#
A Rational Series of Solvents for Use in IncrementalGradient Elution R. P. W. Scott and Paul Kucera Chemical Research Department. Hoffmann-La Roche Inc., Nutley, N.J. 071 10
A rational procedure for choosing a series of solvents for incremental gradient elution in liquid-solid chromatography is described and a practical series of solvents for gradient elution development is given. Examples are included of the use of the solvent system for separating mixtures containing solutes of widely diverse polarities together with the necessary operating conditions. The range of solvents given, commencing with heptane and ending with water, appears to cover a k’ range, relative to heptane of 1 04.
If liquid-solid chromatography is t o become a comprehensive service technique that can handle solutes of all polarities and permit the immediate separation of any type of mixture, then a series of solvents optimized for gradient elution must be chosen. The success of gas chromatography, as a service technique, relies on the use of temperature programming, the counterpart of gradient elution in liquid chromatography, as a means of development. During temperature programming, to a first approximation, the logarithm of the retention volume of a solute is reduced by equal increments with each degree rise in column temperature. Because of the resultant rapid reduction in retention volume with rise in temperature, substances having a wide range of molecular weights and polarities can be separated in one run. Unfortunately, in liquid chromatography, no such scale of solvent properties exists for gradient elution that compares with the centigrade scale in temperature programming. It is the purpose of this paper to determine a series of solvent mixtures for use in gradient elution such that a single solvent change between any consecutive member of the series will result in an approximately constant incremental change in the logarithm of the distribution coefficient of a solutk. Thus, the solvent series will form a scale which can be used to change the retention of a solute regularly and progressively in a manner exactly analogous to the way the temperature scale can be used to effect changes in the retention of a solute in gas chromatography.
THEORETICAL CONSIDERATIONS For satisfactory gradient elution development, it is necessary to employ a large number of solvents having small
differences in polarities and not to attempt the separation by the method of mixing using two or three solvents having large differences in polarity. To understand the reason for this, the processes that occur during gradient elution must be considered. The development processes resulting from gradient elution by the method of mixing using three solvents are depicted diagrammatically in Figure l. It should be stressed that this diagram treats the process of gradient elution in an approximate manner and is generally correct but there are secondary effects that are not discussed or represented in the diagram. The diagram represents the effect of a linear gradient using three solvents, a nonpolar solvent P ( e . g . , heptane), a semi-polar solvent Q ( e . g . , butyl acetate), and a strongly polar solvent R ( e . g . , methanol). The vertical axis represents, in arbitrary units, the effective molecular forces on the solutes in the mobile and stationary phases. Consider the curves representing the forces exerted by the adsorbent on the solutes A to I. While a given solvent flows through the column, the retaining forces remain constant. However, as soon as a more polar solute is introduced into the mobile phase, it is selectively adsorbed and partially deac€ivates the stationary phase so the forces acting on the solutes are all suddenly reduced to a new lower level. The situation is repeated when the third solvent, more polar still, is introduced into the mobile phase producing another step in the curves. These steps representing the different levels of molecular forces that hold each solute onto the adsorbent during the changes of solvent are shown in Figure 1. Now the forces acting on the solutes in the mobile phase are depicted by the heavy line, and it is seen that after an isocratic period of development ( s t ) a linear gradient is used with respect to solvents Q and R ( t u u w x ) . Below the curves, the chromatogram resulting from the gradient elution is shown: the retention volumes of each solute will be explained by discussing the various development processes that occur. Isocratic Process-Elution Development. After injection of the sample, it is seen that solute A has greater forces exerted on it by the mobile phase than the stationary phase, and thus is eluted immediately a t or close to the dead volume of the column. During the rest of the isocratic period between s and t, no further solutes are eluted as all the rest have significantly greater forces acting on them by the adsorbent than by the mobile phase. ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
749
1
SOLJTE I
, Figure 1.
