16
Anal. Chem. 1992, 64, 16-21
Liquid Chromatographic Retention Behavior of Polystyrene Homopolymers on a C4, Bimodal Pore Diameter, Reversed-Phase Column David
M.Northrop, Daniel E. Martire,* a n d R. P. W . Scott
Department of Chemistry, Georgetown University, Washington, D.C. 20057
The lsocratlc retentlon of a series of homopolymer polystyrenes was studled on a C,, M o d a l pore dlameter cdumn wing two dmerent bhary m0Mlephase systems. A slgnlflcant dlfference In retentlon behavkr was observed between data obtalned using the tetrahydrofuran:acetonlttile system, THFACN, as the mobile phase and data acqulred uslng the methylene chlor1de:methanol system, CH2CI,:MeOH, as the moblle phase. Udng the THFACN system It was posdbk to separate lsocratlcally a serles of polystyrenes of dlfferent molecular welght, and the molecular weight range that was separated could be adjusted by changlng the proportions of THF and ACN In the moblle phase. I t was found that In addItlon to a dependence on the nature of the mobile phase, the retentktn behavkr of polystyrenesmay be affected by the excluslon propertles of the column, the ablllty or InaMlIty of the stationary phase to peferentlally adsorb one of the moblle-phase components, and the nature of the stationary phase Itself.
INTRODUCTION Analysis of macromolecules, both synthetic and biological, by liquid chromatographic (LC) techniques has been an area of intense research activity. Two of the most popular methods of analysis are size exclusion chromatography (SEC) and gradient elution liquid chromatography. SEC is a rapid and relatively simple method which provides discriminationbased on size. However, since the exclusion volume, Vex, for any solute is between the accessible interstitial volume, Vi,and the total solvent volume of the column, V,, there is limited peak capacity. Thus,solutes of relatively similar size may not be separable by SEC. In conventional interactive chromatogaphic methods, bonded phases, and mobile phases are chosen such that there are interactions between the solute and the stationary and mobile phases. In this way solutes are retarded in the column such that mobile-phase volumes greater than each solute’s exclusion volume are needed to elute the solute. The disadvantage of this method is that analyses can be very time consuming, particularly if one solute of interest has a small retention volume, V,, while another solute in the same system has a very large retention volume. For this reason gradient techniques have been developed to change the nature of the mobile phase throughout an experiment, thereby reducing the time it takes to elute long-retained peaks by gradually making conditions more favorable for them to elute. One problem that has plagued macromolecular LC analysis until recently, has been the limited molecular weight range that could be analyzed by any one particular column. For SEC, if the pores of the column packing material are too small, all solutes above a certain size will be excluded from the pores and thus all will elute at the interstitial volume. If the pores are too large, then smaller solutes will all elute at the total void volume. For retentive modes of LC, pore size is also 0003-2700/92/0364-0016$03.00/0
important because more than 99%.of the interactive surface area is located within the pores. If solutes cannot enter the pores, their interaction with the stationary phase will be severely limited,Use of wide-pore packing materials is preferred, but under high pressure, the packing material becomes more fragile as the nominal pore diameter increases. Both isocratic and gradient reversed-phase LC analysis of macromolecules has been demonstrated by several groups (1-6). The results show that the retention of macromolecules changes rapidly as a result of small changes in solvent composition. Because of these dramatic changes in retention (sometimesreferred to as “critical behavior”)over a relatively small mobile-phase compositional range, isocratic separation of macromolecules is very difficult. As a result, gradient elution has been necessary to obtain separations of these types of solutes. The purpose of the work described in the present paper is to characterize the behavior of polystyrenes under normal retentive conditions using isocratic elution. Previous studies (4,6)have been somewhat limited because of exclusion effects. In this study these effects have been carefully examined using a column containing a mixture of two silicas of different average pore diameter (this column has been previously described and characterized (7)).This mixed-bed column allowa for the SEC characterization of a wide range of molecular weights (500-20oO OOO), thus allowing for the study of polystyrenes for which the exclusion properties are well