The results show that the commercial agar, which is prepared mainly from Gelidium cartilagineum (?’), contains a cold water-soluble polysaccharide which differs from agarose. The cold watersoluble fraction is probably a mixture of the degradation products of agarose and agaropectin ( 2 ) . Heidelberger (8) shon-rd that this fraction cross-reacts with type XIV antipneumococcal serum. The molar ratio of galactose t o 3.6-anhydrogalactose of 1.15 for washcd agar agrees with ratio of 6 to 5 reported by O’Xeill (I?). It also agrees r+ith the molar ratio for special Agar-Noble (Difco) which is a commercial preparation of n ilshed agar. These preparations differ in that the Noble agar still contains nutrients which will support the growth of Saccharomyces cerevisiae (11 ) . The concentration of galartose in A-carrageenin of 49.0% agrees with values of 46.2 to 48.2% obtained by othcr methods ( I S , 21). Thc molar ratio of 1.55 for galactose and 3,Ganhydrogalactose in the Kcarrageenin suggests that this sample contains some A-carrageenin. O’Seill (15, 91) reported molar ratios of 1.4 and 1.1. The molar ratio of 1.21 for K-furcrllaran is in general agreement nith the conclusion of Painter (18) that this polysaccharitk is .siinilat to carrageenin.
This method may be applied to the determination 3,Ganhydrogalactose in other algal polysaccharides, providing the sugar residues are known. Such information may be obtained by chromatography of the acid hydrolyzates. The interference from unknolm sugars may be detected by a significant difference in thf values of 3,6-anhydrogalactose as determined with the resorcinol and anthrone reagents. Thus, the presence of a high concentration of xylose (5) or fucose (12) will interfere with the va1ucC: obtained v ith anthrone reagmt but not TI ith the rcsor rinol reagent. ACKNOWLEDGMENT
The author acknowledges the helpful suS;gestions of E. G. Young and T. J. Painter. The technical assistance of J. Berrigan is greatly appreciated. The sample of K-furcellarun n-as kindly providcxd by T. J. Painter. LITERATURE CITED
(1) Araki, C., ddz,ances irc Carbohydrate Chem. 8, 315 (1953). (2) Araki, C., PTOC. Intern. Congr. Biochem., 4th Congr. 1958, 15. (3) Araki, C., Hirase, S..J . Chem. SOC. Japan 26,463 (1953). (4) Bacon, J. S. D., Bell, JI. J., Biochem. J 42,397 (1948).
(5) Bonting, S. L., Arch. Bwchern. Biophys. 52, 272 (1954). (6), Brpwn, W. L., Young, ,M. It absorbance of the 0-p.p.m. fluoridr solution prepared simultaneously at 0.400 and determine the absorbances of the other solutions with rc.fercnctc to this solution. Determination of Fluoride Concentration in a Water Sample. Repeat
t h e above procedure through t h e addition of 2 ml. of thorium nitrate solution using a 100-ml. volumetric flask. Then add 50 nil. of a sample passed through a n Amberlite IR-120 ion exchange column. Dilute with distilled water to 100 ml., shake, and let
stand for 25 minutes. Determine absorbance of the solution with reference t o blank solution. For convenience, only the 0-p.p.m. solution and the unknown test solution or solutions need be prepared, once this htandard curve has been established.
0 10
FLUORIDE
WNCENTRITION
- .?W.
Figure 2. Effect of varying pH on absorbance of thorium-phenylfluorone complex solution
EXPERIMENTAL
'The experiments were designed to establish the optimum pH, optimum development time, and best ratio of color-producing reagents, and to ascertain the interferences froni ions normally found in iurface c a h . The rffcct of varying p H R ~ S111vestigated first in a qualitative manntxr Varying amounts of sodium acetate and chloroacctic acid were used in preparing the buffer Folutions eo aL: to produce various pH values in the colord solutions. Five-milliliter portions of the resulting solutions were added to approximately 1-ml. portion. of :i 100 p.p.m. of fluoridr solution. Rcwlts shonn in Table I indicated that the optimum QH range was 3.2 to 4.6. Results of further investigation are shown in Figure 2 . A pH of 3.6 was selected as best, since the absorbancr was more nearly a linew function of the fluoride concentration at this $3. The buffer solution as prepared will produce this pH. Color development appears to be almost instantaneous and absorbance is constant for at least 1 hour. However, necessary manipulation time for 10 or more samples would preclude the use of periods shorter than 25 minutes and, therefore, this was used as the development time in the method. All times are measured from the dilution of the contents of the flasks with distilled water. I n selecting the best ratio of colorproducing reagents, the experiments were directed toward producing a colored solution which would not be so optically dense as to be insensitive t o small amounts of fluoride, not be bleached completely in the 4- to 5p.p.m. range of fluoride concentration, and respond linearly in absorbance to fluoride concentrations over the range 0 to 5 p.p.m. On the basis of the data presented graphically in Figure 3, a ratio of 2 ml. of thorium nitrate solution to 2 ml.
