Anal. Chem. 1981, 53, 1341-1345
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Liquid Chroimatographic Elution Characteristics of Some Bonded Solutes Used To Measure Column Void Volume on Phases Martha J. M. Wells' and C. Randall Clark" Division of Medicinal Chemistty, Depatiment of Pharmacal Sciences, School of Pharmacy, Auburn University, Auburn University, Alabama 36849
The llquld chromatographic elution characteristics of some solutes commonly used to measure column vold volume on hydrocarbonaceous statlonary phases are described. The elution characterlstlcs of tartrazine, sodium benzenesulfonate, sodium nltrate, potassium dichromate, acetone, uracil, N,Ndlmethylformamlde, and the moblle phase components were determlned by using four CI8 columns with aqueous methanol eluents. The results indicate that the volume required to elute acetone, N,N-dlmethylformamide, methanol, and uracil increases as the organlc modlfier in the moblle phase decreases. The elutlon volumes of the lonlc solutes are Independent of mobile phase composition (nonbuffered) but dependent on the concentration of the Injected solute. When the aqueous component of the mobile phase was replaced by a phosphate buffer, the elution volume of the organic lonlc solutes became independent of amount Injected and dependent on organic modlfler content of the mobile phase. The lnorganlc Ionic solutes were independent of both amount Injected and eluent comlposltlon In buffered eluent.
In liquid chromatography (LC), the capacity factor (k') is commonly used to defiie retention ( I ) . If the volume required to elute the peak maxiimum is VR for a retained solute and Vo for a nonretained compound, the capacity factor can be defined by eq 1. The elution of a solute on different columns
VR - VO k'= VO
(1)
is not correctly described by V,, since V, varies with the interand intraparticulate volumes associated with the column packing, as well as with differences in tubing dimensions, guard columns, or other hardware placed between the points of injection and detection. Proper measurement of the column void volume (Vo) is essential for the calculation of solute k' values. The need for research into the problem of determining the column Vo when reversed-phase high-performance liquid chromatographic (RPIHPLC) columns are used has been discussed by Colin et al. (2). Many different solutes are used to determine VOin RPIIPLC; moreover, a large amount of k' data is reported without describing the method used to determine the column void volume. In this study we have investigated the elution characteristics of several solutes used for determining the column VOin RPHPLC. EXPERIMENTAL SECTION Apparatus. The liquid chromatograph consisted of a Waters Model 6000A solvent pump, Model U6K injector, Model 440 UV absorbance detector, an Alltech Associates (Deerfield, IL) HPLC column water jacket, and a Hitachi or a Beckman recorder. RI measurements were determined on an Erma (Tokyo, Japan) rePresent address: USDA Forest Service, Southern Forest Experiment Station, George W. Andrews Forest Sciences Laboratory, Auburn University, AL 36849.
fractometer. Constant temperature in both the HPLC column water jacket and the refractometer was maintained by a Haake (Saddle Brook, NJ) Model FE constant-temperature circulator. Reagents and Chemicals. All chemicals were of reagent grade quality or better and were used as purchased without further purification. Sodium nitrate, N,N-dimethylformamide, spectrophotometric grade methanol, potassium phosphate monobasic, and potassium phosphate dibasic were obtained from Fisher Scientific Co. (Fair Lawn, NJ). Sodium benzenesulfonate and uracil were obtained from Eastman Kodak Co. (Rochester, NY). Potassium dichromate and spectrophotometric grade acetone were purchased from Mallinckrodt, Inc. (St. Louis, MO). Tartrazine was obtained from Aldrich Chemical Co. (Milwaukee,WI). Doubly distilled water was further purified by pumping (Waters Model 6000 solvent pump) through a 7 cm by 2.1 mm i.d. column dry packed with Whatman C0:PELL ODS (30-38 pm) prior to preparation of solvent mixtures. Mobile Phase Characteristics. The mobile phase solvent mixtures were prepared and allowed to equilibrate for at least 1 h before use. The specific gravity and refractive index were measured for each batch of HPLC solvent (methanol-water). The specific gravity of the mobile phase was determined by adding an aliquot of the solvent mixture to a 25-mL Kimax specific gravity bottle equipped with a thermometer, side arm, and a side tube cap with vent. The bottle and contents were weighed (fO.l mg) at 25 "C.The refractometer was calibrated to a value of 1.3325 at 25 "C using the purified water. Refractive index values of the prepared mobile phase solutions were determined at 25 "C. Phosphate buffer solutions (0.1M) were prepared by mixing equal volumes of 0.1 M potassium phosphate monobasic and 0.1 M potassium phosphate dibasic which produced a solution of pH 6.91 prior to mixing with methanol. Chromatographic Procedures. Four analytical HPLC