Chapter 6 Flashcards

To memorise and score the test

1
Q

What is the difference between liquid chromatography (LC) and high performance liquid chromatography (HPLC)?

A

In principle, LC and HPLC work the same way except that HPLC offers superior speed, efficiency, sensitivity, and ease of operation compared to traditional liquid column chromatography.

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2
Q

What are the fundamental concepts of HPLC?

A
  • Understanding the different types/modes of HPLC
  • Distinguishing it from other forms of liquid chromatography
  • Identifying different chromatograms
  • Troubleshooting issues that may arise during analysis.
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2
Q

How does HPLC separate compounds dissolved in a solution?

A

HPLC separates compounds by injecting a small volume of liquid sample into a column packed with tiny particles called the stationary phase. The components of the sample are moved down the column with a liquid mobile phase forced through the column by high pressure.

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2
Q

What are some common chromatography techniques used in HPLC and GC?

A

In modern analytical chemistry, HPLC and GC are the major techniques used. HPLC is suitable for nonvolatile species and offers high performance and speed compared to traditional liquid column chromatography. GC, on the other hand, uses a gas mobile phase and is suitable for volatile compounds.

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2
Q

What is the role of the mobile phase in HPLC?

A

As a carrier to the sample solution and is continuously applied to the column or stationary phase. It facilitates the separation of biomolecules by using two or more solvents in certain ratios

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2
Q

Why is it important to degas solvents/mobile phase in HPLC?

A

To remove impurities. Impurities may interfere with the detection system, especially absorbance below 200 nm. Degas methods include warming, vigorous stirring, vacuuming, ultrasonification, and bubbling helium gas through the eluent reservoir.

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3
Q

What is the difference between isocratic elution and gradient elution in HPLC?

A

Isocratic elution is a separation method where the mobile phase composition remains constant throughout the procedure. In contrast, gradient elution involves gradually changing the mobile phase composition during the sample run. Gradient elution decreases the retention of later-eluting components, resulting in narrower peaks and improved peak shape and height

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4
Q

What is the difference between normal phase and reverse phase chromatography in HPLC?

A

In normal phase chromatography, the stationary phase is polar (e.g., alumina or silica gel), while the mobile phase is non-polar (e.g., hexane). In reverse phase chromatography, the stationary phase is non-polar (e.g., C18) and the mobile phase is polar (e.g., water, methanol, acetonitrile). Non-polar compounds elute faster in normal phase, while polar compounds elute later. Reverse phase chromatography is more commonly used as drugs are usually polar (hydrophilic).

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5
Q

Why are rigid solid particles used as the stationary phase in HPLC?

A

Rigid solid particles, such as microporous, pellicular, or bonded phase particles, are used as the stationary phase in HPLC to reduce space for diffusion. These spherical particles with uniform size provide efficient separation

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6
Q

How does the mobile phase facilitate the migration of components in an HPLC assay?

A

As a sample solution flows through the column with the mobile phase, the components of that solution migrate according to the non-covalent interaction of the compound with the column. The mobile phase carries the sample solution through the column, allowing for separation based on these interactions

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7
Q

What are some degassing methods used for solvents in HPLC?

A
  • Warming
  • Vigorous stirring with a magnetic stirrer
  • Vacuuming
  • Ultrasonification
  • Bubbling helium gas through the eluent reservoir
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8
Q

Why is gradient elution preferred for the analysis of complex samples?

A

The mobile phase composition gradually changes during the sample run. It decreases the retention of later-eluting components, resulting in narrower peaks and improved peak shape and height. This technique is often used in method development for unknown mixtures

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9
Q

What is the purpose of a guard column in HPLC?

A

To protect the main column from impurities

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10
Q

Why are smaller packing particles preferred in HPLC columns?

A

Smaller packing particles lead to better resolution, but they also require high liquid pressure because they resist flow

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11
Q

What is the role of the stationary phase in HPLC?

A
  • Separates the sample components using various physical and chemical parameters
  • Usually made of stainless steel and withstands high pressure caused by the pump
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12
Q

What is the function of HPLC pumps?

