2674
Anal. Chem. 1984, 56, 2674-2681
Modeling of Analyte Behavior in Indirect Photometric Chromatography Dennis R. Jenke Travenol Laboratories, Inc., 6301 Lincoln Avenue, Morton Grove, Illinois 60053
The retention behavior of various organlc and inorganlc anions in indirect photometric chromatography is studled as a functlon of changlng eluant composition for three commercially available chromatographlc columns. The behavlor of all analytes studled can be modeled by a formulation that conslders the thermodynamic equlllbrlum that exlsts between the analyte and multiple eluant ions at the resin slte. Agreement between observed and predlcted behavlor is f5% RSD, which represents the approximate magnitude of the experlmental preclslon. The response of analyte retention to changing eluant composition can be illustrated with wlndow diagrams that allow the analyst to optimize composltion for multlspecles analyses.
Indirect photometric chromatography (IPC), as described by Small and Miller and used for anion and cation determinations ( I ) ,couples analyte separation by ion chromatography with indirect photometric analyte detection. Detection involves measuring the absorbance decrease as the analyte displaces a UV-active component in the mobile phase. Specifically, as a UV transparent analyte travels through the separator column, it replaces an equivalent concentration of the UV active elution in the mobile phase. Thus, the presence of the analyte in the detector causes a decrease in mobile-phase absorbance resulting in a negative peak in the absorbance plot. This single-column technique is a t least as sensitive as the more conventional conductometric detection method (2) and allows the analyst greater control over instrumental sensitivity. Most importantly, however, the IPC procedure does not require dedicated support components; the columns described herein are compatible with conventional HPLC pumps and photometric detectors, which can be freed for other applications by replacement of the column. The success of IPC for determining the ionic content of varied liquid sample types depends upon the development of resin materials capable of resolving analyte species from one another and from matrix components with a mobile phase that contains a UV-active component. Effective utilization of IPC requires (1)accurate characterization of analyte retention behavior, (2) identification of analytical variables that affect the relative behavior of these analyks, and (3) the ability to model analyte behavior so that system's optimization can be achieved. Of particular concern here is the response of anionic species to changes in the chemical nature of the mobile phase. The ability to model or describe how the retention characteristics change in response to changing eluant composition allows the analyst to predict the elution properties of a given analyte suite for a particular eluant composition as well as providing a means of optimitizing the quantitation of such a suite in terms of both interspecies resolution and total analysis times. The behavior of various organic and inorganic anions including C1-, Br-, lactate, acetate, oxalate, gluconate, bisulfite, phosphate, and S042-was studied over a wide range of eluant compositions for three commercially available ion chromatographic columns. The ability of the equilibrium multiple species model developed by Hoover ( 3 ) and applied to ion
chromatography by Jenke and Pagenkopf (4, 5) to describe the behavior of these analytes is documented, and window diagrams are used to optimize typical separations. The nature of the window diagrams is directly related to anabte charge and can be explained in terms of the equilibrium m d e l . EXPERIMENTAL SECTION The chromatographic system consisted of a Kratos (Ramsey, NJ) Model LC250/1 constant volume pump and Spectroflow 773 dual-beam absorbance detector coupled to a Cole Parmer (Chicago, IL) Model K8373-80 strip chart recorder. The effluent from the separator column flowed through the detector sample cell while the reference cell was filled with the eluant being studied. Columns characterized included the AS-1 anion separator manufactured by Dionex (Sunnyvale, CA; No. 030827),the Wescan (SantaClara, CA) 269-001 anion column, and the Vydac (Hespira, CA) No. 302 IC4.6 separator column. Sample loop volume was 0.02 mL, and laboratory temperature was maintained at 22 f 1 "C over the entire course of the experimentation. Eluant flow rate was 2 mL/min for the Wescan and Dionex study and 3 mL/min for the characterization of the Vydac column. Detector wavelength and sensitivity were adjusted for each eluant studied so as