229
Clinica Ckimica Acta, 82 (1978) 229-232 0 Elsevier/North-Holland Biomedical Press
CCA 8958
A SIMPLE AND RAPID THIN-LAYER CHROMATOGRAPHIC METHOD FOR THE IDENTIFICATION OF URINARY CARBOHYDRATES
WOLFGANG PRINZ *, WILLIAM MELDRUM and LYNNE WILKINSON Deputtment of Pkysiological Chemistry, (South Africa)
University of the Witwatersrund, Johannesburg
(Received May 31st, 1977)
Introduction Disturbances in carbohydrate metabolism are reflected in abnormal levels of urinary carbohydrates. Known clinical syndromes include glycosuria, lactose intolerance, fructose intolerance and fructosuria, essential pentosu~a and galactosaemia (galactosuria). Thin-layer chromato~aphy (TLC) offers itself as a simple means of detecting such abnormalities. Although the chromatographic separation of carbohydrates has received a good deal of attention during recent years (cf. refs. l--6), most of the published work lacks the essential element of simplicity. Inherent in the problem of separating carbohydrates by chromatographic means, is the essential similarity in their chemical structures. In order to enhance resolution, some workers have employed special impregnants for the stationary phase [ 1,2]. Others resorted to “dual layer” or 2-dimensional techniques [ 3,4], A further difficulty in the chromatographic analysis of urinary carbohydrates is the gross interference by the normally encountered urinary ions. These have been shown to be the cause of excessive “streaking” and/or i~eproducible results [1,5]. Pre-treatment of urine by ion-exchange column chromatography, followed by eluent concentration [ 2,6] has proved satisfactory, but time consuming. Selective ion-precipitants have also been employed [31We wished to eliminate extensive pre-treatment of urine as well as to avoid the use of 2.dimensional techniques and/or special impregnants. We have confined our study to the separation of physiologically and clinically important carbohydrates, viz.: fructose, galactose, glucose, lactose, maltose, sucrose and xylulose. In view of the pernicious nature of galactosaemia, we focused our efforts on
* Correspondenceshould be addressedto: Dr. W. Prinz,Departmentof PhysiologicalChemistry,
University of the Witwatersrand Medical School, Hospital Street, Johannesburg, 2001 South Africa.
230
obtaining a clear resolution revealed that the separation
of glucose from galactosr. A study of the literature of these two sugars has been extremely difficult.
Materials 1. Reference carbohydrates. Fructose, galactose, glucose, lactose, maltose and sucrose were all readily available commercial samples of A.R. grade. Xylulose was prepared according to the method of Touster [7]. “Marker” solutions (in distilled water) were made up as follows:
EiWctose
28
mM
(500
m&100
ml)
Galactose
28
mM
(500
mg/lOO
mi)
Glucose
28
mM
(500
mgj100
ml)
mM
(250
mgjlO0
ml)
mM
1500
mg,‘lOO
ml)
mM
(250
n&l00
ml)
mM
(250
mg/lOO
ml)
Lactose Maltose SIXUXe Xylulose
7.5 15 7.5 17
2. Ion exchange resins. (i) “Amberlite” IR-120(H) (analytical grade). The commercial product was cleaned by suspending in 2 M HCl and then washed with distilled water until eluent was neutral to pH paper. The resin was dried in a vacuum desiccator and the dried material was stored in a dark bottle. (ii) “Amberlite” IR-4B(OH) (analytical grade). The commercial product was cleaned by suspension in 2 M NaOH, washed with distilled water, dried in vacua and stored in a dark bottle. 3. ,TLC plates. Aluminium backed silica gel “60” TLC plates (Merck Art. No. 5553). Cut to suitable size, these plates were used as supplied by the manufacturers. 4. Chromatographic running medium (‘mobile phase). Methanol/acetone/ water/25% ammonia liquor (100 : 60 : 25 : 15, v/v). This solvent system was prepared freshly, daily. 5. Reagent for visualizing carbohydrate spots [S]. A. 0.2% solution of naphthalene-1,3diol in absolute ethanol. (This solution can be stored in a dark bottle for a week.) B. Concentrated sulphuric acid. A and B are mixed in a propo~ion of 95 : 5 (v/v) respectively, prior to spraying of plate. Method and results To a 4-ml sample of urine were successively added a strong cation exchange resin (“Amberlite” IR-120(H); 1 g dry mass) and a weak anion exchange resin (“Amberlite” IR-4B(OH); 1 g dry mass). The mixture was vortex-mixed for 1 min after each addition. The resin was allowed to settle for approximately 2 min. A suitable voiume (2-10 ~1) of the colourless supernatant liquid was spotted onto the TLC plate. (The use of an “Oxford” or equivalent pipette and a hairdrier is very convenient for the spotting operation.) “Marker” sugar solutions (2 ~1) were spotted on the same plate. The chromatographic development was performed in a vapour-saturated chromatographic tank. Time of run: 75 min; rise of solvent front: 12 cm.
