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Thin-layer chromatographyof free porphyrins on talc
Clin. Biochem. 1, 30-33 (1967)
THIN-LAYER CIIROMATOGRAPHY OF FREE PORPHYRINS ON TALC
T. K. WITH Central Laboratory, Svendborg County Hospital, Svendborg, Denmark (Received January 24, 1967)
SUMMARY 1. A rapid method for separating free porphyrins by thin-layer chromatography on talc is described. The developing fluid is 0.5N HC1 (2 vol) in acetone (3 v01) ; visualization is with Wood's light. 2. Good separations are obtained with mixtures containing 0.1 microgram porphyrins: uroporphyrin, Rt: 0.7-0.8 ; coproporphyrin, R~: 0.35-0.50; dicarboxyl (deutero) porphyrin R,: < 0.1. 3. Application of the method to urine, bile, etc., without previous purification is described.
EXISTING CHROMATOGRAPHICSEPARATION METHODS for porphyrins all require rather laborious purification procedures. In most cases, transforming the porphyrins into ester is necessary followed by extraction of the esters and subsequently by hydrolysis of the purified esters in order to obtain the free porphyrins. The best methods of chromatographic separation of free porphyrins are based on the lutidine-water system introduced by Nicolas and Rimington (4), and generally the modification of Eriksen (2) employing 2,6-1utidine is used. The separation method based on runs on paper with diluted salt solutions developed in our laboratory (6) is more rapid than this lutidine method, but inferior because it produces elongated or even confluent spots. The lutidine method does, however, require considerable purification of the material before the run and at least four hours to give a satisfactory separation. With this method, the number of carboxyl groups determines the R, value of the various porphyrins, i.e. uroporphyrin (UP) has an R, close to zero corresponding to its 8 carboxyl groups; coproporphyrin (CP) with 4 carboxyl groups shows intermediary values; dicarboxyl porphyrins show high R~ values ; esters (no carboxyl groups) have an R~ value of 1.0. In our laboratory we have for 5 years used a modified "lutidine" method involving a mixture of equal volumes of distilled water, pyridine (analytical), and 2,4,6-collidine (analytical) instead of the water--2,6-1utidine mixture. This system gives in our hands quite the same results as the lutidine method with
PORPHYRIN T.L.C. ON TALC
31
ascending technique. Like the lutidine system this system requires saturation of the atmosphere with ammonia. During our work with large-scale preparations of U P and CP from bovine and human urines (7, 8), which has now continued for 15 years, we have used adsorption on talc as our basic prelinqinary separation procedure and have found t h a t utilize this discovery performing c h r o m a t o g r a p h y on columns of talc b u t were unable to develop this technique into a practical method. Recently, using this principle we tried out thin layers of talc on plates and found t h a t this worked with remarkable efficiency even when porphyric urine or bile was applied to the plates without any previous purification. We employed attenuated modifications of the eluting solutions used in our preparatory procedures for development of the chromatograms, i.e. instead of acetone/HC1--9 vol. + 1 vol 1.5N--or 50-/0 v / v sulphuric acid in methanol we used a c e t o n e / H C 1 - - 6 vol -I- 4 vol 0.5N--or 0.05% v / v sulphuric acid in methanol. The acetone/HC1 was the better of the two developers as it gave considerably less streaking of the porphyrin spots than the sulphuric acid/methanol.
DESCRIPTION OF METHOD
Chromatographic Plates The glass plates, 20 X 20 cm, are prepared by means of the T L C apparatus (Desaga). The talc used is of pharmacopea quality* and is passed through a 0.2mm sieve prior to use.t Equal weights of talc and methanol (analytical) are thoroughly mixed in a hand shaker and immediately poured into the trough of the Desaga apparatus after which the plates are prepared. T h e layer thickness is 0.25 rain. The plates are allowed to dry in the air at room temperature for at least 10 minutes before use and are not subjected to further activation procedures. Freshly prepared plates and plates prepared 2 months before use and stored at room temperature in the air are equally effective.
Application of Porphyrin Solutions on Plates About 5-10 ~1 solution containing 0.1-20.0 ~g of porphyrin is employed. T h e fluid is applied to the plates, using a 20/zl micropipette, at least 1 cm from their borders; the distance between neighbouring spots was at least 1.5 cm. Concentrated marker preparations are most practically dissolved in acetone/ HC1--9 vol -}- 1 vol 1.5N--on a watch glass. After use, the fluid rapidly evaporates and the porphyrin on the glass can be re-dissolved and used repeatedly. The spots are allowed to dry, either in the air at room temperature or, more rapidly, by blowing a hot stream of air from an electric hairdryer on the back of the chromatographic plate. For weak solutions several applications of fluid on the same spot are necessary. Often a small "hole" in the talc layer in the centre of the spot is seen, but this has no influence on the chromatography. *Pharmacopea Nordica, Vol. II (ed. Danica), pp. 594-595. Busck, Copenhagen, 1963.
tlbid., Vol. III, pp. 6-7.
