621
Anal. Chem. 1904, 56,621-625
Linearity of Homologous Series Retention Plots in Reversed-Phase Liquid Chromatography Alain Tchapla, Henri Colin,* and Georges Guiochon Laboratoire de Chimie Analytique Physique, Ecole Polytechnique, Route de Saclay, 91128 Palaiseau Cedex, France
Accurate measurements of the retentlon of homologousserles reveal that there Is a discontinuity in the plots of log k’ V.I carbon number (n,) at a polnt ( n c ’ ) correspondlng to the length of the organic ligand of the statlonary phase. The break Is not pronounced and appears more clearly on the plots of methylene group selectlvlty vs. n,. The dlscontlnulty Is observed only wlth monomeric phases, and n,’ depends slightly on the solvent composltlon but Is Independent of the functlonal group of the serles. Thls phenomenon has been studled wlth seven different homologous series and various packlng materlals (Cl, C,, CarC,,, and C18). The Influence of dead volume value on the results was also lnvestlgated. The observed effect Is not due to conformatlonal changes of the solutes and wems to indicate a vertical penetration of the solute molecule Into the bonded layer.
It is generally accepted that retention in reversed-phase liquid chromatography (RPLC) is mainly due to the hydrophobic expulsion of the solute molecules from the hydroorganic mobile phase (1,2). The hydrophobic effect is closely related to the hydrocarbonaceous backbone of the solute molecules (3),and it is clear that the free energy of transfer (AG) from the aqueous to the nonpolar phase depends critically on this parameter. According to the early treatment of Martin ( 4 ) ,the free energy of transfer of a molecule from the mobile to the stationary phase, AG, can be regarded as a linear combination of the free retention energies, AGi, arising from various molecular subunits, according to the following equation: AG = Z A G i i
On the basis of eq 1, one would expect a linear relationship between AG (and log k? and the number of carbon atoms in the alkyl chain of the solute molecule. This linearity is well documented in the literature (5), and it has been used for various purposes, such as the determination of column dead volume (6, 7), the evaluation of solvent eluotropic strength (8-lo),the optimization of separations ( l l ) , and many other investigations (see ref 12-18 for instance). However, it must be pointed out that, a priori, there is no rigorous thermodynamic reason to justify this linear behavior. For instance, it is generally assumed that the surface area of the cavity created in the solvent to accommodate the solute molecule varies linearly with the number of carbon atoms of the homologues. This assumption is reasonable as long as the alkyl chain is linear but it is likely that because of the solvophobic effect, long chains are randomly folded. Thus, the contribution of a methylene group should decrease gradually with increasing chain length, and the log k ’vs. n, plots should be slightly curved with an asymptote having a slope of approximately 213. I t has also been shown that the dispersive interactions (Ed) existing in the stationary phase play a nonnegligible role in the retention process (1). The relationship between Ed and n, is a function of the solute molecule position in the stationary phase. If the molecule is inserted 0003-2700/84/0356-0821$01.50/0
like a piece of string randomly folded between the bonded bristles, then Ed should vary linearly with n,. If the solute alkyl chain is sorbed along a bristle, then a discontinuity in the plot Ed vs. n, should be expected when n, becomes larger than the critical value depending on the length of the bonded moieties. Although such a solute ligand association is entropically unfavorable, Kovats and co-workers have shown that it does exist (19). This is also in agreement with the concept of the critical chain length introduced by Berendsen et al. (12). It must be mentioned that some examples of nonlinear log k’vs. n, plots have been reported in the literature. It is known that the plots log k’ vs. n, often exhibit a departure from linearity for n, values below 3 to 5, depending on the series. This is because the functional group of the series may affect the contribution to the free energy of transfer of a -CH2moiety for the first homologues. Other cases of nonlinearity have also been described. The corresponding log k’vs. n, plots showed either a break (at the intersection of two straight lines) or a regular curvature (6, 20-23). No really convincing explanation has yet been proposed (20, 23). The purpose of this work is to investigate if there is a discontinuity in the log k ‘vs. n, plots occurring a t a certain critical carbon number ( n l )related to the chain length of the stationary phase. It is worth noting that most of the reported studies concerning homologous series have been carried out with a limited number of homologues. Moreover, except in very few cases, the chain length of most solutes investigated was either larger or smaller than that of the bonded phase. It was thus impossible to cover a range of carbon numbers in which the break, which occurs a t a carbon number slightly smaller than that of the stationary phase, could be observed. It must also be indicated that several studies have been carried out by using polymeric phases (3, 21). This point will be discussed below.
