Paper Chromatography of Very Long Chain Phosphates SIR: The paper chromatographic method (4) for separating and qualitatively and quantitatively determining condensed phosphates in mixtures has been successful in the assaying of chain and ring phosphates since this method was first applied (S) to the inorganic phosphate field in 1953. However, for the chromatographic solvents generally employed, the Rf values ( 5 ) are such that separations beyond the decaphosphates corresponding to either the chain or ring (6) varieties are impractical. Berg (1) has shown that h i g h - m o l e c u l a r - w e i g h t phosphates which normally would not move from the origin in an Ebel solvent would moL e appreciably when the solvent was diluted. It is the purpose of this paper to extend Berg's findings with respect
to dilution of the solvent and t o show that paper chromatography can be used in spreading out a distribution of longchain phosphates. The procedure described herein is useful for qualitatively estimating the size distribution of a mixture of high-molecular-weight chain phosphates such as are found, for example, in yeast extracts. EXPERIMENTAL
The undiluted chromatographic solvent used in this study was an Ebel formula, consisting of 735 ml. of isopropyl alcohol, 50 grams of trichloroacetic acid, and 2.5 ml. of concentrated aqueous ammonia in 265 ml. of water. The solvent front was allowed to move about 5 inches in all of the chromatograms-taking from 4 t o 6 hours, with
B
A n
the shorter times corresponding to the more dilute solvents. Quantitative assay of the distribution of PzOsnithin a band was obtained by two procedures. According to the procedure of KarlRroupa (+$), the bands were made several inches wide by starting with a row of spots along the line of origin. The resulting wide chromatographic bands were then cut into strips crossn-ise to the direction of solvent flow. .kccording to the alternate technique, the band chromatograms were developed in such a manner ( 2 ) that a photometric tracing shows the amount of phosphorus. The general technique of Karl-Kroupa was employed. The orthophozphate used in this study was the monopotassium salt which is de5ignated in the captions of the figures as n = 1 . The phosphate designated by n = 10 was a mixture of crystalline monopotasLium phosphate a i t h a 7 itreous potassium phosphate, the molecular weight of which was determined by end-group titration. The values of n = 117 and 230 corresponded to two preparations of Graham's salt (vitreous sodium metaphosphate), the molecular vieights of which were also obtained by end-group titration. The presence of orthophos-
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1 2 3 4 5 6
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Figure 1, Chromatograms of phosphates having chain lengths of 1 ,unity (orthophosphate);2, n = 10; 3, n = 1 17; 4, n = 230; 5, n = 1600; and 6, n = 5000 Chromatograms A, 6, C, D, E, and F correspond to 0, 13.3, 20.0, 26.7, 33.3, and 40.0% excess water, respectively, in the Ebel solvent
1984
ANALYTICAL CHEMISTRY
I
0
Rf Figure 2. Distribution of phosphorus in the chromatograms of chain phosphates for which (A) n = 10, (B)n = 1 17, (C) n = 230, (D) n = 1600, and (E) n = 5000 All chromatograms carried out in the Ebel solvent have 33.3% excess water
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Chziin L e n g t h (log scale)
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1.0
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102
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10'
Figure 3. An empirical estimation of the diminution of Rf value with increasing molecular weight
4
A represents pure water;
1
E, 33% excess water plus the Ebel solvent; and C, the usual concentrated Ebel solvenl
The data of Figure 2 can be put on a semiquantitative basis by relating the Ri value corresponding t o the middle of the bell-shaped curves of Figure 2 to the average molecular weight of the samples studied. This has been done in Figure 3 . The latter figure, which was obtained by a procedure akin to "lifting one's self by the bootstraps," now offers a convenient method for estimating a iize distribution from data such as those of Figure 2 . LITERATURE CITED
0.1
(1) Berg, G. G., ANAL. CHEX 30, 213
phate in the Graham's salt is attributable to a small amount of surface hydrolysis during a period of approximately 7 years that these glasses were stored in sealed contair ers. The longest chain phoqphates ( n = E 1600 and 5000) were potaqsium Ku-rol's salt. The degree of polymerization of 1600 mas obtained from intrinsic-viwosity measurements and the EO00 value from ultracentrifuge data. Figure 1 shows chvomatograms obtained by u 4 n g varioL s dilutions of the Ebel solvent with excess water. It should be noted t h a t when 33.3% excess water is used, the polyphosphate mixture exhibiting an average number of phosphorus atoms of 5000 showed considerable motion from the origin. At 40% excess water, all of the spots moved rap dly and did not
spread out appreciably even though each spot represented a broad distribution of homologs. For assaying high-molecular-weight material, i t appears t h a t a 25 t o 35% dilution of the Ebel solvent is most effective. Preliminary results shown in Figure 2 indicate that, in this concentration range, the paper-chromatographic procedure fractionates the long-chain phosphates and thus gives a measure of the size distribution of the chains. The heavy lines in Figure 2 were obtained b y the Karl-Kroupa procedure of cutting crosswise to the direction of solvent flow, eluting and determining the eluted PBOScolorimetrically. The thin wiggly line was obtained from a photometric tracing. The photometric tracings include the orthophosphate spot which appears at a n Rfvalue of 0.79.
