purposes it is possible to establish the slope with sufficient accuracy using only one mixture. For example, a series of 10 determinations of a sample gave a value of 1.660 with a standard deviation of only 0.5%. Because the line must pass through the origin, the slope can thus be established with sufficient reliability for most purposes. If the concentration of the mixture used for the single calibrating point is known with the requisite accuracy, the averaging effect of three standards is not necessary.
nitrogen (mixture No. 5). The carbon dioxide concentration was found to be as follows:
OTHER APPLICATIONS
Similar accuracy could not be attained on direct analysis of the mixture even if inlet pressure were similarly high. The base peak of methane, mje = 16, would be obscured both by the tailing of the m]e = 14 because of N+ and by mle = 16 because of oxygen,
Any system which can be chemically converted to a mixture of carbon dioxide and nitrogen can be analyzed by this method with equivalent accuracy. An illustration of this is the analysis of nitrogen containing low concentration of methane and other hydrocarbons. A mixture of 0.0207 mol methane in nitrogen was prepared by dilution of a mixture of higher methane content. Enough oxygen was added to the mixture to oxidize the methane. The methane was then oxidized by passage over hot copper oxide and the resulting mixture was analyzed by measuring the mass 44 to 28 ratio. The concentration of carbon dioxide was determined by calculation after determining the slope of the ratio to concentration line using a single known concentration of carbon dioxide in
Average
0.0204 0.0206 0.0206 0.0208 0.0207 0.0206 mol % carbon dioxide
RECEIVED for review October 19, 1967. Accepted January 2, 1968. Certain commercial materials and equipment are identified in this paper in order to adequately specify the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the material or equipment identified is necessarily the best available for the purpose.
Gel Filtration Behavior of Inorganic Salts P. A. Neddermeyer' and L. B. Rogers Department of Chemistry, Purdue University, Lufayette, Ind. 47907
Concentration profiles for small samples of inorganic salts eluted with water from Sephadex G-10 and 6-25 columns were badly skewed, having diffuse front and sharp back edges. The peak volumes increased with sample volume and sample concentration. These behaviors are opposite to those normally found in chromatography and result from a Donnan salt-exclusion effect that arises between the ionic solutes and a small number of fixed negative charges within the gel matrix. Neutralization of the negative sites with acid diminished the salt-exclusion. The effect was eliminated completely and elution behaviors became normal when a moderate concentration of electrolyte was present in the eluent. In spite of the Donnan saltexclusion effect, the inorganic salts eluted in the order of decreasing formula weight as expected from a gel filtration mechanism.
GEL FKTRATION CHROMATOGRAPHY has become a useful method of separating and characterizing molecules on a basis of their molecular dimensions ( I , 2). The theoretical basis for the technique, though still in its infancy, has been dealt with successfully in a number of articles (3-6). Separations in 1 Present address: Research Laboratories, Eastman Kodak Co., Rochester, N. Y. 14650
-
(1) R. L. Pecsok and D. Saunders, Sep. Sci., 1,613 (1966). (2) H. Determan, Angew. Chem. Intern. Ed. Engl., 3, 608 (1964). (3) T. C. Laurent and J. Killander, J . Chromatog., 14, 317 (1964). (4) D. M. W. Anderson and J. F. Stoddard, Anal. Chim. Acta, 34, 401 (1966). (5) G. K. Ackers, Biochem., 3, 723 (1964). (6) J. C. Moore, J . Polymer Sci., A2, 835 (1964).
gel filtration result from the preferential diffusion of small solute molecules into the porous gel structure with the exclusion of large molecules. Those solutes are characterized by the equation
The elution volume, V , , is equal to the sum of the void (interstitial) volume, Vo,and a fraction of volume, V,, which is related to the solvent volume imbibed by the gel beads. Kd is normally derived from the above equation and is similar to a distribution coefficient. It represents the fraction of the imbibed volume available for solute penetration and can take on values between zero (representing complete exclusion from the gel interior) and unity (representing complete penetration). When only the gel filtration mechanism is operative, Kd does not exceed unity, but if it does, adsorption is clearly indicated. However, when adsorption, ion exchange and other interfering processes do not cause a peak to occur beyond a total of (Vo Vt), the interference is often very difficult to discern. Gelotte (7) was one of the first to report on the secondary interactions encountered in gel filtration chromatography with the gel bed material Sephadex. He noted that aromatic and heterocyclic substances tended to adsorb on Sephadex. At low electrolyte concentrations, basic substances also adsorbed while acidic substances were excluded from the gel interiors. Wilk, Rochlitz, and Bende (8) obtained separations of a num-
+
(7) B. Gelotte, J. Chromalog., 3, 330 (1960). (8) M. Wilk, J. Rochlitz, and H. Bende, Zbid., 24, 414 (1966). VOL. 40, NO. 4, APRIL 1968
755
characteristics of ionic solutes are gravely affected by a small number of fixed negative charges within the Sephadex gels. Increasing the eluent ionic strength often eliminates the interferences and normalizes the elution characteristics.
