alarm or printer after the maximum of each peak and is readily compatible with most other 1/0 devices. A 1000-fold expansion analog output is provided. The circuit design contains several unusual features which should, in general, interest designers of new automatic laboratory devices. Efficient automatic attenuation is currently provided by much less simple means on a few sophisticated computing integrators of great cost.
application, although an operator was present, he was unable to reach the recorder range-setting switch while attending the other important controls of the mass spectrometer. The attenuator is also compatible with a repetitive scan demodulating unit recently designed in these laboratories for this mass spectrometer. On another occasion, the unit was used to attenuate the beam monitor output of the mass spectrometer. We also expect to use the attenuator on the output of a Technicon AutoAnalyzer modified to monitor an immobilized enzyme reaction. None of the above instruments are modified to use the autoattenuator. Further, all of the high impedance recorders mentioned could be driven directly from the unbuffered output of the attenuator.
ACKNOWLEDGMENT The author wishes to thank the Barbers, Hazlet, N.J., for assembly tools; teachers, R. I. Edenson, D. E. Smith, F. G. Bordwell, S. S. Chang, J. D. Rosen, and R. Killops, for guidance and incentive, past and present; Williams Electronics, Edison, N.J., H. Cohen of Harwood-Sandler Associates, Woodbury, N.Y ., representing Analog Devices, Inc., and Automatic Laboratory Devices, Bridgeport, Conn., for components, technical suggestions, and printed circuit board artmasters, respectively. Diagrams were executed by James Kiss.
SUMMARY A digital automatic binary attenuator has been described, for use between a number of laboratory instruments and a potentiometric chart recorder, that is portable, efficient, versatile, and of low cost. One of its two displays continuously latches a t the attenuation state of greatest significance for each peak while the other displays the present attenuation state. The unit signals an
Received for review August 9, 1973. Accepted November 2, 1973.
Solvent Selection in Adsorption Liquid Chromatography D. L. Saunders Union Oil Company of California, Research Department, Brea, Calif. 92621
During the past several years, the field of liquid chromatography has been growing at an increasingly rapid rate. Recently, a variety of short courses and monographs (1-5) have appeared describing the theory and practice of the subject. These have gone a long way toward introducing the newcomer to the field; nevertheless, the selection of a solvent for a new problem remains a matter of trialand-error for most newcomers as well as some experienced practitioners. A working theory of adsorption chromatography was developed in the last decade by the prolific work of Snyder who summarized the details in a monograph on the subject ( 5 ) .With this theory, it is possible to determine approximately the appropriate solvent mixture for a specific compound or mixture of compounds. In practice, the application of this theory requires the assembly of a number of sample, adsorbent, and solvent parameters, and a set of somewhat tedious calculations. We have condensed some of Snyder's basic concepts of adsorbent activity, group adsorption strength, and mixed solvent strength into a simplified graphical form. The application of these graphs is rapid and provides a reasonable first approximation to a solvent mixture appropriate for a given sample. It must be stressed that the results are approximate and, in some special cases, the solvent (1) J. J. Kirkland, Ed., "Modern Practice of Liquid Chromatography," Wiley-Interscience, New York, N.Y., 1971. (2) P. R. Brown, "High Pressure Liquid Chromatography,'' Academic Press, New York, N.Y., 1973. (3) S. G. Perry, R. Amos, and P. I. Brewer, "Practical Liquid Chrornatography," Plenum Press, New York, N.Y., 1972. (4) F. Baumann and N. Hadden, Ed., "Basic Liquid Chromatography,'' Varian Aerograph, Walnut Creek, Calif., 1972. (5) L. R. Snyder, "Principles of Adsorption Chromatography," Marcel Dekker, New York, N.Y.. 1968.
470
mixtures indicated will be in error. Yet, in practice, we have found the technique presented below to be a timesaving alternative to both the calculation and the trialand-error approach.