to provide a recognizable analyte response at the concentration level prepared; in all experiments the wavelength was set in the range of 240-260 nm. Eluants used in this study were prepared by dissolution of reagent grade potassium hydrogen phthalate in deionized water with final pH adjustment being obtained by addition of 0.1 M KOH. For the Dionex column, borate was added to the eluant to serve as a buffer as the NaB4O7.10H20salt. Stock solutions (lo00 ppm) of the analytes of interest were prepared by dissolution of their Na salt (KBr for Br-) in deionized water while standard solutions containing a single analyte were prepared by dilution of the appropriate stock with the eluant being used. The limited stability of the bisulfite ion in aqueous solutions (6-9) required that stock and standard solutions containing this ion also contain a preservative. For the low eluant pH study of the Wescan and Vydac columns the bisulfite was preserved as the H S O i ion by the addition of 0.5 v/v 37% formaldehyde solution, while in the high pH study of the Dionex resin the $0:- species was stabilized by the addition of 1.0% by volume isopropyl alcohol. Standard solutions used in all studies contained single analytes present in a concentration range of 20-50 mg/L. Each single ion standard was analyzed in duplicate. THEORY Model. The separation of analytes by an ion-exchange resin can be considered to be mechanistically controlled by the thermodynamic equilibrium that exists at individual resin sites between the analyte species and any ionic component of the mobile phase. Hoover coupled this observation with a plate-type theoretical analysis of column chromatography to produce an equation relating analyte retention, eluant composition, and various resin properties ( 3 ) . When extended to consider mobile-phase, ionic-strength-related effects and limited to the consideration of a mobile phase containing two active components, a monovalent ion 1 and a divalent ion 2, this equation takes the form
u = K*[
"(
(1
4KEE2
0003-2700/84/0356-2674$01.50/00 1984 American Chemical Society
+ --)l' 8QKEE2 z
-
(1)
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
where U = the reduced retention volume of the analyte, El = the activity of species 1 in the mobile phase, E2 = the activity of species 2 in the mobile phase, Q = the column capacity, KE = the selectivity coefficient between eluant species, K A= the selectivity coefficient between monovalent eluant species and analyte, and n = the analyte charge. Mechanistically, the selective coefficients represent equilibrium constants that describe the interaction between the species that occurs a t the resin site. The reduced retention volume U can be obtained from chromatograms by the relationship
u = (t, - t,)F
(2)
where t , = the retention time of the analyte, to = the void volume time equivalent, and F = the eluant flow rate. All three column systems studied include a mobile phase that contains both a monovalent and divalent active component. The Vydac and Wescan resins are based on a silica backbone, making a pH of 7 their practical stability limit. The limited resolving power of the monovalent phthalate ion ( H P ) makes a pH of 4 the practical lower limit for eluant composition. Since the appropriate pK, values for phthalic acid are 3.1 and 5.4 (IO),eluants prepared in the pH range noted above will contain a mixture of both HP- and P2-ions. At lower pH, the concentration of the neutral H,P approaches 10% of the total phthalate present (PT); in this approach it is assumed that the H2P species retains its form as it moves through the column and therefore does not contribute to analyte migration. The Dionex column, based on a polymer support, is less prone to pH-related degradation than the other columns studied and can be operated at higher eluant pH. However, the phthalate system has a limited buffering capacity a t alkaline pH, and therefore eluant preparation a t these pH values becomes poorly reproducible. Borate is thus added to act as a buffer; with a pK,, of 9.24 (IO) the borate ion exists as either H3B03 or H2B03- in the pH range used. Eluant composition and column capacities utilized in the model must be expressed in units that recognize the equilibrium approach upon which the model is based; that is, the volume unit employed should have some significance in terms of the actual eluant/column interaction. The ipteraction equivalent volume (IE) is defined as the amount of resin associated with a given volume of eluant a t any time during the elution process. Although the choice of the eluant volume is somewhat arbitrary, the sample size makes a convenient value for this quantity and adequately describes a particular experimental apparatus. Thus, the exchange capacity Q of the equivalent volume of resin associated with each IE of eluant can be written as
