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Clinical Chemistry 1 > Chromatography and Mass Spectrometry > Flashcards

Chromatography and Mass Spectrometry Flashcards

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1
Q

What is chromatography?

A

A technique for separating complex mixtures based on their solubility and interaction between two phases: a stationary phase and a mobile phase.

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2
Q

What is the stationary phase?

A

Stationary Phase:
- Immobile
- Solid or liquid
Through which the mobile phase flows

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3
Q

What is the mobile phase?

A

Mobile Phase:
- Mobile
- Gas or liquid
Carries the sample

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4
Q

What governs the separation in chromatography?

A

Some molecules are attracted to or have an affinity for the mobile phase, and some have an affinity for the stationary phase = separation.

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5
Q

What is plane chromatography?

A

Plane Chromatography
Takes place on a flat surface or plane
Mobile phase is solvent (water, alcohol, etc.)

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6
Q

What are two types of plane chromatography?

A
  1. Paper chromatography:
    Stationary phase is paper
  2. Thin Layer Chromatography (TLC):
    Stationary phase is a thin layer of sorbent material (silica gel, alumina gel, polyacrylamide gel, or starch gel attached to a glass plate).
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7
Q

Describe how plane chromatography works.

A
  1. Sample spotted on stationary layer
  2. Plate or paper placed in upright container with mobile phase (solvent) at the bottom
  3. Migration of mobile phase through stationary phase via capillary action
  4. Separation occurs due to the difference in time solute spends in mobile versus stationary phase
  5. Solid phase interacts differently with sample compounds during migration
  6. TLC (thin layer chromatography) needs no instrumentation unless quantitating with a densitometer
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8
Q

What are typical clinical applications for plane chromatography?

A

Drug screens, amino acids and L/S ratio

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9
Q

What is the definition/equation for retention factor (with respect to plane chromatography)?

A

Retention Factor:
Defined as the distance travelled by the solute divided by the total distance travelled by the solvent

Rf = Distance traveled by solute (sample)/
Distance traveled by solvent

Controls will have known retention factors

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10
Q

What is done in column chromatography?

A
  1. Samples are loaded into a column with a stationary phase.
  2. A mobile phase is added throughout the process.
  3. Movement through column at different rates based on:
    Gravitational force
    Applied pressure
    Sample affinity to the two phases
  4. Collect and detect eluate emerging from column
  5. Eluate can be quantified using spectrophotometer
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11
Q

What is eluate?

A

Eluate - liquid obtained from a column during separation (e.g. in column chromatography)

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12
Q

What will each eluent fraction contain?

A

Each eluent fraction will contain individual components of the previously mixed sample

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13
Q

What type of stationary phase and mobile phases are used in column chromatography?

A

Stationary phase in column or tube
Support particles such as aluminum silicate gel or resin particles packed into column or coated onto inner surface
Mobile phase can be gas or liquid

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14
Q

What is Adsorption Column Chromatography?

A

Aka liquid-solid chromatography
Mobile phase and sample compete for sites on solid stationary phase (usually silica gel)
Solute will bind to adsorptive sites with attractive forces
Example:
Acidic support adsorbs basic substances

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15
Q

What is Partition Column Chromatography?

A

Aka liquid-liquid chromatography
Separation is based on solubility of solute in an organic solvent and aqueous solvent
Stationary phase is made of inert particles coated with liquid adsorbent
Nonpolar molecules are extracted in the organic solvent
Polar molecules remain in the aqueous solvent

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16
Q

What is Ion-Exchange Chromatography?

A
  1. Separates solutes based on their solubility in two phases as well as different electrical charges
  2. Sample is diluted with a buffer that gives the solute an electrical charge
  3. Interact reversibly with stationary and mobile phases with the opposite charge
  4. Varying pH of the mobile phase allows elution at different times and elution of specifically charged solute particles
17
Q

What is Steric Exclusion Column Chromatography?

A
  1. Aka size exclusion chromatography
    Separated in column by size and shape
  2. Sample containing different sized molecules moves down the column
  3. Larger molecules elute faster from the column while smaller molecules are retained in the gel longer (momentarily trapped)
18
Q

What does HPLC stands for?

A

High-Performance Liquid Chromatography (HPLC)

19
Q

Describe High-Performance Liquid Chromatography (HPLC).

