1454
Anal. Chem. 1983, 55, 1454-1455
Preparation, Titratlon, and Storage of Chloroacetaldehyde for Fluorometric Determination of Adenine and Its Derivatives Wllllam P. McCann,"' Leo M. Hall,2 and William K. Nonldeza University of Alabama In Birmingham, Unlverslty Station, Birmingham, Alabama 35294
Chloroacetaldehyde is used as a reagent which converts adenine and ita nucleosides to fluorescent etheno derivatives which can be determined in chromatographic systems including high-performance liquid chromatography (1-5). This reagent is suitably prepared, for fluorometric procedures, from ita dimethyl acetal (1). The chloroacetaldehyde concentration in such preparations can be determined by a variety of methods. In preparing and using this reagent, we desired a relatively rapid procedure with small volume requirements and needing no specialized equipment. We have adopted a modification of a non-iodometric titration procedure (6)which, in our hands, is more convenient to use than other methods known to us for this purpose. It is suitable for a relatively nonvolatile aldehyde such as chloroacetaldehyde. This method is presented below. Also presented are data on the yields of chloroacetaldehyde obtained in our preparations and on the stability of the reagent on storage, about which we found little information in the literature.
EXPERIMENTAL SECTION Chloroacetaldehyde dimethyl acetal (97%) purchased from Aldrich Chemical Co. (Milwaukee,WI). Chloroacetaldehyde was prepared from the dimethyl acetal by a procedure based on that of Avigad and Damle ( I ) . After 100 mL of chloroacetaldehyde dimethyl acetal mixed with 30 mL of '1.5 M HzSO4 and a few boiling stones was refluxed for 30 min in a 500-mL round bottom flask, heated by an electric mantle, the mixture was then distilled from the same flask at atmospheric pressure, monitoring the temperature of the distillate. The 70 to 80 "C fraction (about 20 mL) was discarded. The 80 to 92 "C fraction (about 70 to 90 mL) was collected, and the remainder, marked by a rapid temperature rise, was discarded. The collected fraction was stored in a glass-stoppered bottle at 4 "C, in the dark. Chloroacetaldehyde is toxic (7)and all contact with liquid and vapor should be avoided. The same applies to ita dimethyl acetal. Three such preparations of chloroacetaldehyde were titrated by a modification of the method of Hanna and Siggia (6). To carry out the titration, a stock solution of 1 M Na2S03is first prepared and adjusted to pH 9.1 with a suitable volume of 1 M NaHS03 or 0.5 M Na2S205. To a 125 mL glass-stoppered flask, 25.0 mL of 1M Na2S03(pH 9.1) is added. Then, 5.00 mL of standardized 0.50 M H2S0, is slowly added with continuous mixing or swirling, followed by 0.50 mL of the chloroacetaldehyde preparation. With the glass stopper in place, the mixture is shaken until no droplets of chloroacetaldehyde are visible. The mixture is transferred quantitatively, with several small water washes, to a 100-mLbeaker. The solution volume is now about 50 mL. With continuous mixing, 1.0 M standard low-carbonateNaOH is slowly added until the pH i s 9.1, determined with a pH meter. Two additional titrations are needed (a) titration of identical aliquots of Na2S03and HzSO4, treated as above but without addition of chloroacetaldehyde; (b) titration of a mixture of 0.50 mL of the chloroacetaldehyde suspended in 50 mL water, without NazSOB or H2S04.The f i t of these additional titrations (a) gives an exact check of the equivalency of the HzS04and NaOH solutions used. The second (b) gives evidence of the oxidation product chloroacetic acid, in the chloroacetaldehyde. If this is found, the calculation of chloroacetaldehyde concentration must include this titration (see below). Department of Pharmacology. 2Department of Biochemistry. Department of Chemistry. 0003-2700/83/0355-1454$01.50/0
RESULTS AND DISCUSSION The principle of the chloroacetaldehyde titration is as follows. A known excess of HS03- is created by adding a known amount of H2S04to a NazSO9solution forming a buffer system of S0?-/HSO3- with a pK, of 6.91 (8). Chloroacetaldehyde, added to such a mixture, forms an addition product illustrated below, binding HS03-. The remaining HSO, can be quantitated with standard NaOH by titration to pH 9.1. The reaction can be written as follows:
HS03-
+ ClCHzCHO
3
ClCHzCH(OH)SO,