1.6-rnm i.dB 3.2-M 0.d.
3-rnm 0.d.
EXPERIMENTAL
Beckmon Teflon Straight Union Reducer, 4 -8-mm
sAir
Bore
Space
>Eluent
Glass Tubing 6-rnrn i.d. Beckrnan Teflon Union T e e , 8 - m Bore
lnje
Po Rubber Septum
I a
Coiurnm 6-mmi.d
Figure 1. Injection port ber of polyaromatics from Sephadex LH-20 in accordance with adsorption or gel filtration mechanisms, when eluting with propanol-2 or with chloroform, respectively. An explanation in terms of relative strengths of solute-solvent and solute-gel interactions accounted well for the observed reversal in elution orders and chromatographic mechanisms. A comparable change from an adsorption mechanism to a gel filtration mechanism was discussed by Kwon (9) for the pH dependent elution of malonaldehyde on Sephadex G-10. The change in mechanism resulted from a molecular transformation of malonaldehyde from an intramolecular hydrogen-bonded species at pH 2.8 to an enolate anion at pH 6.5. Changes in elution volume with sample concentration of a-chymotrypsin and human carboxyhemoglobin on gel filtration columns (10, 11)result from the reversible polymerization of subunits in the solute. Theoretical treatments by Gilbert (12) and by Ackers and Thompson (13) of such systems in rapid equilibrium agree well with experimental data. Anomalous elution behaviors attributed to ionic interactions between charged solutes and the Sephadex gel matrix have been mentioned in the literature (7, 1 4 , but the specific nature of these interactions has not been elucidated. We have studied the elution of a number of simple inorganic salts from Sephadex G-10 and G-25 columns. The elution (9) T. W. Kwon, J. Chromatog., 24, 193 (1966). (10) P. Andrews, Biochem. J . , 91, 222 (1964). (11) D. J. Winzor and H. A. Scheraga, J. Phys. Chem., 68, 338 (1964). (12) G. A. Gilbert, Anal. Chim. Acta, 38, 275 (1967). (13) G. K. Ackers and T . E. Thompson, Biochem., 53, 342 (1965). (14) P. Flodin, Anal. Chim. Acta, 38, 89 (1967).
756
ANALYTICAL CHEMISTRY
Materials. All reagents were analytical grade. Sephadex gels were purchased (Pharmacia Fine Chemicals, Inc., Piscataway, N. J.) as fine powders and subsequently screened into narrow-mesh fractions of standard U. S. sieves. Deionized water was used to make up all solutions and to swell the gel materials. Equipment. Chromatographic columns were constructed of glass and Teflon parts. Columns consisted of 125-cm sections of 6-mm i.d. silanized glass tubing with a stopcock at one end. At the other end, an 8-mm bore Teflon (Beckman Instruments, Inc., Fullerton, Calif.) union tee fitted with a silicone rubber septum was used as a sample injection port to allow direct syringe injections of a sample into a column through which eluent was flowing (See Figure 1). Above the injection port, a 5-cm length of glass tubing served as gel reservoir to allow for bed settling and compression. A Beckman reducing joint (8-mm to 4-mm) allowed the connection of the eluent reservoir to the top of the column by means of 3-mm 0.d. glass and 3.2-mm 0.d. Tygon tubing. The piece of glass tubing, which served as the eluent inlet tube, extended through the reducing joint and the small gel reservoir to the center of the injection port. Construction of the injection port in this manner offered several distinct advantages. Solvent flow did not need to be interrupted for sample introduction. (Experience in our laboratory has shown that after solvent flow was stopped for sample introduction, a steady flow rate was not reobtained during the time of the ensuing chromatogram). Sample injection directly into the gel matrix eliminated all dead space on the front side of the column. In addition, eluent changes were accomplished without solvent flow stoppage by transferring the Tygon tubing on the pump inlet side from one solvent reservoir to another. An air space between the two eluents served not only as a barrier between the two solutions but also as a time marker for the introduction of the new eluent to the column. The small air space did not interfere because it floated to the top of the column upon emerging from the eluent inlet tube. Because the eluent inlet tube was also positioned at the center of the injection port, direct comparison could thus be made between frontal analysis data and sample elution data. Columns were connected to the detector by 0.8-mm i.d. Tygon tubing and to the eluent reservoir by 1.6-mm i.d. Tygon tubing. The total dead volume between the column outlet and the detector cell was 0.66 ml. A Sigmamotor AL-2-E (Sigmamotor, Inc., Middleport, N. Y . ) variable flow kinetic clamp pump was used to feed eluent through the column at a constant flow rate. A differential refractometer (Model R-4, Waters Associates, Inc., Framingham, Mass.) was used as detector and a Sargent SR multi-range recorder (E. H. Sargent and Co., Chicago, Ill.) served to record the elution profiles. The refractometer was powered through a constant voltage transformer and thermostated at 22.00 f 0.01" C with a Sargent thermonitorcontrolled constant temperature bath. The constant voltage transformer, along with a 100 kQ - 1 pf R C filter between refractometer and recorder, served to stabilize the electrical signal sufficiently to allow use of the 25-mV scale on the recorder. Procedures. Sephadex gels, after being swollen in deionized water for at least 24 hours, were packed into columns using the following procedure. A small wad of silanized glass wool was placed in the bottom of the column above the 2-mm bore stopcock. The column was filled with water, and all air bubbles were removed. A funnel on top of the
column was half filled with water, and a slurry of swollen gel added. Then, the stopcock was opened to allow the gel particles to settle under the influence of a slight liquid flow. After the gel had settled, water was pumped through the column at a rate of at least 1 ml/minute for 24 hours, or more, to ensure complete settling of the gel bed under normal operating conditions. Because both the chromatographic column and the refractometer detector were thermostated at 22.0" C (well below ambient temperature), solvents did not require deaeration. The refractometer required at least 2 hours to reach electrical stability and 4 hours to reach maximum thermal stability. Flow rates were measured on the outlet side of the detector by feeding the effluent into the top of a 50-ml buret and monitoring volume with time. Three closely spaced volumetime data points taken at the beginning and at the end of each experimental run sufficed to measure an average flow rate. For example, over one eight-hour period, with flow rate measurements made over 30 minute intervals, a relative standard deviation of 0.17% with a range of 0.60% was obtained for a flow of 0.583 ml/minute. Samples were injected into a column within 1 second using a 50-p1, 100-p1 or 0.50-ml syringe. Peak elution times were measured on the strip chart of the recorder which was operated at 1 inch per minute. Elution times were determined with a 1.0% relative standard deviation in comparison to the 0.2% precision obtained for flow rates. Elution times were then converted to volumes and corrected for extra column dead volume. Void volume, V,, was assumed to be the peak elution volume of Blue Dextran, a commercial polymer (Pharmacia Fine Chemicals, Inc.) having a molecular weight V,), was of 2 million. The total liquid volume, ( V , assumed to be the elution volume of HC1. The imbibed liquid volume, V,, was the difference between these two volumes. Unless stated otherwise, all chromatograms were obtained using deionized water as eluent.
+
RESULTS
Ion Exchange Capacity Determination on Sephadex. Sephadex gels are known (15, 16) to have a small number of fixed ionic charges incorporated in the gel matrix which are believed to be carboxylate groups. After this article was submitted for publication, Eaker, and Porath (16) reported that oleate anions that remain from the manufacturing process of Sephadex G-10 and G-15 also contribute to the number of charges. Washing the gel with 1M pyridine, reportedly extracted the sodium oleate. The nature and number of these fixed charges was checked in the following way. Ten-gram portions of dry Sephadex G-10 and G-25 were swollen in 0.2M NaOH and in 0.2M HC1, washed with deionized water and 1.OM NaCl, and then the amount of acid or base displaced by the NaCl determined. Potentiometric acid-base titrations were attempted on the NaCl washings but found to lack the required sensitivity. A different procedure, which involved treating a column of a 10-gram portion of gel with 0.2M HCI, then flushing it with deionized water until excess acid had been removed and finally monitoring the pH of a 1.OM NaCl wash solution. In all cases, the first 250-300 ml portion of NaCl wash solution became acidic (pH 3.9-4.1) before returning to the original pH 6.5. Integration under pH elution profiles taken in triplicate gave ion exchange capacities for Sephadex G-10 and G-25 of 4.1 i 2.8 and 45 f 23 peq/gram of dry gel, respectively. These capacities are about 1 of those found for ion exchange resins. (15) F. Miranda, H. Rochat, and S. Lissitzky, J . Ckroniatog., 7, 142 (1962). (16) D. Eaker and J. Porath, Sep. Sci., 2, 507 (1967).