characterized. It will be shown that the retentive behavior of polystyrenes is significantly affected by the nature of the binary mobile phase used for elution. Furthermore, it will be shown that proper selection of the mobile phase, and correction for exclusion, makes it possible to obtain separations of polystyrenes using isocratic elution with results that provide a linear relationship between the defined capacity factor, k’, and molecular weight. EXPERIMENTAL SECTION Apparatus. The LC system that was employed consisted of two Waters solvent pumps (Model 510 and Model 6000A), a Waters (Model 680) solvent programmer, a Rheodyne sample injection valve (Model 7010) with a 20-pL sample loop, a Waters variable-wavelength UV absorbance detector (Model 481), a Nealab circulating bath (Model EX-300),and a Linear strip chart recorder (Model 1210). The analytical column used for these experimentswas a 25cm, C4,reversed-phase, binary pore diameter silica column (48% by weight of particles with a nominal pore diameter of 80 A and 52% by weight of particles with a nominal pore diameter of 500 A) as described in our previous work (7). This column provides size exclusion with an exclusion volume versus molecular weight range which is linear from 517 to 2800000. The column temperature was controlled at 30.00 0.02 OC, and the flow rate for all experimenta was held at 1.0 mL/min. The gas chromatographic, GC, system consisted of a Gow-Mac flame ionization detector (Model 40-900), a Hamilton injector (Model 86800), and a circulating bath maintained at 50.0 i 0.1 O C to thermostat the column. The column was a 3 m X 1/8 in. 0.d. stainless steel column packed with 10% Carbowax 20M, 2% KOH on 80/100 Chromosorb WAW from Supelco. A Linear strip
*
0 I99 1 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992
Table I. Polydispersity and Exclusion Volumes solute molecular polydisweight persity, exclusion vol, mL (MW) MJM, usingTHF 517 4000 9000 17 500 30 740 50 000 100 000 390 000
1.03 1.06 1.04 1.04 1.02 1.04 1.06 1.10
2.86 2.61 2.49 2.34 2.26 2.20 2.08 1.86
exclusion vol, mL using CHzClp 2.88 2.64 2.53 2.36 2.28 2.21 2.10 1.87
chart recorder (Model 1210) was used to record the data. Reagents. All solvents were either HPLC grade or SpectraAnalyzed grade from Fisher Scientific (Fair Lawn, NJ). It should be noted that uninhibited tetrahydrofuran (THF) was used in these experiments. This solvent is known to form peroxides which could present a safety hazard if not properly handled. Narrow molecular weight distribution standard polystyrenes (listed in Table I) were obtained from Scientific Polymer Products Inc. (Ontario, NY) and Du Pont. Procedures. An initial, semiquantitative study was made to determine if critical behavior for a polystyrene standard was dependent on solvent strength. The retention volume, Vr, of a MW 30740 polystyrene was measured at a series of different mobile-phase compositions in order to determine its critical composition, defied by Alhedai et al. (6),as the composition at which k' = 1. Isocratic elution was used with a binary mobile phase containing tetrahydrofuran, THF, as the Ygood*solvent and acetonitrile, ACN, as the *poor" solvent. A 20-pL aliquot of a dilute solution (it was later determined to be 20.4 mg/mL) of the polystyrene standard was injected onto the column. The solvent strength of the critical composition was determined by using the solvent strengths of the individual solvents, Si,and calculating a total solvent strength for the mixture, S,, based on the contributions of each individual solvent to the mixture by
ST = Ci(siei)
(1)
where Bi is the volume fraction of each individual solvent (8). These results were compared to those obtained by Alhedai et al. (6). The retention volumes of polystyrene standards from M W 517 to 1OOOOO were measured for a series of mobilephase compositions to determine if a single composition could be used to separate a range of molecular weights. A mobilephase composition of 60% ACN and 40% THF was used to elute isocraticallypolystyrenes from M w 517 to 30740. Using 56% ACN and 44% THF, isoclatic elution of polystyrenes from MW 4000 to 100000was obtained. As will be seen from the retention data, solutes continued to experience exclusion even under retention conditions, so the capacity factor, k', was calculated using k f = (Vr- VeJ/Vex (2) where Vex is the exclusion volume for each individual solute, determined from the elution volume using 100% good solvent, as previously described (7). All measurements were made in triplicate. The third study was a quantitative isocratic examination of five polystyrene standards (MW = 4000,30 740,50 000, 100OOO, and 390000) using two different mobile-phase systems. The fist system was the THF:ACN system, and the second consisted of methylene chloride, CH2C12,as the good solvent and methanol, MeOH, as the poor solvent. Samples of the five polystyreneswere studied at 0.4 and 0.04 mg/mL. Each concentration was also prepared in both the good solvent and in the binary mobile phase. As before, 20-pL injections were made onto the column and detection was made at 254 nm. Again, the retention volume was determined at a series of different mobile-phase compositions, increasing the percentage of the poor solvent until elution of the solute was no longer obtained. When a mobile-phasecomposition was reached, at which the solute had not eluted after passing 20 column volumes of mobile phase, the mobile phase was retuned