32
WITH
T h e fluorescence of the spots is controlled in Wood's light (cf. above), but it is to be emphasized that even if no red fluorescence of the application spot is visible fluorescing spots may develop on chromatography. The diameter of the spots is below 5 mm and the fluid runs out to about twice the diameter of the fluorescing porphyrin spot because of the adsorption of the porphyrins on the talc.
Development of Chromatograms A glass tank with internal measurements 21 X 21 X 10 cm is employed. The development fluid is freshly prepared by mixing 60 ml acetone (analytical) and 40 ml 0.5N HCI. The mixture rapidly turns yellowish but this does not influence the chromatogram. The mixture is poured into the tank, and the plate (eventually two plates at one time) is cautiously placed into the tank, after which the lid is laid on. I t is not necessary to wait for saturation of the atmosphere. Development for one hour gives a good separation of UP and CP, but the progress of the run can be followed by irradiation of the tank with Wood's light in a dark room if a rapid result is required.
Visualization of the Spots Irradiation with Wood's light in a dark room is employed for this purpose. Practical sources of Wood's light have been described previously (1, 5).
Stability of Spots In our experience the fluorescence of the porphyrin spots is apparently unchanged for at least one month, probably considerably longer, if the plates are stored at room temperature in diffuse wintry daylight. DISCUSSION AND RESULTS
UP runs as flattened spots with an R~ of about 0.76--usually 0.70-0.80. CP forms elongated spots with Rf about 0.40--usually 0.35-0.50. Of the dicarboxyl porphyrins deuteroporphyrin was studied and showed an Rf of 0.02. The UP spots are well-defined without tailing even if concentrated solutions are applied, but concentrated solutions of CP form elongated spots. If moderate amounts of porphyrins are employed the separation is, however, perfectly clear even in the CP region. We have studied several porphyria urines of both human and bovine origin (cf. 3) and the bovine urines containing an intermediary porphyrin (5- or 6carboxylic). T h e y were applied directly on the plate without pre-treatment. We have always found clearly defined spots of UP and CP. In one case we used 5 ~1 of a bovine urine containing 0.63 mg CP and 1.32 mg UP per 100 ml--i.e, a porphyrin mixture amounting to about 0.1 ~g--and found three clearly separated spots: UP with R~ of 0.74, intermediary porphyrin with R, of 0.55, and CP with R~ of 0.37. We have also studied the bones of a porphyric calf (see 3). Sawdust from the corticalis of bone--showing bright red fluorescence in Wood's light--was extracted into acetone/HC1 (9 vol + 1 vol 1.5N) which gave an extract with red fluorescence. The extract was applied to the plate, three times on the same spot. After development, one spot with an Rf of 0.73 appeared.
PORPHYRIN T.L.C. ON TALC
33
T h e 2~, values can v a r y from one plate to another, b u t are close to each other for all spots of the same p o r p h y r i n in one run. T o identify the porphyrins with certainty the use of known m a r k e r s is therefore necessary, at least for investigators without special experience with p o r p h y r i n c h r o m a t o g r a p h y . Several crystalline preparations of U P and CP esters of varying degrees of purity were studied both with thin-layer talc c h r o m a t o g r a p h y and with lutidine c h r o m a t o g r a p h y . In several instances more spots were observed with the thinlayer adsorption c h r o m a t o g r a p h y than with the p a p e r partition chromatographic lutidine method. In these cases rather large a m o u n t s of porphyrin were applied to the plates giving a bright red fluorescence of the starting spot. In preparations of U P from h u m a n acute i n t e r m i t t e n t p o r p h y r i a urine (Waldenstr6m t y p e ) - - a n d also in the same urine directly applied to the p l a t e - - t h e r e were three spots in the U P region as against only one on the lutidine p a p e r chromatograms; in one case these spots exhibited Rt values of 0.67 (main spot), 0.57, and 0.54. In coproporphyrin preparations from h u m a n p o r p h y r i a c u t a n e a t a r d a feces three spots were also found in the copro region. These had _R~ values of 0.26 (main spot), 0.13, and 0.09; here comparable spots were observed on lutidine c h r o m a t o g r a p h y if sufficiently large a m o u n t s of p o r p h y r i n were applied to the paper. We also used the method for d e m o n s t r a t i o n of the presence of U P in normal h u m a n urine. Four litres of urine were b r o u g h t to p H 3-4 (universal indicator paper) and adsorbed to talc b y suction through a Btichner funnel (diameter 15 cm). After washing b y sucking through 100 ml water, the p o r p h y r i n was eluted from the talc by passing 50-100 ml acetone/HC1 (9 vol + 1 vol 1.5x) through the funnel. T h e brown eluate exhibited red fluorescence in W o o d ' s light and three to five 5 ~1 volumes of the elnate were applied on top of each other on a prepared plate. After development, two spots with red fluorescence were distinctly visible in Wood's light, a strong one with _R, = 0.30 corresponding to CP and a weaker one with R, = 0.68. corresponding to UP. The technical assistance of Bent Pedersen and K a m m a W i t h is acknowledged. REFERENCES 1. EALES, L., LEVEY, M. J. & SWEENEY, G.D. The place of screening tests and quantitative investigations in the diagnosis of the porphyrias. S. Afr. Med. J. 40, 63-71 (1966). 