Lab Quiz 2 - Intro to Spectrophotometry

Light

a form of energy

electromagnetic radiation

photons of energy emitted

Light can be described as (2):

absorbed, transmitted, or reflected/scattered

Light that strikes a molecule can be:

colorimetric assays

What type of assays/tests measure absorbed light?

**hint: glucose, protein, cholesterol all can be measured as a color

wavelength (nanometers)

What unit of measure is used to measure light?

~400 to ~700nm

wavelength of Visible light

200nm to 400nm

wavelength of UV light

**hint: UV light has a shorter wavelength than visible light

760nm to 3000nm

wavelength of Infrared light

**hint: Infrared light has a longer wavelength than visible light

white light

mixture of all the colors of light

black light

the absence of all colors of light

prism or diffraction grating device

The human eye can see all of the colors if the white light is directed onto what (2)?

violet, blue, green, yellow, orange, red

shortest to longest wavelengths:

lower

Longer wavelengths have _____ energy

higher

Shorter wavelengths have _____ energy

optical density

The most useful measure of light absorption is the absorbance (A), which is commonly called what?

Spectrophotometer

an instrument that measures the intensity of a portion of the spectrum of light

- most commonly used method in automated clinical chem lab

2 main principles of all spectrophotometric methods

1. Substances absorb light at specific wavelengths, therefore the wavelength absorption pattern can be the basis for ID of an analyte

2. The amount of light absorbed is proportional to the amount substance that is present

absoptivity

the amount of absorbance specific for a certain substance

absorbed; transmitted

As the concentration of a solution increases, more light is __________ by the solution and less light is __________

transmittance

the amount of light not retained, but passed through a test solution

Beer's Law

explains the relationship between absorbance, at a given wavelength and concentration

A = abc

A = abc

What is the equation for Beer's Law?

absorbance

A = abc

What does A represent in Beer's Law

absorptivity constant for the substance

A = abc

What does a represent in Beer's Law

length of light path through a substance

A = abc

What does b represent in Beer's Law

concentration of the substance

A = abc

What does c represent in Beer's Law

absorptivity (a) & length of light path (b)

What are the 2 constants in Beer's Law

absorbance (A) is directly proportional to the concentration of the substance (c)

Since absorptivity (a) & length of light path (b) are constants, what is the relationship between absorbance (A) & the concentration of the substance (c)?

A1/C1 = A2/C2

Since absorptivity (a) & length of light path (b) are constants, and absorbance (A) is directly proportional to the concentration of the substance (c), what equation can we use to find the concentration of an unknown substance?

linearity

the extent to which a range over a measurement, such as absorbance, is directly proportional to the concentration

relationship of 2 variables produce a straight line when plotted on a graph

absorbance vs concentration is a linear function

How does linearity relate to the absorbance or transmittance of a substance?

light source/lamp

monochromator

sample holder

photodetector

readout device/meter

power source

The 6 components of a spectrophotometer:

Light source/Lamp

Purpose: Provides wavelengths of light in the visible spectrum or UV range

incandescent tungsten lamp or tungsten-iodide lamp

What type of light source/lamp is used or visible light?

hydrogen deuterium or mercury-arc lamp

What type of light source/lamp is used for UV light?

tunable laser beams

Newer spectrophotometers use what as their light source?

periodic lamp inspections

use lens paper to hold & position lamp during replacement

Maintenance of light source/lamp:

Monochromator

Purpose: Used to eliminate unwanted wavelengths of light & allow the desired light to reach the sample

can be: Filter, Prism, or Diffraction grating

entrance slip

dispersion device

diffraction grating

exit slip

Monochromators are made up of what 4 things?

entrance slip

What is B?

hint: focuses the light where it can be dispersed with a minimum of stray light

dispersion device

What is C & E?

hint: mirrors

diffraction grating

What is D?

hint: light is passed in order to produce a spectrum

exit slit

What is F?

hint: isolates the selected narrow band of wavelengths

bandpass or bandwidth

The range of wavelengths exiting the slit

Indicates the purity of light passing though the sample

higher resolution, lower intensity

What does a smaller bandpass/bandwidth mean in terms of resolution & intensity?

lower resolution, higher intensity

What does a larger bandpass/bandwidth mean in terms of resolution & intensity?

Sample Holder

Purpose: Holds cuvette containing the test solution

Cuvettes can be round or square, and either glass, plastic or quartz

square

Light passes better through what shaped cuvette?

quartz

What type of cuvette is used for a colorless solution?

scratches will interfere and cause light to scatter

What is a disadvantage of glass & plastic cuvettes?

Photodetector

Purpose: Converts the electromagnetic radiation (light) into an electrical signal that is sent to the readout device as an electric current

When light strikes the light-sensitive material in the light detector, molecules are excited and release a stream (current) of electrons

Photocell, Phototube, Photodiode, Photomultiplier tube

What are the 4 types of photodetectors?

Photomultiplier tube

Preferred type of photodetector:

due to a rapid response & more sensitive

true

T or F:

There is a direct relationship between the intensity of the electromagnetic radiation (light) and the electrical signal produced

Readout device/Meter

Purpose: Presents changes in electrical energy numerically and gives the operator a numerical value that represents the amount of %T or A

The computerized device may be linked to the LIS or HIS/EMR for fast delivery of the info to the provider

Power Source

Purpose: Must supply continuous and stable energy to the components

batteries

offer the advantage of direct current & portability, especially in field testing

eliminates interfering susbtances

What is the purpose of blanking an instrument?

set the limits of the instrument from no light transmitted to all light transmitted

What does zeroing and blanking an instrument do?

water blank

Type of blank that cancels out any color found in water

reagent blank

Type of blank that cancels out any color from the reagents

Calibration

a check of an instrument to ensure continued accuracy throughout the reportable range of the test results for the test system

Calibrators

a material of known value use to calibrate, or adjust a measurement procedure, or to compare the results obtained with the response of a test specimen and/or sample

assigned values

commercially prepared calibrators have __________ values for each analyte that have been determined by a reference lab

Standard

reference material whose concentration is determined by assay with a reference method

aka Calibrator

controls

Manufactured using the same base matrix as the substance being tested

stabilized and processed to maintain the native components which may be susceptible to degradation

ranges are established by repeat testing, and then distributed to labs

calibrators

Manufactured to contain a specific amount of a pure analyte and adding it to the appropriate solvent for analysis, so they contain a known amount of analyte

a more accurate calculation is based on the measurement of several standards of different concentration vs one standard

reagent lot number is changed

extensive maintenance performed on instrument

routinely, based on manufacturer's guidelines

Calibration curves must be re-calculated when:

Calibration Verification

Process in which the calibration of the instrument is checked on a periodic basis

6 months

after a major preventative maintenance or repair

whenever recommended by the manufacturer

Time interval/when to perform calibration verification: