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MSC: Analytical Methods > Chromatography and HPLC > Flashcards

Chromatography and HPLC Flashcards

1
Q

What are different detectors for Chromatography and HPLC?

A
  • MS
  • ECD
  • UV/vis spectrophotometry
  • Fluorescence

Batch analysis – not suitable for high throughput/urgent analyses

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2
Q

What are the uses of Chromatography in a clinical lab?

A
  • Drugs of abuse, therapeutic drug monitoring
  • Steroid analysis/profiling
  • Amino acid, organic acids, newborn screening
  • Vitamin analysis
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3
Q

What is Chromatography?

A
  • Separation of dissolved analytes depending on their relative attraction to various solid or liquid phases
  • One phase is fixed “stationary phase”
  • One phase moves “mobile phase”
  • Like attracts like
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4
Q

What are some separation mechanisms?

A
  • Adsorption: Uses electrostatic, hydrogen bonding or dispersive interactions between a molecule and solid support
  • Partition: Based on relative solubility
  • Ion-exchange: Sign and magnitude of ionic charges
  • Steric exclusion: Based on size
  • Affinity: Based on bio-specific interactions e.g. antibody-antigen
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5
Q

What is Partition Co-efficient?

A
  • An analyte is in equilibrium between the two phases. Mobile phase is in equilibrium with Stationary phase
  • The equilibrium constant, k, is termed the partition coefficient;
  • Can be calculated by:

(Analyte(moles) in the stationary phase) / (Analyte(moles) in the mobile phase)

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6
Q

What are the stationary phases and mobile phases in Gas Chromatography?

A
  • Stationary phases: Greases, gums and resins
  • Mobile phases are gases such as helium and nitrogen

Separation based on the relative affinities to the column material and gas

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7
Q

What are stationary and mobile phases in Size Exclusion Chromatography?

A
  • Stationary phase e.g. polyacrylamide gels, sephadex
  • Mobile phase is a buffer

Separation based on the size and shape of molecule

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8
Q

What are stationary and mobile phases in Thin Layer Chromatography?

A
  • Solid phase is silica or alumina
  • Mobile phase is a solvent mixture
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9
Q

What are stationary and mobile phases in Ion Exchange Chromatography?

A
  • Stationary phase can be gels, charged resins
  • Mobile phases include buffers and salts
  • Cation and anion
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10
Q

What is Ion exchange theory?

A

Strong vs Weak Ion exchange

  • To elute from strong ion exchanger you need to replace the analyte with large amounts of salt with the same charge
  • To elute from a weak ion exchanger you can alter the pH, then the analytes are no longer bound. The charge of the ion exchanger alters rather than the analyte
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11
Q

What are stationary and mobile phases in High Pressue Liquid Chromatography?

A
  • Stationary phases are many! Usually silica based with or without functional groups
  • Mobile phases are usually a buffer and a solvent

Also UPLC – Ultra pressure (performance) liquid chromatography

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12
Q

What are features of the solvent in HPLC?

A
  • Often have one aqueous and one organic
  • Can have single mixed solvenet
  • Can use isocratic or gradient elution
  • Can have additive in depending on the application: buffer, salts and ion pain reagent (chromatography on a hydrophobic pair exchange. Make a bridge between analyte and stationary phase)
  • For MS there Adduct promotion and acids – prevent analyte colonisation
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13
Q

What a features of the pump in HPLC?

A

If more than one pump is used, each with a different solvent, mixing of solvents can be achieved

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14
Q

What features of the HPLC columns?

A
  • Usually stainless steel (high pressure <300 bar, ultra pressure <800 bar)
  • Have a packing material inside
  • Held in place by a frit which is a filter to stop particles coming out
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15
Q

What are guard columns?

A
  • Guard columns protect the more expensive analytical column from particulates
  • May have packing material inside or be just a filter
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16
Q

How are chromatography columns labelled?

A

Labelled with

  • Type of column
  • Length of column
  • Diameter of column
  • Particle size

Can use the length and diameter to calculate the volume of the column

17
Q

What is inside the columns?

