1370
Anal. Chem. 1980,
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Panthenol is available in various grades which have specific uses in pharmaceutical preparations; the grade is determined by the amount of free aminopropanol (precursor to panthenol) present in the sample. An existing method for free aminopropanol employs potentiometric titration (11) in which very large amounts of sample are required in order to obtain an accurate result. This method may be used to determine the free aminopropanol content of samples if the hydrolysis step of the procedure is omitted. Figure 3 demonstrates the applicability to the determintion of free aminopropanol at a level of 0.1% on a 4-mg/mL sample solution. The method discussed in this report is specific and rapid for the determination of panthenol and aminopropanol, and can be used to monitor trace levels of these compounds. The procedure may also be applied to the analysis of other amine-containing compounds.
52, 1370-1371
LITERATURE CITED (1) Rogers, G. G.; Campbell, J . A. Anal. Cbem. 1960, 32, 1662. (2) Myszkowska, K.; Tautt, J.; Tuszynska, S.; Wozniak, W. Acta Polon. Pbarm. 1964, 21, 84. (3) Bird. 0.D.; McCready, L. Anal. Cbem. 1958, 30, 2045. (4) Matta, G.; Lopez, E. S . Rev. Farm. 1966, 16, 452. (5) Painier, R. G.; Close, J. A. J . Pharm. Sci. 1964, 53, 108. (6) Wollish, E. G.; Schrnall, M. Anal. Chem. 1957, 2 9 , 1509. (7) Wollish, E. G.;Schrnall, M. Anal. Chem. 1950, 2 2 , 1033. (8) Vacheck, J . Pharmazie 1966, 21, 222. (9) Zappala. A. F.; Simpson, C. A. J . Pbarm. Sci. 1981, 50, 845. (10) Bergmann, F. Anal. Cbem. 1952, 24, 1367. (1 1) Pifer, C. W. Unpublished work, “The Chemical Assay of Bulk Panthenol”; Hoffmann-La Roche Inc.: Nutley, N.J., 1956. (12) Weigele, M.; De Bernardo, S.; Tengi, J.; Leimgruber, W. J . Am. Ctwm. SOC. 1972, 94, 5927.
RECEIVED for review October 10, 1979. Accepted March 27, 1980.
Nomogram for Adjusting Mobile Phase Composition in Reverse Phase High Pressure Liquid Chromatography James L. Meek Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeth’s Hospital, Washington,
One of the first steps in the designing of an assay of a compound by reverse phase high pressure liquid chromatography (HPLC) is to find the mobile phase composition that produces an optimal elution time for the compound. If an initial trial shows that the compound elutes either too close to the time of the void volume or too slowly for convenience, a series of trials is required with decreased or increased, respectively, concentrations of organic solvent until the elution time is in the desired range. If the relationship between retention and concentration of organic solvent were known, it would be possible after the initial trial to calculate just how much to change the percent of organic solvent in order to achieve an optimal retention time. There have been a few studies ( I , 2 ) involving mobile phase composition and retention, but they were limited in the range of polarity of compounds or range of solvent concentrations studied. This paper will show that a simple relationship can be used to describe retention on an octadecylsilyl column vs. mobile phase composition for compounds with a wide range of polarity (catecholamines to anthracene), and a wide range of mobile phase conditions (2.5-80% acetonitrile or methanol in HzO).
EXPERIMENTAL The HPLC apparatus consisted of a Rheodyne sample valve, an Altex model llOA pump, a 25 cm X 4 mm BioRad octadecylsilyl column (10-pm particle size), Altex-Hitachi variable wavelength spectrometer, and Houston Instruments recorder. Mobile phases consisting of 2 5 8 0 % (v/v) organic solvent,0.1% H3P04(to adjust the pH to 2.1 (3))and 0.1 M NaC104 (to prevent tailing of the basic compounds) were pumped at room temperature at 1.5 mL/min through the column. The measure of retention used was k’, defined as ( t , - t o ) / t o where t, is the retention time of the compound,and t ois the elution time of an unretained compound. For an unretained compound, NaN03 was chosen. For this study, mobile phase composition vs. k’data were excluded when the observed k’was less than 0.4 or greater than 30. Regression lines were fitted using the unweighted least squares method. 0003-2700/80/0352-1370$01.00/0
D.C. 20032
