258
Anal. Chem. 1984, 56,256-262
short operation time. The results of UV-visible spectrometry on purified pigments reveal lack of purity control in many of the published works. In particular, we have shown that not only the sample purification but also the proper choice of a solvent (benzene), causing little epimeric interconversion (3), is essential for obtaining reliable CD data. ACKNOWLEDGMENT The authors are grateful to K. Iriyama, The Jikei University School of Medicine, for valuable comments on Chl separation. Registry No. Chla, 479-61-8;Chla', 22309-13-3;Cub, 519-62-0; Chlb ', 22309-14-4;Pheoa, 603-17-8; Pheoa', 75598-38-8;Pheob, 3147-18-0; Pheob', 75498-61-2; benzene, 71-43-2; diethyl ether, 60-29-7; acetone, 67-64-1. LITERATURE CITED (1) Kllmov, V. V.; Dolan, Ed.; Shaw, E. R.; Ke, 8. Proc. Nati. Acad. Sci. U.S.A. 1980, 77, 7227-7231. (2) Hynnlnen, P. H.; Slevers, G. Z. Nafurforsch., 6: Anorg. Chem., Org. Chem. 1981, 366, 1000-1009. (3) Hynninen, P. H.; Wasielewski, M. R.; Katz, J. J. Acta Chem. Scand., Ser. 6 1979, 633, 837-6413. (4) Schwartz, S. J.; Woo, S. L.; von Elbe, J. H. J . Agric. Food Chem. 1981.29, 533-535. (5) Seely, G. R. I n "Primary Processes of Photosynthesis"; Barber, J., Ed.; Elsevier: Amsterdam, 1977; pp 1-53. (6) Strain, H. H.; Svec, W. A. I n "The Chiorophylls"; Vernon, L. P., Seely, G. R., Eds.; Academic Press: New York, 1966; pp 21-66. (7) Svec. W. A. I n "The Porphyrins"; Dolphin, D., Ed.: Academic Press: New York, 1978; Vol. V, pp 341-399. (8) Eskins, K.; Scholfield, C. R.; Dutton, H. J. J. Chromatogr. 1977, 735, 217-220. (9) Shoaf, W. T. J . Chromatogr. 1978, 752, 247-249. (10) Irlyama, K.; Yoshlura, M.; Shiraki, M. J . Chromatogr. 1978, 754, 302-305. (11) Stransky, H. 2.Nafurforsch., C: 6iosci. 1978, 33C, 836-840.
(12) Schoch, S. 2.Naturforsch., C: 6iosci. 1978, 33C, 712-714. (13) Braumann, T.; Grimme, L. H. J. Chromatogr. 1979, 770, 264-268. (14) Iriyama, K.; Shikraki, M.; Yoshlura, M. J. Li9. Chromatogr. 1979, 2 , 255-276. (15) Falkowski, P. G.; Sucher, J. J . Chromatogr. 1981, 273, 349-351. (16) Braumann, T.; Grimme, L. H. Biochim. Biophys. Acta 1981. 637, 8-17. (17) Shioi, Y.; Fukae, R.; Sasa, T. 6iochim. Biophys. Acta 1963, 722, 72-79. (18) Shioi, Y.; Sasa, T. Biochim. Blophys. Acta 198S, 756, 127-131. (19) Strain, H. H.; Thomas, M. R.; Katz, J. J. Biochim. Biophys. Acta 1963, 75, 306-311. (20) Fong, F. K.; Koester, V. J. 6iochim. Biophys. Acta 1976, 423, 52-64. (21) Sauer, K.;Llndsay Smlth, J. R.; Schultz, A. J. J. Am. Chem. SOC. 1966, 88, 2681-2688. (22) Hynnlnen, P. H. Z. Nafurforsch., 6: Anorg. Chem., Org. Chem. 1981, 368, 1010-1016. (23) Scholtz, B.; Ballschmiter, K. J . Chromatogr. 1981, 208, 148-155. (24) Trurnltt, H. J.; Colmano, G. 6iochim. 6lophys. Act8 1959, 37, 434-447. (25) Jeffrey, S.W.; Humphrey, G. F. Blochem. Physioi. Pf/anz. 1975, 767, 191-194. (26) Goedheer, J. C. I n "The Chlorophylls"; Vernon, L. P., Seely, G. R., Eds.; Academic Press: New York, 1966; pp 147-184. (27) Smith, J. H. C.; Benitez, A. I n "Modern Methods of Plant Analysis"; Peach, K., Tracey, M., Eds.; Springer: Heldelberg, 1955; Vol. 4, pp 142-1 96. (28) Zscheile, F. P.; Comar, C. L. Bot. Gaz. 1941, 702, 463. (29) Seely, G. R.; Jensen, R. G. Spectrochim. Acta 1985, 21, 1835-1845. (30) Holt, A. S.; Jacobs, E. E. Am. J . Bot. 1954, 4 7 , 710. (31) Vernon, L. P. Anal. Chem. 1960, 32, 1144-1150. (32) Houssier, C.; S a w , K. J . Am. Chem. SOC. 1970, 02,779-791. (33) Prokhorenko, I.R.; Lobachev, V. M.;Kutyurin, V. M. Zh. Obshch. Khim. 1976 46, 2147-2151. (34) Weiss, C. I n "The Porphyrins": Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. 111, pp 211-223. (35) Petke, J. D.; Maggiora, G. M.; Shipman, L.; Christoffersen, R. E. Photochem. Phofobioi. 1979, 30, 203-223.
RECEIVED for review August 3, 1983. Accepted October 3, 1983.
