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Volume 34
Mai 2014
1
separation
science & applications
■■ UHPLC
■■ HPLC
■■ Sample Preparation
■■ Filtration
brating 30 Ye
Cele f Science ars
o
ww
EST. 1983
w.c a s s s.org
Editorial
Method Screening
The project deadline is approaching. Your promising new compound is almost fully characterized. In the lab a brand new UHPLC is running
but no matter what you do, it seems to be impossible to separate these two diastereomers
that keep you from finalizing the report. Have
you ever encountered a separation problem that
was difficult and time-consuming to solve like
this? Although recent developments in separation technologies save costs and time, method
development remains tedious work. Additionally, the increasing complexity of samples vastly
raises the requirements for specific separation
problems. Several considerations can help in
finding the right UHPLC method for a complex
sample, as described by Frank Steiner in his article (p. 16).
In the pharmaceutical industry, research labs
often address these issues by fast and efficient
method screening which evaluates several if not
many different stationary phases to find a suitable separation setup. To achieve economy of
scale, these processes are bundled in a central
institution that supports several different groups
of researchers within the company.
A recent paper proposes the adaption of this
“industrial” approach to “academic” problems
[1]. In this regard Erik L. Regalado from Merck
Research Laboratories and his co-authors asked
academic groups for challenging separation
problems that were slowing down research
progress. By applying state-of-the-art chromatographic separation tools from the pharmaceutical industry they could easily determine suitable
methods and setups for specific problems and
thereby prove the usefulness of their approach
in academic research.
The establishment of a shared infrastructure
with a single separation science lab featuring
several advanced chromatography setups and
providing services to a university or a whole
geographical area may have
several advantages, especially
when it comes to preparative
chromatography. The authors
also believe that such a facility
would benefit the education of
students who are eager to become
separation specialists.
As this field is still growing, there is a
great demand for dedicated separation scientists. However, the question arises if passing
on separation problems to centralized facilities
would really be an advantage for developing
scientists because a major part of the scientific
education still is the promotion of problem-solving skills. Overcoming such problems with innovative ideas is what fosters the creativity of a
researcher and the ability to think outside the
box. You may argue though that service facilities
and automated separation solutions might give
the researchers more time to think of innovations. But still it can be dangerous not to spend
a thought on the problem and to fully rely on
the simple way. It opens the door to the loss of
practical knowledge, which is a great concern
for many companies.
Luckily separation – science & applications
provides you with up-to-date research results,
new applications and practical considerations
so your knowledge stays right at its peak. Enjoy
reading!
Till von Graberg
Managing Editor
References
[1]Regalado E.L. et al.: Org. Biomol. Chem. 12,
2161-2166 (2014)
© Olivier Le Moal - Fotolia.com
separation 1/2014
3
C O N T E N T Content
Analyzing Complex
Samples Faster
page 16
Editorial3
Dr. T. von Graberg
Detection of Gadolinium
20
Contrast Agents in Surface Water and Plants
Magazine
Towards Unbiased DBS Analysis page 24
One Attendee’s Experience
at CE Pharm 2013
6
“Tribal Knowledge” and other Unexpected
Benefits of the U.S. Government Shutdown
on a Scientific Conference
S. Flores, CASSS, USA
News8
Beyond Appropriateness
and Sustainability
page 26
N. Jakubowski et al., BAM Federal Institute
for Materials Research and Testing,
Berlin, Germany
Sample Preparation
Towards Unbiased DBS Analysis
Sample Preparation of Dried Blood Spots
24
B. Ooms, Spark Holland BV, Emmen, The Netherlands
Filtration
Cover Story
Microscale 2D-RP/RP Peptide
Chromatography An Introduction to the Capabilities
of the Acquity UPLC M-Class System
M. A. Lauber, Waters, USA
HPLC
10
Beyond Appropriateness and
Sustainability26
Universal Self-reliance in Water Supply
C. Noubactep, University of Göttingen, Germany
Ion Chromatography
GPC
Comparison of GPC and Mass
Spectrometry12
Possibilities and Limits for the Control
of Protein Stability
N. Thiessen et al., Bingen University, Germany
Good Health without Bad Breath
Extracting Alliin from Garlic
28
J. Evans, JE Science, UK
Literature
Book Reviews
29
UHPLC
Separation is published as part
of a collaboration between
Analyzing Complex Samples Faster
Practical Considerations for Maximizing
Performance and Productivity in UHPLC
F. Steiner, Thermo Fisher Scientific,
Germering, Germany
4
Separation 1/2014
16
Product Section
Products30
Imprint/Index
Inside Back Cover
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Fig. 1: Andras Guttmann receiving the 2013 CE Pharm Waward.
One Attendee’s Experience
at CE Pharm 2013
“Tribal Knowledge” and other Unexpected Benefits of the U.S. Government
Shutdown on a Scientific Conference
Where were you during the U.S. government shutdown of Oct 2013? I’ll always remember where I was. It was CASSS’
CE in the Biotechnology & Pharmaceutical Industries Conference in Crystal City, Virginia – adjacent to Washington DC.
And, I’ll always remember it as one of the best conferences I’ve ever attended. Though the start was dubious, change
and surprise can turn out to be even better than planned. You see, with our proximity to DC we hoped to have a
particular emphasis on regulatory issues and were looking forward to our Keynote speaker from the FDA, Steven
Kozlowski. The Shutdown threw a monkey wrench into our plan, but the need to improvise resulted in our most
collaborative meeting in years.
CE Pharm strives to be a forum to bring Capillary Electrophoresis practitioners together for
the exchange of ideas and collective learning
through each other’s experiences. The hope is
not only that the program will provide invaluable information, but that the forum itself will
bring together individuals with practical needs
or technical problems and through discussion
they will collaboratively find solutions and create partnerships. As a result, CE practitioners of
all skill and experiences levels will return to
their labs with new information to tackle old
problems and new contacts to consult with as
they encounter CE related issues in the future.
Analytical labs, for years, have spoken of the
6
separation 1/2014
value of “tribal knowledge” as it relates to their
smooth operations; CE Pharm strives to take
“tribal knowledge” to a more global level.
While CE Pharm’s end goal is to meet the
needs of the practitioner of capillary electrophoresis in the biopharmaceutical industry, this goal
can only be fully met by bringing together all the
players in the CE world. This includes the academic labs that push the frontiers of CE by innovating instruments designs and functionality,
developing new chemistries and creating applications in new areas; the CE vendors that provide our instruments, software, kits and supplies;
and finally, and equally as important, are the
regulators with whom we must partner as we
implement practical CE methods that meet all
regulatory expectations.
Academic
On the Academic front, CE Pharm 2013 was incredibly fortunate to have Professor Norman
Dovichi as a keynote speaker. Professor Dovichi’s
energetic presentation struck me like electroshock therapy. For years I had been walking into
the lab under the delusion I know what a CE instrument is and will be in the future. Professor
Dovichi amazed the audience with many designs his lab has created over the years to address unmet needs and push the limits of CE
© rabbit75_fot - Fotolia.com
M a g a z i ne M a g a z i ne
Fig. 2: CE Pharm
strives to achieve
collective leraning
though each other‘s
experiences.
technology and applications. Much of this innovation involved tandem CE mass spectrometry.
This emphasis of CE-MS was also common to
the presentations from Yannis Francois (University of Strasbourg) and Rob Haselberg (Vrije University). Dr. Francois spoke of sheathless CE-MS
for the characterization of n-linked glycans of a
monoclonal antibody. Dr. Haselberg compared
the advantages of affinity capillary electrophoresis (ACE) and CE-MS in the characterization of
a “nanobody”.
Regulatory
As mentioned above Steven Kozlowski, M.D
(FDA Director) was unable to give the keynote
speech for our regulatory session due to the
government shutdown. While that seems like a
terrible loss, and it was, we still had a very full
and diverse regulatory session. The session
opened with long time CE Pharm contributor
Michel Girard representing the regulatory body
of Health Canada. The session also included European perspectives from Guy Auclair (European
Commission) and Ahmad Amini (Swedish Medical Products Agency).
Vendors
Beckman-Coulter (Sciex Separations), Protein
Simple, Perkin Elmer, Polymicro Technologies, a
subsidiary of Molex, Prozyme, Inc., Waters Corporation, and CMP Scientific aptly represented
the instrumentation and equipment aspect of
the conference. Perkin Elmer, Beckman-Coulter
and ProteinSimple each provided an informative
‘lunch and learn’ that enlightened participants
to the range of applications their instruments
support. All the vendors displayed their wares
and made themselves available through the entire conference for meaningful discussion with
conference attendees.
Industry
In her keynote presentation, Genentech’s Stacy
Ma, tied together this relationship of Industry
with academic labs, vendors and regulators
from a historical perspective. Dr. Ma, the 2009
CE Pharm Award winner and longtime advocate
of CE, emphasized that partnering with regulatory agencies was a critical step in implementing
CE for biopharmaceuticals.
Anita Szajek from the US Pharmacopeia who
is USP champion of chapter <129> Analytical
Procedures for Recombinant Monoclonal Antibodies presented “Results from the Round Robin Study: Size Variant and CE Methods”. Another
round robin study was presented in a technical
workshop by Drs. András Guttman (University of
Debrecen) and Sung-Ae Suhr Park (Amgen, Inc.)
entitled: “Multisite N-glycan Mapping Study using Orthogonal Methods: CE and UPLC”. These
multisite studies are a unique aspect of the CE
Pharm Conference and this was the third such
study. In the past CE Pharm organizing committee and participants have facilitated multisite
studies investigating the robustness of CE-SDS
and cIEF between labs across the globe and
have then documented the results in peer reviewed journals. The 2013 study of N-glycan
mapping had enlightening results showing
unique selectivities for each method and demonstrating the value of the orthogonal methods.
Another benefit of the government shutdown
turned out to be that the organizing committee
was able to expand the focus on troubleshooting, a particular area in which the committee
strives to enrich conference participants. Kudos
go out to the 2013 participants, never before
have I seen a more open group. The dialogue
was fantastic, people with great insights, ideas
and opinions, but what impressed me most was
the number of times I heard, “Huh, I never
looked at it that way.” That mindset and the
open dialogue overcame me as a co-facilitator
and prompted me to shut my mouth and listen. I
thought to myself, nothing could be better than
this type of dialogue session and conference
feedback seemed to confirm that.
Concluding Remarks
Not only did the troubleshooting session provide
for lively exchange of ideas and experiences, it
was pervasive throughout the meeting. In addition to traditional presentations, the CE Pharm
meeting is designed to foster this type of ‘organic’ discussion, whether at the breaks, poster
sessions, evening reception or dinners. Both
Beckman-Coulter and ProteinSimple invited participants to dinner, as they have traditionally,
and it was at these dinners that I really got to
know a few colleagues from other companies
making friends and important professional contacts. With capillary electrophoresis, in particular, it is so nice to be able to reach out beyond
the confines of my own company and seek advice based in more extensive experience. It is
“tribal knowledge” at its best.
