2424
Anal. Chem. 1987, 59,2424-2428
Statistical Approach to Solvent Selection as Applied to Two-Dimensional Thin-Layer Chromatography David Nurok* and Sohrab Habibi-Goudarzi Department of Chemistry, Indiana University-Purdue University at Indianapolis, P.O. Box 647, Indianapolis, Indiana 46223
Robert Kleyle Department of Mathematical Sciences, Indiana University-Purdue University at Indianapolis, P.O. Box 647, Indianapolis, Indiana 46223
A statlstkal approach to the gelectlon of solvent systems for two-dknendonal thtn-layer chromatography Is presented. Two functions are used as separation criteria for evaluating 28 two-dimensional solvent systems for the separation of each of 100 subsets of either six or ten steroids, randomly selected from a set of 15 steroids. This approach allows a probability to be asslgned to each solvent system of its being highest ranked for separating any given subset of steroids. This probability varies from 0% to 49% for the solvent systems Considered; it Is influenced by slze of the subset considered and, to a lesser extent, by the selection of separatlon criterion used.
Computer-aided optimization of two-dimensional thin-layer chromatography (TLC) allows the rapid evaluation of a large number of combinations of solvent systems for the two sequential developments. The first report on this topic was by Gonnard and co-workers (2) who introduced two functions for evaluating the quality of a two-dimensional separation of dinitrophenyl amino acids. Ten solvents of fixed composition were considered, which corresponds to 45 possible combinations of solvent for the two developments. Computer-aided optimization has also been used for selecting operational parameters for continuous development two-dimensional TLC with binary solvents of variable composition (2, 3 ) . We have reported on the separation of 15 steroids in which 13 binary mixtures were considered as candidate solvents ( 3 ) . This corresponds to 78 sequential combinations of solvent systems, each of which requires optimization of binary solvent composition. Visual evaluations together with two functions, similar to those introduced by Gonnord and co-workers, were used as criteria to rank the 78 possible combinations of solvent systems. These criteria gave somewhat different ranking of the systems. The highest ranked system was a development on silica gel using butyl acetate/toluene in conjunction with a development on bonded CI8 using 2,2,2-trifluoroethanol/water. This system was ranked first by two of the criteria and fourth by the remaining criterion. The ranking appears meaningful because there is generally a good agreement in spot pattern between the computer-simulated chromatograms and experimental chromatograms. There are however some differences in actual spot positions. The above approach allows the best combination of solvent systems to be identified for separating a given mixture of solutes. I t does not however provide insight as to how the ranking of combinations of solvent systems is affected by the nature of the solute mixture being separated. A statistical approach to answering this question is described in this report. This approach with suitable modification, should also be
Table I. List of Steroids 17a-acetoxyprogesterone
hydrocortisone
androstenediol androstenedione androsterone 7-dehydrocholesterola 176-estradiol estrone ethisterone
lanosterol
mestranol 11-deoxycortisol spironolactone stigmasterol testosterone
This compound yields two spots on TLC. The larger spot was measured. applicable to other forms of chromatography including high-performance liquid chromatography.
EXPERIMENTAL SECTION This report is based on experimental data that has been previously published (3). The steroids used were purchased from the Sigma Chemical Co. (St. Louis, MO) and the solvents used were purchased from the Aldrich Chemical Co. (Milwaukee,WI). The TLC plates were a gift from Whatman Chemical Separations, Inc. (Clifton,NJ). These were K5 silica gel plates, catalog number 4850-820, and KCIBreverse-phase plates, catalog number 4801-800. The silica gel plates were heated at 90 "C for 30 min and then maintained at a relative humidity of 60% until immediately before use by storage over an aqueous 39% sulfuric acid solution in a desiccator. The plates were developed in the Regis SB/CD chamber.