EXPERIMENTAL SECTION Equipment. The apparatus consisted of a Tracor 995 Isochromatographic pumping system (Tracor, Austin, TX), a Rheodyne 7125 injection valve (Rheodyne, Berkeley, CA) and R 401 refractive index detector (Waters Associates, Milford, MA). The columns (300 X 4.6 mm for 10-pm particles and 150 X 4.6 mm for 5-pm particles) were home packed with LiChrosorb RP 2, RP 8, RP 18 (Merck, Darmstadt, FRG), Spherisorb C6(Phase Sep. Queensferry, England), and pBondapak CIS (Waters Associates). Experiments were also carried out with LiChrosorb RP 14 kindly supplied by L. de Galan (Delft University, Delft, The Nederlands). Mobile phases were prepared by pipetting appropriate volumes of the HPLC grade methanol and tetrahydrofuran (Carlo Erba, Milan, Italy) and doubly distilled water. Experiments were carried out at 25.0 f 0.2 OC by water circulation through a jacket using a Haake D3 thermostating unit (Haake, Karlsruhe, F.R.G.). Homologous series were obtained from various sources. Samples of long chain homologues were generally dissolved in the mobile phase or in pure methanol. In some cases (compounds with very long chains), it was necessary to dissolve the solutes in pure acetone. The series investigated and the conditions under which they were studied are summarized in Table I. In order t o avoid overloading of the column (particularly in the case of 0 1984 American Chemical Society
622
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL
1984
Table I. Experimental Conditions homologous series CnH,,+ 1 z Z
-c1
chain length of solutes
chain length of bonded phasea
n,
cbp
3-18
-H
5-20 and 22, 24,28, 32
-COOMe
0-22
-OH -COCH,
2-10 and 10-30 (even) 1-17
-C,H,
1-12 and 18
-CH=CH, -COOHC
4-20 (even) 0-17
mobile phase x% MeOH, (100 - x)% S S = H,O s= T H F ~ X
1, 6
8, 14, 18, 18p 1,6 8, 14, 18, 18p 196 8, 1 4 , 18, 18p 176 8, 1 4 , 18, 18p 1,6 8, 1 4 , 18, 18p 1, 6 8, 14, 18, 18p 18
X
80,90 90,100 80.90
50, 60, 70, 80, 90
90; i o 0
50, 60, 7 0 , 80, 90
80.90 90; 100 80, 90 90,100 80.90 90; 100 80,90 90,100 90,100 90,100 20, 35, 60, 80
50, 60, 80, 80, 90 50, 60, 7 0 , 80, 90
8,18~ 18 a 18p is used for pBondapak C18. Experiments with MeOH-THF mixtures were carried out only with the C,, monomeric packing. ‘ Dicarboxylic acids (4 < n, < 1 6 ) and diaminoalkanes (2 < n, < 18) were simultaneously studied in the same solvent mixtures. strongly retained solutes requiring large injections), two solutions of the solutes at different concentrations (in the ratio 1:10,generally) were injected and the corresponding retention times compared. When they were different, a new solution was prepared from the less concentrated one (10 times dilution) and the experiment repeated until constant retention times were obtained. The peak asymmetry (measured at 10% of peak height) was less than 1.4. A check of one series verified that the use of the mass center instead of the peak maxima did not suppress the discontinuity. Determination of the Dead Time. This is a critical step, particularly since it has been shown by Berendsen that the linearity of log k’ vs. n, plots depends on the value of to (6). The most common methods for dead time (or dead volume) determination in RPLC are (7) (1)injection of DzO,(2) use of column maximum porosity, (3) linearization of convergent homologous series, and (4) injection of “nonretained” compounds. The influence of the method chosen to evaluate the effect of to on the behavior of a homologous series will be discussed. Only the f i t three methods were used. The maximum column porosity was determined by using methanol and carbon tetrachloride (24, 25). Linearizations of homologous series were carried out by using the methodology described previously (7).