17
to a more favorable composition to elute the solute. The excess volume of the mobile phase adsorbed onto the stationary phase was determined by a series of solvent stripping experiments. These were done for both the THF:ACN and CH2C12:MeOHbinary solvent systems. The HPLC column was equilibrated with a fixed composition of mobile phase and then flushed (or stripped) with 15 column volumes of a solvent of higher solvent strength (hexane for the CH2C12:MeOHsystem and 50:W hexanediethyl ether for the THF:ACN system) with quantitative collection of the effluent. A fixed amount of toluene was added as an external standard for quantitative GC analysis. Five replicate GC analyses were done on duplicate runs of the stripping experiment using 0.5-pL injections for each mobile-phase composition examined. Compositionswere studied that spanned the range of mobile-phase compositionsused in the retention studies (75%,65%,55%,md45%CH~Cl~and6O%,50%,4o%,~d30% THF). To take into account any change in the mobile-phase composition due to evaporation during the LC equilibration, standard samples were taken from the solvent reservoir both before and after equilibration and analyzed by GC in the same manner as the samples stripped from the column. Using the layer model, as described by Tani and Suzuki (9)) the measured solvent excess volume can be converted into mole fraction of good solvent adsorbed at the surface, xzn,by the following equation:
+ (alo)(rz(n)) t - (azo- a l o ) ( r p )
(t)(xzl) x28
=
(3)
where t is the number of assumed,adsorbed solvent layers, which for the purpoees of this discussion is assumed to be a single layer, x; is the mole fraction of good solvent in the bulk mobile phase, al0 and azoare the molar cross-sectional areas of the poor and good solvents, which are calculated from u: (m2/mol) = 9200(ui ( ~ m ~ / m o l ) ) ~ / ~ (4) where ui is the molar volume of solvent component i. This calculation assumesthe molecules are rigid spheres in a face-centered cubic structure. The f i i term in eq 3 is the areal reduced surface excess, F2(n),for the solvent component in excess, which is calculated by
r p = (nzu(n))/(m)(S)
(5)
where nzs(")is the moles of excess solvent, m is the mass of the column packing material (2.922 g), and S is the specific surface area of the packing material (123.8 m2/g). Finally, the total amount of adsorbed solvent in the surface layer, ns,can be calculated by ns = n $ ( n ) / ( x { - x21)
(6)
From the total amount of adsorbed solvent, it is possible to calculate the individual contributions from each of the components.
RESULTS AND DISCUSSION The critical behavior of macromolecules occurs when the binary solvent system employs a good solvent and a poor solvent which are very different from each other in solvent character. It was anticipated that the use of two solvents that were more similar to each other in solvent character would provide less dramatic behavior, thus allowing for the isocratic separation of macromolecules. The CH2C12:MeOHsystem has been used as a good/poor solvent system for the fractionation of polystyrenes (I). A comparison of solvent strengths from available tables (8)shows a large difference between these two solvents. THF and ACN are much closer to each other in solvent strength, but THF is a good solvent for polystyrene while ACN is a poor solvent. The Snyder solvent strength parameter for THF is 0.45 and is 0.65 for ACN. For the 30 740 polystyrene the critical composition, where k = 1, was found to be 59% ACN and 41% THF. This mobile phase has a composite solvent strength of 0.57. Work by Alhedai et al. (6) with a CH2C12:MeOHsystem found the critical composition for the same polystyrene to be at 63% CHzClz and 37%
18
ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 195 I
-
1 1
1.0 Ij
0.9 -
20.8
-
+: 206 li
>
050.4
-
03
I
20000
0
40000
60000
80000
100000
Polystyrenes MW (dalton)
5 6 7 8 9 10 Minutes Figure 1. Isocratic elution of polystyrenes. Conditions: sample, 0.4 mg/mL each of the MW 517,4000, 9000, 17 500, and 30 740 polystyrenes: mobile phase, 60% ACN, 40% THF; flow rate, 1.0 mL/min; injection volume, 20 kL.
