PHOTOMETRY – meas.

of radiant energy absorbed/given off by a molecule after illumination of a light source Light source- should always be present Class. of Clin Chem Instrumentation that measures EMR: I. Photometry 1. Spectrophotometry 2. Emission Flame Photometry/ Filter Photometry 3.Atomic Absorption Spectrophotometry 4. Fluorometry 5.Turbidity & Nephelometry I. Electrochemistry EMR (electromagnetic radiation) – radiant energy frm short rays (gamma rays) to WL long rays (UV & microwave) – portions of energy traveling in a wavelength manner – shorter wavelength, higher EM energy Types of EMR: cosmic rays, gamma rays, x-rays, UV rays, infrared, microwaves (radio, tv, radar) Theory of Light Waves – photon (E) or discrete packet of energy traveling in waves – have a peak/crest & trough  Wavelength – distance bet 2 successive peaks/crests

– intensity of color must be proportionate to conc.
of the color-producing subs – simplest colorimetric analysis using a simple device (comparison of unknown w/ standard)

Parts of Spectrophotometer: Source→(EnS)→monochromator→(ExS)→cuvet→detect or→meter/read out device(Galvanometer) 1.Light Source

– ex: Dubosq colorimeter, Sahli’s

– provides radiant energy to monochromator—
separates into discrete WL

hemoglobinometer Dubosq Colorimeter: – refined type of colorimeter parts: 1. light source 2. 2 glass-bottomed cups 3. 2 movable platforms 4. 2 glass plungers 5. optical system 6. 2 identical scales – reading: 20 at platform scales for both cups

– types: Tungsten lamp, Deutenum lamp 1.Entrance slit – minimizes stray light frm light source – prevents scattered light frm entering
monochromator

– Stray light: any WL outside band transmitted &
selected by monochromator

– Bandpass/ bandwidth: range of

– gives light its char. color 1.Nanometer (nm) – 10-9m; SI unit 2.Millimicron (mu) – 1nm 3.Angstrom (Å) – 10-10m, 1nm=10Å
Kinds: 1.visible spectra – 340-700nm

– left cup: standard sol. procedure: 1. 3-5ml of STD to each cup 2. set reading at 20 platform scale 3. Adjust- same color & appear as 1 field 4. Leave left cup 5. Rinse right cup= add unknown 6. Adjust (raise) RC until same intensity(color) 7. Read scale reading Darker→ ↓L Beer’s Law:
conc of color-producing subs X depth of sol through w/c light travels X constant – A=CxLxK

WL allowed to pass by monochromator – Major effect of stray light: Beer’s law won’t be followed = absorbance error – Highest absorbance: 2∞

– To read spm machine: A: R-L (above); %T: L-R
(below) True to stray light:  Analytical error is greatest w/ samples of light conc  More pronounced at extremes of the WL range  Often determines highest conc. Causes of stray light:  Reflection: scratches on optical surfaces (cuvet)  Dust particles in light path  Reflection frm w/in instrument  Extraneous room light: prevented by covering light shield  High colored spectrons (by diffraction gratings) Check:  Cut-off filters – eliminate all radiation at WL beyond one of interest  Liquid chem. (calibrating sol) – absorb short WL - Nickel sulfate(NiSO4), Sodium nitrite(NaNO2), Acetone  Black object – block light path 1.Monochromator

– Absorbance/ optical density of a colored sol =

– A ~ C/L
– A & K are constant, C & L must vary inversely

2.invisible spectra – UV & IR 
Amplitude – distance bet peak & trough Colorimetry – assoc. w/ spectro Primary considerations in analysis: 1. Quality 2. Intensity  Color absorbed – not seen; complimentary to the color seen  White light – all colors are transmitted Kinds of colorimetry: 1. visual 2. Photoelectric: Spectro & Filter photometry VISUAL

– Cu= CsLs/Lu

- Cu = AuCs/As PHOTOELECTRIC COLORIMETRY A.Spectrophotometry

– Meas. light

– Quantitation of subs: meas. amt of light absorbed – Adv: Therefore, it gives a relatively high – High degree of specificity reacts the subs of – meas light intensity of multiple WL
sensitivity, greater ease of rapid meas. compared to visual colorimetry after appropriate treatment

intensity in a much narrower WL using a device (prism/grating) to disperse the light source into a cont. spectrum

– WL selector
Types: a. prism – triangular wedge-shaped piece of glass, quartz, NaCl, KBr –allows transmission of light

– uses eye in determining end pt – very crude & subjective; less sophisticated &
flexible

interest w/ proper rgts = diff colors (analytical separation prior to color formation rxns) A.Filter Photometry – uses filter to isolate part of spectrum

– disperses white light into a cont. color based on
variation of refractive index of the diff WL

– short WL: refracted more
– red end of spectrum: refracted least

– B or V end: refracted most a. Grating – has grooves (3000+) cut into such an angle that each groove behaves like small prism – light is reflected

AsBLK- Au =final reading – dictated by parameters of assay & condition of sample  check for scratches, dirt & fingerprints:

– white light (various color component) – bent as
they pass a sharp corner

– scratches – place cuvets in rubber/plastic coated – dirt & fingerprints – conc HCl, H2O, ethyl
test tubes rack (minimizes scratches/prevent stains); use mild detergent & don’t use TT brush (1:3:4) : wipe dry (lintless tissue paper/ gauze) : rinsed several times in test sol. before reading absorbance

– linear spectrum; provides higher line WL
resolution than is possible w/ a prism & respond to all WL – more clearly defined spectral separation

 A inversely prop to %T  C inversely prop to %T  A=C Incident light – amt of light entering sol. Transmitted light – amt. of light passing through & is absorbed by sol Absorptivity – light absorbed by a molecular spm (specific for each cpd) in a sol

– constant for a pure soln. of any conc. w/in the
readable range of SPM

– may be determined by measuring the absorption
of known conc of a subs. in a cuvet of known path length usually under specific condition

1.Exit Slit- regulate light that is selected by
monochromator

2.Cuvet – analytical cell, sample holder, absorption
cell, optical cell

1.Detector/ Photodetector:

– changes as WL of radiation changes for any
particular molecular type

– holds soln.