RESULTS AND DISCUSSION The effects of several parameters on the linearity of log k’ vs. n, curves have been examined. These parameters include the nature of the homologous series, the mobile phase composition, the type of stationary phase, and the method chosen to determine the column dead volume. The effect of temperature will be reported in another publication. Role of the Homologous Series. During the course of this work, each time the characteristics of the chromatographic system were changed (mobile or stationary phase) as many series as possible were chromatographed in order to investigate the generality of the phenomenon studied. Reported in this section are the results obtained with different series, individually. In the other sections, attention will not be paid to the series themselves since it was observed that, except with the alkylbenzenes, the results were very similar for all the series investigated. The data obtained with the n-alkylbenzenes are discussed in a separate section. It must be kept in mind that in order to observe a discontinuity in the log k’vs. n, curves, a sufficient number of experimental points must be obtained below and above the break point. This means that with a CISpacking, for instance, it is necessary to inject homologues with alkyl chains of at least 25 carbon atoms.
A
.r
1
1.33 1.32 1.31
0
1.201
5
10
15
20
25
33
+\
Figure 1. Mean selectivity log k’vs. n, plots on C,, bonded phase: (A) 9O:lO methanol-water; (B) pure methanol.
Because of the leveling effect of the logarithmic function, it was decided to investigate the linearity of the log k’vs. n, plots in terms of selectivity (k’nc+l/k’pJvalues. A perfectly linear log k’vs. n, plot corresponds to a horizontal a: vs. n, plot. The a: vs. n, plots have the potential advantage of revealing subtle details that cannot appear on a logarithmic scale and minimizing the influence of the tovalue chosen. This will be discussed below. Some experimental results obtained with the CI4phase are shown in Figure 1. Each a: value is the mean of the values obtained with five homologous series: n-alkanes, n-alkyl chlorides, n-methyl esters of carboxylic acids, n-alcohols, and 2-n-alkanones. Examination of Figure 1 reveals several points. First, it is clear that there is a particular phenomenon occurring around n, = 12-14, independent of the series. Secondly, the two plateaus of the plots are not exactly horizontal, the slightly negative slope corresponding approximately to a 0.33% decrease in a value per methylene group. Such a small change in selectivity cannot be observed on a logarithmic plot. It can be concluded that the log k’vs. n, plots seem to be slightly curved (negative curvature) with a discontinuity (a break) at a value of n, independent of the series. The influence of the nature of the mobile and stationary phases on the variation of k’with n, will be discussed in the next sections. Role of the Mobile Phase Composition. These results were obtained with a CIS monomeric packing. They are
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
Table 11.