RESULTS AND DISCUSSION It is obvious, in any mode of chromatography, that the selection of an optimum solvent system (or systems) is dependent on the nature of the solute mixture being separated. Even within a single class of compounds, however, it is often difficult to predict which solvent(s) should be used for separating a given mixture. Choice is usually made on general principles of solvent selection described in textbooks such as ref 4 and 5 or else is based on separations of other mixtures of the same solute class. This report describes a statistical approach to the above problem. Data are obtained for one set of compounds which is divided into a large number of subsets. The information obtained allows quantitative predictions to be made as to which solvent system would be best suited for separating any given subset of these compounds. If the original set of compounds is sufficiently large, the information may possibly be useful for predicting the identity of optimum solvents for solutes that are of the same class as the set, but which are not included within it. This preliminary study is restricted to two-dimensional TLC. Data were available for 15 steroids listed in Table I using the 13 binary solvent systems listed in Table 11. This corresponds to 78 possible combinations of solvent systems for each of the developments in two-dimensional TLC. This
0003-2700/87/0359-2424$01.50/0C 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
Table I1
2425
Table 111. List of 28 Solvent Systems Evaluated Solvents Used as Binaries with Toluene on Silica Gel acetonitrile ethyl formate butyl acetate ethyl propionate 2-butanone nitromethane diethyl ether tetrahydrofuran ethyl acetate
Solvents Used as Binaries with Aqueous 0.5 M Sodium Chloride on Bonded CIS acetone methanol acetonitrile 2,2,2-trifluoroethanol
includes dual phase separations where the first development is performed on silica gel and the second development is on bonded CIS. Dual-phase plates are commercially available. In order to reduce the number of computations, this list was arbitrarily shortened to the 28 solvent systems, listed in Table 111, which are the highest ranked solvent systems for separating the steroids listed in Table I, using the PRF and IDF (see below) as separation criteria. The number of subsets, S, of size m in a set of n solutes is
S=
n! m!(n- m)!
For a set of 15 steroids, there are 3003 subsets of ten and 5005 subsets of six steroids.
COMPUTATIONS The distance migrated by a solute in continuous development TLC, with a binary mixture of solutes is
MD =
1 1 + exp(a In X,
l2 - 21x0 + K t l
+ b)
solvent systema
modeb
acetonitrile/acetonitrile acetonitrile/butyl acetate acetonitrile/methanol acetonitrile/2,2,2-trifluoroethano1 butyl acetate/acetonitrile butyl acetate/2,2,2-trifluoroethanol butyl acetate/tetrahydrofuran 2-butanone/acetonitrile 2-butanone/butyl acetate 2-butanone/2,2,2-trifluoroethanol diethyl ether/acetonitrile diethyl ether/methanol diethyl ether/2,2,2-trifluoroethanol ethyl acetate/acetone ethyl acetate/acetonitrile ethyl acetate/methanol ethyl acetate/2,2,2-trifluoroethanol ethyl formate/acetonitrile ethyl formate/2,2,2-trifluoroethanol ethyl propionate/acetonitrile ethyl propionate/2,2,2-trifluoroethanol nitromethane/acetonitrile 2,2,2-trifluoroethanol/acetone 2,2,2-trifluoroethanol/acetonitrile tetrahydrofuran/acetone tetrahydrofuran/acetonitrile tetrahydrofuran/methanol tetrahydrofuran/2,2,2-trifluoroethanol
NP/RP NP/NP RP/ RP NP/RP NP/RP NP/RP NP/NP NP / R P NP/NP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP NP/RP RP/RP RP/RP NP/RP NP/RP NP/RP NP/RP
'Normal phase solvent is a binary with toluene. Reversed phase solvent is a binary with aqueous 0.5 M sodium chloride. * N P = normal phase TLC on a silica gel layer; R P = reversed phase TLC on a bonded C,, laver.
The inverse distance function (IDF) sums the inverse distance between all pairs of spots in a chromatogram b-1
where a and b are empirically determined constants for each solute, X,is the mole fraction of the strong component in a binary mixture of solvents, I is the solvent path length, x o is the spotting distance, K is the solvent velocity constant, and tl is the analysis time. The spot position of each solute in two-dimensional TLC can be computed by using its MD value for each of the two developments as planar coordinates. The value of 8.3 cm was used for the path length 1 in the computations. This corresponds to the solvent path length that was used for the original data. The value for the analysis time tl was selected such that the most rapidly migrating spot reached the end of the TLC plate. In the previous use of eq 2 for the computer-aided evaluation of two-dimensional TLC, the value of X , was increased over a range of 36 increments of 0.02. This corresponds to 1296 computations for each solute in a combination of two-dimensional solvent systems. The computation that yielded the highest value of either the IDF or P R F (see definitions below) was used for subsequent ranking. In order to decrease the number of computations, the increment size was increased to 0.05. This decreased the number of computations for each solute in a combination of solvent systems to 225. A comparison of the two increment sizes was made by ranking all 78 possible combinations of the solvent systems in Table 11, using both the IDF and P R F (see definitions below) as separation criteria. Only minor differences in ranking were found between the two increment sizes. The highest ranked solvents were identical for both increment sizes. The chromatogram was evaluated a t each mole fraction combination by using the following two functions as evaluation criteria. There is generally a good agreement between visual evaluation of a chromatogram and computer evaluation using these functions (3).
