In the Laboratory
Tablet Analysis Using Gravimetric Dilutions
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Larry A. Simonson Department of Chemistry and Food Science, Framingham State College, Framingham, MA 01701-9101;
[email protected] Early in the development of automated sample preparation devices, it was discovered that direct volume measurements were difficult, whereas direct mass measurements using modern electronic balances were easily done (L. A. Simonson; unpublished work at Zymark Corp., Hopkinton, MA, 1984–1985). This is in contrast to the pre-electronic balance era, when weighing was a slow and tedious procedure. Given this change in convenience, it is interesting to speculate about the current dominance of volumetric methods had modern electronic balances been available for the last 150 years. Would molarity be in moles of solute per kilogram of solution? The use of volumetric glassware in chemical analysis is so well entrenched that challenges to this paradigm are virtually unknown; but this experiment shows how a complete pharmaceutical analysis could be performed without the use of volumetric glassware. The better performance of gravimetric than of volumetric has been recognized for many years (1) and was well generalized by Harris (2, p 839): “Because volumetric dilutions are rarely more accurate than 0.1%, gravimetric dilutions are required for greater accuracy.” Besides accuracy, there are several other reasons for a change from volumetric to gravimetric protocols. We no longer have to be concerned with the temperature dependence of density or the calibration of volumetric glassware. We may be able to conserve reagents because gravimetric procedures are not dictated by the sizes of volumetric glassware, which may be too large and thus lead to waste. Students are shown the correct meaning of the today’s very common unit of solution concentration, ppm, as milligrams of solute per kilogram of solution, in contrast to the widely accepted, but technically incorrect, milligrams of solute per liter of solution. Finally, making solutions gravimetrically is very convenient. There is no need to use expensive glassware and no worry about adding diluent beyond the mark. This experiment was designed to introduce students to the concept of gravimetric dilution using familiar chemicals and measuring devices. The experiment is representative of tablet analysis in general, a process carried out thousands of times every day in pharmaceutical laboratories, and thus will appeal to those students who have a harder time connecting with unfamiliar samples such as oxalates or soda ash. It can be run by students at the high school or college freshman level who have had exposure to Beer’s law, but could also be appropriate at higher levels for students who have never considered gravimetric dilutions. Procedure
Preparation of the Tablet Extract The sample chosen to demonstrate gravimetric dilutions was CVS brand caffeine tablets containing caffeine as an active ingredient in a labeled amount of 200 mg. (Other brands may work as well.) Students tare a clean, dry 250-mL Erlenmeyer flask on a 2-place balance, add a tablet, record the tablet’s
mass (later this will allow a quick calculation of the ratio of active to inert ingredients if desired). While the flask is still on the balance, about 200 mL of distilled water is added and the mass is recorded. A magnetic stirring bar is added and the flask is covered with a plastic film cap. The flask is set on a magnetic stirrer and stirred at a medium speed for about 30 min—a time found to be adequate, but not necessarily optimal. While the dissolution is occurring, students make a series of standards, again using gravimetric dilutions, that cover the expected range of the final diluted sample. Because a 20-ppm solution of caffeine will yield an absorbance at 274 nm (1-cm path length) of about one, the standards are prepared in the range of 10 to 30 ppm to bracket the expected concentration of the sample after a 1:50 dilution of the extract.
Preparation of Standards To facilitate this lab, a standard solution of about 100 ppm of caffeine (aqueous) is carefully prepared in advance by accurately weighing out about 100 mg of pure caffeine on an analytical balance, quantitatively transferring it to a tared 1000-mL beaker, and adding about 1 L of water to bring the final mass to about 1000 g on a 3-place balance. The final concentration of this standard is calculated to the nearest 0.1 ppm by dividing the mass of caffeine in milligrams by the mass of the solution in kilograms. Five diluted standards in the range of 10 to 30 ppm are made by adding an appropriate mass of the 100-ppm standard solution to a tared test-tube on an analytical balance using a disposable pipet, then adding distilled water carefully with a squeeze bottle until the final mass of about 10 g is attained. There is no need to hit target masses exactly, but only to get somewhere close and record the actual masses to 4 decimals. With test tubes, evaporation may yield uncertainty in the 4th decimal, but since 1 g of the standard solution is required for the most dilute standard, and more for the others, excellent accuracy is retained at 3 decimals. Calibration Once the standard solutions are prepared, the absorbance at 274 nm in a 1-cm cell is measured for each and plotted versus the gravimetric concentration.1 These data allow students to find the mathematical relationship between the concentration and the measured absorbance either by using the spreadsheet’s regression line function or by hand, if desired. In our hands, this relationship is very linear. Several students reported the spreadsheet-calculated linear correlation coefficient to be in excess of .99999. Of course the quality of the UV spectrometer will affect these results.2 All absorption measurements are done using an Agilent Technologies 8453E diode array instrument fitted with a 1-cm flow cell. For each standard, students draw about 5 to 10 mL of sample through the cell using a 25-mL plastic syringe, press the measure key, and manually record the absorbance.
