; (C) plug = 3.0 mL, mixer = 1.4 mL, 1 mm i.d.; (D) plug = 5.6 mL, mixer = 1.4 mL, 1 mm i.d. Conditions are given in Figure 2. A
0
iI
-
I=
hm
8 6 4 2 0
15
t me(mini
-
10 5 time(min1
0
Flgure 7. Chromatogram illustrating the (A) isocratic and (B) gradient elution separation of three neutral dyes: V I I , solvent red 23; plug composition, 90/10 (v/v) acetone/H,O; plug volume, (A) 1.4 mL, (B) 3.0 mL, mixer, 1.0 mL, 2 mm i.d.; other conditions as in Figure 3.
the present system. With this combination, the mobile phase changed smoothly in composition from that of the system mobile phase to that of the pure plug over 13.6 column volumes, giving a sufficiently slow increase in the effective mobile phase strength to allow the resolution of closely eluting compounds. For example, the mixture of three neutral colorants presented in Figure 6 was poorly separated in an isocratic plug of 90/10 acetone/water (Figure 7A) but was well-resolved by a 3-mL gradient with this final composition (Figure 7B). The optimum gradient configuration gave a highly reproducible profile. Measurements made by using a dilute acetone solution and UV absorbance showed that the two times at which it assumed half its final value were repeatable within 1.7% from run to run (SD = 0.3, n = 5). CONCLUSION
Transitory mobile phases and mobile phase gradients provide a ready means for the selective elution of different compound classes contained within a single sample. Illustrated in the present study with the cosmetic dyes, the method is general and easily applied to a wide range of compound types, being limited only by the physical constraints of the chromatographic system and the imagination of the chromatographer. While many elution mechanisms remain to be ex-
plored, metal complexation interactions and ionic strength gradients are two potentially important ones that will receive further attention. A distinct advantage of transitory mobile phases is the possibility of rapid change from one separation mode to another, since reestablishing of the original state, and hence readiness for the next separation (plug) principle to be applied, takes place while the separation process still is going on. In addition we profit from the fact that all the problems of changing of the total mobile phase system, such as necessity for rinsing the pump and connections, injecting a new sample etc., can be avoided. The temporary use of relatively agressive mobile phases is also permitted since the contact time with the stationary phase is only very brief. Working with plugs of pH 8.4 has, for example, been carried out over several months without significant deterioration of the column. Another significant aspect of the technique which has not yet been fully exploited is the increased detectability of some compounds in specific solvent systems. In this way, increased sensitivity can be achieved for compounds with poor detectability simply by altering the chemical environment in which they are eluted. A well-known example of this phenomenon is the change in absorption or fluorescence efficiency exhibited by many compounds in solvents of different polarity or pH. Fluorescein, the model weak acid used in this study, for example, is much more fluorescent in the basic elution plug than it is in the pH 3 system mobile phase. In addition, by use of transparent chromatographic columns, such as those of glass or fused silica, it is possible to follow the separation process directly as it occurs on the column. Not only is this helpful in system optimization, but it allows detailed selectivity examinations and peak shape assessments to be made at any stage during the chromatogram. Also, since the chromatogram may be monitored at any point in time and at any location within the column, the analysis of complex samples can be speeded by injecting subsequent elution plugs before peaks resolved in previous plugs have left the column. Further, by scanning the column with a light beam, such as that from a laser source, on column detection ( 3 4 , s )becomes possible for many compounds that are not visible to the naked eye. Finally, coupled with computer evaluation of the generated data, simultaneous detection along the entire column (36) can allow peak distributions to be followed over time, providing additional insight into chromatographic mechanisms and facilitating the quantitation and identification of unknown compounds. Finally, miniaturizing the chromatographic system to one of capillary dimensions will provide several advantages. It will not only simplify on-column detection to give decreased band broadening but also extend the range of feasible elution mechanisms by permitting exotic and expensive transient mobile phases to be used. Gradient generation will also be facilitated since the reduced volume of LC microcolumns greatly simplify the shaping of transient mobile phase plugs (27). On the negative side it should be mentioned that the peak capacity per plug is still rather limited even with the use of gradients, which may place the emphasis on the choice of even more selective separation modes. The usefulness of this approach for routine analysis still has to be proven on a series of real life samples, although from preliminary experience the potential should be quite adequate. ACKNOWLEDGMENT
The authors wish to thank J. W. M. Wegener for his generosity in supplying the cosmetic dye samples and for his advice in the development of a fundamental separation system. Registry No. I, 2321-07-5; 11, 4403-90-1; 111, 587-98-4; IV, 4430-18-6; V, 2425-85-6; VI, 2944-19-6; VII, 85-86-9.