Characterization of Reversed-Phase Liquid Chromatography Columns with Retention Indexes of Standards Based on an Alkyl Aryl Ketone Scale Roger M. Smith Department of Chemistry, University of Technology, Loughborough, Leicestershire LE11 3TU, United Kingdom
The advantages of using a retention Index scale based on the alkyl aryl ketones for reporting retentlons in HPLC are dlscussed and a method is presented to characterize the varlations in the retentlon propertles of reversed-phase liquid chromatography column-eluent combinatlons by using the retentlon Indexes of a set of reference compounds, toluene, nitrobenzene, p-cresol, and 2-phenylethanoi. These compounds were selected, by using multlvarlant analysls, to glve the optimum discrlminatlon between eluents and columns. The method Is compared with alternative methods using capacity factors and selectivity ratios and enables the specific column lnteractlons to be studied.
The rapid spread of high-performance liquid chromatography has largely been due to the availability of stable alkyl-bonded reversed-phase column materials. These materials are prepared by a number of manufacturers (l), but each may use different bonding reactions for the alkyl groups, different
shapes and porosities of silica, and different reactions to cap unreacted silanol groups (2). As a consequence nominally identical columns (e.g., ODs-silicas) from different sources may give markedly different retentions (3-5). Even with the same method, there are batch to batch variations, which together with unannounced changes in manufacturing methods have given rise to concern about column reproducibility (6). These differences between columns also make interlaboratory comparisons difficult and thus limit the use of HPLC techniques in Official and Standards methods as well as hindering studies of the mechanism of retention. A range of techniques have been used to compare different bonded phases, including pyrolysis-GLC (7), but most methods are based on the reversed-phase separation of a test mixture. The capacity factors (iz? or selectivities (a)are then used for manufacturers' quality control (8-10) or to compare different materials (lI,12), although as discussed later many of these tests are inappropriate and may be misleading. More fundamental studies have used nitrobenzene in hexane to test for unreacted silanols (13) or determined the physical pa-
0 1984 American Chemical Society 0003-2700/84/0356-0256$01.50/0
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
rameters of the column such as porosity (14). Most of these retention studies are based on capacity factors k’ = (t - to)/to but there has been a realization in recent years that accurate determination of t o is difficult (15-17). This can cause considerable eirors if k’ < 1 and the reliability of many of the reported abbolute values of k’is uncertain. Although a number of .methods have been proposed for the accurate measurement pf to including deuterium oxide (Is), salts such as sodium nitrate (16),polar nucleosides (In,or extrapolation methods ( I @ , none has gained wide acceptance or is universally employed. Recently two alternative methods for reporting retentions have been proposed wbich use either the alkyl aryl ketones (19) or alkan-2-obes (20) to form retention index scales. Although both methods are based on capacity factors, as will be seen later, indexes are insensitive to uncertainties in the value of toand to small changes in solvent composition. Both scales are based on the linear relationship 1 between the capacity factors and number of carbon atoms (C,) in the standards (21). log
k’ = UC, x 100 + b
The retention indexes of samples (I)are then determined by interpolation log
k’ (sample) = uI
+b
(2)
using eq 2 in the same manner as Kovats’ indexes in GLC. The retention of a compound in chromatography depends on its interaction with both phases and can be expressed by eq 3, in which K is the distribution ratio between the mdbile and stationary phases and a is the phase ratio.
