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A graphical method for the determination of retention times in column liquid-solid chromatography from thin-layer chromatographic data
Jounta~ of Chruma~ogrQphy* :1x (1975) 91-95 0 ?Bevier ScieritiSc Publishing Compzmy, Amsterdam -
A grqshkd Iiquid=-soEd
method for &romatopraphy
the determtn2tEon from thin-kzyer
(First received July 14th. 1975; revised immscript
Printed
in The Netkrhicls
of retentiorn times in colcmm chromatograpfric d&2
received October
lst, 1975)
The advantages of thin-layer chromatography (TLC), stressed by 2 number of workers (e.g., refs. I-4), have frequently been utilized to estimate retention behaviour 2nd to choose 2nd optimize solvent systems in column chromatography+-“. Early investigators did not take into account fimdzment21 dltierences betwsen TLC 2nd column chromatography 2nd thus ivere limited to approximate, qualitative ch2mcteristics of rhe solvent-adsorbent system. More recently, Schfitt 2nd Geiss3s4 derived equations thhatpermit the use of TLC as 2 pilot technique for column chromato_g2phy on a quantitative basis. Moreover, they also defined the experimental conditions that are necessary to secure analo,~ between TLC 2nd column chromato-
@2phy-
The equations derived by Schlitt 2nd Geiss” require the determination of cer‘&in auxiliary parameters 2nd the calculations are time consuming. Therefore, in this work we have attempted to simplify the procedure by the application of a graphicai correlation method, using principles employed esrrlier in a method for the determination of suit2ble solvent systems for coiumn liquid-liquid partition chromatography or countercurrent distribution from paper chromarographic data” and in a comparison of TLC parameters in two difierent experiments I3. The gr2phical correlation method permits the comptison of I2rge sets of experimental data (see 2lso 2 recent paper by Coq er aAis in which grrrphical correlation in another coordinate system is employed). In order to secure linear relationships 2nd 2 wide range of k’ values (k’ = amount of solute adsorbed/amount of solute in the mobile phase), it is advantzgeous to use 2 logarithmic scale for c2pacity f2ctors. Therefore, the R_Wvzlue is csed 2s the TLC parameter : RF.f = log
i -
R
RF
= log
kiTLc,=
f+r4cTLC)
log k” + log c/-
f
For column chromatorz;phy from the equation leg k;,,,
=
iog
W SCiXC)
(CC), the logarithm of the capacity factor is calculated
r, - 1; f” R
=
log k” f
log
WA wx3
Vs (CC)
NOTES
92
folfows from eqns. I 2nd 2 tfiztfor identicd sohentsystems used in the two p same adsorbent of identical a&vi%), the techniques (the same developirg solvent, th, ., k” values are identical and the diEerence in k’ values in TLC 2nd column chromatography results fro-m diEerences in the ratios of the weight of the adsorbent arid +&e volume of the solvent: WA/V,. If the ratio is identicaa2in both methods and the migration rates of solutes are described by eqns. I and 2, then R,< = Iog k’,,,, and the corre!ation line of log kcco versz(.sI?, passes through the origin, the slope being 1.0. Xn the opposite case, when the W,/E’S ratios are digerent for the two methods, then the straight correlation line is shifted (vertically and horizont2hy) in the logarithmic scale by It
To tesr the gaph_ical method, several orgartic nitrogen bases were chromatographed using thir, layers and columcs of silica gel.