Anal. Chem. 1087, 59, 85-90
LITERATURE CITED (1) De$, 2.;Macek, K.; Janlk, J. I n Liquid Column Chromatography: A Survey of M e m Technlques and AppIicatiOns; Elsevier: Amsterdam, 1975. (2) Snyder, L.; Kirkland, J. I n An Introduction to Modern Liquid Chromatography; Wlley: New York, 1981. (3) POOb, C. F.: Schuette, S. A. I n Confempofary PTactice of Chromafcgraphy; Elsevier: Amsterdam, 1984; pp 213-345. (4) Sorei, R. H. A.; Hulshoff, A. I n Advances in Chromatography; Gddings, J. C., Grushka, E., Cazes, J., Brown, P. R., Eds.; Marcel Dekker: New York, 1983; Vol. 21, pp 87-129. (5) Hearn, M. T. W. I n Advances In Chromatography; G i i n g , J. C.; Grushka, E.; Cazes, J.; Brown, P. R., Eds.; Marcel Dekker: New York, 1980; Vol. 18, pp 59-100. (6) "Ion Pair Chromatography" I n Chromatographic Science Series ; Hearn, M. T. W., Ed.; Marcel Dekker: New York, 1985; Voi. 31. (7) Karger, B. L.; Le Page, J. N.; Tanaka, N. I n High-Performance Liquid Chromafography;Horvath, Cs., Ed.; Academic: New York, 1980; Vol. 1, pp 113-206. (8) Berry, V. V.; Shansky, R. W. J. Chromatogr. 1984, 284, 303-318. (9) Berry, V. V. J. Chromatogr. 1084, 290, 143-181. (10) Berry, V. V. J. Chfomafogr. 1985, 321, 33-43. (11) Huber, J. F. K.; Meijers, C. A. M.; Hulsman, J. A. R. I n Advances in Chromatography 1971: Zlatkis, A., Ed.; University of Houston: Houston, TX, 1971; pp 230-235. (12) Snyder, L. R.; Kirkland, J. J. I n Advances in Chromatography 1971; Ziatkis, A., Ed.; Unlverslty of Houston: Houston, TX, 1971, p 328. (13) Berry, V. V.; Engeihardt, H. J. Chromatogr. 1974. 95, 27-38. (14) Crommen, C. J.; Fransson, B.; Schlll, G. J. Chromafogr. 1977, 142, 283-297. (15) Euston, C. B.; Baker, D. R. Am. Lab. (Fairfiekj, Conn.) 1979, 7 1 , 91-92, 94, 98, ?8-100. (16) Slais, K.; Krejci, M.; Kourilovl, D. J. Chromatogr. 1988, 352, 179-197. (17) Nllsson, L. B.; Westerlund, D. Anal. Chem. 1985, 5 7 , 1835-1840. (18) Davis, J. M.; Glddlngs, J. C. Anal. Chem. 1983, 5 5 , 418-424.