Q = Q#/
Vo)
"R
(3)
while the amount of a particular eluant ion contained in this interaction volume of eluant and thus interacting with the resin material noted in (3) becomes
E = E,$
(4) where Q, = the total column capacity, S = sample size, Vo= the void volume of column, and E , = the activity of eluant ion in the mobile phase, Window Diagrams. Window diagrams, as used to characterize and optimize separations in gas (11, 12), a highperformance liquid (I3-16),and ion ( 4 1 7 ) chromatographies, express the relationship between the retention characteristics of a pair of analytes and some operational variable of the chromatographic system. The reduced retention ratio a for analytes A and B is written as (5)
2675 A
I
I
I1
I
1
I
I
3
4
5
6
17
I 0
7
B
1-
I
I
I
~
3
4
5
7 -
16
I
0,
I ~
71
I 02
PH Figure 1. Interpretation of typical window diagrams. Species A and B are monovalent anions; specific C is divalent. The area below the dotted line represents the resolution limits set by the analyst. where tA L tB and thus cy 1 1. One notes that a t cy = 1peak overlap occurs and that as the value of a increases, the separation between the two species also increases. The operational variable of interest herein is eluant composition, which, in the case of the mixed HP-/P2- eluants, is expressed in terms of total dissolved salt concentration and pH and, in the high pH, mixed borate/phthalate eluant example, by the absolute activities of H2B03- and P2-. The interpretation of the resultant three-dimensional plots is made simpler by presenting two-dimensional contours of these response surfaces. The interpretation of a window diagram in this application proceeds as follows: the lowest line in a diagram identifies the analyte pair with the poorest resolution, and the highest line identifies the pair with the best resolution. Thus, the lower line limits optimization in terms of resolution whereas the top line limits the technique in terms of total analysis time. Figure 1 considers the two most common cases observed in ion chromatography. In both cases A and B, the analyst has chosen a = 1.5 to be the minimum acceptable resolution. In case A, all three analyte pairs have an a value greater than 1.5 for the conditions documented, and therefore the separation is analysis time limited. The optimum composition is the one that provides the shortest analysis time, shown in the diagram as the lowest point reached by the upper line. In case B, species C is less strongly retained by the resin than in the previous case, the net result of which is a decrease in aC,*and
2676
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Table I. Eluant Compositions Used in Study
eluant no.
total phthalate (xio-3 M)
activities (xlO+ M) HPP2-
PH
concn (x10-3 M)
P2-
Wescan System 1.91 1.93 1.92 1.01 1.03 1.02 3.96 4.02 3.98 0.50 0.20
1 2
3 4 5 6 7 8 9 10
11
5.02 4.10 5.95 4.14 5.15 5.90 4.15 4.97 5.96 4.11 4.11
1.19 1.60 0.33 0.85 0.60 0.21 3.16 2.50 0.61 0.42 0.18
0.50 0.09 1.17 0.05 0.33 0.65 0.18 0.93
0.63 0.11 1.56 0.05
0.02
0.40 0.80 0.23 1.28 3.31 0.02
0.01
0.01
1.30 1.87 0.68 0.19 1.63
0.47 0.71 0.23 0.61 0.08
0.59 0.95 0.27 0.78
P2-
activities HZBOL
2.21
Vydac System 1.99 2.99 0.99 0.99 1.97
eluant no,
total phthalate (x10-3 M)
4.96 4.98 4.92 5.92 4.11 total borate (x10-3 M)
PH
0.10
concn (xW3M) P2-
Dionex System 1.99 2.93 1.99 0.97 1.93 0.99
3.97 3.74 3.97 3.67 4.12 3.81
a c p If the particular column used is limited to an eluant pH of 7 by its own stability, eluant composition of O1defines the only point in the diagram where the resolution between any species pair reaches the analyst-defined resolution limit. Thus, O1represents the optimum eluant composition in terms of resolution and analysis time. One observes that at pH 6.5 acg = 1 and the species coelute. As the pH continues to increase, species C is affected more than species B, with the result that their a values increase but the elution sequence shifts. To amplify somewhat, at O1the elution order is B followed by C while at O2 the order is reversed. At point O2 the resolution between the two once again reaches the defined limit, and it is observed that O2 represents a more desirable eluant composition than O1since total analysis time is shorter. A third possibility not shown in Figure 1is one in which the a value of one of the species pairs is always less than 1.5. In this case the desired separation could not be accomplished under any analytical conditions.