I '
Eluate Volume (ml)
Figure 2. Elution curves for 25-pl samples of 0.050M solutes eluted with deionized water from a 0.61- X 126-cm Sephadex G-10 column 3. NaCl 4. Glucose
Preliminary Runs. Figure 2 shows chromatograms for several representative solutes. All solutes were eluted between V, and ( V , Vi)and therefore had Kd values between zero and unity. Chromatograms for ionic solutes that did not elute at the void volume, had skewed fronts. Their appearance volumes (the first noticeable deflection from the base line) were equal to or slightly less than V,. The appearance volume for NasP3OIa(12.3 ml) was identical to that for Blue Dextran and hence was clearly less than V,. The chromatogram for glucose, the only nonionic solute, differed in two respects from the others. Its elution profile was symmetrical, and its appearance volume was much greater than V,. Effect of Consecutive Acid Injections. When identical samples of H 3 P 0 4or HC1 were injected consecutively onto the Sephadex G-10 column, elution volumes increased with injection numbers as shown for H 3 P 0 4in Figure 3. The first injection of 25 p1 of 0.01M H3P04 was made after the column had been flushed with 0.2M NaOH and then washed with deionized water. The first chromatogram was symmetrical and eluted at 14.5 ml (Kd = 0.088). With subsequent consecutive injections of identical samples, elution volumes increased, peak heights decreased, and the leading sides of the peaks became extensively skewed. Elution volumes increased almost linearly with injection number. After saturation of the column with 0.10M H3P04and washing with deionized water, a limiting volume of 19.0 ml(& = 0.58) was reached. Although the elution volume shift was 4.5 ml, the appearance volumes did not vary significantly from a value slightly lower than the void volume. Upon washing the column again with 0.2M NaOH and then water, the sequence in Figure 3 was closely reproduced. Qualitatively, identical elution behavior was noted for HCl on the Sephadex G-10 column. The total shift in elution volume for HCl, however, was 6.0 ml to produce a corresponding change in Kd from 0.34 to 1.OO. Later, the behavior observed for the repeated acid injections will be discussed in terms of the charged sites on the resin. Effect of Sample Concentration of Elution Peaks. Elution volumes and peak shapes for the ionic solutes were strongly dependent upon sample concentration and sample volume.
+
VOL 40, NO. 4, APRlL 1968
757
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a
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50.
0 vo
13
1
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15
16
17
Eluate
Volume (mi)
18
19
20
21
Figure 3. Effect on the chromatograms of successive injections of 25 pl of 0.10M HlP04 using a 0.61- X 124-cm column of Sephadex G-10 Peak Sample No. injected Column treatment I. 1st Previously washed with O.2M NaOH and then HzO 2. 6th Successive sample injections 3. 10th Successive sample injections 4. 16th Successive sample injections 5. 22nd Successive sample injections 6. 23rd Previously saturated with 0.10M H3P04and then washed with H 2 0 7. 24th Previously washed with O.2M NaOH and then HzO Figure 4 presents elution behaviors for 50-pl samples of NaCl as a function of sample concentration. The elution volume increased with sample concentration, as did the extent of peak skewing. The chromatograms were superimposed on the front edges but differed significantly in the locations of peak maxima and rear edges. The rear edge of each chromatogram was sharp, dropping from the maximum to the base line within approximately 1 ml. Appearance volumes did not depend on sample concentration and remained equal to the column void
I -'
I
100-
volume. Peak areas but not peak heights, increased linearly with sample concentration. Identical changes in elution peaks were obtained when the solute concentration was kept constant at 0.050M and the sample volume was varied between 10 and 200 p1.
241 --E
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14
-
20-
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12
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&+vi 16
18
20
22
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Figure 4. Variation with sample concentration of chromatograms for 5O-pl samples of NaCl on a 0.61- X 126-cm column of Sephadex G-10 1. 0.010M 4. 0.10M 2. 0.025M 5. 0.2OM 3. 0.050M 758
ANALYTICAL CHEMISTRY
1
0
.O5
.IO
.I5
S a m p l e Concentration
I
.20
(M)
Figure 5. Effect of sample concentration on elution volume for 50-111 samples on a 0.61- X 126-cm column of Sephadex G-10 1. NabPaOla 4. LiCl 2. Na2HPOa 5. NaCl 6. Glucose 3. KCI
Table I. Variation of Peak Symmetry with Sample Concentration Using 50-pl Samples on a 0.61- X 126-cm Column of Sephadex G-10 Sample Front-to-back ratio, F/Bosb concentration, M Na5P3010 NazHPOa LiCl KCI NaCl Glucose
*
1 . 2 0.1 1.2 f 0.1 1 . O f 0.1 1 . 1 =t0 . 1 1.3 f 0.1 1 . 7 f 0.1
0.005 0.010 0.025 0.050 0.100
0.200 a
b
*
1 . 5 f 0.1 1 . 7 + 0.1
1 . 3 f 0.1 1 . 2 f 0.1 1.3 f 0.2 1.7 i 0 . 2 1.9 i:0 . 2 1.9 i 0 . 1
*
1.5 0.1 1.8 f0.1 1.9 f 0.1 3.2 f 0.2 3.7 i:0 . 2 4.6 f 0.7
...
*
3.1 0.6 3 . 4 f 0.2 3.3 + 0.3
2.2 c.2 2.8 f 0.9 4.8 f 0.8 3.7 f 0.1 3.9 f 0 . 2 2.6 i: 0 . 1
1.1 f 0.1 1.2 i0.1 1.1 f 0.1 1.0 f 0.1 1 . 0 zk 0.1 1 . 1 f 0.1
As defined in text. Standard deviations given for duplicate determinations.