1
0
2
3
4
Figure 3. Retention of polystyrenes. Conditions are the same as Flgure 2 except the mobile phase Is 56% ACN, 44% THF and the solutes are MW 4000, 9000, 17 500, 30 740, 50 000, and 100 000 polystyrenes. 0.50
Iy
0.25 0.00
-0.25
0
5 -0.50 L
.-,X -0.75 0 -1 .oo
1.20
9 0.901 1.05
0
L-1.25
h
.’0.75 1
-0
/
-1.50 A
- 1 75
- 2 00
‘
20 0.15
/
0001 0
I
6400
12800
19200
25600
32000
Polystyrenes MW (dalton)
Figure 2. Retention of polystyrenes shown by the plot of k’vs molecular weight of polystyrenes. Conditions are the same as for Figure 1. Data represent the average of three replicate measurements. Standard deviations are smaller than data points in the plot.
MeOH. With the solvent strength parameter for CHzClzat 0.42 and for MeOH at 0.95, the composite solvent strength would be 0.62. To obtain the same solvent strength with THF and ACN, the ratio would have to be 85:15,ACNTHF. The conclusion is that the critical composition is not dependent on the absolute solvent strength of the mobile phase. Using the THF:ACN system the critical behavior of the polystyrenes does not appear to be as abrupt as it is in the CH2Cl2:MeOHsystem studied by Alhedai et al. (6). As a result, the isocratic separation of polystyrenes over a range of molecular weights can be achieved by careful selection of the mobile-phase composition. Polystyrenes from MW 517 to 30 740 were separated isocratically (Figure 1) using a mobile phase consisting of 60% ACN and 40% THF. The peak shapes become increasingly poorer as retention increases. This is in contrast to the well-defined peak shapes demonstrated using gradient elution techniques (2,3). However, as can be seen in Figure 2, the k’values are linear with respect to molecular weight (MW) which is in contrast to SEC on the same column, where the exclusion volume is linear with respect to the log of molecular weight (7). By changing the mobilephase composition it is possible to elute a different molecular weight range, which is demonstrated in Figure 3. Here a separation is obtained for polystyrenes from MW 4000 to 100000, which gives a k’vs MW plot that is linear, by using a mobile phase consisting of 56%
PS30740 in CHtCIz PS50000 in CHtCli
I , 26
44 50 56 62 68 Percent “Good” Solvent (THFor C H Z C I ~ )
32
38
I
74
80
Flgwo 4. Retention as a function of solvent system shown by the plot of log,, k’ vs percent “good” solvent compositlon for both the CH,CI,:MeOH system and the THFACN system. Standard deviations are smaller than the symbols at each point.
ACN and 44% THF. Thus, by judicious selection of the mobile-phase composition it should be possible to separate a wide range of molecular weight using isocratic elution, which would be very useful if gradient instrumentation is not available. In order to determine if the difference in retentive behavior, using the bimodal pore diameter column as compared with the work done on conventional columns was due to column effects, a more detailed retention study was conducted using both the THF:ACN system and a CHzCl2:MeOHsystem. Figure 4 is a plot of the results obtained from the two mobilephases systems using the data acquired for the 0.4 mg/mL concentrations. This graph shows that it is possible to measure retention volumes corresponding to positive values for log k’ with the THF:ACN system. Mobile-phase compositions with lower percentages of good solvent continued to elute the polystyrenes, but the peaks were too broad and distorted to measure accurately retention volumes. In contrast, with the CH2Cl2:MeOHsystem it was not possible to measure retention volumes that corresponded to positive values for log k ! The dashed lines extending upward, after the last data point for each solute, indicate that decreasing the good solvent composition by 1% or 2% resulted in the solutes being retained even after subsequent passage of 20 column volumes of mobile phase. When the mobile-phase composition was returned to one that was more favorable for solute elution, the solutes did elute with normal peak shape. When this procedure was done with the THF:ACN system, no peaks were obse~ed,indicating that the solutes had eluted, but with such diffuse peaks that retention volume determination was not possible.