i
5 1 DISPLI(LYEN1 EFfECl
I
2 ND O I S P L I C T Y E * T EffLCl
Diagram describing the “displacement effect” result-
ing from gradient elution using solvents of widely different polar-
ities
Gradient Process-The First “Displacement Effect.” On the introduction of the semi-polar solvent Q into the mobile phase, the nonpolar solvent P is immediately displaced from the adsorbent resulting in its partial deactivation and the forces between all the solutes and the adsorbent fall to a lower level. The adsorbent will be saturated with new solvent a t quite a low concentration in the mobile phase and, thus, this will occur during the passage of a relatively small volume between ( t ) and ( u ) . Scott and Lawrence (1) determined the adsorbtivity of isopropyl alcohol on silica gel as a function of the concentrations of the alcohol in heptane. They showed that the silica gel is virtually saturated with isopropyl alcohol when its concentration in the heptane is still only about 2%. Returning to Figure 1, it is thus seen that the solvent polarity, and thus the forces between solute and mobile phase, change little during the small volume of mobile phase eluted between ( t ) and ( u ) . However, because of the rapid deactivation of the adsorbent during this volume flow, the molecular forces between solutes B and C exerted by the adsorbent suddenly fall below the level of the forces between the solute and the mobile phase. Thus between t and u, both solutes B and C are eluted simultaneously or very close together and are unresolved. They have in effect been “displaced” from the adsorbent by the solvent Q together with the original solute P. For this reason this process can be considered as the “displacement effect” of the gradient elution. Gradient Process-The First “Elution Effect.” During the interval between u and u, the polarity of the mobile phase can be considered as increasing linearly from a concentration of about 2% v/v of solvent Q in solvent P to 100% solvent Q.During this interval, the forces exerted by the mobile phase on solutes D and E exceed those exerted by the adsorbent and thus solutes D and E are eluted (1) R. P. W. Scott and J. G. Lawrence, J . Chromatogr. Scf.. 8, 619
(1970).
750
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
from the column. Because the relative forces between the solutes and the two phases change gradually between u and u, as opposed to the “displacement effect” between t and u, and further, as the change takes place during the passage of a much greater volume of solvent through the column, solvents D and E are eluted discretely and well resolved. The elution of D and E results almost solely from the gradual change in polarity of the mobile phase and is thus called the “elution effect.” Gradient Process-The Second “Displacement Effect.” The second displacement effect is exactly analogous to the first, on the introduction of the polar solvent R, the semi-polar solvent Q is immediately displaced from the adsorbent which causes its further deactivation, and the forces between the remaining solutes and the adsorbent fall to a new lower level. This process occurs during the passage of a relatively small volume of mobile phase ( u to w) during which the forces between solutes F and G and the mobile phase exceeds that between these solutes and the adsorbent. Thus solutes F and G are displaced and eluted from the column close together and poorly resolved. This is the second “displacement effect” and results from the introduction of the polar solvent R. Gradient Process-The Second “Elution Effect.” The second elution effect commences after the second deactivation step is completed at u! and extends to point x where pure solvent R is flowing through the column. As the polarity of the mobile phase and thus the relative forces between the solutes H and I and the two phases change slowly during the stage wx, the solutes H and I are eluted discretely and well resolved. As this step results again largely from a change in polarity in the mobile phase alone, this stage can be called the second “elution effect .” Now, to separate an unknown mixture of solutes that can cover any part or all of the polarity range, the polarity of the mobile phase must be changed by use of a suitable gradient system from a nonpolar solute such as hexane to a highly polar solvent such as water. From the above discussion, it can also be seen that if this is attempted using a very limited range of solvents ( e . g . , 3 or 4), then the displacement effect a t each solvent change can result in poor resolution and very often no separation whatsoever. To obtain a satisfactory gradient program, a large number of solvents must be employed. Increasing the number of solvents reduces the magnitude of the deactivation steps, and minimizes the “displacement effect.” This will result in a gradual change in mobile phase polarity and adsorbent deactivation and allow solutes having small polarity differences to be effectively separated. The need for a multi-solvent system in gradient elution having been established, it remains to consider the individual properties of each solvent that will permit a rational choice of the solvent series to be used. Before considering the chromatographic properties of the solvents, however, there are certain practical limitations that must be taken into account. These limitations are as follows: Solvents must be readily available either pure or easily purified; solvents should be generally inexpensive; solvents should have low viscosities to ensure rapid mass transfer and thus adequate column efficiency; and solvents must be compatible with the detector employed. Preliminary experiments concluded that all solvents examined having medium polarity, e.g., polychlorinated hydrocarbons, nitroparaffins. esters, ketones, etc., absorbed strongly in the UV. The UV detector was, thus, not considered a suitable detector for use with a series of solvents that covered the complete polarity range. It was, therefore, decided to employ the wire detector, the perfor-
mance of which is independent of the chemical nature of the solvent and restricts the choice of solvent to only those that are reasonably volatile.