Table I result from F and B being small differences between large values. The sum of F and B, which is the peak width at half height, was rarely greater than 2.3 ml. Effect of Gel Porosity and Solute Charge Types on Elution Behavior. Na2S04,NaCI, and CaClz which have cation-toanion charge ratios of 1:2, 1 :1, and 2:1, respectively, were chosen as representative solutes to be eluted from Sephadex columns having different gel porosities. Compared to Sephadex G-25, Sephadex G-10 has a greater degree of crosslinking and smaller imbibed liquid volume. Because the imbibed liquid volumes (pore volumes) differ for the two gels, elution volumes were converted to distribution coefficients ( K d ) in order to compare elution data for Sephadex G-10 and G-25 columns. The behavior of the three salts on Sephadex G-25 columns was qualitatively identical to that already discussed for NaCl and other ionic solutes on Sephadex G-10. However, differences in magnitudes did exist, as is illustrated in Figure 6. On both columns, distribution coefficients increased with sample concentration to limiting values in the order Na2S04< NaCl < CaCL For the more porous Sephadex G-25 gel, Kd values were greater in magnitude and their limiting values were approached more rapidly. For example, at the 0.050M concentration level on Sephadex G-10, the salts Na2S04,NaCl, and
Similar behavior for the other ionic solutes is illustrated in Figure 5 . The extent to which elution volume changed with sample concentration depended on individual solutes, but it covered more than half of the available range, Vi,for LiCl, NaC1, and KCl. However, the relative elution order always remained the same (Na5P3010< Na2HP04< KC1 = LiCl < NaCl) at all concentration levels. In contrast, glucose behaved in a nearly ideal way--e.g., its peak was symmetrical and its maximum was independent of sample size and concentration. In an attempt to quantitate the degree of peak skewing, the ratio of the front portion, F,to the back portion, B , of the peak width was determined in milliliters at 50% of the peak maximum. The front-to-back ratio, FIB, is unity for symmetrical peaks and is greater than unity for peaks skewed like those in Figures 2, 3, and 4. Table I gives data for peak skewing with sample concentration for the ionic solutes in Figure 5. The front-to-back ratios generally increased with sample concentration and remained in the relative order Na5P3010< NaaHP04 < LiCl < KCl < NaCl. The elution sequence is followed except for LiCl, which comes before KCl rather than being equal to it. (The maximum in the NaCl front-to-back ratio relationship has been reproduced.) The relatively large standard deviations in
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E l u a t e Volume
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Figure 6. Effect of sample concentration and gel porosity on dis- Figure 7. Effect of background electrolyte concentration on tribution coefficients ( K d )for 25-pI samples of the solutes 1. Na2S04, the chromatograms of 5O-pl samples of 0.050M NaCl using a 0.61- X 126-cm column of Sephadex G-10 2. NaCl, and 3. CaCl,. Eluents : 0.61- X 124-cm Sephadex G-10; V, = 13.7 ml, Vi = 8.74 ml b) 0.61- X 125-cm Sephadex G-25; V, = 14.8 ml, Vi = 15.1 ml a)
1. Deionized H 2 0 2. 10-4MNaCl
3. 10-JMNaCl 4. 10-2MNaCl
VOL 40, NO. 4, APRIL 1968
759
Na,SO,
A A 3
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Sample Concentration (M)
L
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a
Figure 8. Effect of sample concentration and background electrolyte concentration on (a) the elution volume and (b) the appearance volume for 50-pl samples of NaCl using a 0.61X 126-cm column of Sephadex G-10 Eluents : I . (0)Deionized H2O 2. (a) 10-4MNaCl
3. ( 0 ) 10-3M NaCl 4. ( 0 )10-2MNaCl
CaClz had reached only 33 %, 36%, and 44% of their limiting values, respectively, whereas, on Sephadex G-25, these percentages were 70 %, 82 %, and 84 %, respectively. Effect of Eluent Electrolyte Concentration on Elution Peaks. In Figure 7 are shown chromatograms for identical NaCl samples added to eluents having different background concentrations of NaC1. A higher background electrolyte concentration increased the elution volumes and produced more nearly symmetrical chromatograms. At the same time, as shown in Figure 8, the elution volume dependence on sample concentration diminished until it was totally eliminated at the 0.010M NaCl electrolyte level. Although appearance volumes in a given background electrolyte were not dependent on sample concentration, they increased dramatically with background concentration. For the chromatograms in Figure 7, where the electrolyte concentration was increased from zero to 0.010M NaCI, the elution volume increase was from 18.2 t o 21.9 ml, whereas the appearance volume increased from 13.4 t o 19.7 ml. More data are shown in Figure 8.
Table 11. Variation of Peak Symmetry with Sample Concentration and Background Electrolyte Concentration Using 50-pl Samples on a 0.61- X 126-cm Column of Sephadex G-10. Sample concentration, M 0.005 0.010
0.025 0.050 0.100 0.200 a
Front-to-back ratio, F/Balb Eluent electrolyte concentration (NaC1) Zero (H20) 2.2zk0.2 2.8 f 0.9 4.81t0.8 3.7f0.2 3.9zk0.2 2.610.1
10-4M 2.5f0.4 2 . 5 f 1.0 2.710.4 3.3zkO.1
3.610.5 2.210.1
As defined in text.
