Nucleic Acid Analysis with UV-vis and NMR - Spectroscopy

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November 2009 Volume 24 Number 11 

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Nucleic Acid Analysis 

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The Sharp New Teeth 

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The Importance of 

Pressure and Vacuum

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Contents 

Columns 

Volume 24 Number 11 

November 2009 

November 2009 

Volume 24 Numb er 11 

MASS SPECTROMETRY FORUM 16 

Pressure and Vacuum: Not Really Trivial 

Good vacuum system design is a crucial underpinning for high performance instrumentation. 

The important aspects of pressure and vacuum need regular teaching, and here Ken Busch 

discusses them. 

Kenneth L. Busch 

The Tiger Has Sharp New Teeth 

The new FDA Commissioner wants a strong FDA and is backing her words with action by 

initiating a program that cuts the time that firms must respond to 483 observations from 

30 to 15 business days. Not only is the time halved but the response must be complete! 

Therefore, it is better and cheaper to be compliant than not. 

R.D. McDowall 

FOCUS ON QUALITY 

23 

Cover image courtesy of 

Getty Images. 

DEPARTMENTS 

From the Editor 8 

News Spectrum 14 

Product Resources 44 

Calendar 47 

Ad Index 50 

ATOMIC PERSPECTIVES 

Speciation Analysis by LC–ICP-MS 

Speciation analysis by LC-ICP-MS has been growing rapidly in popularity and application 

over the past several years. Not only have people begun looking at different elements and 

species, but there has been an increase in the variety of matrices that speciation analysis is 

being performed on. 

Kenneth Neubauer 

Articles 

Interaction of Indigo Carmine with Nucleic Acids in the Presence 34 

of Cetyltrimethylammonium Bromide: Spectral Studies and the 

Confirmation of Combined Points 

The interaction of indigo carmine with nucleic acids in weak acid medium was studied in the 

presence of cetyltrimethylammonium bromide (CTMAB) using resonance light scattering, 

UV-vis, and NMR. 

30 

Changqun Cai, Xiaoming Chen, and Hang Gong 


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From the Editor 

A Stronger FDA: What Does It Mean for You? 

The FDA, like the IRS or the DMV, is one of those governmental agencies that by its 

very nature can inspire many with at least a hint of dread. For one thing, it can often 

seem that they are only heard from when something bad is happening (that is, you 

haven’t paid your taxes, you haven’t paid a speeding ticket . . . or there is an inspection of 

your facility on the way). 

Here at Spectroscopy, we leave the politics of the “Bush FDA” versus the “Obama FDA” to 

others, and instead focus on what is currently happening and how it impacts the laboratories 

and the daily work of our readers. In other words, we try to answer the question, “What 

does it all mean for me?” With this in mind, columnist Bob McDowall, one of the foremost 

experts on not only the FDA and its policies and regulations, but compliance in general, 

presents his take on the recent FDA Modernization Act and the posture of the FDA and 

its new commissioner, Margaret Hamburg, in general. Admittedly, the title of his column 

gives away his point of view to a large degree, as “The Tiger Has Sharp New Teeth” tells you 

where he thinks the FDA is headed. Tougher enforcement, shorter compliance times, and 

more appear to be on deck, but as always, Bob has answers and advice on how to succeed 

in this new environment. For whether you believe tougher regulation and enforcement is a 

good thing or a bad thing, the fact is that the laboratories of many readers will be forced to 

confront the coming changes regardless. 

At Spectroscopy, we have always made it our mission to bring readers practical, nuts-andbolts 

information to help them in their daily work, and this column, aimed at those on the 

frontlines of materials analysis, is just one more example of this mission in action. We hope 

you find this column and the other columns and technical research in this issue useful, and 

as always, feel free to contact myself or any one of our staff members at the e-mail addresses 

listed. 

Enjoy the issue. 

