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A semiautomatic multispotting apparatus for thin-layer chromatography
ANALYTICAL
BIOCHEMISTRY
79, 639-642 (1977)
A Semiautomatic Multispotting Apparatus for Thin-Layer Chromatography A semiautomatic multispotting apparatus for simultaneously applying up to 12 samples can conveniently be constructed with a minimum of investment in materials, and no extraordinary equipment is required to build it. Use of the apparatus for routine analysis results in considerable savings of the technician’s time and enhances the replicability of the analysis.
In our laboratory, we have done a large number of adenine determinations which involve one-step separation of the mono-, di-, and triphosphate nucleotides on polyethyleneimine (PEI) cellulose thin-layer chromatography. In order to obtain reproducible results and to spot multiple samples simultaneously, we devised a multispotting apparatus which would accomplish both purposes. We would now like to report how easily this apparatus can be made and to show the results of a thin-layer chromatogram utilizing the multispotting apparatus. METHODS The apparatus was made from wood and metal, although it could have just as easily been constructed of Plexiglas or similar materials. The syringe-holding assembly (Fig. la) was constructed from 0.75in. board, 20.2 cm wide and 9 cm long: Grooves (0.8 cm wide x 0.4 cm deep) were cut the entire length of the board, separated by l-cm spaces. As many as 12 grooves, allowing for the simultaneous spotting of 12 different samples, can be cut conveniently in a 22.6-cm-wide board. A butt plate, 2.8 x 20.2 cm, with 3/64-in. holes drilled to coincide with the center of each groove, was placed at the base of the grooved piece. A hold-down plate, composed of a 1.9 x 20.2 x 5 cm board with a 5mm-wide strip of soft synthetic sponge material (obtained from lumber yards), was used to hold the tops of the syringe barrels in place. This was held down by two thumbscrews in each end of the holddown plate. The upper half of the syringe-holding assembly consists of a 13 x 21.5cm frame to hold the turn-screw and a sliding bar which is responsible for compressing the syringe plungers simultaneously. The turn-screw bar was made from a %-in. chrome-plated rod, fastened at both ends of the grooved board by wood screws. A hole was drilled in the center, and a 3/16-in. tap was welded at the top to provide threads for the turn-screw which runs through the frame. A flat washer was affixed to the bottom of the turn-screw which rests against the sliding bar. The sliding bar consisted of a flat plate with two 0.25-in. rods, 9.7 cm long 639 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0@33-2697
640
SHORT COMMUNICATIONS
FIG. 1. Semiautomatic multispotting apparatus base assembly and syringe-holding assembly. (a) Syringe-holding assembly is constructed of wood and stainless steel; the assembly is shown from a top view, with one syringe in place to show the relation between the butt plate, the grooves holding the syringe, and the sliding bar. (b) Base assembly constructed from 0.75 and 0.25in. wood. Unless otherwise noted, all dimensions are in centimeters.
and at 90” angle, located at each end. These rods slide in 0.25in. holes drilled in the grooved board 2 cm from each end of the board (see Fig. la). The base assembly of the multispotter was also made from a 0.75in. lumber cut in a 20.8 x 11.5cm rectangle (Fig. lb). The complete assembly is shown in Fig. 2. Rubber pads, made from serum-bottle stoppers, were glued on the bottom of the base plate. An 8.25-in. square of 0.75-in. plywood was used to maneuver the thin-
SHORT COMMUNICATIONS
641
layer plate under the tips of the syringe needles. We found this arrangement more convenient than adjusting the multispotting apparatus itself. In order to spot multiple samples, the syringes (Teflon-tipped 50~1 syringes obtained from Scientific Products) were loaded to the desired amount and were placed in the multispotting apparatus. These were then placed in the desired position, next to the thin-layer plate (PEIcellulose, Brinkman Instrument, Inc.), and a dryer was arranged to blow over the plate. The turn-screw was then advanced slowly until the desired quantity of liquid was extruded and allowed to dry, before the next aliquot was spotted on the plate. RESULTS AND DISCUSSION
Figure 3 shows repeated samples of AMP and ATP being separated on a PEI-cellulose plate. Thirty-microliter samples were placed in each syringe and applied to the origin. The time required to spot all 10 samples was 30 min, as compared to 2 hr when spotting manually. After the samples have been applied, the syringes can be removed easily by lifting the hold-down plate. A minimum time is required to assemble the apparatus (less than 10 min) and disassemble it for further applications and cleaning of syringes. The cost of the apparatus is minimal. Our cost was less then ten dollars for parts, excluding syringes, and 4-5 hr of labor to construct it. Equip-
FIG. 2. Photograph of multispotting
apparatus in position to apply sample to thin-layer plate.
642
SHORT COMMUNICATIONS
FIG. 3. Results of simultaneous spotting of identical mixtures of AMP and ATP. The compound with the larger&is AMP, and the other slower moving material is ATP. Streaking of the ATP fraction indicates impurities in the commercial product.
ment needed to construct the apparatus included a drill press, table saw, and, for the case in which it was made of metal and wood, welding capabilities. If it were to be constructed of Plexiglas or a similar material, even less equipment would be required. The only commercial apparatus we are aware of that will perform similar feats is completely automated and costs from 800 to 1000 dollars. Thus, constructing the apparatus and making it semiautomatic results in considerable savings of money and time. L. D. R. B. W.H. R. L. H. L. Snyder Research Foundation 1407 Wheat Road Winfield, Kansas 67156 Received November 8, 1976; accepted
1 To whom correspondence
January
5, 1977
should be addressed.