Author
Tim Blanc, ImClone Systems, a wholly-owned subsidiary of Eli Lilly & Company
Contact
Stephanie Flores
Executive Director, CASSS
Emeryville, CA, USA
sflores@casss.org
separation 1/2014
7
N e w s Nanotube Coating Helps Shrink Mass Spectrometers
Nanotechnology is advancing tools likened to Star Trek’s “tricorder” that perform on-the-spot chemical analysis for a range of applications including medical testing, explosives detection and food safety. Researchers found that when
paper used to collect a sample was coated with carbon nanotubes, the voltage
required was 1,000 times reduced, the signal was sharpened and the equipment was able to capture far more delicate molecules. A team of researchers
from Purdue University and the Indian Institute of Technology Madras performed the study, which is detailed in a designated “very important paper” by
the journal Angewandte Chemie.
According to R. Graham Cooks, Purdue’s Henry B. Hass Distinguished Professor
of Chemistry, this is a big step in their efforts to create miniature, handheld
mass spectrometers for the field. Further he says that the dramatic decrease in
power required means a reduction in battery size and cost to perform the experiments. The entire system would become lighter and cheaper, which will
bring it that much closer to being viable for easy, widespread use.
Cooks and Thalappil Pradeep, a professor of chemistry at the Indian Institute of
Technology Madras, Chennai, led the research. Eberhard-Gerstel Prize 2014 Awarded
Jacob Haun, Institute for Energy and Environmental Technology, Duisburg, Germany, has been awarded the 2014 Eberhard–Gerstel Prize by the Working
Group Separation Science of the Section for Analytical Chemistry of the Gesellschaft Deutscher Chemiker (GDCh; German Chemical Society). The award is endowed with 2,500 € and is presented biennially for an outstanding publication
in the field of analytical separation techniques.
Haun is awarded for his article entitled “Online and Splitless NanoLC × CapillaryLC with Quadrupole/Time-of-Flight Mass Spectrometric Detection for Comprehensive Screening Analysis of Complex Samples”, which was published in
Analytical Chemistry in September 2013. The award was presented at the Analytica Conference, Munich, Germany, on April 2, 2014, where Jacob Haun also
gave a presentation.
Award winning article:
Haun J. et al.: Anal. Chem., 2013, 85 (21),
pp 10083–10090. DOI: 10.1021/ac402002m
www.chemistryviews.org
www.gdch.de
www.gerstel.de
Danaher Announces CEO Transition
In April Danaher Corporation announced that Executive Vice President Thomas
P. Joyce, Jr. will succeed H. Lawrence Culp, Jr. as President and Chief Executive
Officer upon Mr. Culp’s retirement on March 1, 2015. Mr. Culp will continue in
an advisory role into the first quarter of 2016. He began his career at Danaher
in 1990 and has been President and Chief Executive Officer since May 2001.
While there is still more he intends to accomplish, he believes this is the right
time to start this transition. Mr. Joyce began his career at the Company in 1989
as a Marketing Project Manager in and has a demonstrated track record of success in a wide range of positions over the past 25 years. He will become just the
fourth CEO in the Company’s 30-year history.
www.danaher.com
A carbon nanotube-coated paper triangle placed on an ionization source
charged by a small battery is held in front of a mass spectrometer. Purdue
University photo/Courtesy of Thalappil Pradeep
Original publication:
Narayanan R. et al.: Angewandte Chemie. Article first published online: 18 MAR
2014. DOI: 10.1002/anie.201311053
www.purdue.edu
Michel Spagnol
Novasep announced the appointment of Michel Spagnol as chairman of the
Supervisory Board, following the departure of Roger-Marc Nicoud, the founder
of the group. Michel Spagnol joined Novasep as its CEO in June 2013. Since
then, he has reinforced the executive management team with the appointments of Christian Thiry as chief financial officer, Thierry Van Nieuwenhove as
president of the Synthesis Business Unit and the promotion of Nadège Laborde
as president of the Industrial Biotech Unit.
www.novasep.com
Pittcon 2014 Editors’ Awards
The Pittcon Editors’ Awards are fast becoming an important event at Pittcon.
For a product to be considered for this award, the requirements are that it be
the first time to be on display at an exposition and must be a functioning instrument. Typically, winning products feature innovations in technology or industrial design or may enable new analytical applications. This year’s winners
are: Gold: Texas Instruments (Dallas, TX) for its DLP NIR scan evaluation module,
Silver: Waters (Milford, MA) for its Acquity QDa, Bronze: AB Sciex (Framingham,
MA) / Beckman Coulter (Brea, California) for their CESI-MS.
Videos on these products may be found here: http://vimeo.com/user12663057
Acquisition
Chromatography specialist Altmann Analytik broadened its product offering by
integrating Dinkelberg, which is a company in the field of general lab supplies.
Dinkelberg analytics has a strong foothold in southern Germany. It was found in
1922 and is reknown for the development and production of water baths, vacuum package testers and other lab equipements used in food laboratories. At
www.dinkelberg.de the customers will still find equipment and consumables
for general laboratory uses. It is expected to develop a common “go-to-market”
by mid 2014.
http://pittcon.org
www.altmann-analytik.de
Partners in Metabolomics
Numerous scientists today use metabolomic approaches to answer the questions posed by their routine work. Often this requires the use of both GCMS and
LCMS platforms to interrogate the bigger picture, which can lead to a potential
data processing bottleneck. To meet these needs, AB Sciex, Genedata, and Leco
have joined forces to offer a collaborative hardware/software bundle, enabling
metabolomics researchers to comprehensively integrate, process, and analyze
experimental data across different GCMS and LCMS platforms.
“Pharmaceutical Analysis”
No other industry is as thoroughly regulated as the pharmaceutical industry.
The active ingredients are legion and so are the standards to test for them.
Metrohm’s new brochure “Pharmaceutical analysis” is a valuable compendium
to get an overall view of the most important parameters determining the quality of pharmaceuticals and the methods best suited to check them. Methods
described include near-infrared spectroscopy (NIRS), potentiometric, colorimetric and Karl Fischer titration, ion chromatography, stability measurement,
voltammetry, atline and online process analysis.
http://uk.leco-europe.com
http://pharma.metrohm.com
8
separation 1/2014
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Cover story Microscale 2D-RP/RP
Peptide Chromatography
An Introduction to the Capabilities of the Acquity UPLC M-Class System and Columns
This article describes the performance capabilities and reproducibility of microscale 2D-RP/RP peptide
chromatography
with
an
Acquity UPLC M-Class System and
Column.
Microscale LC-MS methods have been used extensively in the field of proteomics. Recently,
these techniques have become increasingly attractive as orthogonal methods alongside immunoassays for the analysis of host cell protein
impurities in biotherapeutics products. Narrow
(300 μm ID) columns can be employed in these
and other such applications as a means to derive an abundance of information from a relatively minimal amount of sample. Obtaining
high peak capacity peptide separations is desirable in such work, because better separation efficiency means trace analytes can be more easily
resolved.
This can be achieved through multidimensional chromatography wherein the combination of orthogonal separations results in greater
resolving power. Two-dimensional reversedphase (2D-RP/RP) chromatography is a unique
example. The utility of 2D-RP/RP involving high
pH fractionation with a highly stable, organosilica hybrid stationary phase (BEH Technology)
followed by gradient separation on a sub-2-μm
particle analytical column has been reported
previously [see references at the end of this article].
Here, we demonstrate that 2D-RP/RP chromatography operated at ≥680 bar with an
­Acquity UPLC M-Class and the related 300 μm
analytical column can be a robust solution for
obtaining high peak capacity. It will be shown
that such a system affords high resolving power
and outstanding chromatographic reproducibility and performance with extended use.
Setup
A tryptic digest mixture derived from 4 different
proteins (Massprep Digestion Standard Mix 1,
p/n 186002865) was studied by 2D-RP/RP using
the configuration outlined in Figure 2A. Peptides
from 400 fmoles of sample were separated by a
10
separation 1/2014
Fig. 1: Acquity UPLC
M-Class System and
Columns
linear gradient at a flow rate of 8 μl/min (689
bar) using an Acquity UPLC M-Class and detected by ESI-MS with a micro-probe outfitted SYNAPT G2-S at a resolution of ca. 20,000. Figure 2B
presents base peak intensity chromatograms
typical of this analytical strategy, wherein a
5-step fractionation was combined with bidirectional flow trapping and a 30 min 2nd dimension gradient with an HSS T3 column (300 μm x
150 mm).
Theoretical Peak Capacity
and Reproducibility
Based on peak widths observed in the extracted
ion chromatograms of 15 different peptides in
this mixture (Figure 3A), the average peak capacity (10% peak height) for the 2nd dimension
separations was estimated to be 277. As these
analyses were completed with 5 step fractionation, the multiplicative resolving power of the
two dimensions indicates that the apparatus is
capable of producing a theoretical peak capacity
of 1385. This demonstrates that remarkably high
peak capacity peptide separations can be
achieved by means of 2D-RP/RP with this system.
The reproducibility of this chromatography
proved to be equally impressive. In studying the
performance of three different column sets, it
was found that from 6 replicate analyses the retention times of the 15 monitored peptides
could be reported with a standard deviation of
≤0.11 min. Moreover, the largest difference in
average retention time between the column sets
for a particular peptide was only 3.2%, and the
peak capacities obtained with each column set
were in agreement to within 20%.
Checking the Lifetime
Lifetime testing of this 2D-RP/RP application
also revealed the chromatography to be robust.
Performance under extended use was investigated by effectively subjecting the columns to
over 100 2D analyses. To expedite this life-time
study, full gradient elution of the 2nd dimension
column was only performed for 2 of every 20
Cover story
cycles. This accelerated the aging of the high pH
1st dimension column as well as the trapping
column, which is expected to represent the predominant mode of failure in the system. Data
obtained in this manner for runs 1 to 121 are
displayed in Figure 3B. Notably, the 2D-RP/RP
method exhibited consistent peak capacities
throughout the lifetime study (variation in peak
capacity ≤5%), an average retention shift of a
thousandth of a minute per run (0.1 min for 100
runs), and an average system pressure ranging
from 9,500 psi to 11,100 psi, well below the
15,000 psi maximum operating pressure of the
Acquity UPLC M-Class System.
Summary
Two dimensional RP comprised of high pH fractionation with a highly stable, organo-silica hybrid stationary phase (BEH Technology) followed
by gradient separation on a sub-2-μm particle
analytical column is a superb example of 2D
chromatography. Its utility is expanded upon
here, with an Acquity UPLC M-Class System and
the related 300 μm ID analytical column. Not
only is the resolving power of such a system
noteworthy, so too is its reproducibility and robustness, which has been evaluated through
analysis of multiple column sets and accelerated
lifetime testing. Two dimensional RP with this
system holds significant promise as a means to
reliably generate the high peak capacity separations needed to study complex peptide samples,
such as those faced during host cell protein
analysis.
Fig. 2: Microscale two dimensional reversed phase (2D-RP/RP). (A) Fluidics configuration. (B) Base peak
intensity chromatograms of Massprep Digestion Standard Mix 1 as obtained using an Acquity UPLC MClass, Synapt G2-S, 5 high pH fractionation steps, Acquity UPLC M-Class HSS T3, 300 μm x 150 mm Column, and a sample load of 400 fmoles.