David Walsh 

Editor-in-Chief 

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Editorial Advisory Board 

Ramon M. Barnes University of Massachusetts 

Paul N. Bourassa Lifeblood 

Chris W. Brown University of Rhode Island 

Kenneth L. Busch National Science Foundation 

Ashok L. Cholli University of Massachusetts at Lowell 

David M. Coleman Wayne State University 

Patricia B. Coleman Ford Motor Company 

Bruce Hudson Syracuse University 

Kathryn S. Kalasinsky Armed Forces Institute of Pathology 

David Lankin University of Illinois at Chicago, College of Pharmacy 

Barbara S. Larsen DuPont Central Research and Development 

Ian R. Lewis Kaiser Optical Systems 

Steve Lowry ThermoFisher Scientific 

Howard Mark Mark Electronics 

R.D. McDowall McDowall Consulting 

Linda Baine McGown Rensselaer Polytechnic Institute 

Robert G. Messerschmidt Rare Light, Inc. 

Nancy Miller-Ihli M–I Research 

Francis M. Mirabella Jr. Equistar Technology Center 

John Monti Shimadzu Scientific Instruments 

Thomas M. Niemczyk University of New Mexico 

Anthony J. Nip CambridgeSoft Corp. 

John W. Olesik The Ohio State University 

Richard J. Saykally University of California, Berkeley 

Basil I. Swanson Los Alamos National Laboratory 

Jerome Workman Jr. Luminous Medical, Inc. 

Contributing Editors: 

Fran Adar Horiba Jobin Yvon 

David W. Ball Cleveland State University 

Kenneth L. Busch National Science Foundation 

John Coates Coates Consulting 

Howard Mark Mark Electronics 

Volker Thomsen Consultant 

Jerome Workman Jr. Luminous Medical, Inc. 

Process Analysis Advisory Panel: 

James M. Brown Exxon Research and Engineering Company 

Bruce Buchanan Sensors-2-Information 

Lloyd W. Burgess CPAC, University of Washington 

James Rydzak Glaxo SmithKline 

Robert E. Sherman CIRCOR Instrumentation Technologies 

John Steichen DuPont Central Research and Development 

D. Warren Vidrine Vidrine Consulting 

European Regional Editors: 

John M. Chalmers VSConsulting, United Kingdom 

David A.C. Compton Industrial Chemicals Ltd. 

Spectroscopy’s Editorial Advisory Board is a group of distinguished individuals 

assembled to help the publication fulfill its editorial mission to promote the effective 

use of spectroscopic technology as a practical research and measurement tool. 

With recognized expertise in a wide range of technique and application areas, board 

members perform a range of functions, such as reviewing manuscripts, suggesting 

authors and topics for coverage, and providing the editor with general direction and 

feedback. We are indebted to these scientists for their contributions to the publication 

and to the spectroscopy community as a whole.


News Spectrum 

Research 

Scientists using planetary spectroscopy recently reported 

that the basic ingredients for life have been found around 

a second extrasolar planet. Although the planet itself 

is uninhabitable for any forms of life found on Earth, 

the discovery shows that the basic components of life 

are widespread in the atmospheres of many kinds of 

exoplanets. 

To make this discovery, researchers trained the Hubble 

and Spitzer Space Telescopes on HD 209458b, a hot 

Jupiter that orbits very close to its sunlike star. Located 

150 light years away in the Pegasus constellation, this 

star has been the subject of previous studies, such as that 

undertaken in December of 2008 by researchers from the 

Jet Propulsion Laboratory, Pasadena, California. At 

that time, a similar Jupiter-like planet, HD 189733b, was 

found to have carbon dioxide in its atmosphere. 

Planetary spectroscopy is easiest to do for systems in 

which a large exoplanet orbits very close to its home star. 

With smaller planets orbiting farther from their star, it is 

harder to detect the minute changes in the star’s light. 

Researchers from Forschungszentrum Jülich, an 

interdisciplinary research center located in Kreis Düren, 

Germany, have utilized a new form of spectroscopy 

to identify explosive liquids, or liquid components for 

the fabrication of explosives, in plastic bottles almost 

instantly. This novel method uses electromagnetic 

radiation to identify materials, and a novel nanoelectronic 

device to detect signals. 

The researchers have suggested a fast and reliable 

way to increase the range of frequencies that their 

spectrometer can analyze, thereby verifying the molecular 

signature of the liquid and creating a much more detailed 

“thumbprint” that can be checked against the range of 

possibly dangerous liquids available to terrorists. 

