**Determination of Compound Content Using the Spectrophotometric Standard Curve Method**
Key words: spectrophotometer; standard curve; US instrumentation; UV-1100; UV-1200
When using a UV-visible spectrophotometer for quantitative analysis, the pure sample to be tested is typically prepared into a series of standard solutions. A standard curve is then created by measuring the absorbance at specific wavelengths. By comparing the absorbance of an unknown sample to this curve, the concentration of the compound can be determined.
For example, in the case of aspirin (A.P.C.), which contains three components—aspirin (A), phenacetin (P), and caffeine (C)—the maximum absorption peaks are at 277 nm and 275 nm, which are relatively close. To accurately determine their concentrations, they must be separated first. The method involves solving simultaneous equations based on the absorbance values measured at different wavelengths.
The tablets were crushed and dissolved in chloroform. They were then extracted twice with a 4% aqueous sodium carbonate solution, washed once with distilled water, and the aqueous layers were combined. Aspirin entered the aqueous layer, while phenacetin and caffeine remained in the chloroform. The aqueous layer was further extracted three times with chloroform to remove any remaining 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 these measurements, the concentration of aspirin was calculated as follows:
If the absorbance of 100 mg/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 per 100 mL.
For the standard phenacetin solution, 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 values were K₂₅₀ = 0.0177 L/(mg·cm) and K₂₇₅ = 0.0518 L/(mg·cm). Using these values, a system of equations was solved to find the concentration of phenacetin, resulting in 1.55 mg/L, or 155 mg/100 mL. Since the unknown solution was diluted 250 times, the actual content in the tablet was found to be 1.01 × 250 = 252 mg for phenacetin and 0.155 × 250 = 38.8 mg for caffeine.
Similarly, in the case of cresol, the different positions of methyl and carboxyl groups lead to ortho, meta, and para isomers. Each isomer has distinct absorption bands, and their concentrations can be determined by measuring absorbance at 277 nm, 273 nm, and 268 nm, respectively. Using simultaneous equations, the content of each component can be accurately calculated.
Keywords: spectrophotometer; standard curve; US instrumentation; UV-1100; UV-1200
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