References
[1] Gilar M. et al.: Anal Chem 77 (19), 6426-34. (2005)
[2] Gilar M. et al.: J Sep Sci 28 (14), 1694-703 (2005)
[3] Doneanu C. E. et al.: MAbs 4 (1), 24-44 (2012)
Authors
Matthew A Lauber, Stephan M Koza, and Kenneth J
Fountain, Waters Corporation
Contact
Matthew A. Lauber
Waters Corporation
Tel.: +1 508 482 3017
matthew_lauber@waters.com
www.waters.com
Waters, The Science of What‘s Possible, Acquity UPLC,
Acquity, Synapt Symmetry, and XBridge are registered
trademarks of Waters Corporation. Massprep and BEH
Technology are trademarks of Waters Corporation.
Fig. 3: Chromatographic performance testing results. (A) Extracted ion chromatograms of the 15
different peptides monitored in the analysis.
(B) Metrics monitored during lifetime testing of
a microscale 2D-RP apparatus. System pressure
was measured after 3 min into the 2nd dimension gradient and averaged for each of the 5 runs
in a full 2D-RP/RP cycle.
▶
separation 1/2014
11
G P C Comparison of GPC and
Mass Spectrometry
Possibilities and Limits for the Control of Protein Stability
©
Gel permeation chromatography (GPC) is often used for the chromatographic
characterisation of protein formulations. GPC is simple to use and detects
high molecular weight contaminants (e.g. aggregates) and low molecular
weight degradation products. Here, GPC is used for the stability examination
of haemoglobin preparations. This article compares two common GPC columns
and considers their efficiency by means of mass spectrometry investigations.
Up to now, a validated method based on the
Tosoh TSKgel SuperSW2000 GPC column has
been used for these measurements. The present
measurements are intended to investigate
whether the method can be transferred to the
Agilent Bio SEC-3 GPC column and whether the
measurements can be continued with this new
column. According to information from the manufacturer, the stationary phase features a coated
silica gel, for which a different stability can be
expected.
The investigations present a detailed comparison with regard to the linearity, detection
limit, reproducibility and selectivity of the two
GPC columns.
In addition, the results are compared with
MS examinations, which have a considerably
greater separation performance / selectivity.
With this, the importance (correctness) of GPC
for the investigation of protein stability is to be
evaluated.
12
separation 1/2014
so pronounced as with the TSKgel SuperSW2000
column. An outlier at the 9th measurement
(15.97 min.) with the Bio SEC-3 column was not
taken into account in the comparison.
Selectivity
The GPC is to detect the formation of higher molecular weight oligomers or low molecular weight
degradation products. For the evaluation of the
two systems, the same samples were analyzed
and compared with the two columns. The Bio
SEC-3 column showed a different differentiation
Stability of the Elution
Statistical evaluation of the retention showed
good reproducibility for both columns (Srel <
0.1%, see Table 1). However, it was established
that retention of the main component increases
significantly if the concentration is lower (from
15.56 min with 1000 µg/ml to 16.15 min with
5 µg/ml). At present this cannot be explained.
Because of this, samples with a constant, low
concentration have to be used.
For a detailed comparison, the retention
times or peak heights of the individual measurements are compared graphically in Figure 1. Examination of the peak areas of the main components showed no significant trend for the two
columns and this is therefore not included in the
graphic evaluation.
The Bio SEC-3 column also required several
measurements in order to achieve a constant retention or peak height. However, the trend is not
1-
ign
es
d
ps
om
a.c
oli
t
Fo
Bio SEC-3
TSKgel
SuperSW2000
Outlier
1
1
Trend
No
Yes
Skew / Kurtosis
not
significant
not
significant
Srel [%]
0.06
0.05
Outlier
None
1
Trend
No
No
Skew / Kurtosis
not
significant
not
significant
Srel [%]
0.31
0.37
Retention
Area
Table 1: Reproducibility of retention / area
(TSKgel / Bio SEC column)
Thinking Forward.
GPC/SEC
Theory or practice?
If there is
one thing
we can do,
it’s both.
PSS are world leaders in macromolecular characterization and have the expertise to help you with your
analysis requirements. We offer a range of products
and services from instruction courses and training thru
contract analysis, consulting, method development
and qualification services all the way up to supplying
turnkey GPC/SEC and LC/2D systems.
All this comes with the personal and direct support
of our dedicated team of innovative and pioneering
specialists. Is there any better way of achieving your
analysis goals?
Driving GPC/SEC forward
Phone + 49 6131 962390
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G P C of the high molecular weight component. The low
molecular weight contamination can also not be
detected in the expected way.
Fig. 2 shows the separation of the same haemoglobin batch for various storage conditions.
This was also measured with the two columns.
With the Bio SEC-3 column, the high molecular
weight component can only be differentiated
from the main peak with storage at 35 °C. With
the other two samples, the high molecular weight
component cannot be definitely differentiated
from the main peak and therefore cannot at present be calculated as a percentage of the main
component using the TSKgel column. In contrast
to this (Fig. 2b) the high molecular weight component can be explicitly determined for all 3 samples using the TSKgel Super SW2000 column.
Reproducibility
Essential statistical parameters were calculated
for a series of 12 measurements (Table 1).
The results for the reproducibility do not
show a significant difference between the two
columns. The only conspicuous feature is the
proof of the retention time trend of the TSKgel
SuperSW2000, which has already been described above, occurs with the first measurements before a constant elution is achieved in
the course of subsequent measurements.
Fig. 1: Comparison graph of retention time and peak height (TSKgel/Bio SEC)
Linearity
By dilution of a standard haemoglobin preparation
(1000 µg/ml) 5 further calibrators were obtained
(5, 10, 50, 100, 500 µg/ml). In addition, the blank
buffer injections were used as a 0 µg/ml calibrator.
a)
b)
Fig. 2: Comparison of storage stability at 7 °C, 25 °C, 35 °C with a) Bio SEC- b) TSKgel column
14
separation 1/2014
After linear regression verified, by variance
analysis, the straight line was rejected as the calibration model for the Bio SEC column; the 2nd degree polynomial was accepted for the entire calibration range. In the lower concentration range
from 0-10 µg/ml a straight line was accepted.
G P C
For the Super SW column, both the straight
line and the 2nd degree polynomial model were
rejected. A straight calibration line could only
be used in the range 10 – 1000 µg/ml (without
the 0 µg/ml calibrator).
From the calibration data follows that in the
range from 0 to 100% the peak area is not
based on a straight correlation with the concentration of the components; however in the range
from 0-1%, so that the detection limit can be
calculated with a straight line model.
Detection Limit
From the registration of the 1st calibrator, the detection limit of the Bio SEC column can be estimated as 0.5 µg/ml (signal/noise = 3:1, confirmed linearity in the range < 10 µg/ml). For the
Super SW column this is 1µg/ml.
Recovery
Recovery was determined by means of 2-dimensional HPLC. In the GPC measurement of 2 different proteins, the main peak of the sample
was passed to a reversed phase cartridge (heartcut) during elution of the main signal and quantified via a reversed phase chromatography
(RPC) column by means of UV detection. Direct
RPC measurement of the protein sample was
used as a 100% reference. The two columns do
not show any serious differences in terms of recovery. The Bio SEC column appears to have a
slightly better recovery due to its coating (Table
2).
Separation Efficiency
Separation was illustrated on the basis of chromatograms of two proteins (ribonuclease A and
haemoglobin) with new columns, and is depicted in Table 3.
The equivalent theoretical plate heights of
the two columns are comparable in spite of
Bio SEC-3 Recovery [%]
SuperSW2000 Recovery [%]
Ribonuclease A
121
110
Haemoglobin
93
75
Table 2: Recovery of haemoglobin / Ribonuclease A (TSKgel / Bio SEC column)
Bio SEC-3
TSKgel SuperSW2000
1.501
0.962
Ribonuclease A
Symmetry ¹
Height equivalent of one theo- 43
retical plate [µm] ²
33
Haemoglobin
Symmetry ¹
1.519
0.876
Height equivalent of one theo- 80
retical plate [µm] ²
97
Table 3: Symmetry / Peak capacity heamoglobin / Ribonuclease A (TSKgel / Bio SEC column)
(¹ USP-Tailing, ² Height equivalent to one theoretical plate according to the tangent method)
different particle sizes (Bio SEC-3: 3 µm, TSKgel: 4 µm).
Accuracy
Alternatively to GPC, mass spectrometry was
used to check the protein stability (ESI-TOFMS with online desalination without HPLC
separation). In the mass spectrum (Fig. 3), in
addition to the two subunits of haemoglobin
(m/z = 15037.68 and 16033.56) a further mass
was detected in the relevant range (m/z =
16195.61) The concentration of this component increases with longer incubation periods,
especially at higher temperatures. It is assumed that in this case there is an adduct with
a low molecular weight component of the
pharmaceutical formulation.
This component could not be detected with
GPC. In addition, several masses within the
Masse
Intensität [%]
2460,27
19
2503,29
22
2531,50
20
2675,43
35
3562,88
19
3690,99
18
15037,63
82
16033,56
85
16195,61
27
range 2500-5000 m/z (see mass list Table 4) are
not differentiated in GPC.
The correctness of GPC analyses of protein
stability must therefore be regarded critically.
Conclusion
In general, it must be taken into consideration that
the signal area percentages of the GPC measurement do not allow any conclusions to the present
concentrations due to the (usually unknown) absorption coefficients. The GPC analysis with the
Bio SEC-3 column did not show any clear difference with regard to the detection limit and the reproducibility. There are also no significant differences according to linearity and recovery.
The selectivity of the elution is a different
matter. In contrast to the TSKgel column, with
the Bio SEC column shortly before and after the
main peak no or only poorly separated components (high or low molecular weight) elute,
which could be assessed as indicators of protein
degradation. Therefore, the TSKgel column is
preferable for this task.
Regardless of this, it must not be ignored
that the separating performance of GPC is very
low (peak capacity ~10). Therefore it cannot be
expected that all of the protein components will
be detected after storage. This is impressively
demonstrated by the MS measurements. An assessment of protein stability merely on the basis
of GPC examinations must therefore be rejected.
Authors
N. Thiessen, M. Müller, E. Reh
Contact
Fig. 3: Mass spectrum of haemoglobin formulation after maximum entropy deconvolution.
Dipl.-Ing. Michael Müller
Center of Proteinanalysis
University of Applied Sciences, Bingen
Tel.: +49 6721 409-362
muellermicha@fh-bingen.de
separation 1/2014
15
U H P L C Analyzing Complex Samples Faster
Practical Considerations for Maximizing Performance and Productivity in UHPLC
All progress in HPLC goes along with column development, but only its combination with appropriate instrument technology and software enables true
user value. Understanding fundamental relationships is a pre-requisite to
take full advantage of related potentials, which may even enable workflows
with fewer stages and less operator interference, like e.g., less sample preparation. Column and instrument technology progress combined with the use of
appropriate detection technology and powerful software support can tremendously fuel benefits for user workflows.