The method is called Hilbert spectroscopy, and it 

works over a wider range of frequencies, from a few 

gigahertz to a few terahertz. With the incorporation of 

a Josephson junction, which is a nanoscale electronic 

device, the researchers have undertaken practical 

detection experiments that can directly transform the 

electromagnetic spectrum received by the spectrometer 

into an electrical signal that warns of suspicious fluids. 

A team of researchers from Duke University’s 

Marine Laboratory, Durham, North Carolina recently 

used atomic force microscopy and mass spectrometry 

to solve the mystery of what makes barnacles adhere 

so tenaciously to various surfaces while underwater. 

Scientists have long known the chemical properties of the 

glue these crustaceans use, but not how these chemicals 

interact to create a sticky effect. 

The team discovered how to gently remove this glue 

Market Profile: Portable Fluorometers 

A small niche market within fluorescence spectroscopy 

is portable fluorometers. Primary applications are in the 

agricultural industry, but there is considerable potential 

elsewhere. The market landscape is small and fragmented, 

with no dominant leader, and significant potential 

for growth. 

Environmental 

testing (21%) 

By far the most common application 

for portable fluorometers 

is as the chlorophyll fluorescence 

system. Such analysis can 

provide information for a number 

of agriculturally and environmentally 

important applications, 

such as identification of 

genetically modified crops, plant 

health, pesticide levels, and 

more. Other uses include evaluation 

of water quality by way of algae levels. An area 

of significant potential that has yet to be realized is in 

clinical and biological analyses, where there is currently 

significant work underway in exploring the potential of 

portable and handheld fluorometers. 

There are quite a few competitors in the market, with 

many of them based in Europe. However, almost all of 

Other (17%) 

Portable fluorescence demand in 2009. 

these companies are small, independent manufacturers. 

None of the major diversified spectroscopy vendors currently 

compete in the portable fluorescence market. 

The worldwide market for portable fluorometers is 

less then $10 million, but is expected to see double-digit 

annual growth due to the continued 

growth of agricultural 

and environmental applications, 

as well as the potential demand 

from clinical and biological 

applications. 

The foregoing data were based 

upon SDi’s market analysis 

and perspectives report 

Agriculture (62%) 

entitled Global Assessment 

Report, 10th Edition: The 

Laboratory Life Science and 

Analytical Instrument Industry, September 2008. 

For more information, contact Stuart Press, Vice 

President – Strategic Analysis, Strategic Directions 

International, Inc., 6242 Westchester Parkway, Suite 

100, Los Angeles, CA 90045, (310) 641-4982, fax: 

(310) 641-8851, www.strategic-directions.com.


November 2009 Spectroscopy 24(11) 15 

from the barnacles (Amphibalanus 

amphitrite) as they secreted it, wihch 

enabled them to deconstruct the glue 

and find out exactly how it worked. 

They initially compared the glue 

to another substance that clots in 

solution, red blood cells, expecting 

the mechanism by which glue 

particles bind, and red blood cells 

bind, to be different. 

However, they found they are 

strikingly similar. In blood, a number 

of enzymes work to create long 

protein fibers that bind red blood 

cells together into a clot and create a 

scab. Using atomic force microscopy 

and mass spectrometry among other 

techniques, the team found that very 

similar enzymes, known as trypsinlike 

serine proteases, are at work 

in barnacle glue. One of these glue 

enzymes is very similar to Factor XIII, 

an essential blood clotting agent in 

human blood. 

However, these results do make 

evolutionary sense, says team 

member Professor Dan Rittschof: 

“Virtually no biochemical pathway is 

brand new. Everything is related and 

really important pathways are used 

over and over,” he explains. 

Scientists and engineers from 

NASA’s Langley Research Center, 

Hampton, Virgina, recently 

deployed an instrument named 

the “Far-Infrared Spectroscopy of 

the Troposphere (FIRST)” in order 

to measure the effect of highaltitude 

water vapor on the Earth’s 

atmosphere. Deployed in the “driest 

place in the world,” the Chilean 

desert of Atacama, FIRST is one of 

only four instruments of its kind in 

the world. 

