Week 1: Intro to Chem/Spectrophotometry

Question 1

What is clinical chemistry?

Answer 1

About soluble mediators in liquid portion of body, be it plasma or serum. Serum preferred

Question 2

How long does it take for clot to form in whole blood?

Answer 2

30 minutes

Question 3

Which is the most common cause of metabolic disease in the West?

Answer 3

Obesity

Question 4

Which tests are done in BMP (basic metabolic/mini)?

Answer 4

• Glucose
• Albumin
• Total protein
• Sodium
• Potassium
• Chloride
• BUN (blood urea nitrogen)
• Creatinine
• CO2

Question 5

Which tests are done in CMP (complete metabolic panel)?

Answer 5

• BMP + liver panel
  Liver panel:
• Alanine aminotransferase (ALT)
• Aspartate aminotransferase (AST)
• Alkaline phosphatase (ALP)
• Total bilirubin

Question 6

What is the matrix?

Answer 6

• Plasma or serum
• Urine
• Feces
• Other body fluids (CSF, peritoneal fluid, pleural fluid, synovial fluid, amniotic fluid)

Question 7

Which type of system is best for quantitating color change of reaction between reagent + compound?

Answer 7

Closed systems utilizing focused light

Question 8

How do photometric instruments measure light intensity?

Answer 8

Without consideration of wavelength. Most instruments use filters (photometers), prisms, gratings (spectrometers) to isolate narrow range of incident wavelength

Question 9

What is Beer’s law in words?

Answer 9

The concentration of a substance is directly proportional to amount of light absorbed or inversely proportional to logarithm of transmitted light

Question 10

How do spectrophotometric instruments work?

Answer 10

Measure light transmitted by solution to find concentration of light-absorbing substance in solution

Question 11

List the components of a spectrophotometer

Answer 11

• Light source
• Monochromator
• Sample cell (cuvette)
• Photodetector

Question 12

What is the range of visible spectrum on the EM spectrum? Which colors?

Answer 12

400 nm (violet) to 700 nm (red)

Question 13

What are the two most common incandescent light sources? Which parts of EM spectrum do they cover?

Answer 13

1. Tungsten lamp: Visible + IR
2. Deuterium: UV region

Question 14

Purpose of the monochromator? What types are there?

Answer 14

Purpose is to disperse light. Can use prism

Question 15

What is the Beer’s law equation? What do the variables stand for?

Answer 15

A = Ebc

A= absorbance
E = molar absorptivity
b = length of light path through solution (cm)
c = concentration

Question 16

What is the equation for percent transmittance? What do the variables stand for?

Answer 16

%T = T/I X 100
T = radiant energy transmitted (that exits cuvette)
I = Incident radiant energy on sample

Question 17

What is the %T of water and why?

Answer 17

100% because all energy goes through to other side of cuvette. Nothing is absorbed.

I(incident) = I(absorbed) + I(transmitted)

Question 18

What does the exit slit do?

Answer 18

Selects wavelength of interest

Question 19

List the types of photodetectors

Answer 19

1. Photocell (self-propelled)
2. Phototube (external power supply)
3. Photomultiplier (amplifies light)

Question 20

How does photomultiplier work?

Answer 20

Has dynode chain made up of anodes with successively increasing voltages.

Light -> electrons -> Amps -> Volts -> Absorbance

Question 21

What are ways to maintain wavelength accuracy?

Answer 21

• Holmium oxide
• Didymium
• Mercury

Question 22

Difference between reference range and linear range?

Answer 22

Reference range: what’s normal for population

Linear range: how high or low you can test

Question 23

How to test for stray light effect?

Answer 23

Measure absorbance against true absorbance. Should have straight line

Question 24

Linear dynamic range also known as…?

Answer 24

Sensitivity range

Question 25

What are other types of analytical measurement?

Answer 25

• Atomic absorption spectrophotometry
• Fluorometry
• Chemiluminescence
• Turbidimetry
• Nephelometry

Question 26

What is atomic absorption spectrophotometry?

Answer 26

Used to measure concentration by detecting the absorption of EM radiation by atoms rather than by molecules

Question 27

What is fluorometry? Advantages + disadvantages when compared to spectrophotometry?

Answer 27

• Uses fluorescence to determine concentration of analyte
• Advantages: higher specificity + sensitivity
• Disadvantage: Very sensitive to environmental changes (pH, temp)

Question 28

What is chemiluminescence? Advantages + disadvantages?

Answer 28

• Emission of light by molecules in excited states produced by chem rxns. No excitation radiation required like in fluorescence
• Advantages: sub-picomolar detection limits, speed, ease of use, simple instrumentation
• Disadvantage: needs more calibrations bc impurities cause background signal

Question 29

What is turbidimetry?

Answer 29

• Spectrophotometry that measures transmitted light 180°C from the incident light, to determine concentration
• Sample handling critical
• Depends on concentration + particle size

Question 30

What is nephelometry?

Answer 30

• Measures transmitted/scattered light by placing light detector at 90°C from incident light
• Minimizes errors from colored solutions and increases sensitivity