b
4
(3) S D is
the separation distance between spot centers. S D is set at 1 mm for pairs of spots separated by 1 mm or less in order to prevent the value of the function from being extremely large for very close spots and being undefined for overlapping spots. Optimization is effected by minimizing the value of the IDF. The IDF is based on D g , a function proposed by Gonnord and co-workers (I),which sums the inverse of the square of the distances. The planar response function is defined as k-1
b
(4) where SDswis a specified separation distance. Pairs of solutes with separations greater than SDsPec are assigned separations of SDSpec. Thus only pairs separated by less than SDSPec contribute to the PRF. The value of SD8pec could, in principle, be a function of the plate area available and the number of compounds separated, such that the value of the PRF would never be zero. In the present study SDsPec was arbitrarily assigned values of 15 and 30 mm, respectively, for the subsets of ten and six. These values are sufficiently large that the P R F does not have a value of zero for any of the two-dimensional systems. The PRF is based on the chromatographic response function which was used by Morgan and Deming (6) in a simplex optimization of a gas chromatographic separation. The latter function is in turn based on a function introduced by Kaiser (7). The P R F approaches zero from a negative direction as the quality of a separation improves. Ideally these separation functions could b e used to first optimize and then rank each of the 28 solvent system com-
2426
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
Table IV. Steroids
2. Values
According to the PRF for the Three Highest Ranked Solvent Systems for Separating Subsets of Ten
solvent systema
modeb
first 25 subsetsC
second 25 subsetsC
value third 25 subsetsc
butyl acetate/2,2,2-trifluoroethanol butyl acetate/acetonitrile
NP/RP NP/RP NP/RP
3.1 6.4 9.0
2.5 5.4 7.2
2.7 6.6 8.6
tetrahydrofuran/acetonitrile
fourth 25 subsetsc 2.2 6.9 8.8
all 100 subsetsd overall ranking 2.6 6.3 8.4
1 2 3
Normal phase solvent is a binary with toluene. Reversed phase solvent is a binary with aqueous 0.5 M sodium chloride. NP = normal phase TLC on silica gel layer; RP = reversed phase TLC on a bonded C,, layer. Subsets are randomly selected from a population of 3003. dThe four groups of 25 subsets constitute the 100 subsets. Table V. Steroids
P Values
According to the PRF for the Three Highest Ranked Solvent Systems for Separating Subsets of Six
~~
solvent systema
modeb
first 25 subsetsC
butyl acetate/2,2,2-trifluoroethanol butyl acetate/acetonitrile ethyl acetate/2,2,2-trifluoroethanol
NP/RP NP/RP NP/RP
7.8 9.0 7.8
second 25 subsetsc 8.1 12.0
6.6
i; value third 25 subsetsc
fourth 25 subsetsC
5.0 7.3 9.6
5.2 8.3 10.2
all 100 subsetsd overall ranking 6.2 8.2 9.9
1
2 3
Normal phase solvent is a binary with toluene. Reversed phase solvent is a binary with aqueous 0.5 M sodium chloride. NP = normal phase TLC on a silica gel layer; RP = reversed phase TLC on a bonded C,, layer. Subsets are randomly selected from a population of 5005. dThe four groups of 25 subsets constitute the 100 subsets. Table VI.