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(2) Chess, W. B., Bernhart, D. N., ANAL. CHEM.31, 1116 (1959). (31 Ebel. J. P.. Bull. SOC.Chim. France 20, pp: 991,998 (1953). (4) Karl-Kroupa, E., Ax.41,. CHEM. 28,
1091 (1956). (5) Van Wazer,,,J. R., "Phosphorus and Its Compounds, Interscience, Yew York, 1958, Vol. I, pp. 662-5, 702-4. Also see H. Grunze and E. Thilo. Sitzher. Deut. Akad. Wiss. Berlin, Kl. Math. u. Allgem. Naturw. 5 , 1 (1953). (6) Van Wazer, J. R.. Karl-Kroupa, E., J . Am. Chem. SOC.78, 1772 (1956). SHIGERU OH AS HI^ JOHX R. VAN WAZER Inorganic Chemicals Division Monsanto Chemical Co. St. Louis, Mo. This work was carried out during leave of absence from Kanazawa University in Japan, 1957-58 academic year.
Cali bra tion of Nuclear Magnetic Resonance Chemical Shift Scale SIR: With the adv3nt of the T'arian hIodel A-60 analytica, KRIR spectrometer, it has become possible to use precalibrated chart pa3er. This permits the direct reading of resonance line positions without the necessity of establishing a scale b y means of two known reference poin ts-e.g., an internal reference and a side band of known separation. However the reliability of the readings from the A-60 chart paper must be eitablished before accurate chemical shifts and spin-spin coupling constants can be measured or calculated. The 6-60 can be adjusted so that accurate readings can be obtained on each setting of sweep width (1000, 500, 250, 100. and 50 c.P.s.). One waj- of checking the adjustments and measuring line positions is the side-band technique of Arnold and Packrvd (1). I n this technique, the radiofr3quency magnetic field applied t o the sample is modulated with a low-amplitude signal of known audiofrequency. This causes side bands t o appear on each side of the resonance lines in the spectrum The separation of the side bands from the parent peak (in c.P.s.) is equal t o the modulating
frequency. While this is a valuable technique and is standard procedure for other NhfR instruments, audiofrequency oscillators and frequency counters are not always available to owners of A-60's. The present note describes a simple method for checking the calibration by use of a reference sample. A similar method using the various resonance lines of p-anisaldehyde has been proposed ( 2 ) .
Each of the solutes in this solution produces a single sharp line at a characteristic position of the NMR spectrum. The concentrations were selected such that the lines are of about the same intensity. The position of each line was measured approximately 40 times at 37" C. by the side-band technique (1). The mean values are shown in Table I.
EXPERIMENTAL
Table I. Chemical Shifts of Solutes in Reference Sample at 37" C.
A solution was made up of 3% tetramethylsilane, 2% cyclohexane, 3y0 acetone, 9% l,l,l-trichloroethane, 275 pdioxane, 8% dichloromethane, and 18% chloroform (by volume) in carbon tetrachloride. So t h a t i t could be reproduced by others, the solution was prepared by pipeting each of the seven solutes into about 40 ml. of carbon tetrachloride in a 100-ml. volumetric flask, then making u p to the mark with additional carbon tetrachloride. This procedure gives reproducible results for chemical shifts. A sample of this solution was placed in a n A-60 sample tube, degassed by freeze-thawing under vacuum, and sealed.
C.P.S. at 60 Mc./
see." (std. dev.
8,
p.p.m."
(std. dev. 0.001)
Compound 0.03) Tetramethylsilane 0 0 (by def.) Cyclohexane 85.98 1.433 Acetone 126.72 2.112 l,l,l-Trichloroethane 163.84 2.731 p-Dioxane 217.24 3.621 Dichloromethane 318.06 5.301 Chloroform 439.82 7.330 a Downfield from tetramethylsilane
VOL. 35, NO. 12, NOVEMBER 1963
1985