SMITH' TRIPLETT ARCHER EMERSON
BIOCHEMISTRY
79, 639-642 (1977)
A Semiautomatic Multispotting Apparatus for Thin-Layer Chromatography A semiautomatic multispotting apparatus for simultaneously applying up to 12 samples can conveniently be constructed with a minimum of investment in materials, and no extraordinary equipment is required to build it. Use of the apparatus for routine analysis results in considerable savings of the technician’s time and enhances the replicability of the analysis.
In our laboratory, we have done a large number of adenine determinations which involve one-step separation of the mono-, di-, and triphosphate nucleotides on polyethyleneimine (PEI) cellulose thin-layer chromatography. In order to obtain reproducible results and to spot multiple samples simultaneously, we devised a multispotting apparatus which would accomplish both purposes. We would now like to report how easily this apparatus can be made and to show the results of a thin-layer chromatogram utilizing the multispotting apparatus. METHODS The apparatus was made from wood and metal, although it could have just as easily been constructed of Plexiglas or similar materials. The syringe-holding assembly (Fig. la) was constructed from 0.75in. board, 20.2 cm wide and 9 cm long: Grooves (0.8 cm wide x 0.4 cm deep) were cut the entire length of the board, separated by l-cm spaces. As many as 12 grooves, allowing for the simultaneous spotting of 12 different samples, can be cut conveniently in a 22.6-cm-wide board. A butt plate, 2.8 x 20.2 cm, with 3/64-in. holes drilled to coincide with the center of each groove, was placed at the base of the grooved piece. A hold-down plate, composed of a 1.9 x 20.2 x 5 cm board with a 5mm-wide strip of soft synthetic sponge material (obtained from lumber yards), was used to hold the tops of the syringe barrels in place. This was held down by two thumbscrews in each end of the holddown plate. The upper half of the syringe-holding assembly consists of a 13 x 21.5cm frame to hold the turn-screw and a sliding bar which is responsible for compressing the syringe plungers simultaneously. The turn-screw bar was made from a %-in. chrome-plated rod, fastened at both ends of the grooved board by wood screws. A hole was drilled in the center, and a 3/16-in. tap was welded at the top to provide threads for the turn-screw which runs through the frame. A flat washer was affixed to the bottom of the turn-screw which rests against the sliding bar. The sliding bar consisted of a flat plate with two 0.25-in. rods, 9.7 cm long 639 Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0@33-2697
640
SHORT COMMUNICATIONS
FIG. 1. Semiautomatic multispotting apparatus base assembly and syringe-holding assembly. (a) Syringe-holding assembly is constructed of wood and stainless steel; the assembly is shown from a top view, with one syringe in place to show the relation between the butt plate, the grooves holding the syringe, and the sliding bar. (b) Base assembly constructed from 0.75 and 0.25in. wood. Unless otherwise noted, all dimensions are in centimeters.
and at 90” angle, located at each end. These rods slide in 0.25in. holes drilled in the grooved board 2 cm from each end of the board (see Fig. la). The base assembly of the multispotter was also made from a 0.75in. lumber cut in a 20.8 x 11.5cm rectangle (Fig. lb). The complete assembly is shown in Fig. 2. Rubber pads, made from serum-bottle stoppers, were glued on the bottom of the base plate. An 8.25-in. square of 0.75-in. plywood was used to maneuver the thin-
SHORT COMMUNICATIONS
641
layer plate under the tips of the syringe needles. We found this arrangement more convenient than adjusting the multispotting apparatus itself. In order to spot multiple samples, the syringes (Teflon-tipped 50~1 syringes obtained from Scientific Products) were loaded to the desired amount and were placed in the multispotting apparatus. These were then placed in the desired position, next to the thin-layer plate (PEIcellulose, Brinkman Instrument, Inc.), and a dryer was arranged to blow over the plate. The turn-screw was then advanced slowly until the desired quantity of liquid was extruded and allowed to dry, before the next aliquot was spotted on the plate. RESULTS AND DISCUSSION
Figure 3 shows repeated samples of AMP and ATP being separated on a PEI-cellulose plate. Thirty-microliter samples were placed in each syringe and applied to the origin. The time required to spot all 10 samples was 30 min, as compared to 2 hr when spotting manually. After the samples have been applied, the syringes can be removed easily by lifting the hold-down plate. A minimum time is required to assemble the apparatus (less than 10 min) and disassemble it for further applications and cleaning of syringes. The cost of the apparatus is minimal. Our cost was less then ten dollars for parts, excluding syringes, and 4-5 hr of labor to construct it. Equip-
FIG. 2. Photograph of multispotting
apparatus in position to apply sample to thin-layer plate.
642
SHORT COMMUNICATIONS
FIG. 3. Results of simultaneous spotting of identical mixtures of AMP and ATP. The compound with the larger&is AMP, and the other slower moving material is ATP. Streaking of the ATP fraction indicates impurities in the commercial product.
ment needed to construct the apparatus included a drill press, table saw, and, for the case in which it was made of metal and wood, welding capabilities. If it were to be constructed of Plexiglas or a similar material, even less equipment would be required. The only commercial apparatus we are aware of that will perform similar feats is completely automated and costs from 800 to 1000 dollars. Thus, constructing the apparatus and making it semiautomatic results in considerable savings of money and time. L. D. R. B. W.H. R. L. H. L. Snyder Research Foundation 1407 Wheat Road Winfield, Kansas 67156 Received November 8, 1976; accepted
1 To whom correspondence
January
5, 1977
should be addressed.
SMITH' TRIPLETT ARCHER EMERSON