231
After allowing the plate to dry, the sugar spots were visualized with “developing reagent” and heating to 105°C for 5 min.
by spraying
Results Criteria employed for the identification of carbohydrates were R, values and the characteristic colours produced by different sugars. The following carbohydrates are clearly resolved: galactose, glucose, lactose, maltose, sucrose and xylulose. Fructose has an R, value too near that of glucose to claim a satisfactory separation of these two sugars. However, they are easily differentiated by their colours. R, values, colour and sensitivity limits of the sugars studied are summarized in Table I. A typical run with “standard” carbohydrate solutions in normal
Fig. 1. From left to right: glucose *: sucrose *: galactose *; (lactose + xyhllose) *; (glucose + galactose) *; (glucose + galactose) **; (glucose + sucrose + galactose + lactose + xylulose) *; (glucose + sucrose + galactose + lactose + xylulose) * *. * Resin-treated “standard” carbohydrate solutions in normal human urine. ** Untreated “standard” carbohydrate solutions in normal human urine. (Original in colour.)
23”
TABLK
1
Sugar
Fruc tow Galactose Glucose Lact.ow Maltose Sucrose Xylulosr
Xf x 100
40 32 42 21 27 37 65
COlOUl
PLUplt?-L=d Blue Dark purple Violet Blue-purple Red Orangt~
Sensitivity
limit ’
mmol/l
mg/100
0.55 0.85 0.55 0.15 0.30 0.08 0.65
10 15 IO 5 10 3 10
* Values given refer to: Concentration in original urine solution resin-treated urine sample is spotted onto tht TLC platti,.
ml
if 10 ~11 of thr supernatant
human urine * is depicted in Fig. 1. The R, values of carbohydrates treated urine are identical to those of “marker” sugar solutions. Untreated urine samples tend to appear as a smear. Furthermore, (where discernible) of carbohydrates in untreated urine, are severely (Fig. 1).
liquid from
in resin-
Rf values depressed
Discussion The TLC method for the identification of urinary carbohydrates which we have developed required no complex operations. no special materials and little time. The entire procedure should take less than 2 h, most of which time being required for the development of the chromatogram. Individual runs and Rf values are reproducible to within 5%. The only critical requirement is a freshlyprepared solvent system. We have found that retention of the same solvent system for longer than one day results in progressive distortion of chromatograms. Xylulose exhibits a slight tendency to streak **. However, this is not a serious disadvantage, since its R, value is very much higher than those of any of the other carbohydrates studied. Although the use of “marker” solutions is not essential, it is to be recommended. We have never found the Rf value of a “marker” carbohydrate to differ by more than 3% from that of a resin-treated “standard” solution of the same carbohydrate in normal human urine, when run on the same plate. References Young. D.S. and .Jackson, A.J. (1970) Clin. Chem. 16, 954 Lato, M., Brunelli. B., Ciuffini, G. and Muzetti, T. (1968) J. Chromatogr. 36, 191 Ghebregzabher. M., Rufini, S., Ciuffini, G. and Late. M. (1974) J. Chromatogr. 95, 51 Mezzetti. T., Ghehregzabher. M., Rufini, S., Ciuffini. G. and Lato, M. (1972) J. Chromatogr. 74, 273 Saini, A.S. (1971) J. Chromatogr. 61, 378 Vitek. V. and Vitek. K. (1971) J. Chromatogr. 60, 381 Touter, 0. (1962) Methods of Carbohydrate Chemistry, Vol. 1 (Whistler, R.L. and Wolfram. M.L., eds.), p. 98, Academic Press. New York Lewis, B.A. and Smith, F. (1969) Thin-layer Chromatography (Stahl, F.. ed.), Springer, Berlin
* Same concentrations as recommended for “marker” solutions, see Materials. ** This is possible due to some degree of epimerization taking place during development in the basic solvent system. This sugar is, in fact, synthesized by the epimerization of xylose in hot pyridine solution [71.
