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Chromatography Fundamentals > Chromatography (EN) > Flashcards

Chromatography (EN) Flashcards

1
Q

What is HPLC?

A

High-Performance Liquid Chromatography (HPLC; formerly referred to as high-pressure liquid chromatography),
is a technique in analytical chemistry used to separate components in a mixture, to identify each component, and to quantify components.

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2
Q

What is resolution (R) in HPLC?

A

Resolution describes the ability of a column to separate the peaks of interest. It describes whether you have achieved base line separation or not.

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3
Q

Which parameters are influencing resolution?

A

Resolution takes into consideration

  • Efficiency
  • Selectivity
  • Retention
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4
Q

Why is good resolution important?

A

Baseline separated peaks ensure the most accurate quantitative result.

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5
Q

Which parameter has the highest influence on resolution?

A

Selectivity

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6
Q

Definition of resolution

A

Resolution describes the ability of a column to separate the peaks of interest.

Resolution describes whether you have achieved base line separation or not.

  • tri* Retention time compound i
  • Wbi* Peak width at baseline
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7
Q

What means Retention time in Liquid Chromatography?

A

The time between the instant of injection and the detection of the component is known as the retention time (tr).

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8
Q

What is Chromatography?

A

Chromatography is a physical method of separation that distributes components to separate between two phases, one stationary (stationary phase), the other (the mobile phase) moving in a definite direction.

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9
Q

What is efficiency (N)?

A

It describes the separation power of the column. Column efficiency is used to compare the performance of different columns.

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10
Q

Which parameters are influencing colum efficiency?

A

Column length ⇒ increasing colum length increases efficiency

Particle size ⇒ decreasing particle size increases efficiency

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11
Q

Which parameters are influencing the Retention factor (k)?

A
  • Stationary phase
  • Mobile phase
  • Temperature
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12
Q

What is the retention factor (k)?

A

The Retention factor measures the period of time that the sample component resides in a stationary phase relative to the time it resides in the mobile phase. It is calculated from the Retention time (tr) divided by the time for an unretained peak (t0).

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13
Q

What is selectivity or separation factor?

A

Selectivity is a measure of the time or distance between the maxima of two peaks. If a = 1, the two peaks have the same retention time and co-elute. It is defined as the ratio in capacity factors.

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14
Q

Which parameters are influecing the retention factor?

A
  • Stationary phase
  • Mobile phase
  • Temperature
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15
Q

Why is a high plate number (N) desired in chromtography?

A

High plate number provides sharp and narrow peaks, better detection and peak capacity to resolve complex samples.

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16
Q

What kind of diffusion lead to peak broadening inside the column?

A
  • Eddy diffusion
  • Axial or longitudinal diffusion
  • Resistance to mass transfer
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17
Q

What does the Van Deemter Equation describe?

A

The Van Deemter equation relates the resolving power of a chromatographic column to flow and kinetic parameters which are responsible for peak broadening.

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18
Q

What is peak capacity?

A

Peak capacity is the number of peaks (n) that can be separated in a given time with a given resolution.

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19
Q

What is influencing peak capacity?

A

The peak capacity depends on different factors like column length and particle size.

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20
Q

Smaller column material particles improve …

A

…resolution and efficiency

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21
Q

HPLC is used for which compounds?

A
  • non volatile such as pharmaceuticals (aspirin)
  • proteins (allergens)
  • organic chemicals (polymers)
  • natural products (ginseng)
  • thermally unstable compounds (TNT)
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22
Q

What would be the best technique for volatile compounds?

A

Gas Chromatography (GC)

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23
Q

How is quantitation in HPLC working?

A

The substance passes between a light source and a detector. The detector measures the intensity of light. More light absorbed by the substance means less light is transmitted to the detector. Reducing light intensity produces a detector response, a so-called „peak“.

24
Q

Can HPLC be used for quantitation of a substance?

A

Yes.

More or less light is absorbed depending on the quantity of the substance. The amount of light absorbed is proportional to the quantity of the substance, which means twice the amount of substance will absorb twice the amount of light.

25
Q

Which modules are typically stacked in a HPLC system?

A
  • Pump
  • Injector
  • Column
  • Detector
26
Q

What is the pump doing in HPLC?

A

The role of the pump is to force a liquid (called the mobile phase) through the liquid chromatograph at a specific flow rate.

27
Q

An isocratic run means….

A

…a constant mobile phase composition (solvent A and B are premixed upfront).

Best for simple separation e.g. quality control

28
Q

A gradient run means….

A

… a variable mobile phase composition (solvent B increases over time in %).

