Instrumentation Lab Practical I

Front 1

Chromatography is:

Back 1

a separation technique based on differing affinities of compounds for the mobile and stationary phase

Front 2

Chromatography can be used for

Back 2

• Separation
• Purification
  • Identification

Front 3

Liquid chromatography can be used on

Back 3

a soluble compound

Front 4

Affinity chromatography

Back 4

• selectively and reversibly binding proteins to a solid support matrix
• Based on structural properties
  • Forms ligands

Front 5

Liquid chromatography is useful for

Back 5

• Separating ions
• Uses lower temperatures than GC
  • Stationary phase: solid support within a glass or plastic column

Front 6

Size exclusion

Back 6

• Larger molecules elute first
  • Based on molecular size

Front 7

Adsorption Chromotography

Back 7

• Separated in a series of adsorption/desorption steps with the stationary phase

Front 8

Partition Chromotography

Back 8

• Partitioning and relative solubility between two liquid phases
  • Normal: polar stationary phase
• Reverse: non-polar stationary phase
• Elution: changing polarity of the mobile phase

Front 9

Ion exchage

Back 9

• Form of adsorption
• Cation exchanger: positive molecule
  • Anion exchanger: negative molecule

Front 10

Bed volume

Back 10

total volume of solvent and resin taken up by the column

Front 11

Void volume

Back 11

• volume taken up by the liquid phase in the column

Front 12

elution volume

Back 12

amount of solvent required to remove a particular solute from the column

Front 13

effluent/eluate

Back 13

the mobile phase that has exited the column

Front 14

eluting/developing

Back 14

movement of solvent and solute through the loaded column

Front 15

Flow rate in LC elution

Back 15

Hydrostatic pressure

The longer the column the faster the flow rate

Front 16

LC Elution Factors

Back 16

Mobile phase solvent polarity greatly affect retention

Front 17

LC modes of Elution

Back 17

• Isocratic- solvent composition remains constant throughout the analysis
• Stepwise- solutes are eluted tru incremental changes in solvent
  • Gradient-gradual change in the solvent composition

Front 18

HPLC

Back 18

• High performance liquid chrom.
• Stationary phase: small particle with large surface area
• Mobile phase: under high pressure
• Closed system
  • Flow-tru detector

Front 19

Advantages of HPLC

Back 19

• High resolving power and short analysis time
• reuse columns
• improved reproducibility
• readily automated
  • used analytically or preparatively

Front 20

Retention time

Back 20

time for the maximum elution of a particular solute

Front 21

Retention volume

Back 21

amount of solvent volume required to elute the solution

Front 22

resolution

Back 22

degree of solute separation; measure of column efficiency

Front 23

Instrumentation of HPLC

Back 23

• Solvent reservoir
• high pressure pump
• injection port
• packed column
• detector
• recorder
  • fraction collector

Front 24

HPLC packing materials

Back 24

• Usually silica
• Pore size: 50-500 Angstroms
  • Surface area: 30-800 m^2

Front 25

HPLC eluent in reverse phase

Back 25

• acetonitrile or methanol in water
  • Add hydrocarbon chains to stationary phase to increase non-polar

Front 26

HPLC solvents

Back 26

• Filtered to remove/prevent gases/bubbles
  • eluting power related to its polarity

Front 27

Larger values of resolution mean

Back 27

better separation

Effected by:

• column length
• packing particle size
• stationary phase
  • mobile phase

Front 28

Nucleon

Back 28

portons and neutrons in the nucleus

Front 29

Z

Back 29

atomic number; number of protons

Front 30

A

Back 30

Mass number; total number of nucleons

Front 31

N

Back 31

neutron number; number of neutrons in the nucleus

Front 32

Nuclide

Back 32

nucleus with a particular A and Z

Front 33

Isotope

Back 33

nuclides with the same Z but different A (same element various nuclear species)

Front 34

Isobar

Back 34

Nuclides with the same A but different Z (different elements same mass)

Front 35

Nuclear radiation

Back 35

Release of energy or matter during the transformation of an unstable atom to a more stable

Front 36

Line of stability

Back 36

Shows stable nuclides are clustered

Below: nuclide deficient in protons

Above; nuclides deficien in neutrons

Front 37

Half-life

Back 37

Time required for 1/2 of the radioactive atoms originally present in the sample to decay