A
  • Most often silica particle based.
  • Older columns were irregular size particles but now very regular
  • HPLC particles are usually 2.5 – 15 micrometres
  • UPLC particles are smaller than this. Alternative types are available
18
Q

What are functional groups that can be added to particles?

A
  • C18
  • C8
  • C4
  • Phenyl
  • Amide
  • CN
  • PFP
  • HILIC
19
Q

What are the benefits of pores in Chromatography?

A
  • The particles are very porous, like the pores of a sponge – 99% of chromatographic surface is inside the pores
  • Mobile phase mustbe allowed into the pore in order for chromatographic retention of the analyte to take place.
20
Q

How does reversed phase chromatography get carried out?

A

Mobile phase is polar and stationary phase is non-polar

  • Most non-polar species like the stationary phase best and comes out last
  • Somewhat polar species like the stationary phase somewhat and comes slows down a little
  • Most polar species like the mobile phase best and comes out first
21
Q

What are principles of reversed phase separation?

A
  • The greater the concentration of organic substrate on the packed bed, the stronger the retention: 29%C > 20%C > 12%C
  • The longest alkyl bonded phase gives the greatest retention: C18 > C8 > C4
  • Non polar groups on sample increase retention: toluene elutes after benzene
  • Polar functional groups on sample reduce retention: phenol elutes before benzene
22
Q

What is back pressure?

A
  • Back pressure is the pressure generated as the mobile phase moves through the column
  • The smaller the particle, the greater the back pressure
  • Other factors include Flow rate, Column dimensions and Solvent composition
23
Q

What are the detectors used in chromatography?

A

Can be:

  • UV-visible spectrometer
  • Fluorescence detector
  • Mass spectrometer
24
Q

What is the plate model of Chromatography separation?

A
  • This assumes that the column contains a large number of separate layers or theoretical plates.
  • In each of these plates, there is equilibration of the analyte between the mobile and stationary phases.
  • The analyte moves through the column by transfer between mobile and stationary phase at each plate
25
Q

How does changes in number of plates affect the operation?

A
  • The more theoretical plates, the better separation you get
  • Another way to express this is to look at the height equivalent to a theoretical plate (HETP)
  • HETP = Length of column / number of theoretical plates
  • This is reported in millimeters, and the shorter the HETP, the more plates are ‘contained’ within a given length of column and the more efficient the column
26
Q

What is the Rate theory of Chromatography?

A
  • The HETP is dependent on different properties of the column. These are described in the Van Deemter equation

HETP = A + B/u + Cu

  • A = Eddy diffusion
  • B = Longitudinal diffusion
  • C = Mass transfer
  • u = average velocity of the mobile phase
27
Q

What are factors that affect seperation?

A
  • Eddy diffusion
  • Longitudinal diffusion
  • Mass Transfer
28
Q

What is Eddy diffusion?

A
  • Analyte molecules take different paths through the stationary phase at random.
  • This causes peak broadening as the different paths are different lengths.
  • The more regular the particle size and packing of the column, the less Eddy diffusion is observed
  • Modern UPLC columns with very small particle sizes have much less Eddy diffusion than more traditional columns
29
Q

What is Longitudinal Diffusion?

A
  • The concentration of analyte is less at the edges of the band than at the centre, so analyte diffuses from the centre to the edges.
  • This causes band broadening
  • Increasing mobile phase velocity can decrease the effect of longitudinal diffusion as the analyte spends less time on the column.
  • Higher mobile phase velocity increases the back pressure on the system, so a UPLC system may be needed to take advantage of higher velocities
30
Q

What is Mass transfer?

A
  • The analyte takes time to equilibrate between mobile and stationary phases
  • If the velocity of the mobile phase is high and the analyte has a strong affinity for the mobile phase, the analyte in the mobile phase will move ahead of the analyte in the stationary phase
  • This causes band broadening
  • The higher the mobile phase velocity, the worse the band broadening
31
Q

How can mass transfer effect be altered?

A
  • Fused core columns are aimed at reducing mass transfer effect
  • The analytes can’t travel as far into the particle as it would in a fully porous particle
  • They are also faster to re-equilibrate after changes in mobile phase as this doesn’t travel into the particle as far either

MSC: Analytical Methods (13 decks)

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