Best for complex samples e.g. method development, unknown mixtures

29
Q

What is the injector in HPLC used for?

A

The injector serves to introduce the liquid sample into the flow stream of the mobile phase.

30
Q

What is Carryover?

A

Compounds of a previous run that appear in a subsequent run, can often be reduced with needle wash.

31
Q

What does area prescision means (injection)?

A

Repeatability of injection volumes

32
Q

What is linearity (injection)?

A

Precision over a defined range of injection volumes

33
Q

What is the heart of a chromatograph and why?

A

The column is considered the “heart” of the chromatograph. The column’s stationary phase separates the sample components of interest using various physical and chemical parameters.

34
Q

What are the main separation modes in HPLC?

A
  • Reversed-phase
  • Normal phase
  • Ion exchange
  • Size exclusion
  • HILIC
35
Q

Size exclusion chromatography is mainly used for….

A

…polymer characterisation and proteins.

36
Q

Name common detection technologies used in HPLC

A
  • Spectroscopic (UV)
  • Refractive Index (RI)
  • Fluorescence (FL)
  • Mass spectrometry (MS)
37
Q

What is Gas Chromatography (GC)?

A

Gas chromatography (GC) is an analytical method to separate and analyze compounds that can be vaporized without decomposition.

It is used to test the purity of a particular substance, or to separate the components of a mixture to determine the relative amounts of each.

38
Q

Can GC be used for qualitative and quantitative analysis?

A

Yes

39
Q

What are typical characteristics of a compound to be suitable for GC analysis?

A

The compound must have sufficient volatility and thermal stability. If all or some of the components of a sample are volatile at around 400°C or below, and do not decompose at these temperatures, the compound can probably be analyzed using a gas chromatograph.

40
Q

What is the main difference in GC analysis to HPLC?

A

In GC analysis, the sample is vaporized and transported via a carrier gas into a column.

In HPLC, the sample is dissolved and liqid is moving it onto the column.

41
Q

Name minimum three typical applications for GC

A
  1. Food and flavor analysis
  2. Environmental analysis (PAH, pesticide, herbicides, benzene)
  3. Industrial chemical analysis (alcohol, halogenated hydrocarbons, aromatic solvents, phenols)
  4. Petroleum industry analysis (gasoline, volatile sulfur compounds, refinery gases)
  5. Pharmaceutical analysis (narcotics, barbiturates, medications)
42
Q

Can peptides be analyzed with GC?

A

No, peptides are either volatile nor thermal stabil.

HPLC or electrophoresis are the methods of choice for peptide analysis.

43
Q

What happend inside the GC column?

A

Gas chromatography uses a gaseous mobile phase to transport the sample through the column, which can be packed or coated on its inside surface. As the GC column is heated, the compounds begin to separate based on boiling point. Changing the column to polar stationary phase will change the separation capabilities. Compounds will separate by both boiling point and polarity characteristics.

44
Q

Which components are typically found in a GC system?

A
  • Gas source
  • Sampler
  • Inlet
  • Column
  • Detector
45
Q

Which carrier gases are used in GC?

A
  • Helium
  • Nitrogen
  • Hydrogen
  • Mixture of argon and methane
46
Q

What is a very important characteristic of a carrier gas?

A

Purity.

Contaminants may react with the sample and the column, create spurious peaks, load the detector and raise the baseline, and so on.

47
Q

How is a sample introduced into a GC analyser?

A

The inlet introduces the vaporized sample into the carrier gas stream. The most common inlets are injection ports and sampling valves.

48
Q

What factors are impacting a GC separation?

A
  • Selectivity
  • Polarity
  • Phenyl content
49
Q

The column diameter influences …

A

… efficiency, solute retention, head pressure, and carrier gas flow rate.

50
Q

GC column length affects …

A

… solute retention, head pressure, bleeding, and costs.

51
Q

Which GC detector do you know and what is the basic detection principle?

A
  • Thermal conductivity: detects compounds with thermal conductivity that differs from carrier gas
  • Flame ionization: detects compounds that burn or ionize in a flame
  • Eletron capture: detects electron-capturing compounds (for example, halogenated compounds)
  • Mass selective: identifies components from mass spectra (when combined with GC, the most powerful identification tool available)
52
Q

The peak size corresponds to…

A

… the amount of compound in the sample. As the compound`s concentration increases, a larger peak is obtained.

53
Q

What are the strengths of GC analysis?

A
  • Easy-to-use
  • Robust
  • Many detectors available
  • Low cost analysis
54
Q

What is the main limitation in GC analysis?

A

The compounds must be thermally stable.

55
Q

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Chromatography Fundamentals (1 decks)

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