QC II LAB - Module 5: UV Spectrophotometric Assay of Acetaminophen, USP 29

190-380 nm

Range of the UltraViolet region

380-780 nm

Range of the Visible region

Electronic Transition

This happens when the absorption of UV/visible radiation occurs through the excitation of electrons within the molecular structure to a higher energy state.

UV

Near to Violet

Infrared

Near to red

Bathochromic Shift

• Red Shift

• Shift to a longer wavelength

Spectrometers

Instruments that measure the ratio of the radiant power of two beams at selected wavelengths within the spectral range.

Source

Continuous radiation over the wavelength of interest

Monochromator

For selecting narrow band of wavelengths from the source spectrum

Detector (or Transducer)

For converting radiant energy into electrical energy

Device

To read out the response of the detector

Cuvette

Transparent container where the sample may be held

UH5300 double-beam UV/visible spectrophotometer (Hitachi)

The instrument that will be used in the UV spectrophotometric determination of sulfadiazine, which employs a silicon photodiode as its detector.

Deuterium Lamp

Lamp for UV region (190-380)

Tungsten Lamp

Lamp for visible region (380-above)

Xenon Arc Lamp

Lamp for UV-visible region (190-780)

Photodiode Detector

• Semiconductor Diode

• With a junction that is accessible to light exposure

Double-Beam In-Time Instrument

• The beam from the source strikes a vibrating or rotating mirror that alternately passes the beam through the reference cell and the sample cell and, from each, to the detector.

• In effect, the detector alternately sees the reference and the sample beam, and the output of the detector is proportional to the ratio of the intensities of two beams.

Double-Beam In-Space Instrument

• Stationary beam splitter

• Used to divide the light into two separate portions that go separately to two matched detectors

• The sample beam travels through the sample

Acetaminophen

• Less toxic alternative to the coal-tar analgesics, acetanilide and phenacetin

• Analgesic-antipyretic, weak anti-inflammatory

N-acetyl-p-aminophenol

Chemical name of Acetaminophen

Principle of the Assay

• Acetaminophen absorbs UV light, and the amount of light is proportional to its concentration in solution.

  • Based on Beer-Lambert Law:

    • Absorbance ∝ Concentration

      • Higher acetaminophen Concentration → Higher Absorbance

HPLC (UV Detection)

Official Method of the Assay of Acetaminophen

UV Spectrophotometry

Unofficial Method of the Assay of Acetaminophen

Single-Point External Standard Method

• This is involved in the unofficial assay of acetaminophen

• A standard methanolic solution containing known drug concentration will be prepared

  • To determine the absorptivity of the drug at the wavelength used

Nitrate Titration

International Pharmacopoeia Method of the Assay of Acetaminophen

Plastic/Glass Cuvette

Cuvette used for Colorimetry (Visible Spectroscopy)

Quartz Cuvette

Cuvette used for UV Spectroscopy