Podcast
Questions and Answers
In chromatography, what is the primary role of the mobile phase?
In chromatography, what is the primary role of the mobile phase?
- To dissolve the stationary phase.
- To provide a solid support for the stationary phase.
- To remain static and allow components to bind.
- To carry the mixture's components through the stationary phase. (correct)
Which of the following is a characteristic common to all types of chromatography?
Which of the following is a characteristic common to all types of chromatography?
- The use of a gaseous stationary phase.
- The presence of both a stationary and a mobile phase. (correct)
- The absence of a mobile phase.
- The exclusive use of solid stationary phases.
What distinguishes the mobile phase from the stationary phase in chromatography?
What distinguishes the mobile phase from the stationary phase in chromatography?
- The stationary phase moves, while the mobile phase remains fixed.
- The mobile phase is always a liquid, while the stationary phase is always a solid.
- The stationary phase carries the components, while the mobile phase does not.
- The mobile phase flows, while the stationary phase remains fixed. (correct)
If a substance interacts strongly with the stationary phase, what is the expected outcome in a chromatographic separation?
If a substance interacts strongly with the stationary phase, what is the expected outcome in a chromatographic separation?
In chromatography, how does the mobile phase facilitate the separation of different components in a mixture?
In chromatography, how does the mobile phase facilitate the separation of different components in a mixture?
In quantitative analysis, which factor is most crucial for ensuring the reproducibility of peak responses?
In quantitative analysis, which factor is most crucial for ensuring the reproducibility of peak responses?
Which of the following HPLC detectors is based on the principle of light absorption?
Which of the following HPLC detectors is based on the principle of light absorption?
A researcher is developing a new method for quantifying a compound with low UV absorbance. Which detector would be the MOST suitable alternative to a UV-Vis detector?
A researcher is developing a new method for quantifying a compound with low UV absorbance. Which detector would be the MOST suitable alternative to a UV-Vis detector?
A scientist aims to identify and quantify various unknown compounds in a complex sample. Which HPLC detector would offer the most comprehensive information for both identification and quantification?
A scientist aims to identify and quantify various unknown compounds in a complex sample. Which HPLC detector would offer the most comprehensive information for both identification and quantification?
A compound's peak area in HPLC analysis shows poor reproducibility across multiple injections. Which of the following is the MOST likely cause?
A compound's peak area in HPLC analysis shows poor reproducibility across multiple injections. Which of the following is the MOST likely cause?
In chromatography, what does $t_R$ typically represent?
In chromatography, what does $t_R$ typically represent?
Which parameter is most directly related to the quantity of analyte detected in chromatography?
Which parameter is most directly related to the quantity of analyte detected in chromatography?
What information can be derived from the peak width at half height ($W_{1/2}$) in a chromatographic peak?
What information can be derived from the peak width at half height ($W_{1/2}$) in a chromatographic peak?
In the context of UV-Vis chromatography, what does AU generally represent on the y-axis of the graph?
In the context of UV-Vis chromatography, what does AU generally represent on the y-axis of the graph?
If a chromatographic peak exhibits significant tailing, which parameter is most likely affected?
If a chromatographic peak exhibits significant tailing, which parameter is most likely affected?
Under which conditions are optical activity measurements most effectively conducted?
Under which conditions are optical activity measurements most effectively conducted?
Why is temperature control important in optical activity measurements?
Why is temperature control important in optical activity measurements?
What would be the most likely consequence of not maintaining a controlled temperature during optical activity measurements?
What would be the most likely consequence of not maintaining a controlled temperature during optical activity measurements?
For what type of work is temperature control most crucial in optical activity measurements?
For what type of work is temperature control most crucial in optical activity measurements?
In a scenario where precise temperature control is not possible, what is the best approach to ensure reasonably reliable optical activity measurements?
In a scenario where precise temperature control is not possible, what is the best approach to ensure reasonably reliable optical activity measurements?
What change would cause the optical rotation angle, $\alpha$, to increase, assuming all other variables remain constant?
What change would cause the optical rotation angle, $\alpha$, to increase, assuming all other variables remain constant?