P
Values and Overall Ranking of Solvent Systems According to the IDF for Subsets of Ten and Six Steroids solvent system”
modeb
butyl acetate/2,2,2-trifluoroethanol butyl acetate/acetonitrile diethyl ether/acetonitrile
NP/RP NP/RP NP/RP NP/RP RP / RP RP / RP RP/RP
acetonitrile/acetonitrile 2,2,2-trifluoroethanol/acetonitrile 2,2,2-trifluoroethanol/acetone
acetonitrile/ methanol
i;
subsets of ten steroids valueC overall ranking 2.9 5.9 9.7 14.1 16.2 22.8 23.1
1 2 5 14 18 27 28
f
subsets of six steroids valueC overall ranking 6.1 7.6 11.6 14.2 14.5 19.7 17.4
1 2 8 14 16 27 21
NP = normal a Normal phase solvent is a binary with toluene. Reversed phase solvent is a binary with aqueous 0.5 M sodium chloride. phase TLC on a silica layer; RP = reversed phase TLC on a bonded C1, layer. ci; value is for 100 randomly selected subsets. binations for each of the 3003 subsets of ten solutes and each of the 5005 subsets of six solutes. This would consume a considerable amount of computer time due to the large number of computations for each subset. This number was reduced by sampling 100 randomly selected subsets for each of the two subset sizes. The solvent systems for each of the subsets were ranked from 1 to 28 according to the two evaluation criteria. This is referred to as q ranking in the context of this paper. Each solvent system was assigned an average value P=
I n
-Cqr n1=1
(5)
for the given solvent system and given subset size, where n is the number of subsets of steroids considered. The T values for subsets of ten range from 2.6 to 23.1 depending primarily on the solvent system used and to a very much lesser extent on whether the IDF or PRF is used as an evaluation criterion. These P values are used to determine the overall ranking of solvent systems in the range of 1 to 28. Table IV lists the three best solvent systems for subsets of ten steroids according to values of T with n of 25 or 100, using the PRF as a ranking criterion. For a given solvent system the groups of 25 subsets have similar 7 values. This justifies the random sampling of subsets. There is some fluctuation in the 25 subset averages within each individual solvent combination, but this variability (s2 = 0.42 with 9 degrees of freedom) is significantly less, at the 1% significance level, than
the variation among the 100 subset averages for the three different solvent systems in this table (s2 = 8.5 with 2 degrees of freedom). A similar analysis based on all 28 solvent systems leads to the same conclusion. In a similar manner, Table V lists the three best solvent systems for subsets of six steroids. While the differences among the P values for “all 100 subsets” are less substantial than those in Table IV, it should be noted that the ordering of the two highest ranked solvent systems is the same in the two tables. The number of steroids in a subset affects the i; values and can affect the overall ranking as is shown in Table VI which ranks solvent systems according to the IDF. The same two solvent systems have the highest overall rankings for subsets of six and ten. Inspection of the table shows that all solvent systems do not have the same overall ranking for the two sizes of subset. There is a substantial difference in 7 values between the two subset sizes for the highest ranked solvent system in Table VI. The value of 2.9 for subsets of ten, compared to the value of 6.1 for subsets of six, illustrates that the selection of a solvent system for a satisfactory separation becomes more critical as a mixture of solutes becomes more complex. However, the correlation between the average ranks for ten and six steroid subsets over all 28 solvent pairs is 0.949 which is quite strong. Spearman’s rank correlation (8), which is computed by using the overall ranks, is also 0.949. The IDF and PRF yield somewhat different 7 values and overall ranking of solvent systems, as is shown in Table VII,
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
Table VII.
P Values
2427
and Overall Ranking of Solvent Systems According to the IDF and PRF for Subsets of Ten Steroids PRF
IDF
solvent system"
modeb
F valueC
overall ranking
butyl acetate/ 2,2,2-trifluoroethanol
NP / RP NP/RP NP/RP NPjRP NPjRP RPjRP RPjRP
2.6 6.3
1 2 3 4
butyl acetatejacetonitrile tetrahydrofuran/acetonitrile
diethyl ether/acetonitrile ethyl acetatejmethanol 2,2,2-trifluoroethanol/acetone
acetonitrile/methanol
I
8.4 9.7 15.1 21.6 22.1
F valueC
overall ranking 1 2 11
15
2.9 5.9 11.6 9.7 13.9
27
22.8
28
23.1
5 13 21 28
ONormal phase solvent is a binary with toluene. Reversed phase solvent is a binary with aqueous 0.5 M sodium chloride. *NP = normal Dhase TLC on a silica gel laver; RP = reversed uhase TLC on a bonded C,, laver. ' 7 value is for 100 randomlv selected subsets.
XI
I!