10-JM 1.310.1 1.8 1 0 . 2 2.2fO.2 2.5fO.2 2.510.2 2.010.3
10-2M 1.1fO.2
1.410.2 1,210.1 1.2fO.1 1.010.1
* Standard deviations given for duplicate determinations. 760
ANALYTICAL CHEMISTRY
Eluate
Volume (ml)
Figure 9. Chromatograms for 20-4 samples of Na2S04 and NaCl mixtures eluted from a 0.61- X 124-cm Sephadex G-10 column Eluents:
A. B. Samples: 1. 2. 3. 4.
10 -2M NaCl Deionized H20 0.0125M Na2SOaand 0.0125M NaCl 0.025M Na2S04and 0.025M NaCl 0.050M Na2S04and 0.050M NaCl 0.010M Na2S04and 1.00M NaCl
Equally dramatic changes were brought about in the peak shapes. At higher electrolyte levels, the chromatograms generally sharpened and became more nearly symmetrical curves (Table 11). For example, the peak widths at half height for 50p1 samples of 0.10M NaCl were 2.22 =t0.15, 2.15 f 0.10, 1.78 f 0.05, and 1.70 f 0.03 ml, for eluents of water, lOW4M, lO-3M, and 10-2MNaC1, respectively. At the same time, the diminishing difference between appearance and elution volumes in Figure 8 also indicates that the chromatogram was sharpening. Similar effects of background electrolyte were noted for the chromatograms of all ionic solutes, used either as solute or background electrolyte. Figure 9 shows the effect of eluent composition on the peak resolution for a mixture of NazS04and NaCl. For Figure 9A,- 20 p1 samples of equimolar mixtures of the salts were eluted with 0.010M NaC1. The two peaks were well separated, with Na2S04eluting at 14.7 ml and NaCl eluting at 21.1 ml. Both salts behaved ideally. Elution volumes remained constant and peak height and area increased linearly with sample concentration. Identical mixtures of salts, when eluted from the same column with water as eluent, did not give complete separations. Sodium sulfate eluted over the range 14.2-14.4 ml while sodium chloride eluted at 15.4 ml and beyond, SO that the peaks overlapped. Elution volumes for both salts increased with increasing concentration, but that for NaCl more
so than for Na2S04. The peaks drew apart at higher sample concentrations, decreasing the relative height of the troughs. For the three sample concentrations 0.0125, 0.025, and 0.050M, the trough heights were 88 %, 73 and 58 % of the NaCl peak heights and 3 3 z , 2 7 x , and 2 0 z of the NaaSOa peak heights, respectively. Although the absolute peak overlap increased, the relative overlap decreased. Fractionation for a mixture 0.010M in Na2S04and 1.00M in NaCl was also accomplished (curve 4 , Figure 9B), although a comparable fractionation of a hundred-fold excess of Na2S04over NaCl could not be obtained.
z,
DISCUSSION
The nonideal chromatographic behavior of ionic solutes on Sephadex gels strongly resembled that t o be expected from a Type 111 isotherm of Brunauer et al. (17). Although the theoretical treatment of the Type I11 isotherm was based upon a n adsorption mechanism, its qualitative features are not strictly limited t o that mechanism. Furthermore, it seems unlikely that adsorption contributed significantly t o the retentions of ionic solutes on Sephadex columns in view of the fact that none of the distribution coefficients exceed unity. From the work of Ohashi, Yoza, and Ueno (18), who reported that polyphosphate salts eluted from Sephadex G-25 in less than the internal liquid volume, (Vo Vf),and from the data in the present study, the gel filtration mechanism appears to govern the elution of ionic solutes in spite of the anomalous elution behaviors noted. This is seen from the increasing elution order of Blue Dextran < Na5P5Ol0< Na2HP04 < KCl = LiCl < NaCl < HCl with decreasing formula weight or, more correctly, solvated ionic or molecular dimensions. Correlations between logarithm of molecular weight and elution volume for protein homologs on more porous Sephadex gels have been reported by Andrews (10) and Whitaker (19). The observed elution sequence is expected from a gel filtration mechanism. Thus, the process of solute diffusion into and out of the gel pores is probably the chief contributor t o the hold up for the ionic solutes. The skewing observed when water was the eluent, the changes in elution behavior observed for successive injections of acid, the effects of sample size and concentration, the effect of background electrolyte concentration, and the normal behavior of glucose, however, all point t o interactions between the ionic solutes and the fixed negative charges within the gel matrix. These ionic interactions result from a Donnan salt exclusion effect (20) which restricts the penetration of ions into the charged gel matrix. The degree of salt exclusion decreases with increasing salt concentration. A parallel can be found in the work of McKelvey, Spiegler, and Wyllie (21) on ultrafiltration (reverse osmosis) of salt solutions through high-capacity cation-exchange membranes where the degree of desalting was greater (more efficient salt exclusion) when the solute concentration was lower. In our study, the fact that appearance volumes for ionic
+
(17) S. Brunauer, L. S. Deming, W. E. Deming, and E. Teller, J . Am. Chem. Soc., 62, 1723 (1940). (18) S . Ohashi, N. Yoza, and J. Ueno, J . Chromatog., 24, 300 (1966). (19) J . R. Whitaker, ANAL.CHEM., 35, 1950 (1963). (20) W. Meyer, R. S . Olsen, and S. L. Kalwani, Ind. Eng. Chem., Process Design Decelop., 6 , 55 (1967). (21) J . G. McKelvey, Jr., K. S . Spiegler, and M. R. J. Wyllie, Chem. Eng. Progr. Symp. Ser., 55, (24), 199 (1959).