k’= Ka
(3)
The distribu\ion ratio is determined by the cumulative interaction of each component (x)of the compound with each phase (j and k ) according to the Martin equation (22). X
log K =
C f(x - j , k )
(4)
Thus the capacity factor and retention indexes (from eq 2 and 3) can be related to the interactions by eq 5 and 6. log k’ = log a
I =A
+ Cf(x - j,k)
+ BCf(x - j,k)
The latter can be subdivided into interactions betweeh the mobile phase (m)or stationary phase (s) with alkyl (CH,) and functional groups (3t) in the sample (7).
I = constant
+ Cf’(CH, - rn) + Cf’(CH, - s) + Cf’b - rn) + Cf’b - s)
(7)
In reversed-phase systems the major contribution to retention comes from the solvophobic (hydrophobic) interaction of the sample with the mobile phase, and the stationary phase plays a largely passive role. Because of its low polarity, the interaction with the alkyl chains (CH,-s) should be negligible. However, because of the unprotected silanol groups on the stationary phase, it possesses small but significant “specific” interactions (23) with functional groups represented by ( x s). Therefore with the same mobile phase, differences in column materials and hence in ( x - s) should be reflected by changes in retention indexes. In many HPLC studies this last term is ignored if the same column is used throughout but is an essential factor in intercolumn studies and prompted Halasz to note that “It is extremely dangerous to discuss the mechanism of chromatography with reverse-phase without defining with great accuracy the quality of stationary phase used” (24).
257
The present paper describes the establishment of a practical system for the characterization of column/eluent combinations by the comparison of the retention indexes of a selected set of standards and its application to a number of nominally identical chromatographic systems. It extends earlier studies in which columns with different bonded groups (2-2, C-18, (2-22, and phenyl could be differentiated (25, 26). By use retentipn indexes rather than capacity factors the interactions are normalized to the influence of the methylene group. The same intention was achieved by Tanaka et al., who compared different mobile phases on a single column by adjusting the eluent composition to give the same methylene selectivity (27). In order to compare the magnitude of the interactions, hexane-water can be used as a reference system, the differences in retention indexes between it and the test chromatograms giving column retention constants similar to the Rohrschneider constants in GLC. The method potentially forms an alternative method for determining interaction indexes for functional groups to that proposed by Jandera, Colin, and Guiochon (28).Recently this study has led to a comparison of k hpLcand k iLE for hexadecane/water (23). An extension of this study has unfortunately described these interaction indexes as “retention indexes” (29) which could cause confusion with conventional usage. EXPERIMENTAL SECTION Chemicals. Solvents for chromatography were HPLC grade (Fisons,Loughborough, Leics., U.K.). Alkyl aryl ketones and test compounds were laboratory grade. The retention index standard solution was prepared by mixing 25 or 50 pL of the alkyl aryl ketones in methanol (10 mL), which was diluted 100 pL to 10 mL with the mobile phase. The solutions of test compounds were made up with 1-10 pL in 100 mL of eluent. Equipment. HPLC separations were carried out with a Pye Unicam PU 4010 Pump, Altex 153 absorbance detector at 254 nm and 0.08 aufs, and Hewlett-Packard 3390 integrator. Tenmicroliter samples were injected by use of a Rheodyne 7125 valve and separations were carried out at ambient temperature. Shandon Southern columns were slurry packed with commercially available stationary phases. The value of t owas determined by using a solution of sodium nitrate. Retention Indexes. Retention indexes were calculated as ref 19, using triplicate values of k’ and a least-squares routine. Multivariant analysis, principal components and hierachical cluster, was carried out by using GENSTAT V routines (Lawes Agricultural Trust) (30) at the University of Manchester, England. RESULTS AND DISCUSSION Determination of Retention Indexes. To establish a method for the practical characterization of a chromatographic system (column/eluent combination), it is desirable to gain the maximum discrimination between systems with the minimum