PHOTOMETRY – meas. of radiant energy – intensity of color must be proportionate to conc.

Parts of Spectrophotometer:
absorbed/given off by a molecule after illumination of Source→(EnS)→monochromator→(ExS)→cuvet→detect
of the color-producing subs
a light source or→meter/read out device(Galvanometer)
– simplest colorimetric analysis using a simple
Light source- should always be present [Link] Source
device (comparison of unknown w/ standard)
Class. of Clin Chem Instrumentation that measures – provides radiant energy to monochromator—
EMR: – ex: Dubosq colorimeter, Sahli’s separates into discrete WL
I. Photometry hemoglobinometer
1. Spectrophotometry Dubosq Colorimeter: – types: Tungsten lamp, Deutenum lamp
2. Emission Flame Photometry/ Filter Photometry – refined type of colorimeter [Link] slit
[Link] Absorption Spectrophotometry parts: – minimizes stray light frm light source
4. Fluorometry 1. light source
2. 2 glass-bottomed cups – prevents scattered light frm entering
[Link] & Nephelometry 3. 2 movable platforms monochromator
I. Electrochemistry
EMR (electromagnetic radiation) – radiant energy frm
4. 2 glass plungers – Stray light: any WL outside band transmitted &
5. optical system selected by monochromator
short rays (gamma rays) to WL long rays (UV & 6. 2 identical scales
microwave)
– reading: 20 at platform scales for both cups – Bandpass/ bandwidth: range of WL allowed to
– portions of energy traveling in a wavelength pass by monochromator
manner – left cup: standard sol. – Major effect of stray light: Beer’s law won’t be
– shorter wavelength, higher EM energy procedure: followed = absorbance error
Types of EMR: cosmic rays, gamma rays, x-rays, UV 1. 3-5ml of STD to each cup – Highest absorbance: 2∞
rays, infrared, microwaves (radio, tv, radar) 2. set reading at 20 platform scale
Theory of Light Waves 3. Adjust- same color & appear as 1 field
– To read spm machine: A: R-L (above); %T: L-R
(below)
– photon (E) or discrete packet of energy traveling 4. Leave left cup
True to stray light:
in waves 5. Rinse right cup= add unknown
– have a peak/crest & trough 6. Adjust (raise) RC until same intensity(color)  Analytical error is greatest w/
samples of light conc
 Wavelength – distance bet 2 successive 7. Read scale reading
 More pronounced at extremes of the
peaks/crests Darker→ ↓L
Beer’s Law: WL range
– gives light its char. color  Often determines highest conc.
[Link] (nm) – 10-9m; SI unit – Absorbance/ optical density of a colored sol = Causes of stray light:
conc of color-producing subs X depth of sol  Reflection: scratches on optical surfaces (cuvet)
[Link] (mu) – 1nm through w/c light travels X constant
 Dust particles in light path
[Link] (Å) – 10-10m, 1nm=10Å – A=CxLxK
 Reflection frm w/in instrument
Kinds: – A ~ C/L  Extraneous room light: prevented by covering light
[Link] spectra – 340-700nm – A & K are constant, C & L must vary inversely shield
[Link] spectra – UV & IR – Cu= CsLs/Lu  High colored spectrons (by diffraction gratings)
 Amplitude – distance bet peak & trough - Cu = AuCs/As Check:
PHOTOELECTRIC COLORIMETRY  Cut-off filters – eliminate all radiation at WL beyond
Colorimetry – assoc. w/ spectro [Link] one of interest
Primary considerations in analysis:  Liquid chem. (calibrating sol) – absorb short WL
1. Quality – Meas. light intensity in a much narrower WL
using a device (prism/grating) to disperse the - Nickel sulfate(NiSO4), Sodium
2. Intensity nitrite(NaNO2), Acetone
 Color absorbed – not seen; complimentary to the light source into a cont. spectrum
 Black object – block light path
color seen – Quantitation of subs: meas. amt of light absorbed [Link]
 White light – all colors are transmitted after appropriate treatment
– WL selector
Kinds of colorimetry: – Adv: Therefore, it gives a relatively high
1. visual sensitivity, greater ease of rapid meas. compared Types:
2. Photoelectric: Spectro & Filter photometry to visual colorimetry
a. prism – triangular wedge-shaped piece of glass,
VISUAL – High degree of specificity reacts the subs of quartz, NaCl, KBr –allows transmission of light
– uses eye in determining end pt interest w/ proper rgts = diff colors (analytical
separation prior to color formation rxns) – disperses white light into a cont. color based on
– very crude & subjective; less sophisticated & [Link] Photometry variation of refractive index of the diff WL
flexible
– meas light intensity of multiple WL – short WL: refracted more
– uses filter to isolate part of spectrum – red end of spectrum: refracted least
 – B or V end: refracted most AsBLK- Au =final reading  A inversely prop to %T
a. Grating – has grooves (3000+) cut into such an – dictated by parameters of assay & condition of  C inversely prop to %T
angle that each groove behaves like small prism sample  A=C
 check for scratches, dirt & fingerprints: Incident light – amt of light entering sol.
