FOTOMETER

Foto : Cahaya
Meter : Pengukuran

Fotometer adalah metode pengukuran suatu zat terlarut

dengan mengunakan cahaya
Electromagnetic radiation characteristic
Wave length interval, where the type of EMR begins

rays X rays u.v. visible I.R. microwaves radiowaves

A 1 10 1800 3400 7000
Nm / m 180 340 700
0,7 400
cm 0.04 25

Its important in clinical chemistry :

* U.V. light
* Visible light
Unit of the wavelength :
o
Angstrom = A = 10-10m
Nanometer = nm / milimicron = m = 10-9m
Micrometer = micron = = 10-6m

Clinial chemistry determinations :

* U.V. light
* Visible light
Visible light :
Red
Orange
Yellow
Green
Blue
Bluish violet ( indigo )
Violet
R.O.Y.G.B.I.V.
Uv, visible and short IR spectrum characteristic

Wavelength Region name Color observed

< 380 Ultra violet Not visible
380 - 440 visible Violet
440 - 500 visible Blue
500 - 580 visible Green
580 - 600 visible Yellow
600 - 620 visible Orange
620 - 750 visible Red
750 - 2000 infra red Not visible
Colors and complementary colors of the u.v. and visible spectrum

( mm ) Region name Colour observed Complementary

or sol. color
180 - 220 Short u.v. Not vis -
220 - 340 u.v. Not vis -
340 - 430 Vis Violet Yellow green
430 - 475 Vis Blue Yellow
475 - 495 Vis Green blue Orange
495 - 505 Vis Blue green Red
505 - 555 Vis Green Purple
555 - 575 Vis Yellow green Violet
575 - 600 Vis Yellow Blue
600 - 620 Vis Orange Green blue
620 - 700 Vis Red Blue green
Pengukuran secara Fotometer
Principles :

X solution

I A I
o

b
An incident light beam with intensity Io passing through a
square curette containing a solution of a compound that
absorbs light of a certain wavelength ( ).
The intensity of the transmitted light beam Is will be less than
Io and we defined the transmittance ( T ) of light as Is / Io.
The absorbance light of a certain wavelength is related to the
concentration of the substance being measured.

A
A max
A1 Figure I
Max = 500nm
A2
A1=<<

= 400nm
At
A2 = >>

100 500
nm
( max)
A Max

Figure II
max = 490 -510

490 510
 490 510 nm
A
Figure III
min = 550 nm

A min

500 min 600 nm

Bagan dari Fotometer

Filter :
340, 405, 492. 546, 578
m
Spectrum
Light source :
* Tungsten lamp Cuvette :
* Halogen lamp * Round / square
* Mercury lamp * Material : glass, silica / quartz, plastic
* Hydrogen lamp
Detectors :
Monochromator : * Barrier layer cell
* Filter * Photomultipleir tube
* Grating
* Prism
Meter = readout devices
* Direct reading : output photocell, directly to the meter
* Digital readout devices, absorbance concentration
Hg - line spectrum

Energy

250 300 350 400 nm

Continued spectrum of wolfram/ tungsten lamp

Energy

400 500 600 700 800 nm

Beers law

Io = incident light
I = transmitted light
b =the distance of the light
passed through

Io A I
A = a.b.c = log 10 / % T =
2 log % T
B

A = absorbance
a = absorptivity
b = distance of the light passed through = diameter of the cuvette
c = concentration of the subtance in a solution
%T = percent transmittance ( T = I / Io )
 Filter photometer:
Light source:
Lamp/ halogen lamp

Tungsten/ diafragma filter slit cuvette

Halogen
 Fotometer:
Lensa
Lensa Lensa
Filter

Source lamp

Cuvette adaptor

Kaca penahan panas Diafragma

 Spectrophotometry nomenclatur

Name Symbol Defenition

Absorbance A - Log T = Log Io / I
Absorptivity a A / bc ( c in g.L )
Molar absorptivity A / bc ( c in mol / L )
Path length b Internal cell = sampel length in cm
Transmittance T I / Io
Wavelength m M = 10A = 10-9m
Absorption max max Wavelength at wich max
absorption occurs
 b = 1 cm A = A1% = absorption coef. 1 cm
c = 1 g / dl ( = 1% ) E = E1% = extinction coef. 1 cm

I II
%T A

Conc. Conc.
 Absorbance

2.0 1.0 .80 .70 .60 .50 .40 .30 .20 .10

0 10 20 30 40 50 60 70 80 90 100
Transmmitance

Figure I : %T - concentration
Figure II : A - concentration
Figure III : scale of the relation A -%T
Beers law application
Solution I
Subtance = p
Concentration = Cl mg / 100 ml
Reading of absorbance = A1
Absorptivity of max = a
cuvette = b cm
I
A1 = a.b.cl
Solution II
Subtance = p
Concentration = C2 mg / 100 ml
Reading of absorbance = A2
Absorptivity of max = a
cuvette = b cm
II A2 = a.b.c2
I / II = A1 / A2 = a.b.c1 / a.b.c2 A1 / A2 = c1 / c2

Requirement :
1. Max
2. Standard solution
3. Blank solution

Photometric measurement
I. End point reaction
Color appeared = calorimetric measurement
Turbidity appeared = turbidimetric measurement
II. Kinetic reaction
* the measurement is conducted during the reaction occurs
* the absorbance changing in a define time unit, is the
function of a substance concentration in the solution
Calculation
* Beers law : A1 / A2 = C1 / C2
* Transmission - concentration calculation
* Calibration curve

Calibration curve

A 5
4
3
2
1

0
50 100 Cx C = mg / 100 ml
Beers law requirement :
1. Monochromatic light : max
2. Reading at the 20 - 70%T
3. No fluorescent subtance
4. The standard concentration being used is in the range
concentration of linearity with the concentration of the subtance
being subtance being measured.
Serum/Standard Reagensia

Baca Absorbans