What is photometry?

* used most freq in clin chem

*consists of absorbance or reflectence technology

*uses color and color variation to determine concentration

*installed in many of our automated instruments

Light is what kind of energy?

Radiant energy

 How does light travel?

Distance between waves is called?

Wave length

The human eye responds to....

light (radiant energy) with wavelengths between:

380-750 nm

What determines the color of light seen by the eye?

the wavelength of the light

Combo or mixture of light energy that has dif wave legnths is known as

daylight or white light

Red

Orange

Yellow 

Green

Blue

Violet

The color of light see in the vis spectrum depends on wavelength that is..

When light is not absorbed it is...

blue solution appears blue because...

the concentration of the substance

The intensity of color is _______ to the conc. of the substance

proportional

What is beers law?

concentration of a substance is directly proportional to the amount of light absorbance inversily proportional to the logarithim of the transmitted light

Absorbance and transmittance are INVERSILY PROPORTIONAL to eachother

ex. 40 absorbance = 60 transmittance

what do the letters in...

A=abc

...stand for?

A=absorbance

a=absorbtivity

b=sample light path(width of test tube)

c= concentration of absorbing material

1.molar absorbtivity of the chemical

2.stray light

3.light scatter

What are factors that effect abc

b,path length?

1.poor quality cuvette (scratch)

2.size of holder bigger then cuvette

3.not optically pure

4.unequal light path length across the light beam

1. shifts in chemical equilibria as a function of concentration (soln may need to be buffered to prevent this)

2. light scattering by the sample matters especially in turbid sample

Another word for absorbed light is 

optical density (OD) 

What is %T

%T

Is the amount of light that passes through a colored substance 

A graph of absorbance of light by a “kind” of solution TO the concentration is ......

LINEAR

Does transmitted light decrease in direct proportion to the concentration or color intensity of the solution being measured?

NO!

Will %T give a straight line when plotted on a graph?

NO!

What is the equation to finding absorbance from %T?

ABS=2-log%T

What is a spectrophotometer used for..

*Incandescent Tungsten

*Tungsten

*Iodide lamp(visible, IR)

UV lamps are deuterium discharge lamp, what does this provide

Mercury lamps emit a sharp line spectrum with both......

Important factors for a light source are..

1. Range
2. Spectral distribution within a range
3. Source of radiant production
4. Stability of the radiant energy
5. Temperature

What does a monochromator do?

*isolates individual wavelengths

*uses width of entrance and exit slits to allow wavelengths through

What is the bandpass of a monochromator?

defines the range of wavelengths transmitted and is calculated as width at more than half the maximum transmittance

(narrowre the bandpass, the more expensive)

Spectral Bandpass

*shows absorbtion spectrum of the analyte over a wavelength  range from 200-400nm

*never passes single color/wavelength, passes small range of wavelengths

* Range of wavelengths=spectral Bandpass

*Smaller slit=smaller spectral bandpass

1.Colored Glass

2.Intereference Filters

3.Prism

4.Diffraction gratings

Colored glass filters

*least expensive

*pass a wide range of radient energy

*not precise but simple, cheap, and somewhat useful

Interference filters

*produce monochromic light(one color) using principle of constructive intereference waves

*2 peices of glass, mirrored on one side are seperated by transparent spacer that is precisely 1/2 the desire wavelength

*light wave enter 1 side of filter and reflected at 2nd surface

*wavelengths twice the space between the glass surfaces reflect back and forth reinforcing others of the same wavelength finally passing through

*other wavelenghts will cancel out bc of phase differences (destructive interference)

*fiters transmit multiples of the desired wavelengths, they require acessory filters to get ride of harmonic wavelengths

*made to pass a very narrow range of wavelengths, have good efficiency

Prism monochromator

*Narrow beam of light is focused on a prism and refracted as it enters the more dense glass

*Shortwave lengths are refracted more than long wavelengths, which disperses the white light into a spectrum.

the prism can be rotated allowing only the desired wavelength to pass through an exit slit

Diffraction gratings

*most commonly used

*many parallel grooves (15,000-30,000 inch) are etched into a polished surface. Based on the principle that wavelengths bend as they pass a sharp corner, the degree of bending depends upon wavelength

Name 3 properties of a sample cell..

1. Cuvette either round or square

2. has to be optically pure, free from scratches, no writing bc it has to absorb light

3.Square preferred bc plane parrallel optical surfase and constant light path

* convert transmitted energy into electrical energy

*least expsenive =

1.Barrier-layer cell or photocell, contains selenium when light hits this, excites E, silver and iron gives resistance and forms barrier proportional to radiation

Photocells

*require no external voltage source but use internal electron transfer to produce current in an external circuit.

*this type of cell is used mainly in filter photometers with wide band pass

*durable but temp sensitive, nonlinear at very low and high levels of light

Phototube

*Type of photodetector

*similar to barrier cell in that it gives off electrons when light energy strikes it

*differs in that outside voltage it required

*contains negativr charged cathode and positive charged anode in a glass case

*Cathode made of rubidium or lithium that acts as a resistor in the dark but emites electrons when exposed to light

*emitted electrons jump over the + anode where they are collected and return to measureable circuit