number of measurements. It is therefore necessary to determine which reference test compounds are most sensitive to changes in column or eluent but also to avoid duplication by identifying those test compounds which yield unique information. In the previous study, which: compared columns with different bonded groups, six reference compounds were proposed (toluene, phenetole, nitrobenzene, methyl benzoate, benzyl alcohol and p-cresol) (25). These compounds were chosen as the aromatic equivalents of the standards used by Rohrschneider (31)and McReynolds (32) so that they could be detected by ultraviolet spectrometry. Subsequently, it was decided to replace benzyl alcohol by 2-phenylethanol, which had also been measured earlier, as the retention times of the former compound were often much shorter than those of the retention index standards. Although amines are particularly sensitive to uncapped silanol groups, their chromatography is very critically dependent on pH, metal ions on the column
258
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
surface, and water content of the mobile phase and thus they would have only limited applicability (33). In order to be able to test the ability of the method to distinguish between nominally identical bonded phases, eight octadecylsilyl bonded silica columns were chosen for the study. They include two very different materials: ODs-Zorbax, a fully capped spherical phase, and ODS-Partisil 10, a low coverage uncapped irregular phase as well as three ODSHypersil columns which would be expected to be very similar, two batches of 5 pm and one of 3 pm material. The efficiences of the columns were not compared as this should not effect the relative retentions. The capacity factors of the six reference compounds and the retention index standards were measured by use of three eluents, 7030 methanol-water, 5050 acetonitrile-water, and 4050 tetrahydrofuran-water, which have similar elution strengths (33) (Table I). The capacity factors for each sample were calculated by use of sodium nitrate to determine toand varied markedly between columns. However for each system there was a linear relationship between log k' and carbon number of the index standards. For each mobile phase the slopes of the relationships were very similar confirming that the methylene group selectivity (aCH2)is primarily dependent on the hydrophobic interaction with the eluent. By use of these results the retention indexes were calculated (Table 11). In order to confirm the insensitivity of retention indexes to errors in the accurate value of to,the results for the ODS-Hypersil column (HI) with 70% methanol-water were recalculated assuming towas f15%. The indexes of the standards showed only minor changes (less than 1%)for the different to values whereas the capacity factors changed markedly (e.g., toluene I = 1044 and 1044 and p-cresol I = 803 and 797). The slope of the standard curve also changed suggesting that earlier small differences in slopes could be due to inexact values for to. Detailed examination of the retention indexes showed that the values for each standard were much more consistent than the capacity factors and changing the eluent produced systematic changes in indexes. In particular the indexes of 2phenylethanol were markedly lower with acetonitrile-water whereas those of p-cresol were highest in tetrahydrofuranwater. The order of elution of methyl benzoate and nitrobenzene reversed on changing from methanol-water to tetrahydrofuran-water as also noted by Tanaka et al. (27). For some standards the ODS-Zorbax and ODS-Partisil columns showed larger specific changes in indexes compared to the other columns which can be attributed to differences in specific column interactions. Differences of this order between columns were also observed by Baker during an interlaboratory (and hence intercolumn) collaborative study of the reproducibility of retention indexes based on the alkan-%one scale (34). Optimization of Reference Standards. Because of the difficulty of analyzing the variations in the indexes of reference standards in order to identify which compounds were most significant as indicators of differences in retention properties and also to recognize which provide essentially the same information, the data were examined by a multivariant analysis. This technique has previously been employed in the examination of GLC stationary phases both to identify phases with essentially similar properties (35) and to identify the standards which characterize phase differences (36) as in the present study. Before the analysis was carried out, it was desirable to examine the expected types of interaction of the reference standards. In his studies on the properties of compounds frequently used as solvents, Snyder concluded that the interaction of a solvent was based on its overall polarity and