RESULTS AND DISCUSSION Table I summarizes the composition of the eluants used for the characterization of the three chromatographic columns. It is particularly important to note that the high ionic strength of these eluants ((1-8) X M) is reflected in a significant difference between species activities and concentrations. This difference is illustrated in the disparity between total and the sum of the HP- and P2phthalate concentration (PT) activities. The activities of each species are calculated by using the extended Debye-Huckel relationship, in which the value of the activity coefficient (y) is expressed as the product of the ionic strength (I) of the eluant and the charge of the ion (2)by
8.50 8.50 9.10 8.50 9.55 9.10
1.43 1.99 1.40 0.75 1.30 0.74
0.60 0.57 1.60 0.56 2.58 1.55
1.98 2.93 1.98 0.96 1.94 0.99
where A , B , and a are solvent and analyte defined constants. The use of activities is particularly important in the case of P2-which by virtue of its larger charge is both more susceptible to ionic-strength-related effects and has a greater impact on the chromatographic separation. The retention characteristics of the analytes with all three columns studied are summarized in Table 11. For the Vydac and Wescan columns, retention time decreases as the total salt content and pH of the eluant increases. Similarly, in the Dionex system, retention times decrease as P2- activity increases (by changing eluant pH); however, the effect of changing H2B03-activity is much smaller than that of P2-. It is interesting to note that in the Wescan study HSO, and the HSOf/formaldehyde stabilization complex have significantly different retention characteristics, while in the Dionex system 502- and the SO:-/isopropyl alcohol complex do not. While the addition of formaldehyde is necessary to assure species stability, chromatographicallythis addition complicates the determination of HSO, by shifting its retention downfield to a time very near that of C1-. Indeed, the retention time observed for the HS0,-/formaldehyde complex reflects that time expected for the formate ion. Modeling of the observed behavior with eq 1requires that the KA and KEselectively coefficients be known. Retention times, eluant composition, and column capacity can be substituted into this expression for each set studied, thereby producing a sequence of equations in which the coefficients are the only unknowns. This set of equations is solved by iterative minimization of the variability of KA for a given value of KE. The resultant values of the calculated selectivity coefficients are shown in Table 111. The values of KE,which reflect the selectivity difference between the divalent and monovalent eluant species, confirm that the P2-species plays a more important role in the chromatographic behavior than both HP- or HzBOS-. Both the Wescan and Vydac columns
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
2677
Table 11. Retention Times of Analytes Studied species/eluant no.
1
2
3
4
retention time, min 5 6
7
8
9
10
11
1.15 1.55 1.95 2.65 1.75 1.00 5.20 2.05 3.15 2.45 0.60
3.50 4.65 5.50 4.10 2.75 4.95 5.90 19.7 4.35 3.35 0.60 0.60
5.15 6.50
Wescan System (Flow Rate = 2 mL/min) c1BrNO3lactate acetate HzSO3HzPOd-
1.60 2.70 3.40 1.85 1.35 2.00 2.35 5.50
2.65 3.80 4.65 1.25 1.00 2.75 1.60 11.85
1.55 2.20 3.15 2.50 1.70 1.45 3.65 3.80
3.55 5.05 6.20 1.75 1.80 4.10 2.30 20.15
2.00 3.00 3.75 1.55 1.50 2.55 2.30 9.20
1.65 2.60 3.40 1.35 1.25 2.50 1.90 6.00
1.80 2.30 2.60 1.15 1.20 2.00 1.50 6.05
1.40 1.65 2.30 1.10 1.00 1.35 1.45 3.15
HS03gluconate void vol equiv dextrose
0.50
0.60
0.60
0.50
0.55
0.55
0.55 0.65
0.60 0.65
so:-
0.70
8.00
0.45 0.50
