CAMLPR: Light measuring systems

cards 1- 24: reflectance and absorbance spectrophotometry; cards 25 -33: nephelometry/ turbidimetry/ emission spectrophotometry

what is the bougher lambert law

the longer the cuvette, the less light is transmitted thru and picked up by the detector

what is beers law?

concentration of a substance is directly proportional to the amount of light absorbed, while conc of substance is inversely proportional to transmittance.

what is the abs of dH2O

(beers law: conc=abs) 0

what is %T (transmittance) of dH2O?

(beers law: transmittance is inversely proportional to conc) 100%

what is the relationship between Abs and transmitance (%T). give formula

A= 2- log%T

in an assay measuring the transmittance (%T) of a coloured end product spectrophotometrically, readings of 70%T for the highest std and 98%T for the sample would indicate what ab the sample conc?

beers law indicates that conc is inversely proportional to abs. bc 98%T is high, that would indicate tht conc would be low

what is the beer lambert law

absorbance is directly proportional to conc (A=c) if light path and absorptivity are constant

how is the wavelength picked to measure a specific substance

the WL selected for analysis is at peak of max absorbance to achieve max analytical sensitivity. (It means the instrument chooses the wavelength where the substance absorbs the most light so the test can detect even small amounts of that substance more accurately.) LAMBDA MAX

what happens if the wavelength selected for a specific substance is not lambda max?

imprecision and inaccuracy

difference between bandpass and bandwidth

bandpass is the number of wavelengths passing thru the exit slit. bandwidth is the width of the opening of the exit slit (nm)

what is better, a smaller or larger bandwidth

the smaller the bandwidth the better the resolution

is the quality of a spect better or worse with a smaller stated bandpass

better

would you buy a spect w a bandpass or bandwidth that was higher or lower to achieve better spectral resolution

lower

how do you determine bandwidth of a solution

Bandwidth=λ2​−λ1​

Where:

• λ1\lambda_1λ1​ = lower wavelength at half max absorbance

• λ2\lambda_2λ2​ = upper wavelength at half max absorbance

Example:

• λmax = 500 nm

• Half-max absorbance occurs at 490 nm and 510 nm

Bandwidth:

510−490=20 nm510 - 490 = 20 \text{ nm}510−490=20 nm

So the absorption band is 20 nm wide.

if you are purchasing a new spect, the bandwidth of the monochromator should not exceed ___% of the natural bandwidth

10%

what is NADH natural bandwidth? whats the bandwidth the monochromator must have when measuring it

58 nm.

spects bandwidth should not exceed 10% therefore 10% of 58 is 5.8 nm. The monochromator must be 6nm or less

spectral isolation typical bandwidths: glass filters are referred to as _____ filters. how many bandwidths is it usually?

wide band pass filters; 20-50 nm

spectral isolation typical bandwidths: interference filters are referred to as _____ filters. how many bandwidths is it usually?

narrow bandpass filters; 5-15 nm

spectral isolation typical bandwidths: diffraction gratings how many bandwidths is it usually?

you get what you pay for. they range from 0.5 nm to 20 nm.

when performing manual testing, what is the purpose of a sample blank

eliminate absorbance from sample color

when performing manual testing , whats the purpose of a rgt blank?

eliminates abs from rgt color

example of reflectance spectrophotometry

urine test strips (light reflects off of coloured surface)

difference in diffuse and specular reflectance spectrophotometry. which do we use in the lab?

diffuse= light is reflected off an irregular surface allowing light to scatter in different directions. specular= light produces “mirror” image

we use diffuse reflectance spectrophotometry

t/f in refelctance spectrophotometry, analyte conc is directly proportional to reflection density

false: reflection density is the measure of the extent to which a surface reflects light.

ex: the more light absorbed = the less light reflected

difference between turbidimetry and nephelometry

turbidimetry= measure of light that passes thru the turbid sltn

nephelometry= measure of light that is reflected/ scattered off of a turbid sltn (amt of scatter is directly proportional to size and number of particles in the sltn)

in turbidimetry, amt of light abs is directly or indirectly proportional to conc?

directly proportional

in nephelometry, whereis the detector placed t read the light scattered?

at an angle 30-90 deg from incident light

what happens if you move the detector where the most amt of light is scattering in nephelometry

increased sensitivity

where in the lab is nephelometry used

measuring Ag-Ab complexes (determines size and number)

most sensitive light measuring system

chemiluminesence

what is fluoresence

occurs when a molecule absorbs light at one wavelength and re emits light at a longer wavelength (think glow in the dark stuff)

why is the 2 ary filter of a fluorometer at a right angle?

so that it only detects emitted light and not the excitation source.

what is the relationship between fluorescence intensity and the concentration of the analyte (fluorophore)

directly proportional