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Chemistry > Chem Exam 2 (Instrumentation Part II) > Flashcards

Chem Exam 2 (Instrumentation Part II) Flashcards

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1
Q

What is the definition of electrophoresis?

A

Migration or separation of charged compounds based on their electrical charges

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2
Q

Where do anions migrate? Are anions positive or negative ions?

A

Anions are negative ions that move toward the anode (positive electrode)

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3
Q

Where do cations migrate? Are cations positive or negative ions?

A

Cations are positive ions that migrate towards the cathode (negative electrode)

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4
Q

What is the term for a molecule that can carry positive or negative charges depending upon the environment?
What is an example of a molecule that can do this?

A

Ampholyte or zwitterion
Ex. Proteins

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5
Q

what is the Isoelectric point (pI)? What happens when a protein reaches pI?

A

The point at which a protein has a net zero charge.
When a protein reaches pI, it will not move any further.

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6
Q

What are the two types of support media for electrophoresis based on what they separate out? Give examples of each.

A

Charge only separation - starch, cellulose acetate, agarose gel
Charge and size separation - polyacrylamide gel (PAGE)

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7
Q

What are the two types of support media for electrophoresis based on what they separate out? Give examples of each.

A

Charge only separation - starch, cellulose acetate, agarose gel
Charge and size separation - polyacrylamide gel (PAGE)

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8
Q

What is zone electrophoresis? What is an example of it?

A

Separation and migration of a mixture of compounds into different zones using electrical charge
(ex: serum protein electrophoresis)

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9
Q

What are the steps in the procedure for zone electrophoresis?

A
  1. sample application
  2. separation
  3. detection
  4. quantitation
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10
Q

What is wick flow?

A

Movement of water from the buffer reservoir toward the center of the media.
Water in the buffer moves up both wicks and toward the center of the gel as electrophoresis generates heat and buffer in the media evaporates.

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11
Q

Do proteins move against or with wick flow on the anode side?

A

With the charge and AGAINST wick flow

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12
Q

Do proteins move against or with wick flow on the cathode side?

A

With the charge and WITH wick flow

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13
Q

What is electroendosmosis? What support media does this usually occur in?

A

The tendency of support media to take on a negative charge, giving the buffer a positive charge. This can cause the buffer to move toward the cathode and carry some large, low-mobility molecules like immunoglobulins to move away from the anode.
This can mostly occur in paper and cellulose acetate.

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14
Q

What types of support media does electroendosmosis not usually occur in and why?

A

Agarose gel and PAGE because they are essentially neutral so electroendosmosis is minimal.

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15
Q

What are some sample considerations that may affect electrophoresis?

A

Hemolytic or lipemic samples, age of samples, therapeutic drugs, use of plasma instead of serum

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16
Q

What factors are most important about sample application for electrophoresis?

A

Accurate amount of sample added, allowed time for sample to equilibrate to media prior to electrophoresis

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17
Q

What is the typical pH used for serum protein electrophoresis?

A

Alkaline pH (8.6)

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18
Q

What are the five fractions that serum proteins separate into and in what order are they found?

A
  1. Albumin
  2. alpha-1 globulins
  3. alpha-2 globulins
  4. beta globulins
  5. gamma globulins (immunoglobulins)
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19
Q

Which of the serum protein electrophoresis fractions moves the least distance? The most distance?

A

The least - gamma globulins
The most - albumin

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20
Q

How does isoelectric focusing electrophoresis work?

A

A mixture of compounds in a sample (frequently proteins) are added to a support media which has a built in pH gradient. Proteins move to their pI (isoelectric point) and do not migrate any further.

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21
Q

What is a benefit of isoelectric focusing?

A

Difference in pI (isoelectric point) allow for distinct separations of many proteins into distinct, sharp bands. It is good for separating mixtures of many proteins.

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22
Q

What is two-dimensional electrophoresis?

A

Bands separated during an initial electrophoresis are further resolved using a second electrophoresis. The gel from the first electrophoresis is turned 90 degrees and then electrophoresed a second time. It gives excellent separation of even extremely complex mixtures.