CHROMATOGRAPHIC PROPERTIES OF THE SOLVENT SERIES The forces holding a solute on the surface of an adsorbent are reflected in the value of its partition coefficient which in turn is a function of the excess free energy of the solute adsorbed on the solid. Due to competition between solute and solvent for the active surface, if the excess free energy of the solvent is significantly greater than that of the solute with respect to the adsorbent, the solute will no longer be adsorbed and will be eluted from the column. At a constant temperature, the retention volume of a solute will change linearly with the exponent of the excess free energy of adsorption. Thus, if the solvent series is to behave analogously to the Kelvin scale in temperature programmed gas chromatography, then the excess free energy of adsorption must increase linearly along the solvent series. The evaluation of such an ideal series of solvents based on the experimental measurement of excess free energies of adsorption would require an inordinately large amount of experimental work. It is, therefore, necessary to compromise between what is theoretically desirable and what is practically possible. The excess free energy of adsorption of a solvent, A G n will be a function of Kn the partition coefficient of solvent (n) between solvent ( n - 1) and the adsorbent for a given series of solvents. For a column of constant phase ratio and constant temperature, -AGn = R T In (ak’,), where ( a ) is the phase ratio of the column, k ’ n the capacity ratio of solvent (n)and k ’ , = K n / a and T i s the temperature. If the column is operated a t constant flow rate, then k ’ , = (Vn/Vo) - 1 = ( t n / t o ) - 1, where Vn and Vo are the retention volume of solvent ( n ) and the dead volume respectively and t , and t o are the retention time of solute (n)and the dead time, respectively. It follows that WAG, = R T l n { a [ ( t , / t o ) - l]}. Thus if a solvent series is chosen such that [(t,/to) - 11 for any solvent n measured in solvent (n - 1) is constant, then AG, measured in solvent ( n - 1) will also be constant. Hence for the solvent series (tn/to - l)(n-ll = hn’measured in solvent
(n-1)
A
constant (1)
Although this condition provides a rational basis for a choice of solvents it does little to indicate a useful and practical value for [(t,/to) - 11. From preliminary work with a number of solvents, it became apparent that the magnitude of the change in retention volume of a solute from solvent to solvent could form the basis for chosing a rational series of solvents. In order to restrict the number of solvents to practical limit, results indicated that the corrected retention volume of any solute ( A ) chromatographed in solvent n should be between two and three times that when chromatographed in solvent ( n 1). Thus for a given column operated a t constant temperature and flow rate.
+
i.e.
Akn‘/Ah(n+i)
’ A 2.5
In practice this means that the corrected retention volume of any solute is about halved each time the solvent is changed. Because of the limited choice of solvents avail-
able resulting from the practical constraints placed on them by column and detector considerations, the conditions imposed on the series by Equation 2 cannot be obtained precisely. However, as will be seen later, the conditions can be met sufficiently close to provide an extremely effective solvent series. The conditions given in both Equations 1 and 2 involve some function of A G , and as a result suffer the limitations associated with bulk property measurements. The distribution coefficient K which is a direct function of AG is a measure of the net forces holding the solute or in this case the deactivating solvent, on the adsorbent. These forces can be polar or nonpolar in nature and neither K nor A G differentiates between the two. Depending on the chemical nature of the solutes, two substances having the same partition coefficient in a given liquid/solid system may be held on the adsorbent, one by predominately nonpolar forces and the other by predominately polar forces. It follows that if the solvent series is to elute substances in order of increasing polarity, then the dispersion force effect on each solute must decrease progressively along the solvent series. Now the dispersion force effect of each solvent can be considered as approximately proportional to its molecular weight and thus a third condition of the solvent series will be. M n < M,,-I and M , - M , , - l , s constant, where M , and M n - l l are the effective molecular weights of solvents (n)and ( n - l), respectively. Hence the three conditions that have to be met for an effective solvent series are 1) ( t n i t o
- 1)(n-I) = hn’
- constant,
measured in solvent (n-1)
3)
