chem lab 3 a photo metric equipment

8/14/2019 Chem Lab 3 a Photo Metric Equipment

1/6MLAB 2401 - Clinical Chemistry Lab Manual CB 15

UNIT: Electromagnetic Radiation and

Photometric Equipment 3photo.wpd

Instrumentation ITask

To review the theory of electromagnetic radiation and the principle and use of common laboratoryinstruments using electromagnetic radiation.

Objectives

Upon completion of this exercise, the student will be able to:

1. State the wave and particle theory of electromagnetic radiation.2. Know what colors correspond to visible wavelengths.3. Identify the regions of the electromagnetic spectrum occupied by gamma, x-rays, UV, visible,

IR, and microwaves indicating relative wavelength, frequency, and energy.

4. Know basic principles of how instruments determine results.

5. List and compare the principles/applications of different photometric equipment.

Principle

1. Properties of light and radiant energy.

Radiant energybehaves as if it has electric and magnetic fields and is found asdiscrete

bundles of energy (photons) traveling inwave form (particle and wave theories). The more

energy contained, the more frequent the wave and therefore, the shorter the wavelength.

Wavelengths are measured in nanometers (nm) or 10 meters.-9

2. Interactions of light and matter.

A. Absorption (absorption spectroscopy UV, visible, infra-red, atomic)

When an atom, ion, or molecule absorbs a photon, the additional energy results in analteration of state (it becomes excited). Depending on the individual species, this maymean that a valence electron has been put into a higher energy level, or that thevibration or rotation of covalent bonds of the molecule have been changed.

In order for a ray of radiation to be absorbedit must:

1. have the same frequency of the rotational or vibrational frequency in the

molecules it strikes, and;2. be able to give up energy to the molecule it strikes.


2/6

UNIT: Electromagnetic Radiation and Photometric Equipment (continued)

B 16 C MLAB 2401 - Clinical Chemistry Lab Manual

Energy FrequencyWavelength

(nm)Type of Radiant

EnergyReflects Absorbs

>**

**********

>**

**********

25 x 10 radiowaves (uwaves)7 continuousnon-visible

The energy absorbed is distributed throughout the structure. The absorption pattern ofcomplex organic molecules will be the cumulative sum of the absorption of all of theindividual covalent bonds as a result of the energy's distribution throughout the molecule.

As the energy is released from the atoms, molecules, or incandescent solids, an emissionspectra is formed.

B. Emission spectra are ofthree types.

1. Line spectrum is light of one wavelength formed as the result of excited electrons

ofatoms returning to their ground state. Theatom emits the same wavelengths

as those absorbed by the atom.

2. Band spectrum is a group(s) of wavelengths formed from the return of excited

electrons inmolecules returning to their ground state. An array of wavelengths are

emitted as the process is complicated by the internal energy of vibration androtation (all substances except rarefied gas and atoms).

3. Continuous spectrum is a very broad and continuous group of wavelengths

emitted by incandescent solids (tungsten lamps).

Electromagnetic radiation in its various forms is measured in common laboratory instruments:

A. Absorption of EM radiation1. atomic absorption2. spectrophotometry

B. Emission of EM radiation1. flame photometer2. fluorometer 3. scintillation counter


3/6


4/6



Equipment Clinical Applications PrincipleEnergy

Source

Mono-

chromater

Sample

Holder

Colorimetry/photometry

any proceduremeasuring colordevelopment or loss

absorption/transmissionof light through a coloredsolution providing ameans of measuring lightabsorbing particles

tungsten(deuterium,

Hg, He)

filter(interferenc

e filter)

cuvetteslit

Spectrophotometry visible change in Abs.between 400-700 nm

UV change in Abs. at340nm of NAD : NADH

tungsten/UV(deuterium,

Hg, He)

prism ordiffraction

grating

ReflectanceSpectrophotometry

Same as above Filtered light is focused ona flat test surface. Someof the light is absorbed,the remainder is reflectedonto a photodetector. Theabsorption of light bychromophores (reflectiondensity) is inverselyrelated to the intensity ofreflected light.

tungsten filter

variespaper,plastic

strip, dryfilm orslide

Turbidimetry-------------------

lipids, CSF protein,serum proteins,amylase

light transmission througha turbid solution

tungsten/UV filter

cuvetteslitNephelometry degree of light scattered UV filter

fluorometer

Fluorometry drugs, hormones,intermediarymetabolites

absorbs high intensity UVand emits longerwavelength visible light(lower energy)

UV source 2filters/prism

Primaryexciter filterSecondaryfluorescenc

e filter

cuvetteslit

Emission FlamePhotometry

electrolytes, traceminerals (Na, K, Li)

The energy absorbed asheat is released in the

form of light energy of awavelength specific forthe element proportionalto its concentration

hot flame specificfilter for Na,

K, Li

burner-aspirator

slit

Atomic AbsorptionSpectrometry

Ca, Mg, Cu, Mn, Fe,Pb, Hg, etc.

A hot flame releasesmetallic atoms from mole-cules. The ground statemetallic atoms absorbmonochromatic light,generated by a hollowcathode tube, proportionalto their concentration

hollowcathode tubeof measured

metal

prism ordiffraction

grating

burner-aspirator

slit

Densitometry(very basicphotometer)

Hb, protein electro-phoresis, CPK, LDHisoenzymes

photometer scans acolored strip

tungsten/UV filter stage slit

Refractometry TP, specific gravity light bends withconcentration; refractiveindex

tungsten none stage slit

ScintillationCounters

a. gammab. beta (liquid)

drug assays,hormonesimmunoassays (TSH,

12T3, T4, B , Folate)

EM radiation countsdirectly proportional toconcentration. Sample inliquid phosphor.Fluorescence convertedinto longer wavelength

atomicnucleus

none cuvetteslit


5/6


MLAB 2401 - Clinical Chemistry Lab Manual CB 19

Study Questions

Name

Instructions: Legibly write your answers in the space provided. Unless otherwise indicated, each

question is worth one point.

1. Describe / define wavelength.

2. In clinical laboratory work, wavelength is most commonly measured in what unit?

3. The discrete bundles of energy emission in the electromagnetic spectrum are called

__________________________________________.

4. Ultraviolet light has shorter / longer(circle one) wavelength than visible light and would

therefore have higher / lower(circle one) energy than visible light.

5. Gamma rays have a very short wavelength. You would, therefore, expect them to have

higher / lower(circle one) frequency and energy.

6. What type of emission spectra is expected from:

a. atoms

b. molecules

c. solids

7. According to information presented in this lab, briefly summarize what happens whenelectromagnetic radiation is absorbed by an atom or molecule. (2 points)

8. A spectrophotometer and an atomic absorption spectrophotometer can be grouped togetheras they measure light _________________________________________.

9. A flame photometer and scintillation counter (gamma counter) measure light/radiation_________________________________.

10. From the Table of Photometric Equipment included in the lab, list two (2) types of chemistryequipment that measure change in the electromagnetic radiation.


6/6



After reviewing the lab, provide a brief explanation of the principles of the following in your ownwords. (2 points each)

11. photometry

12. emission flame photometry

13. atomic absorption spectrophotometry

14. reflectance photometry -

15. fluorometry -

chem lab 3 a photo metric equipment

Documents