L7 - UV-Visible Spectrophotometry

objectives

• Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solution.
• The basic principle is that each compound absorbs or transmits light over a certain range of wavelength.
• This measurement can also be used to measure the amount of a known chemical substance.
• Spectrophotometry is one of the most useful methods of quantitative analysis in various fields such as chemistry, physics, biochemistry, material and chemical engineering and clinical applications.

Introduction

• Every chemical compound absorbs, transmits, or reflects light (electromagnetic radiation) over a certain range of wavelength.
• Spectrophotometry is a measurement of how much a chemical substance absorbs or transmits.
• Spectrophotometry is widely used for quantitative analysis in various areas (e.g., chemistry, physics, biology, biochemistry, material and chemical engineering, clinical applications, industrial applications, etc).
• Any application that deals with chemical substances or materials can use this technique.
• In biochemistry, for example, it is used to determine enzyme-catalyzed reactions.
• In clinical applications, it is used to examine blood or tissues for clinical diagnosis.
• There are also several variations of the spectrophotometry such as atomic absorption spectrophotometry and atomic emission spectrophotometry.
• A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution.
• With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected.
• Depending on the range of wavelength of light source, it can be classified into two different types:

1. UV-visible spectrophotometer: uses light over the ultraviolet range (185 - 400 nm) and visible range (400 - 700 nm) of electromagnetic radiation spectrum.

2. IR spectrophotometer: uses light over the infrared range (700 - 15000 nm) of electromagnetic radiation spectrum.

• In visible spectrophotometry, the absorption or the transmission of a certain substance can be determined by the observed color.
• For instance, a solution sample that absorbs light over all visible ranges (i.e., transmits none of visible wavelengths) appears black in theory.
• On the other hand, if all visible wavelengths are transmitted (i.e., absorbs nothing), the solution sample appears white.
• If a solution sample absorbs red light (~700 nm), it appears green because green is the complementary color of red.
• Visible spectrophotometers, in practice, use a prism to narrow down a certain range of wavelength (to filter out other wavelengths) so that the particular beam of light is passed through a solution sample.
• A chemical that absorbs light in the UV/Vis range is called a CHROMOPHORE

Devices and Mechanism

• Figure 1 illustrates the basic structure of spectrophotometers.
• It consists of a light source, a collimator, a monochromator, a wavelength selector, a cuvette for sample solution, a photoelectric detector, and a digital display or a meter.
• Detailed mechanism is described below.
• Figure 2 shows a sample spectrophotometer

A spectrophotometer, in general, consists of two devices; a spectrometer and a photometer. A spectrometer is a device that produces, typically disperses and measures light. A photometer indicates the photoelectric detector that measures the intensity of light.

• Spectrometer: It produces a desired range of wavelength of light. First a collimator (lens) transmits a straight beam of light (photons) that passes through a monochromator (prism) to split it into several component wavelengths (spectrum). Then a wavelength selector (slit) transmits only the desired wavelengths, as shown in Figure 1.
• Photometer: After the desired range of wavelength of light passes through the solution of a sample in cuvette, the photometer detects the amount of photons that is absorbed and then sends a signal to a galvanometer or a digital display, as illustrated in Figure 1.

• You need a spectrometer to produce a variety of wavelengths because different compounds absorb best at different wavelengths.
• For example, p-nitrophenol (acid form) has the maximum absorbance at approximately 320 nm and p-nitrophenolate (basic form) absorb best at 400nm, as shown in Figure 3.
• ƛ_max or (wavelength max) is the wavelength at which most absorption of that species occurs. This can be used to calculate concentration or (and just like in infra-red spectroscopy) it can be compared with a known UV-VIS spectrum of that chemical - the absorption should correlate and this comparison with a reference sample will help us identify compounds.

Summary videos and practical exercises