Flgure 2. Frequency distribution of q ranking according to the IDF for 100 subsets of six steroids for the system butyl acetate/toluene (silica gel)-aqueous 2,2,2-trifluoroethanol(bonded C,J. Flgure 1. Frequency distribution of q ranking according to the IDF for 100 subsets of ten steroids for the system butyl acetate/toluene (silica gel)-aqueous 2,2,2-trifluoroethanol (bonded CIJ.
for subsets of ten steroids. However, for this subset size, solvent systems rank highest (1 or 2) or lowest (27 or 28) irrespective of whether the IDF or P R F is used as a criterion of overall ranking. The system tetrahydrofuran/tolueneaqueous acetonitrile is included in the table to illustrate that relatively large discrepancies between the two ranking criteria are possible. However, the discrepancy of eight in the overall ranking for this system is atypical since 25 of the 28 solvent systems differ by 2 or less in the overall rankings. The correlation between the average ranks for the IDF and P R F is 0.947 for all 28 solvent pairs, while the correlation between overall ranks (i.e. Spearman's rank correlation) is 0.958. A similar statement is true for subsets of six. The P value for a given solvent system is relatively insensitive to whether the P R F or IDF is used as a ranking criterion for a given subset size. Figure 1 shows the frequency distribution of q ranking according to the IDF for the highest ranked solvent system, butyl acetate/ toluene-aqueous 2,2,2-trifluoroethanol, for the separation of subsets of ten steroids. The frequency distribution is fairly narrow. A similar plot is found for the frequency of q ranking according to the PRF. Figure 1 shows that when the IDF is used as a separation criterion, this is the best solvent system for separating 49 of the 100 subsets of steroids. It is reasonable to expect that this would be the best solvent system for about 50% of the 3003 possible subsets of ten. The plot also shows that this solvent system is unsuitable for a few of the subsets of ten. I t ranks 13th out of the 28 solvent systems for the two subsets that are least suited for separation by this solvent system. The solvent system, second in ranking according to the IDF for the separation of subsets of ten steroids, is butyl acetate/tolueneaqueous acetonitrile. The frequency distribution of this system is wider than that of the highest ranked system
shown in Figure 1. This is the best system for separating 15 of the 100 subsets but is a poor system for several of the subsets being ranked 21st for one of these. It is interesting to note that the same system was very highly ranked for separating a mixture of 30 steroids of which the 15 considered in this study is a subset (2). Figure 2 is for the same solvent system as in Figure 1, but with subsets of six instead of ten. This results in a wider frequency distribution illustrating that the identity of the solvent system becomes less critical as the complexity of a solute mixture decreases. I t is the highest ranked solvent system for 20 subsets of six as compared to 49 subsets of ten. It is a poorly ranked solvent for several of the subsets and is 24th for the subset that is least suited for separation by this solvent system. The lowest ranked of all 28 solvent systems for the separation of subsets of ten steroids is aqueous acetronitrileaqueous methanol. While it is the worst solvent for 27 of the 100 subsets, it is nevertheless the best solvent system for one of the subsets. Clearly a good separation of a single mixture of solutes is no guarantee that a solvent system will be useful for separating other mixtures of the same class of solutes. A solvent system ranked higher by q ranking according to a particular separation criterion will yield a better separation than a solvent system with a lower ranking. It should however be emphasized that for simple mixtures, a solvent system with even a low ranking may provide an adequate separation. In the case of a complex mixture, even the highest ranked solvent system may provide an inadequate separation. The probability of obtaining a complete separation of a mixture of solutes can be estimated for each of the two-dimensional systems by assigning a value to SDsPec corresponding to a complete spot separation. A complete separation is then found when the P R F has a value of zero.
CONCLUSION For the 15 steroids listed in Table I, a random sampling of the 3003 subsets of ten or the 5005 subsets of six indicates:
2428
Anal. Chem. 1987, 59,2428-2432
that probabilities of highest ranking can be estimated for all two-dimensional systems considered, according to the size of the subset, and using either the PRF or the IDF as a ranking criterion; that r values are substantially influenced by the size of the subsets considered but that in most cases are not substantially influenced by whether the PRF or IDF are used as ranking criteria; that the identity of either the very highest ranked or very lowest ranked solvent systems for separating subsets of a given size is not affected by which function is used as a ranking criterion although the ranking of intermediate solvent systems is influenced by which function is used. A similar study is currently being performed with the same two-dimensional systems but with a different mixture of steroids, as well as with other classes of compounds. This statistical approach to the selection of separation systems should be applicable, with suitable modification, to solvent selection in one-dimensional TLC, to stationary phase selection in gas chromatography, and to solvent selection in column liquid chromatography. It should apply to any class of compounds for which there is an empirical relationship
between solute capacity factor and solvent composition.