columns were used in this study. A 25 cm by 4.6 mm i.d. Partisil ODS (10 pm, irregular, porous particle) column, 113583, and a 25 cm by 4.6 mm i.d. Partisil ODs-2 (10 pm, irregular, porous particle) column, 1H3213, were purchased from Whatman, Inc. (Clifton, NJ). Two 15 cm by 4.6 mm i.d. Ultrasphere ODS (5 Fm, spherical, porous particle) columns (UE795 and UE2588N) were obtained from Altex Scientific, Inc. (Berkeley, CA). In operation, the Partisil ODs-2 and Ultrasphere ODS columns were preceded by guard columns 7 cm by 2.1 mm i.d. dry packed with Whatman C0:PELL ODS (30-38 wm). The guard column and analytical column were contained inside a column jacket and maintained at 25.0 f 0.2 "C by circulating water from a constant-temperature bath through the column jacket. The Partisil ODS column was operated at ambient temperature and without a guard column. The mobile phase consisted of mixtures of water and methanol or phosphate buffer (0.1 M) and methanol at a flow rate of 1.5 mL/min. The ultraviolet detector was operated at 254 nm and varied over five sensitivities (0.5,0.1, 0.02,0.01, and 0.005AUFS) for studies of retention as a function of solute concentration. Solutions of sodium nitrate, N,N-dimethylformamide, sodium ben-enesulfonate, potassium dichromate, acetone, and tartrazine were prepared in water. Solutions of uracil were prepared in methanol. RESULTS AND DISCUSSION The movement and band spreading of unsorbed solutes on glass beads have been discussed by Horvath and Lin (3). Even though a solute is unsorbed, measurements of the column void
0003-2700/81/0353-1341$01.25/00 1981 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
P
0 CH,-l--CH, I
Flgure 1. Compounds included in this study: (1) sodium nitrate; (2) potasslum dichromate; (3) sodium benzenesulfonate; (4) tartrazine; (5)uracil; (6) N,N-dimethylformamide; (7) acetone. volume can vary according to the molecular size of the solute and its ability to penetrate the intraparticulate pores of the column packing material. This situation is then described by eq 2. The elution volume of an unsorbed solute, V,, is the
v, v,+
= $Vi (2) sum of two factors, the interstitial fluid volume, V,, and the intraparticulate fluid volume explored by the solute, 4Vi. In the extreme case in which 4 = 0, the unsorbed solute is completely excluded from the pores of the packing material, and when 4 = 1, the solute undergoes total pore penetration. When there is incomplete pore penetration by the solute, 0 < $ C 1. The ability of silica gels which are used for adsorption chromatography to also exhibit exclusion properties has been discussed by Scott and Kucera ( 4 ) . For the unsorbed solutes, the shortest elution time will be exhibited by solutes for which $ = 0, and the longest elution time by those having 4 = 1,according to eq 2. Solutes which undergo only partial pore penetration elute at volumes between the two extremes of V , and (V, VJ. A search of the literature for a solute suitable to use in determining Vo for RPHPLC columns reveals that many materials have been used for this purpose. Several authors have used components of the mobile phase to determine the column Vo,Le., water ( 5 ) ,deuterium oxide (6), methanol (7, 8), acetonitrile (9), or water-organic modifier solutions of a composition different to that of the mobile phase ( 1 0 , I I ) in nonbuffered solvent systems, or the solvent front (12) in buffered solvents. Among the organic solutes (other than components of the mobile phase) used to measure Vo in nonbuffered eluents are uracil (10) and acetone (13),while N,N-dimethylformamide (14) has been used in buffered solvents. The organic salts sodium benzenesulfonate (15) and tartrazine (16) as well as the inorganic salts potassium dichromate (17) and sodium nitrate (18) have been used in nonbuffered eluents. Sodium nitrate (19) has also been used with buffered mobile phases to determine V,,. (Structures are shown in Figure 1.) Hemetsberger et al. (20,21) have used an equation developed by Halasz to calculate the elution time of a nonretained component. This relationship has also been used by Lochmder and Wilder (18)to verify the elution time they obtained by using sodium nitrate. Initial experiments with nonionic organic compounds (acetone, uracil, N,N-dimethylformamide, and methanol) showed the elution characteristics of these solutes to be dependent on mobile phase composition and independent of the amount injected. A plot of measured elution volume vs. mobile phase composition for acetone, uracil, and methanol is shown in Figure 2. The average elution volume for all solute concentrations injected is plotted for acetone and uracil. A 1 0 - ~ L sample was consistently injected for methanol. Methanol is not detectable over the entire mobile phase composition range. The elution behavior of N,N-dimethylformamide is almost identical to that of acetone in this experiment. Similar results
+
P
10
20
30
40
50
60
70
80
90
100
% % MeOH Flgure 2. Elution volume vs. eluent composition on Partisll ODs-2: (1) acetone; (2) uracil; (3) methanol.