16
separation 1/2014
Column Technology and Instrument
Capabilities in UHPLC
With the advent of UHPLC, smaller particle diameters packed into columns of smaller dimensions were introduced [1]. Shorter columns with similar or higher plate counts
operated at higher mobile phase velocities
nowadays account for significantly faster analyses. At the same time the column diameter
reduction from typically 4.6 mm to 2.1 mm allows fast eluent velocities at even lower flow
rates than in conventional HPLC. A benefit
next to higher analysis speed is reduced solvent consumption, better compatibility to
mass spectrometric detection, and greater
amount sensitivity in optical detection. All this
could be further improved with the use of 1
mm columns or capillary columns. To take full
advantage of particle and column miniaturization UHPLC instruments must meet these requirements:
UHPLC
Table 1: Example for the attempt to improve resolution through efficiency increase when transferring from HPLC to
UHPLC. All numbers are theoretically modeled on the basis of the artificial method #1 (*method #2 is not feasible in
practice).
Method
dp / µm
LColumn / mm
F / ml / min
N / plates
tAnalysis / min
Rs
Δp / bar
#1
5 FullyPorous
100
0.25
6,000
10
1.0
100
#2*
1.7 FullyPorous
100
0.75
18,000
3.3
1.7
2,700
#3
1.7 FullyPorous
50
0.625
9,000
2
1.2
1,125
#4
2.5 SolidCore
250
0.625
~45,000
10
2.5
1,000
#5
2.5 SolidCore
150
0.625
~30,000
6.7
2.0
670
Smaller particles run at higher
linear velocities (required due to
van Deemter characteristics) demand significantly higher operation pressures. Rule of thumb: operational pressure increases with
the same factor as the speed of
analysis at equivalent peak resolution, from HPLC to UHPLC typically
5 to 10-fold.
Highly efficient and smaller volume columns require smaller injection volumes to avoid overloading.
At constant column plate number,
the maximum injection volume decreases by the same factor as column volume, typically 10- to 15fold for UHPLC on 2.1 mm columns.
This increases amount sensitivity
by the same factor.
Significantly higher frictional
heating in columns can change the
effective column temperature relative to the set value, and it can induce radial temperature gradients
that affect separation efficiency.
While adverse efficiency effects are
less pronounced with narrow bore
columns, this issue needs to be addressed by the thermostatting
technology.
Highly efficient small volume
columns generate smaller peak
volumes which must be considered
for all system extra column volumes including detector flow cells.
The corresponding peak volume
decreases with the column volume
factor (10- to 15-fold for 2.1 mm
columns). This can be a challenge
with optical detectors where light
path reduction is unwanted, because of signal reduction due to
Beer’s law.
The detector must record transient signals with a steep slope
and a narrow base width (changes
with factor of analysis speed increase). These parameters typically
change by factor 10, but up to 50
when using extreme linear velocities on short columns.
These system requirements are
mandatory to fully translate UHPLC
column performance into user benefit. Obviously all system volume requirements (gradient dwell volume,
injection volume, tubing volume,
flow cell volume) become challenging to adapt, especially when column diameters below 2.1 mm are
applied.
Importance of Retention
Time Stability and System
Technology
The primary indicator for compound identification in chromatography is retention time. Better column efficiencies enable more
peaks in shorter time and the requirements on retention time precision to unequivocally identify analytes increase accordingly. Even
with highly selective detection devices like tandem mass spectrometry, precise control of retention
times is needed to correctly schedule ion transitions. Only a perfect
flow and gradient control combined with appropriate column
thermostatting truly accounts for
this. Effects resulting from noncompressed liquid in the sample
loop at high operating pressure
must also be considered.
creased by the factor of the particle
size decrease (to maintain efficiency) this approach induces a tremendous pressure increase. At constant
column length the pressure increases with the 3rd power of the particle
size reduction, while N and the
analysis speed increase linearly. The
characteristic figures of this method transfer are shown in Table 1,
methods #1 and #2. We consider
method #1 operating a typical conventional column that generates a
back pressure of 100 bar (for the
sake of simplicity), an analysis time
of 10 minutes and a poorly resolved
critical peak pair with Rs=1.0. From
method #2 we see that the constant column length approach for a
5 µm to 1.7 µm transfer yields
3-fold speed-up (not required!),
1.7-fold resolution boost (RS increases with square root of N) at
the price of 27-fold pressure increase. As the 2,700 bar are in no
way feasible on commercial instrumentation, the only resolution is to
shorten column length and slightly
decrease flow rate (method #3).
While this brings the pressure into
operational range (~ 1,100 bar),
the related plate number increase
only translates into Rs=1.2, still insufficient for practical requirements.
Let us now look at an approach
with superficially porous particles
[2] (2.5 µm for simplicity) run at 2.5
times the mobile phase velocity and
the fair assumption of similar efficiency increase as with a 1.7 µm totally porous particles. As speed-up is
not the goal, we increase the column length to 250 mm by the same
factor as we increase the eluent velocity. This results in method #4 and
yields Rs=2.5 (more than required)
with the same analysis time as
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New Methods: Column Efficiency or Column Selectivity?
Speed-up by shortening column
length is only one user benefit in
UHPLC. To increase plate number N,
columns can also be kept at equivalent length when reducing stationary phase particle size. Chromatographers may want to do this when
they have poorly resolved peaks in
their original method, but do not
know how to improve selectivity
(relative analyte retention). Let us
see how this looks in practice. As
the linear velocity still has to be in-
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U H P L C method #1 and still at reasonable column back
pressure (1,000 bar). Moving on to method #5 finally yields the perfect resolution (Rs=2.0) combined with a nice 33% speed-up in the pressure
comfort zone of UHPLC (670 bar).
These observations allow for three principal
conclusions:
▪▪ It is much more effective to tune selectivity
to achieve resolution improvements in LC.
▪▪ If resolution increase and not speed increase
is the goal, go for a moderate particle size
reduction and use the required increase of
mobile phase velocity to run a longer column at similar analysis time.
▪▪ As superficially porous particles with diameters between 2.5 µm and 3 µm can provide
similar efficiency like fully porous sub- 2 µm
ones, they offer a great potential for kinetical resolution boost.
The question whether selectivity or efficiency is
the better choice must be answered in favor of
selectivity, though UHPLC potentials may encourage the opposite.
Fig. 1: Overlaid chromatograms for the separation of green tea extract on Thermo Scientific Accucore
XL HPLC column chains of 300 mm and 450 mm lengths. Peak resolution and peak capacity increase by
22% with 50% column length increase following theory.
Fig. 2: Comparison of column chains from 300 mm of 1.9 µm totally porous packings (Thermo Scientific
Hypersil Gold HPLC columns) and 450 mm of 2.6 µm superficially porous packings (Accucore HPLC columns) for the analysis of Traditional Chinese Medicine extracts [7].
18
separation 1/2014
How to Leverage UHPLC Potentials to
Deal with Truly Complex Samples?
While the importance of selectivity for peak resolution became obvious, there are scenarios
where selectivity increase at a distinct part of
the chromatogram comes at the expense of another part of the chromatogram. If a high number of substances must be separated in complex
samples, this issue is inherent. Any chromatographic method can be assessed for a theoretical number of compounds it can separate at
best. This is done by the so-called theoretical
peak capacity and considers perfectly equidistant retention at a resolution of 1.0. Peak capacity nC increases with the square root of N and is
best in shallow gradients with the widest possible elution window. In such methods with gradient volumes of more than 30 times the column
void volume, theoretical peak capacities of twice
the square root of N can be obtained. To achieve
a peak capacity of 1000, a column needs to generate 250,000 plates. Even a well packed sub-2
µm phase requires a column length above 700
mm for this efficiency. At a feasible mobile phase
viscosity and with appropriate linear velocity it
will not be possible to operate such columns
within a 1,200 bar pressure restriction. Packings
with excellent efficiency but better permeability
are superior. Superficially porous materials with
diameters below 5 µm fulfill this and with zero
dead volume column couplers they allow to run
500 mm or higher column lengths easily [3]. Figure 1 shows the potential of 4 µm solid core column chains for the analysis of green tea. With
extending the column by a factor of 1.5 and
adopting the gradient, the resolution can be increased by more than 20% and even complex
samples can be resolved. Operating a 500 mm
column length of a 2.6 µm solid core stationary
phase with 5 mm/s at pressures not significantly
above 1,000 bar is feasible. Figure 2 shows the
application of 300 mm 1.9 µm totally porous
versus 450 mm 2.6 µm solid core column chains
for the analysis of Traditional Chinese Medicine
(TCM) samples, both running at 1,200 bar pressure. While the 1.9 µm particles yield the better
production rate of 17 peaks/minute, the 2.6 µm
solid core method with 14 peaks/minute
achieves 25% higher peak capacity thanks to
the extended column length.
Peak capacity optimization in combination
with adequate detection techniques is a crucial requirement for successful analysis of
complex samples. Such samples are often
found in natural substance analysis, e.g., in
TCM, or even simple tea extracts. Unlike proteomics analysis workflows where samples
can be extremely complex but are chemically
quite homogenous, natural substances [4] can
contain compounds from tens of different
chemical classes. On the detection side the
two key requirements are compound identification and quantification. While identification
is normally based on MS/MS techniques or MS
providing high resolution accurate mass,
UHPLC
quantification remains challenging. Ideally, a
close-to-universal technology can be applied,
with no need for individual calibration.
Charged aerosol detection (CAD) [5] in combination with inverse gradient solvent effect
compensation [6] comes closest to this. An important prerequisite is a strong software support to tune the method and calibration functions for universal quantification and to
translate very data rich chromatograms adequately into analytical answers.
Helpful to Know
for Best Success in UHPLC
© javier brosch/Fotolia.com
UHPLC can deliver more theoretical plates in
shorter time than HPLC and has potential to
save solvent and sample thanks to column miniaturization. Method speed up can be achieved
with the same factor as pressure increase. Kinetic peak resolution increase of only 50% is accompanied by a 5-fold pressure increase, so selectivity improvement is the better way to
increase resolution. For truly complex samples
peak capacity has to be maximized which can
be carried out most effectively in regard to the
pressure by using 2.5 to 4 µm superficially porous particles in long columns or column chains.
Method transfer between systems is another
challenge which is discussed in the extended
electronic version of this article which can be
found at http://bit.ly/Steiner1.
References
[1] Meyer V.R.: G.I.T. Laboratory Journal 17, 13-15
(2013)
[2] Gritti F.: Chromatography Today 5, 4-11 (2012)
[3] Eeltink S. et al.: J. Sep. Sci. 33, 2629-2635 (2010)
[4] Koehn F.E and Carter G.T.: Nature Reviews Drug
Discovery 4, 206-220 (2005)
[5] Hutchinson J.P. et al.: J. Chromatogr. A 1217,
7418-7427 (2010)
[6] Górecki T. et al.: Anal. Chem. 78, 3186-3192
(2006)
[7] Heidorn M. et al.: Dionex Application Note, http://
bit.ly/Steiner2
Contact
Dr. Frank Steiner
Manager, HPLC Solutions Marketing
Thermo Fisher Scientific
Germering/Germany
frank.steiner@thermofisher.com
Complete electronic version:
http://bit.ly/Steiner1
enter the world
of science
AL.com
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R
U
O
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Y
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O
T
A
www.LABOR
www.gitverlag.com
H P L C Detection of Gadolinium
Contrast Agents in Surface Water and Plants
For years an increased concentration of gadolinium has been observed in the environment. This can
be traced back to its use in medicine, as gadolinium has been used
for about 25 years in hospitals as a
contrast agent for magnetic resonance imaging (MRI).