19
ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992
Table 11. Effects of Sample Concentration and Solvent on Retention solute dissolved in mobile phase
solute dissolved in 'good" solvent
k'
"good" solvent
%
100 54 52 50 48 46 44 42 41 40 39
0.04
0.4
0.04
mg/mL
mg/mL
mg/mL
mg/mL
50 000-Da Polystyrene in THF:ACN System 0.00 0.00 0.00 0.00 0.08 0.08 0.08 0.08 0.11 0.11 0.11 0.11 0.14 0.14 0.14 0.14 0.20 0.20 0.20 0.20 0.32 0.33 0.31 0.31 0.58 0.58 0.56 0.56 1.21 1.21 1.15 1.15 1.94 1.95 1.87 1.87
peak visible but not determinable
no peak-switchover to 45% THF gave no peak
0.00 0.06 0.08 0.09 0.10 0.11 0.16 0.20 0.27 0.36
0.00 0.06 0.07 0.08 0.10 0.11 0.14 0.18 0.22 0.28
/
-0.66
0.4
0.00 0.06 0.08 0.09 0.10 0.11 0.16 0.21 0.27 0.36
-0.1 2
k'
50000-Da Polystyrene in CH2Cl2:MeOHSystem 100 70 69 68 67 66 65 64 63 62 61
R2=0.9540
0.00 0.06 0.08 0.08 0.10 0.11 0.15 0.18 0.22
not visible peak visible but not determinable not visible 60 no peak-switchover to 65% CHzClzgave large peak
Work by Glhkner (10) and Stadalius et al. (11)indicates that solute concentration effects need to be considered when LC analysis of macromoleculesis performed. The reason for this is that when the mobile phase in which the polystyrenes are dissolved becomes unfavorable, the solutes may either collapse on themselves or with each other, effecting a phase separation such that one phase is rich in polystyrene while the other phase is deficient in polystyrene. If this occurs, the point at which it occurs may change as the concentration of solute is changed; i.e. as the solute is diluted, the point at which phase separation occurs will be at a mobile-phase composition that is richer in poor solvent. For this reason our work was done at two different solute concentrations 1order of magnitude apart. The results for the MW 50000 polystyrene, which are representative of the results seen for all of the solutes, are shown in the first two columns of Table I1 and indicate that there was no significant effect of concentration on the capacity factor. The only problem encountered with some of the solutes was that the 0.04 mg/mL concentration was too small to be distinguished from the solvent disturbance peak (when retention volumes corresponded to values near the solvent peaks) or the baseline (when retention was long and peak shape began to deteriorate). This would seem to indicate that experiments were run at dilute enough solute concentrations to be operating under "normal chromatographic conditions". Recent work by Lochmtiller and McGranaghan (5) has indicated that the environment of the sample before it reaches the column may contribute to the solute's behavior on the column. Their work suggested mixing of the solute with the mobile phase by using a "serpentine" tubing or "crocheted reactor" prior to the column. In order to determine if the solute's mobile phase environment had any effect on retention in our work, we prepared two different solutions of the polystyrenes a t each concentration. The first solution contained the polystyrene dissolved in the mobile phase, while the second solution consisted of the polystyrene dissolved in
Rz = 0.9974
l A
c -1.20
I
j
I
;
THFACNSystem CHzC12 MeOH System
0
-390
"
"
"
"
'
"
8
'
I
"
'
8
"
350 4 0 0 4 5 0 5 0 0 550 600 650 700 750 8 0 0 8 5 0 In M
Flgure 5. Plot of In S vs In M for MW 4000, 30 740, 50 000, 100 000, and 390 000 polystyrenes. The flow rate is 1.OO mllmin. Data r e p resent the average of three replicate measurements,and the standard deviations are smaller than the data points in the plot.