Front 38

Basic unit of energy used in radiation

Back 38

eV

Front 39

Non-ionizing radiation

Back 39

• Heat
• Light
• sound
• infrared
• radiowaves
  • microwaves

Front 40

Ionizing radiation

Back 40

• Alpha rays
• Beta rays
• gamma
  • x-rays

Front 41

Ionization

Back 41

The removal of an electron from the atom

The remaining atom is an ion

Positive charge

Front 42

Excitation

Back 42

The addition of energy to an atom, thereby exciting it from its ground state to a higher energy state

Excess energy is liberated as a photon

Front 43

Alpha particles

Back 43

• Highly charged
• Heavy particles
• Energy range 3 to 8 MeV
  • Short range in air <10 cm

Front 44

Beta particles

Back 44

• May either be positively or negatively charged
• electrons emitted from the nucleus
• Range in skin less than 1mm
• Must be 70 keV to penetrate skin
  • More penetrating then alpha particles

Front 45

Bremsstrahlung Radiation

Back 45

• If beta particles interact with the nucleus they give rise to x-rays
  • Need a low Z material like plexiglass

Front 46

Gamma radiation

Back 46

• Electromagnetic radiation emitted from the nucleus
• produced after the release of energy after alpha, beta, or electron capture bombardment
  • Greater penetrating power
    • unstable nucleus

Front 47

X-ray

Back 47

• Electron cloud
• produced by bombardment an appropriate metal target with accelerated electrons
  • Also produced by Bremsstrahlung radiation

Front 48

Gas filled Detectors

Back 48

• Ion chambers- alpha, beta, gamma, and x-ray, dose rate information
  • GM counters- beta, gamma, x-ray, count rate information

Front 49

Scintillators detector

Back 49

• NaI (Thallium doped sodium iodide)
• X-ray
  • Some gamma ray

Front 50

Geiger-Mueller Counter

Back 50

• Filled with 98% helium and 1.3% butane
• best at detecting beta
• quality check
• approx count rate
  • high sensitivity

Front 51

Scintiillation Detections

Back 51

• X-ray or lower energy gamma ray fields
  • Scintillator- creates a pulse of light when radiation interacts with it
• PMT- converts to an electrical signal

Front 52

Gamma Counter

Back 52

• Measures gamma and x-ray radiation
  • radiation interacts directly with a NaI crystal doped with thallium

Front 53

Curie Ci

Back 53

Quantity of radioactive material decaying at a rate of 3.7X10^10 Bq

Front 54

Becquerel Bq

Back 54

Quantity of radioactive material decaying at a rate of one transformation per second

Front 55

Movement of a charges particles in an electrical field

Back 55

V=Eq/f

Front 56

Densitometer

Back 56

• Read electrical pattern
• light source
• filter
  • the denser bands absorb more light

Front 57

Electroendosomosis

Back 57

uncharged or weakly negative charged molecules are carried towards the cathode by the electroendosmotic movement

Front 58

Disc gel electrophoresis

Back 58

• Upper layer stacking gel: large pore size
  • Lower layer resolving gel: smaller pore size
• Different ionic strength

Front 59

2-D Electrophoresis

Back 59

• First dimension separated by charge in IEF
  • Second dimension proteins are separated by size in SDS-PAGE

Front 60

Immunoelectrophoresis (IEP)

Back 60

• Separate protein on agarose gel electrophoresis
  • Second step, separated proteins interacted with specific antibodies to examine proteins antigenic properties
    • If antibody: antigen reaction occurs, a visible insoluble precipitin is formed

Front 61

Immunofixation Electrophoresis

Back 61

• Protein electrophoresis followed by immunoprecipitation on an agarose gel
• incubated with nonspecific antiserum
  • Gel washed stained and destained to visualize protein

Front 62

Isoelectric focusing (IEF)

Back 62

• use of carrier ampholytes
• maintain pH zones; have good buffering capacity
• zero mobility at their pIs
• electrophoresis in pH gradient
  • ampholytes move faster

Front 63

CE capillary electrophoresis

Back 63

• Performed in capillary tubes
• permits the usage of high voltages
• moves towards the electrode
• in-line detection