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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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.
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 8, JULY 1983
shows the chromatogram for the same column after being cut to 10 cm in length. Reducing the length of the column resulted in a substantial decrease in column back pressure and the decrease in retention time led to an increase in sensitivity. As expected, the retention time and the number of theoretical plates are reduced in proportion to the reduction in column length. Hence, the ratio of height equivalent of a theoretical plate (HETP) before and after cutting for three columns was 1.08, 0.93, and 1.09. The cutting of the column per se is accomplished in a few minutes because of the softness of nickel. Use of a tubing cutter permits shortening of columns with no disturbance of the column bed; the unused portion of a column is saved and fitted out as a second column. We have found this procedure to be suitable for routine assay methods because it allows us to easily adjust our chromatographic results without altering overall chromatographic conditions. Because of the relative hardness of stainless steel, we have not tried this technique on conventional stainless steel columns. As most liquid chromatographers do not use soft nickel tubing, a description of our other impressions of it may be useful. It is easy to handle with respect to cutting and attachment of column end fittings. The efficiency of columna packed in nickel tubing appears to be at least as good as
conventional stainless steel, although we have not studied this in detail. A potential disadvantage that we initially considered was the possibility of the reduced wall thickness leading to column distortions under pressure. This has not been a problem in the year or more that we have been using these columns even though they have sometimes been operated at high pressures (250-300 bar). Although nickel columns can be bent because of their dimensions and softness, this is not a problem with normal column handling procedures. As our experience with nickel has been so positive, we would like to encourage more liquid chromatographers and manufacturers to consider its use.
Registry No. Nickel, 7440-02-0. LITERATURE CITED (1) Majors, R. E. Anal. Chem. 1972, 4 4 , 1722-1726. (2) Nakatsu, K.; Owen, J. A.; Scully. K. C/ln. Blochem. 1978, 7 1 , 148-1 49.
RECENEDfor review January 10,1983. Accepted March 10, 1983. This work was supported by the Medical Research Council of Canada.
CORRECTION Thermal Vaporization for One-Drop Sample Introduction into the Inductively Coupled Plasma Eiichi Kitazume (Anal. Chem. 1983,55, 802-805). There is a typographical error in one of the names in the Acknowledgment. The correct spelling of the name is Hiroshi Kawaguchi.