on three selective factors, proton donation, proton acceptance, and dipole interactions (37). On the basis of the original data from Rohrschneider, he classified 65 compounds into seven interaction groups on the basis of related properties. With this table it can be determined that the alkyl aryl ketones (represented by acetophenone) (group VIa), cresols (VIII), and 2-phenylethanol (IV) fall into different groups, whereas toluene, nitrobenzene, and phenetole are in the same group (VII). Methyl benzoate was not in the original table but aliphatic esters are in the same group (VIa) as the aryl ketones. Snyder suggested that as three interaction factors were involved it should be possible to define any system with three standards from different interaction groups and a retention index scale (37). Following the same argument it has been successfully demonstrated that considerable flexibility in elution order can be obtained with a ternary solvent system (based on three distinctly different groups) and water as an overall polarity modifier (38). These conclusions suggest that a liquid chromatographic system can be characterized by using a limited range of standards as long as each responded to a particular interaction. In order to test the discrimination of the reference compounds in the present study, a multivariant factor analysis (39) was carried out on the indexes from all three eluent systems which should reveal those standards most sensitive to differences in the mobile phase composition. The comparison of all the sets of data from Table I11 showed that 95% of the variances in the indexes of the standards could be expressed by the two principal components, whose major contributions were derived from p-cresol and 2-phenylethanol, respectively (Table 111). The remaining components contributed less than 5% to the overall differences. A graphical representation of the scores of the indexes on the two principal components showed that the three eluent systems were fully resolved (Figure 1). As expected from the hydrophobic model of reversed phase separations, the differences in the eluent had a greater influence than the differences between the columns, although clearly there was a greater variation in indexes from the methanol-water system than from the tetrahydrofuran-water system. The individual contributions of the standards to the principal components also showed that the influence of methyl benzoate to the overall discrimination was very small (Table 111). From this analysis it can be seen that an overall separation system (column/eluent) can be best characterized by measuring the polar interactions expressed as the indexes of p cresol and 2-phenylethanol but that the dominant influence on the separation is caused by the differences in the mobile phase. In order to compare the different column packing materials, it is therefore necessary to study each mobile phase separately to remove the effects of the eluent-sample interaction. When the indexes of the standards were analyzed (Table IIIb, c, and d), it could be seen that in each case toluene was the major contributor to the first two principal components and that phenetole and nitrobenzene showed a similar effect in the same direction. Again methyl benzoate made only a minor contribution to the discrimination. It was concluded that methyl benzoate therefore has little diagnostic value and should not be used as a reference compound because it responds to the same interactions as the retention standards. This agrees with its tentative assignment to the same Snyder interaction group as the alkyl aryl ketones. The influences of toluene, nitrobenzene, and phenetole were similar and as they are in the same interaction group it was felt that possibly only one should be needed. Reexamination of the data using only limited sets of reference compounds confirmed this conclusion. It was therefore decided that
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
h -
a:E -
;$O
CN
n
259
260