M , - M+l)
constant
EXPERIMENTAL Capacity ratios were obtained using a 50-cm long column, 5-mm i.d., fitted with a septum injection system, and packed with Bio-si1 A silica gel having a particle diameter of 20-44 microns. The solvent pump was a Milroyal Model D and the detector a LDC Refractomonitor. In all experiments, a constant flow rate of 0.5 ml/min was employed. Using the same column at all times, the retention volumes of a range of practical solvents were measured using the solvents themselves as the mobile phase. The dead volume of the column was also measured using a non-adsorbed solute and the k’ value for each solvent determined. The first solvent used was heptane and a number of other solvents chromatographed and, from the values of k’ obtained, the second solvent was chosen. The k’ values of solvents chromatographed using the second solvent allowed the choice of the third solvent and so on. The results obtained from the solvents selected for use with incremental gradient elution are shown in Table I. DISCUSSION It is seen from Table I that the average value for k ’ , chromatographed in solvent ( n - 1 ) was 0.32 but individual values varied from a minimum of 0.14 for carbon tetrachloride in heptane to 0.51 for acetone chromatographed in methyl acetate. This range of values for k , had to be tolerated if solvents that conformed to the criteria previously discussed were to be used. It is also seen from Table I that the average value for A h ’ n / A k ’ ( n + l j was 2.33 but individual values ranged from a maximum of 3.34 to a , the value of h’ for solute minimum of 1.56 [where ~ k ’ is ANALYTICAL CHEMISTRY, VOL. 45, NO. 4 , APRIL 1973
0
751
Table I . Basic Solvents Used for Incremental Gradient E1utio.n. Values for k r ( n +1 ) and k r ( n + * l $ Determined in Solving n
Chlorcf o m
3
0.651
0.286
0
Ethylene Dichloride
4
1.800
3.750
3.233
0
2-Nltropropa"n
5
c.450
0.303
c:
Nltrmethane
6
2.15
0.612
3.148
0
Propyl Acetate
7
2.812
1.003
0.485
0
Methyl Acetate
8
1.162
3.565
0.232
3
Acetone
9
1.082
3.638
3.370
0
Ethanol
1;
1.483
:.E63
3.512
C
Methanol
11
1.161
3.812
0.402
0
water
12
1.463
0.882
0.355
Average Value Of
I I
I
! 2.34
3.22
3.34
' 2 . 4 4
12.06
2.06
1.72
1.56
11.96
12.48
0
2.48
k ' , " ) A '( n + l l
Table I I. Physical Properties of Solvent/Solvent Mixtures Used for Incremental Gradient Elution
\
5
3
0
0.684
752
0
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, APRIL 1973
LIKELY PEAK IDENTITY I SOUALANE
I5
2 ANTHRACENE
16 ACETYLSILICYLIC ACID
3 METHYL STEARATE
17 BENZOIC ACID
4
BENZWHLNWE
I8 t-BOC LEUCINE
0 CHLOROANILINE
10 1-BOC GLYCINE
6
OUININE
7
1
2 0 ALANINE
NITROANILINE
? p.UNITRWENZENE 8
p-NITROPHENOL
0
DlHIDllOCHOLESTEROL
21 GLUCOSE I
10 CATECHOL I 1 PHENACETIN
I 2 ADENINE
2
13 PHEHOLPHTHALEIN
I
n
EEOQ
Figure 2. Chromatogram of a mixture containing solutes of widely differing polarities using incremental gradient elution
A chromatographed in solvent n and A h ’ ( n + l is l t h e value of h’ for solute A chromatographed in solute ( n + l)]. It should be noted t h a t the ratio for water to methyl alcohol was obtained from the ratio of the k’ values for acetic acid in methyl alcohol and water, respectively. Consider a solute that was eluted a t a k’ of unity with water as the mobile phase, then its potential k’ value, (Z), if chromatographed using heptane as the mobile phase can be calculated from the following equation. 2 = ( , , h * ’ / ’ { h 2 ’ x) ( * h 2 ‘ / & 3 ’ ) x ( c h , ’ l c h 4 ’ ) x ...(3) Using the values for A k ‘ n / A k ‘ l n -1 , from Table I1 it can be seen t h a t Z = 2.34 X 3.22 x 3.34 X 2.44 X 2.06 X 2.06 X 1.72 x 1.56 x 1.96 x 2.48 x 2.48 = 8429. Thus the solvent series can cover a k’ range based on heptane as the mobile phase of about 104. If the actual 12’ range employed during the elution procedure is Y, then Y = 2.34 3.22 + 3.34 t. 2.44 2.06 2.06 + 1.72 1.56 1.96 2.48 + 2.48 = 25.6. Thus a mixture of solutes that contained substances that were eluted by heptane at one extreme t o water at the other would have the total analysis time effectively reduced by a factor of 8429125.6 = 329 compared with the theoretical analysis time if separated on heptane alone. In the series, the solvent carbon tetrachloride is allowed t o raise the mean molecular weight of the eluant t o 154 and for the subsequent 10 solvents, the molecular weight of the eluant was arranged to fall linearly t o a molecular weight of 18 for water in accordance with condition 3. The increment of decrease in molecular weight from solvent to solvent was 13.6 and to achieve this, mixtures were made u p using solvents previously employed in the series to achieve the correct mean molecular weight. The basic solvents and their properties together with the composition of the actual solvents used are shown in Table 11. It is seen that only solvents 3, 4, 5, 6, and 7 needed t o be mixtures, the molecular weight of the remaining solvents being sufficiently close to the required value to be acceptable. Where solvent mixtures were employed to obtain the desired mean molecular weight, the new more polar solvent introduced in the mixture, was always maintained in excess of 25%. This ensured that the polarity effect of the