DISCUSSION The sample partition ratio k' is defined as the ratio of the amount of sample in the stationary phase to the amount in the mobile phase. It can be calculated from retention data using Equation 1. tr #3-
- to to
where tr is the sample retention time and t o is the retention time of an unadsorbed component. In adsorption chromatography, the value of k' for a given component can change by a factor of -lolo from a very strong solvent to a very weak one. Nearly always we seek a solvent mixture which will give 12' > 1 to obtain separation of the component of interest from other sample components of similar composition. Generally, we also seek a solvent mixture which will give k' < 10 in order to reduce the total separation time and minimize the sample dilution which occurs at high k'. Snyder (5) defines the solvent strength parameter eo as the adsorption energy per unit area of standard adsorbent. For a given sample and adsorbent, log k' varies linearly with eo. We define the E3 value of a sample as the solvent strength required to give 12' = 3. Figure 1 shows the E3 values for a variety of compounds on a typical silica (300 m*/gram). We will describe how to approximately determine the solvent strength for a very broad variety of mono- and poly-func-
ANALYTICAL CHEMISTRY, VOL. 46, NO. 3, MARCH 1974
E3 VALUES SOLVENT STRENGTH REQUIRED FOR K ' * 3
HYDROCARBONS
HALIDES
ALKENES MERCAPTANS
DISULFIDES
SULFIDES
ETHERS
NITROS
ESTERS
NITRILES
KETONES
ALDEHYDES R-CHO SULFONES
R-SO-CH3
ALCOHOLS PHENOLS AMINES R-NH2 ACIDS
I
AMIDES
Figure 1. E3 values on silica gel
tional compounds, and how to obtain several alternative solvent mixtures for any solvent strength using only two simple graphs.
Figure 1 shows the E3 values for a series of aromatic hydrocarbons, substituted benzenes, . and aliphatic compounds (R = &HIS-). Consider naphthalene in Figure 1;
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the range of E3 values extends from 0.09 for fully activated adsorbent (equilibrated with dry solvent) down to -0.13 for highly deactivated adsorbent. This means that with an active silica column and a solvent strength of 0.09, naphthalene will elute with k' = 3. With deactivated columns, weaker solvents will be needed to give k' = 3 down to a lower limit of about -0.13 for highly deactivated silica. Adsorbent columns are often deactivated to reduce tailing, increase sample capacity, and minimize other problems. Most practical systems will be partly deactivated and will exhibit E3 values intermediate between the extremes indicated in Figure l. Extention to More Complex Molecules. By the grouping of various parameters and comparison of their relative effects on E3, it was possible to formulate the following approximate rules for estimating E3 in a broad variety of polyfunctional compounds. In the examples accompanying these rules, E3 values cited are for fully activated columns. The rules also apply to column-solvent systems of equivalent deactivation. 1. Saturated hydrocarbons and simple olefins are eluted with k' < 3 with all solvents. Fluorinated solvents are an exception; see ref. (6). 2. Alkyl and halogen substituted aromatics have E3 values within f 0.05 unit of the parent compound. 3. Aliphatic compounds with 2-20 carbons will have E3 values within ~k0.05unit of the model compound (R = C,jH13-) used in Figure 1. 4. Difunctional compounds will have E3 values larger than either of the monosubstituted compounds. For example, for benzaldehyde E3 = 0.23 and for methoxybenzene E3 = 0.15, but the E3 value for methoxybenzaldehyde will be larger than 0.23 by an increment which depends upon the difference between E3 values ( A E 3 ) of the mono substituted compounds as follows: AE3
Increment
0-0.1 0.1-0.2 >o . 2
0.15 0.07 0.00
In our example AE3 = 0.23 - 0.15 = 0.08; thus for methoxybenzaldehyde, the increment is 0.15 and E3 = 0.23 + 0.15 = 0.38. Similarly, for diaminohexane AE3 = 0.0, E3 = 0.54 0.15 = 0.69. 5 . Additional functional groups will add successively smaller increments to Es, but the size of these increments cannot be easily predicted. 6. Addition of an aliphatic halogen or mercaptan moiety will increase E3 by an increment 50 to 100% larger than the value indicated in 4 and 5 above. For example, for chlorohexyl ethyl ether E3 = 0.15 2 (0.07) = 0.29. 