Possible Future Extensions. The applicability of the proposed idea can be broadened in several ways. First, NaCeEDTA 8Ht0 and other analogous lanthanide salts might be used with xylenol orange in the development of a general procedure for the determination of a wide variety of metals at microgram levels. Second, ytterbium-EDTA (log Kj ‘U 19.9) might be used to determine selectively those metals that form highly stable EDTA complexes. Finally, substitution of DTPA (diethylenetriamine pentaacetic acid) for EDTA
may permit the determination of those elements that currently are not determinable because of their tendency to hydrolyze and to react irreversibly with the indicator. YtterbiumDTPA, for example, has been used effectively for the titration of thorium in pyridine-acetate medium to a xylenol orange end point. RECEIVED for review February 5, 1968. Accepted June 25, 1968.
Determination of Inorganic Phosphate in the Presence of Adenosine Triphosphate by the Automatic Reaction Rate Method S. R. Crouch and H. V. Malmstadt Department of Chemistry and Chemical Engineering, University of Illinois, Urbana, Ill.
I N A RECENT article ( I ) , we presented a new analytical method for inorganic orthophosphate based on automated measurements of the initial rate of formation of molybdenum blue from phosphate, molybdate, and ascorbic acid. In comparison to the classical molybdenum blue procedure for phosphate (2), which requires 5-10 minutes for color development, the reaction rate procedure requires only 20-30 seconds of reaction time per sample. Thus, the rate procedure should be advantageous for the determination of inorganic phosphate in biological samples which contain readily hydrolyzable phosphate esters. Several authors have discussed the problems of phosphate determinations in such samples (3, 4). The hydrolysis of phosphate esters, such as adenosine triphosphate (ATP), is both acid and molybdate catalyzed ( 4 , 5). One common modification of the molybdenum blue procedure (6) allows the reaction to be carried out at pH 4 minimizing the acidcatalyzed hydrolysis. However, as has been pointed out (3, pH 4 is optimum for the molybdate-catalyzed hydrolysis of the terminal phosphate of ATP. Because of the slowness of the color development, almost all the common methods for the determination of phosphate utilize 5-10 minutes of reaction time during which extensive hydrolysis of phosphate esters can occur. EXPERIMENTAL
The spectrophotometric reaction rate measuring system has been previously described ( I ) . Samples were analyzed by both the reaction rate method ( I ) and the method of Fiske and Subbarow (2). A 10-minute color development period was used with the latter method. A Spectronic 20 colorimeter (Bausch & Lomb) was used for analyses by the method of Fiske and Subbarow. Solutions containing ATP were prepared from the disodium salt (Sigma Grade, Sigma Chemical Co., St. Louis, Mo.). (1) S. R. Crouch and H. V. Malmstadt, ANAL.CHEM.,39, 1090 (1967). (2) C . H. Fiske and Y . Subbarow, J . Biol. Chem., 66, 375 (1925). (3) M. Blecher, Anal. Biochem., 7, 383 (1964). (4) B. B. Marsh, Biochim. Biophys. Acta, 32,357 (1959). (5) H. Weil-Malherbe and R. H. Green, Biochem. J., 49, 286 (1951). (6) 0.H. Lowry and J. A. Lopez, J.Biol. Clzem., 162,421 (1946).
61801
I[d G/
15 sec
,
TIME
Figure 1. Recorded reaction rate curves for inorganic phosphate and for ATP (a).
(b).
4 ppm P 10-ZM ATP
Blood serum samples were deproteinized with trichloroacetic acid and centrifuged as previously described ( I ) . For the rate measurements, neutralization of the serum supernatant is necessary because of the strong dependence of the rate on sample acidity (7). Serum supernatant analyzed by the Fiske and Subbarow method was not neutralized. RESULTS AND DISCUSSION
In Figure 1, recorded reaction rate curves for the formation of phosphomolybdenum blue from inorganic phosphate and from ATP are shown. After an initial increase of absorbance upon starting the reaction with ascorbic acid, ATP shows only a very small rate even at the 10-2M level. After about
(7) S. R. Crouch and H. V. Malmstadt, ANAL.CHEM.,39, 1084 (1967).
