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The hazards of knotting dialysis bags
SHORT COMMUKICATIOiV'S
556
Aluminum chloride, which has been used in the characterization of flavonoids (1) and free phenolic acids (2)) produces spectral shifts in molecules capable of chelating it. Thus the carboxyl groups of the free cinnamic acids and their glucosides respond to this reagent strongly; the carboxyl of quinic acid derivatives, being removed from the aromatic portion of the molecule, produces only a slight shift; and glucose esters show none at all. The o-dihydroxy group of caffeic acid. also chelates aluminum chloride, so an additional degree of shift is apparent in this acid and its esters. These shifts are summarized in Table 2. All the spectra of Table 2 were obtained using natural mixtures of cis and trans isomers but may be taken to represent the tran.s compounds. Such mixtures can be resolved if necessary by chromatography in 2% aqueous acetic acid (Table 1) (3). The glucosides used here were prepared synthetically by procedures described elsewhere (4). The glucose esters were isolated from tobacco plants after feeding of the aglycones. ACKNOWLEDGMENTS Thanks are extended to Dr. 0. L. Gamborg for the use of authentic samples of 3-p-coumaroylquinic acid (from Dr. E. Haslam) and 3-feruloylquinic acid (from Dr. J. Come) ; and to Mr. B. K. Bailey for technical assistance. REFERENCES 1. Juan, L., in “The Chemistry of Flavonoid Compounds” (T. A. Geissman, ed.), p. 119. Macmillan, New York, 1962. 2. NAKAGAWA, Y., SHETLAR, M. R., AND WENDER, S. H., Anal. Biochem. 7, 374 (1964). 3. CHALLICE, J. S., AND WILLIAMS, A. H., J. Chromatog. 24, 357 (1966). 4. STECK, R., Can. J. Biochem. 45, 889 (19671. WARREN STECK Prairie
Regionnl Research
Laboratories
National Council Saskatoon, Canada Received May 1’7,196?
of Canada
The Hazards
of Knotting
Dialysis
Bags
Tappan’s simple dialysis method (1)) although possibly a useful improvement over the classically knotted dialysis bag, still has one
SHORT
COMMUNICATIOSS
1. Dialysis of normal pooled human plasma (containing 5% dextrose) FIG. against distilled water, after a 22 hr dialysis and two changes of water. Note how the solution has forced itself past the first knot and is in the process of passing the srcond one. The other rnd was similarly knotted ;mrl had wry much the same aspect.
important flaw. That flaw is the other knot. The vast majority of workers using dialysis likes to believe that a knot in ceIlophane tubing is impermeable to solute as well as to solvent. It is not. When a dialysis bag is closed with several knots (with about 1 in. interval) on each side, it quickly becomes apparent that the liquid to be dialyzed very easily passes the first knot and often also the second one (see Fig. 11. Three knots cd each end (or when used as a U-tube, three knots with both ends knotted toget’her). nre the minimum safeguards for th’e prevention of large, but generally unnoticed, losses of solution (2, 3). It would be interesting to learn if there are data about the impermeability of Tappan’s clamp system (1). If these clamps are really impermeable, his system (1) could be used to clamp off both ends of the cellophane simultaneously and thus not only facilitate repeated sampling but also assure the integrity of the dialysis bags. REFERENCES 1. TAPPAN, D. V.. ANLZ. Biochenl. 18, 392 (196i). 2. v.ax Oss, C. J., in “8eminars in IlllIllunogenctics” Lippincott, Philadelphia, 1967.
(T.
J. Greenwalt,
ed.),
p. 1,
558
SHORT COMMUNICATIONS
3. VAN Oss, C. J., in ‘(Advances in Separation Wiley, New York, 1967.
and Purification,”
(E. S. Perry, ed.),
CAREL J. VAN Oss Serum and Plasma Departments, Department Milwaukee, Received
of Biology,
Marquette
Wticonsin May
Thin-Layer
Milwaukee University
Blood
Center
and
6%33
23, 1967
Chromatographic and
Corresponding
Separation
of Hexitols
Hexoses
The hexitols, sorbitol and galactitol, have become of increasing biological importance, the former because of its accumulation in nervous tissue of diabetic animals (1) and the latter because of its presence in the tissues and urine of patients with congenital galactosemia (2) and in the tissues of animals fed a high galactose diet (3). Galactitol has been considered the etiological factor in galactose cataract formation (4). Metabolic studies with galactitol have necessitated a convenient method of separation of this polyol from galactose as well as from other common hexitols. Paper chromatographic procedures have failed to achieve the desired results (5). Gas chromatography has resolved this difficulty but requires laborious procedures and expensive equipment (6). Recently Nemec et al. (7) employed silica gel G on glass plates to separate sorbitol, galactitol, and mannitol from their related aldoses but were unable to separate these hexitols from each other. This communication describes the use of borate impregnated silica gel G on thin layer to effect the separation of these polyols from their corresponding hexoses and from each other. Materials and Methods. 0.02 M borate buffer, pH 7.85, was made up fresh as a 3% solution of 0.02 M sodium borate, pH 9.2, in 0.02 M boric acid, pH 6.6; 120 ml of the resulting solution was added to 60 gm silica gel G, according to Stahl (Brinkmann Instruments). The mixture was shaken manually for 90 set and spread onto 20 X 20 cm glass plates to a thickness of 250 ti using the Desaga spreader. After drying overnight the plates were placed into a 126°C oven for 60 min, then stored at room temperature in a wooden box with desiccant. Before using, the plates were reactivated for 30 min in the 126’ oven. Unlabeled sugars were obtained from Phanstiehl Laboratories, Inc.
