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A simple pre-equilibration apparatus for thin-layer chromatography
MICROCHEMICAL
JOURNAL
15,
74-77 (1970)
A Simple Pre-equilibration Thin-Layer ANTHONY Central
Apparatus
for
Chromatography
V. DETHOMAS
AND MARK
ZDANKIEWICZ
Connecticut State College, Department of Chemistry, New Britain, Connecticut 00650 Received July 14, 1969
Good resolution in TLC requires the presaturation of the developing chamber atmosphere with solvent vapor (la, b). Presaturation is commonly assistedby lining the inner walls of the chamber with filter paper wicks (2, 3). By scaling down the size of the developing chamber, it should be possible to eliminate the use of these wicks and also to decrease the time required for pre-equilibration of the chamber. As shown in Fig. 1, a modified apparatus may be easily constructed from one-hole neoprene stoppers (A and B), a suitable length of 7-mm glass rod (C), and a 2 X 20-cm test tube (D). The liquid sample is applied by means of a spotting pipet at a point about 3 to 6 mm from one edge of an Eastman 1 Chromagram sheet (E) that has been previously cut to about I X 12 cm. Then the opposite edge of the sheet is inserted into the slit in the lower side of stopper (B). About 10 ml of benzene is added to the test tube which is then immediately closed with stopper (A) and maintained in the vertical position shown in Fig. 1 for approximately 20 minutes, which is presumably ample time for chamber saturation. At this point, glassrod (C) is carefully pushed downward until the solvent level is about 2mm below the spotted sample (F). After development, the chromatogram is removed from the apparatus and allowed to dry in the air for several minutes. Then the R, values are calculated in the usual way. The results in Table 1 were obtained by using a test solution containing yellow, red, and blue dyes.* Unresolved zones are obtained when either presaturation of the chamber is ignored (No. 1) or when migration distances are too large (Nos. 4 and 7). In the latter instances, the developing time is also relatively large. Increasing the sample size from 2 to 4 PLIshows an adverse effect upon 1Silica gel adsorbentthickness,100 p Type K 301R. Obtainedfrom Distillation ProductsIndustries,Division of EastmanKodak Co., Rochester,New York. * CamagDye Mixture No. 1. Obtainedfrom Aruthr H. ThomasCo., Vine St., at Third, P.O. Box 779, Philadelphia5, Penn. 74
7
6
5
4
3
2
~~
2.0 2.0 2.0 2. 0 4 0 4.0 4.0
size (j.J!
dist.
(cm) 5.0 5.0 7.0 10.0 5.0 7.0 10.0
Solvent
1
ca. 1 .O
0.02 0.01 ca. 1.0
imprecise.
0.57 f 0.01 0.55 f 0.08
ca. 1.0
ca . I . 0
0.88 f O.YO f
0.50 * 0.04 0.67 rt 0.04
Av Rf valuesll'a --. ~~-._ -‘..--~~ Red ca. 1.0 .... .‘. Blue 0.3.-0.5 0.31 f 0.04
0.42 f 0.04 0.4 0.5 0.45 f 0.02 0.38 f 0. IO 0.3-0.5
RESCJLTS
ca. 1.0 0.85 f 0.03 0.97 f 0.01
-~----.-..------~~~ Yellow
OF EXPERIMENTAL
5eventeen runs for No. 2: 6 for all others. fJPre-equilibration conditions used for all entries except No. I. “Poor resolution of yellow and red zones. dBest resolution of all zones. “Good resolution of all zones; R, value of yellow zone close: to unity. “Fair resolution of all zones. QGood resolution of all zones; Rj values of red and blue zones relatively
-.II,-
.-. 1
_ _~
Sample
NO.
SUMMARY
TABLE
develop (min)
20-25 >60
>60 11-13
30 -3.5
IO- I5
20 25
Approx time
.
/
L7 -~c
7d K
ci
2
cn
$ 2
-c
% 2
-,I
-c
Comments
76
DETHOMAS
AND
ZDANKIEWICZ
_
FIG.
1. The
3cm
apparatus.
the resolution of the colored zones (Nos. 2 and 5) or upon the precision of the measurements (Nos. 3 and 6). By allowing the migration distance to increase, the Rf values for all components also increase at the 2 ~1 level, whereas at the 4~1 level, a decrease in the Rf value of the blue component may be observed. The results of these studies demonstrate the necessity of meticulously controlling the variables involved in the chromatographic process. Presaturation of the chamber, sample size, and solvent migration distance are critical to the attainment of suitable resolution and precise R, measurements. ACKNOWLEDGMENTS
The authors are indebted to Professor Arts) for drawing the figure and also
Alfred to the
Caputo Division
(Department of Chemical
of Industrial Education-
APPARATUS
DuPont Small investigation.
Grants
Program
for
FOR
partial
77
TLC
financial
support
received
during
this
REFERENCES I.
2. 3.
Stahl, E., (0) Thin layer chromatography. IV. Scheme, border-effect, acidic and basic layers, step technique. Arc/r. P/rtrr/~~. 292/64, 41 l-416 (1959): (h) Pllurr~l. Rltrldsc/rarr l(2), 1 (I 959). Palmer, J. K.. Corm. Agr. Expr. Sftr., NW HNWII. Bd. 589, 12-14 (1955). Zollner, N. and Wolfram. G.. K/i,!. Woc~/~.cc/rr. 40. 1098 ( 1962).