were obtained on all columns tested and show that the elution volumes for these solutes increase with decreasing concentration of organic modifier. Thus, these compounds appear to be retained according to the typical reversed-phase process and would not be suited for use in measuring the column void volume. Chromatograms illustrating the elution upon variation of the mobile phase composition (nonbuffered) are given in Figure 3. These chromatograms further illustrate that nonionic organic solutes such as acetone, uracil, N,N-dimethylformamide and methanol are not suitable for determining Vo in RPHPLC. Figure 3 illustrates the elution of sodium nitrate, uracil, and acetone over the entire range of mobile phase composition. Figure 3A was obtained in pure methanol and Figure 3E was produced by using pure water as eluent. The increased separation observed between these solutes as the concentration of organic modifier is decreased would indicate reversed-phase retention. Furthermore, Figure 3E shows a peak for methanol (solvent for the uracil sample) between peaks 1and 2. On this column, acetone and N,N-dimethylformamide were inseparable over the entire water-methanol composition range. The elution volume for sodium nitrate remains constant in this series of chromatograms. The elution behavior of the ionic solutes was observed to be extremely dependent on the background electrolyte composition in the mobile phase. In nonbuffered eluent, determination of individual elution volumes suggests these solutes should be separable when injected concomitantly. The separation of similarly detectable amounts of sodium nitrate and sodium benzenesulfonate from the water in which they were dissolved (Figure 4) illustrates this point. It can be observed that the separation between peaks 1and 2 is greater in Figure 4B. The observed elution volume for sodium nitrate was 2.00 mL and 1.78 mL for sodium benzenesulfonate. When these ionic species are injected together in a nonbuffered eluent, it is not possible to obtain a separation. Furthermore, the results in Figure 4 are misleading due to the difference in detectability of these two solutes. When similar concentrations are injected, the resulting retention volumes are the same for both solutes. Water is not detectable over the entire solvent composition range. Chromatograms presenting the results from both buffered and nonbuffered mobile phases are shown in Figures 5 and 6. Figure 5A represents the chromatography of tartrazine, sodium nitrate, sodium benzenesulfonate, and potassium dichromate in nonbuffered 50% v/v methanol, while Figure 5B,C was obtained in 50% v/v methanol buffered with 0.1
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
1343
2
)I
2
e
P
N ln 1
a
?
0
1
2
3
0
1
2
3
T i m e (Minutes)
Figure 4. RPHPLC elution on Partisil ODS in methanol/water (50:50): A (1) sodium nitrate, (2) water; B (1) sodium benzenesulfonate, (2) water.
:i 2
0
1
2
3
4
5
e
7
I3
9
10
3
Time (Minutes)
Flgure 3. RPHPLC elution1 on Ultrasphere ODS (UE795) in methanol-water: (A) 100:0, (B) 80:20, (C) 5050, (D) 20:80, (E) 0:lOO; (1) sodium nitrate, (2) uracil, (3) acetone.