556
Aluminum chloride, which has been used in the characterization of flavonoids (1) and free phenolic acids (2)) produces spectral shifts in molecules capable of chelating it. Thus the carboxyl groups of the free cinnamic acids and their glucosides respond to this reagent strongly; the carboxyl of quinic acid derivatives, being removed from the aromatic portion of the molecule, produces only a slight shift; and glucose esters show none at all. The o-dihydroxy group of caffeic acid. also chelates aluminum chloride, so an additional degree of shift is apparent in this acid and its esters. These shifts are summarized in Table 2. All the spectra of Table 2 were obtained using natural mixtures of cis and trans isomers but may be taken to represent the tran.s compounds. Such mixtures can be resolved if necessary by chromatography in 2% aqueous acetic acid (Table 1) (3). The glucosides used here were prepared synthetically by procedures described elsewhere (4). The glucose esters were isolated from tobacco plants after feeding of the aglycones. ACKNOWLEDGMENTS Thanks are extended to Dr. 0. L. Gamborg for the use of authentic samples of 3-p-coumaroylquinic acid (from Dr. E. Haslam) and 3-feruloylquinic acid (from Dr. J. Come) ; and to Mr. B. K. Bailey for technical assistance. REFERENCES 1. Juan, L., in “The Chemistry of Flavonoid Compounds” (T. A. Geissman, ed.), p. 119. Macmillan, New York, 1962. 2. NAKAGAWA, Y., SHETLAR, M. R., AND WENDER, S. H., Anal. Biochem. 7, 374 (1964). 3. CHALLICE, J. S., AND WILLIAMS, A. H., J. Chromatog. 24, 357 (1966). 4. STECK, R., Can. J. Biochem. 45, 889 (19671. WARREN STECK Prairie
Regionnl Research
Laboratories
National Council Saskatoon, Canada Received May 1’7,196?
of Canada
The Hazards
of Knotting
Dialysis
Bags
Tappan’s simple dialysis method (1)) although possibly a useful improvement over the classically knotted dialysis bag, still has one
SHORT
COMMUNICATIOSS
1. Dialysis of normal pooled human plasma (containing 5% dextrose) FIG. against distilled water, after a 22 hr dialysis and two changes of water. Note how the solution has forced itself past the first knot and is in the process of passing the srcond one. The other rnd was similarly knotted ;mrl had wry much the same aspect.
important flaw. That flaw is the other knot. The vast majority of workers using dialysis likes to believe that a knot in ceIlophane tubing is impermeable to solute as well as to solvent. It is not. When a dialysis bag is closed with several knots (with about 1 in. interval) on each side, it quickly becomes apparent that the liquid to be dialyzed very easily passes the first knot and often also the second one (see Fig. 11. Three knots cd each end (or when used as a U-tube, three knots with both ends knotted toget’her). nre the minimum safeguards for th’e prevention of large, but generally unnoticed, losses of solution (2, 3). It would be interesting to learn if there are data about the impermeability of Tappan’s clamp system (1). If these clamps are really impermeable, his system (1) could be used to clamp off both ends of the cellophane simultaneously and thus not only facilitate repeated sampling but also assure the integrity of the dialysis bags. REFERENCES 1. TAPPAN, D. V.. ANLZ. Biochenl. 18, 392 (196i). 2. v.ax Oss, C. J., in “8eminars in IlllIllunogenctics” Lippincott, Philadelphia, 1967.
(T.
J. Greenwalt,
ed.),
p. 1,
558
SHORT COMMUNICATIONS
3. VAN Oss, C. J., in ‘(Advances in Separation Wiley, New York, 1967.
and Purification,”
(E. S. Perry, ed.),
CAREL J. VAN Oss Serum and Plasma Departments, Department Milwaukee, Received
of Biology,
Marquette
Wticonsin May
Thin-Layer
Milwaukee University
Blood
Center
and
6%33
23, 1967
Chromatographic and
Corresponding
Separation
of Hexitols
Hexoses
The hexitols, sorbitol and galactitol, have become of increasing biological importance, the former because of its accumulation in nervous tissue of diabetic animals (1) and the latter because of its presence in the tissues and urine of patients with congenital galactosemia (2) and in the tissues of animals fed a high galactose diet (3). Galactitol has been considered the etiological factor in galactose cataract formation (4). Metabolic studies with galactitol have necessitated a convenient method of separation of this polyol from galactose as well as from other common hexitols. Paper chromatographic procedures have failed to achieve the desired results (5). Gas chromatography has resolved this difficulty but requires laborious procedures and expensive equipment (6). Recently Nemec et al. (7) employed silica gel G on glass plates to separate sorbitol, galactitol, and mannitol from their related aldoses but were unable to separate these hexitols from each other. This communication describes the use of borate impregnated silica gel G on thin layer to effect the separation of these polyols from their corresponding hexoses and from each other. Materials and Methods. 0.02 M borate buffer, pH 7.85, was made up fresh as a 3% solution of 0.02 M sodium borate, pH 9.2, in 0.02 M boric acid, pH 6.6; 120 ml of the resulting solution was added to 60 gm silica gel G, according to Stahl (Brinkmann Instruments). The mixture was shaken manually for 90 set and spread onto 20 X 20 cm glass plates to a thickness of 250 ti using the Desaga spreader. After drying overnight the plates were placed into a 126°C oven for 60 min, then stored at room temperature in a wooden box with desiccant. Before using, the plates were reactivated for 30 min in the 126’ oven. Unlabeled sugars were obtained from Phanstiehl Laboratories, Inc.