Spectrophotometry Part 1

Question 1

speed of light (c)

Answer 1

2.998x108 m/s

Question 2

PS-mass spectrometry

Answer 2

With ties to Electrochemistry (REDOX) and Equilibrium (Reversibility of reaction)
* (a) Initially MB+
* (b) Some LMB was formed upon addition of zinc and sulfuric acid
* Redox reaction SHIFTS RIGHT (LeChatelier)
* (c) Return to MB+
* Cease adding acid; stir vigorously (to mix O2 from air, which re-oxidizes LMB to MB+)
* Redox reaction SHIFTS LEFT (LeChatelier)

Question 3

How is wavelength and frequency calculated?

Answer 3

c=(lambda)(v)
* lambda=m
* v=s^-1=Hz

Question 4

Planck’s constant (h)

Answer 4

6.626x10^-34 Js

Question 5

E per photon

Answer 5

E = hv [=] J

Question 6

Per mol of photons

Answer 6

*E = hvNA [=]J/mol
*Na 6.022x10^23/mol

Question 7

Types of wavelength

Answer 7

See picture

Question 8

Types of light

Answer 8

See pic

Question 9

Light

Answer 9

SEE PICTURE

Question 10

Fluoresce (shining)

Answer 10

Blue light shining on antifreeze solution; fluoresced light proportional to
concentration of fluorescein

Question 11

Instrument Components

Answer 11

-Produces continuous spectrum from some minimum around 350 nm to some maximum around 2.5 um – including blue, green, yellow, red (see below)

Question 12

Beers Law

Answer 12

A = (epsilon)bc
* where epsilon is molar absorptivity, units M-1cm-1 – varies with wavelength!
* b is path length, units cm
* c is concentration, units M
* A has no units (i.e. dimensionless), sometimes called AU (absorbance units)
* Plot of A versus c at fixed b and fixed epsilon (i.e. fixed wavelength) is called Beers Law Plot1
* Generally, you are calculating A, epsilon or c; whereas b is usually specified
-Beers Law plot should specify b, lambda, instrument

Question 13

Example of Methylene Blue

Answer 13

• The spectrum of methylene blue in aqueous solution shown below was measured in a 1.00-cm cuvette.

Question 14

Transmitence, absorbance, and concentration relationship

Answer 14

See pic

Question 15

Precautions in the use of Beers Law

Answer 15

Beers Law is convenient:
* A = (Epsilon)bc
* Here are some reasons you must be careful when using Beers Law:
-Beers Law is only strictly valid for monochromatic light
*Polychromatic light will cause curvature in Beers Law plot
* Particulate light scattering will introduce positive error (falsely high absorbance)
* Stray light will introduce error (falsely high transmittance)
* Chemical effects will introduce negative error (falsely low concentration)
* Example: Acid-Base indicator speciation depends on pH
* Example from Fluorescence: fluorescein quantum yield depends on pH
* Interfering species will introduce positive error (falsely high concentration)

Question 16

Practical Considerations For Spectrophotometry

Answer 16

Fill cuvette above light level
* Don’t fil cuvette to top, it could spill
* Dry outside of cuvette with lint-free tissue
* Do not hold by optical surface – fingerprints scatter light
* Ensure reproducible positioning of cuvette
* Special protocol for high precision measurements
* Do not attempt to interpret absorbance measurement at the stray light limit of the instrument
* Instead, perform quantitative dilution or use shorter path length or different (lamda)max
* Solutions may need filtering
* Remove any gas bubbles such as air bubbles from filling; CO2 in soda; other product gases
* Visually inspect sample-filled cuvette before placing in instrument
* Blank is either pure solvent or method blank (all components sans analyte)

Question 17

Why is Fluorescence more sensitive than Absorbance?

Answer 17

Consider a low concentration of analyte
* Absorbance: A = (epsilon)bc, small c means small A. %T = 100x10^-A
* %T ~ 100% which means P ~ P0
* Subtraction of two very large numbers in search of a very low number
* Detecting a flashlight on the western horizon right as sun is setting
* Fluorescence:
* Fluorescence = Φ^f + (epsilon)bc + Iexcitation
* where Φf is the fluorescence quantum yield
* Absolute signal, not a difference signal
* Detecting a flashlight on a dark, moonless night

Question 18

Hyphenated techniques involving
spectrophotometry

Answer 18

Hyphenated techniques are combinations of two (or more) analytical methods linked together, often to improve separation, identification, or quantification. When spectrophotometry is involved, it usually acts as the detection step.

Question 19

Examples of hyphenated techniques

Answer 19

1. HPLC-UV (High-Performance Liquid Chromatography – Ultraviolet Spectrophotometry)
   * HPLC separates the components in a mixture, and UV spectrophotometry detects them based on how they absorb UV light.
2. GC-UV (Gas Chromatography – Ultraviolet Spectrophotometry)
   * GC separates volatile compounds, and UV is used for detection — although this combo is less common than GC-MS.
3. CE-UV (Capillary Electrophoresis – UV Detection)
   * Capillary electrophoresis separates small ions and molecules based on charge and size; UV detects them at specific wavelengths.
4. IC-UV (Ion Chromatography – UV Detection)
   * IC separates ionic species (like fluoride, chloride, nitrate), and UV detects certain ions that absorb light (some require chemical derivatization to be UV-detectable).
5. Flow Injection Analysis (FIA)-UV
   * A sample is injected into a flowing stream of reagent, and reactions produce a color change detected by UV-Vis spectrophotometry.