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23
Q

Give a general description of how two-dimensional electrophoresis is performed.

A

2 electrophoresis procedures:
First electrophoresis is usually isoelectric focusing- a charge dependent procedure
Gel is then turned 90 degrees
Second electrophoresis uses a different media which separates by charge as well as molecular size (PAGE)

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24
Q

What does southern blotting separate?

A

DNA fragments

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25
Q

What does northern blotting separate?

A

RNA fragments

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26
Q

What does western blotting separate?

A

protein fragments

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27
Q

Briefly describe the process used for performing an immunoblotting procedure.

A

protein/dna/rna fragments are electrophoresed to separate them. separated bands are transferred onto a membrane.

the membrane is treated with different antibodies to particular fragments of interest.

labeled detection antibody is overlaid. if the target fragments are present, they bind with detector antibody.

detector antibody is detected

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28
Q

Name two characteristics of capillary electrophoresis that make it unique from other forms of electrophoresis.

A
  1. No physical support media is used
  2. No stains used and no densitometer is necessary
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29
Q

What are some advantages of capillary electrophoresis?

A

Very small sample size, able to analyze multiple samples simultaneously, fast separation and good resolution, excellent heat dissipation, automation of procedural steps, no stains or densitometer needed

30
Q

What direction does liquid flow in capillary electrophoresis ?

A

Liquid flows towards the cathode

31
Q

What is stronger in capillary electrophoresis - electrical field or elecro-osmotic flow?

A

electro-osmotic flow is much stronger

32
Q

What is electroimmunoassay electrophoresis also known as?

A

Rocket electrophoresis, Laurell rocket electrophoresis, or electroimmunodiffusion

33
Q

What does electroimmunoassay electrophoresis produce?

A

A precipitin line in the shape of a rocket in the gel

34
Q

What do immunoelectrophoresis and immunofixation techniques have in common?

A

Both use aliquots of patient serum that are treated with specific anti-sera and electrophoresed

35
Q

What are both immunoelectrophoresis and immunofixation techniques designed to confirm?

A

The presence of a monoclonal antibody and provide information about the type of monoclonal antibody (IgG, IgA, IGM, and does it have Kappa or Lambda chains)

36
Q

What technique is used in capillary electrophoresis to confirm and characterize possible monoclonal proteins?

A

Immunosubtraction or immunotyping

37
Q

What is different about immunosubtraction/immunotyping versus immunoelectrophoresis and immunofixation electrophoresis?

A

IEP and IFE are gel techniques that we look for the presence of a band to identify and classify a monoclonal protein, while in immunotyping we look for the disappearance of a monoclonal protein on the electrophoresis tracing

38
Q

What is chromotography?

A

The separation of a mixture of solutes between two phases: a stationary phase and a mobile phase

39
Q

What are the two basic forms of chromatography?

A

Planar (paper or thin-layer chromatography) and columnar (gas and liquid chromatography)

40
Q

What are the four types of separation mechanisms in chromatography?

A

Adsorption, partition, steric exclusion, and ion exchange

41
Q

How does the separation mechanism adsorption work in chromatography?

A

Sample and mobile phase compete for adsorptive sites on the stationary phase

42
Q

How does the separation mechanism partition work in chromatography?

A

Dissolved substances are separated based upon differences in solubility in polar and nonpolar solvents

43
Q

How does the separation technique steric exclusion work in chromatography?

A

Molecules are separated by size and shape using a stationary phase with various pore sizes

44
Q

How does the separation technique ion exchange work in chromatography?

A

Uses a charged resin and a mobile phase of the opposite sign to exchange either cations or anions

45
Q

What is known as liquid-liquid chromatography?

A

Partition chromatography

46
Q

What is known as solid-liquid chromatography?

A

Adsorption chromatography

47
Q

What is retention time?

A

The time it takes from sample injection to the time a specific compound elutes off a column. Can be viewed as the time from injection to the time a particular peak appears on a chromatogram

48
Q

What is retention volume?