The equilibrium of the reaction appears to lie well to the right, as illustrated above. When titrations are done with ordinary speed the reverse or leftward reaction rate appears to be slow in the case of chloroacetaldehyde (6), even as HS03- is converted back to S032-by NaOH addition. The molarity of the chloroacetaldehyde is calculated from the difference between the initial millimoles of HSO, formed and the final millimoles of HS03- found after reaction with the chloroacetaldehyde, correcting for acid in the chloroacetaldehyde if necessary, and the volume of the aliquot titrated. An illustrative titration of the first chloroacetaldehyde preparation is as follows: Using the method described above, 3.60 mL of 1.00 M NaOH was needed to bring the pH of the titration solution from slightly over 7 to 9.10 when 0.50 M H2S04was used and when 0.50 mL of the chloroacetaldehyde preparation was present. Without chloroacetaldehyde, an identical mixture of NasS03 and HzS04required 5.00 mL of 1.00 M NaOH. Separate titration of 0.50 mL of chloroacetaldehyde in 50 mL of water required 0.03 mL of the same NaOH, indicating very little acid contamination. The calculated molarity of the chloroacetaldehyde from these data is (5.00 - 3.60 - 0.03)/0.50, or 2.74 M. The only problems known to us in the titration procedure are: (1)a sufficient amount of chloroacetaldehyde must be used to ensure the precision of the titration; (2) this amount must be less than the molar equivalent of HSO, in the system; (3) the NazS03solution must be continuously mixed during the slow addition of HzS04. Otherwise, local high concentrations of the acid will form HzSO3 and liberate SOz into the air which impairs accuracy, and is very unpleasant for nearby personnel unless a hood is used. To evaluate the accuracy and reproducibility of the method, the following trials were carried out. The first chloroacetaldehyde preparation was titrated by an iodometric method (9) as well as by the method given above. An average value from each type of titration indicated 2.75 M chloroacetaldehyde. The iodometric method, however, was more difficult to perform as the reagents were not easily prepared or stadardized, the end points of the titrations, using starch indicators, were not easily determined, and the range of values from four individual titrations was 2.55-2.95 M. In contrast, five replicate titrations with the noniodometric method, using 0.5-mL aliquots of the chloroacetaldehyde, gave values ranging from 2.68 to 2.82 M, with a standard deviation of 0.055. At a mean value of 2.75 M, this is a relative standard deviation of 2%. The method was also tested by titrating 0.25 mL and 1.0 mL aliquots of the chloroacetaldehyde solution. 0 1983 American Chemical Society
Anal. Chem. 1983,
The results of these titrations agreed within 2% of the mean value of 2.75 M, above. A second chloroacetaldehyde preparation, titrated with the non-iodometric procedure, was found to be 1.77 M. A third preparation similarly titrated was found to be 3.96 M. Examination of the volumes collected during distillation indicates that molarity of the chloroacetaldehyde solutions increased with incrleasing volume of distillate collected within the 80-92 "C temperature range. As judged by serial titrations, the first preparation has remained stable for 15 months to date, the second for 3 months, ,end the third for 2 months, all stored as described (Methodfn). No preparation was found to contain significant amounts of chloroacetic or other acid, even after 15 months of refrigerated storage of the first preparation. We have used these chloroacetaldehyde preparations to form etheno derivatives of adenine, adenosine, arabinosyladenine, and the corresponding 5'-nucleotides using the method of Kutteseh et al. (3). These solutions were tested in a Perkin-Elmer 650 10s spectrophotofluorometer by using an exciting wavelength of 315 nm and emitted wavelength of 415 nm. Our findings ( l o ) ,not otherwise given here, are comparable with the high sensitivity of the methods described previously (1-5). In addition, the etheno derivatives appear to be stable for at least 3 weeks when stored in neutral aqueous solution a t 4 OC in the dark, after diethyl ether extraction to remove excess chloroacetaldehyde as described by others (3).
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55, 1455-1456
Reagent fluorescence blanks are low when suitable water and reagents are used. Registry No. Chloroacetaldehyde, 107-20-0; chloroacetaldehyde dimethyl a'cetal,97-97-2;bisulfite, 15181-46-1;adenine, 73-24-5.