If a substance is levorotatory, how would the plane of polarization be rotated relative to the incident beam?
If a substance is levorotatory, how would the plane of polarization be rotated relative to the incident beam?
A sample with a length of 2 cm is placed in a polarimeter. When plane-polarized light is passed through the sample, the observed rotation is +30 degrees. What does the '+' sign indicate?
A sample with a length of 2 cm is placed in a polarimeter. When plane-polarized light is passed through the sample, the observed rotation is +30 degrees. What does the '+' sign indicate?
How is the optical rotation,$\alpha$, affected if the light path, $l$, is halved and the wavelength, $\lambda$, is doubled, assuming the refractive index difference, $\Delta_n$, remains constant?
How is the optical rotation,$\alpha$, affected if the light path, $l$, is halved and the wavelength, $\lambda$, is doubled, assuming the refractive index difference, $\Delta_n$, remains constant?
What condition causes different retardation of circularly polarized light components in an optically active medium?
What condition causes different retardation of circularly polarized light components in an optically active medium?
Which factor does not directly influence the observed angle of optical rotation?
Which factor does not directly influence the observed angle of optical rotation?
In an optically active medium, what directly leads to the retardation of circularly polarized light components to varying degrees?
In an optically active medium, what directly leads to the retardation of circularly polarized light components to varying degrees?
If an optically active substance has $n_l > n_r$, what does this indicate about the behavior of light passing through it?
If an optically active substance has $n_l > n_r$, what does this indicate about the behavior of light passing through it?
In an optically active medium where circularly polarized light components are retarded differently, what would be observed if $n_l$ was approximately equal to $n_r$?
In an optically active medium where circularly polarized light components are retarded differently, what would be observed if $n_l$ was approximately equal to $n_r$?
Consider two optically active materials, A and B. Material A has a greater difference between $n_l$ and $n_r$ than Material B. How will the retardation of circularly polarized light differ between these materials?
Consider two optically active materials, A and B. Material A has a greater difference between $n_l$ and $n_r$ than Material B. How will the retardation of circularly polarized light differ between these materials?
Flashcards
Stationary Phase
Stationary Phase
The non-moving phase in chromatography. It's either a solid or a liquid supported on a solid.
Mobile Phase
Mobile Phase
The moving phase in chromatography. It's either a liquid or a gas.
Chromatography
Chromatography
A technique that separates mixtures by using a stationary and mobile phase.
Mobile Phase Function
Mobile Phase Function
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Stationary Phase Role
Stationary Phase Role
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Optically Active Medium
Optically Active Medium
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Refractive Index Difference
Refractive Index Difference
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Differential Retardation
Differential Retardation
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nL
nL
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nR
nR
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Reproducibility
Reproducibility
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Precision of sample introduction
Precision of sample introduction
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HPLC
HPLC
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UV-Visible detectors
UV-Visible detectors
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Fluorescence detectors
Fluorescence detectors
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What does 'tR' represent?
What does 'tR' represent?
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What does 'Area' represent?
What does 'Area' represent?
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What is 'W1/2'?
What is 'W1/2'?
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What is 'Wb'?
What is 'Wb'?
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What do ‘h1/2’ and ‘h’ indicate?
What do ‘h1/2’ and ‘h’ indicate?
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Optical Rotation (α)
Optical Rotation (α)
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Light Path (l)
Light Path (l)
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Wavelength (λ)
Wavelength (λ)
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Dextrorotation
Dextrorotation
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Levorotation
Levorotation
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Optical Activity
Optical Activity
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Sodium D Light
Sodium D Light
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Standard Temperature for Optical Activity
Standard Temperature for Optical Activity
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Importance of Temperature Control
Importance of Temperature Control
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Consequence of Temperature Variation
Consequence of Temperature Variation
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Study Notes
- Pharmaceutical Instrumental Analysis covers topics like Separation Techniques (HPLC) and Optical Activity (Polarimetry).
- The labs associated are Lab 5 and Lab 6.
Chromatography
- A separation technique with Greek roots: chroma (color) and graphein (to write).
- Initially developed by a Russian botanist for separating plant pigments using a column of calcium carbonate.
