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John Dolan is best known as one of the worlds foremost troubleshooting authorities. Separation Science and John Dolan have collaborated to
offer this digital learning platform providing valuable advice on everyday issues, problems and challenges faced by LC practitioners.
Importantly, you will also have the opportunity to interact with John through our online questions submission system.
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can adjust the peak width settings so, for example, very broad peaks in the
background are ignored.
Excessive baseline noise can make it hard to distinguish a peak from the
baseline or may just make it look bad. The data system and/or detector usually
has the capability of adding some kind of noise filtering or peak smoothing
so that the peaks look better. The time-constant is one description of such a
filter and can be thought of as a running-average filter that reports the average
response for a defined number of data points. As a rule of thumb, the time
constant should be no larger than about one-tenth of the peak width. So a peak
width of 10 sec could tolerate a 1-sec time constant. This would smooth the peak,
making it easier to visualize as well as to integrate.
Data systems sample the detector output at a specific frequency. It is important
that enough points are gathered across a peak so that it is well defined and
the reported area is reliable. With the introduction of UHPLC, peak widths are
narrower than with conventional HPLC systems, so the data rate available often
is faster up to 200 Hz (points per second). This makes sure that all peaks have
enough data points for good definition. However, it only takes 15-20 data points
across a peak to adequately determine the peak area and retention time more
points than this tend to collect noise faster than enhance the signal. For example,
a 1-sec wide peak would need a data rate of 20 Hz (20 points per second x 1 sec
= 20 points across the peak) for adequate performance. A system set to 100 Hz
would collect five times as much data as needed, which also unnecessarily uses
data storage space. A good practice is to collect data at a high frequency during
Figure 1
Figure 1: Summary of some of the integration settings that are commonly available for adjustment.
R&D activities so you dont miss anything, but when the method is set up for
routine use, reduce the data rate to 15-20 points per peak to save disk space while
maintaining adequate data for good peak definition.
Because it is hard to outsmart a modern data system, I recommend that you
set up your method and run representative chromatograms across the range of
sample concentrations you expect, allowing the data system to adjust itself. If you
dont like the results, you can tweak the settings, but my guess is that you will be
happy with the default settings most of the time.
John Dolan is best known as one of the worlds foremost HPLC troubleshooting
authorities. He is also known for his ongoing research with Lloyd Snyder,
resulting in more than 100 technical publications and three books.
Contact John at TechTips@sepscience.com
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Determination of Ephedra Alkaloids & Synephrine by Strong CationExchange SPE and LCMS/MS Detection
Using a Pentafluorophenylpropyl HPLC Column
Company: UCT
Ephedra alkaloids are phenethylamines that occur naturally in plants. They are potent CNS stimulants and have
a sympathomimetic effect on the peripheral nervous system. The ephedra alkaloids are small, hydrophilic, basic
analytes that are difficult to retain on traditional HPLC columns. This application note outlines an alternative approach
using a fluorinated stationary phase. High capacity strong cationexchange SPE cartridges were used for the
extraction of phenethylamines from dietary supplements.
Method Development for Enantiomers
Company: Agilent Technologies
This application note describes the quantification of human IgG with the Agilent Bio-Monolith Protein A HPLC Column
using the Agilent 1260 Infinity Bio-inert Quaternary LC.
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Separation of statistic MMA-MAA Copolymers using
Gradient SEC
Application Note Pharmaceutical Analysis
Author
Isocratic GPC/SEC is a powerful tool to separate macromolecules based on their hydrodynamic volume. In case of homopolymers GPC/SEC allows the fast, precise and easy
determination of the complete molar mass distribution.
Unfortunately many modern polymeric materials are copolymers and isocratic GPC/SEC does
not provide any information about the chemical composition. Here SEC-gradients or polymer
HPLC is the method of choice.
Introduction
Statistic copolymers of methyl methacrylate (MMA) and methacrylic acid (MAA) are widely used
in pharmaceutical applications. Besides the molar mass distribution the chemical composition
and the amount of comonomers in the copolymer is of importance. Separations by conventional
gradient HPLC failed, since the polar eluents required to dissolve polymers of high acid content
prevent adsorption onto the stationary phase, resulting in pronounced breakthrough peaks. The
problem can be avoided applying SEC-gradients, resulting in the desired separation according
to the amount of methacrylic acid. The system can be calibrated using reference materials of
known composition. This allows determining the average copolymer composition as well as the
compositional heterogeneity.
System Requirements
Conditions
Pump
Injection system
Columns
Calibration
Loading
Detector
Software
Polymer Standards
Service-USA, Inc.
160 Old Farm Rd, Suite A
Amherst | MA 01002 | USA
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A High-Throughput SPE Method to Support the Biomonitoring of Phthalate Metabolites in Human Urine Using
ISOLUTE ENV+ Columns Prior to LC-MS/MS
Company: Biotage
This application note describes the extraction of nine phthalate metabolites from human urine using ISOLUTE ENV+
solid phase extraction columns. Proof-of-concept for this sample preparation method was determined on a set of real
patient samples (n=5).
InertSustainSwiftTM
Company: GL Sciences
This note explains how InertSustainSwiftTM C18 columns are designed to enhance your high throughput strategy due
to increased detection sensitivity, high plate count, very low bleed and rapid re-equilibration ensuring accurate results
are met for LC-MS and LC-MS/MS.
Separation of Statistic MMA-MAA Copolymers using Gradient SEC
Company: PSS Polymer
Isocratic GPC/SEC is a powerful tool to separate macromolecules based on their hydro-dynamic volume. In the case
of homopolymers GPC/SEC allows the fast, precise and easy determination of the complete molar mass distribution.
Here SEC-gradients or polymer HPLC is the method of choice.
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Practical HPlC for biopharmaceuticals
Although small molecule and biopolymer
separations have traditionally been considered
as separate activities, analysts in the
biopharmaceutical industry regularly have to
deal with both. Fortunately, the underlying
principles of chromatography apply equally well
in both situations when interpreted
appropriately.
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