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79 How To Buy HPLC Technology 2019 PDF
79 How To Buy HPLC Technology 2019 PDF
2019
th
How to Buy
HPLC Technology EDITION
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2019 HPLC Technology
1. Introduction to High-Performance
Liquid Chromatography
High-performance liquid chromatography reducing solvent consumption.
(HPLC) is one of the most powerful analytical The higher pressure allows for the use of a
tools available today and works by separating smaller particle size for the column packing
individual compounds based on their polarity. material. Columns with smaller particles
HPLC separation is distinct from liquid generate sharper peaks with increased
chromatography (LC), with solvent passing resolution, due to reduced diffusion distances
through the column under high pressure, for analytes.
making separations much faster and, in turn, By comparison, ultra-high-performance
liquid chromatography (UHPLC) uses an
even smaller particle size than HPLC for
the packing material. This helps to produce
increased resolution and sensitivity. UHPLC
requires even higher pressures than HPLC,
which means systems capable of handling
TOP TIP
Figure 1 : Discover how scientists are bridging the gap between When choosing your HPLC
HPLC and UPLC with the groundbreaking ACQUITY Arc System f
rom Waters Corporation column, consider seeking
advice from chromatography
experts such as Restek, who
have helped analysts around
the world with the innovative
tools they need.
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2019 HPLC Technology
Figure 2 : Find out how Adam Rosebrock is advancing metabolomics with adaptive uses of HPLC and mass spectrometry to
“
determine biochemical pathways
this increased pressure can be relatively In selecting the most appropriate column,
expensive. It is worth noting that methods a number of considerations must be taken
developed for HPLC do into account. These are
not readily transfer to summarized in Table
UHPLC and vice versa.
Methods developed 1, and the sections to
However, it can be noted
that some systems have
been developed to bridge
the gap between the two,
as shown in this video
interview on ACQUITY
“
for HPLC do not
readily transfer to
follow. For insights into
how one laboratory has
adapted its HPLC analysis
to advance metabolomics,
watch this exclusive
interview with Dr. Adam
Arc System from Waters UHPLC. Rosebrock on The
Corporation. Scientists’ Channel.
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2019 HPLC Technology
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2019 HPLC Technology
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2019 HPLC Technology
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Figure 3 : Discover how separation technology is being used for the analysis of petroleum and the impact of oil spills on the
natural environment
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2019 HPLC Technology
L8 NH2 - Aminopropyl silane Aminopropyl silane bonded to totally porous silica, 3-10 µm
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2019 HPLC Technology
Key Separation
Separation Mode Separation is based on Application
Characteristic
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2019 HPLC Technology
The next considerations are size, shape and Many leading manufacturers
chirality of the particles that make up the
have LC experts on hand
column packing material.
The majority of new HPLC methods are to offer assistance when it
performed on spherical shaped or spheroidal comes to selecting the right
(almost spherical) particles. Spherical
column for you regarding
particles provide higher efficiency, better
column stability and lower back-pressures problematic separations
compared to irregularly shaped particles. (e.g. ColumnMatch.com
Phenyl columns (USP L11) are an alternative
to C18 columns and provide a unique
or Hamilton application
selectivity for aromatic compounds through index). By informing the
pi-pi interactions. Biphenyl columns, such as manufacturer what type
the Raptor™ Biphenyl LC columns by Restek
(Figure 4), are the next generation of phenyl
of separation you require,
columns. They offer both aromatic selectivity whether chiral, achiral or
and an increase in hydrophobic retention. aromatic, they can suggest
These phenyl phases are of particular
interest when analyzing drug compounds the best column for you, or
and metabolites, such as fentanyl and its even design one that fits your
analogues.
needs. This will ensure fast
If all other factors are constant, the
smaller the particles are, the more efficient and efficient analysis of your
the separation. The major problem with a compounds.
decrease in particle size, from 10-, 7-, 5-, and
3-micron diameters, is that the backpressure
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2019 HPLC Technology
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columns can be quite expensive and you will
The retention capacity is the time samples therefore want to ensure they last as long
spend in the column. It is of importance to as possible. Guard columns can be used to
both the quality and speed of protect your analytical column
separations. and prolong its lifetime. HPLC
The retention capacity
is influenced by surface
The pore size guard columns (or cartridges) are
installed in front of an analytical
area and carbon load (the
percentage of carbon in and volume of column and protect it from
strongly retained impurities.
the packing material). The
pore size and volume of
the packing material is also
important in determining
retention capacity. This is
because surface area is
the packing
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material is also
important.