M phosphate (pH 6.91). Comparison of Figure 5B and Figure 5C (both obtained with buffered mobile phase) demonstrates that under these conditions potassium dichromate elutes after tartrazine and prior to siodium nitrate thereby obscuring the separation of these two solutes. The elution characteristics of these solutes in nonbuffered and buffered mobile phases containing 20% v/v methanol are presented in Figure 6. The separations in 50% v/v methanol-phosphate buffer and in 20% v/v methanol-phosphate buffer are quite different (Figures 5B,C and 6B). In the buffered mobile phase, tartrazine and sodium benzenesulfonate exhibit a dependence upon the concentration of the eluent which implies that these two solutes are being chromatographed under these conditions. Explanation of these results is facilitated by conclusions drawn from gel permeation (or size exclusion) chromatography (GPC). Experiments on the electrolyte effects of salts in aqueous GPC (22, 23) have established that when an unbuffered solution contains two or more different ionic species, a Donnan salt-exclusion effect occurs, and one of the solutes is blocked from regions penetrable by the others. The presence of a salt in the eluent is used to prevent interference from the Donnan equilibrium. Thus, in the concomitant injection of sodium nitrate and sodium benzenesulfonate in unbuffered eluents, the Donnan salt-exclusion effect results in the equilibration of the level of sodium ions inside and outside
I 0
1
2
0
1
2
0
1
2
Time (Minutes)
Figure 5. RPHPLC elution on Ultrasphere ODS (UE2588N) in: A, methanol-water (5050)(1) tartrazine, (2) sodium nitrate, sodium benzenesulfonate, and potassium dichromate; B, methanol-phosphate buffer (5050)(1) tartrazine, potassium dichromate, and sodium nitrate, (2) sodium benzenesulfonate; C, methanol-phosphate buffer (5050) (1) tartrazine, (2) sodium nitrate, (3) sodium benzenesulfonate.
the bonded silica particles. This causes these solutes to elute together. When the Donnan effect was masked by the presence of phosphate buffer, it was possible to obtain a separation of the ionic solutes. Comparisons of the elution data as a function of the amount of solute injected are presented in Figures 7 and 8. These
1344
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
3.0 '
2.8
'
2.7
1 2.8
2.6 '
2.5
.
2.4
I
. 2.3 .
2.2
-I
2.1
;I 0
1
'
2.0
'
1.8
'
1.8
E Q
-5 >
E
1.7
.E
1.6
ij
a
'
1.5 1.4 1.3 1. 2
2
0
1
2
3 1.1
T i m e (Minutes) '
1.0
Figure 8. RPHPLC elution on Ultrasphere ODS (UE2588N) in: A, methanol-water (20:80) (1) tartrazine, potassium dichromate, sodium nitrate, and sodium benzenesulfonate; 0, methanol-phosphate buffer (20:80) (1) potassium dlchromate, (2) sodium nitrate, (3) tartrazine, and (4) sodium benzenesulfonate.
-12
-11
-10
-8
-8
-7
-6
-5
-4
-3
-2
-1
log Moles Injected
Figure 8. Elution volume vs. moles of solute injected on Ultrasphere ODS (UE2588N). Curves were determined in methanol-water (20:80), while horizontal lines were obtained in methanol-phosphate buffer (20:80): (-) sodium nitrate; (- -) sodium benzenesulfonate; (-e) potassium dichromate: (.- .) tartrazine.
-
'
1.4
'
1.3 1.2 1.1
'
1.0
10.9 -12
-11
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
log Moles Injected
Figure 7. Elution volume vs. moles of solute injected on Ultrasphere ODS (UE2588N). Curves were determined in methanol-water (50:50), while horizontal lines were obtained in methanol-phosphate buffer (5050): (-) sodium nitrate; (- - -) sodium benzenesulfonate; (.-) potassium dichromate; (. .) tartrazine.