7. Compounds containing polynuclear aromatic moieties should be considered benzene compounds substituted with additional aromatic rings. For example, nitrophenanthrene should be considered a nitrobenzene (E3 = 0.19) substituted with two additional rings-ie., naphthalene ( E 3 = 0.09). Therefore, AE3 = 0.1 and the corresponding increment is 0.15, thus E3 = 0.34. Deactivated and Low Surface Area Silicas. The parameters used to calculate Figure l were for 300 m2/gram silica ( e . g . , Porasil A, Porasil T, Zorbax, LiChrosorb). Silicas with lower surface areas, silanized silicas, or those permanently deactivated by other means, and pellicular (solid core) silicas will all show E3 values lower than those indicated in Figure 1 for dry silica. Insufficient data are available to predict the extent of this effect for these adsorbents, but we suspect for a given adsorbent the extent
+
+
(6) L. R . Snyder. J. Chromatogr., 36,476 (1968) 472
of the shift will be reasonably constant for all compound types. Water deactivated silica gels are somewhat more predictable and have several other advantages ( 7 ) . As we have mentioned for a given compound, E3 will be largest on columns equilibrated with dry solvents. The low e'' end of the bands plotted in Figure 1 corresponds to columns equilibrated with 75% water saturated solvent, while the center of the scale corresponds to about 50% water saturation. Fifty per cent water saturation is recommended for general work. Water saturated solvents may be prepared by stirring the solvent mixture vigorously for 2-3 hours with 20 grams/l. of a mixture of equal quantities of water and silica gel. The silica serves to provide a high surface area to speed equilibration. Solvents containing acetonitrile or methanol cannot be water saturated in this manner. We have found that preparing these solvents from acetonitrile or methanol containing 3% vol water provides sufficient deactivation equivalent to about 50% saturation. Solvent Mixtures. Once the E3 values have been estimated from Figure 1 and the preceding rules, we can choose an appropriate solvent mixture from Figure 2. The six solvents used in Figure 2 are among the most useful for adsorption chromatography for several reasons: Viscosities are low allowing maximum resolution per unit time; UV cutoffs are low permitting the use of UV detectors; boiling points are low for use with transport detectors and for sample recovery; and the whole range of the solvent strengths is covered. In this figure, we plot solvent strength (to) across the top U S . various binary solvent compositions in a manner similar to Neher (8).Each horizontal line corresponds to a range (0-100% vol) of binary solvent mixtures. The first five lines represent mixtures of pentane with other solvents. The top line of this series of five corresponds to mixtures of pentane and isopropyl chloride. For any solvent strength intermediate between pentane ( t o = 0) and isopropyl chloride ( e " = 0.22), we may read the required solvent composition by dropping p vertical line from the eo scale. Thus, for e o = 0.10, we read 26% vol isopropyl chloride in pentane and for t o = 0.20, we read 80% isopropyl chloride in pentane. The next four lines in this series correspond to mixtures of pentane with methylene chloride ( e o = 0.32), ethyl ether ( c " = 0.38), acetonitrile ( c " = 0.501, and methanol ( e " = 0.73). The second series of lines correspond to binary mixtures of isopropyl chloride and solvents of higher t o . For any given solvent strength, Figure 2 indicates several binary mixtures. For example, the dashed line in Figure 2 indicates solvent mixtures with t o = 0.30. One of these mixtures (arrow) is 76% vol methylene chloride in pentane. Similarly, 49% vol ethyl ether in pentane or 37% vol ethyl ether in isopropyl chloride, etc. would also give t o = 0.30. Any of the indicated solvent mixtures would be an appropriate first choice for a sample with E3 = 0.30. Adjusting the Solvent. Because of the assumptions and approximations used in the derivation of Figures 1 and 2, and Rules 1-7, and the limitations of the basic theory, our initial choice of solvent is seldom exactly correct-ie., k' f 3 . However, it is usually close enough to allow a rapid adjustment to the optimum. We have found that the following rules are helpful in adjusting k' and improving selectivity (the ratio of k' for two partly resolved components). a. An increase in solvent strength c " of 0.05 .will decrease k' by a factor of 2-4 ( I ) . (7) J. J. Kirkland. J . Chromatogr., 83,149 (1973). (8) R. Neher, in "Thin Layer Chromatography,'' G. 6. Marini-Bettolo, Ed., Elsevier, Amsterdam, 1964.P 75.