– borosilicate, quartz or plastic  borosilicate: strongly alkaline  soft glass: acidic soln; don’t etch glass  quartz/plastic: more expensive, don’t absorb UV radiation, for WL below 320nm Types: a.round b.square – plane parallel optical surfaces & constant light path – less error form lens effect & refraction – prevents light more likely to occur in round Common causes of error:  failure to position properly (frosted markings)  failure to match absorbent readings of cuvet (inexpensive unmatched cuvets) Resolve by:  using blank reading: to meas. tolerance of each cuvet at each WL used (set at OA or 100%T) – should be used for each determination

– electron tube amplifying current that convert radiant energy to equivalent amt of electrical pr photoelectric energy

– absorbance of 1g/L of a chemical specific at a
given WL Calaculations of conc. From absorbance Measurement 1.Ratio of STD to unknown (x or u)

– Ex:Barrier-layer cells (less expensive)
Photoemission tube/ phototube Photomultiplier 1.Meter/Data Read out device – simplest method of displaying output of the detecting system Read out devices: a.meters b.digital display c.printed read outs d.recorder Beer’s Law (Beer-Lambert’s or Beer-Bouguer’s law)

– gives the calcu of conc from absorbance
reading/meas. – one-pt calibration

– used if absorptivity constant is known for a – Std meas. should result in A readings falling – >.900 = ↑ relative error assoc. w/ meas.
Cu = AuCs/As approx bet 0.100-0.900 particular conc, absorbance can be calculated directly using Beer’s Law

– mathematical basis of colorimetry – conc. of a subs is directly proportional to the amt

– 1 purpose: read out absorbance due to the rgt – treated like specimen/analyte – Reference sol: electrical readout of the
O

 

instrument is arbitrarily at 100% T Distilled H2O – 0.000 A (Rgt stability) Rgt blank – compensate for any unwanted light absorption due to other mat./ interference in the rxn

– checks rgt for determination: high rgt blk value =
Rgt deterioration

– good inexpensive QC procedure Sample blank – sample is added to mixture containing all components of the rxn except the rgt w/c reacts to form final colored prod. – absorbance is measured & subtracted from entire reaction system (accuracy)

of light absorbed or inversely proportional to the logarithm of transmitted light A = abc = log 100%T A = 2-log%T  A = absorbance  a = absorptivity  b = length path of the sol. In cm  c = conc of the subs of interest/ cons. of absorbed molecule  %T = % transmittance Transmittance – ratio of transmitted light to incident light potted y-axis 100%T= all light transmitted Absorbance – optical density

1.STD curve/ STD graph/ Calibration curve – if absorptivity is not known, A of analayte being – if A is plotted against the conc. Of
measured must be compared to the absorbances of at least 3 diff known conc. of subs/analyte (STDS) these subs (STD), result is a graph (STD curve)

– Standard curve: plotting the conc. Of the STDs – Since A ∞ C = Straight Line Calibrated curve
– Performed before running analysis of unknown STD curve glucose on the x axis (abscissa) against their respective A readings in the Y axis (ordinate)

– amt of light absorbed/blocked by a sol. – Difference bet. amt of incident light & transmitted
light = conc – No unit

– Intercept at O – to show proportionality
(semilog: inversely proportional) – Linearity is demonstrated & Beer’s Law is followed & analysis adheres to BL

– Higher absorbance readings deviating from being
linear, samples would be diluted & reassayed. New value: multiplied by the dilution factor (reciprocal of dilution) to correct the conc.

– Indicator of how low one can meas accurately
(accuracy of labortorian)

– Eliminated the need to run known mat/STD for
standardization

1.Molar Absorptivity Values
SPM QA Procedures – ensures (check instrument fxn)

– performed when an instrument is initially placed
into operation – periodically Spectral Absorbance Curve (SA graph/ST curve) made calibrating sol SPM QA/ Check/ Calibration: 1.WL calibration 2. absorbance/ T accuracy 3. linearity 4. stray light 5. electronic stability check WL Calibration Evan’s Blue dye- WL of max A- 610nm Filters w/ intense A maxima of known __: 1. didymium 2. holmium oxide Spectral Band Width:

– verify the WL of max absorbance/ min T for newly

– designates degree of monochromicity of a filter – of more sophisticated monochromator – obtained by measuring intensity of light emerging
– – – – from monochromator against the WL then measuring the peak width at ½ the height filters have wide band widths of 20-60nm Diffraction grating have much narrower band widths on the order of 1-5 nm Narrower spectral band width – more closely the instrument records the true absorption pattern of the sample Narrow BP instruments are more sensitive & precise

In cuvets 1. Blank 2. STD 3. QCS 4. SBlank 5. Unknown(X)

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