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Anal. Chem. 1981. 53. 1142-1 144
DC level to that recorded in the wavelength in the base line. RESULTS AND DISCUSSION The extremely simple stepping motor scheme for controlling the monochromator which is outlined above is constructed and wired from commercially available components. The pulse generation for the motor steps is done completely in software and there is no provision for hardware encoding of the actual wavelength. Once the current wavelength setting is entered in the program, the computer calculates all subsequent wavelengths from the number and direction of step pulses which have been executed. We routinely acquire as many as 10 scans of the CD spectrum for the purposes of signal averaging without any observable discrepancy arising between the actual wavelength setting of the monochromator and that stored in the computer. The scanning speed is limited by the mechanical properties of the McPherson Model 218 vacuum monochromator. A typical scan covers a range of 100 nm a t a speed of 20 nm/min. For archival storage of CD data on the floppy disk, we have chosen to store 32 complex Fourier coefficients for each scan. In practice we have not needed more than 8-10 coefficients for data of the type described here and in a previous effort (2). This indicates that further economy of storage space would be possible using this scheme. Storage of 32 Fourier coefficients requires approximately lo00ASCII characters per
scan allowing data for more than 200 scans to be stored on a single density floppy disk. The absorption measuring scheme we have outlined above is approximately equivalent to a double beam spectrophotometer only if the output of the light source in the instrument is stable over the time necessary to scan the base line and sample. We have observed that the output of the Hanau 200-W deuterium source is stable to within *0.5% over time periods of at least 1h. Thus our spectrophotometric method is found to give results in agreement with those of an accurate double beam instrument within an accuracy of f0.05 absorbance units for solutions of less than unit absorbance. At higher optical densities, the stray light of the McPherson monochromator becomes significant. Listings of the computer programs used in this work are available from the authors on request. They are written almost entirely in FORTRAN, with a few subroutines in PDP-11 assembly code.
LITERATURE CITED (1) Duben A,; Bush, C. A. Anal. Chem. 1980, 52, 835. (2) Bush, C. A. Anal. Chem. 1974, 46, 890-895.
RECEIVED for review November 3,1980.
Accepted March 23, 1981. This research was supported by NSF Grant PCM7918887.
Quantitative Determination of Compounds in Aqueous Methylcellulose by Reversed-Phase Liquid Chromatography Linda L. Ng Merck Sharp and Dohme Research Laboratories, West Point, Pennsylvania 19486
Methylcellulose is a cellulose ether (1) CH.OH r H PH CH,OH 1 9" HO ~~~~~H ii
C e l l u l o s e chain
0
OH
C&OH
H
H
CHsOH
that has no ill effects on experimental animals (2,3)or man (4) after consumption for extended periods. Hence, it has been used widely by the food, pharmaceutical, and cosmetic industries in products such as swelling, coating, molding, bulking, granulating, and suspending agents, thickeners, stabilizers, emulsifiers, surfactants, and adhesives (5). In toxicological studies of drugs, aqueous methylcellulose at a level of 0.5 or 1.0% is frequently employed to obtain uniform suspensions of new/investigational drugs for gavage dosing. The drugs of interest are usually polar and lend themselves readily to determination by reversed-phase highperformance liquid chromatography (HPLC). However, methylcellulose as a suspending agent has a major drawback since direct analysis of diluted samples frequently causes immediate HPLC column blockage. Even extensive cleaning will not salvage the blocked column. The enormous number of samples to be processed in such studies often necessitates a rapid and efficient cleanup procedure. Consequently, we have developed a simple method using sodium chloride to eliminate most of the methylcellulose. The method has the required precision, accuracy, and ease of sample processing. EXPERIMENTAL SECTION Materials. All drug substances were obtained from Merck & Co., Inc. (West Point, PA). Acetonitrile was from Burdick and Jackson Laboratories, Inc. (Muskegan, MI). Purified formic acid (go%), primary standard potassium phosphate, and laboratory 0003-2700/81/0353-1142$01.25/0
grade granular sodium chloride were purchased from Fisher Scientific Co. (Fair Lawn, NJ). Methylcellulose USP 400 CP s was obtained from Callahan Chemicals (Palmyra, NJ). Instrumentation. The instruments used were a Branson sonic bath, IEC Model HN tabletop centrifuge,Hewlett-Packard Model 1084B high-pressureliquid chromatograph with auto sampler and Waters pBondapak/C18 reversed-phase column attached to a Brownlees RP-8 guard column, V W R vortex mixer Model 550-G, Beckman research pH meter, and an Ubbelhode viscometer with MGW Landa Model B-1 thermostat control water bath. Viscosity Measurements. To 4 mL of 0.5% (w/v) methylcellulose, various weights of sodium chloride were added. After Vortex mixing, the sample was centrifuged. Two milliliters of the clear supernatant was removed by pipet and diluted