pure good solvent. Table I1 shows these results which appear to confirm the aforementioned observations. Again, concentration had little effect, but as the retention of a solute increased, the difference between values for the solute dissolved in the mobile phase and the solute dissolved in the good solvent increases. This would suggest that injection of the good solvent into the system may change the local environment around the solute in the mobile phase and, perhaps, at the stationary-phase surface such that, as the retention changes more rapidly with changing mobile-phase composition, the effect is more dramatic. The BMAB theory (12)predicts that, when only the good solvent is adsorbed in the surface phase, isocratic plots of In S vs In M should be linear, with unit slope, for homopolymers of higher molecular weight, where (7) and M is the degree of polymerization, which is found by dividing the molecular weight of the polystyrene by the molecular weight of the monomer unit which is 104.15. Figure 5 demonstrates that, for both systems studied, linear ln S-ln M behavior is obtained with slopes, however, less than unity. It is also interesting to note that the plot remains linear even for lower molecular weight (4000) polystyrene in the THF: ACN system while in the CH2C12:MeOH system it is only linear for molecular weights higher than 30 000. The results of the S vs M measurements can also be expressed by an empirical relationship of the form
s
= C'M" (8) which was first utilized by Stadalius et al. (13),where C'is a constant term and n is determined experimentally. Figure 6 shows a plot of our results with the best linear fits being given by setting n = 0.55 for the THF:ACN system and n = 0.35 for the CH2C12:MeOHsystem. These values correspond to the slopes of the In S vs In M plots. As is predicted, S increases with an increase in molecular weight. However, the change is more dramatic for the CH2C12:MeOHsystem than for the THFACN system. It should be noted that in both Figures 5 and 6 the plots are linear even for the MW 390000 polystyrene, which other workem (4,6)have shown to deviate for columns with 100 1\ diameter pores. This would indicate that exclusion effects are important, suggesting that a column that does not produce pore exclusion of these higher molecular weight polymers is necessary for proper analysis by this method. The BMAB (14)theory also predicts that I#Jc will increase with an increase in M. Figure 7 shows that this does indeed occur and that I#Jc changes linearly with In M for the higher molecular weight solutes. Note once again that the slope is
ANALYTICAL CHEMISTRY, VOL. 64, NO. 1, JANUARY 1, 1992
20
I1
slope = 0.201 R2 = 0.9 733
4.80 0
M(0.35)
0
0
-
slope = 0.0054
10
20
30
40
50 M"
60
70
80
90
i
hI1 3 6 0 1
"." 100
Flgure 8. Slope vs degree of polymerization shown by the plot of S vs W where n is determined from the slope of the In S vs In M plots in Figure 5. All other conditions are the same as in Figure 5. 70 0 65 5
THF:ACN System CHzCIz System
4.35 -
2 4.05 4.20
0
1
$465 u : : 4 50 -
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
'
Table 111. Results from the Solvent-Stripping Experiment
CH2C12:MeOHSystem Excess CH2Cl,
56 5 52 0
@C 47 5 43 0
mobile-phase comp CH2C12:MeOH
excess vol of CH2Clz,mL
75:25 6535 5545 4555
0.0192 f 0.0010 0.0199 i 0.0017 0.0236 f 0.0015 0.0241 f 0.0011
38 5
R2=0.999 1
29 5 35
'
'
F W e 8. Solvent poleritv parameters as a functlon of Solvent system shown by the pbt of Pvs In M for the MW 30 740,50 000, 100 000, and 390000 polystyrenes. P' Is determined for the mobllaphase compositlon at 4 based on literature values for P'.
