LFS 251 Spectrophotometric assay of riboflavin Lab Assessment

Laboratory Practical 1 Spectrophotometric assay of riboflavin

Introduction Spectrophotometry is the most widely used and versatile of all the analytical tools in biochemical laboratories. It allows particular components (solutes) in a solution to be examined without interference from other molecules present, the sample examined is not destroyed in the process, and the measurements are made quickly. The selectivity of the method is achieved by examining a sample solution at a particular wavelength in the electromagnetic spectrum which corresponds to the absorption of light by a chemical grouping (chromophore) which is characteristic of the component to be analysed and not of other components in the solution.

This phenomenon is extremely useful in biochemistry and use of this property for measurement is called spectrophotometry. Biochemists usually refer to the coloured compounds as being chromophores. In other words, a chromophore is a compound that has the ability to absorb light at certain wavelengths

When spectrophotometry can be used in experiments, it will often be the first choice because:

(1) It is generally a sensitive method and thus requires very little material.

(2) It generally does not damage or change the sample. 27

(3) It can be highly selective and identify chemical changes in real time. (That is, it is possible to follow some processes and reactions as they occur.)

The Electromagnetic Spectrum: As spectrophotometry uses light (electromagnetic radiation), it is worthwhile becoming familiar with the frequency of wavelength relationships of the electromagnetic spectrum. This is shown in Figure 1. For spectrophotometry, the wavelengths used are in the visible and ultraviolet sections of the electromagnetic spectrum. You should also familiarise yourself with the wavelengths and colours of the visible spectrum.

Understanding that white light is made up of a spectrum of colours (the visible spectrum) explains why some things are coloured. If white light is passed through a solution containing a coloured compound, some wavelengths are selectively absorbed by the compound. The reason a solution appears to be, say yellow, is that some of the wavelengths of visible light (such as blue) are absorbed, or filtered out, and the colours that are not absorbed create a yellow impression. The resultant colour that can be seen is a mixture of the remaining wavelengths of light that are not absorbed. Classic examples of this include haemoglobin (the red colour of blood) and chlorophyll (the green colour of leaves).

Beer-Lambert Law: The degree of absorption of radiant energy occurring when light of a specific wavelength passes through a solution depends on the wavelength used, the solute (chromophore) responsible for absorption, its concentration and the length of the light path through the solution. Quantitatively, this is expressed by the Beer-Lambert Law for monochromatic light.

Hence, provided the thickness of the solution (l) remains constant, at any given wavelength a linear relationship exists between Absorbance, A, and concentration, c.

Spectrophotometers are instruments that can measure the amount of light absorbed by a coloured compound (chromophore) at a selected wavelength of light. The wavelength of light is expressed in terms of nanometres (nm). A spectrophotometer has two light sources: - A deuterium lamp that emits light mostly in the ultraviolet light spectrum (200 - 400 nm). - A tungsten lamp which emits light mostly in the visible light spectrum (400 - 1100 nm). Biochemists often refer to these instruments as being UV-VIS spectrophotometers. Figure 2 provides a simplified overview of a spectrophotometer. The essential components of any photometer include a source of monochromatic light (light of one wavelength), something to hold the absorbing solution, a sensitive device (usually a photoelectric cell) to convert light to the electric current of the signal, and finally a device to convert the signal to a direct reading on a galvanometer

fill a cuvette, take an absorbance reading, empty it, wash it and fill it with your next sample. In most experiments, you are interested in the change of absorbance of one or more specific compounds. Other compounds, solvent, and even cells or other particulate material will contribute to the absorbance reading. Thus, it is always important to consider what is needed in each experiment for blank readings. A blank sample (often called a reagent blank) is used in most cases to set the instrument to zero absorbance.

Not all chemicals have a colour that humans can see, but we can still use a spectrophotometer to find their concentration in solution. Chemicals very often absorb 'light' of a shorter wavelength than blue, called ultraviolet (UV), or a longer wavelength than red (infrared). Both of these wavelengths are outside of the visible spectrum. The chemical appears to be colourless but the spectrophotometer knows better! A measurement of absorbance is made by setting the spectrophotometer to the correct 'colour'; for example, 280nm in the ultraviolet to measure protein (all proteins absorb UV light at this wavelength).

If more than one chemical in your solution absorbs light at the same wavelength, it can be very difficult to accurately determine their individual concentrations. A common method to overcome this is to react the compound of interest with another chemical and generate a different colour.

A note of caution: Glass absorbs UV light and cuvettes are often made of glass! If measuring below 340 nm, make sure that either plastic or quartz cuvettes are used. Another note of caution: Do not use plastic cuvettes if using organic solvents as the cuvettes will dissolve!

Wavelength Scan of a Chromophore: It is often necessary to select a particular wavelength of light at which a chromophore will absorb strongly. At this wavelength, interference from other chromophores that may be present can be minimised. To do this, it is essential to undertake a wavelength scan of the chromophore. This involves testing the chromophore at all wavelengths in the ultraviolet (UV) and/or visible light spectrum with the spectrophotometer, and then selecting a particular wavelength at which the chromophore absorbs strongly. Figure 3 shows a wavelength scan for the chromophore, riboflavin, in part of the UV and visible light region. The spectrophotometer in the middle of the room shows a full wavelength scan of a 100 µM solution of riboflavin. At which wavelength of light does the chromophore absorb the strongest in the visible light region (380-750 nm)? Use this wavelength in the following procedure.

Standard Curves: Absorbance values obtained from a spectrophotometer do not tell us the concentration of the compound of interest (chromophore), only the amount of colour. Therefore, all biochemical assays must have a standard curve. A set of standards must be prepared that contain a known concentration of the compound being measured. The standards are treated in the same way as the biological specimen being tested, and then the absorbance of the standards is measured on the spectrophotometer. A typical standard curve is shown in Figure 4

By preparing a standard curve, the absorbance of the compound developed in the test sample can be compared to the absorbance of the standards, and thus the concentration determined. In Figure 4, a test sample (unknown) with an absorbance of about 0.3 is marked on the graph. What would the concentration of this test sample be?

Reagent Blank: An important part of all standard curves is the reagent blank (see earlier). A reagent blank contains all reagents except the compound being measured. This allows the determination of any absorbance in the final solution that may arise from the reagents themselves. Reagent blanks are also called the “reference” or ‘zero” solution.

Riboflavin (vitamin B2): Riboflavin (vitamin B2) is an important vitamin which humans cannot synthesise, and therefore has to be obtained from the diet. In the body it is an integral part of the cofactors FMN and FAD, which bind to specific enzymes to transfer electrons in reactions of oxidation and reduction. The flavin-enzyme combination is known as a flavoprotein. Riboflavin has a characteristic absorption spectrum, which provides the basis for a spectrophotometric assay. You will be investigating the spectrophotometric properties of riboflavin in this practical.