Toxic to Humans
In order to protect patients from the toxic effects of the free gadolinium ion, the metal is
bound and administered as a highly stable, less
toxic polyaminocarboxylate-chelate complex [1]
Fig. 1 shows some typical Gd-based contrast
agents. The toxic effect of free gadolinium is
based on the fact that gadolinium is a competitor of calcium and can therefore block cellular
processes in the organism [2].
Most of the administered gadolinium complex is excreted by the patient a few hours after
the examination without any serious side effects
[3]. Because of this, these contrast agents are
considered to be harmless. However, it has been
known for some time that in rare cases the intake of gadolinium-based contrast agents may
cause NSF (Nephrogenic Systemic Fibrosis). NSF
is a disorder with symptoms such as irreversible
hardening of the skin and organs, which may be
fatal [4]. This primarily applies to patients suffering from kidney damage. It is suspected that
through the intake of preparations containing
iron, the transmetallation process in the body is
accentuated and toxic gadolinium is released. In
case of renal failure, excretion of the contrast
agent is delayed, so that a longer retention of
the complex in the organism could lead to increased transmetallation and decomposition of
the gadolinium chelate [5, 6].
Toxic to the Environment
As the gadolinium-based contrast agent is not
collected in hospitals after excretion and there is
20
separation 1/2014
no adequate purification of the waste water by
the sewage treatment plants, Gd-based contrast
agents can be detected in rivers and lakes, in
some cases in the range of µg l-1. An increased
concentration of gadolinium in surface water
was first recorded by Bau and Dulksi in 1996 [7]
and was described as a gadolinium anomaly. It
can be found everywhere where gadoliniumbased contrast agents are used in hospitals and
clinics [8 – 14].
It is estimated that annually, 1,100 kg of
gadolinium complexes are released into the environment in Germany [10,15]. In a baseline
study it could be demonstrated by means of
measurements in a sewage treatment plant,
that only about 10 % of the contrast agent is
decomposed or retained during the sewage
treatment process, while the remainder enters
the surface water unchanged. Little is known
about the quantity of this input and the whereabouts of the contrast agent in the environment.
Analysis
The determination of the concentration of an element does not present a challenge to analysis;
however this is not the case with the analysis of
the species of bound gadolinium in the various
contrast agents. Due to the very low concentrations of Gd compounds in environmental samples, mass spectroscopy with inductively coupled plasma (ICP-MS) is a suitable choice for
analysis. This method is characterized by the
very low detection limits (sub-ng/l), which are
necessary for this investigation. The especially
large linear dynamic measurement range and
simple preparation of the samples as well as
calibration by means of liquid standards are
other convincing features. As in the plasma
(5,000 – 10,000 K) of ICP-MS information about
the species is completely lost due to atomization
and ionization of the compounds, leaving only
information about the elements, high performance liquid chromatography (HPLC) is used for
the analysis of the species. Among the various
HPLC methods in particular hydrophilic interaction chromatography (HILIC) has proved to be
effective for these investigations due to its high
separation performance. In this case, ICP-MS is
used as a highly sensitive HPLC gadolinium detector. With the aid of HILIC, above all hydrophilic, polar species such as Gd-based contrast
agents can be separated, for which the usual
Reverse-Phase (RP-HPLC has proved to be inadequate [17 – 19]). However, a disadvantage of
HILIC with ICP-MS is the high input of organic
solvents, which may cause the formation of carbon and carbon deposits due to the combustion
of the solvent in ICP-MS. Because of this, in spite
of a low flow rate of only 150 µl min-1 in HPLC,
oxygen must be added as an auxiliary gas in order to burn the excess carbon.
Gd in Surface Water
The section of the Teltow Canal near to Stahnsdorf was selected as a suitable model system for
a surface water, as the waste water from Berlin
Joanna Simpson
comfortable with on the
spot sampling at home
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BETTER SAMPLE CARE
minutes, providing maximum sensitivity without any manual intervention.
H P L C is discharged into this canal and the quantities
of water are well recorded. For the analysis,
samples of the surface water were taken over a
distance of 5 kilometers downstream of the inlet
point of a sewage treatment plant in Stahnsdorf.
Over this distance there are no further inflows
into the canal. The sewage treatment plant accepts the waste water from several clinics, so
that a high input of Gd contrast agents could be
expected. This was confirmed by means of concentration measurements using ICP-MS. The Gd
input at the sewage treatment plant discharge
point was approx. 990 ng l-1 on the date of sampling. Within the first kilometer, the Gd concentration reduced rapidly and then remained constant at 99 ± 16 ng l-1 over the further course of
4 km. Mathematically this reduction in concentration can be explained simply by the dilution
of the water from the sewage treatment plant
by the water in the Teltow Canal. Figure 3 shows
a typical HPLC-ICP-MS chromatogram from the
surface water of the Teltow Canal. In this sample, the two contrast agents Dotarem and Gadovist (see Fig. 1) were detected.
Bioaccumulation
The very high concentrations of gadolinium
measured in the Teltow Canal and the gadolinium-based contrast agents which were identified
in this inevitably give rise to the question as to
whether the contrast agent can enter the food
chain or can be taken in by plants, fish or other
organisms, or possibly can even be accumulated.
To investigate this question an examination using the model of cress plants (Lepidium sativum)
Fig. 2: Surface water, Teltow Canal in Berlin 22
separation 1/2014
Fig. 1: Chemical structures of frequently used Gd-based magnetic resonance imaging contrast agents
was carried out to determine whether gadolini
um-based contrast agents can be absorbed via
the root system. For this, the irrigation water of
the plants was dosed with various contrast
agents for several days (3 and 5). Subsequently
the plants were decomposed and the gadolinium concentration was determined using ICP-MS
(plasma mass spectrometry). With this it could
be confirmed that there is absorption of contrast
agents by plants and that the root system does
not block the intake. The concentrations which
were found in the leaves corresponded to the
concentration in the irrigation water. Interestingly, the concentration in the roots and stems
of the plants was considerably lower (Factor
5 – 10). The same plants were also subjected to
a gentle aqueous extraction process in which
the species of contrast agent are retained. With
© Haldir
essential questions with regard to
the whereabouts, decomposition
or deposition, transport and input
or accumulation in the biosphere
still remain unanswered. In our
opinion there is a need for action
and with the highly sensitive HPLCICP-MS we have the necessary tool
to pursue these questions.
Further literature is available from the
authors.
Literature
Fig. 3: HPLC Chromatogram (of the mass / charge of 158Gd) of surface water
from the Teltow Canal (Samples taken from the discharge point of the
Stahnsdorf sewage treatment plant) – dominant contrast agents Dotarem
(Gd-DOTA) and Gadovist (Gd-BT-DO3A)
the aid of the HPLC-ICP-MS method described above, all of the contrast agents used could be found in
the extracts. This means that the
plants completely absorb the contrast agents (without metabolisation or decomposition).
Summary
These investigations of environmental samples provide a first insight, which is intended to show
how easily these contrast agents
can enter the food chain. However,
[7] Bau M.und Dulski P.: Earth and
Planetary Science Letters 143 245255 (1996)
[8] Elbaz-Poulichet F. et al.: Water Research 36 1102-1105 (2002)
[9] Hennebrüder K. et al.: Talanta 63
309-316 (2004)
[10]Knappe A. et al.: Chemie der Erde
[Soil Chemistry] 65 167-189
(2005)
[1] Weinmann H. J. et al.: American
Journal of Roentgenology 142
619–624 (1984)
[2] Darrah T. H. et al.: Metallomics 1
479-488 (2009)
[3] Guggemos D. B. (2005): Tübinger
Ergebnisse Dissertation. EberhardKarls-Universität zu Tübingen
[Tübingen results dissertation. Eberhard-Karls University, Tübingen]
[4] Marckmann P. et al.: Journal of the
American Society of Nephrology
17 2359-2362 (2006)
[5] Idée J.-M. et al.: Fundamental &
Clinical Pharmacology 20 563-576
(2006)
[6] Kunnemeyer J. et al.: Analytical
Chemistry 81 3600–3607 (2009)
Authors
Uwe Lindner, Jana Lingott, Norbert
Jakubowski,
Ulrich Panne, BAM Federal Institute for
Materials Research and Testing
Contact
Norbert Jakubowski
BAM Federal Institute for Materials
Research and Testing
Department of Inorganic Trace Analysis
Berlin, Germany
norbert.jakubowski@bam.de
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www.gilson.com/spotprep
HIGH
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Gilder Prep Software
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S a mp l e P r ep a r a t i o n Towards Unbiased DBS Analysis
Sample Preparation of Dried Blood Spots
© Digipic
- Fotolia.c
om
Variation of the blood hematocrit (Ht) level still is a serious issue for analyte
quantitation in dried blood spot (DBS) analysis [1, 2]. It influences spot size and
consequently causes deviations in the sample aliquot when only a part of the
spot is analyzed. The most obvious approach to solve this problem is using the
entire spot for analysis. A multi-dispenser prototype was evaluated in this regard with respect to ease of use and volumetric precision. As not only spot size,
but also analyte recovery varies with blood Ht [1], temperature-enhanced desorption conditions were applied to re-dissolve the entire dried blood sample.
Online FTD-SPE-MS/MS
DBS analysis was carried out using a DBS autosampler (DBSA) (Fig 1). The DBS card is transferred from the card rack to the sliding card
holder by the x-y-z robot of the DBSA. A picture
is taken of the card for determining the exact
spot position, as well as storing information.
Then, the blood spot is clamped for flow-through
desorption (FTD, patented technology). The
clamp has an internal rim diameter of 6 mm (optional 2 and 4 mm) enabling the desorption of
the entire blood spot. Internal standard is added
via loop and is flushed over the DBS card together with the desorption solvent. The entire
sample is subsequently flushed towards a SPE
cartridge for clean-up. A heater placed upstream of the clamp allows heating of the desorption solvent up to 80 °C. Afterwards, the
same SPE cartridge is used as a “mini LC column” eluting the analytes directly by gradient
towards the MS (work has partly been carried
out with an experimental device instead of
DBSA prototype; the principle of these measurements is the same as described) (Fig. 2).
Spiked Blood of Different Ht Levels
The Ht level of human K3-EDTA blood was determined (approximately) after centrifugation (15
min, 3000 rpm) by dividing the volume of red
blood cells by the total blood volume. Blood of
different Ht levels was then prepared by addition of plasma (low Ht) or red blood cells (high
Ht).