A[A(AG)]
623
Values Calculated from a vs. n, Plots
cbp
mobile phase x% MeOH, (100 - x)% s %S A [ A ( G ) ] , calimol
6 S = H,O
8 S = H,O
14
18
18
S = H,O
S = H,O
S = THF
20,lO
10,o 23, 27
10,o 10,18
10, 0 13, 13.5
10, 20, 30, 40, 50 15, 1 7 , 16, 12, 11
20,27
I
I
d
1.40 1.39
k‘
::$
@
1.36
0
-
4 -
1.30
5
0
- I,U
L -5- - 7 d 0-
, nc 5
x)
15
x,
T
1.28,
+
10
,
15
20
25
,
I
,
15
20
25
30
25
Figure 2. log k’vs. n, plots on CI8 bonded phase for n-alkanes in methanol-tetrahydrofuran mixtures. Tetrahydrofuran concentration is as follows: (1) 0%; (2)10%; (3)20%; (4) 30%; (5)40%; (6)50%. 0
qualitatively similar to those obtained with monomeric adsorbents of different chain lengths. The critical problem associated with the use of hydroorganic eluents is the dramatically large values of the capacity ratios. For instance, the measured k’ values of octacosanol (Czg), docosane (C2&and methyl tricosanoate (C23)are 342,302, and 131, respectively, in the methanol-water 90:lO mixture. With the 80:20 methanol-water mixture, the calculated k ’ values (8,9) for the same compounds would be 9082,6820, and 1891, respectively. This obviously limits the range of solvent compositions in which experiments can be carried out. In this respect, the use of nonaqueous eluents is advantageous. Attention has been particularly focused on the methanoltetrahydrofuran system since it gives low selectivities and at the same time rather large log 0 values (8) (log 0 is the intercept of the line log k’vs. n,, assuming a linear relationship). Consequently, homologues with a short alkyl chain are sufficiently retained for accurate measurements of their capacity ratios, and homologues with a long chain are not too strongly adsorbed. The results obtained are presented in Figure 2. They are similar for n-alkyl chlorides and n-methyl esters. It is clear from this figure that, regardless of the solvent composition, there is a change in selectivity at n,‘ around 15-16. This result suggests that the break does not seem to be associated with a change of the solute conformation in the mobile phase, since it would be reasonable to observe a different behavior in such different eluents as the mixtures 9O:lO methanol-water and 5050 methanol-tetrahydrofuran. This conclusion is supported by the following results: Conformational effects have been reported in hydroorganic solvents with bifunctional homologous series of cuw-dicarboxylic acids and cuw primary and tertiary diaminoalkanes (26). Therefore the behavior of these series was investigated with a C1g phase and the selectivity change was observed around nd = 11 (instead of 14-15 for a monofunctional series). Its magnitude was much larger than that of a monofunctional series. Thus, when the conformational change is known to occur, the selectivity plots are different from those obtained under “regular” conditions. Role of the Stationary Phase. In order to evaluate the role of the stationary phase, systematic experiments have been
5
10
30
7
i 1.13
[
,
0
5
, -, 10
15
20
“c
25
,J 30
Figure 3. Mean selectivity vs. n, plots on various bonded phases: (A) monomeric C,B bonded phase; solvent, methanol-water 90: 10; (B) monomeric C8 bonded phase; solvent, methanol-water 9O:lO; (C) monomeric CB bonded phase; solvent, methanol-water SO:10; (D) monomeric C1 bonded phase; solvent, methanol-water 90: 10. carried out with several monomeric bonded phases (Cbp = 1, 6, 8, 14, and 18) and with a presumed polymeric phase (pBondapak c18,c b p = 18). (We have obtained contradictory information about this material. Considering the procedure used to prepare the phase, it is most likely to be polymeric.) With each bonded phase, two mobile phase compositions have been used (see Table I). It must be mentioned that the data obtained with short bonded phases (C, = 1,2,6, and 8) must be interpreted with caution. This is because a critical role is given to homologues with short chains for which the measured selectivity may be affected by the nature of the functional group. Moreover, short homologues were weakly retained under these conditions, which makes their k’ very sensitive to experimental errors and to the value of the void volume chosen. Examination of Figure 1,3, and 4 reveals that a step change in selectivity occurs at a carbon number depending on the bonded phase chain length. The n,’ numbers are around 12-18, 12-14, 8-11, and 4-8 for the c18, Cl4, cg, and c6 packings, respectively. The results obtained with the Cs material are in agreement with the work of Engelhardt and Ahr (23). The selectivity changes correspond to a 3-8% decrease in cu value per methylene group. The values of the corresponding free energy change (AG) are reported in Table 11. The magnitudes of the steps are obtained by extrapolating the two lines corresponding to the plateau regions and by drawing a vertical line at the inflection point of the step. They are approximately 15-20 cal/mol with a precision of 20-30%. The observation of Figures 3D and 4 also reveals that there is no step with the C1 and the polymeric c18 phases.