EXPERIMENTAL
Column chromatography was carried out using a DuPont Model 830 liquid chromatograph with a UV detector. A steel column, 50 cm x 2.1 mm L-D., was packed with wide-pore silica (average pore diameter 120 A, particle size 1%501*m, produced by Polish Reagents, Ghwice, Polaod). Small portioss of silica (dried at 140” for 2 h) were introduced into the tube, the column being tapped 50 times against a wooden bench between each addition. Reageat-grade solvents dried uver molecular sieve 13X were used. The column was developed with binary solvent mixtures; solutions of single solures (3-lO$ of 2.50/o_w/v) were injecred after equilibration of the column. The experiments were carried out at 25 i I’, the flow-rate being kept constant at 0.7 mi/min. Ascending TLC was carried out using 10 x 20 x 3O-cm glass tanks lined with ‘fiber-paper. Glass plates, I8 i: 29 cm, were covered with O.25-mm layers of the same sihca used in the co!umn experiments. The samples (solutions in acetone or methanol) were spotted 2 cm from the edge and dried with a stream of warm air so as to prevent adsorption of water vapcur. The solvents, prepared as in column chromatography, were poured into the tanks and after conditioning in the vapour (3CMO min) the plates were deveIoped to a distance of 16 cm. The spots were reveaied with DragendcrE reagent (KBiI,) or bisdiazotized benzidine. RESULTS
AND
OlSCiJSSfON
The results are presented in Figs. i-3. For practical reasons, double scales are marked OI? the two coordinates: on the RAtIaxis a subordinate scale of RF values’5S’6 (which can be found directiy from the thin-layer chromatograms) and the relative retention time 1,/r: on the log k’ axis. The plot thus allows clie to estimsrte relative retention tines (or relative retention volumes, V,/ Vi) directly from experimentally determined bi, values.
93
NOTES
1.4
L -0.4
~-_-(J2 OJ i7.0
0.2 43
t0.s
02 +0.4
0.4
1.8.
702
0.6
RF
-0.2
Rt.+
-+02
Fig. 1. Cor_r&6~n of lo_grithms of ~paciiy ratios obtained by TLC (abscissa) sod coitrmn chrom2tography (ordinate). System: si!icz+thyI acetzte (10 moL”?) f cyclohexane. Solutes (Figs. l-3): aniline (A) and its 2-nitro-(ZNA), Gnitro- (4NA) and 3,4dimethyl- (XMA) derkztiues; l-amino(IAN; and 1,5-diulrincnaphthaiene (I5AN); 1,2-diaminobenzene (12AB); quinoline (Q); 6-nitroquinoiine (@IQ); a-naphthoquino!ine (aNQ); and acridine (ACR).
The equations of the correlation
lines determined by the least-squares method
2re : Fig. I (20 mole-% ethyl acetate): Iog k&c, = 0.98R,, f 0.34 Fig. 2 (IO mole- % ethyl methyl ketone) : Iog k& = 1_10 R,, i 0.17 Fig. 2 (60 mole- y/,ethyl methyl ketone) : log k&-, = I .07 &?%I t 0.25 Fig. 2 (both compositions) : log k& = 0.99 R,, f 0.19 Fig. 3 (10 and 38 mole- 7: ethyl acetate): log k&-, = 1.01 RA,c-+ 0.22 The stopes of the correlation lines are thus close to unity, in accordance with theoreticat expectation. The lines are shifted from the origin by ra. 0.2-W unit, which could have been due to higher rados of adsorbent weight to solvent vo!tnme in column chromatography or other causes, slrch as difFerences in adsorbent activities in the two techniques, kinetic e!Tects, demiting of the solvents and non-equilibrkm conditions in TLC. It is worth mentioning that linear correlations are observed even for different compositions of the developing solvent of $ given type (Figs. 2 and 3) and even for different solvent systems (the last Cwo correlation equations for Figs. 2 and 3 are virtually ideAcz1). III the theoretical considerations, idealized chromatographic processes were assumed; in reality, the re!ationships can be distorted by additional effects, such as demking of the solvents and non-ecpi!ibrirrm conditions in TLC. It has been demonstrated” that under comparable conditions (identicaf adsorbent, conditioning of
1
1.6 i-O.2
/
e
Ace
ethy_y!mecAy! ke;oC : (S) f Fig. 2. As Fis. 1, with deveiopin, = co!veat _ nob % S; dased circles, @I mob % S.