Blood was mixed with a standard solution in
40% acetonitrile (ratio 20:1) to obtain blood
spiked with Chlortalidone (3.0 μg/ml), Hydrochlorothiazide (0.5 μg/ml), Acebutolol (0.1 μg/
24
separation 1/2014
ml), Haloperidol (0.1 μg/ml), Verapamil (0.1 μg/
ml) and Propranolol (0.2 μg/ml). After the spiked
sample was equilibrated for at least 1 hour 5-μl
blood was applied to filter paper cards and allowed to dry at room temperature for ≥ 2 hours.
Sample Application Using a Multi-dispenser Prototype
The capillary was held horizontally into a blood
drop and filled by capillary suction. Blood was
then dispensed in 5 serial volumes of 5 μl (dispenser strokes are pre-set) onto the DBS card.
Touching the card with the capillary was uncritical; in fact, spot size was smaller when touching
the card. Spot size of sample volumes up to 10
μl can thus be kept small enough to allow fullspot analysis using the 6-mm clamp.
Standard mixture for loop injection
The volume of the “20 μl” loop was accurately
determined to be 21.87 μl. For loop injection of
equivalent amounts of compounds as present in 5-μl blood spots the following standard mixture is prepared in 10% acetonitrile with
0.2% FA: Chlortalidone (0.68 μg/
ml), Hydrochlorothiazide (0.11
μg/ml), Acebutolol (0.023 μg/ml), Haloperidol (0.023 μg/ml), Verapamil (0.023
μg/ml), Propranolol (0.46 μg/ml).
Measurement of recovery and
MS matrix effects
Recovery of the analytes from the blood
spot and MS matrix effects were measured using the loop attached to the 10-
port valve. The procedure involves three subsequent experiments:
▪▪ A “normal” analysis of a spiked blood spot
was performed.
▪▪ The loop was filled with the standard mixture and a blood spot analysis is performed
using blank blood as the sample. By switch-
Fig. 1: DBS Autosampler
S a mp l e P r ep a r a t i o n
Fig. 2: FTD-SPE-MS/MS workflow
ing the loop into the desorption solvent
stream at the moment of starting the desorption, blank blood and analyte mix are
flushed over the SPE cartridge simultaneously. By comparing 1 and 2, any loss of
analyte due to incomplete recovery from
the blood spot can be determined independently of SPE recovery and MS ionization
suppression.
▪▪ See step 2, but now a totally blank card (no
blood spots) was clamped. By comparing 3
and 2, signal loss or enhancement due to
matrix effects on MS ionization could be determined.
Results:
Optimization and Exploration
Effects of hematocrit and spot aging on analyte recovery
Dried blood spots were initially analyzed using
“standard” conditions for desorption (1 ml water 0.2% FA at 2ml/min; no heater used). Recoveries were determined for spots made of blood
with Ht levels 0.3 and 0.7 after 1 and, respectively, 4 days of storage. For comparison blood
spots (Ht 0.7) are analyzed using optimized desorption conditions.
Under standard conditions, recovery was
significantly lower for DBS of high Ht level.
Spot-aging also slightly decreased analyte recovery for DBS of high Ht level. Under optimized temperature-enhanced desorption conditions (heater at 80°C) there was ≥94%
recovery for DBS of high Ht level even after 4
days aging.
Precision of sample application by multi-dispenser prototype
The 5-μl spotting precision of a prototype multidispenser was investigated taking the FTD-SPEMS/MS measurement of Haloperidol for calculation. Five capillaries were filled with blood and
each one was used to dispense 5 x 5 μl onto a
DBS card
Fig. 3: Example of online FTD-SPE-MS/MS separation for blood spiked at
250 ng/ml
RSDs were acceptable for all individual capillaries in case the first spot was not used for calculation.
A good overall precision was obtained for a
series of 20 spots from all 5 capillaries.
Recovery of the analytes spiked to blood of different Ht levels was determined as described in
experiments 1 and 2 (see Measurement of Recovery and MS Matrix Effects). Experiment 2 was
carried out with blank blood at Ht level 0.45.
Recovery for all Ht levels ranged between
88.9% and 107.9% with no dependency of recovery on the Ht observed. The dried blood spot
was efficiently removed from the card by optimized desorption at high temperature.
wards onto DBS cards. Touching the DBS card
does not result in any analytical issues; in contrast, it helps to keep the spot diameter small.
The entire bloodspot is analyzed so that no sample is wasted. Sample clean-up by online SPE reduces MS matrix effects to an acceptable level.
By using temperature-enhanced desorption conditions recoveries close to 100% are attained
independent of the blood Ht level. FTD at high
temperatures combined with online SPE-MS/MS
of full blood spots overcomes the effect of Ht on
recovery in DBS analysis. This micro-sampling
concept therefore enables a robust and reliable
quantitative DBS analysis. In addition, the completely automated workflow omits the cumbersome and error prone practice of punching-out
discs from DBS.
Matrix Effects
Acknowledgement
FTD Recovery
Matrix effects were within the bioanalytical acceptance range (+/- 15% signal reduction or enhancement), except for Chlortalidone with a
signal loss of just over 16%. Further optimization of the extraction conditions or an additional
wash after trapping the analytes on the SPE cartridge will likely bring the matrix effect of Chlortalidone within 15%, but this was considered
beyond the scope of the present study
Further detailed results can be found at
http://bit.ly/DBS-Analysis.
Discussion
A new technology for automated analysis of
DBS samples has been investigated and reported previously [3]. This is based on patented
flow-through desorption of the blood spot,
which can be coupled online to MS/MS analysis
via a disposable SPE cartridge for sample cleanup and separation. Here we report how a DBS
Autosampler can overcome critical issues in DBS
analysis.
The multi-dispenser showed that it can be
conveniently handled to sample blood from a
finger prick and to dispense 5-μl aliquots after-
Drummond Scientific is gratefully acknowledged
for loan of the prototype multi-dispenser.
References
[1] Denniff P. and Spooner N. Bioanalysis, 2 (8), pp
1385-1395 (2010)
[2] de Vries R. et al.: Bioanalysis, 5 (17), pp 21472160 (2013)
[3] Ooms JA et al.: Bioanalysis, 3(20), pp 2311-2320
(2011)
[4]http://bit.ly/DBS-Analysis
Authors
Christel Hempen, Lena Knegt, Bert Ooms
Spark Holland BV
Contact
Bert Ooms
Spark Holland BV
Emmen, The Netherlands
bert.ooms@sparkholland.com
separation 1/2014
25
Alexey Protasov-Fotolia.de
F i l t r a t i o n Beyond Appropriateness
and Sustainability
Universal Self-reliance in Water Supply
The world is on track to achieve the millennium development goals for safe
drinking water. However, the current paradigm for water supply in low-income communities is not satisfying. This article presents a universal solution
for self-reliance in safe drinking water provision by filtration on packed beds
containing metallic iron.
Worldwide industrialization and urbanization
result in increased water pollution. Sources of
chemical contamination include agricultural, domestic, industrial, mining activities as well as
medical and municipal wastes. Chemical contamination is leached from various solid wastes
and transported into aquifers and rivers. Nonprotected surface water could be regarded as a
cocktail of pollutants, which should be treated
to meet relevant standards.
The number of groups of chemical species
that are potential contaminants is huge: chlorinated organic compounds, dyes, heavy metals,
nitroaromatic compounds, pharmaceuticals,
phenols etc. [1-3]. Each class of compound is
made up of individual substances with different
chemical and physical properties. For example,
dyes are of various molecular sizes and solubilities, they are either anionic, cationic or neutral.
Some of them are redox active. In other words,
treating dyes as a class of substances with regard to remediation is not appropriate. In fact,
the remediation technologies rely on specific interactions with the contaminants: adsorption,
co-precipitation, coagulation, ion-exchange, oxidation, reduction, size-exclusion. From these
processes, adsorption, co-precipitation, and sizeexclusion are the methods of choice to remove
aqueous microbial contamination.
26
separation 1/2014
Appropriate Technology
for Safe Drinking Water
Considering natural waters as a cocktail of
chemical and microbial contaminants implies
that appropriate technologies for their treatment should address several types of contaminants. This is conventionally achieved through
a combination of processes including screening, coagulation, filtration, and disinfection.
Such treatment chains are found in centralized
waterworks, where raw water is collected,
treated and distributed to the population by
pipeline network. Centralized systems are expensive to install, operate and maintain, especially for low-income communities. Membrane
technology combining ultra-filtration and reverse osmosis has been proven the sole compact method to free water from chemical (e.g.
arsenic, pesticides), microbial (e.g. bacteria,
viruses) and physical (e.g. colour, turbidity)
contamination because it works on a pure
size-exclusion basis. However, this technology
needs high pressure, thus electricity, to operate.
The term “appropriate technology” emphasized that solutions in the developing world
should be small-scale, affordable, energy efficient, environmentally sound, use locally avail-
able resources, and be capable of being controlled and maintained by the local community
[4]. These criteria make small-scale, decentralized membrane-based water treatment systems simply non appropriate. There are voices
calling for a revision of basis criteria for an
“appropriate technology”. However, proponents of re-evaluation are mostly interested in
money making as “the design of technology
appropriate for developing countries is an increasingly profitable business for manufacturers and distributors” [4]. The proponents of
self-reliance regard membrane technology as a
bridge or an emergency solution. This article
presents the concept of filtration on packed
beds of metallic iron (Fe0 filters) as a universally appropriate technology for safe drinking water provision.
Fe0 filters for safe drinking water
Fe0 as removal and recovery agent for dissolved metal is known to hydrometallurgists
for more than 100 years. The concentrations of
metal ions are in the range of some 100 mg/l
and the pH of the solution is flexibly adjusted
to pH ≤4.5. For natural waters, contamination
levels are in the range of μg to a few mg/l and
the pH of water is in the range 6.0 ≤ pH ≤ 9.5.
Accordingly, water treatment with Fe0 occurs
in a domain of low Fe solubility. In other
words, Fe0 is corroded mostly by the solvent
(H2O) and its surface is covered by layers of
corrosion products. Corrosion products include
two other reducing agents, Fe2+ and H/H2. The
reductive properties of these reagents are enhanced by adsorbing onto nascent iron hydroxides [5]. The major consequence is that
contaminants, present in trace amounts (μg/l)
must migrate through a multi-layer oxide scale
to reach the Fe0 surface. Accordingly, although
Filtration
Fig. 1: Flow chart of water treatment involving metallic iron (Fe0). Water is
first filtered through a conventional filter, e.g. biosand with sand of various
particle sizes. The filtrate is affined in two Fe0-based beds. The ideal vol. Fe0
ratio is 25% [6].
Fig. 2: Flow chart of an alternative water treatment using Fe0 mostly as Fe2+
source. The O2 level of raw water is reduced in a biosand. The filtrate leaches
Fe2+ from the Fe0 bed (Fe2+ is labile under anoxic conditions). Fe2+ is oxidized
by aeration to enhance contaminant removal in the subsequent sand filter.
Fe0 corrosion by water is an electrochemical reaction, contaminant reduction (if applicable) is
not the simultaneous cathodic
process [1].