to 25 mL with water. Viscosity was measured at 25 0.03 "C. The process was repeated at 25,37, and 50 O C with 0.40 g of sodium chloride and at pH 1.5, 3.3, 6.2, 8.0, and 11.8 with 0.65 g of sodium chloride. The pH was adjusted with hydrochloric acid and sodium hydroxide. The standard curve was obtained by dilution of specified volumes of 0.5% methylcellulose to 25 mL with water. Chromatographic Measurements. Chromatographic conditions are described with each chromatogram in Figures 1-3. Sample Preparation. Solution. To 4 mL of an aqueous methylcellulose solution of the drug in a 50-mL centrifuge tube, 20 mL of acetonitrile and 0.65 g of sodium chloride were added with immediate vortex mixing after each addition. The upper layer was transferred to a volumetric flask and 20 mL of acetonitrile added for a second extraction. The solution was Vortex mixed and centrifuged for 5 min. The organic layer was then transferred to the same volumetric and diluted to the desired concentration with water or water-acetonitrile mixture as needed. Suspension. Acetonitrile was added to the suspension of drug in methylcellulose (about 2:3 v/v) in a 25-mL volumetric flask and reduced to a solution by ultrasound. Eight milliliters was
*
0 1981 Amerlcan Chemical Soclety
ANALYTICAL CHEMISTRY, VOL. 53, NO. 7, JUNE 1981
1143
10
0
I
I
I
05
10
15
lVaCl ~n g per 4 mLof 0 5 % Methylcellulose diluted 10 25 rnl
JU i
Flgure 1. Quantitation of MK-421 after removal of methylcellulose
Figure 4. Effect of added sodium chloride on methylcellulose viscostty.
using 33% acetonitrile in flormic acid-water (1:70), 2 mL/min flow rate, 50 OC oven temperature, and 215-nm detector.
6
20
30
40
50
O C
100064
' I1
~
Flgure 5. Effect of temperature on precipitation of methylcellulose in
' I
-..-
Flgure 2. Quantitation of mevinolin after removal of methylcellulose using 60% acetonitrile in 0.01 M pH 4 potassium phosphate buffer,
2 mL/min flow rate, 30 O C oven temperature, and 238-nm detector.
a system of 0.40 g of NaCl in 4 mL of 0.5% methylcellulose diluted to 25 mL. phase was transferred to a volumetric flask, and an additional 16 mL of acetonitrile was added to the centrifuge tube. The solution was vortex-mixed and centrifuged for 5 min. All the organic layers were combined and diluted to the required concentration with water or water-acetonitrile mixture as needed.
RESULTS AND DISCUSSION The experimental data indicated that viscosity of aqueous methylcellulose (y) is linearly proportional to concentration (X).The coefficient of determination is 0.998 and fits the straight line
Y = 1.005 + 0.09X
2 :
.z , E l m
2 E : k n
? o
I1 I
CYCLOBENZEPRINE . HC I
Figure 3. Quantltatlon of dlflunisal and cyclobenzaprine hydrochloride after removal of methylcellulose using 65 % acetonitrile in formic acid-water (1:70), 2 mL/min flow rate, 40 O C oven temperature, and 290-nm detector.
then pipetted into a 50-mL centrifuge tube. Twelve milliliters of acetonitrile and 0.5 g of sodium chloride were added with immediate vortex mixing after each addition. The upper organic
where the Y intercept is the viscosity of water. Sodium chloride is one of the few compounds that decreases the solubility of methylcellulose in water (6). By employing the linear relationship that exists between viscosity and concentration of methylcellulose in solution, the graph on Figure 4 depicts a rapid decrease in concentration of methylcellulose with increasing amounts of sodium chloride. The minimum of the curve is equal to the optimum condition of the process, whereby at least 95% of the methylcellulose has precipitated. The slight increase of viscosity with further addition of sodium chloride is due to the increase in concentration of the salt solution. Methylcellulose solubility decreases with increasing temperature which allows use of a lower amount of sodium chloride a t higher temperatures (Figure 5 ) . On the other hand, changes in pH have no effect on the quantity of me-
1144
Anal. Chem. 1981, 53, 1144-1145 -
Table I. Effect of pH on Precipitation of Methylcellulose viscosity, CP s 1.015 1.014
1.016
PH 1.5 3.3 6.2
viscosity CP s 1.016 1.016
PH 8.0
11.8
Table 11. Recovery Study of Pharmaceutical Compounds from 0.5% Methylcellulose
generic name or MK no. aspirin hydrochlorothiazide MK-4 2 1 mevinolin cyclobenzaprine HCl diflunisal plus cyclobenzaprine HCI
mg/mL of methylcellulose recovered spiked concn concn 0.54 7.93 0.55 7.93 0.51 8.30 0.48 7.93 8.89 6.61 0.10
0.51 7.83 0.55 8.14 0.48 8.44
indicated close to 100% efficiency for the process (Table 11). Both columns performed well after the analysis and no significant column pressure change was observed after separate cleaning of the columns. The compounds either are already on the market or are undergoing clinical trials and were selected for the diversity of their functional groups. Application of this technique to compounds with other functional groups, though not yet attempted, is very likely possible. Acidic or alkaline compounds will not affect the amount of methylcellulose removed. The sensitivity is limited by the detection capability of the instrument. However, since Samples are not quantity restricted, this aspect has not been explored. The type and quantity of compounds using this cleanup procedure will be restriced by their solubility in acetonitrile.
