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50 Key Topic Related to HPLC
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High-Performance Liquid Chromatography (HPLC) is an
analytical technique used for separating, identifying, and
1 HPLC Definition
quantifying components in a mixture, used widely in
pharmaceuticals, environmental testing, and food analysis.
Based on compounds interacting differently with stationary
and mobile phases, leading to separation. Compounds
2 Principle of HPLC
interacting more with the stationary phase move slower,
while those with the mobile phase move faster.
Difference Between Normal- Normal-phase HPLC: polar stationary phase and nonpolar
3 Phase and Reverse-Phase mobile phase. Reverse-phase HPLC: nonpolar stationary
HPLC phase and polar mobile phase.
Includes solvent reservoir, pump, injector, column, detector,
Main Components of an
4 and data system. Each plays a role in effective sample
HPLC System
separation and detection.
Material inside the column, often silica or polymer particles,
5 Stationary Phase in HPLC that interacts with analytes. Determines separation efficiency
based on analyte properties.
The solvent that carries the sample through the column. Can
6 Mobile Phase in HPLC be a single solvent or a mixture, chosen to optimize
separation.
Includes UV-Vis, PDA, fluorescence, refractive index (RI), and
7 Common HPLC Detectors mass spectrometry detectors, each suited to different
analytes.
Site where analytes are separated based on their interaction
8 Purpose of the HPLC Column with the stationary phase. Different column chemistries (e.g.,
C18, C8) cater to different separation needs.
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50 Key Topic Related to HPLC
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Includes reverse-phase (C18, C8), ion-exchange, size
9 Types of HPLC Columns exclusion, and chiral columns, each suited to specific
applications depending on analyte characteristics.
Ensures consistent flow of the mobile phase through the
10 Role of the HPLC Pump column at a set pressure, crucial for accurate
chromatographic results.
Introduces the sample into the system, typically through a
Purpose of the Injector in
11 sample loop, ensuring accurate volume and minimal
HPLC
contamination.
Involves changing the mobile phase composition during
12 Gradient Elution in HPLC separation, allowing analytes of different polarities to be
separated efficiently.
Uses a constant mobile phase composition throughout the
13 Isocratic Elution in HPLC
analysis, ideal for analytes with similar polarities.
Choosing a Suitable Mobile Depends on analyte solubility and interactions with the
14
Phase stationary phase, often optimized through trial and error.
Monitors analytes as they elute from the column, converting
Purpose of the HPLC
15 signals into data for quantification. Detector choice depends
Detector
on analyte properties.
Includes choice of stationary phase, mobile phase
Factors Affecting HPLC
16 composition, flow rate, column temperature, and sample
Separation
size, all essential for peak resolution.
The time it takes for an analyte to pass through the column
17 Retention Time
and be detected, used to identify and quantify analytes.
Refers to how well two analyte peaks are separated, with
18 Peak Resolution
higher resolution needed for accurate analysis.
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50 Key Topic Related to HPLC
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Baseline in HPLC The detector response when no analytes are present,
19
Chromatogram providing a reference point for analyte detection.
Protects the analytical column from contaminants and
20 Purpose of Guard Column
particulate matter, prolonging its lifespan and performance.
UV-Vis detectors measure absorbance at one wavelength,
Difference Between UV-Vis
21 while PDA detectors measure across a range, offering more
and PDA Detectors
data.
The time for an unretained compound to pass through the
22 Dead Time in HPLC
column, used as a reference point for other analytes.
Describes how well a column separates analytes, often
23 Column Efficiency expressed in terms of theoretical plates. Higher efficiency
results in sharper peaks.
Concept used to measure column efficiency; more plates
24 Theoretical Plates
mean better separation.
Occurs when the trailing edge of a peak is elongated, usually
25 Peak Tailing in HPLC due to strong analyte-column interactions. Prevented by
adjusting mobile phase or pH.
Occurs when the leading edge of a peak is extended, typically
26 Peak Fronting in HPLC caused by sample overload or strong analyte-column
interactions.
Refers to the separation degree between adjacent peaks.
27 Resolution in HPLC
Higher resolution means clearer, distinguishable peaks.
A compound added to normalize sample variations,
28 Internal Standard in HPLC
improving precision and accuracy.
Involves a nonpolar stationary phase and polar mobile phase,
29 Reverse-Phase HPLC useful for separating a wide range of organic compounds,
widely applied in diverse sample types.
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50 Key Topic Related to HPLC
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Applied to complex mixtures with analytes of different
Gradient Elution Usage in
30 polarities, often improving resolution and reducing analysis
HPLC
time compared to isocratic elution.
Involves adjusting mobile phase, flow rate, column type, and
HPLC Method Development
31 temperature to achieve optimal separation, requiring a
Optimization
systematic approach and experimentation.
A measure of how long an analyte is retained compared to
32 Retention Factor (k’) in HPLC the mobile phase; a higher retention factor indicates stronger
interaction with the stationary phase.
Refers to the ability to distinguish between analytes based on
33 Column Selectivity
their interactions with the stationary phase.
Includes solvents like water, methanol, and acetonitrile, often
Common Mobile Phases in
34 mixed with buffers (e.g., phosphate) to control pH and
Reverse-Phase HPLC
optimize separation.
Stabilizes pH in the mobile phase, improving reproducibility
Purpose of Buffers in HPLC
35 and preventing unwanted interactions between analytes and
Mobile Phases
stationary phase.
Refers to the resistance mobile phase encounters in the
Column Backpressure in
36 column. High backpressure may indicate blockages or
HPLC
increased solvent viscosity.
Degassing Mobile Phase in Removes dissolved gases that can form bubbles, disrupting
37
HPLC flow and detection.
Theoretical plates are calculated using the equation N =
Calculating Theoretical Plates
38 16(tR/W)^2, where tR is retention time and W is peak width
in HPLC
at the base.
Ensures that the system is functioning properly before
39 System Suitability in HPLC analysis, checking parameters like retention time, resolution,
and detector sensitivity.
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Causes of High Backpressure Can be due to blockages, degraded packing material, or
40
in HPLC viscous solvents.
Role of Column Temperature Affects interactions between analytes and stationary phase,
41
in HPLC influencing retention time and peak shape.
Symmetrical peaks are essential for accurate quantification,
Significance of Peak
42 with asymmetry indicating potential problems in the sample,
Symmetry in HPLC
column, or mobile phase.
Effect of Flow Rate on HPLC Flow rate influences retention time and resolution; higher
43
Separation rates reduce run time but may compromise resolution.
Void volume is the volume of mobile phase needed to elute
Void Volume and Void Time
44 an unretained compound, while void time is the
in HPLC
corresponding time, useful for system calibration.
Carryover can be minimized through proper injector cleaning,
Minimizing Carryover in
45 optimized sample preparation, and effective wash steps
HPLC
between runs.
Flushing with strong solvents (e.g., methanol, acetonitrile)
46 Cleaning an HPLC Column removes residues and contaminants, maintaining
performance and prolonging column life.
Proportional to analyte concentration, used for quantification
47 Peak Area in HPLC
by comparing to standards.
Linearity indicates how well detector response correlates
Linearity in HPLC Method
48 with analyte concentration, essential for accurate
Validation
quantification.
Significance of Ensures consistent results across multiple analyses, critical for
49
Reproducibility in HPLC method validation and regulatory compliance.
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50 Key Topic Related to HPLC
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Poor resolution can be addressed by adjusting mobile phase
Troubleshooting Poor
50 composition, column type, or flow rate, and optimizing
Resolution in HPLC
sample preparation.