While regarding contaminant
reduction as the cathodic reaction
of iron corrosion, an abundant literature is available on the feasibility of using Fe0 filters as universal material for safe drinking
water provision and proper sanitation in low-income communities [5,6]. The idea is to reproduce
conditions available in subsurface
permeable
reactive
barriers
(PRBs) for sustainable Fe0 filters
for safe drinking water provision
in waterworks [6]. Fe0 PRBs have
been efficient for more than 15
years. The main parameter to be
controlled is the dissolved O2 level which should be lowered to ≤
1.5 mg/l. Basically, the O2 level
can be controlled by biosand filters or by sacrificial Fe0 beds (e.g.
containing just 5 to 10 vol% Fe0).
In this case it could be necessary
to introduce a flow equalizing
bed (e.g. gravel, sand) between
the sacrificial and the treatment
beds (Figure 1 and 2).
Contact
Universality of Fe0 filters
The universality of Fe0 filters arises
from the fact that they do not need
electricity to operate. They can be
coupled to all other existing devices as refinement stage. For example, if a charcoal filter after Kearns
[7] is not efficient enough for the
removal of micro-organisms or
heavy metals, complementary Fe0
filters can be designed. They can be
customized to meet the requirements of single households and
small communities. They can also
be tailored for seasonal use only. In
the developing world, these filters
give local researchers a unique opportunity to solve the long lasting
problem of water supply on a selfreliant basis. Once a sustainable
solution for safe drinking water is
established, the won self-confidence will be the weapon to face
remaining developmental challenges.
Chicgoua Noubactep(a-c)
(a) Angewandte Geologie
Universität Göttingen, Germany
Tel.: +49 551 39 3191
cnoubac@gwdg.de
(b) Kultur und Nachhaltige
Entwicklung CDD e.V.,
Göttingen, Germany
(c) Comité Afro-européen,
Namur, Belgium
References
[1]Ghauch A.: Iron-based metallic
systems: An excellent choice for
sustainable water treatment. Habilitation Thesis, University of
Grenoble, France.
[2] Gheju M.: Water Air Soil Pollut.
2011, 222, 103–148 (2013)
[3] Wang H.et al.: Clean – Soil, Air, Water, DOI: 10.1002/clen.201300208
(2014)
[4] Sima L.C. and Elimelech M.: Environ. Sci. Technol. 47, 7580–7588
(2013)
[5] Noubactep C.: Clean - Soil, Air, Water 41, 702–710 (2013)
[6] Rahman M.A. et al.: J. Appl. Solution Chem. Model. 2, 165–177
(2013)
[7] Kearns J.: Water Conditioning &
Purification, October 2012, 7–12
separation 1/2014
27
I o n C h r o m a t o g r a ph y Good Health without Bad Breath
Extracting Alliin from Garlic
Garlic may have an undesirable effect on your breath, but it has a very desirable effect on your
health, able to reduce cholesterol levels, lower blood pressure and kill bacteria. These health benefits are primarily due to the presence of sulfur-containing compounds in garlic, particularly a sulfoxide derivative of the amino acid cysteine known as alliin, which is also responsible for the
characteristic aroma of garlic.
Salt and Alcohol
For their ATPE method, Bo Cui and his colleagues at Qilu University of Technology in Jinan
used an ammonium sulfate solution and the alcohol propanol as their two immiscible liquids,
both of which had already proved effective at
extracting various different proteins. First off,
though, they conducted numerous experiments
to optimize the conditions for this method.
Eventually, they alighted on using a 19% ammonium sulfate solution and a 20% propanol solution at pH 2.35 with sodium chloride as an additive to improve the yield.
They needed to conduct this exhaustive optimization process because many biomolecules, including alliin, are soluble in both salt solutions
and alcohol. The precise level of solubility depends on the balance between competing
chemical interactions, particularly hydrophobic
interactions and electrostatic interactions, taking place between the biomolecules and the two
liquids. Cui and his colleagues needed to make
sure they set up the conditions such that alliin
28
separation 1/2014
was much more soluble in one of the liquids
than the other.
Pure Health
The conditions they finally alighted on
caused alliin, together with various amino acids and polysaccharides in the
garlic powder, to dissolve preferentially in the ammonium sulfate
solution, driven by hydrophobic
interactions. When Cui and his
colleagues mixed garlic powder into this two-phase system, they were able to extract alliin with a yield of
20.4 mg/g. In contrast, a
conventional
ultrasonicbased method for extracting alliin from garlic powder was only
able to produce yields
of 15 mg/g.
However, this alliin is
still contaminated with
various amino acids and
polysaccharides from the
garlic powder, so to purify
the alliin Cui and his team turned
to cation-exchange chromatography. Using
an acetic acid solution at pH 1.5 as the mobile phase gives the alliin a positive charge, allowing it to be separated from the uncharged
polysaccharides. The alliin can then simply be
recovered with water, separating it from the remaining amino acids due to their different isoelectric points and producing alliin with a purity
of 80%.
So now you can have all the benefits of garlic
without the associated social embarrassment.
Reference
[1] Jian X.-M. et al.: J. Chrom. B 957, 60-67 (2014)
© yvdavid - Fotolia.com
Obtaining these health benefits without the corresponding bad breath requires a method for
extracting alliin from garlic and now a team of
Chinese food scientists has come up with the
most effective way for doing this yet developed.
Their method involves first using aqueous twophase extraction (ATPE) to separate the alliin
from other components of the garlic and then
purifying the alliin with cation-exchange chromatography.
ATPE is a form of liquid-liquid extraction, in
which compounds are separated based on their
relative solubility in two immiscible liquids. Traditionally, these two liquids are water and some
kind of organic solvent, but this does not work
for biomolecules such as proteins, because the
harsh solvent tends to degrade them. Hence, the
development of ATPE, which replaces the water
and organic solvent with two water-based solutions that are still immiscible.
Contact
Jon Evans
JE Science
Chichester, UK
www.jescience.co.uk
L i t e r a t u r e
Book Reviews
Separation Process Principles
This book is a comprehensive and up-to-date
treatment of the major separation operations in
the chemical industry. The 3rd edition is renamed Separation Process Principles – Chemical
and Biochemical Operations to reflect the inclusion of bioseparations in several chapters. Extraordinary advances that are being made in the
biological fields could significantly help solve
world problems in the energy, environmental,
and health areas. To help provide instruction in
the important bioseparations area, a new author
has been added for this edition, D. Keith Roper,
who has industrial and academic experience in
this field.
The book provides review chapters on thermo and mass transfer, comprehensive discussion
of many separation processes, photos, diagrams,
and descriptions of process equipment, and
challenging, realistic problems. Improved clarity,
study questions, boxed equations and examples
are especially helpful for students encountering
separation processes for the first time.
Seader, J. D. / Henley, Ernest J. / Roper, D. Keith
3. Edition September 2013
ISBN 978-0-470-48183-7 – John Wiley & Sons
Pitfalls and Errors of HPLC in Pictures
The third edition of this popular problem-solving
guide for this widely-used method includes eleven completely new examples and several updated ones, adding up to 100 contributions about
pitfalls and errors in HPLC. Each example is presented on a double page with the text on the
left-hand and a figure on the right-hand side,
true to the motto ‚a picture says more than a
thousand words‘. In addition, the author presents essential fundamentals as well as helpful
strategies, such as equipment tests or quality assurance processes.
New in this edition are for example
the topics:
Get the Mobile
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‘Variability of the standard deviation’, ‘Influence
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phase water’.
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3., revised and enlarged Edition – January 2013
ISBN 978-3-527-33293-9 – Wiley-VCH, Weinheim
www.wiley-vch.de
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P r o d uc t s Streamline Sample Preparation
Development
High-Throughput MS
Waters Corporation unveiled its new web-based Oasis Method Development
Tools designed to help customers reduce their sample preparation method development time as part of the Simple Prep campaign. Based on the customers’
sample requirements, the tools will recommend the optimized solid-phase extraction (SPE) protocol to develop robust methods with high recovery for liquid
chromatography and mass spectrometry applications. Oasis features basic and
advanced capabilities like the the Micro Sample Volume Tool, the Maximum Selectivity Tool and the General Purpose Tool.
Waters Corp.
www.waters.com
Merit HPLC Systems
Cecil Instruments have launched a
newly developed low cost easy to use
HPLC range. The new Merit systems expand its comprehensive Adept HPLC
range and are designed to provide ease
of use, reducing the time for software
familiarization. The fully PC controlled
isocratic and binary systems equipped
with the new software, provide automatic integration of chromatography peaks without operator intervention. The
systems make operation easy for analysts and are a good match for screening,
quality assurance, method development, teaching and use by novices.
Agilent Technologies launched the Rapidfire 365 High-throughput Mass Spectrometry System. It offers increased plate capacity, full integration with the
companies` automation technology and improved productivity for researchers
working on drug discovery, clinical research and forensic toxicology. Alongside
the full compatibility with Agilents entire line of mass spectrometers, the system has numerous advancements like unattended runs for up to 60 hours, facilitating more than 20,000 injections and enabling over-the-weekend runs,
multiple assays in a single run, automated method development for novel analytes, and intuitive data review and visualization. The system also identifies active compounds against challenging targets, confirm the activity of those compounds, and assess absorption, distribution, metabolism, elimination and
physiochemical properties of those compounds.
Agilent Technologies Inc.
www.agilent.com
Cecil Instruments
www.cecilinstruments.com
Unique HPLC Solution for Enhanced Laboratory
Workflow and Efficiency
Thermo Scientific Accucore HPLC columns use Core Enhanced Technology to maximize your workflow.
They provide fast and highly efficient separations in a robust format. The sixteen phase options available are compatible with most HPLC instrumentation and a wide range of applications. Each bonded
phase is manufactured using our advanced bonding technology.
Accucore HPLC Columns contain solid core particles and are engineered to a diameter of 2.6 μm
and a very narrow particle size distribution. They allow high speed, high resolution separation, with
back pressures significantly lower than those associated with U
­ HPLC.
Accucore HPLC Columns for Biomolecules are packed with 150 Å pore diameter particles and
allow an optimum combination of retention and resolution for peptides and proteins.
Accucore XL HPLC Columns use 4 μm solid core particles, and allow users of conventional HPLC
methods to enjoy performance far beyond that of columns packed with 5 μm, 4 μm or even 3 μm fully
porous particles.
Key Features
Thermo Fisher Scientific Inc.
analyze@thermofisher.com
www.thermoscientific.com/accucore
30
separation 1/2014
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P r o d uc t s Beckman Coulter Brings New Functionalities to Avanti Centrifuges
Metrohm has introduced a method to determine Chromium (VI) in toys by ion
chromatography. Children are exposed to intakes of heavy metals from a wide
variety of sources. Chromium (VI) in particular represents a potential hazard, as
it is absorbed from food and drinking water, from the air that is breathed, from
textiles, from utensils that contain metal and from toys. The analytical determination of chromium (VI) content in toys for compliance with limit values is described in the European standard DIN EN-71-3-2013 (Safety of toys Part 3 – Migration of certain elements). The company has developed an ion
chromatography method for this application using preconcentration and Inline
Matrix Elimination.