– light is reflected
Transmitted light – amt. of light passing through &
– white light (various color component) – bent as – scratches – place cuvets in rubber/plastic coated is absorbed by sol
they pass a sharp corner test tubes rack (minimizes scratches/prevent
Absorptivity – light absorbed by a molecular spm
stains); use mild detergent & don’t use TT brush
– linear spectrum; provides higher line WL – dirt & fingerprints – conc HCl, H2O, ethyl
(specific for each cpd) in a sol
resolution than is possible w/ a prism & respond
([Link])
– constant for a pure soln. of any conc. w/in the
to all WL readable range of SPM
: wipe dry (lintless tissue paper/ gauze)
– more clearly defined spectral separation – may be determined by measuring the absorption
: rinsed several times in test sol. before
reading absorbance of known conc of a subs. in a cuvet of known path
[Link] Slit- regulate light that is selected by length usually under specific condition
monochromator
– changes as WL of radiation changes for any
[Link] – analytical cell, sample holder, absorption [Link]/ Photodetector: particular molecular type
cell, optical cell – electron tube amplifying current that convert – absorbance of 1g/L of a chemical specific at a
– holds soln. radiant energy to equivalent amt of electrical pr
given WL
– borosilicate, quartz or plastic photoelectric energy
Calaculations of conc. From absorbance Measurement
 borosilicate: strongly alkaline – Ex:Barrier-layer cells (less expensive) [Link] of STD to unknown (x or u)
 soft glass: acidic soln; don’t etch glass Photoemission tube/ phototube
Photomultiplier – gives the calcu of conc from absorbance
 quartz/plastic: more expensive, don’t absorb UV [Link]/Data Read out device reading/meas.
radiation, for WL below 320nm
– simplest method of displaying output of the – one-pt calibration
Types:
[Link] detecting system – used if absorptivity constant is known for a
Read out devices: particular conc, absorbance can be calculated
[Link] – plane parallel optical surfaces & [Link] directly using Beer’s Law
constant light path [Link] display
– less error form lens effect & refraction [Link] read outs – Std meas. should result in A readings falling
– prevents light more likely to occur in round [Link] approx bet 0.100-0.900
Common causes of error: – >.900 = ↑ relative error assoc. w/ meas.
 failure to position properly (frosted markings) Beer’s Law (Beer-Lambert’s or Beer-Bouguer’s law) Cu = AuCs/As
 failure to match absorbent readings of cuvet – mathematical basis of colorimetry
(inexpensive unmatched cuvets)
Resolve by: – conc. of a subs is directly proportional to the amt
of light absorbed or inversely proportional to the [Link] curve/ STD graph/ Calibration curve
 using blank reading: to meas. tolerance of each
cuvet at each WL used (set at OA or 100%T) logarithm of transmitted light – if absorptivity is not known, A of analayte being
– should be used for each determination A = abc = log 100%T measured must be compared to the absorbances
A = 2-log%T of at least 3 diff known conc. of subs/analyte
– 1 purpose: read out absorbance due to the rgt
O
 A = absorbance (STDS)
– treated like specimen/analyte  a = absorptivity – if A is plotted against the conc. Of
these subs
– Reference sol: electrical readout of the  b = length path of the sol. In cm (STD), result is a graph (STD curve)
instrument is arbitrarily at 100% T  c = conc of the subs of interest/ cons. of – Standard curve: plotting the conc. Of the STDs
 Distilled H2O – 0.000 A (Rgt stability) absorbed molecule on the x axis (abscissa) against their respective A
 Rgt blank – compensate for any unwanted light  %T = % transmittance readings in the Y axis (ordinate)
absorption due to other mat./ interference in the rxn Transmittance – ratio of transmitted light to incident
light
– Since A ∞ C = Straight Line Calibrated curve
– checks rgt for determination: high rgt blk value = potted y-axis
– Performed before running analysis of unknown
Rgt deterioration STD curve glucose
100%T= all light transmitted
– good inexpensive QC procedure Absorbance – optical density – Intercept at O – to show proportionality
 Sample blank – sample is added to mixture – amt of light absorbed/blocked by a sol. (semilog: inversely proportional)
containing all components of the rxn except the rgt – Linearity is demonstrated & Beer’s Law is
w/c reacts to form final colored prod.
– Difference bet. amt of incident light & transmitted followed & analysis adheres to BL
light = conc
– absorbance is measured & subtracted from entire
reaction system (accuracy) – No unit
 – 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
[Link] 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)
– verify the WL of max absorbance/ min T for newly
made calibrating sol
SPM QA/ Check/ Calibration:
[Link] 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:
– 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)