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
_____1_1___-___---1l___________l__
Table 11. Retention Indexes of Reference Compounds on ODs-Columns retention indexes PhNO, MeOBz
l _ s _ l l _ _ _ _ _ l _ l l l l _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
eluent/column 70:30 MeOH/H,O HI H,
? S
T Z P 50:50 MeCN/H,O HI H,
E3
S T
Z P 40:60 THF/H,O HI H,
E3
S T Z P
PhMe
PhOEt
PhEtOH
p-cresol
1062 1043 1070 1055 9 88 1046 1040 965
1025 1009 1026 1013 96 1 1014 1005 948
862 859 857 86 9 838 875 843 84 9
90 9 903 910 913 895 902 906 902
783 779 779 758 743 754 702 767
810 799 794 77 7 759 776 703 7 53
1027 1015 1030 1026 1009 1019 1027 984
1003 997 1005 988 997 998 968
873 878 874 874 873 880 859 870
891 884 888 8 90 889 890 887 881
6 94 701 6 94 691 697 692 673 696
76 0 768 76 6 747 745 751 719 720
1001
1088 1067 1076 1083 1051 1069 1086 1042
1042 1029 1034 1043 10 28 1033 1041 1035
913 918 921 927 924 926 922 956
888 884 887 897 888 893 888 893
727 744 741 717 726 738 713 745
885 882 88 5 873 873 87 7 860 88 9
Table 111. Factors Calculated with Principal Components Analysis of the Retention Indexes of Six Reference Standards eluent a all sets
compo- weightnent inga 76.1 14.2 8.2 66.6 30.3
1 2
3 b 70:30 MeOH/H,O
1 2
3
1.8
c 50: 50 MeCN/H,O
1 2
67.8 30.3 1.3 66.0 20.2 10.6
3 d 40:60 THF/H,O
1
2 3
contribution from reference standard PhOEt PhNO, MeOBz PhEtOH
-______l___ll__l_ I _
PhMe 0.35 -0.02 0.75 0.62 -0.46 -0.17 0.51 -0.55 -0.08 0.71 0.23 -0.50
0.29 -0.10
0.46 0.49 -0.35 -0.01 0.45 -0.32 -0.04
0.16 -0.16 -0.45
0.36 -0.37 -0.27 -0.17 -0.02 -0.97 0.13 0.29 -0.78 -0.46 -0.52 -0.53
0.15 0.90 -0.06 0.30 0.58 -0.03 0.75 0.50 -0.04 -0.43 0.6 3 -0.15
-0.01
0.21 0.15 0.07 -0.05 -0.07
0.09 -0.08 -0.53 -0.02 -0.21 -0.24
p-cresol 0.80 0.05 -0.36 0.49 0.58 -0.12 0.71 0.50 0.30 -0.25 0.45 -0.43
a Percent of overall variance attributed to this comnonent. Components 4-6 have been omitted because they contribute so little (< 4%) to overall variance. - - - - - - - - - - -_- - I _-_-_- - I _- - - - -_ 1 _ _ 1 _ _ 1 _ _ _ _ _ _ _ 1 1 _ 1 _ 1-_ _ _ _ _ _ _ _ _ _
_---___-__-1______________11_______11__1__1-11--_--1--1--
Table IV. Factors Calculated with Principal Components Analysis of the Retention Indexes of Four Reference Compounds contribution to component from each reference standard eluent component weighting PhMe PhNO, PhEtOH p-cresol a all
1
2 3 4 b 70:30 MeOH/H,O
c 50: 50 MeCN/H,O
1 2
3 4 1 2
3 d 40:60 THF/H,O
4 1
2 3 4
77.2 15.1 6.8 0.9 66.2 30.5 2.3 1.0
67.4 31.i 1.3 0.3 69.1 20.5 8.1 2.1
0.3 5 0.02 0.93 -0.13 0.57 -0.79 0.16 0.15 0.46 0.78 -0.16 -0.40 0.72 0.28 0.60 -0.20
0.38 0.40 -0.26 -0.79 0.19 -0.06 -0.98 0.00
0.19 -0.26 -0.94 0.01
-0.47 -0.46 0.73 -0.17
0.17 -0.92 -0.09 -0.36 0.45 0.48 0.06 0.75 0.17 -0.51 0.09 -0.84 -0.43 0.66 0.00
-0.62
0.84 -0.01
-0.25 0.48 0.66 0.38 0.10
-0.64 0.85 -0.25 0.28 0.36 0.26 0.52 0.33 0.74
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984 * 261 Cpt
2
MeOH
Flgure 1. Comparison of column-eluent combinations. Plot of values of first and second components from the muitivariant analysis of the retention indexes of six reference compounds separated on eight columns (key, Table I) with 7030 MeOH-H,O, 5050 MeCN-H,O, and 40:60THF-H,O
as eluents. phentole could be omitted as it duplicated the information available from the other compounds. Nitrobenzene is also unnecessary in the present study but has been retained because in earlier work with phenyl bonded silica columns it appears to demonstrate a specific charge transfer interaction (26, 40). The principal component analyses were then repeated by using only toluene, nitrobenzene, 2-phenylethanol, and p-cresol as standards (Table IV) and the results were very similar to those obtained with six standards. The discrimination between the column materials can be demonstrated by a plot of the scores of the indexes on two principal components for each eluent (Figure 2). As expected the three ODs-Hypersil columns were very similar in each case and the ODs-Partisil and ODs-Zorbax columns differed markedly from each other and from the other columns. The four reference compounds also enabled the different column/mobile phase combinations to be discriminated in almost exactly the same way as previously and informal examination of the results from the previous study (25) clearly showed that these reference compounds would be equally applicable to the comparison of columns with different