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In the Laboratory
Diluting the Tablet Extract The stirring is stopped after 30 min and the slurry is allowed to settle until the top inch or so is clear. In the case of our CVS tablets, no filtration is needed, as the slurry settles in a few minutes; but other samples may require filtration of a small portion of extract before dilution. Since this tablet extract solution is expected to contain about 200 mg in about 0.2 kg, it has a concentration of about 1000 ppm and will need dilution by a factor of 50 to get it to the desired 20 ppm for measurement.3 This is accomplished by taring a small beaker or Erlenmeyer flask on an analytical balance, adding about 1 g of the clear extract using a disposable pipet, and adding about 49 g of distilled water. All steps involve weighing to nearest 0.1 mg. As in any gravimetric dilution, it may be necessary to cap vessels to reduce evaporative losses, but this was not found necessary in our labs. From the mass data, the dilution ratio for the extract is calculated for later use in determining the tablet contents. The absorbance of diluted extract is then measured using the same conditions as were used for the standard. Triplicates of the extract are run to give students a measure of repeatability and a dose of confidence in their results, but because of the ease in which replicates can be run, some instructors may elect to do more. Calculating the Tablet’s Contents To get the tablet’s contents, in milligrams, students first calculate the gravimetric concentration of the diluted extract by reference to the calibration curve. Then by mentally “undiluting” this extract using the extract dilution ratio, they calculate the gravimetric concentration of original extract, which when multiplied by the mass of the original extract in kilograms yields the desired result. Apart from the main pedagogic aspects of this experiment, the data make a nice example of the usefulness of an electronic spreadsheet in analytical chemistry.
The error in concentration, based on actual calibration data, is estimated using a least squares spreadsheet designed by Harris (2, p 106). This was found to be 0.06 ppm, which in relative terms is about 3‰. This is the major error. The error in the dilution ratio was estimated by assuming an uncertainty of ± 1 unit it the least significant digit of the balance (i.e., ± 0.0001 g on a 4-place balance). Since two weighings are required for each term in the ratio, the worstcase error in each is ± 0.0002 g. The total error in this term is due essentially to the 1-g mass of extract used and is not affected significantly by the 50 g of diluted extract produced. This results in an uncertainty of ± 0.0002 g in a 1-g sample or a relative error of 0.2‰. The error in the total mass of extract is determined for the most part by the accuracy of the estimate of the amount of undissolved solids in the extract, and insignificantly by the weighing of the 200-g sample on a 2-place balance. The relative error associated with the weighing is estimated, using the same criterion as above, to be ± 0.02 g in 200 g or 0.1‰. The relative error in the correction for undissolved solids is estimated by assuming a maximum error of 100 mg in the amount of sample that does not dissolve. (Students are allowed to assume that only the 200 mg of caffeine in the 600-mg tablet dissolves and that the remaining 400+ mg remains as solids.) A reasonable estimate of this error is 100 mg in 200 g of extract or 0.5‰, which thus dominates the error in the 3rd term in the calculation of tablet contents. For the product of three independent terms, the total relative error propagates as the square root of the sum of the squares of the individual relative errors (2, p 63). Letting R x be the relative error in x, we get 2 2 1/2 R tot = (R concn + R 2diln ratio + Rmass extract)
Using the values discussed above as reasonable estimates of relative standard deviations we get R = [(3‰)2 + (0.2‰)2 + (0.5‰)2]1/2
Hazards There are no significant hazards in this experiment. Discussion of Results Tablet analysis is generally done to satisfy regulatory requirements such as those published in the United States Pharmacopoeia (USP) where compliance in content uniformity is established for most tablets by showing that each tablet of a group of ten contains between 85 and 115 percent of the labeled amount. By pooling the data from ten students or pairs, it is possible for the class to judge the USP compliance of the lot of tablets used. However, until this gravimetric method has been validated by an appropriate authority, the results are not official and cannot be used in any claims for or against the tablet producer. In spite of this disclaimer, students get some sense of the importance of understanding the regulatory aspects of analytical work.
Errors There are errors associated with each of the three terms in the equation used to calculate the mass of caffeine in the tablet: mass caffeine = [concn diluted extract × dilution ratio × mass extract]
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Thus the estimated relative error in mass of caffeine in the tablet is R = 3‰. This corresponds to an error ±0.6 mg in the amount of caffeine in a tablet nominally containing 200 mg of caffeine. W
Supplemental Material
A handout containing detailed instructions for students is available in this issue of JCE Online. Notes 1. Depending on their instruments, instructors may find better performance at wavelengths slightly different from 274 nm. Instructors should have a recent UV spectrum of aqueous caffeine available for students so they will have a basis of understanding the choice of the measuring wavelength. 2. For best results, it is important that the effective slit width (nm) of the spectrometer not exceed 10% of the bandwidth of the sample. For aqueous caffeine the absorbance bandwidth is about 30 nm, so the spectrometer should have an effective slit width of no more than 3 nm.
Journal of Chemical Education • Vol. 78 No. 10 October 2001 • JChemEd.chem.wisc.edu
In the Laboratory 3. The dilutions in this experiment were chosen to yield absorbances near 1.0 because that is where the instrument we used has optimum performance. Instructors are encouraged to modify all the dilutions as necessary to obtain solutions that yield optimum signal-to-noise ratios.
Literature Cited 1. Kratochvil, B.; Nolan, J. E. Anal. Chem. 1984, 56, 585–589. 2. Harris, D. C. Quantitative Chemical Analysis, 5th ed.; Freeman: New York, 1999.
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