Combining high performance and
application versatility, Avanti JXN-26
centrifuges from Beckman Coulter
Life Sciences offer laboratories an intuitive interface and advanced data
management features that expand
functionality and flexibility. The instruments can be run from an Apple
iOS or Android device using Mobilefuge, a mobile application to run and
manage laboratory centrifuges. Researchers and facility managers can
leverage the instrument’s data management, user tracking, networking
and remote capabilities for ease-ofuse and efficiency in biological sample preparation, and to meet quality
control standards. Safety, logistics
and quality challenges in multiuser labs and Good Manufacturing Practice
(GMP) environments are met with software designed to improve daily operations across applications.
Metrohm
www.metrohm.com
Beckman Coulter Life Sciences
www.beckman.com
Chromium (VI) in Toys
Sieve Shakers
HPTLC Software
Camag announced the availability of a new version of its Vision Cats HPTLC
software (V1.4) for qualitative HPTLC analysis that supports chromatographers
using HPTLC for quality control, identification, and detection of adulteration, as
well as stability studies in various fields of application. The company has implemented several tools, i.e. a clipboard function or annotations integration into
reports, that make every HPTLC analysts life easier. The latest release integrates
the company’s Method Library, where users can download validated methods
showing examples incl. reference standards and execute analyses accordingly,
which makes HPTLC a useful tool for quality control, particularly for identification. While saving costs and achieving reproducible results through the use of
standardized procedures the company’s Method Library makes own method
development redundant.
Camag
www.camag.com
32
separation 1/2014
The Fritsch sieve shakers are designed for dry, wet and micro-precision sieving, measuring the quantitative particle size distribution of solids
and suspensions, and separating and
fractioning. Their operation is simple
and ergonomic. The shakers offer fast
and reproducible results, and can
manage sample quantities between
0.05 g and 15 kg. Sieve diameter
from 100 mm to 450 mm, mesh
widths from 5 μm – 125 mm. They
can be used as testing equipment in
accordance with DIN EN ISO 9001.
The high performance Vibratory Sieve
Shaker Analysette 3 ro offers everything you need for fast determination
of quantitative particle size distribution in the laboratory. As a shaking
sieve system with an electromagnetic drive oscillates the sieve stack into
regulated vertical oscillations, and is
a suitable solution for sieving sample quantities up to 2 kg and a measurement
range from 5 μm to 63 mm.
Fritsch
www.fritsch.de
P r o d uc t s
Solution for Western Blotting
Flow Control Solution
Readytector is an all-in-one detection solution for Western
blotting developed by Candor
Bioscience. It contains all ingredients for a quick one-step immunodetection. Only a specific
primary antibody has to be added. “All-in-one” stands for all
ingredients in one solution as
well as for all-in-one step.
Blocking and binding of primary
and secondary antibodies are
carried out simultaneously. The
only additional step is washing
with the specific Readytector Wash Buffer. Bottle sizes of 40 ml, 120 ml and 500
ml are available.
With Bronkhorst’s mounting
concept Flow-SMS a variety
of components for mass
flow and pressure measurement and control can be assembled to constitute a very
compact gas delivery system. On a lightweight and
rugged mounting rail system one or more mass flow
(or pressure) sensor/control
modules can be combined
with mixing chambers, (pneumatic, electrical or manual) shut-off valves, filters
or any other functional module as per customer’s request. Flow ranges can be
selected between approx. 5 mln/min up to 50 ln/min or even higher. In case a
pressure sensor or controller is included, the pressure range can be chosen between 0-100 mbar and 0-10 bar absolute or gauge. Space efficiency, servicefriendliness, and flexibility of expansion or modification are realized within a
compact, top-mount design.
Candor Bioscience
www.readytector.com
MS Calibration
Bronkhorst High-tech B.V.
www.bronkhorst.com
Cerno Bioscience introduces version 4 of its flagship Massworks software product. It comes with an extensive array of capabilities and features including Direct Read support for three Hires MS systems including e.g.: Thermo Orbitrap or
FT ICR, Waters TOF/qTOF, and Agilent TOF/qTOF; ion mixture analysis capable of
simultaneous quantitation of more than a dozen mutually overlapping ions, for
applications ranging from deamidation / deaminiation impurity determination
to hydrogen-deuterium exchange (HDX MS); Elemental composition analysis
with weak or no observable monoisotope; Direct Read support for two unit
mass resolution data systems, Agilent Masshunter and Advion CMS.
Sample Preparation
Cerno Bioscience
www.cernobioscience.com
Biotage
www.biotage.com
KNF Lab Presents Its First Rotary
­Evaporator
Quality Control in Polypropylene Manufacturing
KNF Lab’s first rotary evaporator, the
RC900, was presented for the first time
at Analytica 2014. The easy-to-clean
design offers a range of novel features
that focus on easy and reliable operation and increasing operator safety - including a remote control unit, simple
one button functions for routine tasks
such as changing flasks, easily adjustable rotation speeds and dynamic temperature control. Dr Alexander Scherer,
Chair of Organic Chemistry at the Friedrich-Alexander-Universität ErlangenNürnberg, Germany, has beta-tested
the new system in his laboratory and made further suggestions on how to
make the system even easier to operate. KNF Lab took these suggestions on
board and the result is a high-performance system that features a user-friendly,
intuitive touch screen and automated functions.
The new Polymer Char Crystex QC instrument represents a step forward in
technology for automation of the amorphous phase determination, previously
measured as Xylenes Solubles; in Manufacturing Quality Control laboratories of
Polypropylene (PP). It overcomes all difficulties of the old-style gravimetric
methods based on traditional wet chemistry. This modern quantitative chemical
analysis method eliminates the use of any laboratory glassware, external filtration or extraction devices, as well as all the associated tedious manual operations. It is designed to control PP production measuring individual samples and
requiring minimum bench space and utilities. This solution covers this
way the need of manufacturers to measure the
amount of soluble or amorphous fraction,
which has become a reference in
the PP Production Quality Control.
For the improvement of sample preparation the “Hints, Tips and Troubleshooting” Section in Biotage’s updated Isolute SLE+ User Guide informs on ways to
improve and extend the range of compounds the user can extract. It also contains specific information for challenging biological matrices, including whole
blood. Topics are: Multi-suite drugs of abuse applications; applications using
Isolute SLE+ on automation platforms, Method Selection Chart; Load-WaitElute Procedure.
Polymer Characterization
www.polymerchar.com
KNF Neuberger
www.knf.de
separation 1/2014
33
P r o d uc t s Higher Loads for Pharmaceutical Lab-Scale
Purification
Phenomenex ha introduced a 30 mm I.D.
(internal diameter) Kinetex core-shell
5-micron column in Axia hardware for
preparative HPLC and SFC in pharmaceutical lab-scale purification.
Also offered in a 21.2 mm I.D., the 30
mm column enables increased sample
loading and throughput. With the addition
of this larger diameter column, the company offers the
only core-shell media line that can be used from analytical
scale through scale-up to purification. The Axia preparative format delivers long column lifetime, high efficiencies, performance, and high reproducibility. 5-micron is the largest particle in the core-shell family and delivers better
performance than 5-micron fully porous offerings, with no increase in backpressure. According to the company 5-micron media provides 60 to 90 percent higher
average efficiencies compared to the same size fully porous columns with little to
no method development.
Phenomenex
www.phenomenex.com
Saving You Hours of Tedious
GC–MS Data Processing
Targetview is an software package allowing accurate and automatic identification of both target and ‘unknown’ compounds in GC–MS profiles. Load in your
data file and your library, and within seconds the software will have located
your compounds of interest, saving you hours of tedious manual processing.
This updated version now features an improved user interface and new fastsearch option, speeding up target searches against very large libraries, and
minimizing memory usage for complex data. Use the company’s new secure
upload facility to send an example data file to receive an example report completely free of charge.
Blot Detection
Molecular Devices has introduced its
“Scanlater Western Blot Detection System”. With a user-installable cartridge,
researchers can turn the Spectramax i3
or Paradigm Multi-mode Platform into a
western blot detection system in minutes and realize compressed protein detection workflow, extended signal stability, and expanded dynamic range.
Molecular Devices
www.moleculardivices.com
separation 1/2014
Merck Millipore has introduced the Clarisolve depth filter for single-stage clarification of pretreated feed streams. The depth filter is a clarification device with a
gradient density structure specifically designed to the particle size distributions
of pretreated feed streams. Delivering improved volumetric capacity and reduced turbidity they process pretreated feeds in a significantly reduced footprint
without the need for the secondary stage of clarification. The use of the filters
eliminates the need for centrifugation which enables implementation of a fully
single-use process train, and also reduces the pre-use flushing requirements.
Merck Millipore
www.merckmillipore.com/clarisolve
Accelerated Vacuum Filtration
Vacuubrand’s range of environmental
friendly diaphragm vacuum pumps offer
different vacuum performance and application-oriented features for all typical
needs. The manufacturer’s oil free diaphragm pumps feature quietness, robustness and long service intervals. They are
also available in chemical-resistant versions for handling of aggressive vapors.
Single-stage pumps with an ultimate vacuum down to 70 mbar are also suitable
for filtration of clear and particle free liquids, e.g., for the determination of sources
of microbial contaminations by ‘membrane filtration’. For vacuum regulation as
prescribed in the procedure for colony counting of drinking water there is an optional upgrade to include vacuum gauge and manual regulation valve. Here the ME
1 pump series with 100 mbar ultimate vacuum and a gas throughput of 0.7 m³/h
offers a flexible solution. For multiport filtrations with 3- and 6-place vacuum manifolds also stronger single-stage pumps of the bigger NT series are available.
Vacuubrand
www.vacuubrand.de
Advion Expands its Line of
Compact Mass Spectrometers
Markes International Ltd.
www.markes.com
34
Depth Filters
Advion launched a new compact mass spectrometer, the Expression L CMS. The
new spectrometer has an extended mass range designed for chemists focused
on larger molecule applications such as peptide synthesis, polymer chemistry
and natural products. It is used in a variety of application areas. This spectrometer is compact enough to fit in fume hoods or on cramped benches and has a
2000 m/z mass range. Lab and chemist deployed, instead of core facility protected, ensures that assays are available 24/7, and that the expression line of
mass spectrometers is available to work as hard as the chemists who synthesize and purify the samples.
Advion Inc.
www.advion.com
ts at …
more produc
urnal.com
laboratory-jo
Advion 34
Dr. A. Maisch 27
Novasep Agilent Technologies 30
Fritsch 32
Phenomenex 34
BAM Federal Institute for Materials 20
Gerstel 8
Polymer Char 33
Beckman Coulter Life Sciences
32
Gilson 23
Polymer Standards Service 13
Biotage 33
JE Science 28
Robu Glasfiltergeräte Bronkhorst High-Tech 33
Dr. Ing. H. Knauer 29
Shimadzu KNF Neuberger 33
Spark Holland Leco Instruments
8
Camag 17, 32
Candor Bioscience 33
8
9
Inside Front Cover
Thermo Fisher Scientific 21, 24
16, 30, Outside Back Cover
6
Markes 34
University of Applied Sciences Bingen 12
Cecil Instruments 30
Merck 34
University of Göttingen 26
Cerno Bioscience 33
Metrohm Vacuubrand 34
CASSS - International Separation Science Society Danaher Corporation
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