61 .O
34 0
'
550 580 610 640 670 700 730 760 790 820 850 In M, M = degree of polymerization
40
45
50
55
60
65
70
75
80
85
Surface Composition of CH2C12:MeOHSystem mobile-phase comp CH,Cl,:MeOH
V(CH2C12), mL
V(MeOH), mL
vol % of surface covered with CHzClp
7525 65:35 5545 45:55
0.129 0.113 0.101 0.086
0.024 0.038 0.048 0.061
84*1 75 1 68 1 59 f 1
In M
Flgure 7. Critical composition vs degree of polymerization shown by the plot of 4 vs In M . The values for 4 are taken as the "good" solvent composition at the critical compositlon. Conditions are the
same as Figure 5. greater for the CH2C12:MeOHsystem than for the THF:ACN system, indicating that changes in 4, are more dramatic for the former system. This is reflected in the inability to obtain ismatic separations of a range of molecular weights by elution with the CH2C12:MeOHsystem, whereas it is possible with the THF:ACN system. As was mentioned earlier, no correlation was found between a particular solvent strength and &. However, as can be seen in Figure 8, there is a specific relationship between solute molecular weight and the solvent polarity at 4,. Note that the slope for the CHZClz:MeOHsystem is slightly greater than for the THF:ACN system and that, as was noted before, there is a difference in the polarity for a given solute in the two different systems. All of this suggests that some of the behavior of macromolecules in retentive LC analysis is due to mobile-phase effects per se. As was noted by Armstrong and Boehm (2),the good solvent should be one which will preferentially interact with the stationary phase. Unpublished work in this laboratory has suggested that for the CH2C12:MeOHsystem on a C18column, the preferential adsorption of CHzClzmay be such that if monolayer adsorption onto the stationary phase is assumed, that monolayer would be close to 100% CH2C12.This means that the stationary phase would provide a much better environment for the polystyrene solute molecules than the mobile phase, for compositions smaller than 4,. For the solvent systems that we have been using, it was thought that some of the differences in retentive behavior might be due to differences in the adsorbed solvent. Since THF and ACN are more similar to each other, preferential adsorption could
**
THFACN System Excess THF mobile-phase comp THF:ACN
vol of excess THF, mL
6040 5050 4060 3070
0.0061 i 0.0005 0.0048 i 0.0005 0.0090 i 0.0005 0.0070 f 0.0008
Surface Composition of THFACN System mobile-phase comp THFACN
V(THF), mL
V(ACN), mL
vol % of surface covered with THF
60:40 5050 4060 3070
0.094 0.077 0.067 0.051
0.052 0.068 0.079 0.096
64 1 53 f 1 46 f 1 35 f 2
*
~
~~
be rather limited. Table 111shows the results of the stripping experiments. Because the excess quantities of adsorbed solvent were so small compared to the bulk mobile phase, errors in the measurements are relatively large. Therefore, the results are presented only to make a qualitative &tinction between the two systems. As can be seen there are signifcant differences. The amount of preferentially adsorbed good solvent for both systems is not enough to account for 100%
ANALYTICAL CHEMISTRY, VOL. 04, NO. 1, JANUARY 1, 1992
of the assumed monolayer; however, the excess of adsorbed CH2C12is at least twice as large as that of adsorbed THF. It is also known from the work of Flory (15) that polystyrenes prefer a good solvent environment. Thus if there is a better solvent environment at the stationary-phase surface than in the bulk mobile phase, the amount of preferential adsorption will affect the nature of the solute’s preference for the mobile or stationary phase. If the preferential adsorption is significantly less, as appears to be the case with the THF:ACN system, then changes in the solute’s retention behavior with changes in mobilephase composition will be leas dramatic than in a system, such as the CH2C12:MeOHsystem, where preferential adsorption is more significant. One final observation can be made with respect to the nature of the stationary phase. A comparison of 4, for the MW 30 740 polystyrene in our work and that of Alhedai et al. (6) shows a significant difference. In our work, done on a C4 phase, 4, was at 56% CH2Clzand 44% MeOH, while in the previous work, done on a Cla phase, 4, was at 63% CH2Clz and 37% MeOH. This may reflect differences in preferentially adsorbed solvent. According to Welsch et al. (161, columns with similar phase densities and silanol concentrations but different alkyl chain lengths show differences in their wettability. The longer the bonded-phase chain, the more difficult it is to wet the phase. This indicates that solvent penetration of the bonded phase is more difficult for a Cla phase than for a C4 phase. Therefore, MeOH is more likely to be adsorbed onto unreacted silanols on the C4phase than on the Cla phase. Also the shorter C4 phase may not shield solutes from any MeOH which might be adsorbed onto unreacted silanols. As a result, the difference between the environment of the stationary phaseladsorbed solvent surface and the mobile phase will be smaller for the C4phase than for the C18phase. Thus, it would be expected that the transition from low to high retention would be more abrupt on the C18column than on the C4 column and the critical composition (4,) would be higher for the column with the C18phase than with the C4 phase.
