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Analytical Chemistry Fundamentals > Chromatography Fundamentals (EN) > Flashcards

Chromatography Fundamentals (EN) Flashcards

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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 (N), selectivity (α) and 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 (α)

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6
Q

What means Retention time (tr) 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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7
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 (mobile phase) moving in a definite direction.

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8
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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9
Q

Which parameters are influencing colum efficiency?

A

Column length (increasing colum length increases efficiency) and particle size (decreasing particle size increases efficiency).

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10
Q

Which parameters are influencing the Retention factor (k)?

A
  1. Stationary phase
  2. Mobile phase
  3. Temperature
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11
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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12
Q

What is selectivity or separation factor (α)?

A

Selectivity is a measure of the time or distance between the maxima of two peaks. If α = 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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13
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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14
Q

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

A
  1. Eddy diffusion
  2. Axial or longitudinal diffusion
  3. Resistance to mass transfer
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15
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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16
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.

17
Q

What is influencing peak capacity?

A

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

18
Q

Smaller column material particles improve …

A

…resolution and efficiency.

19
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).

20
Q

What would be the best technique for volatile compounds?

A

Gas Chromatography (GC)

21
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“.

22
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.

23
Q

Which modules are typically stacked in a HPLC system?

A
  • Pump
  • Injector
  • Column
  • Detector
24
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.

25
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

26
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

27
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.

28
Q

What is Carryover?

A

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

29
Q

What does area prescision means (injection)?

A

Repeatability of injection volumes

30
Q

What is linearity (injection)?

A

Precision over a defined range of injection volumes

31
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.

32
Q

What are the main separation modes in HPLC?

A
  • Reversed-phase
  • Normal phase
  • Ion exchange and size exclusion
  • HILIC
33
Q

Size exclusion chromatography is mainly used for….

A

…polymer characterisation and proteins.

34
Q

Name three detection technologies used in HPLC

A
  • Spectroscopic (UV)
  • Refractive Index (RI)
  • Fluorescence detection (FLD)
  • Mass spectrometry (MS)
35
Q

Definition of resolution

A

tri Retention time compound i

Wbi Peak width at baseline

Analytical Chemistry Fundamentals (1 decks)

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