0.3 7lD
-0s
02
03
+0.5
f 0.4
0.4 )
i.6 i
‘IAN
0.5 -016
+02
0.6
i7F RM
cycioherrure. Open circles, 10
Nm33
95
the plates in TLC, use of sutiiciently large tanks satmated r&h solvent vapour) the mechanisms of adsorption processes in TLC and column chromatography are identical or very simiIar_ For adsorbents of the same type difFeri;ngin specific surface arez (ao,,,, a,,,,) the correfation be &o&2 be shifted by !og (qrrc,/qcc,)_
Thanks are due to Docent Dr. Jerry S. Kowakzyk, Head of the Laboratory of LiqrriciChromatography at the Institute of Industrial _4nalysis, Polytechnic University, Gdazisk, Poland, for valuable discussions dming ihe experimental work and for permission to use the equipment of the Laboratory. REFERENCES i 2 3 4 S 6 7 8 9 IO 11 12 I3 14 !S 16 17
R. _AXIOS.zsd S. G. Perry, J. Chranmogr., 83 (1973) 245. R. M. Scott, J. Ckroamogr. Sci., 11 (1973) 129. H. S&&t and F. Geis, 4. Ckrmzarogr., 67 (1972) 261. F. Geiss, Parameter dcr Diinnsck~ch~-~kronia~ogra~k~~, Vieweg, Brzttinschweig, 1972. K. D&m and &I. Ftichs, ZfeIv_ Ckinz. AC&, 45 (1952) 261. A. G. Nettisg, J. Cfrromzrogr., 53 (197G) 507. G. R. Duncan, J. Ckrumatogr., 8 (1962) 37. W. Gdkiewicz 2nd T. Wawrzynowicz, Ckromctogrcpkia, 3 (1970) 356. A. Stdyhwo and 0. S. Privet& J. Ckromarogr. Sci., iI (i973) 20. G. Ghenini 2nd E_ Cemi, in T. BP-UP, r;nd G. Gheisini (Editors), E_xmzciion Ckromarograpky, AkzdCmizl KY&b and Ekevier, Buditpest and Amsrerdzm, 1975, Ch. 14, p. 366. I. B. Ruppet, L. F. Cunco and J. G. Krsruse, J. Ckem. Educ., 43 (1971) 635. E. Soczewikki, Advaz. Ckroniarogr., 8 (1969) 91. L. R. SEyder, Adran. Ckrom~~rogr., 4 (1967) 3. B. coq, 3. P. Nhlas, A. L.xr!otte 2nd M. Porti-Imlt, J. ChfoJnLzrogr_,97 (197&) 137. E. Soczewtiki end W. GoIkiewicz, Ckromarograpkia, S (1979 4331. E. Sacze~~~ki znd W. Gdkiewicz, Ckromatograpkia, 6 (1973) 269. W. G&ciewicz, Ckromatograpkicz, irz press.
A grqshkd Iiquid=-soEd
method for &romatopraphy
the determtn2tEon from thin-kzyer
(First received July 14th. 1975; revised immscript
Printed
in The Netkrhicls
of retentiorn times in colcmm chromatograpfric d&2
received October
lst, 1975)
The advantages of thin-layer chromatography (TLC), stressed by 2 number of workers (e.g., refs. I-4), have frequently been utilized to estimate retention behaviour 2nd to choose 2nd optimize solvent systems in column chromatography+-“. Early investigators did not take into account fimdzment21 dltierences betwsen TLC 2nd column chromatography 2nd thus ivere limited to approximate, qualitative ch2mcteristics of rhe solvent-adsorbent system. More recently, Schfitt 2nd Geiss3s4 derived equations thhatpermit the use of TLC as 2 pilot technique for column chromato_g2phy on a quantitative basis. Moreover, they also defined the experimental conditions that are necessary to secure analo,~ between TLC 2nd column chromato-
@2phy-
The equations derived by Schlitt 2nd Geiss” require the determination of cer‘&in auxiliary parameters 2nd the calculations are time consuming. Therefore, in this work we have attempted to simplify the procedure by the application of a graphicai correlation method, using principles employed esrrlier in a method for the determination of suit2ble solvent systems for coiumn liquid-liquid partition chromatography or countercurrent distribution from paper chromarographic data” and in a comparison of TLC parameters in two difierent experiments I3. The gr2phical correlation method permits the comptison of I2rge sets of experimental data (see 2lso 2 recent paper by Coq er aAis in which grrrphical correlation in another coordinate system is employed). In order to secure linear relationships 2nd 2 wide range of k’ values (k’ = amount of solute adsorbed/amount of solute in the mobile phase), it is advantzgeous to use 2 logarithmic scale for c2pacity f2ctors. Therefore, the R_Wvzlue is csed 2s the TLC parameter : RF.f = log
i -
R
RF
= log
kiTLc,=
f+r4cTLC)
log k” + log c/-
f
For column chromatorz;phy from the equation leg k;,,,
=
iog
W SCiXC)
(CC), the logarithm of the capacity factor is calculated
r, - 1; f” R
=
log k” f
log
WA wx3
Vs (CC)
NOTES
92
folfows from eqns. I 2nd 2 tfiztfor identicd sohentsystems used in the two p same adsorbent of identical a&vi%), the techniques (the same developirg solvent, th, ., k” values are identical and the diEerence in k’ values in TLC 2nd column chromatography results fro-m diEerences in the ratios of the weight of the adsorbent arid +&e volume of the solvent: WA/V,. If the ratio is identicaa2in both methods and the migration rates of solutes are described by eqns. I and 2, then R,< = Iog k’,,,, and the corre!ation line of log kcco versz(.sI?, passes through the origin, the slope being 1.0. Xn the opposite case, when the W,/E’S ratios are digerent for the two methods, then the straight correlation line is shifted (vertically and horizont2hy) in the logarithmic scale by It
To tesr the gaph_ical method, several orgartic nitrogen bases were chromatographed using thir, layers and columcs of silica gel.