solutes were equal to, or slightly less than, the void volume indicates that, at salt concentrations near zero, the salts were completely excluded from the gel interior. With increasing salt concentration, penetration into the gel interior increased. In addition, the fact that the front portions of peaks of different sample concentrations and volumes overlapped shows that concentration gradients were established reproducibly. It is reasonable that the peak front slope for an electrolyte be flattened and the back sharpened compared t o the peak for a nonelectrolyte (see Figure 2). O n the front side of the peak, the charged sites hindered diffusion of ions into the gel, whereas, on the back side, they enhanced diffusion out of the gel interior. It is evident from Figures 5 and 8, that when enough salt is present in either the sample or the eluent, the exclusion effect diminished to the vanishing point and allowed values of elution volume to be reached which depended only upon the gel filtration mechanism. After a limiting value has been obtained, any extra solute only builds up the concentration in the gel interior rather than increasing the extent of penetration as was the case for smaller sample amounts. Because salts of greater hydrodynamic volume are exposed to fewer of the fixed ionic charges in the gel, their behavior is influenced less by the exclusion effect. Thus, both the front-to-back ratio and dependence of elution volume on sample concentration were than for NaCl (Table I, Figures 2 and 5). smaller for Na5P3OI0 Figure 3 shows that the effects of the stationary negative charges on eluting solutes can be modified by a n exchange of hydronium ions for the sodium counter ions on the fixed charges. Upon successive acid injections, the elution volume for phosphoric acid increased to its limiting value, showing that the acid had penetrated progressively farther into the gel interior. The skewness of the peak and a n appearance volume equal t o V , showed that the exclusion force was still present, but at a much reduced level. McKelvey, Spiegler, and Wyllie (21) have predicted that in membranes with fixed negative charges, the degree of salt exclusion should increase over the series CaCI2, NaCI, and Na2S04. In addition, they suggested that the degree of exclusion would be less in membranes of larger pore size since the electrostatic effect of the negative wall charge extends over a smaller fraction of the pore. Figure 6 shows that the experimental results on Sephadex G-10 and G-25 bear out the predictions of McKelvey, Spiegler, and Wyllie. The degree of salt exclusion (smaller Kd) increased from CaC12 to NaCl t o Na2S04,as expected on the basis of the cation-to-anion charge ratio. This sequence held on both Sephadex G-10 and the more porous Sephadex G-25 (Vf = 8.74 and 15.1 ml, respectively). The rates at which the limiting K d values were attained with increasing sample concentration can be used as measures of the degree of ionic interaction between gel and solutes. O n Sephadex G-25,at 0.10M sample concentration, CaC12, NaCl, and Na2S04had already attained 70 72 and 84 respectively, of the limiting Kd value. These percentages were approximately double those found for Sephadex G-10. Although the elution orders on both gels were Na2S04< NaCl < CaCh, as expected from a n electrostatic interaction, it does not fit a mechanism based upon formula weight. Smith and Kollmansberger (22), Siegel and Monty (23), and Casassa (24) have suggested that hydrodynamic volume or Stokes radius, rather than formula weight, might be better parameters
z, z, z,
(22) W. B. Smith and A. Kollmansberger, J . Phys. Chem., 69, 4157 (1965). (23) L. M. Siegel and K. J. Monty, Biochim. Biophys. Acta, 112, 346 (1966). (24) E. F. Casassa, J . Polymer Sci., B5, 773 (1967). VOL. 40, NO. 4, APRIL 1968
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to use for hydration spheres contribute to molecular dimensions. Grubisic, Rempp and Benoit (25) report that viscometric hydrodynamic volume determines retention in gel permeation columns. Figures 7 and 8 show that the Donnan salt exclusion effect can be eliminated in gel filtration by the presence of sufficient electrolyte in the eluent. The same has been done in osmometry (26) to eliminate osmotic pressure contributions from small membrane diffusable ions. In gel filtration, the presence of sufficient inert electrolyte enables ionic solutes to elute as sharp, symmetrical peaks having elution volume independent of sample amount. The beneficial effect of electrolyte upon separations is illustrated in Figure 9 for equimolar mixtures of Na2S04and NaCl. In comparison, identical elutions with water did not give as nearly complete separations. The behavior of salt mixtures eluted with water from Sephadex G-10 (Figure 9B) is in good agreement with the data for the individual salts presented in Figures 4, 5, and 6. That the fractionation improved with sample loading, although opposite to normal chromatographic experience, was to be expected from the divergence of the Na2S04 and NaCl curves in Figure 6. (Similar behivior would be expected for mixtures of Na5P3010or Na2HP04with any of the alkali metal chlorides shown in Figure 5.) Because the concentration gradients on the lower front side of the NaCl peaks were reproducible and independent of sample amount (Figure 4)) it was possible to predict that a small amount of Na2S04and a large excess of (25) Z. Grubisic, P. Rempp, and H. Benoit, J. Polymer. Sci.,B5, 753 (1967). (26) C. Tanford, “Physical Chemistry of Macromolecules,” Wiley, New York, 1961 p 221.