ACKNOWLEDGMENT Sincere thanks to colleagues who have offered helpful comments and assistance and to the library staff of Merck Sharp and Dohme Research Laboratories for their literature search.
0.47
7.90 8.70 6.62 0.10
thylcellulose precipitated (Table I). T o test the applicability of the above observations, we modified the procedure to analyze suspensions as well as solutions of compounds in 0.5% aqueous methylcellulose. A number of drugs were assayed individually or in combination. Acetonitrile acted as a solvent and later as an extraction medium to separate the drug from the aqueous phase and precipitate. The recovery data using an external standard
LITERATURE CITED (1) Whistler, R. L., Smart, C. L., Ed. “Polysaccharide Chemistry”; Academlc Press: New York, 1953; p 76. (2) Isshiki, Y. Kagawa Dabaka Nogakubo Gakujutsu Hokoku 1977, 28 (60), 33-36. (3) McCollister, S. E.; Kociba, R. J.; McColiister, D. D. Fd Cosmet. ToxlCOl. 1973, 11, 943-953. (4) Lehman, A. J. Quant. Bull. Assoc. Fd. Drug Officials U . S . 1950, 74, 3, 82-96. (5) Greminger, G. K., Jr.; Savage, A. E. I n “Industrlal Gums”; Whistler, R. L., BeMlller, J. N., Eds.; Academic Press: New York, 1959; p 565. (6) Spurlin, H. M. I n “Cellulose and Cellulose Derivatlves: Hlgh Polymers Part 11”; Ott, E., Spurlin, H. M., Grafflln, M. W., Eds.; Interscience: New York, 1954; Vol. 5, p 935.
RECEIVED for review January 1, 1981. Accepted March 23, 1981.
Determination of Trace Solvent in Waxes and Lubricating Oils Bachan S. Rawat” and Guru Prasad Indian Institute of Petroleum, Dehra Dun, India
Solvent dewaxing of lubricating oil stocks with methyl ethyl ketone (MEK)-toluene mixture is well-known in the petroleum industry. Lubricating oil and wax stocks after solvent removal should not normally contain more than 50 and 150 ppm of toluene and 50 and 100 ppm of MEK, respectively. Determination of solvents in small quantites in these stocks is, therfore, necessary. The gas-liquid chromatographic (GLC) method devised for this purpose (1) uses a solvent-stripping manifold system consisting of a freeze-out trap at -50 OC to retain solvents before they are flashed to the GLC column. Although the precision reported in this technique is good, the method is cumbersome and needs special care to resolve such small quantities of toluene/MEK from these high boiling and viscous stocks. In the present study, a simple technique was used which consists of azeotroping toluene and MEK with methanol. As wax and lube oil stocks are not miscible with methanol, the solvent molecules trapped within wax or lube oil molecules cannot be easily brought in contact with methanol during 0003-2700/81/0353-1144$01.25/0
distillation. For this purpose moderately polar solvents like cyclohexanol, cyclohexanone, hexanol, and hexyl acetate were tried which dissolve both methanol and wax or lube oil. Such a solvent, however, should not form an azeotrope with methanol (e.g., carbon tetrachloride, 55.7 O C ) . Carbon tetrachloride and chloroform otherwise also do not form azeotropes with toluene. Methanol-toluene and methanol-MEK form azeotropes (2) at 63.8 and 63.5 “C, respectively. Toluene (bp 110.6 OC) and MEK (bp 79.6 “C) do not form azeotropes.
EXPERIMENTAL SECTION All the chemicals used had purities of more than 99% as checked by GLC. The wax (congealing point 58.60 O C ) and lubricating oil (370-500 O C ) were free from toluene and MEK. About 800 g of wax or lubricating oil was placed in a distillation flask along with about 400 g of cyclohexanone or cyclohexanol, 10 mL of methanol, and known quantities of toluene/MEK. The quantities of toluene and MEK were taken in such a way that wax or lube oil contained them in the range of about 30-200 ppm. The flask was connected to the distillation assembly as shown 0 1981 American Chemical Society
