INSTRUMENTATION

Photometry

Measurement of light

Light

a form of energy that travels in waves

nanometers (nm)

Light is expressed in

Peak/ Crest

Highest point of the light wave

Trough

Lowest point of the light wave

Wavelength

Distance between 2 consecutive peaks of a light wave

inversely proportional

Wavelength of light is ________ to the energy

↓ energy

↑ Wavelength =

↑ energy

↓ Wavelength =

heat or electrical energy

Light is convertible into other forms of energy like

Law of Conservation of Mass and Energy

Energy is not created nor destroyed. It can only be transferred and transformed from one into another

1. Visible / White Light

2. Invisible Light

Types of Light

Visible / White Light

Range: 400 - 700 nm (or 380-720 nm)

Violet

Color with the highest energy

Red

Color with the lowest energy

Red

Color with the longest wavelength

Violet

Color with the shortest wavelength

1. Ultraviolet light

2. Infrared light

Types of Invisible Light

Ultraviolet light

Range: 4 - 400 nm

e.g. x-rays, γ-rays

Ultraviolet light

Infrared light

Range: 700 nm - 0.3 cm

e.g. microwaves, radiowaves

Infrared light

Caraway Method

Used for uric acid

Phosphotungstic acid

Caraway Method reagent

Caraway Method

(+) color: Tungsten-blue

Caraway Method

Range: 650 - 700 nm (660 nm)

Visible light

Caraway Method light source

Enzymatic Method

Used for uric acid

Uricase

Enzymatic Method reagent

Enzymatic Method

Uric acid → Allantoin

Enzymatic Method

Range: 293 nm

Ultraviolet light

Enzymatic Method light source

Dubowski Reaction

Aka Condensation/ Ortho-toluidine method

Dubowski Reaction

Used for glucose

o-toluidine

Dubowski Reaction reagent

Dubowski Reaction

(+) color: Bluish-green

Dubowski Reaction

Range: 620 - 630 nm

Visible light

Dubowski Reaction light source

Wacker

LDH Forward reaction

Wroblewski La Due

LDH Reverse reaction

LDH method

(+): NAD/ NADH

LDH method

Range: 340 nm

Ultraviolet light

LDH method light source

Cyanmethemoglobin

Used for hemoglobin

Cyanmethemoglobin

Range: 540 nm

Visible light

Cyanmethemoglobin light source

OD450

Amniotic fluid

Amniotic fluid: OD450

for HDFN

OD650

Lamellar body

Lamellar body: OD650

for FLM

Visible light

Amniotic fluid & Lamellar body light source

Tanzer-Gilvarg

CK Forward reaction

Oliver-Rosalki

CK Reverse reaction

1. Light source

2. Monochromator

3. Cuvet

4. Detector

5. Readout system

Spectrophotometer components

Tungsten-halogen (iodide) lamp

Light source for Vis-IR range

1. Mercury arc lamp

2. Xenon-arc lamp

3. Deuterium discharge lamp

Light source for UV range

Monochromator

Isolates a specific wavelength of interest from the light source

1. Prisms

2. Diffraction gratings

3. Interference filters

Monochromators

Cuvet

Holds the sample solution

Cuvet

Normally transparent

Reflectance Photometer

Has non-transparent cuvet

Cuvet

Square-end or round-end

Photodetector

Convert the transmitted light energy into an equivalent amount of electrical energy

1. Barrier layer cell

2. Photodiode

3. Photomultiplier tube

Photodetectors

Photomultiplier tube

Most sensitive photodetector

Readout system

Measures the magnitude of the current generated by the detector

1. Digital read-out device

2. Ammeter

3. Galvanometer

Readout systems

1. Double Beam Spectrophotometer

2. Fluorometer

3. Flame Emission Photometer

4. Atomic Absorption Spectrophotometer

5. Reflectance Photometer

6. Nephelometer

7. Turbidimeter

Light-based instruments

Double Beam Spectrophotometer

2 cuvettes

Double beam in-space spectrophotometer

2 photodetectors

Double beam in-time spectrophotometer

1 photodetector

Ultraviolet light

Fluorometer light source

Fluorometer

Involves excitation light and fluorescent light

Fluorometer

Has 2 monochromators set at right angles

Quenching

Fluorometer disadvantage

Flame Emission Photometer

Based on measurement of light emitted by the analyte upon exposure to the flame

Flame Emission Photometer

For analytes that are easily excited by flame

1. Na

2. K

3. Li

Analytes easily excited by flame

1:100

In FEPS Na and K – specimen must be diluted at

Li or Cs

FEPS internal standard

Atomic Absorption Spectrophotometer

Based on absorption of atoms instead of molecules

Atomic Absorption Spectrophotometer

For analytes that aren’t easily excited by flame

Atomic Absorption Spectrophotometer

Reference method for Ca and Mg

Tetany

caused by ↓ Ca or ↓ Mg

Hollow cathode lamp

Atomic Absorption Spectrophotometer light source

Atomizer

In AAS, flame serves as an

Reflectance Photometer

Measures light reflected

Reflectance Photometer

Uses pads or slide as cuvet

Reflectance Photometer

Principle of Kodak Ektachem/ Vitros and automated dipstick readers

1. Reflectance Photometry

2. Dry slide Photometry

Kodak Ektachem/ Vitros are based on

1. Spreader layer

2. Scavenger layer

3. Reagent layer

4. Indicator layer

5. Support/ basal layer

Layers of dry slide

Scavenger layer

Removes specific interferences

Indicator layer

Contains chromogen/ pH indicator

Nephelometer

Measures light that has been scattered

Nephelometer

Quantitation of immunoglobulins

Nephelometer

Automated WBC differential count

Size/ volume

Forward scatter in automated WBC differential count detects

Internal cell complexity

Side scatter in automated WBC differential count detects

Turbidimeter

Measures light that has been blocked by the particles in solution

Turbidimeter

3% sulfosalicylic acid (SSA)