Clinica Ckimica Acta, 82 (1978) 229-232 0 Elsevier/North-Holland Biomedical Press
CCA 8958
A SIMPLE AND RAPID THIN-LAYER CHROMATOGRAPHIC METHOD FOR THE IDENTIFICATION OF URINARY CARBOHYDRATES
WOLFGANG PRINZ *, WILLIAM MELDRUM and LYNNE WILKINSON Deputtment of Pkysiological Chemistry, (South Africa)
University of the Witwatersrund, Johannesburg
(Received May 31st, 1977)
Introduction Disturbances in carbohydrate metabolism are reflected in abnormal levels of urinary carbohydrates. Known clinical syndromes include glycosuria, lactose intolerance, fructose intolerance and fructosuria, essential pentosu~a and galactosaemia (galactosuria). Thin-layer chromato~aphy (TLC) offers itself as a simple means of detecting such abnormalities. Although the chromatographic separation of carbohydrates has received a good deal of attention during recent years (cf. refs. l--6), most of the published work lacks the essential element of simplicity. Inherent in the problem of separating carbohydrates by chromatographic means, is the essential similarity in their chemical structures. In order to enhance resolution, some workers have employed special impregnants for the stationary phase [ 1,2]. Others resorted to “dual layer” or 2-dimensional techniques [ 3,4], A further difficulty in the chromatographic analysis of urinary carbohydrates is the gross interference by the normally encountered urinary ions. These have been shown to be the cause of excessive “streaking” and/or i~eproducible results [1,5]. Pre-treatment of urine by ion-exchange column chromatography, followed by eluent concentration [ 2,6] has proved satisfactory, but time consuming. Selective ion-precipitants have also been employed [31We wished to eliminate extensive pre-treatment of urine as well as to avoid the use of 2.dimensional techniques and/or special impregnants. We have confined our study to the separation of physiologically and clinically important carbohydrates, viz.: fructose, galactose, glucose, lactose, maltose, sucrose and xylulose. In view of the pernicious nature of galactosaemia, we focused our efforts on
* Correspondenceshould be addressedto: Dr. W. Prinz,Departmentof PhysiologicalChemistry,
University of the Witwatersrand Medical School, Hospital Street, Johannesburg, 2001 South Africa.
230
obtaining a clear resolution revealed that the separation
of glucose from galactosr. A study of the literature of these two sugars has been extremely difficult.
Materials 1. Reference carbohydrates. Fructose, galactose, glucose, lactose, maltose and sucrose were all readily available commercial samples of A.R. grade. Xylulose was prepared according to the method of Touster [7]. “Marker” solutions (in distilled water) were made up as follows:
EiWctose
28
mM
(500
m&100
ml)
Galactose
28
mM
(500
mg/lOO
mi)
Glucose
28
mM
(500
mgj100
ml)
mM
(250
mgjlO0
ml)
mM
1500
mg,‘lOO
ml)
mM
(250
n&l00
ml)
mM
(250
mg/lOO
ml)
Lactose Maltose SIXUXe Xylulose
7.5 15 7.5 17
2. Ion exchange resins. (i) “Amberlite” IR-120(H) (analytical grade). The commercial product was cleaned by suspending in 2 M HCl and then washed with distilled water until eluent was neutral to pH paper. The resin was dried in a vacuum desiccator and the dried material was stored in a dark bottle. (ii) “Amberlite” IR-4B(OH) (analytical grade). The commercial product was cleaned by suspension in 2 M NaOH, washed with distilled water, dried in vacua and stored in a dark bottle. 3. ,TLC plates. Aluminium backed silica gel “60” TLC plates (Merck Art. No. 5553). Cut to suitable size, these plates were used as supplied by the manufacturers. 4. Chromatographic running medium (‘mobile phase). Methanol/acetone/ water/25% ammonia liquor (100 : 60 : 25 : 15, v/v). This solvent system was prepared freshly, daily. 5. Reagent for visualizing carbohydrate spots [S]. A. 0.2% solution of naphthalene-1,3diol in absolute ethanol. (This solution can be stored in a dark bottle for a week.) B. Concentrated sulphuric acid. A and B are mixed in a propo~ion of 95 : 5 (v/v) respectively, prior to spraying of plate. Method and results To a 4-ml sample of urine were successively added a strong cation exchange resin (“Amberlite” IR-120(H); 1 g dry mass) and a weak anion exchange resin (“Amberlite” IR-4B(OH); 1 g dry mass). The mixture was vortex-mixed for 1 min after each addition. The resin was allowed to settle for approximately 2 min. A suitable voiume (2-10 ~1) of the colourless supernatant liquid was spotted onto the TLC plate. (The use of an “Oxford” or equivalent pipette and a hairdrier is very convenient for the spotting operation.) “Marker” sugar solutions (2 ~1) were spotted on the same plate. The chromatographic development was performed in a vapour-saturated chromatographic tank. Time of run: 75 min; rise of solvent front: 12 cm.
