Instrumentation and Analytical Principles

Question 1

How is light classified? what is visible light?

Answer 1

according to wavelength: UV light has very short wv, infrared has very long; Visible light is that between 400 and 700 nm, which when combined produces white light

Question 2

What does a spectrophotometer measure?

Answer 2

the light transmitted by an analyte in solution, in order to determine the concentration. Analytes may absorb, transmit, and reflect light to varying degrees but always characteristic to the analyte

Question 3

what types of lamps are most common in spectrophotometers, and for which regions?

Answer 3

Tungsten: for visible and IR
Deuterium: for UV

Question 4

what must be done when the lamp, is changed in a spectrophotometer?

Answer 4

recalibration bc changing the light source changes the angle of the light striking the monochromator

Question 5

what is the monochromator?

Answer 5

a device that allows selection of a particular set of wavelengths at the input: allows polychromatic light to be separated into narrow bands of monochromatic light

Question 6

what types of monochromators are used in photometers vs spectrophotometers?

Answer 6

glass and interference filters vs diffraction gratings and prisms

Question 7

How does the monochromator allow wavelength selection?

Answer 7

the exit slit is used to select the bandpass which allows the light of the selected wavelength to pass through the cuvette onto the detector

Question 8

what is atomic absorption spectrophotometry based on, principle-wise?
what is it used to do?

Answer 8

the fact that ground-state ATOMS (not molecules) absorb light at defined wavelengths/ each metal has a specific line spectrum

put another way: measure concentrations by detecting absorption of electromagnetic radiation by atoms

Question 9

what are the basic steps of atomic absorption spectrophotometry?

Answer 9

1. sample in sample cell is atomized in a flame or by electrothermal method; the atoms to be measured are kept at ground-state
2. beam from hollow cathode lamp is passed through a chopper to the flame
3. the ground-state analyte atoms in the flame absorb the same wavelengths of light from the lamp as they would emit when excited
4. the light NOT absorbed by the atoms is measured as a decrease in light by the detector (photomultiplier tube)

Question 10

what is measured in nephelometry?

Answer 10

light scattered by particulates in solution; amount of scatter is directly proportional to number and size of particles present in solution

Question 11

what does turbidimetry measure? what instrument does it use?

Answer 11

the light blocked by particles in solution, as a decrease in transmittance; uses a spectrophotometer

Question 12

briefly describe fluorescence

Answer 12

a type of luminescence in which atoms absorb energy at a particular wavelength, their electrons are raised to higher-energy orbitals and then when return to ground state they emit light of longer wavelength and lower energy than the exciting wavelength

Question 13

what is chemiluminescence?

Answer 13

the process where the chemical energy of a reaction produces excited atoms and when electrons return to ground state, emit light

Question 14

chromatography is a technique in which solutes in a sample are separated for ID based on:

Answer 14

physical differences that allow their differential distribution between a mobile phase and a staionary phase

Question 15

describe some mobile phase and stationary phase types in chromatography

Answer 15

mobile: liquid or gas
stationary: can be silica gel bound to the surface of a glass plate or plastic sheet, or silica/polymer bound to a column

Question 16

IN Gas-Liquid chromatography, will a more volatile solute elute faster or slower? what other solute attribute causes faster elution?

Answer 16

faster; a solute that has less interaction for the solid phase (the column)

Question 17

When a sample is injected into a gas-liquid chromatographer, what happens to it? How does it move through the column?

Answer 17

It is vaporized bc the interior is much higher temp; then an inert carrier gas carries it into the column

Question 18

what are some inert carrier gases used in chromatography?

Answer 18

helium, hydrogen, nitrogen, argon

Question 19

what determines separation of solutes in GLC?

Answer 19

both the differences between vapor pressures of solutes and amount of interaction with the column

Question 20

What is HPLC and what governs separation of solutes?

Answer 20

High Performance liquid chromatography; separation of solutes is governed by selective distribution of solutes between mobile and solid phases (using polarity and nonpolarity)

Question 21

what is the diff btw isocratic and gradient elution in HPLC?

Answer 21

In isocratic, the solvent strength remains constant; In gradient it continually increases during separation

Question 22

What is the diff btw normal-phase and reversed-phase liquid chromatography?

Answer 22

IN normal phase, the stationary phase is polar and the mobile phase is nonpolar
In reverse……obviously they are the opposite

Question 23

If a solute is introduced to the detector for HPLC before all the other solutes, and appear first, were they faster or slower to be eluted? What does this mean regarding their properties?

Answer 23

they are faster to elute if detected first; this means they were more volatile and/or less attracted to the solid phase.

Question 24

what is the principle of mass spectrometry?

Answer 24

electrons bombard a sample and ionizes it into fragment ions that are separated by their mass-to-charge ratios; a spectrum is produced that is unique for a compound so it can be id’d, as well as the number of ions produced relates to quantity

Question 25

What are the most important applications of Mas spectrometry in clinical labs?

Answer 25

identification of drugs or drug metabolites, steroids, as well as the amino acid composition of proteins

Question 26

what can be used in conjunction with mass spec to identify the eluate?

Answer 26

both gas-liquid and HPL chromatography

Question 27

what is anodic stripping voltammetry used to assay and what is it based on (a technique using an electrochemical cell and the voltage at which sharp rise in current id’s the analyte electrochemically and the wave height reflects its concentration)

Answer 27

it is used to assay heavy metals, such as lead in blood; The description of this method is polarography

Question 28

what method of assay common to electrolytes in serum is based on potentiometry? what is the principle

Answer 28

Ion-selective electrodes; the concentration of a substance in solution is determined using an electrochemical cell made of two half-cells and the potential diff btw an indicator electrode and reference electrode is measured

Question 29

what antibiotics are used as neutral carriers for Iion exchange (selective) electrodes for potassium, and for ammonium?

Answer 29

K: valinomycin

NH4+: nonactin and monactin

Question 30

what determines the charge of proteins in a solution?

Answer 30

the pH of the solution, bc they are amphoteric

Question 31

In buffer solutions of pH 8.6 for electrophoresis, what net charge do serum proteins take on?

Answer 31

a net negative charge, and they migrate toward the POS ANODE

Question 32

what are some stains that are used to visualize separated serum proteins in agarose or cellulose gel?

Answer 32

coomassie blue, amido black B, Ponceau S

Question 33

what stains are used for visualizing proteins separated by electrophoresis from CSF, for lipoproteins, and LD isoenzymes?

Answer 33

CSF: silver nitrate
lipoproteins: 7B and oil red O
LD: nitrotetrazolium blue