2. ERIKSEN,L. Paper chromatography of porphyrin pigments. Scan& J. Clin. Lab. Invest. 5, 155-157 (1953). 3. J~RGENSEN, S.K. Studies in congenital porphyria in cattle in Denmark. Brit. Vet. J. 117, 61-73 (1961). ~. NICOLAS, R. E. H. & RIMINGTON, C.
5. 6. 7. 8.
Qualitative analysis of the porphyrins by partition
chromatography. Scan& J. Clin. Lab. Invest. 1, 12-18 (1949). WITH, T . K . A portable source of Wood's light in porphyrin analysis. Scand. J. Clin. Lab. Invest. 9, 208-209 (1957). WIT~I,T . K . Paper chromatography of free porphyrin with neutral salt solution. Scand. J. Clin. Lab. Invest. 9, 395-397 (1954). WITH, T . K . Preparation of crystalline porphyrin ester from bovine porphyrin urine. Biochem. J. 68, 717-720 (1958). WITH, T . K . Preparation of crystalline porphyrin esters from porphyria urines and normal human urine. Scand. J. Clin. Lab. Invest. 10, 297-302 (1958).
THIN-LAYER CIIROMATOGRAPHY OF FREE PORPHYRINS ON TALC
T. K. WITH Central Laboratory, Svendborg County Hospital, Svendborg, Denmark (Received January 24, 1967)
SUMMARY 1. A rapid method for separating free porphyrins by thin-layer chromatography on talc is described. The developing fluid is 0.5N HC1 (2 vol) in acetone (3 v01) ; visualization is with Wood's light. 2. Good separations are obtained with mixtures containing 0.1 microgram porphyrins: uroporphyrin, Rt: 0.7-0.8 ; coproporphyrin, R~: 0.35-0.50; dicarboxyl (deutero) porphyrin R,: < 0.1. 3. Application of the method to urine, bile, etc., without previous purification is described.
EXISTING CHROMATOGRAPHICSEPARATION METHODS for porphyrins all require rather laborious purification procedures. In most cases, transforming the porphyrins into ester is necessary followed by extraction of the esters and subsequently by hydrolysis of the purified esters in order to obtain the free porphyrins. The best methods of chromatographic separation of free porphyrins are based on the lutidine-water system introduced by Nicolas and Rimington (4), and generally the modification of Eriksen (2) employing 2,6-1utidine is used. The separation method based on runs on paper with diluted salt solutions developed in our laboratory (6) is more rapid than this lutidine method, but inferior because it produces elongated or even confluent spots. The lutidine method does, however, require considerable purification of the material before the run and at least four hours to give a satisfactory separation. With this method, the number of carboxyl groups determines the R, value of the various porphyrins, i.e. uroporphyrin (UP) has an R, close to zero corresponding to its 8 carboxyl groups; coproporphyrin (CP) with 4 carboxyl groups shows intermediary values; dicarboxyl porphyrins show high R~ values ; esters (no carboxyl groups) have an R~ value of 1.0. In our laboratory we have for 5 years used a modified "lutidine" method involving a mixture of equal volumes of distilled water, pyridine (analytical), and 2,4,6-collidine (analytical) instead of the water--2,6-1utidine mixture. This system gives in our hands quite the same results as the lutidine method with
PORPHYRIN T.L.C. ON TALC
31
ascending technique. Like the lutidine system this system requires saturation of the atmosphere with ammonia. During our work with large-scale preparations of U P and CP from bovine and human urines (7, 8), which has now continued for 15 years, we have used adsorption on talc as our basic prelinqinary separation procedure and have found t h a t utilize this discovery performing c h r o m a t o g r a p h y on columns of talc b u t were unable to develop this technique into a practical method. Recently, using this principle we tried out thin layers of talc on plates and found t h a t this worked with remarkable efficiency even when porphyric urine or bile was applied to the plates without any previous purification. We employed attenuated modifications of the eluting solutions used in our preparatory procedures for development of the chromatograms, i.e. instead of acetone/HC1--9 vol. + 1 vol 1.5N--or 50-/0 v / v sulphuric acid in methanol we used a c e t o n e / H C 1 - - 6 vol -I- 4 vol 0.5N--or 0.05% v / v sulphuric acid in methanol. The acetone/HC1 was the better of the two developers as it gave considerably less streaking of the porphyrin spots than the sulphuric acid/methanol.