bonded groupings. The sets of indexes for the reference standards can therefore be used in two ways. Firstly by use of the same eluent they can be used to compare the specific interactions of different columns in order to recognize which columns have closely similar properties and which are markedly different. It should then be possible to classify columns in the same way as the McReynolds constants are used in GLC and enable columns with different bonded phases to be related and for the effect of different eluents to be studied. The second function of the constants is that they can potentially be used to define the elution conditions required for a particular separation, as a set of indexes or column retention
constants, which should be independent of a specific make or brand of packing material. Small variations in eluent composition can then be used to compensate for differences in overall retentions and together with the use of retention indexes to define sample retentions should enable methods to be readily defined in Standard Methods of Analysis. The contributions of the standards to the principal components showed some differences between the mobile phases but as all the standards appeared to be providing significant information it is felt that they probably represent the minimum set required to give good discrimination between the columns. Because the values of the contributions in Table IV are applicable only to this set of data, they cannot be used for further additional columns and therefore to describe the specific interactions of a column the retention indexes would have to be quoted. Alternatively the indexes themselves can be compared to standard values based on the hexane-water partition and expressed as column retention constants (25). Comparison with Related Studies. The present study confirms the assumption that compounds in the same interaction group will respond in similar ways to changes in columns and that most discrimination between columns will be obtained by the comparison of compounds from different groups. In particular homologous and nonpolar compounds will show small relative changes because their retentions are primarily influenced by the hydrophobic interactions with the mobile phase. However, the standards used in many previous comparison studies have often been closely related, including naphthalene/biphenyl (8),naphthalene/anthracene (9),and anthracene/phenanthrene (8, 11), or homologues such as dimethyl and diethyl phthalates (9, 11), benzoic and toluic acids (9, l l ) ,and benzene and toluene (12). Although Goldberg found a wide range of capacity factors for the last three of these pairs on different columns, the selectivity ratios showed only small variations even when columns with different
ANALYTICAL CHEMISTRY, VOL. 56, NO. 2, FEBRUARY 1984
262
Cpt
a)
2
E,B
!
P; P
:.:: s
::.:
MeOH-
Water
secondary amine (11)). In a few cases more appropriate comparisons have been made such as between androstenedione/testosterone (8,9) and methyl benzoate/anisole (1I) in which compounds expected to be in different Snyder interaction groups were used. A number of authors have also used a proposed ASTM test mixture, benzaldehyde, acetophenone, methyl benzoate, dimethyl terphthalate, benzyl alcohol, and benzene, to demonstrate separation on a column (9). However the first four compounds are from the same interaction group and should behave in the same way on changing conditions. In the present studies the first three have almost constant indexes (respectively 760 (19),800, and 890 so that in effect they create an “index scale” with constant differences against which the last two compounds can be compared.
z LITERATURE CITED Cpt
b)
2
6 81 I
MeCN-Water
xz
-68
I
Cpt 2
(I/ -68
I
TH F-W a t er
Flgure 2. Comparison of columns using the same eluent: plot of values of first and second components from the multivariant analysis of the retention Indexes of four reference compounds separated on eight columns (key, Table I) using respectively (a) 7090 MeOH-H,O, (b) 5050 MeCN-H,O, or (c) 4030 THF-H,O as eluent. bonded phases from C-1 to phenyl were compared (11). The most sensitive pair of the compounds he studied was caffeine and theophylline (also studied by Gonnet (12)),relatively polar compounds with different functional groups (tertiary and
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RECEIVED for review June 22, 1983. Accepted October 10, 1983.
