Nov., 1047
CALCIUM I O N
ACTIVITIESI N SOLUlIONS STABILIZED BY SODIUM hfETAPHOSPH.4TE
184'. A mixture of this sample with p-aminobenzoic acid resublimed to needles a t 150" and melted at 187-188'. Formylation of the Hydrolysis Product (11) (Resynthesis of Rhizopterin (I)).-Twenty-five milligrams of the hydrolysis product (11) was treated with a mixture of 2 ml. of formic acid (88q7,) and 0.7 ml. of acetic anhydride. The mixture was heated at reflux temperature until all dissolved (about twenty minutes). T h e solution was filtered and concentrated t o dryness in vacuo. The soliii residue was dissolved in 1 N ammonium hydroxide; the solution was filtered and diluted to ahout ten volumes with water. I t was then warmed on a steam-bath and acidified slowly with acetic acid to about PH 4. The rhizopterin crystallized in yellow platelets. I t was collected in a centrifuge tube, washed with water, and dried. Microbiological assays of the product, which was resynthesized in this manner, using S. lactis R . showed i t t o have the biological activity of rhizopterin. For analyses, samples were dried in a weighing-pig at 140". A n a l . Calcd. for CljHl?hT~04: C, 52.94; H, 3.56; N, 24.70. Found: C, 52.57; €1, 3.24; N, 24.70. Formylation of Pteroic Acid (11) (Synthesis of Rhizop.terin (I)).-One hundred fifty milligrams of pteroic acid (11), prepared as describecl,ll was treated with 10 ml. of formic acid (98%) and heated on a steam-bath. All solid dissolved after five minutes, but heating was continued for one hour. The solution was concentrated to dryness under rcduccd pressure. The residue was dissolved in 10 ml. of 1 N ammonium hydroxide. The solution was diluted to about 10 volumes, warmed to 70" and acidified with acetic acid to pH 4. Rhizopterin ( I ) crystallized from the solution in light yellow leaves. For analyses, samples were dried in a weighing-pig a t 140". Anal. Calcd. for C ~ S H I ~ N C, ~ O52.91; ~: Ilishccl, but its early designatioii :IS :I heskinictaphosphate liolynier, has bcen retained. 4 stock solutiori coiitaiiiiiig 1000 p . p . rn. w a s uscd. A solutioii of c;tlciurn bicarbonate was prepared by passing carboil dioiitle gas through :t zuspension of powdered calcium carbonate. After the excess carl.toiiate h:td settled the clear supernatalit liquid was reirioved by a siphon and the calcium conceritr;iriori tleterinined 1)y :I gravimetric calcium oxulatc igiiitioii riirthotl to be 0.0171 -V. This solution was employed i n two >cries of trextincnts designed to convert bicarbonate to carbonatc by rernt)v:il of carbon dioxidc, namely: (a) bubbling of atniospllcric .iir under a partial v x u u m ; ( b ) boiling. (a) I n seven 125-rnl. Pyrex Brlcnrncyer flisks, 100- -. (11) Xarshall,
J. P h y s . Chrm., 43, 1155 (1930); 48, 67 ( 1 9 4 4 ) ; 5oil .SL~. S O L .Arne,. Z'voc., 7, 192 (1043). ( 1 2 ) Rfarshall a n d B e r g m a n , THISJ O U R N A L . 63, 1911 ( 1 9 4 1 ) ; 1. f ' h y s . C h ~ m .46, , 2 2 , 325 (1042). (13) X1ar:;hall and Krinhill, i b i d . , 64, 1814 (10428, J . Phys. C h c m . , 46, 1077 (1042). ( 1 4 ) Marshall a n d Ayrrh, Soil S c i . S O C . A m r r . P m c . , 11, 171 (1947).