CONCLUSION It has been shown that with proper selection of operating conditions it is possible to obtain an isocratic separation of a wide range of solute molecular weights. A plot of k’values versus molecular weight has been shown to be linear for this type of separation. The advantage of using a bimodal pore diameter column is that it allows for the analysis of high molecular weight macromolecules while providing a physical stability that is greater than that of large pore diameter columns and also allowing for the analysis of smaller molecular weight solutes. The so-called critical composition has been shown to increase linearly with the logarithm of solute molecular weight. As has been demonstrated previously, the slope, as defined by eq 7, can be fit to a functional dependence on M of the form C’M”.
21
One of the most important results of this work is that the mobile phase plays an important role in the separation of macromolecules. By selecting good and poor solvents which are similar to each other in solvent character, it is possible to moderate the critical behavior of the solutes of interest. It would appear that this is related to the ability of the stationary phase to preferentially adsorb the good solvent. If thisadsorption is moderated, then the difference between the stationary-phase environment and that in the bulk mobile phase will be decreased, thus decreasing the solute’s phase preference. These effects may also be stationary-phase dependent in that long-chain phases may increase the effect of preferential adsorption while short-chain phases may moderate this process. All of these results suggest that retentive LC analysis of macromolecules is more complex than originally thought. In order to describe more accurately the behavior of these solutes in an LC system, it will be necessary to understand in detail the microscopic environments at the conditions under which these molecules are analyzed.
ACKNOWLEDGMENT We thank Tom Beesley (ASTEC) for providing the silica for the mixed-bed, reversed-phase columns. We also acknowledge Lane Sanders (NIST) for assistance in preparation and packing of the column and Richard Boehm (Georgetown University) for helpful discussions. bd6tl3‘ NO. THF, 109-99-9; ACN, 75-058; P h C H 4 H 2 (homopolymer),9003-53-6; CH2Cl2,75-09-2;MeOH, 67-56-1.
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378985. (3) Snyder, L. R.; Stadallus, M. A.; Quarry, M. A. Anal. Chem. 1983, 55, 1412A- 1430A. (4) (Yackner, 0.; van der Berg, J. H. M. J . chrometogr. 1988, 352, 511-522. (5) . . LochmWler. C. H.: McGranacrhan. M. B. Anal. CY”. 1989. 87. 2449-2455. (6) 1990, 29. . . Alhedai, A.; Boehm, R. E.; Martire, D. E. ChromatOerephle _ . 313-321. (7) Northrop, D. M.; Scott, R. P. W.: Marthe. D. E. Anal. Chem., 1091, 63,1350-1354. (8) Pode, C. F.; Schuette, S. A. Contemporary Ractfce of Chromatogrephy; Elsevier: New York, 1984. (9) Tanl, K.; Suzukl, Y. J . Chromatogr. Sc/. 1989. 27, 698-703. (IO) Gkckner, G. W e Appl. Chem. 1983, 55, 1553-1556. (11) . . Stadallus. M. A.; Quaw,M. A.; Mourey, T. H.;Snyder, L. R. J . ChroM t O g r . 1988, 358. 1116. (12) Boehm, R. E.; Martire, D. E.: Armstrong. D. W.; Bui, K. H. Mewom&-
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Culed lB89. . -, 16. . . 466-478. ... .. . .
(13) Stadallus, M. A.; Quamy, M. A.; Mourey,T. H.;Snyder, L. R. J . Chmmat-. 1986. 358. 17-37. (14) Boehh, R. E.;’Ma&e, D. E. Anal. Chem. 1989, 67, 471-482. (151 . . Florv. P. J. Mclnles ofPo&mw Chemlstrv: Cornell Universitv Prese: I#’&, NY, 1971. (16) Welsch, T.; Frank, H.; Vlgh, G. J . Chromatogr. 1990. 506. 97-108.
RECEIVED for review July 8,1991. Accepted October 3,1991. This material is based upon work supported by the National Science Foundation under Grant CHE-8902735.
