Spectroscopy Techniques

Spectrophotometers in the chemical/biological laboratory 1. What is the identity or nature of the component(s) of a sample? (qualitative assessment) 2. How much of an analyte is present in a sample? (quantitative assessment)

When light strikes “matter” it can be Transmitted Reflected Scattered Absorbed

Absorption is the basis of spectrophotometry Samples are in a liquid form and can either absorb or transmit light. Spectrophotometers allow us to quantify how much light is absorbed and transmitted at a particular wavelength Need textbook figures 19.8 etc.

This is expressed as a ratio called transmittance (t) Spectrophotometers compare the amount of light transmitted through a sample with that transmitted through the blank. This is expressed as a ratio called transmittance (t) t = light transmitted through the sample light transmitted through the blank Often, %T is used, i.e. (t X 100%) Transmittance can range from 0-1 and %T from 0-100% I.ei no light through the sample to ALL light through the sample.

Absorbance (A) = -log10 (t) Analysts are more interested in the amount of light absorbed by the sample……………..why? Absorbance (A) = -log10 (t) Absorbance is also called optical density (OD). It does not have units. Abbreviations sometimes used are 1.6A or OD1.6 FIG 19.9 Absorbance versus concentration is linear therefore it is more conventient

Spectrophotometers allow us to graphically display the pattern of light absorption by a solution The extent to which a particular material absorbs different wavelengths of light is called an absorbance spectrum

The Spectrophotometer Radiation source (visible, UV or IR) Monochromator (frequency/wavelength selector) Sample holder Detector (convert radiation to electrical signal) Recorder (graph the signal)

Which radiation source? UV spectra can be similar for many substances. IR spectra are different for each substance. IR spectrophotometry is one of the few tests available for specific identification. 1. Forensic scientists have identified and cataloged thousands of organic compounds to serve as REFERENCE materials.

Sample preparation & the importance of “blanks” Samples are dissolved in a solvent that may absorb light and then placed in a cuvette that may also absorb light. “ Blank sample” should contain no analyte but should contain everything else - i.e. solvent, reagents common to other samples being read. The blank is used to set the instrument to 100%T (or zero absorbance) every time the wavelength is changed.

Performance verification Calibration is part of the routine quality control/maintenance for a spectrophotometer There are two parts to calibration Wavelength accuracy – ensure the wavelength selected agrees with the actual wavelength that exits the monochromator and shines on the sample. Photometric accuracy (absorbance sale accuracy) – measured absorbance (or transmittance) agrees with the value of the accepted reference standard.

Quantitation with spectrophotometry A standard curve (calibration curve) is a graph of analyte concentration in standards of known concentration (X axis) versus absorbance (Y axis) Given a standard curve it is possible to determine concentration of analyte in unknown samples if the absorbance can be measured. PAGE403 of textbook.

Evolution of Beers’ Law The line on the standard/calibration curve has an equation: General equation for any line is Y = mX + a Substitute A (for absorbance) and C (for concentration then equation becomes: A = mC + a, where a =0 because the blank is used to zero the instrument THUS, A = mC or absorbance = (slope of the line)(concentration of analyte)

Thus the equation becomes: A = (b)C 2 main factors affect the slope of the line (m): The tendency of the compound of interest to absorb light at the wavelength used (called absorptivity) The path length (the distance light passes through the sample; typically measured as the length of the cuvette in mm or cm) Thus the equation becomes: A = (b)C Where: A = absorbance,  = absorptivity, b = path length, C = concentration sometimes “K” is used in place of (ab) as path length is the same for the same cuvettes used for an application

In short, Beers law states that ..” absorbance of a compound is directly proportional to its concentration”…. This is the basis for all quantitation using UV/Vis spectroscopy

Deviations from Beers’ Law 1. At high absorbance levels, stray light causes spectrophotometers to have a nonlinear response 2. At very low levels of absorbance a spectrophotometer may be inaccurate 3. There may be one or more components in the sample that interfere with the measurement of absorbance of a particular analyte.