EXPERIMENTAL
Column chromatography was carried out using a DuPont Model 830 liquid chromatograph with a UV detector. A steel column, 50 cm x 2.1 mm L-D., was packed with wide-pore silica (average pore diameter 120 A, particle size 1%501*m, produced by Polish Reagents, Ghwice, Polaod). Small portioss of silica (dried at 140” for 2 h) were introduced into the tube, the column being tapped 50 times against a wooden bench between each addition. Reageat-grade solvents dried uver molecular sieve 13X were used. The column was developed with binary solvent mixtures; solutions of single solures (3-lO$ of 2.50/o_w/v) were injecred after equilibration of the column. The experiments were carried out at 25 i I’, the flow-rate being kept constant at 0.7 mi/min. Ascending TLC was carried out using 10 x 20 x 3O-cm glass tanks lined with ‘fiber-paper. Glass plates, I8 i: 29 cm, were covered with O.25-mm layers of the same sihca used in the co!umn experiments. The samples (solutions in acetone or methanol) were spotted 2 cm from the edge and dried with a stream of warm air so as to prevent adsorption of water vapcur. The solvents, prepared as in column chromatography, were poured into the tanks and after conditioning in the vapour (3CMO min) the plates were deveIoped to a distance of 16 cm. The spots were reveaied with DragendcrE reagent (KBiI,) or bisdiazotized benzidine. RESULTS
AND
OlSCiJSSfON
The results are presented in Figs. i-3. For practical reasons, double scales are marked OI? the two coordinates: on the RAtIaxis a subordinate scale of RF values’5S’6 (which can be found directiy from the thin-layer chromatograms) and the relative retention time 1,/r: on the log k’ axis. The plot thus allows clie to estimsrte relative retention tines (or relative retention volumes, V,/ Vi) directly from experimentally determined bi, values.
93
NOTES
1.4
L -0.4
~-_-(J2 OJ i7.0
0.2 43
t0.s
02 +0.4
0.4
1.8.
702
0.6
RF
-0.2
Rt.+
-+02
Fig. 1. Cor_r&6~n of lo_grithms of ~paciiy ratios obtained by TLC (abscissa) sod coitrmn chrom2tography (ordinate). System: si!icz+thyI acetzte (10 moL”?) f cyclohexane. Solutes (Figs. l-3): aniline (A) and its 2-nitro-(ZNA), Gnitro- (4NA) and 3,4dimethyl- (XMA) derkztiues; l-amino(IAN; and 1,5-diulrincnaphthaiene (I5AN); 1,2-diaminobenzene (12AB); quinoline (Q); 6-nitroquinoiine (@IQ); a-naphthoquino!ine (aNQ); and acridine (ACR).
