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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.

Peak Integration, Part 2: If You Dont Like The


Default Conditions

In HPLC Solutions #127 we took a brief look at how HPLC integrators


detect and measure a peaks height or area. Much of the time the default
settings for the data system are sufficient for reliable operation, but you
may find that the integration process isnt quite the way you want it.
Here well look at some of the options available to adjust the integration
process.
Click here to read>>
www.sepscience.com

HPLC Solutions Video Tutorials

Visit the new video tutorial section, where articles are


also provided in video format.
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Featured Applications

Determination of Ephedra Alkaloids & Synephrine by


Strong CationExchange SPE and LCMS/MS Detection
Using a Pentafluorophenylpropyl HPLC Column
Method Development for Enantiomers
A High-Throughput SPE Method to Support the
Biomonitoring of Phthalate Metabolites in Human Urine
Using ISOLUTE ENV+ Columns Prior to LC-MS/MS

HPLC Solutions on ChromForum

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HPLC Solutions articles on Chromatography Forum.
A specific topic category has been set up...
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Peak Integration, Part 2: If You Dont Like the Default


Conditions
In HPLC Solutions #127 we took a brief look at how HPLC integrators detect and
measure a peaks height or area. Much of the time the default settings for the data
system are sufficient for reliable operation, but you may find that the integration
process isnt quite the way you want it. Perhaps the system misses small peaks
or it may pick too many peaks. Well look here at some of the options available to
adjust the integration process. Note that the names of these functions will vary
depending on the brand of system you are using.
Figure 1 summarizes some of the integration settings that are commonly
available for adjustment. Be sure to consult the documentation for your data
system to see the details on how to use these settings. As mentioned last time,
the integration algorithms do a pretty good job of determining the starting
and ending points for a peak if the peak is large enough, the baseline is smooth
enough, and there isnt too much drift on the baseline. Change any of these and
you may miss the start or end of the peak or miss the peak altogether. On the
other hand, if the sensitivity is too high, you may end up integrating many more
small peaks than you are interested in. The slope sensitivity is the adjustment that
may help take care of this problem. By making the system more or less sensitive
to a change in baseline slope, you can adjust whether or not peaks are found or
missed. An alternative technique, if you are picking up too many peaks, is to turn
on integration only for a certain time segment of the chromatogram or you can
ignore peaks that are below a specific height or area.
Peaks in a normal isocratic run will gradually get broader throughout the run.
For gradients, the peaks are all about the same width throughout the run. You

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

FEATURED APPLICATIONS

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request copy

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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request copy

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InertSustainSwiftTM C18 columns are designed to enhance your High Throughput


strategy by controlling pore size, 20 nm and surface area, 200m2/g of Evolved Surface
Silica (ESS) and carbon loading, 9.0 %. The inherent characteristics of ESS also enable
rapid re-equilibration even with gradients.

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request copy
Separation of statistic MMA-MAA Copolymers using
Gradient SEC
Application Note Pharmaceutical Analysis
Author

Dr. Wolfgang Radke


contact: WRadke@pss-polymer.com

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

PSS SECcurity GPC1260 binary pump


flow rate [mL/min]: 1.0
mobile phase:
Gradient: Chloroform/DMAc

Injection system

PSS SECcurity GPC1260 Autosampler


Injection interval: 32 min

Columns

PSS PROTEEMA precolumn (8*50 mm)


PSS PROTEEMA, 3 m, 100 (8x300 mm)
Temperature: 60/C

Calibration

PSS MMA-MAA copolymers of different acid content


(MAA: 9%, 25%, 31%, 42%, 48%wt)

Loading

Samples dissolved in DMAc


1 mg/mL, 100 L injection volume

Detector

PSS SECcurity ELS1000


Gas flow: 1.5 SL/min
Nebulizer temperature: 100 /C
Evaporator temperature: 200 /C.

Software

PSS WinGPC UniChrom with ChromPilot and Chemical


Heterogeneity module

PSS Polymer Standards


Service GmbH
In der Dalheimer Wiese 5
55120 Mainz | Germany

Phone +49 6131 96239-0


Fax
+49 6131 96239-11
E-Mail info@pss-polymer.com
Web www.pss-polymer.com

Polymer Standards
Service-USA, Inc.
160 Old Farm Rd, Suite A
Amherst | MA 01002 | USA

Phone +1 413 835-0265


Fax
+1 413 835-0354
E-Mail usa@pss-polymer.com
Web www.pss-polymer.com

Click to
request copy

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
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