(22) (23) (24) (25) . . (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (38)
85
Council Dkectlve of 27 July 1976 on the Approximation of the Laws of the Member States relating to Cosmetic Products (76/768/EEC), Official Journal No. L262, 27.9.1976, pp 189-200. Modifled by the Ammendment Directive of 17 May 1982 (82/288/EEC), Official Journal NO. L167, 15.6.1982, pp 1-32. Bailey, J. E., Jr. J. Chromafogr. 1985, 347, 163-172. Wittmer, D. P.; Nuessle, N. 0.; Haney, W. G., Jr. Anal. Chem. 1975, 47, 1422-1423. Gloor, R.; Johnson, E. L. J. Chromatogr. Sci. 1977, 15, 413-423. Jandera, P.; Engelhardt, H. Chromatograph& 1980, 13, 18-23. Jandera, P.; Churicek, J.; Bartosovl, J. Chromatograph& 1980, 73, 485-492. Wesener, J. W. N. Vriie Universheit. Amsterdam, 1983; unDublished data. DiBussolo, J. M.; Gant, J. R. J. Chromafogr. 1985, AC8605293 327, 67-78. Slais, K.; Frei, R. W., Anal. Chem. (In press). Knox, J. H.; Laird, G. R. J. Chromatogr. 1978, 722, 17-34. Handbook of Chemistry and phvsics; CRC: Boca Raton, FL, 1981; VOl. 62, p D-130. Van de Venne, J. L. M.; Hendrlx, J. L. H. M.; Deelder, R. S. J. Chromatogr. 1978, 767, 1-16, Bartha, A.; Vigh, Gy.; Billlet, H.; De Galan, L. Chromatographia 1985, 2 0 , 587-590. Bartha, A.; Vlgh, Gy.; Biiilet, H. A. H.; De Galan, L. J. Chromafogr. 1084, 303, 29-38. Bldllngmeyer, B. A.; Deming, S. N.; Prlce, W. P., Jr.; Sachok, B.; Petrusek, M. J. Chromatogr. 1979, 786, 419-434. Shelly, D.; Gluckman, J. C.; Novotny, M. V. Anal. Chem. 1984, 56, 2990-2992. Guthrle, E.; Jorgenson, J. Anal. Chem. 1084, 5 6 , 483-486. hlderloos, D. 0.; Rowlen, K. L.; Blrks, J. W.; Avery, J. P.; Enke, C. 0. Anal. Chem. 1088, 5 8 , 900-903.
RECEIVEDfor review June 9, 1986. Accepted September 8, 1986.
Performance of Annular Membrane and Screen-Tee Reactors for Postcolumn-Reaction Detection of Metal Ions Separated by Liquid Chromatography R. M. Cassidy* and S. Elchuk General Chemistry Branch, Chalk River Nuclear Laboratories, Atomic Energy of Canada Limited, Chalk River, Ontario, Canada KOJ 1JO
Purnendu K. Dasgupta Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas 79409-4260 The experlmentai factors that contrlbute to base ilne nolse In the postcolumn-reactiondetectlon of metal ions eluted from hlgh-perfonnance dynamic ion exchangers have been evaluated and compared for a screen-tee reactor (- 1 pL internal volume) and three annular membrane reactlons having Internal volumes of 1.5, 5.3, and 9.6 pL. Measurements of mixing homogeneity, performed with pulseless gas-pressure pumping, showed that both reactor deslgns gave a mixing homogeneity that was 99.983% of that theoretically possible (perfect mixing) for two solutions differing In absorbance by 2.38 units. For normal operation with high-performance reciprocating pumps for eluent delivery, pump pulsations were responsible for 90-100% of the observed peak-tcqeak noise. Column efficiency measurements with a series of ianthanlde metal Ions gave similar curves for both designs (HETP values of 0.01-0.04 mm). Both reactors gave reproducible peak areas, had good peak shapes, and operated rellabty. With the membrane reactors some leakage of eluent into the reagent solution occurred at high eluent or reagent flow rates, but thls was not a problem for normal operating conditlons.
Postcolumn-reaction (PCR) systems have played an im-
portant role in the extension of liquid chromatographic techniques to the determination of a wide range of analytes that are otherwise difficult to monitor with common detector systems. One of the earliest reported dedicated liquid chromatographs, an amino acid analyzer, relied on a PCR system (I), and the continued attractiveness of such approaches is reflected in the growing commercial availability of PCR systems. Extensive information on PCR design, operation, and chemistry is available in a recently published monograph (21, and reviews of different aspects of PCR systems appear on a frequent basis in the literature. Advances in PCR design have been particularly important to the development of inorganic high-performance liquid chromotography (HPLC). Recent comparisons of HPLC-PCR and isotope-dilution mass spectrometry for the determination of metal ions has shown that the HPLC-PCR can offer significant advantagesfor some metal-ion determinations,even when accuracies and precisions of 1 % are required (3, 4). The PCR system used in the above studies ( 3 , 4 ) involved the reaction of a colorimetric reagent with the eluted metal ions and subsequent detection with a variable-wavelength UV-visible detector. Similar PCR chemistry has been used by many workers and necessary equipment is now offered commercially by a number of manufacturers. While the
0003-2700/87/0359-0085$01.50/00 1986 American Chemical Society