The SecurityGuard™ Universal
HPLC Guard Cartridge System
by Phenomenex Inc offers a
convenient and economical
way to extend HPLC column
lifetimes. This column guard is
inversely proportional to pore also universal so can effectively
size. A larger pore size results protect all analytical columns
in lower retention. The particle pore size is without effecting results. An alternative to
measured in angstroms and generally ranges using a guard system is the use of an in-
between 100-1000 angstroms; 300 angstroms line filter. Filters are a simple, cost-effective
is the most popular pore size for proteins means of providing a quick, efficient clean-up
and peptides and 100 angstroms is the most for occasional ‘dirty’ samples.
common for small molecules.
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2019 HPLC Technology
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The physical dimensions of the HPLC column required to achieve the same linear velocity
hardware should also be optimized for the through the column. The major disadvantage
desired separation. Larger associated with smaller
columns are useful in the diameter columns is
scale-up process, but
smaller columns generally
It is best to choose that the sample loading
capacity is reduced.
offer greater sensitivity and
are therefore more useful in
analytical applications.
The column length is
determined by how many
– and what type – of
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the shortest column
possible for your
application.
In summary:
• Larger ID columns (over
10 mm) are mainly used to
purify large-scale amounts
of material because of
compounds you have to their relatively large
separate. Long columns loading capacity.
provide better resolution and sensitivity but
require longer retention times and higher • Analytical scale columns (4.6 mm) are the
pressure. It is best to choose the shortest most common type. They are most often used
column possible for your application, without in quantitative analysis.
affecting resolution.
The internal diameter (ID) of an HPLC • Narrow-bore columns (1–2 mm) are used
column influences both detection sensitivity for applications where more sensitivity is
and separation selectivity (resolution). The required.
most commonly used ID for HPLC columns
is 4.6mm. Small column diameters provide • Capillary columns (under 0.3 mm) are used
higher sensitivity than larger column almost exclusively with alternative types of
diameters for the same injected mass, detection, such as mass spectrometry.
because the concentration of the analyte
in the mobile phase is greater. Smaller Choosing the physical dimensions of your
diameter columns also use less mobile phase column will therefore depend on your
per analysis, because a slower flow rate is application and throughput requirements.
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The number of uses for HPLC is constantly Canabis Testing
growing and, like any other analytical tool, We have seen enormous growth in the
the realms of application are vast. cannabis market, particularly detailed
Current hot topics can cannabinoid profile analysis,
be seen on SelectScience to ensure accurate potency
in the HPLC/UHPLC
section of the website.
Chromatography is testing. This application note
highlights a LC-UV method
Key topics to keep an
eye on over the coming also finding itself for the resolution of 16
cannabinoids, using Restek
months are indicated
below, with some
excellent examples of
their applications.
Industry professionals
highlight glycan mapping
at the heart of
cannabis analysis
laboratories.
“ columns.
In this webinar (Figure 5),
produced in partnership
with Restek, discover
how both HPLC and gas
chromatography are being
and biotherapeutic used to ensure product
applications as two of the quality and consumer safety.
most hotly discussed applications currently.
Read this article to find out how UHPLC has
been used accelerate glycobiology. Other
examples include the use of HPLC for the
characterization of biosimilars and analysis of
mAbs, ADCS and impurities.
We are also seeing increasing interest in
the use of HPLC columns for the analysis of
persistent environmental pollutants, such
as PFOA and PFOS. Find out how scientists
in California have developed systems for
efficient sediment analysis using Kinetex
Core-Shell HPLC/UHPLC Columns from
Figure 5: Discover how HPLC columns are being used in cannabis analysis in
Phenomenex in this article. this webinar
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• Mixed mode stationary phases for multiple retention mechanisms in the same run
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Acknowledgements
• Susan M. Steinike, LC Columns Product Marketing Manager – Restek Corp
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