LITERATURE CITED Avigad, G.; Damie, D. Anal. Blochem. 1972, 5 0 , 321-323. Barrio, J. R.; Secrist, J. A,, 111; Leonard, N. J. Blochem. Biophys. Res. Commun. 1872, 46, 597-604. Kuttesch, J. F.; Schmalstieg, F. C.; Nelson, J. A. J . Llq. Chromatogr. 1978, I , 97-109. Nelson, J. A,; Kuttesch, J. F.; Goidblum, R. M.; Goidman, A. S.; Schmalstieg, F. C. I n "Physiology and Regulatory Functions of Adonoshe and Adenine Nucleotides"; Baer, H. P., Drummond, G. I., Eds.; York, 1979 pp 417-427. Raven Press: NW\N Leonard, N. J.; Barrio, J. R.; Secrist, J. A,, 111 Biochim. Biophys. Acta 1972, 269, 531-532. Hanna, J. G.; Siggia, S. I n "Treatise on Analytical Chemistry" (Part 11); Kolthoff, I.M., Elving, P. J., Eds.; Interscience: New York, 1966; Voi. 13 (Functional Groups), pp 204-205. Lawrence, W. H.; 13iillngham, E. 0.; Turner, J. E.; Autian, J. J . Pharm. Scl. 1972, 61, 19-25Weast, R. C., Ed. "Handbook of Chemistry and Physics", 54th ed.; Chemical Rubber Publishing Co.: Cleveland, OH, 1973-1974 p D130. Willard, H. H.; FurMan, N. H.; Brlcker, C. E. "Elements of Ouantltaiive Analysis", 4th ed.; Van Nostrand: Princeton, NJ, 1956; Chapter 15. McCann, W. P.; Hail, L. M.; Siler, W.; Whitley, R. J.; Furner, R. L.; Hafer, L. M.; Barton, N. J. Fed. Proc., Fed. Am. SOC.Exp. 8/0/. 1883,42, 1140.
RECEIVED for review January 6, 1983. Accepted April 4,1983. This work was supported by a Grant (AI-16444) from the U,S. Public Health Service.
Shortening of Liquid Chromatography Columns for Reduced Retention Time KanJiNakatsu," R. Andrew R. Tasker, and Maria BukowskyJ Department of Pharmacology and Toxlcology, Queen's Unlv@rsi@,Kingston, Ontario, Canada K7L 3N6
We have packed our own HPLC columns for approximately 6 years for use in the assay of various drugs and toxicants in biological samples. Because of curiosity and convenience, we started to use nickel column blanks in place of stainless steel blanks about 2 years ago. Occasionally, our packed columns have retention times for analytes that are longer than desired and resoluition that is greater than required. In such cases, we have found it convenient to leave the chromatographic conditions unaltered and to cut the column to an appropriate length.
EXPERIMENTAL SECTION The column hardware consisted of nickel tubing, in. 0.d. (3.175 mm),,with an internal diameter of 2.1 mm (Chromatographic Specialties,Brockville, Canada) and stainless steel column end fittings (SS-200-6-IZV, Swagelok) with 0.5 wm porosity, stainless steel frits. Columns were packed by the method of Majors ( 1 ) . To reduce the length of columns, the columns were cut with an ordinary tubing cutter and fitted with another column end fitting. For lthe data reported here, the column was used for the assay of theophylline in biological samples by the method of Nakatsu et al. (2).
RESULTS AND DISCUSSION Typical results of cutting an HPLC column are shown in Figure 1. The lower tracing (A) shows the chromatogram for an uncut column 25 cm in length and the upper tracing (B) 0003-2700/63/0355- 1455$01.50/0
A
1 1 1 1 1 1 1 0
2
4
6
8 1 0 1 2
TIME (min)
Flgure 1. Chromatograms before and after cutting the HPLC column Chromatographic conditions were room temperature, flow rate 1 mL/min, stationary phase 5 Mm silica gel (Partisil 5), mobile phase hexane/2-propanol/water (80: 19: l), detection UV at 280 nm. Injections oftheophylline (1 pg) were at the triangles. The lower tracing (A) is for the uncut 25 cm long column and the upper traclng (6)is for the cut 10 cm long colurrin.
0 1983 American Chemical Society