824
ANALYTICAL CHEMISTRY, VOL. 56, NO. 4, APRIL 1984
d
2.0
I
0
5
10
15
20
30
25
,.I9t 1 .I8
i
1.17 1.16 . 0
I
I
“c
5
10
15
M
25
,30
Figure 4. Mean selectivity vs. n , plots on pBondapak C,8 bonded
phase: (A) solvent, 9O:lO methanol-water; (B) solvent, pure methanol.
These results suggest that the structure of the bonded phase plays a critical role. A possible explanation of this phenomenon is that, with monomeric phases, the alkyl chains of solute molecules are sorbed along the bonded bristles. In other words, solute molecules penetrate into the “fur” of the bonded bristles via their alkyl chains. As long as the length of the alkyl chain of the homologues is shorter than that of the bonded phase, an increase in the number of methylene groups in the solute molecule results in a constant increment of the dispersive forces associated with the soluteligand complexes: the contribution of the stationary phase to the selectivity is constant. When the length of the alkyl chain of the solute exceeds that of the bonded phase, a certain number of methylene groups are necessarily “out” of the stationary phase. These outer groups undergo weaker dispersive interaction with the stationary phase than the inner groups. The corresponding solutes are thus less retained than expected, and there is a concomitant decrease in selectivity. As far as the C1 phase is concerned, it is clear that the penetration cannot occur and that the solute molecules are simply lying on the phase. For this bonded phase, a vs. ne plots must not exhibit any step change in selectivity (see Figure 3D). The situation is different with polymeric materials. In this case, there is no reason to consider one particular bonded moiety since there exists a complex network in which the chains lose their identities. When a solute molecule penetrates this network, its alkyl chain is intertwined within the polymeric network. Thus there is no reason to observe any particular phenomenon when increasing the solute chain length (see Figures 4 and 5). The results obtained with pBondapak c1((suggest a polymeric structure. Role of the Dead Volume Marker. A change in VOvalue entails a significant change in k’ for weakly retained compounds (k’< 2), whereas for moderately or strongly retained solutes, the effect is almost negligible. However, the results discussed previously concern very small changes in selectivity, and it is important to investigate if they are not a consequence of the method used to determine Voand if a slightly different Vo value would generate a different value of the carbon number at which the break occurs. In order to clarify these points all previous calculations have been repeated by using the various methods for Vodetermination mentioned in the Experimental Section. We will only comment on the results obtained with the C18 monomeric phase. Analogous conclusions have been reached with all other stationary phases investigated. The range of column porosities corresponding to the different Vo estimations is 0.65-0.75. In all cases, the break
Flgure 5. log k’vs. n, plots on pBondapak C18phase (solvent 9O:iO
methanol-water): (+) n-phenylalkanes; (0)n-alkanes; ( 0 ) n-alkyl chlorides; (A)n-methyl esters of carboxylic acids; (0)n-alcohols; (0) 2-n-alkanones.