A
  • Force the mobile phase through the liquid chromatography at a specific flow rate
  • Ensure a stable and pulse-free flow of the mobile phase
13
Q

What are the two types of HPLC pumps commonly used?

A
  • Constant pressure pump
  • Constant volume pumps
14
Q

How do constant pressure pumps and constant volume pumps differ in maintaining flow rate?

A
  • Constant pressure pumps maintain a constant pressure irrespective of stationary phase resistance
  • Constant volume pumps maintain a constant flow rate through the stationary phase and increase the pressure if there is an increase in stationary phase resistance within the pre-set limits of the pump
15
Q

What is the purpose of sample preparation in HPLC?

A
  • Compatibility to further analysis
  • Simplifying complex samples
  • Removing interferences from the matrix
  • Concentrating or diluting the sample
  • Speeding up the analysis process
  • Ensuring system robustness
16
Q

What are some commonly used detectors in HPLC?

A
  • UV detectors
  • Fluorescence detectors
  • Mass spectrometry detectors
  • Refractive index detectors
17
Q

What are the characteristics and applications of a UV detector in HPLC?

A

A UV detector in HPLC responds to substances that absorb light. It is mainly used to separate and identify the principal active components of a mixture. UV detectors are versatile, sensitive, and widely used in various industries

18
Q

What are the advantages of using mass spectrometry as a detector in HPLC?

A
  • Can analyze and provide the molecular identity of a wide range of components
  • Widely used in pharmaceutical laboratories and research and development
19
Q

What factors should be considered when choosing a sample preparation product for analysis?

A
  • Specific analysis requirements
  • The nature of the sample should be considered
20
Q

What is the role of an HPLC detector in the analysis process?

A
  • Detects individual molecules that elute from the column and converts the data into an electrical signal
  • Measures the amount of those molecules and provides an output to generate a liquid chromatogram
21
Q

What are the advantages and limitations of using a UV detector in HPLC?

A
  • Advantages: Versatile, sensitive, and cost-effective
    Disadvantages: Cannot be used for substances that are low in chromophores and do not absorb light at low range
22
Q

What is the role of the data processing unit in an HPLC instrument?

A

Controls all the modules of the HPLC instrument, takes signals from the detector to determine the retention time of sample components for qualitative analysis and calculates the concentration of each detected component for quantitative analysis

23
Q

How is the concentration of each detected component calculated in HPLC analysis?

A

Calculated from the area or height of the corresponding peak in the HPLC chromatogram

24
Q

What are some common abnormalities that can occur in HPLC peaks?

A
  • Baseline spikes due to air bubbles
  • Negative peaks caused by larger mobile phase absorbance than sample absorbance
  • Peak doubling/tailing due to co-elution of interfering compounds, column overload, or channeling in the column
25
Q

What are some applications of HPLC?

A

clinical quantification of drugs in body fluids, environmental identification of chemicals in drinking water, forensic analysis of textile dyes, industrial stability testing of compounds in food products, pharmaceutical quality control and shelf-life determination, and research for separation and isolation of components from natural samples

26
Q

What are some advantages of HPLC compared to other separation techniques?

A
  • Fast and efficient separation
  • Continuous monitoring of column effluent
  • Applicability to complex mixtures
  • Accurate quantitative measurements
  • Repetitive and reproducible analysis using the same column
  • Versatility in choice of mobile and stationary phases
27
Q

What are some limitations imposed by high pressure in HPLC instrumentation?

A

High pressure imposes constraints on HPLC instrumentation, such as the cost of generation and the need for instruments that can withstand high pressure

28
Q

Why is HPLC not commonly used for the purification of complex biopolymers like proteins?

A
  • The denaturation of proteins by organic solvents used in some HPLC formats
  • The expense of large capacity HPLC columns
29
Q

How does Fast Protein Liquid Chromatography (FPLC) differ from HPLC?

A

FPLC is a form of liquid chromatography specifically designed for the separation of biopolymers like proteins and DNA. It operates at lower pressures compared to HPLC and uses reciprocating pumps with two pistons to avoid pulses