Vydac System (Flow Rate = 3 mL/min) c1BrNO3lactate acetate HSOfa HzPOL SO4-
gluconate dextrose glycine void vol equiv
2.70 3.57 4.10 2.12
2.33 2.30 2.12
10.55 1.95 1.00 1.00 0.90
2.20 2.90 3.30 1.87 2.04 1.92 1.90 6.27 1.65 1.04 1.02 0.90
3.27 4.31 5.15 2.60 2.88 2.78 2.92 16.26 2.25 0.97 0.96 0.80
1.76 4.49 5.97 0!77 0.77 1.69 0.98 3.55 5.49 0.71 1.10 0.71 3.57 4.70 0.65
1.84 5.51 6.50 0.81 0.81 12.45 1.31 4.48 7.98 0.68 1.85 0.70
2.85 3.90 4.60 2.22 2.55 2.37 3.27 10.34 2.00 0.94
4.06 5.36 6.15 2.85 2.02 3.47 3.51 20.40 2.56 0.93 0.90 0.75
Dionex System (Flow Rate = 2 mL/min)
c1-
BrNOc lactate acetate
2.00 5.93 7.12 0.88 0.91 2.42 1.18
oxalate dextrose gluconate glycine S032-
so:-
void vol equiv
5.48 8.94 0.73 1.57 0.62 6.92 0.65
6.34 0.62
2.06 6.73 8.84 0.72 0.72 3.69 1.21
7.96 1.19 2.19 0.65 7.85 11.31 0.50
1.63 4.94 5.97 0.65 0.70 2.08 1.38 3.59 6.89 0.55 0.55 0.55
2.65 9.10 10.80
5.48 0.52
13.34 0.60
1.00 4.45 1.80 9.12
" In 0.5% formaldehyde. * In 1% isopropyl alcohol. have a somewhat different relative affinity for the HP-/P2pair. All K Aselectivity Coefficients calculated accurately reflect the observed elution order with a larger K Abeing indicative of a longer retention time. Given the selectivity coefficients from Table I11 and the eluant composition, retention times can be calculated. The agreement between the observed and calculated retention time is summarized in Table IV and Figures 2-4. As can be seen in Table IV, the average absolute magnitude of the difference between calculated and observed values (av IRSDI) is generally around 5 % , which approaches the magnitude of the experimental precision. In general, the agreement for the monovalent analytes is slightly better than that observed for the divalent ions. The average magnitude of the signed difference between calculated and observed behavior is generally small, suggesting that no significant skew exists in the comparison. This point is reflected by the near unity slopes and near zero intercepts of the plots of observed vs. predicted behavior. It is observed that while the fit for the Dionex column is nearly equivalent over the entire range of eluant composition studied, the behavior of the other columns deviates by a larger absolute amount from its predicted nature as retention time increases (Le., as the eluant becomes more diluted). This effect has been
Table 111. Selectivity Coefficients Wescan KE(P~-/HP-) 0.03 f 0.003 KE(P2-/HzB03-) KA
Brc1NO3lactate
acetate HZPOL HP0:HS03HS03-" S032-b 50:-
2.55 f.0.26 1.53 f 0.15 3.25 f 0.40 1.08 f 0.12 0.91 f 0.20 1.69 f 0.17
Vydac
Dionex
0.075 f 0.007 0.10 f 0.01 9.29 f 0.66 6.29 f 0.45 11.31 f 0.80 4.33 f 0.05 5.09 f 0.28 4.98 f 0.57
13.72 f 1.01 3.52 f 0.23 17.30 f 0.67 0.49 f 0.08 0.53 f 0.10 0.14 f 0.01
1.81 f 0.14 1.36 f 0.10
4.83 f 0.19
0.44 A 0.04
0.70 f 0.08
0.32 f 0.01 0.47 f 0.03 0.61 f 0.04 1.54 f 0.52
oxalate gluconate 1.85 f 0.10 3.59 f 0.18 "In 0.5% formaldehyde. *In 1% isopropyl alcohol.
attributed to changes in effective column capacity in response to resin swelling triggered by changes in eluant composition (5). Clearly, for the conditions studied, the Dionex column
2678
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
12 11
.-
10
C
E
Y
9
0
.-E
8
I .
1
2
3
4
5
6
7
8
9
10
11
Actual retention time (min)
Actual retention time (min) Figure 2. Comparison between calculated and observed retention times-Wescan column. Species represented include Br-, NO3-,CI-, HzPO,-, HS0,-/formaldehyde, and SO,'-.
Figure 4. Comparison between calculated and observed retention times-Dionex column. Species represented include Br-, NO3-, CI-, HP04'-, S0,2-/isopropyl alcohol, and SO,'-.
8
9
7 8
.-cE
7
6
1
-
t a
I-
c
;.
