Analyst, March, 1974, Vol. 99, pp. 149-152 149 Gel Supports for Electrophoresis with Pyridine - Acetic Acid Media BY D. LEIGHTON, G. J. MOODY AND J. D. R. THOMAS (Chemistry Department, University of Wales Institute of Science and Technology, Cardif, CFl 3N U, Wales) Agar gels that are suitable for use with an 80 per cent. solution of pyridine in water and cross-linked polystyrene gels in pyridine - acetic acid are effective support media for electrophoresis. They provide the means for widening the application of electrophoresis t o materials themselves insoluble in aqueous media but soluble in pyridine - acetic acid. Illustrative examples of the electrophoresis of methylene blue, rhodamine €3, alizarin red, alizarin blue and eosin are given. ZONE electrophoresis, although usually performed in aqueous buffer - electrolyte systems, has also been effected in non-aqueous systems, and the work recorded to date indicates a wider perspective for electrophoresis.For example, Paul and Durruml have separated oil- soluble dyes by electrophoresis in absolute ethanol, nitromethane - glacial acetic acid and pyridine - glacial acetic acid systems on filter-paper supports. Also, the electrophoretic behaviour of arsenazo I11 and its analogues on filter-paper in glacial acetic acid and mixed formic acid - chloroacetic acid media has been reported,2 and several inorganic ions that were difficult to separate in aqueous solutions were readily resolved in a methanol - acetone - hydrochloric acid m e d i ~ m . ~ Apart from filter-paper, thin-layer supports (as prepared for chromatography) are suitable for electrophoresis in non-aqueous media (Leighton, D., Moody, G.J., and Thomas, J. D. R., unpublished work), but the enormous capacity for resolution achieved with the various gel supports in aqueous systems4 cannot always be extended to non-aqueous media because of gel solubility and stability problems, especially with polyacrylamide. The characteristics of electrophoretic systems based on agar gels for aqueous pyridine media, and cross-linked polystyrene gels for pyridine - acetic acid buffer electrolytes, are described in this paper. EXPERIMENTAL PREPARATION OF AGAR GELS- The appropriate amount of special Noble Agar (Difco Laboratories, Detroit), but ideally 2.25 g, was dissolved in 30 cm3 of water by heating the mixture on a boiling water bath and 120 cm3 of pyridine were added.After cooling it on ice until it became viscous, the mixture was poured into a O.6-cm deep horizontal trough formed by an aluminium frame sealed to a 20 x 16-cm glass plate with high-vacuum silicone rubber grease. The agar surface was levelled flush with the top of the aluminium frame with a glass rod and the whole kept on ice until the gel was firm. The frame was removed and slits were cut midway along the gel layer for sample application, PREPARATION OF CROSS-LINKED POLYSTYRENE GELS- A mixture of styrene and divinylbenzene (50 to 60 per cent. m/m in ethylvinylbenzene) monomers in appropriate proportions, and containing 0-60 g of benzoyl peroxide initiator, was added to 120 cm3 of a pyridine - acetic acid - perchloric acid mixture (Table I) and the whole poured into a trough similar to that described above for agar gels, except that the top side of the aluminium frame was now covered with a glass plate.In order to facilitate filling of the trough, one 16-cm edge of the frame was cut away. For gelling, the assembly, which was held together by strong rubber bands, was placed in a boiling water bath with the open end just above the water level. Between 1 and 3 hours were usually required for gelling, depending on the concentration of the divinylbenzene. After gelling, the top glass plate and the aluminium frame were removed, thus leaving the gel resting on the other plate. Samples for electrophoresis were placed into slits cut midway along the gel layer.