appears at the same carbon number, and the selectivity shifts are identical. Moreover, simulations made after choosing different column porosities revealed that it is necessary to have c < 0.5 or c > 0.8 in order to make the break disappear. Such porosity values are not realistic, and the results suggest that, in a large range of c values, the same phenomenon can be observed. It must be mentioned that, if a change in Vovalue does not affect the nature of the phenomenon, the linearity of the log k’ vs. ne plots for the first few homologues is, however, affected in each series. Particular Behavior of the 11 -Alkylbenzenes. This series has not been included in the results discussed previously since it was observed that the break in the selectivity curves occurs at a carbon number smaller than that corresponding to the other series. For the Cg, C14, and C18 packings, the critical carbon numbers are 4 , 7 , and 9, respectively, and for the C6 packing the break cannot be observed. With the C1 and the polymeric C18 packings the behavior is identical with that of all other series investigated. These results are not contradictory to those reported above, and they can be explained in terms of penetration of the phenyl group “into” the stationary phase. Stated differently, this group behaves as a short chain of methylene moieties. Thus, it is reasonable that the break occurs at a carbon number smaller than that corresponding to the polar series, for which it is likely that the functional group stays “out” of the stationary phase to maximize the polar interactions with the solvent. From the results given it can be estimated that a phenyl group corresponds approximately to three methylene groups. CONCLUSIONS The systematic investigation of the behavior of homologous series in RPLC using various types of stationary phases and different eluent compositions suggests the two following conclusions. First, the log k’vs. n, plots show a break which appears more clearly on the plots of selectivity vs. ne. This probably results from the penetration of the solute alkyl chain “into” the
Anal. Chem. 1984, 56,625-628
stationary phase, followed by an association with the ligands. Secondly, the plateaus of the selectivity decrease slightly with increasing chain length of the solute molecule. However, the magnitude of this effect is extremely small. This is probably due to the conformational changes resulting from the progressive transition of the solute conformation from a straight chain (small n, values) to a random coil (large n, values).
LITERATURE CITED Howath, Cs.; Melander. W. R.; Molnar, I . J . Chromatogr. 1078, 125, 129-156. Karger, B. L.; Gant, J. R.; Hartkopf, A.; Welner, P. H. J . Chromatogr. 1076, 128, 65-78. Tanaka, N.; Thornton, E. R. J . Am. Chem. SOC. 1077, 9 9 , 7 . 3-0 - 0- 7. 3- 0- 7 Martin, A. J. P. Biochem. SOC. Symp. 1040, No. 3 , 4-10. Melander, W. R.; Horvath, Cs. Chromafographia 1982, 15,86-90, and references contained therein. Berendsen, G. E.; Schoenmakers, P. J.; de Galan, L.; Vigh. G.; VargaPuchony, 2.; Inczedy, J. J . Llq. Chromafogr. 1080, 3, 1669-1686. Krstulovic, A. M.; Colin, H.; Gulochon, G. Anal. Chem. 1082, 54, 2 138-2 143. Colin, H.; Krstulovic, A. M.; Gonnord, M. F.; Gulochon, G.; Yun, 2.; Jandera, P. Chromatographla 1083. 17, 9-16,
-
625
Colin, H.; Yun, 2.; Gulochon, 0 . ; Jandera, P.; Dlez-Masa, J. C. J . Chromatogr. Sci., in press. Grushka, E.; Colin, H.; Gulochon, G. J . Chromatogr. 1082, 248, 325-341. Colin, H.; Guiochon, G.; Dlez-Masa, J. C. Anal. Chem. 1981, 53, 146- 155. Berendsen, G. E.; de Galan, L. J . Chromatogr. ISSO, 196, 21-37. Karch, K.; Sebestian, I.; Halasz, I . J . Chromatogr. 1078, 122, 3-16. Colin, H.; Guiochon, G. J . Chromatogr. 1078, 158, 183-205. Vlgh, Jy.; Varga-Puchony, 2. J . Chromatogr. 1980, 196, 1-9. Lochmuller, C . H.; Hangac, H. H.; Wilder, D. R. J . Chromatogr. Sci. 1981, 19, 130-136. Sleight, R. B. J . Chromatogr. 1974, 83, 31-36. Rehak, V.; Smolkova, E. J . Chromatogr. 1080, 191, 71-79. Rledo, F.; Kovats, E. Sz.; Czencz, M.; Liardon, 0. Helv. Chlm. Acta 1081, 61, 1912-1941. Steudel, R.; Mausle, H. J.; Rosenbauer, D.; Mockel, H.; Freyholdt, T. Angew. Chem., I n t . Ed. Engl. 1081, 2 0 , 394-395. Baker, J.; Ma, C. Y. J . Chromatogr. 1070, 169, 107-115. Schoenmakers, P. J., private communication. Engelhardt, H.; Ahr, G. Chromafographia 1081, 1 4 , 227-233. Slaats, E. H.; Kraak, J. C.; Brugman, W . J. T.; Poppe, H. J . ChromafOgr. 1078, 149, 255-270. van de Venne, J. L. M. Thesis, Elndhoven, 1979. Tchapla, A.; Fabre, C. Tetrahedron 1982, 38, 2147-2155.