5
c
e
2
n e + m
-
-m 3
5 4
3
u
a
4
3 ~~~
1
~
2
3
~~
4
~
5
6
7
8
9
Actual retention time (min)
Figure 3. Comparison between calculated and observed retention times-Vydac column. Species represented include Br-, NO3-, CI-, H,PO,-, HS0,-/formaldehyde, and SO,,-.
is less prone to swelling effects, although the reason for this relative immunity is presently unclear. Window diagrams for the three systems of interest are shown in Figures 5-10. The diagrams are constructed to consider the separation of C1-, NO3-, the bisulfite stabilization Perhaps the most significant feature of complex, and Sod2-. these diagrams is the relationship between the relative charge of the species pair and the slope of its line in the window diagram. For species of similar charge, the slope of the line representing a for that pair approaches zero (Le., no concentration-related effect on a). For species of dissimilar charge, a changes as the inverse of changing eluant composition. This result is conceptually consistent with the math-
2
1
Figure 5. Constant pH contour of window diagram for the Wescan column, pH 4.11. Documented species pairs include (A) NO,-/CI-, (6) S0,2-/CI-, (C) CI-/HSO,--formaldehyde, (D) SO,'-/HSO,--formaldehyde, (E) NO,-/HSO,--formaldehyde, and (F)S04'-/N03-.
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
2679
'i
5
I
1
1
I
4
5
6
2 -
PH Figure 6. Constant PTcontour of of window diagram for the Wescan M. Documented species pairs are the same column, P, = 4 X as those in Figure 5.
E
I
1
I
I
4
5
6
PH
7
Figure 8. Constant P, contour of window diagram for the Vydac M. Documented species pairs are the same column, PT = 2 X as those in Figure 7.
6
5
3
't
4
3
2 C 1
1
2
3
P~(X 10-3 M)
Flgure 7. Constant pH contour of window diagram for the Vydac column, pH 4.94. Documented species pairs include (A) NO,/CI-, (B) S042-/CI-, (C) CI-/HSO,--formaldehyde. (D) S0,2-/HS0,--formaldehyde, (E) NO,-/HSO,--formaldehyde, and (F) S04*-/N03-.
ematical model. For similarly charged species, the ratio of reduced retention volumes simplifies to the ratio of the selectivity coefficients of the individual species. Because the magnitude of the selectivity coefficient is independent of
eluant composition, so should a be. For dissimilarly charged species, the ratio of reduced retention times obtained from the model reduces to an expression that retains some eluant composition dependence. Thus, cy remains a function of eluant composition. For the separation of C1-, NO;, HS03-/formaldehyde, and Sod2by the silica based columns, the pair HS03--formaldehyde C1- represents the limit-to-interspecies resolution as shown by its position in Figures 5-8. With a typical t, of approximately 2.5 min for C1- and peak widths a t the base line of 0.2 min for C1- and HS03-/formaldehyde at 20 mg/L, an a value of 1.2 indicates baseline resolution and should be the analyst-defined resolution limit. Diagram 5 through 8 show that this criteria is just barely met for the eluant compositions studied; clearly then, the separation of these four analyses is an analysis-time-limited process. As shown by these diagrams, the eluant composition represented by the most concentrated electrolyte is the optimum. One notes, however, that as the concentration of either C1- or HSO,/ formaldehyde increases past 20 ppm and peak broadening occurs, no eluant composition shown can produce the desired chromatographic separation. The behavior of the C1-, NO3-, SO?-/isopropyl alcohol, and SO-: analytical suite for the Dionex column is somewhat more complicated that those for the silica-based materials. From Figures 9 and 10, one observes that significant peak interference occurs between the NO3- and both the S032-/isopropyl alcohol and SO,2- ions. In Figure 9, as the activity of the P2in the eluant increases, first the NO;/ S0,2--isopropyl alcohol
2680
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
Table IV. Fit of Calculated vs. Observed Behavior Wescan ClNO,HZPOHP02HSOc
Dionex
Vydac
av IRSDl
av RSD
av IRSDl
av RSD
av IRSDl
av RSD
5.35 6.48 3.84
+0.48
-0.21 +0.06 +0.46
3.74 2.07
+0.20 -0.41
t0.29
4.37 3.95 5.54
4.91
+.50
4.65
-0.28
2.56
-0.28
7.19 5.09
+0.35 t0.46
7.15 4.28
-0.22 -0.22
+0.13 +0.12 -0.12 -0.31
6.15 14.71 3.75
+om
1.09 3.44 4.92 5.87
+0.52 +0.07
0.97 3.27 2.98
-0.46 -0.32 -0.15
fO.10
so32-
so*2-
Br-
oxalate lactate acetate gluconate
5 B
4
3 oi
2
F
1
I
I
I
1
2
3
AH,BO,-
(X
lo-’ M I
Flgure 10. Constant contour of window diagram for the Dionex colM. Documented species pairs are the same umn, A p = 1.4 X as those in Figure 9.