ANALYTICAL CHEMISTRY, VOL. 46, NO. 3, MARCH 1974
T a b l e I. E s t i m a t e d C o n t r i b u t i o n s to P a r a m e t e r A Group
Aliphatics
Aromatics
10 7
9
-COOH -S-S-C Ha
2
b. A decrease in e o of 0.05 will increase k' by a factor 2-4 (I). c. Selectivity will be greatest if the concentration of the stronger component of the solvent mixture is 6 % vol or >50% vol. For example, a t t o = 0.30, either 76% methylene chloride in pentane or 1.7% acetonitrile in isopropyl chloride would be expected to give maximum selectivity (9). d . Substitution of ethyl ether or methanol for one of the other strong components can often improve selectivity by the formation of hydrogen bonds (9). e. If a dry solvent results in tailed peaks or t , which varies with concentration, a more highly deactivated solvent should be used. It should be remembered that this will shift E3 to a lower value. Calculations and Assumptions. The basic equation for the prediction of retention in adsorption chromatography ( 5 )can be written
log K = c
+ d(S - AP) + f
(2)
where K is the distribution coefficient, c and d are constants which depend upon the adsorbent and the water content, S and A are sample parameters made up of contributions from component functional groups, and f is a secondary absorption effect function. In the following discussion the function f was ignored since it is generally small and usually unpredictable. The partition ratio is related to the distribution coefficient by Equation 3.
k'
=
W -K
(3)
V"
where W is the weight of silica per unit column length, and column V is the void volume per unit column length. For porous silica W / V = 1, and we may substitute k' directly for K in Equation 2. Then solving for e', we obtain =
By definition E3 =
s
- (log k'
- c)/d
A to
(4)
when k' = 3, therefore
E3 =
s - C' ~
A
(5)
where C' = (0.477 - c ) / d . For dry 300 m2/gram silica, c = -0.96 and d = 0.83 ( 0 ) ; hence C' = 1.74. For highly water deactivated silica, c = -2.00, d = 0.69 ( 5 ) , giving C' = 3.58. In the preparation of Figure 1, values of S #and A were calculated from functional group contributions given by Snyder ( 5 ) .Values for carboxyl and disulfide contributions to A were not available, so the estimated values listed in Table I were used. For the extension of Figure 1 to more complex molecules, Rule 1 is simply a statement of the fact that nonaromatic hydrocarbons are unadsorbed with all the solvents listed in Figure 2 . Fluoroalkanes and fluoroethers have solvent strengths less than 0 resulting in some ad(9) L R Snyder, J Chrornatogr, 63,15 (1971)
Figure 2. Mixed solvent strengths on silica gel. CCI4 must be substituted for part of the pentane to achieve miscibility for mixtures indicated ( * )
sorption of olefins and saturated hydrocarbons (6). However, these solvents have found little use in adsorption chromatography presumably because of their high cost. Rules 2 and 3 arise from the very small group adsorption energies of alkyl groups generally, and halogens substituted on aromatics. For the formulation of Rules 4 and 5, consider a monofunctional molecule RX with adsorption energy S and area A. T o a first approximation, the difunctional molecule RX2 will have adsorption energy S' = 2 s and area = 2A. Thus, the difference in E3 values is given by
- C' s - C' - C' 2A A 2A If we use an average value of C' = 3 and A = 10, then AE3 = 0.15. If the strength of the second group is less than the first S 5 S' I 2S, then the increment well be less than 0.15. This decrease in increment is accentuated by the so-called localization effect ( 5 ) . Rule 6 accounts for the fact that in aliphatic molecules, halogens and mercaptans have small group area contributions in proportion to their adsorption energies. Finally, Rule 7 simply allows for the adsorption of aromatic carbon atoms in polynuclear aromatics. The graphical and rule of thumb approach presented here is an approximate but convenient method for quickly finding the solvent mixture appropriate for the separation of a given mixture. Those who require more accurate prediction can refer to the detailed calculations presented in reference ( 5 ) . AE, =
2s
Received for review August 15, 1973. Accepted October 29, 1973.
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