PHOTOMETRY – meas. of radiant energy
Parts of Spectrophotometer:
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
– 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
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
– uses eye in determining end pt
– very crude & subjective; less sophisticated &
flexible
– 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)
– ex: Dubosq colorimeter, Sahli’s
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
– 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:
– Absorbance/ optical density of a colored sol =
conc of color-producing subs X depth of sol
through w/c light travels X constant
– A=CxLxK
– A ~ C/L
– A & K are constant, C & L must vary inversely
– Cu= CsLs/Lu
- Cu = AuCs/As
PHOTOELECTRIC COLORIMETRY
A.Spectrophotometry
– Meas. light intensity in a much narrower WL
using a device (prism/grating) to disperse the
light source into a cont. spectrum
– Quantitation of subs: meas. amt of light absorbed
after appropriate treatment
– Adv: Therefore, it gives a relatively high
sensitivity, greater ease of rapid meas. compared
to visual colorimetry
– High degree of specificity reacts the subs of
interest w/ proper rgts = diff colors (analytical
separation prior to color formation rxns)
A.Filter Photometry
– meas light intensity of multiple WL
– uses filter to isolate part of spectrum
Source→(EnS)→monochromator→(ExS)→cuvet→detect
or→meter/read out device(Galvanometer)
1.Light Source
– provides radiant energy to monochromator—
separates into discrete WL
– 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 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
– WL selector
Types:
a. prism – triangular wedge-shaped piece of glass,
quartz, NaCl, KBr –allows transmission of light
– 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
– white light (various color component) – bent as
they pass a sharp corner
– linear spectrum; provides higher line WL
resolution than is possible w/ a prism & respond
to all WL
– more clearly defined spectral separation
1.Exit Slit- regulate light that is selected by
monochromator
2.Cuvet – analytical cell, sample holder, absorption
cell, optical cell
– 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
– 1 purpose: read out absorbance due to the rgt
O
– treated like specimen/analyte
– Reference sol: electrical readout of the
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)
AsBLK- Au =final reading
– dictated by parameters of assay & condition of
sample
 check for scratches, dirt & fingerprints:
– scratches – place cuvets in rubber/plastic coated
test tubes rack (minimizes scratches/prevent
stains); use mild detergent & don’t use TT brush
– dirt & fingerprints – conc HCl, H2O, ethyl
(1:3:4)
: wipe dry (lintless tissue paper/ gauze)
: rinsed several times in test sol. before
reading absorbance
1.Detector/ Photodetector:
– electron tube amplifying current that convert
radiant energy to equivalent amt of electrical pr
photoelectric energy
– 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)
– mathematical basis of colorimetry
– conc. of a subs is directly proportional to the amt
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
– amt of light absorbed/blocked by a sol.
– Difference bet. amt of incident light & transmitted
light = conc
– No unit
 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
– changes as WL of radiation changes for any
particular molecular type
– 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)
– gives the calcu of conc from absorbance
reading/meas.
– one-pt calibration
– used if absorptivity constant is known for a
particular conc, absorbance can be calculated
directly using Beer’s Law
– Std meas. should result in A readings falling
approx bet 0.100-0.900
– >.900 = ↑ relative error assoc. w/ meas.
Cu = AuCs/As
1.STD curve/ STD graph/ Calibration curve
– if absorptivity is not known, A of analayte being
measured must be compared to the absorbances
of at least 3 diff known conc. of subs/analyte
(STDS)
– if A is plotted against the conc. Of
these subs
(STD), result is a graph (STD curve)
– Standard curve: plotting the conc. Of the STDs
on the x axis (abscissa) against their respective A
readings in the Y axis (ordinate)
– Since A ∞ C = Straight Line Calibrated curve
– Performed before running analysis of unknown
STD curve glucose
– 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)
– verify the WL of max absorbance/ min T for newly
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:
– 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)