I
I
coelute (line F at a = 1). Clearly, H2B03-has a limited effect on the retention characteristics of the four analytes shown. The slightly upward trend of lines E and B is believed to represent a manifestation of the limited experimental precision. Registry No. Lactate, 113-21-3; acetate, 71-50-1; oxalate, 338-70-5; gluconate, 608-59-3; Br-, 24959-67-9; C1-, 16887-00-6; NO,, 14797-55-8;HZPO,, 14066-20-7;HPO?!, 14066-19-4;HSO,, 15181-46-1; SO?-, 14265-45-3; SO-:, 14808-79-8.
LITERATURE CITED (1) Small, H.; Miller, T. E., Jr. Anal. Chem. 1982, 54, 462-469. (2) Jenke, D. R.; Mitchell, P. K.; Pagenkopf, G. K. Anal. Chlm Acta 1983, 55, 279-285. (3) Hoover, T. B. S e p . Sci. Techno/. 1982, 77,295-305 (4) Jenke, D. R.; Pagenkopf, G. K. J. Chromatogr. 1983, 269, 202-207. (5) Jenke, D. R.; Pagenkopf, G. K. Anal. Chem. 1984, 56, 88-91. (6) Steven, T. S.;Turkelson, V. T.; Albe, W R. Anal. Chem. 1977, 4 9 , 1176-1 178. (7) Hansen, L. D.; Richter, 6. E.; Rolllns, D. K.; Lamb, J. D.; Eatough, D. J. Anal. Chem. 1979, 57,633-637. (8) . . DasauDta. P. K.: De Lesare. K.; Ullrey, J C Anal. Chem 1980, 52, 19 11-’1922. (9) Lindgren, M.; Ledergren, A.; Lindberg, J. Anal. Chim. Acta 1982, 747, 279-286 - -_.
(10) Pagenkopf, G. K. “Introductlon to Water Chemistry”; Marcel Dekker: New York, 1978; pp 245-246. (11) Laub, R. J.; Purnell, J. H. J. Chromatogr. 1975, 772,71-79. (12) Laub, R. J.; Purneil, J. H. J. Chromatogr. 1978, 4 8 , 1720-1724.
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268 I
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RECEIVED for review July 5, 1984.
Accepted September 4,
1984.
New System for Delivery of the Mobile Phase in Supercritical Fluid Chromatography Tyge Greibrokk,* Ann Lisbeth Blilie, Einar J. Johansen, and Elsa Lundanes
Department of Chemistry, University of Oslo, P.O.Box 1033, Blindern, 0315 Oslo 3, Norway
The delivery of pressurized mobile phases for supercritical fluid chromatography (SFC) has usually been achieved by pumping the fluids in the liquid phase either by syringe pumps or by membrane pumps. The only commercial SFC instrument available so far makes use of a diaphragm pump (1). For the formation of pressure gradients, pressure control rather than flow control has been recommended in SFC (2, 3). However, in systems with added modifiers and partiylarly with modifier gradients, flow control rather than pressure control could well be preferable for the same reasons as in HPLC. Since the retention in supercritical chromatography systems is 46t a linear function of the pressure, but directly related to the density and to the dielectric properties of the fluids ( 4 ) , the pumping system should contain both flow control and pressure control, in our opinion. With the present development toward microbore columns, the possibility of forming modifier gradients at low flow rates should also be considered. This would require a second pump with reproducible delivery of 10 pL/min and less. Today the majority of the HPLC pumps are of the standard reciprocating type (piston pumps), due to easy and rapid solvent shift, unlimited solvent delivery and good properties for gradient elution. Most modern piston pumps include both flow and pressure control, as well as microflow ability. Previous reports have contained claims that piston pumps are not suitable for SFC, either due to a lack of pressure control or due to a lack of pulse-free operation (2). The purpose of this investigation was to examine whether piston pumps can deliver supercritical fluids at reproducible flow rates and whether the pulses created by such a system are acceptable or not. Carbon dioxide was chosen as the supercritical fluid, since this seems to be the most commonly used fluid in SFC today.