RESULTS AND DISCUSSION Retention was measured for 10 compounds listed in Figure 1 including neutrals, acids, and bases a t mobile phase concentrations of acetonitrile in water or methanol ranging from 2.5 to 80% (v/v). When log k’was plotted vs. log % acetonitrile (Figure l),essentially straight lines were obtained for each compound. The correlation coefficient r for the compounds ranged between 0.992 and 0.999. However, the slope and y intercept differed for each compound (increasing with increasing lipophilicity). Qualitatively similar data (not shown) were obtained with methanol, the methanol giving longer retention times than acetonitrile for each compound a t any given percent solvent composition. Since log k’ was approximately a linear function of log 70 organic solvent in this range of concentrations, these two parameters were used as the scales for the construction of a nomogram (Figure 2) ( 4 ) . For each compound, lines were drawn connecting each mobile phase composition tested with its observed k’. A point was placed at the intersection of these lines which represents for that compound the relationship between k’and 70 organic solvent. The points for these 10 compounds (Figure 2) lie near a straight line regardless of the compounds’ polarity for both the organic solvents tested. The actual location of a point along that line depends on the polarity of both the solute and the organic solvent, but neither quantity needs to be known to use the nomogram. T o use the nomogram (Figure 2), connect with a straightedge the observed k’ and known mobile phase concentration and note the intersection with the center line. Rotating the straightedge around this point will show what mobile phase conditions would be needed to achieve a given k’ for that compound. As an example (dotted lines), suppose that a k’ of 3 was desired for a compound. In an initial trial a t 10% acetonitrile, the observed k‘was 10. After connecting these points, rotating the straightedge around the point of intersection with the center line reveals that a mobile phase of approximately 20% acetonitrile should be tried to achieve a Q 1980 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. !52,NO. 8, JULY 1980
1371
70 O r g a n i c
k'
Solvent
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k'
\
0 5
0 4
7
e
1 5
5
10
10
40
-
60
BO
70 A c e t o n i t r i l e Figure 1. Linearity of log k'with log % acetonitrile (v/v) on a Bic-Rad "ODs-10'' column. Compounds: (1) epinephrine, (2) dopamine, (3) tyrosine, (4) 5-hydroxytryptophan, (5) benzyl alcohol, (6) phenylacetic acid, (7) benzaldehyde, (8) methyl benzoate, (9) naphthalene, (10)
anthracene
0 . 0I 0.9
k'of 3. Caution: ionic compounds may not show this relationship unless the mobile phase contains salts to reduce adsorption. In both adsorption and partition chromatography, the retention of a compound can be empirically expressed (5) by an equation of the form log k ' = a log b c (1)
+
where the value of coefficient a is dependent on the nature of the compound, and b and c are dependent on the chromatographic system. When a mixed solvent is used, the value of b must depend on the relative amounts of the 2 solvents and their nature. For reverse phase chromatography, log k ' was found to be linear with percent organic solvent over the range 20-90% (2). For very nonpolar compounds in reverse phase HPLC, a better fit was found for log k ' vs. log concentration of organic over a similar range of concentrations (1).
In the present study, the data fit an equation of the form log k' = a log % organic log c (2) where a and c both depend on the nature of the compound and the nature of the organic solvent for concentrations ranging between 2.5 and 80%. Outside of this range (below 2.5% organic), observed and predicted values for k 'diverge since the equation predicts that retention will increase without limit as the percentage approaches zero, whereas in reality the k'at 0 % acetonitrile are only 2-3 times greater than a t 2.5% acetonitrile. Although a rational equation might be derived from basic principles which could cover the complete range of solvent concentra-
+
t
5
1 2 . 5
Figure 2. Nomogram relating retention (k') to the concentration of
acetonitrile or methanol in the mobile phase. Data points used in constructing the nomogram were obtained with acetonitrile (m) or methanol ( 0 ) tions, the empirical relationship described here should be sufficient for the purposes of mobile phase selection. The data used in preparing this nomogram were obtained on only a single Bio-Rad octadecylsilyl column with a limited number of sample compounds. Examination of data taken from the graphs in several reports of k'vs. % organic solvent (1, 6, 7) suggests that the nomogram should be useful with any octadecylsilyl column with high carbon loading. For other types of columns, a nomogram could be readily made using a piece of semilog graph paper and the k'at 3 different organic solvent concentrations for 3 compounds of interest.
LITERATURE CITED (1) F. Murakami. J . Chromafogr., 178, 393 (1979). (2) L. R . Snyder and J. J. Kirkland, "Introduction to Modern Liquid Chromatography", John Wiley and Sons, New York, 1974, p 466. (3) W. S . Hancock, C. A. Bishop, R. L. Prestidge, D. R. K. Harding, and M. T. W. Hearn, Science, 200, 1168 (1979). (4) D. S. Davis "Nomography and Empirical Equations". Reinhold, New York, 1962, p 211. (5) L. R. Snyder "Principles of Adsorption Chromatography", Marcel Dekker, New York, 1968, p 138. (6) P. Lindroth and K. Mopper, Anal. Cbem.. 51, 1667 (1979). (7) A. P. Graffeo and B. L. Karger. Clin. Cbem., 22, 184 (1976).
RECEIVED for review January 28,1980. Accepted April 7,1980.