A

High pressure is used to push the mobile carrier through the short packed column. The column is the stationary phase is a packed with tiny particles, usually silica. The mobile liquid phase are the solvents. Separated molecules are then read by a detector and a chromatogram is created.

20
Q

What different principles can be employed with the stationary column?

A

Column can employ adsorption, ion-exchange, etc.

21
Q

What are the typically types of detectors used in HPLC?

A

UV-VIS or Mass spectrophotometer

22
Q

What is a chromatogram?

A

Visible record showing the result of separation of components in a mixture by chromatography

  1. Produces a characteristic graph for each component separated by chromatography
  2. A chromatogram plots detector output versus time.
  3. Can provide qualitative results in which the column retention time correlates to the identity of the compound.
23
Q

How does HPLC work?

A
  1. Separation based on polarity of compounds (polar or nonpolar)
    “Like attracts like” in polarity-based chromatography
  2. The mobile phase and stationary phase will have different polarities
  3. Compounds in the sample with similar polarity to the stationary phase will be delayed (attracted to packing material in column)
  4. Compounds in the sample with similar polarity to the mobile phase will move through the column quickly
24
Q

What is the difference between normal phase HPLC and reversed-phase HPLC?

A

Normal Phase HPLC
Stationary phase is polar (example: silica); mobile phase is nonpolar
Reversed-Phase HPLC
Stationary phase is nonpolar (example: silica C18); mobile phase is polar

25
Q

What is gas chromatography?

A

Gas Chromatography
Used to separate mixtures of compounds that are volatile or can be made volatile
Stationary phase is in a long column
Mobile phase is a gas such as nitrogen, helium, or argon

26
Q

What are the process steps in gas chromatography?

A

GC Steps:
1. Sample is rapidly heated and becomes vaporized
2. Flows with the carrier gas through the heated column
3. Separation of molecules occurs as they pass through the column at different rates
4. Effluent passes through detector
Can be a mass spectrophotometer
5. Chromatogram is read and interpreted
6. Compare peaks to standards and calculate unknown concentration

27
Q

How can mass spectrometry be used with GC and HPLC?

A

Can be used as a detector following GC and HPLC

28
Q

Can mass spectrometry be used with direct injection?

A

Yes, can also have direct injection instruments (no separation beforehand).

29
Q

Describe the steps and how a mass spectrometer works.

A
  1. Sample is introduced to MS
  2. Sample is vapourized and then ionized to form charged ions and fragments
    Ionized into unique mass spectrum
    Ions are accelerated between two charged plates
  3. Ions move through a magnetic field.
    4 Charged particles are deflected and separated according to mass-to-charge ratio (m/z) forming a mass spectrum
  4. Ions are measured by a detector which gives the intensity of the ion current for each fragment
30
Q

What does a mass spectrum tell us? Describe the graph.

A

A mass spectrum is:
The m/z ratios of the ions present in a sample plotted against their intensities
The heights of the peaks denote the relative abundance of the various components in the sample

31
Q

In a chromatogram what does the peak area mean?

A

Peak area:

Quantitative result by calculating the area under the peak

32
Q

In a chromatogram what does the retention time mean?

A

Retention time

Time between sample injection and recorder peak signal

33
Q

In a chromatogram what does resolution mean?

A

Resolution: Describes the separation and width of the peak Conditions of the column.

34
Q

What does MALDi-ToF stand for and what is it?

A

Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry

Laser excitation to ionize chemical groups in the proteins of organisms to create a specific protein profile

35
Q

What are the basic process steps of MALDi-ToF?

A
  1. Isolated organism from culture is applied directly onto plate
  2. Sample is mixed with a chemical matrix
  3. Matrix molecules absorb laser light energy, creating an excited energy state and ionizing the organism proteins
    a) Charged ions drift through the free field
    b) The speed of travel (time of flight) is proportional to the ion’s mass
    - ->Smaller ions detected first
  4. Ions are measured by a detector and a protein spectrum for that specific organism is obtained
  5. The protein spectrum (or mass spectrum) is compared to a database of thousands of different organisms
36
Q

What is the advantage of MALDi-ToF?

A

Only takes a matter of minutes for each sample and needs relatively low amount of supplies

Clinical Chemistry 1 (11 decks)

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