JOURNAL
15,
74-77 (1970)
A Simple Pre-equilibration Thin-Layer ANTHONY Central
Apparatus
for
Chromatography
V. DETHOMAS
AND MARK
ZDANKIEWICZ
Connecticut State College, Department of Chemistry, New Britain, Connecticut 00650 Received July 14, 1969
Good resolution in TLC requires the presaturation of the developing chamber atmosphere with solvent vapor (la, b). Presaturation is commonly assistedby lining the inner walls of the chamber with filter paper wicks (2, 3). By scaling down the size of the developing chamber, it should be possible to eliminate the use of these wicks and also to decrease the time required for pre-equilibration of the chamber. As shown in Fig. 1, a modified apparatus may be easily constructed from one-hole neoprene stoppers (A and B), a suitable length of 7-mm glass rod (C), and a 2 X 20-cm test tube (D). The liquid sample is applied by means of a spotting pipet at a point about 3 to 6 mm from one edge of an Eastman 1 Chromagram sheet (E) that has been previously cut to about I X 12 cm. Then the opposite edge of the sheet is inserted into the slit in the lower side of stopper (B). About 10 ml of benzene is added to the test tube which is then immediately closed with stopper (A) and maintained in the vertical position shown in Fig. 1 for approximately 20 minutes, which is presumably ample time for chamber saturation. At this point, glassrod (C) is carefully pushed downward until the solvent level is about 2mm below the spotted sample (F). After development, the chromatogram is removed from the apparatus and allowed to dry in the air for several minutes. Then the R, values are calculated in the usual way. The results in Table 1 were obtained by using a test solution containing yellow, red, and blue dyes.* Unresolved zones are obtained when either presaturation of the chamber is ignored (No. 1) or when migration distances are too large (Nos. 4 and 7). In the latter instances, the developing time is also relatively large. Increasing the sample size from 2 to 4 PLIshows an adverse effect upon 1Silica gel adsorbentthickness,100 p Type K 301R. Obtainedfrom Distillation ProductsIndustries,Division of EastmanKodak Co., Rochester,New York. * CamagDye Mixture No. 1. Obtainedfrom Aruthr H. ThomasCo., Vine St., at Third, P.O. Box 779, Philadelphia5, Penn. 74
7
6
5
4
3
2
~~
2.0 2.0 2.0 2. 0 4 0 4.0 4.0
size (j.J!
dist.
(cm) 5.0 5.0 7.0 10.0 5.0 7.0 10.0
Solvent
1
ca. 1 .O
0.02 0.01 ca. 1.0
imprecise.
0.57 f 0.01 0.55 f 0.08
ca. 1.0
ca . I . 0
0.88 f O.YO f
0.50 * 0.04 0.67 rt 0.04
Av Rf valuesll'a --. ~~-._ -‘..--~~ Red ca. 1.0 .... .‘. Blue 0.3.-0.5 0.31 f 0.04
0.42 f 0.04 0.4 0.5 0.45 f 0.02 0.38 f 0. IO 0.3-0.5
RESCJLTS
ca. 1.0 0.85 f 0.03 0.97 f 0.01
-~----.-..------~~~ Yellow
OF EXPERIMENTAL
5eventeen runs for No. 2: 6 for all others. fJPre-equilibration conditions used for all entries except No. I. “Poor resolution of yellow and red zones. dBest resolution of all zones. “Good resolution of all zones; R, value of yellow zone close: to unity. “Fair resolution of all zones. QGood resolution of all zones; Rj values of red and blue zones relatively
-.II,-
.-. 1
_ _~
Sample
NO.
SUMMARY
TABLE
develop (min)
20-25 >60
>60 11-13
30 -3.5
IO- I5
20 25
Approx time
.
/
L7 -~c
7d K
ci
2
cn
$ 2
-c
% 2
-,I
-c
Comments
76
DETHOMAS
AND
ZDANKIEWICZ
_
FIG.
1. The
3cm
apparatus.
the resolution of the colored zones (Nos. 2 and 5) or upon the precision of the measurements (Nos. 3 and 6). By allowing the migration distance to increase, the Rf values for all components also increase at the 2 ~1 level, whereas at the 4~1 level, a decrease in the Rf value of the blue component may be observed. The results of these studies demonstrate the necessity of meticulously controlling the variables involved in the chromatographic process. Presaturation of the chamber, sample size, and solvent migration distance are critical to the attainment of suitable resolution and precise R, measurements. ACKNOWLEDGMENTS
The authors are indebted to Professor Arts) for drawing the figure and also
Alfred to the
Caputo Division
(Department of Chemical
of Industrial Education-
APPARATUS
DuPont Small investigation.
Grants
Program
for
FOR
partial
77
TLC
financial
support
received
during
this
REFERENCES I.
2. 3.
Stahl, E., (0) Thin layer chromatography. IV. Scheme, border-effect, acidic and basic layers, step technique. Arc/r. P/rtrr/~~. 292/64, 41 l-416 (1959): (h) Pllurr~l. Rltrldsc/rarr l(2), 1 (I 959). Palmer, J. K.. Corm. Agr. Expr. Sftr., NW HNWII. Bd. 589, 12-14 (1955). Zollner, N. and Wolfram. G.. K/i,!. Woc~/~.cc/rr. 40. 1098 ( 1962).