231
After allowing the plate to dry, the sugar spots were visualized with “developing reagent” and heating to 105°C for 5 min.
by spraying
Results Criteria employed for the identification of carbohydrates were R, values and the characteristic colours produced by different sugars. The following carbohydrates are clearly resolved: galactose, glucose, lactose, maltose, sucrose and xylulose. Fructose has an R, value too near that of glucose to claim a satisfactory separation of these two sugars. However, they are easily differentiated by their colours. R, values, colour and sensitivity limits of the sugars studied are summarized in Table I. A typical run with “standard” carbohydrate solutions in normal
Fig. 1. From left to right: glucose *: sucrose *: galactose *; (lactose + xyhllose) *; (glucose + galactose) *; (glucose + galactose) **; (glucose + sucrose + galactose + lactose + xylulose) *; (glucose + sucrose + galactose + lactose + xylulose) * *. * Resin-treated “standard” carbohydrate solutions in normal human urine. ** Untreated “standard” carbohydrate solutions in normal human urine. (Original in colour.)
23”
TABLK
1
Sugar
Fruc tow Galactose Glucose Lact.ow Maltose Sucrose Xylulosr
Xf x 100
40 32 42 21 27 37 65
COlOUl
PLUplt?-L=d Blue Dark purple Violet Blue-purple Red Orangt~
Sensitivity
limit ’
mmol/l
mg/100
0.55 0.85 0.55 0.15 0.30 0.08 0.65
10 15 IO 5 10 3 10
* Values given refer to: Concentration in original urine solution resin-treated urine sample is spotted onto tht TLC platti,.
ml
if 10 ~11 of thr supernatant
human urine * is depicted in Fig. 1. The R, values of carbohydrates treated urine are identical to those of “marker” sugar solutions. Untreated urine samples tend to appear as a smear. Furthermore, (where discernible) of carbohydrates in untreated urine, are severely (Fig. 1).
liquid from
in resin-
Rf values depressed
Discussion The TLC method for the identification of urinary carbohydrates which we have developed required no complex operations. no special materials and little time. The entire procedure should take less than 2 h, most of which time being required for the development of the chromatogram. Individual runs and Rf values are reproducible to within 5%. The only critical requirement is a freshlyprepared solvent system. We have found that retention of the same solvent system for longer than one day results in progressive distortion of chromatograms. Xylulose exhibits a slight tendency to streak **. However, this is not a serious disadvantage, since its R, value is very much higher than those of any of the other carbohydrates studied. Although the use of “marker” solutions is not essential, it is to be recommended. We have never found the Rf value of a “marker” carbohydrate to differ by more than 3% from that of a resin-treated “standard” solution of the same carbohydrate in normal human urine, when run on the same plate. References Young. D.S. and .Jackson, A.J. (1970) Clin. Chem. 16, 954 Lato, M., Brunelli. B., Ciuffini, G. and Muzetti, T. (1968) J. Chromatogr. 36, 191 Ghebregzabher. M., Rufini, S., Ciuffini, G. and Late. M. (1974) J. Chromatogr. 95, 51 Mezzetti. T., Ghehregzabher. M., Rufini, S., Ciuffini. G. and Lato, M. (1972) J. Chromatogr. 74, 273 Saini, A.S. (1971) J. Chromatogr. 61, 378 Vitek. V. and Vitek. K. (1971) J. Chromatogr. 60, 381 Touter, 0. (1962) Methods of Carbohydrate Chemistry, Vol. 1 (Whistler, R.L. and Wolfram. M.L., eds.), p. 98, Academic Press. New York Lewis, B.A. and Smith, F. (1969) Thin-layer Chromatography (Stahl, F.. ed.), Springer, Berlin
* Same concentrations as recommended for “marker” solutions, see Materials. ** This is possible due to some degree of epimerization taking place during development in the basic solvent system. This sugar is, in fact, synthesized by the epimerization of xylose in hot pyridine solution [71.