@ SAC and the authors.150 LEIGIITON d U l . : GEL SUPPORTS FOR ELECTROPHORESIS [A?UdySt, VOl. 99 ELECTROPHORESIS- Electrophoresis was carried out in tlie same way as for aqueous systems by using a Shandon power pack (Catalogue No. SAE 2525) and a Bairct and Tatlock horizontal electro- phoresis tank (Catalogue No. C40/4000) constructed of silica. Contact between the gel and buffer - electrolyte was effected with wicks made of filter-paper. The test solutions used were 1 per cent. solutions of the dye in the appropriate buffer. TABLE I PARAMETERS OF POLYSTYRICSE GEL FORMATION Gel constituents r 1 Buffer composition : Divinyl- pyridine to acetic Styrene benzene Renzoyl acid ratio, V / V monomer/ monomer/ peroxicle/ (120 cn13 usctl in I3uffcr GelNo. cm3 1 1 2 3 4 5 6 7 8 9 36 10 48 11 60 12 72 13 84 18 24 19 ;: J 22 cm3 19.2 - 14.4 12.0 9.6 7.3 6.0 4.8 3.6 ] 7.2 4.8 7.2 1 i 4*8 J .. g each instance ) pH partof 71 t o 73 per cent. 6.45 perchloric acid i Om6 I, 100 : 0 J 100 : 1.0 100: 7.16 100 : 19.2 100 : 33.8 100 : 40.6 100 : 48.7 100 : 66.7 9.2 8-46 6.7 5-98 5.46 5.33 5.09 4.73 Gelling time/ hours Gel character 1.0 Cloudy and 1.25 } rubbery 1.5 Yellow and 1.5-2.0 rather brittle Clear yellow but no gel 1.6 -) 1.0 1.0 0.75 0.75 1-0 2-0 1.0 1.0 1.6 + Clear yellow 1.5 J 1.5 Translucent 1.0 White opaque 0.5 } precipitate * Following 4-t-butylcatechol inhibitor extraction (by shaking with four times the monomer volume o 0.06 M sodium hydroxide solution) from styrene and divinylbenzene monomers, the gelling times were 1.25 (a) 2 (b) and 6 (c) hours.RESULTS AND DISCUSSION AGAR GELS- The development of agar gels was directed towards a purely non-aqueous pyridine system but the agar would not dissolve in water containing more than 80 per cent. of pyridine; the study of agar gel parameters was therefore based on varying the amount of agar in a mixture of 30 cm3 of water and 120 cnl3 of pyridine. The qualities of gels obtained after 1 hour, with the various amounts of agar used parenthesised, were as follows: no gel formed even after 8 hours (0.9 g), very soft gel (1.2 g), firm gel (1.5, 2.25, 3.0 and 3.75 g), very firm gel (4.5 g). Firm and very firm gels were suitable for testing by electrophoresis and typical results for dye systems are summarised in Table 11. Migration of methylene blue and rhodamine I3 to the cathode, and of alizarin blue and eosin to the anode, is as expected from the cationic and anionic residues of these respective pairs of dyes.Although the zero migration of alizarin red in the buffers with high pyridine contents is rather surprising, agar gels based on aqueous pyridine mixed solvent media clearly provide suitable supports for zone electrophoresis in buffer media containing very substantial amounts of pyridine, especially for the gel based on 2-25 g of agar in 30 cm3 of water plus 120 cm3 of pyridine (Table 11).TABLE I1 ,Y % Migration distanceslcm *% ELECTROPHORETIC MOBILITIES OF DYES ON GEL SUPPORTS I N PYRIDINE - ACETIC ACID MEDIA I A Buffer Gel constituents Buffer PH Agar gels- (1 + 4 v/v) (4 + 1 v / v I Water (30 cm3) + agar (1.5 g) -+ pyridine (120 cm3) Water (30 cm3) + agar (2.25 g) + pyridine (120 cm3) Water (30 cm3) + agar (4.5 g) $- pyridine (1 20 cm3) Water (120 cm3) + agar (2.25 g) + pyridine (30 cm3) Pyridine (120 cm3) + acetic acid (1.2 cm3) -t 71-73 per cent.perchloric acid (0.6 cm3) + Water - pyridine 8.8 Water - pyridine 8.5 Cross-linked colystyrene gels- benzene (6.0 cm3) f- benzoyl Pyridine (120 cm3) 4- acetic 5-45 styrene (24 cm3) -k divinyl- peroxide (0.6 g) (gel No. 6) acid (2.25 d -t 71-73 ver cent. 9.2 i 5*45 Pyridine - acetic 1 5.45 Pyridine - acetic 5-45 Pyridine - water 8.6 (1 4- 4 v/v) acid (3 + 1 V / V ) * Tailing. i perchloric :&id (0.6 cm;) + styrene (24 cm3) + divinyl- benzene (4.8 cm3) + benzoyl peroxide (0.6 g) (gel No. 7) Pyridine (120 cm3) + styrene (24 cm3) + divinylbenzene (4.8 cm3) -+ benzoyl peroxide (0.6 g) (gel.No. 14) Pyridine (120 cm3) + styrene (24 cm3) + divinylbenzene (7.2 cm3) + benzoyl peroxide (0.6.g). (gel No. 15) Pyridine (90 cm3) + acetic acid (30 cm3) + styrene (24 cm3) f divinylbenzene acid (3 + 1V/V) (4-8 cm3) + benzoyl peroxide (0.6 g) (gel No. 19) Pyridine Silica gel- { Silica gel G (Merck) Constant voltage/ V 1000 1000 1000 300 1000 600 600 600 600 600 1000 300 Starting current/ mA 35 35 35 5 8 7 4 3 5 5 1 4 Time/ minutes 40 45 45 180 180 180 45 45 90 240 240 180 Cathode Origin Anode - 1 Methylene blue 0.5 5.5 0.5 2.0 3.5 3.5 9.0 1.8 6.5 9-6t 0.1 0.4 Rhoda- mine B 0.3 1.5 0.3 1.2 2.0 2.0 2.2 0.4 