DESCRIPTION OF METHOD
Chromatographic Plates The glass plates, 20 X 20 cm, are prepared by means of the T L C apparatus (Desaga). The talc used is of pharmacopea quality* and is passed through a 0.2mm sieve prior to use.t Equal weights of talc and methanol (analytical) are thoroughly mixed in a hand shaker and immediately poured into the trough of the Desaga apparatus after which the plates are prepared. T h e layer thickness is 0.25 rain. The plates are allowed to dry in the air at room temperature for at least 10 minutes before use and are not subjected to further activation procedures. Freshly prepared plates and plates prepared 2 months before use and stored at room temperature in the air are equally effective.
Application of Porphyrin Solutions on Plates About 5-10 ~1 solution containing 0.1-20.0 ~g of porphyrin is employed. T h e fluid is applied to the plates, using a 20/zl micropipette, at least 1 cm from their borders; the distance between neighbouring spots was at least 1.5 cm. Concentrated marker preparations are most practically dissolved in acetone/ HC1--9 vol -}- 1 vol 1.5N--on a watch glass. After use, the fluid rapidly evaporates and the porphyrin on the glass can be re-dissolved and used repeatedly. The spots are allowed to dry, either in the air at room temperature or, more rapidly, by blowing a hot stream of air from an electric hairdryer on the back of the chromatographic plate. For weak solutions several applications of fluid on the same spot are necessary. Often a small "hole" in the talc layer in the centre of the spot is seen, but this has no influence on the chromatography. *Pharmacopea Nordica, Vol. II (ed. Danica), pp. 594-595. Busck, Copenhagen, 1963.
tlbid., Vol. III, pp. 6-7.
32
WITH
T h e fluorescence of the spots is controlled in Wood's light (cf. above), but it is to be emphasized that even if no red fluorescence of the application spot is visible fluorescing spots may develop on chromatography. The diameter of the spots is below 5 mm and the fluid runs out to about twice the diameter of the fluorescing porphyrin spot because of the adsorption of the porphyrins on the talc.
Development of Chromatograms A glass tank with internal measurements 21 X 21 X 10 cm is employed. The development fluid is freshly prepared by mixing 60 ml acetone (analytical) and 40 ml 0.5N HCI. The mixture rapidly turns yellowish but this does not influence the chromatogram. The mixture is poured into the tank, and the plate (eventually two plates at one time) is cautiously placed into the tank, after which the lid is laid on. I t is not necessary to wait for saturation of the atmosphere. Development for one hour gives a good separation of UP and CP, but the progress of the run can be followed by irradiation of the tank with Wood's light in a dark room if a rapid result is required.
Visualization of the Spots Irradiation with Wood's light in a dark room is employed for this purpose. Practical sources of Wood's light have been described previously (1, 5).