The equations of the correlation
lines determined by the least-squares method
2re : Fig. I (20 mole-% ethyl acetate): Iog k&c, = 0.98R,, f 0.34 Fig. 2 (IO mole- % ethyl methyl ketone) : Iog k& = 1_10 R,, i 0.17 Fig. 2 (60 mole- y/,ethyl methyl ketone) : log k&-, = I .07 &?%I t 0.25 Fig. 2 (both compositions) : log k& = 0.99 R,, f 0.19 Fig. 3 (10 and 38 mole- 7: ethyl acetate): log k&-, = 1.01 RA,c-+ 0.22 The stopes of the correlation lines are thus close to unity, in accordance with theoreticat expectation. The lines are shifted from the origin by ra. 0.2-W unit, which could have been due to higher rados of adsorbent weight to solvent vo!tnme in column chromatography or other causes, slrch as difFerences in adsorbent activities in the two techniques, kinetic e!Tects, demiting of the solvents and non-equilibrkm conditions in TLC. It is worth mentioning that linear correlations are observed even for different compositions of the developing solvent of $ given type (Figs. 2 and 3) and even for different solvent systems (the last Cwo correlation equations for Figs. 2 and 3 are virtually ideAcz1). III the theoretical considerations, idealized chromatographic processes were assumed; in reality, the re!ationships can be distorted by additional effects, such as demking of the solvents and non-ecpi!ibrirrm conditions in TLC. It has been demonstrated” that under comparable conditions (identicaf adsorbent, conditioning of
1
1.6 i-O.2
/
e
Ace
ethy_y!mecAy! ke;oC : (S) f Fig. 2. As Fis. 1, with deveiopin, = co!veat _ nob % S; dased circles, @I mob % S.
0.3 7lD
-0s
02
03
+0.5
f 0.4
0.4 )
i.6 i
‘IAN
0.5 -016
+02
0.6
i7F RM
cycioherrure. Open circles, 10
Nm33
95
the plates in TLC, use of sutiiciently large tanks satmated r&h solvent vapour) the mechanisms of adsorption processes in TLC and column chromatography are identical or very simiIar_ For adsorbents of the same type difFeri;ngin specific surface arez (ao,,,, a,,,,) the correfation be &o&2 be shifted by !og (qrrc,/qcc,)_
Thanks are due to Docent Dr. Jerry S. Kowakzyk, Head of the Laboratory of LiqrriciChromatography at the Institute of Industrial _4nalysis, Polytechnic University, Gdazisk, Poland, for valuable discussions dming ihe experimental work and for permission to use the equipment of the Laboratory. REFERENCES i 2 3 4 S 6 7 8 9 IO 11 12 I3 14 !S 16 17
R. _AXIOS.zsd S. G. Perry, J. Chranmogr., 83 (1973) 245. R. M. Scott, J. Ckroamogr. Sci., 11 (1973) 129. H. S&&t and F. Geis, 4. Ckrmzarogr., 67 (1972) 261. F. Geiss, Parameter dcr Diinnsck~ch~-~kronia~ogra~k~~, Vieweg, Brzttinschweig, 1972. K. D&m and &I. Ftichs, ZfeIv_ Ckinz. AC&, 45 (1952) 261. A. G. Nettisg, J. Cfrromzrogr., 53 (197G) 507. G. R. Duncan, J. Ckrumatogr., 8 (1962) 37. W. Gdkiewicz 2nd T. Wawrzynowicz, Ckromctogrcpkia, 3 (1970) 356. A. Stdyhwo and 0. S. Privet& J. Ckromarogr. Sci., iI (i973) 20. G. Ghenini 2nd E_ Cemi, in T. BP-UP, r;nd G. Gheisini (Editors), E_xmzciion Ckromarograpky, AkzdCmizl KY&b and Ekevier, Buditpest and Amsrerdzm, 1975, Ch. 14, p. 366. I. B. Ruppet, L. F. Cunco and J. G. Krsruse, J. Ckem. Educ., 43 (1971) 635. E. Soczewikki, Advaz. Ckroniarogr., 8 (1969) 91. L. R. SEyder, Adran. Ckrom~~rogr., 4 (1967) 3. B. coq, 3. P. Nhlas, A. L.xr!otte 2nd M. Porti-Imlt, J. ChfoJnLzrogr_,97 (197&) 137. E. Soczewtiki end W. GoIkiewicz, Ckromarograpkia, S (1979 4331. E. Sacze~~~ki znd W. Gdkiewicz, Ckromatograpkia, 6 (1973) 269. W. G&ciewicz, Ckromatograpkicz, irz press.