1.6 4.8 0 0.8 Aliza- rin red 0 0 0 0.8 (to anode) 0 0 0 0 0 0 0 0 Aliza- rin blue 0.3 1.6 0-3 0.8 0.3 1.0 5.7* 0.7 2.0 6.5 0.6 0.3 Eosin 0.8 3.0 0.6 1.2 0.4 0.8 4*5* 0-9 1.5 4.5 0.8 1.9 t M o d to end of gel (9.6 cm) in 166 minutes.152 LEIGHTON, MOODY AND THOMAS CROSS-LINKED POLYSTYRENE GELS- The several parameters examined for polystyrene gel formation are summarised in Table I.An alternative photopolymerisation process that involved gelling under an ultraviolet fluores- cent lamp gave gels of similar quality to those given by the thermal method described above but the required gelling times of at least 3 days are rather too long for convenience. The general features for successful gel formation are bound by the requirements that the ratio of divinylbenzene to polystyrene should not be less than 1 : 5 (gel No. 7) or as high as 2 : 5 (gel No.3), although the former stipulation can be relaxed when the amount of poly- styrene monomer is increased (gels Nos. 9 to 13). With respect to pH (a relative, empirical quantity only for the mixed solvent media used in this study), stable gels were obtained for the pH range 9.2 to 5-45 (gels Nos. 14 to 19), but under more acidic conditions a precipitate formed during the thermal treatment stage (gels Nos. 21 and 22). Despite the presence of 4-t-butylcatechol inhibitor in the styrene and divinylbenzene monomer constituents, gelling times were of the order of 1 to 3 hours, but removal of the inhibitor reduced the times by about one third [see (a) and (b) in Table I for gels Nos. 5 to 71. Such a reduction was considered to be an insufficient advantage for the removal of the inhibitor to be introduced into the procedure for the preparation of standard gels.A further variant assessed with the view of simplifying gel preparation was the pre- forming of cross-linked polystyrene slabs followed by soaking in buffer electrolyte. Even though translucent slabs were obtained, these slabs, when swelled by floating on the buffer electrolyte, became brittle and snapped readily and were therefore unsuitable as supports for electrophoresis. Gels Nos. 4 to 7 and 9 to 19 (Table I) were of suitable quality for electrophoresis and the most economical in terms of monomer materials are gels Nos. 6 and 7, and 14 to 19. Repre- sentative samples of these gels were tested for the electrophoresis of dyes and typical results are summarised in Table 11.The migration behaviour is the same as for agar gels, with methylene blue and rhodamine B migrating to the cathode, alizarin blue and eosin migrating to the anode and alizarin red remaining immobile. Migration distances are rather less for the more cross-linked gel No. 6 than for gel No. 7 and also less for gel No. 15 than for gel No. 14. The conditions corresponding to gel No. 14 provide for the efficient separation of all five dyes. Reducing the pH from 9.2 (gel No. 14) to 5.45 (gel No. 19) lowers the migration rates, especially for the anionic dyes, but the separation is still effective. Migration rates were even less for a buffer of similar acidity on a silica gel G support, This feature and the reduced diffusion for the polystyrene gel confirm the suitability of the polymer gel as a support medium. CONCLUSION Although not as convenient or as elegant to use as purely aqueous systems, agar gels make suitable stable supports for electrophoresis in media of high pyridine content. Similarly, cross-linked polystyrene gels that are stable in pyridine - acetic acid provide another electro- phoresis gel support medium for the separation of materials that are difficultly soluble in water and for which pyridine and acetic acid can emphasise the differences in the polar character of such materials. The authors thank the Esso Petroleum Company Ltd. for financial support, including a research award (to D.L.). REFERENCES 1. 2. 3. 4. Paul, M. H., and Durrum, E. L., J , Amer. Chew. Soc., 1952, 74,4721. Savvin, S. B., Akimova, G., Krysin, E. P., and Davydova, M. M., Zh. Analit. Khim., 1970, 25, 430. Maki, M., Japan Analyst, 1955, 4, 156. Moody, G. J., and Thomas, J. D. R., “Practical Electrophoresis,” Merrow Press, Watford, to be Received July 6th, 1973 Accepted October 17th, 1973 published.