VOL 40, NO. 12, OCTOBER 1968
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Table I. Determination of Inorganic Phosphate in Control Blood Serum with and without Added ATP Inorganic phosphorous, mg/100 ml of serum Found by Found by Reporteda F-Sb R.R.O 4.6 4.6d 3.8 3.8e
4.5 7.1 3.8 6.9
4.48 4.54 3.95 3.98
Manufacturer’s value. Fiske-Subbarow analysis (2). Reaction rate analysis (1). d Blood serum doped with solid ATP to make 1@1M ATP. e Blood serum doped with solid ATP to make 10-2 ATP. a
b
30 seconds, phosphate can be determined with very little interference from ATP hydrolysis, even when ATP is present in about 100-fold molar excess. The experiment illustrated in Figure 1 indicates that the
h a 1 step in the analysis of a biological sample can be accomplished by the reaction rate procedure with very little hydrolysis of ATP. To test the entire analysis procedure, blood serum samples with accurately known amounts of inorganic phosphate were doped with ATP and analyzed by the rate method and the Fiske-Subbarow method. Table I shows the results obtained on control serum with and without added ATP. The highest ATP content represented about a 100-fold molar excess over phosphate. These results indicate that the reaction rate procedure for phosphate is capable of high specificity. Hydrolysis of ATP is seen to contribute only a very small error even when ATP is present in large excess. Because of convenience and the availability of control samples, only blood serum was utilized as the biological sample. However, these results can be directly related to other biological materials for the same analysis steps (deproteinization, centrifugation, and analysis of the supernatant) are required. RECEIVED for review May 13, 1968. Accepted June 17,1968.
On-Column Formation and Analysis of Trimethylsilyl Derivatives George G . Esposito U.S . A r m y Coating and Chemical Laboratory, Aberdeen Proving Ground, M d . 21005
TRIMETHYLSILYL (TMS) reagents are used extensively in gasliquid chromatography (GLC) to improve the chromatographing properties of various chemical types. The reaction is relatively simple and rapid; reactive species yield compounds which are more volatile, less polar and thermally stable. A number of methods have been employed for TMS derivatization. These include procedures for alcohols and amines (I), phenols (Z), sugars (3),amino acids (4) and polyols (5); Mason and Smith (6) investigated the quantitative aspects of the technique. Even though the above methods have found great utility in GLC analysis, they all suffer from one deficiency; they are not applicable to compounds mixed with reactive solvents. In his review on reaction gas chromatography (7), Beroza referred to the on-column mode of derivative formation, but the methods were primarily concerned with the “peak-shift” technique and did not mention the on-column formation of TMS derivatives. A method is presented which describes an on-column technique for the formation of TMS derivatives of certain classes of compounds and their subsequent chromatographic analysis, The principal feature of the technique is the ability to accommodate aqueous and alcoholic solutions, thus avoiding excessive sample manipulations. The concept is illustrated by application to fatty acids, carboxylic acids, and polyhydric alcohols. ~~
(1) S. H. Langer, S. Connell, and I. Wender, J. Org. Chem., 23, 50 (1958). (2) S . Friedman, C. Jahn, M. Kaufman, and I. Wender, Bur. Mines BUM.609 (1963). (3) C. C. Sweeley, R. Bentley, M. Makita, and W. W. Wells, J. Amer. Chem. SOC.,85,2497 (1963). (4) K. Ruhlman and W. Gresecke, Angew. Chem., 73,113 (1961). (5) B. Smith and 0. Carlsson, Acra Chem. Scad., 17,455 (1963). (6) P. S. Mason and E. D. Smith, J. Gas Chromatog., 4, 398 (1966). (7) M. Beroza and R. A. Coad, ibid., p 199.
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ANALYTICAL CHEMISTRY
A solution containing the sample is injected onto the chromatographic column and is immediately followed by an injection of TMS reagent. The interval between injections is all the time required for the water and alcohol to separate from the rest of the sample. TMS derivatives are formed as the reagents pass through the zones occupied by reactive compounds and are chromatographed as they traverse the remaining segment of the column. EXPERIMENTAL
Reagent. The trimethylsilation reagent used throughout the investigation was a commercial product recommended for the conditioning and treating of chromatographic columns. It is distributed by the Pierce Chemical Co. (Rockford, Ill.) under the trade name, SILYL.8 and consists of three different TMS donors, N,O-bis-(trimethylsily1)-acetamide, trimethylsilyldiethylamine, and hexamethyldisilazane. The details of the method used to obtain the experimental data are presented in Table I. The test is conducted by preloading the sample and reagent syringes; the sample is introduced first, immediately followed by the reagent.
RESULTS AND DISCUSSION A 1 solution of polyols in a mixture of alcohol and water was analyzed and produced the chromatogram shown in Figure 1. When a water solution of the same mixture was tested, the results were essentially the same. There is an obvious decrease in yield as the boiling point of the poly01 increases. A probable explanation is that the time duration of contact between reagent and poly01 is greatest with the lower boiling polyols. . When a synthetic mixture of saturated fatty acids was injected onto the column from an alcohol solution, the oncolumn reaction with reagent produced the chromatogram