NaCl would fractionate as shown in Figure 9B. In addition, the NaCl impurity level under any given NazSOl peak would be independent of the total amount of NaCl in the mixture. The need of inert electrolyte for the separations of ionic solutes on Sephadex gels has been demonstrated. In choosing the proper electrolyte for a particular separation, a number of factors need to be considered. The electrolyte should be highly ionized to give a level of ionic strength sufficient to overcome the Donnan salt exclusion effect. For completely ionized salts, the concentration need not be greater than 10-2M. The electrolyte should have its cation or anion in common with that of one of the salts in the sample in order to reduce the total number of cation-anion combinations that might result from exchanges. The hydrodynamic volume of the electrolyte should be small enough to allow penetration into essentially all of the gel interior. For instance, a salt such as Na5P3010would not penetrate Sephadex G-10 sufficiently (see Figure 5) to neutralize the effect of all ionic charges within the gel interior. For the separation of certain chemical systems, background electrolyte concentrations as high as 10-2M might not be desired or tolerated. In those cases, compromises between background impurity level and peak resolution would have to be made. Optimum conditions would need to be determined empirically. RECEIVED for review November 1, 1967. Accepted February 2, 1968. This work was supported in part by the U. S. Atomic Energy Commission under contract AT (11-1)-1222. Presented at the Division of Analytical Chemistry, 154th National Meeting of the American Chemical Society, Chicago, September 1967.
Quantitative Infrared Analysis of Mixtures of Isotopically Labelled Gases Mark M. Rochkind Bell Telephone Laboratories, Inc., Murray Hill, N.J. A new spectrophotometric method based on an unconventional technique of low-temperature sample preparation provides a general method of qualitative and quantitative analysis for isotopically labelled gases. The method, infrared pseudo matrix isolation, involves condensing specially diluted gas samples onto a cold infrared transmitting substrate by controlled-pulse deposition. The simple vibrational spectra which result are recorded using commercial instrumentation. Data for deuterated methanes and ethylenes as well as methyl ether, propionaldehyde, and neopentane are presented. The latter, which represent a range of molecular polarity and bulk, serve to support the general quantitation of the method. As little as 0.2 pmole of certain gases may be detected; acute selectivity is shown by the successful spectrometric separation of the 3 ethylene-d2 isomers. Pseudo matrix isolation proves far superior to gas chromatography for molecular isotope analysis. As such, the most promising application appears to be in the area of photochemistry.
A NEW SPECTROPHOTOMETRIC technique (infrared pseudo matrix isolation) was recently proposed (1, 2 ) for qualitative analysis of multicomponent gas mixtures. The technique requires conventional spectrometric instrumentation but uses cryogenic temperatures to effect an unusual method of sample 762
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preparation. The latter results in condensed-phase spectra characterized by relatively sharp absorptions, few in number, occurring at highly reproducible frequencies. Such spectra contrast markedly with normal gas-phase spectra and encourage use of low temperature sample preparation for gas analysis. In work previously reported ( I ) , pseudo matrix isolation spectra of thirteen C1-CI hydrocarbons were recorded and multicomponent mixtures containing random collections of these were analyzed. That work constituted a feasibility study, for satisfactory gas chromatographic methods already existed for the analysis of light hydrocarbon mixtures which matched the sensitivity and qualitative selectivity of the experimental infrared technique. Nonetheless, low-temperature sampling proved a practicable means of spectrochemical analysis and we engaged in further studies. A large number of gases and volatile liquids representing widely differing classes of chemical compounds have been investigated and synthetic multicomponent mixtures containing isomeric molecules as well as molecules of distinct structural classes have (1) M. M. Rochkind, ANAL.CHEM., 39,567 (1967). (2) M. M. Rochkind, Enairoti. Sci. Techtiol., 1, 434 (1967).