+
+ +
+
+
OAK MOSS RESIN OIL
+
SANCAL WOOD
OIL
Figure 3. Separation of two resin oils using incremental gradient
new solvent was maintained a t a sufficiently high level to achieve the necessary deactivation of the adsorbent and the elution of the appropriate solutes. Examples of Use of Solvent System. The apparatus employed was that previously described for use with incremental gradient elution (2) and the solvent system suggested in this paper was employed t o separate a complex mixture, containing a t the extremes, the solutes squalane and glucose. The apparatus also included additional reservoirs for the solvents, ethanol, acetone, ethyl acetate, and dichloroethane which were used sequentially a t the end of each run for reconditioning the column. The elution and the reconditioning programs ensured t h a t the column was (2) R P W Scott and P Kucera, J Chromatogr Sci in press
ANALYTICAL CHEMISTRY, VOL. 45, NO. 4, A P R I L 1973
0
753
automatically reconditioned for the next analysis. The solutes separated and the operating conditions are included with the chromatogram shown in Figure 2. The choice of flow rate, solvent period and mixing vessel volume depends on the column characteristics and the solvents employed, and these three variables together condition the concentration profile of the mobile phase entering the column. The choice of the values of these three variables is critical and their effects on the resulting separation will be discussed elsewhere. It is seen from the chromatogram in Figure 2 that twenty-one individual peaks are shown and that an effective separation is achieved for a mixture containing solutes having widely different polarities. The time taken for the separation was seven hours which might be thought to be excessive but it should be remembered that during the separation a total range of 8026 k’ values is being developed which is equivalent in isocratic terms of about 19 k’ values per minute. The actual k’ values employed in this development were about 26 and thus the chromatogram was developed a t an actual rate of one k’ value every 16 minutes. The separation of synthetic mixtures is, however, of academic interest and serves only to demonstrate the scope of the system. To demonstrate its applicability to unknown complex samples of the type often met with in analytical laboratories, two resins used in the perfume industry were separated on the same column using the identical conditions employed in the separations of the synthetic mixture. The samples examined were sandal wood resin oil and oak moss resin oil. two quite different samples containing quite different compounds. The results obtained are shown in Figure 3. An effective and useful separation was immediately obtained demonstrating the
754
ANALYTICAL CHEMISTRY, VOL. 4 5 ,
NO. 4 , APRIL 1973
efficiency of the solvent system and the apparatus as a whole.
CONCLUSION It is possible to choose a series of solvents for incremental gradient elution on a rational basis to provide a solvent system that can be used to separate mixtures containing solutes of diverse polarities. The present series of twelve solvents is practical and will provide effective and useful separations, but it is possible that the polarity steps between some solvents may be still excessive for ideal gradient development. It may be necessary, ultimately, when more experience with the system has been gained, to increase the total number of solvents to sixteen or perhaps even twenty. Once the solvents are chosen, the gradient conditions, particularly with respect to column flow rate, solvent .period, and mixing vessel volume, need careful study if the solvent series is to be used with columns of different types and geometries. Such studies are presently being carried out. It is interesting to note that all separations described in this paper were carried out on a column having only 150 theoretical plates. The use of the solvent system with columns of high efficiency, specially designed stationary phases and supports, and operated a t high pressures provides exciting possibilities for the future.
ACKNOWLEDGMENT The authors would like to thank B. Buglio for help in the development of the solvent series. Received for review November 29, 1972. Accepted January 8, 1973.