A

The amount of a specific compound that elutes. Reflected in peak height- the higher the peak, the more of a substance present

49
Q

What is a retention factor?

A

The distance a component migrates compared to the distance the solvent front moves in paper or thin-layer chromatography

50
Q

How is retention factor calculated?

A

Rf = distance a component moved/distance the solvent front moved

51
Q

What is efficiency with respect to chromatography?

A

Optimal flow rate resulting in smaller width peaks

52
Q

What is resolution in chromatography?

A

Complete separation between peaks (no overlapping of peaks)

53
Q

What is the difference between an isocratic and a gradient elution?

A

Isocratic means the eluent strength remains the same through separation.
Gradient means the eluent concentration changes through separation.

54
Q

Why might a gradient elution be used?

A

Gradient elution often results in better resolution and efficiency - overall better separation of compounds

55
Q

What is the difference between normal phase and reversed phase chromatography?

A

Normal phase: polar stationary phase, less or non-polar mobile phase
Reversed phase: nonpolar stationary phase, more polar mobile phase

56
Q

What molecular characteristic does a mass spectrometer use to measure analytes?

A

Mass to charge (m/z) ratio

57
Q

What can we use a mass spec for?

A

Identify unknown compounds
Quantify unknown compounds
Give information about the structure and chemical properties of compounds

58
Q

What are the two basic steps in mass spec measurement?

A

Sample is volatilized and then ionized

59
Q

What are the three types of ionization?

A

Electron ionization (EI)
Electrospray ionization (ESI)
Atmospheric pressure chemical ionization (APCI)

60
Q

What types of ionization are used for gas and liquid chromatography?

A

Gas chromatography: electron ionization
Liquid chromatography: electrospray and atmospheric pressure chemical ionization

61
Q

What are some advantages of GC/MS and LC/MS?

A

Can be used to definitively identify compounds
Very sensitive and specific - often considered a gold standard method for detecting/confirming the presence of a particular compound

62
Q

What are some applications of chromatography?

A

Toxicolgy confirmation testing
Detection of low concentration analytes (such as testosterone and other fertility hormones)
Pathogen identification in microbiology

63
Q

What are some benefits of automation?

A

Cost savings
Reduction of repetitive tasks by techs
Increased reproducibility of test results
Decreased human errors during analytical process
Ability to expand testing menu using fewer techs

64
Q

What is something automation cannot change?

A

Any inherent flaw in particular methodology

65
Q

What type of analyzer configuration are most modern clinical chemistry analyzers?

A

Random-access analyzers - any test, any sample, any time

66
Q

How can automation increase throughput?

A

Automation can offer the lab the ability to add modules or different methods to a testing system to increase the number of tests that can be performed per hour without adding additional staff

67
Q

What is dead volume or dead space?

A

Sample volume that is necessary but is not used during testing. Dead volume is the bit of fluid in the bottom of a sample tube or cup that is needed to provide sufficient volume for testing

68
Q

What is carryover? What are some methods of reducing or eliminating carryover?

A

Carryover is the transport of residue from one sample to another. We test our analyzers for carryover at least every 6 months. Washing probes between samples decreases or ideally eliminates carryover. Use of disposable tips completely eliminates the possibility of carryover.

69
Q

What are the steps in chemistry analyzer operations?

A
  1. Sample identification
  2. Determine tests to perform
  3. Delivery of reagents
  4. Delivery of specimen
  5. Chemical reaction phase
  6. Measurement phase
  7. Signal processing and data handling
  8. Transmission of results to LIS
70
Q

What are some ways that automation can help with the chemistry analyzer steps?

A

Automation can usually speed up or standardize all these steps, allowing the tech more free time to concentrate on tasks that require technical attention

71
Q

Why is maintenance important?

A

Regular instrument maintenance is crucial to proper instrument function and quality test results

72
Q

What are the steps to organizing troubleshooting?

A

Recognition, interpretation, implementation, and evaluation

Chemistry (12 decks)

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