Table 1. Effect of Varying pH on Absorbance of Thorium-Phenylfluorone Complex Solution
Buffer Composition, Meq. _____ Fluoride, C2H3- C&- Ahsorbance P.P.R?I. O * S B OICl 0 1 2 3 4
5
0 1 2
3 4 5
0 1 2 3 4
5
0 1 2
3 4
5 0 1 2 3 4
5
10 10 10 10 10 10
8 8 8 8
10 10 10 10 10 10
9 9 9 9
0,180 0,160 0.142
:. . 4
9 9
10
10
0,300
10
10 10 10 10
0.260 0.210
10
0.178 0.149 0,131
4.1 4.1 4.1 4.1 4.1 4.1
9 9 9 9 9 9
10 10 10 10 10 10
0.300 0.259 0.223 0.185 0 162 0 , 1:io
4.0 4.0 4.0 4.0 4.0 4.0
8 8 8 8 8 8
10 10
0,300
10
0 218 0.218 0.157 0.153
3.6 3.6 3.6 3.6 3.6 3.6
10 10 10 10
4
5
7
0
G
1 2 3 4
6 6 G 6 6
5
1 6 4 6 4.6 4 (i 1 6
0,300 0.252 0.210 0.176 0.143 0.136
7 7 7 7 7
0 1 2 3
8 8
irH
1 (i
0.300 0,254 0,210
10 10 10
10 10 10
4.4 4.4 4.4 4.4 4.1
0.283
0,300 0,278
10 10 10
0.217 0.204 0.172 0.110
3.4 3.4 3.4 3.4 3.4 3.4
10 10 10 10 10 10
0.300 0.282 0.250 0 .207 0.170 0.145
3.2 3.2 3.2 3.2 3.2 3.2
VOL 32, NO. 10, SEPTEMBER 1960
0
1331
phate ion is present in a concentration greater than 1 p.p.m., it must be removed by distillation. Interference due to iron is shown in Figure 5 . This interference is undoubtedly caused by the formation of a Fe(II1)-phenylfluorone comples. At a concentration above 0.5 p.p.ni., iron produces a deep blue as opposed to thti deeu rose of the thorium coniiilex. Since it became apparent that iron would interfere seriously, it was dccided to eliminate completely all cations through the use of an ion exchange resin. Xielsen (8) also has reported the successful use of such method.
0 30
I
b
Y
\
Ot{
3
i
03j 0
2
I
FLUORIDE
0 0
0 001 0
I
2 FLUORIDE
3 CONCEHTRATION
-
I FRM.
5
Figure 3. Effect of changing ratio of color producing reagents
0 Feu.
mlphole
I eeM,RwMte
4
-
5
P?M.
Figure tion
5. Effect of iron on determina-
Table
II.
0 301
I
i
Determination of Fluoride Concentration
Found, P.P.11. _ _ _ ~ !ifc,gregianYhenylfluorone Naier met hod method
Fluoridc of phenylfluoroiie solution b e 4 satisfied the conditions imposed. This method, like most other methods for fluoride determination, 11as expected to suffer from interferences due to certain cations and anions. Experiments were undertaken to establish ivhich ions commonly found in sui face water r$ ould interfere. The bicarbonate ion was eliminated since its existence would be precluded by the pH of the medium. Sulfate and nitrate were not investigated because of their presence in appreciable amounts in the reagents used. The anions investigated Ivere phosphate and chloride. Chloride did not interfere up to a concentration of 100 p.p.m. The extent of the interference of phosphate is shown in Figure 4. This phenomenon may be due to the formation of thorium phosphate which would destroy the thorium-phenylfluorone lake. If phos-
3
CONCENTRATION
Present, 1' .P.XI. 1. 0 2.0 3.0
1.1 2.0
1 .o 1.8
2 $1
2.7
3001 2 FLUORlOE
3 CONCENTRATION
-
I P?M
Figure 4. Effect of phosphate ion on determination
., l o tcst the effectiveness of
the resin, a solution containing Fe(II1) ( 5 p.p.ni.). Ca(I1) (50 p.p.m.), pllg(I1) (20 p.p.ni.), Al(II1) (10 p.p.m.), RIii(I1) ( 5 p.p.m.), and fluoride (20 p.p.m.) was prepared. Fifty-milliliter portions of this solution were passed through the column, which has already been described. Analj-sis of three such samples gave 19.0. 19.5] and 19.0 p.p.m. of fluoride. This method is compared nith the Negregian-Maier method ( 7 ) in Table 11. Interfering ions present were those listed above.
LITERATURE CITED
(1) Urownlcy, F. I., Sellers, E. E., J . A m , W a t e r Tt-orks Assoc. 49, 1234 (1957). (2) Bumstead, H. E., Wells, J. C., .4sa1,. CHEM.24, 1595 (1952). ( 3 ) Curry, R. P., Mellon, RI. G.> Ibid., 28, 1567 (1956). (4) Damodaran, V., J . Sci. Ind. Research ( I n d i a ) 16B,366 (1957). (5) Fenton, H. J. H., J . Chem. Soc. l',xizs. 93, 1064 (1908). (6) Hines, E., Baltz, D. E., ASAL. CIIEX. 24, 947 (1952). ( 7 ) Megregian, S., Maier, F. J., J . Ana. W a f e r W o r k s -1ssoc. 4 4 , 239 (1952). (8) Sielsen, H. XI,> ASAL, C H E l f . 30, 1009 (1958). RECEIVED for review December 14, 1959. Accepted June 16, 1960.
Determination of Piperazine as Piperazine Diacetate G. R. BOND, Jr. Houdry Process Corp., Marcus Hook, Pa.
b A rapid, yet accurate, method i s presented for the determination of the piperazine content of crude reaction mixtures encountered in its manufacture, as well as in its refined form. The method i s based upon ihe fact that piperazine is precipitated quantitatively and selectively (with the exception of certain of its homologs) from dilute solution in acetone upon
1332
ANALYTICAL CHEMISTRY
addition of at least the theoretical amount of glacial acetic acid required to form piperazine diacetate. cornOf the sample Other than piperazines either do not form pre-
ciPitates Or form Oils which are removed. This procedure has been used successfully to determine piperazine content ranging from 1 to 100%.
W
rapidly increasing use of piperazine and its salts in anthelmintics, antihistamines, surfaceactive agents, stabilizers, catalysts, and other pharmaceutical and agricultural products, the need has arisen for a rapid, yet accurate, method for the determination of the piperazine content of crude reaction mixtures encountered in its manufacture, as well ITH THE