RECEIVED for review April 18,1983. Resubmitted December 21, 1983. Accepted December 21,1983.
Oxygen Removal in Liquid Chromatography with a Zinc Oxygen-Sc rubber Column W. A. MacCrehan* and W. E. May National Bureau of Standards, Organic Analytical Research Section, Center for Analytical Chemistry, Washington,D.C. 20234
A slmple and effectlve method has been developed for oxygen removal from llquld chromatographic eluents, based on a zinc scrubber column. The mechanism of the oxygen reductlon has been verlfled by differential pulse polarography. The scrubber column has been applled to remove the oxygen Interference In two llquld chromatographlc detectlon systems, reductive amperometry and molecular fluorescence, and Its advantages are demonstrated In the detectlon of nitro polynuclear aromatlc hydrocarbons.
The removal of atmospheric oxygen from liquid chromatographic (LC) mobile phases may be desirable or necessary for several analytical reasons. Most frequently, solvents are degassed to avoid the physical problem of ”outgassing” on the low pressure end of the analytical column. Molecular fluorescence detection of certain compounds is also affected by the dissolved oxygen. The presence of oxygen can cause intersystem energy transfer from the excited state of the fluorophore resulting in “quenching”of the fluorescence signal through this nonradiative decay pathway (1). Oxygen removal to very low levels is an absolute requirement for most reductive electrochemical detection (2-4) since the oxygen reduction current contributes deleteriously to residual current and detector noise. Finally, some analytes are sensitive to oxygen oxidation during the chromatographic process [i.e., ascorbate (5)] and either oxygen removal or the addition of antioxidants to the mobile phase is necessary to prevent decomposition. Several approaches have been used to remove oxygen from LC mobile phases. Coarse degassing is routinely performed by either evacuation, warming, or purging the solvent reservoir
with helium. Tejada and co-workers (6) have developed an on-line method for elimination of oxygen quenching in fluorescence detection based on catalytic reduction with a packed bed of crushed “three-way automotive catalyst”. More rigorous deoxygenation is required for reductive electrochemical detection and consequently several approaches have emerged. The most simple approach involves continuous purging of the solvent reservoir with high-purity argon or nitrogen. Kissinger and co-workers (3,7)recommend purging at elevated temperature with continuous reflux of the solvent. One very effective approach (4)uses alternate evacuation and purging in a sealed mobile phase reservoir. Electrochemical reduction of the oxygen with a special high-pressure coulometric cell has also been proposed (8) to remove oxygen “on-line” between the pump and injector, although conventional purging of the solvent reservoir was still required, due to less than 100% oxygen reduction. Recently, two similar “on line” approaches have been reported for oxygen removal from mobile phases. A postcolumn deoxygenator has been developed based on the diffusion of oxygen through gas permeable tubing under the influence of a vacuum (9). However, the device has an internal volume of 205 FL which would contribute to chromatographic band broadening. Another membrane oxygen-scrubber described by Warner et al. (IO)used a Cr(I1) solution as a trap to create the concentration gradient through the permeable tubing. However, the internal volume of the tubing required for complete oxygen removal would be too large to be suitable for LC use. All of these deoxygenation procedures either are quite laborious or require additional specialized equipment to be added to the LC system. This work presents a simple and effective chemical method for oxygen removal based on the
This article not subject to U.S. Copyright. Published 1984 by the American Chemical Society