-
figures are complicated by the differences in UV detectability among the solutes. Plots were prepared for all mobile phase compositions and columns studied and only a representative example is presented. These plots show that the elution volumes of the ionic solutes increased with increasing amounts injected as long as the mobile phase was not buffered. Experiments in GPC have shown that exclusion can occur with molecules of the correct size if they possess the same charge as groups on the surface of the support (24). This results in limited pore penetration. Buytenhuys and van der Maeden (23)conducted gel permeation chromatography on unmodified
silica gel with aqueous solvents and demonstrated an ion exclusion effect resulting from a negative charge on the surface of silica gel. The effect was suppressed by using an aqueous buffer to shield the negative charges of the silica. The concentration dependence observed in Figures 7 and 8 in unbuffered eluents can then be understood on the basis of this ion exclusion effect. On the basis of studies with reversedphase supports, Berendsen et al. (25) suggested that a t low electrolyte concentration in unbuffered eluents, a salt is excluded from the pores of the packing, presumably due to electrical charges on the phase surface. With increased electrolyte concentration in the mobile phase, the ion exclusion effect is suppressed and the pores become accessible to the salt. Thus, injection of trace amounts of a salt in nonbuffered eluents yields the interstitial or exclusion volume, V,, in eq 2. When the mobile phase is buffered with 0.1 M phosphate, the ionic solutes do not exhibit any solute concentration dependence, yet sodium benzenesulfonate and tartrazine are dependent upon eluent composition as shown in Figures 5B,C and 6B. From these studies it is apparent that the solutes can be separated into three groups: group I, methanol, uracil, acetone, N,N-dimethylformamide; group 11, sodium nitrate, potassium dichromate; group 111,sodium benzenesulfonate, tartrazine. For those solutes in group I, elution is dependent on the mobile phase composition yet independent of the amouqt of solute injected. The solutes in group I1 are relatively independent of solvent composition in both buffered and nonbuffered eluents but demonstrate a concentration dependence in nonbuffered eluents which disappears in the presence of phosphate buffer. Group I11 solutes exhibit a concentration dependence-solvent composition independence
Anal. Chem. 1981,
in nonbuffered methanol solutions, but they are concentration independent -solvent composition dependent in methanolphosphate buffer mixtures. From the foregoing discussion, solutes in groups I and I11 can be eliminated as suitable for use in determining the column void volume. Discrimination between the group I1 solutes, sodium nitrate and potassium dichromate, can be made on the basis of the elution order for equimolar quantities of these solutes in buffered eluent. That solute which undergoes a greater pore penetration of the silica gel backbone is expected to have a greater elution volume, e.g., sodium nitrate. Therefore, according to eq 2, sodium nitrate more nearly represents the case of C#I = 1 than does potassium dichromate.
CONCLUSIONS On the basis of the evidence presented for the solutes examined, acetone, N,N-dimethylformamide, methanol, and uracil appear to be unsuitable for determining column void volume on hydrocarbonaceous stationary phases. When buffered methanol eluents are used, the injection of any detectable amount of sodium nitrate produces a good estimate of the column void volume in RPHPLC. In unbuffered aqueous methanol, the column void volume can be estimated by an injection of approximately 3 x 10+ mol or greater of sodium nitrate. Perhaps the most important conclusion is that the identity and concentration of the solute used to determine the column void volume must accompany all reported k'data in RPHPLC studies.
53, 1345-1350
1345
Horvah, Csaba; Lin, HungJye J . Chromafogr. 1976, 726,401-420. Scott, R. P. W.; Kucera, P. J . Chromafogr. 1977, 725, 251-263. Scott, R. P. W.; Kucera, P. J . Chromafogr. 1877, 742, 213-232. McCormick,.R. M.; Karger, 8. L. Anal. Chem. 1980, 52, 2249-2257. Schabron, J. F.; Hurtubise, R. J.; Silver, H. F. Anal. Ch8m. 1978, 50, 1911-1917.
Roumeliotis, P.; Unger, K. K. J. Chromafogr. 1978, 749, 211-224. Melander. Wayne R.; Chen, Bor-Kuan; Horvath, Csaba J . Chromatogr. 1979, 785, 99-109.
Karger, Barry L.; Gent, J. Russel; Hartkopf, Arleigh; Weiner, Paul H. J. Chromafogr. 1978, 728, 65-78. Gant, J. R.; Dolan, J. W.; Snyder, L. R. J . Chromfogr. 1979, 785, 153- 177.
Baker, John K.; Ma, Cheng-Yu J . Chromatogr. 1979, 769, 107-115. Johnson, Howard J., Jr.; Cernosek, Stanley F., Jr.; GutierrezCernosek, Rose Mary J . Chromafogr. 1979, 777, 297-311. Unger, Stefan H.; Feuerman, Tony F. J . Chromatogr. 1979, 776, 426-429.
