**Determination of Compound Content Using the Spectrophotometric Standard Curve Method**
**Keywords:** spectrophotometer; standard curve; US instrumentation; UV-1100; UV-1200
When using a UV-visible spectrophotometer for quantitative analysis, the sample to be tested can be prepared into a series of standard solutions. A standard curve is then plotted based on these solutions. The absorbance of the unknown sample is compared against this curve to determine its concentration.
For example, aspirin (A.P.C.) contains three components: aspirin (A), phenacetin (P), and caffeine (C). These compounds have absorption peaks at 277 nm and 275 nm, which are close to each other. To distinguish them, they must be separated first. By measuring absorbance at two different wavelengths, the concentrations can be calculated using simultaneous equations.
The tablets were ground and dissolved in chloroform. They were then extracted twice with a 4% aqueous sodium carbonate solution and washed once with distilled water. The aqueous layers were combined, and aspirin was transferred to the water layer, while phenacetin and caffeine remained in the chloroform. The aqueous layer was further extracted three times with chloroform to ensure complete extraction of phenacetin. The chloroform layers were combined, filtered, and diluted to 250 mL with chloroform. Then, 1 mL of this solution was transferred to a 100 mL volumetric flask and diluted to the mark with chloroform. The absorbance was measured at 250 nm and 275 nm, yielding values of 0.795 and 0.280, respectively.
The aqueous layer was acidified to pH 2 and extracted with chloroform. The extract was transferred to a 100 mL volumetric flask, diluted to the mark, and the absorbance was measured at 277 nm, giving a value of 0.78.
Based on the standard curve, the concentration of aspirin was determined as follows:
If the absorbance of 100 mL/L aspirin at 277 nm is 0.72, then the content of aspirin in the sample is:
(0.78 / 0.72) × 100 = 108 mg/L, or 10.8 mg/100 mL. This means that the tablet contains 10.8 g of aspirin.
For phenacetin, the specific absorption coefficients were measured as K250 = 0.0767 L/(mg·cm) and K275 = 0.0200 L/(mg·cm). For the standard solution, the coefficients were K250 = 0.0177 L/(mg·cm) and K275 = 0.0518 L/(mg·cm). Using these values, the simultaneous equation yields a concentration of 1.55 mg/L for phenacetin, or 10.15 mg/100 mL. Since the unknown solution was diluted 250 times, the actual content of phenacetin in the tablet is 1.01 × 250 = 252 mg, and the caffeine content is 0.155 × 250 = 38.8 mg.
Similarly, cresol has different ortho, meta, and para isomers due to the positions of methyl and carboxyl groups. These isomers exhibit distinct absorption bands, allowing their concentrations to be determined by measuring absorbance at 277 nm, 273 nm, and 268 nm, respectively. The same approach of using simultaneous equations can be applied to calculate the individual component contents.
This method is widely used in pharmaceutical and chemical analysis, especially when multiple components are present in a sample. It provides accurate results when the spectrophotometer is properly calibrated and the standard curves are correctly established.
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