Stability of Spots In our experience the fluorescence of the porphyrin spots is apparently unchanged for at least one month, probably considerably longer, if the plates are stored at room temperature in diffuse wintry daylight. DISCUSSION AND RESULTS
UP runs as flattened spots with an R~ of about 0.76--usually 0.70-0.80. CP forms elongated spots with Rf about 0.40--usually 0.35-0.50. Of the dicarboxyl porphyrins deuteroporphyrin was studied and showed an Rf of 0.02. The UP spots are well-defined without tailing even if concentrated solutions are applied, but concentrated solutions of CP form elongated spots. If moderate amounts of porphyrins are employed the separation is, however, perfectly clear even in the CP region. We have studied several porphyria urines of both human and bovine origin (cf. 3) and the bovine urines containing an intermediary porphyrin (5- or 6carboxylic). T h e y were applied directly on the plate without pre-treatment. We have always found clearly defined spots of UP and CP. In one case we used 5 ~1 of a bovine urine containing 0.63 mg CP and 1.32 mg UP per 100 ml--i.e, a porphyrin mixture amounting to about 0.1 ~g--and found three clearly separated spots: UP with R~ of 0.74, intermediary porphyrin with R, of 0.55, and CP with R~ of 0.37. We have also studied the bones of a porphyric calf (see 3). Sawdust from the corticalis of bone--showing bright red fluorescence in Wood's light--was extracted into acetone/HC1 (9 vol + 1 vol 1.5N) which gave an extract with red fluorescence. The extract was applied to the plate, three times on the same spot. After development, one spot with an Rf of 0.73 appeared.
PORPHYRIN T.L.C. ON TALC
33
T h e 2~, values can v a r y from one plate to another, b u t are close to each other for all spots of the same p o r p h y r i n in one run. T o identify the porphyrins with certainty the use of known m a r k e r s is therefore necessary, at least for investigators without special experience with p o r p h y r i n c h r o m a t o g r a p h y . Several crystalline preparations of U P and CP esters of varying degrees of purity were studied both with thin-layer talc c h r o m a t o g r a p h y and with lutidine c h r o m a t o g r a p h y . In several instances more spots were observed with the thinlayer adsorption c h r o m a t o g r a p h y than with the p a p e r partition chromatographic lutidine method. In these cases rather large a m o u n t s of porphyrin were applied to the plates giving a bright red fluorescence of the starting spot. In preparations of U P from h u m a n acute i n t e r m i t t e n t p o r p h y r i a urine (Waldenstr6m t y p e ) - - a n d also in the same urine directly applied to the p l a t e - - t h e r e were three spots in the U P region as against only one on the lutidine p a p e r chromatograms; in one case these spots exhibited Rt values of 0.67 (main spot), 0.57, and 0.54. In coproporphyrin preparations from h u m a n p o r p h y r i a c u t a n e a t a r d a feces three spots were also found in the copro region. These had _R~ values of 0.26 (main spot), 0.13, and 0.09; here comparable spots were observed on lutidine c h r o m a t o g r a p h y if sufficiently large a m o u n t s of p o r p h y r i n were applied to the paper. We also used the method for d e m o n s t r a t i o n of the presence of U P in normal h u m a n urine. Four litres of urine were b r o u g h t to p H 3-4 (universal indicator paper) and adsorbed to talc b y suction through a Btichner funnel (diameter 15 cm). After washing b y sucking through 100 ml water, the p o r p h y r i n was eluted from the talc by passing 50-100 ml acetone/HC1 (9 vol + 1 vol 1.5x) through the funnel. T h e brown eluate exhibited red fluorescence in W o o d ' s light and three to five 5 ~1 volumes of the elnate were applied on top of each other on a prepared plate. After development, two spots with red fluorescence were distinctly visible in Wood's light, a strong one with _R, = 0.30 corresponding to CP and a weaker one with R, = 0.68. corresponding to UP. The technical assistance of Bent Pedersen and K a m m a W i t h is acknowledged. REFERENCES 1. EALES, L., LEVEY, M. J. & SWEENEY, G.D. The place of screening tests and quantitative investigations in the diagnosis of the porphyrias. S. Afr. Med. J. 40, 63-71 (1966). 2. ERIKSEN,L. Paper chromatography of porphyrin pigments. Scan& J. Clin. Lab. Invest. 5, 155-157 (1953). 3. J~RGENSEN, S.K. Studies in congenital porphyria in cattle in Denmark. Brit. Vet. J. 117, 61-73 (1961). ~. NICOLAS, R. E. H. & RIMINGTON, C.
5. 6. 7. 8.
Qualitative analysis of the porphyrins by partition
chromatography. Scan& J. Clin. Lab. Invest. 1, 12-18 (1949). WITH, T . K . A portable source of Wood's light in porphyrin analysis. Scand. J. Clin. Lab. Invest. 9, 208-209 (1957). WIT~I,T . K . Paper chromatography of free porphyrin with neutral salt solution. Scand. J. Clin. Lab. Invest. 9, 395-397 (1954). WITH, T . K . Preparation of crystalline porphyrin ester from bovine porphyrin urine. Biochem. J. 68, 717-720 (1958). WITH, T . K . Preparation of crystalline porphyrin esters from porphyria urines and normal human urine. Scand. J. Clin. Lab. Invest. 10, 297-302 (1958).