Bakalyar, Stephen R.; Mcliwrick, Rod; Roggendorf, Elizabeth J . Chromatogr. 1977, 742, 353-365. Bakalyar, Stephen R.; Henry, Richard A. J . Chromafogr. 1976, 726, 327-345.
Schoenrnakers, P. J.; Billlet, H. A. H.; Tijssen, R.; de Galan, L. J . Chromatogr. 1978, 749, 519-537. Lochmuiler, C. H.; Wilder, D. R. J . Chromafogr. Scl. 1979, 77, 574-579.
Horvath, Csaba; Melander, Wayne; Molnar, Imre J . Chromafogr. 1976, 725, 129-156.
Hemetsberger, H.; Maasfeld, W.; Ricken, H. Chromafographie 1978, 9 , 303-310.
Hemetsberger, H.; Kelierman, Marlene; Ricken, H. Chromafographie 1977, 10, 726-730.
Neddermeyer, P. A.; Rogers, L. B. Anal. Ch8m. 1969, 41, 94-102. Buvtenhuvs. F. A.; van der Maeden, F. P. B. J . Chromatoor. 1978. 749, 489-500.
Krejci. M.; Kourilova, D.;Vespalec, R.; Slais. K. J . Chromafogr. 1980, 191 . - . , 3-7 - ..
Berendsen, a r t E.; Schoenmakers, Peter J.; de Galan, Leo; Vigh, Gyula; Varga-Puchony, ZRa; Inczedy, Janos J . Llq. Chromafogr. 1980, 3 , 1669-1686.
LITERATURE CITED (1) Haftmann, Erich "Chromatography: A Laboratory Handbook of Chro-
RECEIVED for review December 29, 1980. Accepted April 27,
matographic and Electrophoretic Methods"; Van Nostrand Reinhold: New York, 1975; p 49. (2) Colin, Henri; Ward, Norman, Guiochon, Georges J . Chromfogr 1978,
1981. We sincerely appreciate the support of the American Foundation for Pharmaceutical Education Silas M. Burroughs Memorial Fellowship for M. J. M. Wells.
749, 169-197.
Preparative Liquid Chromatography for Fractionation of Petroleum and Synthetic Crude Oils James W. Vogh' and Jane S. Thomson Barflesville Energy Technology Center, U S . Deparlment of Energy,
P.0. Box 1398, Bartlesville,
The procedures for routine preparative liquid chromatographic separation of hydrocarbon classes in high boiling petroleum and coal liquid samples have been improved by use of highperformance liquid chromatography techniques. Separations were carried out on alumina and silica gel to produce sample fractions equivalent to those obtained with older methods. The columns are reusable foilowing suitable solvent backwashing and provide stable performance and good hydrocarbon class resolution over an extended series of runs. The complete operating time, including regeneration to starting conditions, is 80 min for the alumina column and 25 min for the silica gel column. Capacity ranges from 1 to 6 g of sampie, depending on its composition.
One of the primary steps in the detailed characterization of petroleum and similar materials has been the adsorption column separation of hydrocarbon classes. The principal methods have been the silica-alumina gel procedure developed
Oklahoma 74003
under the API-60 program or variations of this procedure (1). The subsequent steps through gel permeation and characterization by mass spectrometric and other methods depend largely on the success of the adsorptioii column separation. The silica-alumina procedure in common use is a conventional open column chromatographic method. It was developed through a procedure of step gradient displacements of a series of model compounds. This became a set procedure depending on the dimensions of the column, the quality and activation of the adsorbants, and the volumes and flow rate of the eluting solvents. Sample capacity varied from 4 to 15 g depending on the amount or nature of the more strongly adsorbed components. The procedure does not permit an easy change of scale of equipment unless the column is coupled to a liquid chromatography monitor. The silica-alumina column method usually provides a good separation of the hydrocarbon classes; however, there are difficulties and inconvenient aspects in this procedure. As a method conceived for routine use, the most severe problem is the requirement for an unbroken 50-h operation. Other
This article not subject to U.S. Copyright. Published 1981 by the American Chemical Society
