Analytical Techniques

measure the intensity of electromag radiation transmitted by a solution to determine the concentration of light-absorbing substance in solution

I/ Io where I is the intensity of transmitted light and Io is the intensity of incident light

Abs= -log (T)

number of absorption bands corresponding to structural groups within molecule

concentration of a substance is directly proportional to right amt of light absorbed or inversely proportional to log of transmitted light

A= a * b * c
a=absorptivity coefficient (constant)
b=path length
c=conecntration

abs is directly proportional to b and c
A=a*b*c
therefore, absorbance is proportional to inverse log of transmittance
A=-logT
T=log(1/ T)

a solution transmits light corresponding in wavelength to its color

solution absorbs light of wavelengths COMPLEMENTARY to its color

1. light source
2. monochromator
3. sample cell
4. photodetectors

UV region
190-420 nm

part of the UV and visible

deuterium gas

halogen-iodide and bromide

xenon gas

190-800 nm

isolates light within a narrow region of spectrum

diffraction gratings
prisms
filters

diffraction gratings and prisms

produce monochromatic light based on the principle of constructive interference of waves

2 pieces of glass each mirrored on one side, separated by transparent spacer that is 1/ 2 of the desired wavelength

filters (photometers), prisms, and gratings (spectrometers) select/ isolate narrow range of incident wavelength

defines the range of wavelengths transmitted

as width at half the max transmittance

a bandpass less than 1/ 5 of the natural bandpass of the chromophore

to convert the transmitted radiant energy into an equiv amount of electrical energy

photocell
phototube
photomultiplier tube
photodiode

photodiode+array → photocell → phototube → pmt

photocell / photodiode → phototube → pmt

photocell → phototube / pmt → photodiode

photocell → photodiode → phototube → pmt

photocell

photocell

temperature sensitive and nonlinear at very low and very high levels of illumination

uses dynodes of increasing voltage to amplify the current significantly

10,000X as sensitive as barrier-layer cell, which has no amplification

because of lack of internal amplification.
Linearity, speed, and small size are excellent

has linear array that allows it to respond to specific wavelength resulting in complete UV/ visible spectrum analysis

each photodiode responds to specific wavelength--a complete UV/ visible spectrum can be obtained in less than 1 sec

wavelength accuracy
stray light
linearity

The wavelength indicated on the control dial is actual wavelength of light passed by the monochromator

using standard absorbing solutions or filters with absorbance maxima of known wavelength

didymium or holmium oxide glass (stable)

any wavelengths outside the band transmitted by monochromator

reflection of light from scratches on optical surfaces or from dust particles in light path

absorbance error, esp in high absorbance range.
Places limits on upper range of linearity

when a change in concentration results in a straight-line calibration curve, as discussed under Beer's Law

concentrations of solutions that contain fluorescing molecules

short wavelength high energy excitation light

mechanical attenuator

greater specificity and sensitivity

very sensitive to environmental changes

excitation wavelength is held constant and emission intensity is measured as a function of emission wavelength

the emission is measured at one wavelength while the excitation wavelengths are scanned

Stokes shift (idk)

1. measure emitted light
2. 2 filters
3. detector position
4. advantage / disadvantage

2 light sources
need chopper
detecter -- PM tube

power supple
electrodes
buffer
gel
imaging analysis

1. protein with negative charges
2. electric circuit