LITERATURE CITED (1) Gonnord, M.-F.; Levi, F.; Guiochon, G. J . Chromatogr. 1983, 264. 1-6. (2) Johnson, E. K.; Nurok, D, d . Chromatogr. 1984, 302, 135-147. (3) Steinbrunner, J. E.; Johnson, E. K.: Habibi-Goudarzi, S.; Nurok. D. I n Planar Chromatography; Kaiser, R. E., Ed.; Heuthig Verlag: Heidelberg, 1986; Vol. 1: p 239. (4) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; Wiley: New York, 1979. (5) Poole, C. F.; Schuette, S. A. Contemporary Practice of Chromafogra phy; Elsevier: Amsterdam, 1984. (6) Morgan, S. L.; Deming, S. N. J . Chromatogr. 1975, 772, 267-285. (7) Kaiser, R. E. Gas Chromatographie; Geest 8 Portig: Leipzig, 1960; p 33. (8) Hollander, M.; Wolfe. D. A. Nonparametric Stafistical Methods ; Wiley: New York. 1973. ~
RECEIVED for review December 9, 1986. Resubmitted June 2, 1987. Accepted June 2, 1987. This work was supported by a grant from the Dow Analytical Laboratories University Support Program. The TLC plates were a gift from Whatman Chemical Separations, Inc.
Preparation and Evaluation of Slurry-Packed Capillary Columns for Normal-Phase Liquid Chromatography Franca Andreolini, Claudio Borra, and Milos Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Effective procedures have been developed to pack fused slllca (Capillary) columns with 5-pm particles of slllca adsorbent and other polar materials (diol-, cyano-, and amlnobonded phases). Mlcrocdumns exhlbltlng overall efflclencles between 70 000 and 90 000 theoretical plates were prepared. The reduced plate heights, separatlon Impedance, and sample capaclty of such columns were further evaluated.
The sample complexity often encountered with biological, environmental, and technologically important nonvolatile mixtures demands high-efficiency separation techniques. Consequently, the interest in microcolumns of capillary dimensions for use in liquid chromatography (LC) has grown rapidly during the last several years. The column technology of highly efficient and stable slurry-packed capillary columns is a subject that deserves primary attention. While several reports have now been published concerning the preparation and evaluation (1-5) of reversed-phase, slurry-packed capillary columns, virtually no investigations have been performed on the packing techniques and quantitative aspects of normal-phase columns with similar dimensions. Straight-phase separations encompass adsorption chromatography on silica gel and alumina (although the latter is rarely used) and partition chromatography on polar chemically bonded phases. Some desirable features of normal-phase chromatography are (a) the ability to obtain a class separation selectively, (b) the capability of resolving certain isomers, which are of great significance in the analysis of natural products and in pharmaceutical chemistry, (c) the ability to separate hydrophilic species that cannot be easily retained 0003-2700/87/0359-2428501.50/0
in the reversed-phase systems, (d) the ability to differentiate solutes based on the differences in hydrophilic rather than hydrophobic structure, (e) compatibility of organic phases with molecules that have either low stability or aggregation problems in aqueous phases, and (0 the availability of a wide range of stationary-phase selectivities. All these features make normal-phase liquid chromatography with high-efficiency capillary columns quite attractive. The use of capillary columns, characterized by flow rates of 1-3 ML/min, allows the routine use of expensive, ultrapure mobile phases, which are demanded for a reliable practice of adsorption chromatography. In addition, “exotic” mobile phases (e.g. deuteriated or chiral solvents) could also be used in this type of chromatography. And, moreover, the lower polarity, higher volatility, lower gas expansion volumes and wetting characteristics of organic mobile phases, combired with a low flow rate, confer an advantage in coupling straight-phase capillary columns to mass spectrometers, infrared instruments, and the detection techniques that require solvent elimination. This communication describes the packing procedures developed for slurry-packed capillary columns, using silica gel and polar chemically bonded stationary phases of the diol, cyano, and amino types. Markedly different packing procedures for each material had to be employed in order to obtain optimum results. Performance of the prepared columns has been evaluated through the reduced plate height vs. reduced velocity plots and the separation impedance values. In addition, reproducibility of the packing procedures and the column loading capacities has been assessed.
EXPERIMENTAL SECTION Column Packing. After a porous PTFE frit was fixed at the
column end ( 6 ) ,fused silica capillaries (PolymicroTechnologies, 0 1987 American
Chemical Society
