1. INTRODUCTION TO SPECTROPHOTOMETRY

2. BACKGROUND
Spectrophotometry is a method of analyzing that involves how light interacts with the atoms (or molecules) in a sample of matter.
Visible light is only a small portion of the entire electromagnetic spectrum and it includes the colors commonly observed (red, yellow, green, blue and violet). The visible spectrum consists of electromagnetic radiation whose wavelengths range from 400 nm to nearly 800 nm.

3. Why do some substances appear colored?
BACKGROUND
white light is observed, what is actually seen is a
mixture of all the colors of light
Why do some substances appear colored?
When this light passes through a substance, certain energies (or colors) of the light are absorbed while other color(s) are allowed to pass through or are reflected by the substance.
If the substance does not absorb any light, it appears white (all light is reflected) or colorless (all light is transmitted). A solution appears a certain color due to the absorbance and transmittance of visible light. For example, a blue solution appears blue because it is absorbing all of the colors except blue.

4. BACKGROUND
A sample may also appear blue if all colors of light except yellow are transmitted. This is because blue and yellow are complementary colors. (See the color wheel above.)

5. BACKGROUND
The amount of light absorbed by a solution is dependent on the ability of the compound to absorb light (molar absorptivity), the distance through which the light must pass through the sample (path length) and the molar concentration of the compound in the solution.
If the same compound is being used and the path length is kept constant, then the absorbance is directly proportional to the concentration of the sample.

6. Spectrophotometer
A spectrophotometer is used to provide a source of light of certain energy (wavelength) and to measure the quantity of the light that is absorbed by the sample.
Light Bulb Prism Filter Slit Sample Detector

7. Spectrophotometer
The basic operation of the spectrophotometer includes a white light radiation source that passes through a monochromator. The monochromator is either a prism or a diffraction grating that separates the white light into all colors of the visible spectrum. After the light is separated, it passes through a filter (to block out unwanted light, sometimes light of a different color) and a slit (to narrow the beam of light--making it form a rectangle). Next the beam of light passes through the sample that is in the sample holder. The light passes through the sample and the unabsorbed portion strikes a photodetector that produces an electrical signal which is proportional to the intensity of the light. The signal is then converted to a readable output that is used in the analysis of the sample.
Light Bulb Prism Filter Slit Sample Detector

8. Spectrophotometer Absorbance -- a number between 0 and 2
The spectrophotometer displays this quantity in one of two ways:
Absorbance -- a number between 0 and 2
(2) Transmittance -- a number between 0 and 100%.
The sample for a spectral analysis is prepared by pouring it into a cuvette which looks similar to a small test tube. A cuvette is made using a special optical quality glass that will itself absorb a minimal amount of the light. It is also marked with an indexing line so that it can be positioned in the light beam the same way each time to avoid variation due to the differences in the composition of the glass

9. Viewing the Visible Spectrum
The spectrophotometer is designed to detect absorbances of light at different wavelengths when the light passes through a solution of some given concentration. Some compounds absorb more light at one wavelength than another, so the wavelength must be changed every time a different compound is being analyzed to achieve optimum results from a spectrophotometer. The wave-length of light is selected by adjusting the wavelength dial and read on the wavelength display.
Please note that the accepted symbol for wavelength is the Greek letter lambda ()

10. A visible spectrophotometer can be used to learn why colored solutions appear a particular color. For example, WHY does blue food coloring appear blue? Simply put, the solution is blue because it transmits (and reflects) blue visible light more than it transmits other colors of visible light. In other words, blue food coloring absorbs blue visible light the least and absorbs other colors of light more.
When white light is observed, what is actually being seen is a mixture of all the colors of light. When this light passes through a substance, certain energies (or colors) the light are absorbed while other color(s) are allowed to pass through or are reflected by the substance. This is why some substances appear colored. The color that we see is the combination of energies of visible light which are not absorbed by the sample. If the substance does not absorb any light, it appears white or colorless.

11. Two ways to make colors
A solution appears a certain color due to the absorbance and transmittance of visible light. For example, the blue solution appears blue because it is absorbing all of the colors except blue. A sample may also appear blue if all colors of light except yellow are transmitted (yellow is absorbed). This is because blue and yellow are complementary colors. Any two colors opposite each other on the color wheel (see figure above) are said to be complementary. The wavelength (numbers outside the wheel) associated with the complementary color is known as the wavelength of maximum absorbance. This is because in a colored solution the maximum amount of light is absorbed by the complementary color. Note: cyan = green.

12. Basic Principles of Gas Chromatography

13. THE CHROMATOGRAPHIC PROCESS - PARTITIONING
(gas or liquid)
MOBILE PHASE
Sample
out
Sample in
STATIONARY PHASE
(solid or heavy liquid coated onto a solid or support system)

14. Columns Packed Capillary
Packed - As suggested by the term, it is filled with a coated inert solid support such as fire brick, alumina, and graphite with a specific mesh size. The coatings are called phases and for best results are chemically bonded to the support. Chemical bonding provides for longer column life and less bleeding (major source of background noise) contributing to lower sensitivity. Column dimensions 1/8” - 1/4” ID x up to about 6’ using glass or stainless steel. Advantages - higher capacity (higher conc). Disadvantages: low resolution and low S/N.
Capillary - Here the phase (film) is coated on the inside diameter of the capillary wall with film thickness range of 0.1 to 5μ where the ticker film provides for better resolution but also allows for more bleed. Typical dimensions .25mm - .53mm ID x up to 60m made of fused silica coated with polyamide. Advantages: high resolution and better S/N. Disadvantages: low capacity and cost.

15. Polarity - Non-polar Polar +
Non - Polar : Equal distribution of electrons over the entire molecule. Look at the structure of fluorene.
Polar: Non-equal distribution of electrons in a molecule causing one size of the molecule to be more positive or negative thus creating poles of charges. Look at 2,4,5-T (2,4,5-trichlorophenoxyacetic acid).

16. Gas Chromatography gas system inlet column detector data system
Filters/Traps
Data system H
RESET
Regulators
Syringe/Sampler
Air
Hydrogen
Gas Carrier
Inlets
Detectors
gas system
inlet
column
detector
data system
Column

17. Schematic Diagram of Gas Chromatography

18. Injector

19. Phases Solid phase loops
Here are some of the commonly used phases. They range in polarity from non-polar such as low polarity DMS to the higher polarity of mixtures with DPS with any combination available. There are specialty phases with very high polarity such as cyanopropylphenyl siloxane or trifluoropropyl methyl siloxane for fluorinated compounds. Some of these columns are used for separation confirmation such as the cyanopropylphenyl siloxane column for method

20. Schematic Diagram of Flame Ionization Detector
Exhaust
Chimney
Collector Electrode
Igniter
Hydrogen
Column
Inlet
Effluent

21. Electron Capture Detector

22. Chromatograms
Note peaks 15, & 18 on the DB-5 column and note the same peaks on the DB-1701 column.
This shows the need for confirmatory columns (columns with different phases) so that separation of the compounds can be verified.