Vol. 69
ml. samples were prepared containing 100 p. p. m. of calcium as bicarbonate and the following concentrations of sodium hexametaphosphate: 0, 0, I/*, 1 , 2 , 3 and 5 p. p . m. Five of the flasks were connected to a vacuum-line manifold and air was bubbled through the solutions for six hours a t room temperature. The remaining two flasks, containing 0 and 2 p. p. m. of hexametaphosphate, were stoppered and reserved for reference measurements. After six hours an appreciable quantity of precipitate had formed in the aerated 0 p. p . m. hexametaphosphate system but none was observed in the remainder of the series. Differential weighings indicated a loss of 1.5 ml. of water during aeration. PH values were determined by a glass electrode and calcium ion activities by 465" calcium bentonite clay membrane electrodes on separate subsamples pipetted from the seven flasks. (b) A series of six similar bicarbonate solutions were prepared containing 0, 0, 1, 2, 4 and 6 p. p. m. of sodium hexametaphosphate. The solutions were heated separately for ten minutes over a bunsen burner, boiling vigorously during the second five-minute period. One of the 0 p . p. m. sodium hexametaphosphate solutions was boiled an additional twenty minutes; this flask lost 32 ml. of water. The loss of water from the other flasks ranged from 5.5 to X ml. The six solutions were restored to their initial weights by addition of water. Calcium ion activities and pH values were determined on separate subsamples. Heating at 105" for twenty hours effected the loss of 19 g. of water from 100 g. of powdered C a S 0 ~ 2 H 2 0 . Nine grams of the dried salt was added t o 700 ml. of water in a flask and shaken for three minutes by hand. The mixture was filtered rapidly on a Buchner funnel under a partial vacuum. Immediately, 80 ml. of the clear filtrate was added t o each of six 125-ml. Erlenmeyer flasks containing amounts of water and sodium hexametaphosphate required to provide sodium hexametaphosphate concentrations of I), 1, 2 , 3, 4 and 6 p. p. m. in a final volume of 100 ml. I t was known from previous experiments that immediate dilution of the filtrate by 20% furnishes a calcium sulfate concentration that can be stabilized by 4 p. p. m. of sodium hexametaphosphate. After the solutions had stood four days at room temperature without further agitation samples were pipetted out for measurement of calcium ion activity. The calcium ion activity of each system was determined from the potential difference across a clay membrane electrode between two saturated calomel electrodes as descril)etl i n tlvtail clsewhere.i*~1:'~14 The clay membranes were prepureti from calcium-saturated bentonite heated a t ~ E for J ' twenty-four hours. This material is not specific for c:ilciuin ions, but the systems studied intentionally contained 110 other cations except a negligible concentration of sodium ions from the sodium hexametaphosphate. The poteiitial incasured is attributed to calcium ion activity ~tloite. Poteiitials were measured a t 25' with a Lee& : t i i d Sortlirup type K potentiomcter and a high resist:ince gulwrionieter. The solutions were sufiicieritly dilutc that thc Seriist equation was applicable. The activity values reported were c,tlculutcd from the mean of 1 ht, potentids iiidiv.itcd Iiy duplicate clay elrctrodcs.
Results I V 'I'able I are listed thc PH values a l i d calcium iori a&\-ities uf the carbonate systems aiid visual estimates uf the relative exteiit of 1)recipitatiori. The determined activities of the aeratcd carbonate systems havc been reduced by 1.5% to correct for evaporation. When carbon dioxide was iivt renioved, nietaphosphate affected neithcr the fiH nor the calcium activity. The average activity of the controls, 0.00164, is also the maximum activity of the aerdtioii x i d boiling series. In the aeration series, metaphosphate over the range 0.5-5 p.p.in. caused
‘rAULIc I PRECIPITATION, ptI L’ALIII~S,A N D CALCIUM I O N ACTIVITIES o r ? CARUONATE SYSTEMS -- No treatment Aerated t i IIOII~J---.Ihiled 5 minutesI’wC.L I’rec:, l’re-
--
7
(NaF011,
OH
act i v i 1 y , a
