1-3 - 2024 4th Chem 448

Advanced separation techniques
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An Introduction to Chromatographic Separations
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Retention time
Distribution Constants
The analyte has been retained because it spends a time tS in

the stationary phase. The retention time is
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High-Performance Liquid Chromatography
High-performance liquid chromatography (HPLC) is

the most versatile and widely used type of elution
chromatography. The technique is used by scientists for
separating and determining species in a variety of
organic, inorganic, and biological materials. In liquid
chromatography, the mobile phase is a liquid solvent
containing the sample as a mixture of solutes. The types
of HPLC are often classified by separation mechanism
or by the type of stationary phase. The varieties include
(1) partition, or liquid-liquid, chromatography; (2)
adsorption, or liquid-solid chromatography; (3) ion-
exchange, or ion, chromatography; (4) size-exclusion
chromatography; (5) affinity chromatography, and (6)
chiral chromatography.
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Instrumentation
➢ Mobile-Phase Reservoirs and Solvent Treatment Systems

➢ Pumping Systems
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Basic HPLC System Components
Solvent Degasser – removes air gases from the solvents as they flow to the HPLC pump
HPLC Pump – provides solvent flow and proportioning
Autosampler – draws samples from vials and injects them into the solvent flow provided by the pump.
Detector – responds to the separated analytes emerging from the HPLC column and produces a signal output for the software
Column Oven – houses the HPLC column and keeps a stable temperature for reproducible separations.
Isocratic vs. Gradient Elution Modes

Isocratic Elution
•A single composition of solvents is used for the duration of the separation
•Later eluting peaks are broader than earlier eluting peaks because of dispersion
•Steps must be taken to periodically flush the column at higher solvent strength to clean it of intractable materials that build up
from sample injections
Gradient Elution
•The composition of solvents is changed either continuously or stepwise
•In general, peaks are sharper throughout the chromatogram when compared to isocratic elution
•Some separations may be achieved which are not possible using isocratic elution
•Chromatogram run times may be shorter when compared to isocratic elution
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Isocratic and Gradient Elution Pumping Modes
❑ Isocratic Pump
A single channel pump which requires the user to pre-mix the mobile phase. Composition remains constant with
time.
❑ Quaternary Low Pressure Gradient Pump
A 4 channel pump which creates mixtures of separate solvent channels under software control. Mixing is done
before the pump heads. Composition may be changed with time.
❑ Binary High Pressure Gradient Pump
A 2 channel pump which creates mixtures of 2 solvents under software control. Mixing is done after the pump
heads. Composition may be changed with time.
However, isocratic elution is often favoured over gradient elution because the gradient process requires greater care
and regulation on the part of the operator. Isocratic elution also requires less specialised chromatographic
equipment.
Detectors for HPLC
UV/Vis Detectors
• Responds to chromophoric analytes in the range 190 – 800nm

• Single wavelength monitoring
• Good for the majority of organic analytes
• Gradient elution compatible 11
Photodiode Array (PDA) Detectors Diode Array HPLC Detectors
Are most commonly used to record the ultraviolet
• Responds to chromophoric analytes in the range 190 – 800nm
and visible (UV-vis) absorption spectra of samples that
• Multi-wavelength monitoring
are passing through a high-pressure liquid
• Complete spectral profiles of chromatographic peaks
chromatograph. This enables qualitative information to
• Good for the majority of organic analytes
be gathered about the samples.
• Gradient elution compatible
Fluorescence Detectors Refractive Index Detectors
• Responds only to analytes which fluoresce naturally or can • Considered a universal detector for all analytes
be made to fluoresce through derivatization • Comparitively insensitive
• Extremely sensitive • Not compatible with gradient elution
• Gradient elution compatible
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Sometimes, multiple detection techniques are employed, e.g., LC-UV-Vis-MS, LC-MS-MS, and LC-NMR-MS.
A UV-Vis spectroscopic detector is a universal detector for any HPLC system. The photo-diode-array (PDA)
detector is another form of UV-Vis detector that allows analysis of compounds containing chromophores,
coumarins, flavonoids, isoflavonoids, etc. A PDA detector facilitates the analysis of individual HPLC peaks after
completion of a run and helps to obtain complete UV-Vis spectrum of individual compounds. Independent
chromatograms can also be constructed at various wavelengths to enhance selectivity of the data. PDA is one of
the most popular detectors, but compounds with no chromophores are not UV-Vis sensitive, and cannot be
detected. Nowadays, an MS detector, generally in combination with a UV-Vis or PDA detector, is probably the
most sought-after detection method employed for HPLC, especially when analyzing complex mixtures of
compounds like natural product extracts or herbal products. When an MS detector is used, separated compounds
emerging from the column can be identified on the basis of their mass spectral data. The ionization techniques
used in HPLC-MS are generally soft ionization techniques, e.g., electrospray ionization mass spectrometry (ESI-
MS) that display mainly the molecular ion species with only a few fragment ions are generally used in HPLC-
MS. Sometimes, tandem mass spectrometry (MS-MS), which provides fragments through collision-induced
dissociation of the molecular ions, is also employed.
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What are the different types of HPLC?
❖ Normal-Phase HPLC
In normal phase chromatography, the stationary
phase is non- polar and the mobile phase is polar.
This means that any non-polar substances in the
sample elute more quickly as they are more similar
to the mobile phase and move quickly.
❖ Reverse Phase
Reverse phase High Performance Liquid
chromatography is the opposite of normal phase.
namely, a polar mobile phase, such as water and
a polar organic solvent, is used with a non-polar,
hydrophobic stationary phase.
Reverse phase HPLC is often preferred over
normal phase HPLC as the use of water as the
solvent eliminates the danger of analyte retention
times being skewed due to absorption of water into
the atmosphere. Reverse High Performance Chromatography is also considered to be more
flexible as the hydrophobic stationary phase can be used in conjunction with hydrophobic,
hydrophilic, ionic and ionisable compounds to separate out their different compounds.
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Column Chemistry Choices Based on Sample Solubility
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Advanced separation techniques

The analyte has been retained because it spends a time tS in

High-performance liquid chromatography (HPLC) is

➢ Mobile-Phase Reservoirs and Solvent Treatment Systems

Isocratic vs. Gradient Elution Modes

• Responds to chromophoric analytes in the range 190 – 800nm

Fluorescence Detectors Refractive Index Detectors

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