- Used in labs for separating substance mixtures into components and identifying compounds.
- All forms use a stationary phase (solid or liquid on a solid) and a mobile phase (liquid or gas).
- The mobile phase flows through the stationary phase, carrying mixture components at varying rates.
High Performance Liquid Chromatography (HPLC)
- Physical separation technique in liquid phase.
- A sample is separated by distributing between the stationary and mobile phases, with a flowing liquid pumped at high pressure.
- The flowing liquid can be an organic solvent.
- The stationary phase may consist of porous silica particles packed in a column.
- The essential components of a complete HPLC system are a solvent delivery system (pump), fixed volume injector loop or autosampler, packed column, solvent reservoirs, detector, data system, and recorder.
- The column is central to the HPLC system as they are particle-packed.
- Solvent delivery systems, commonly called pumps, deliver the mobile phase and samples are typically introduced via syringe injection or autosamplers.
- Reproducible and precise injections are important if using the HPLC for quantitative analysis.
- Detectors used include UV-Visible, fluorescence, electrochemical, and mass spectrometry.
- Chromatographic efficiency (N) is expressed as the number of theoretical plates: N = 16 (tR)2/Wb where tR = retention time, w = peak width at base.
- Resolution (Rs): Rs = t2-t1/Wb2-Wb1/2 which represents the degree of separation between two peaks.
- Asymmetry factor (AF) = A/B at 10% of peak height (A and B are the two half widths at each side of the peak centre).
HPLC Types of Chromatography
- Normal phase chromatography: uses a stationary phase with polar functional groups and a non-polar mobile phase.
- Reversed phase liquid chromatography (RPLC): uses a non-polar stationary phase and polar mobile phase.
- Retention in RPLC occurs due to hydrophobic interactions between the solute and stationary phase.
- Most organic compounds can interact with the stationary phase, giving RPLC a wide range of applications.
- RP-HPLC columns are often silica-based for mechanical stability.
- A typical stationary phase involves chemically bonding a long-chain hydrocarbon group to porous silica.
- Typical ligands are n-octadecyl (C18), n-octayl (C8), and n-butyl (C4).
- Separation in RPLC is affected by the stationary phase type, column length, type and % of organic solvent in the m. p and mobile phase pH.
- Flow rate and temperature can also affect separation.
HPLC Types of elution modes
- Isocratic elution: uses a single solvent (or a mixture in one reservoir) as the mobile phase.
- Gradient elution: uses two or more solvents separately as a mobile phase, with the solvent ratio varied in a programmed manner.
- Gradient elution can take longer for column equilibrium and is incompatible with some LC detectors and can have baseline problems.
Practice Questions
- Pyridine and t-butylbenzene peaks by HPLC, with retention times of 1.3 min and 3.5 min respectively and peak widths of 0.4 min and 0.6 min respectively.
- To calculate: resolution between peaks, retention factor for pyridine (tm 0.45), and efficiency of butylbenzene
Optical Activity (Polarimetry)
- When plane-polarized light goes through a medium, it's slowed down to an extent shown by the refractive index of the medium.
- In an optically inactive medium, both circularly polarized components are slowed down a bit, and the beam comes out polarized in the original plane.
- In an optically active medium, components are slowed to different extents because the refractive indices differ for left (nī) and right (np) circularly polarized light.
Optical activity
- The rotation angle (α) of plane-polarized light is given by: α = (1800 / λ) * Δn where l is the light path, λ is wavelength, α is optical rotation.
- When looking towards the light source, optical rotation is positive (dextrorotation) if the plane of polarization rotates clockwise, and negative (levorotation) if it rotates anticlockwise.
- Sodium D light is often used at 20°C for optical activity measurements.
- Temperature control is needed for precise work.
- Instruments used include Visual Polarimeters and Spectropolarimeters.
- Optical rotation can be used to assess the purity of optically active materials.
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Description
Explore separation techniques for pharmaceutical analysis, including High Performance Liquid Chromatography (HPLC). Learn how HPLC separates samples by distributing them between stationary and mobile phases using a flowing liquid pumped at high pressure. This includes Labs 5 and 6 on Chromatography.