turning the cylinder upside down) and a thermally insulated f in. stainless steel transfer line. A 2-pm filter was inserted in the line to prevent particles in the cylinder from entering the system. A Waters Associates Model 6000 A pump with square pump heads was modified in the following way: (1)Two additional check valves (SSI-02-0129Scientific Systems Inc., State College, PA) were connected both to the inlet and to the outlet. The valves were placed in a homemade block of stainless steel containing cooling channels (Figure 1). (2) Cooling channels were drilled in the solid outer part of both pump heads (Figure 2). (3) The pump heads and check valves were connected with thermally insulated tubing to a Julabo F 10 V refrigeration unit pumping methanol at -8 O C through the whole system (Figure 3). (4) The electronics module was removed from the pump housing in order not to risk any problems from condensed humidity. A purifier column (500 X 7 mm) filled with 28 mesh activated carbon (Alfa Products, Danvers, MA) was inserted prior to the injector (Figure 3) in order to improve the C 0 2 quality. The purifier column acted as an efficient pulse damper as well, in addition to a Orlita PDM 3.350 pulse damper (Orlita Dosiertechnik, 63 Giessen, FRG). A stop valve was inserted prior to the injector. Thus, the pump and the check valves could be held at the equilibrium pressure of the fluid during stops, column replacement, etc. Modifiers were added to C02 by using a microflow pump (Waters Model 590) and a T-piece of tubing or, better, by using a homemade high-pressure low-volume solvent mixer. Injectors. Samples were injected in standard micro HPLC injectors (Rheodyne 7410 or Valco C1 4W). The Rheodyne injector was used at room temperature, while the Valco injector was heated to temperatures above the critical temperature by a block heater. With a flame ionization detector, the samples were dissolved in dichloromethane or in carbon disulfide. Columns. The columns (CP-Spher C18,250 X 1.3 mm) were kept at 40-60 "Cin a gas chromatograph oven or with a separate column heater. All connections were made from thermally insulated 1/16 in. stainless steel tubing with 0.1 mm inner diameter. Detectors and Restrictors. Three detectors were utilized, two UV detectors (Perkin-ElmerLC-55 and Shimadzu-SPD-2AM) and one flame ionization detector (Hewlett-Packard5790 A). The UV detectors could be used at pressures up to approximately 200 bar without leaks or other problems. The restrictor following the UV detector was made by crimping the end of the 1/16 in. stainless steel tubing and keeping it at 80-100 "C in order to avoid blocking by solid COz. The restrictor in the FID was made by crimping the end of a piece of platinum tubing 0.4 mm 0.d. and 0.1 mm i.d. (Goodfellow Metals, Cambridge, UK). The restrictor was located 4-6 mm below the jet tip of the FID.
EXPERIMENTAL SECTION Delivery of Fluid. Liquid COz (standard grade) was transferred from the cylinder to the pump by an eductor tube (or by
RESULTS AND DISCUSSION COz Delivery. The actual delivery of COSwas measured as gas at atmospheric pressure by connecting a tube from the
A study has been undertaken to examine the propertles of reciprocal HPLC pumps In supercrltlcal fluld chromatography. After small modifications, Including cooled-down pump heads and extra check valves, a stable and reproduclble flow of supercritical COz was obtained. With an efflclent pulse damper and a UV detector the pumping contribution to the baseline noise was measured to be less than absorbance unlts at most wavelengths. The short-term reproduclblllty of retentton tlmes and peak helghts was measured to be 1-2% (coefficient of variation).
0003-2700/84/0356-2661$01.50/0
0 1984 American Chemical Society