It is hoped that the instrument will 

help researchers predict changes in 

the Earth’s climate more effectively, 

as it measures a band of radiation 

linked to the absorption of water 

vapor through the greenhouse effect. 

This radiation activity is a significant 

climate factor and many believe that 

it may comprise up to half of the 

Earth’s natural cooling mechanism. 

Even though other major factors have 

been studied from orbit, up until 

now, the technology has not existed 

to do the same with water vapor. If 

successful, the FIRST equipment will 

deliver the first precisely measurable 

insights into the effect of water vapor 

on the Earth’s climate. 

Industry 

The Institute for Systems Biology 

(ISB) (Seattle, Washington), and 

Agilent Technologies, Inc. (Santa 

Clara, California) announced a 

collaboration to create the Human 

Multiple Reaction Monitoring (MRM) 

Atlas, a comprehensive resource 

designed to enable scientists to 

perform quantitative analysis of 

all human proteins. The project is 

expected to fuel research gains in 

biomarker discovery and validation, 

the search for protein-based 

diagnostic tests, personalized 

medicine, and human health 

monitoring. 

The program is supported by grants 

totaling $4.6 million to ISB’s Robert 

Moritz and Leroy Hood for developing 

the “Complete Human Peptide 

and MRM Atlas” by the National 

Human Genome Research Institute 

of the National Institutes of Health, 

under “The American Recovery 

and Reinvestment Act - Grant 

Opportunities.” Ruedi Aebersold 

of the Swiss Federal Institute 

of Technology (ETH), Zurich, 

Switzerland is collaborating as well, 

with additional funding from the 

European Research Council. 

“We believe this will be a 

revolutionary development in protein 

analysis,” said Rob Moritz, ISB faculty 

member and director of Proteomics, 

“one that will accelerate and 

catalyze the routine use of protein 

quantitation for immensely important 

breakthroughs in the understanding, 

early detection, and monitoring of 

human disease.” 

The atlas is designed to enable 

scientists to quantitatively access the 

approximately 20,000 proteins in 

human tissues, cell lines, and blood, 

potentially transforming many areas 

of human health research. 

Waters Corporation (Milford, 

Massachusetts) recently held the 

2nd Annual Global Food Safety Policy 

Forum with the Center for Science 

in the Public Interest (CSPI) in 

Washington, DC, to bring together 

policymakers and private experts 

from around the globe to discuss 

solutions that improve food supply in 

the U.S. and worldwide. 

U.S. Rep. Rosa DeLauro, the House 

of Representative’s leading food 

safety champion, delivered the 

keynote address. She called for 

a robust U.S. traceability system, 

equivalency with trading partners, 

and greater reliance on science and 

technology to improve the safety of 

food imports. “This is about food 

safety science and where we want to 

go,” she said. 

CSPI Director of Food Safety 

Caroline Smith DeWaal said Congress 

has taken steps to improve U.S. 

food safety but new legislation 

is needed because the country is 

operating under antiquated laws 

and consumers are increasingly 

concerned. 

The Government Accountability 

Office released a new report at the 

Forum, concluding that FDA and 

USDA have taken steps to address the 

challenges in ensuring the safety of 

food imports but the agencies need 

to make better use of data available 

to them to target riskier imports and 

learn from the European Union’s 

system. 

HORIBA (Edison, New 

Jersey) has entered into a 

purchase agreement of land for 

the construction of a new research 

facility on the campus of Ecole 

Polytechnique Paris, France) as 

part of the PARIS Saclay cluster. 

The new facility will welcome the 

headquarters of the recently created 

holding for all corporate activities in 

Europe, HORIBA Europe Holding SAS, 

consolidating 350M EUR and 1700 

employees in France, Germany, UK, 

Italy, and Spain. It will also serve as 

HORIBA’s European Research Centre. 

The facility will be initially 7500 m 2 

with a late 2011 move in, with the 

possibility of expanding to 18,000 m 2 

in later phases. ◾


Mass Spectrometry Forum 

Pressure and Vacuum: 

Not Really Trivial 

Achieving and maintaining vacuum in a mass spectrometer, and measuring and monitoring 

the vacuum pressure, is fundamental for proper instrument operation. This installment of 

“Mass Spectrometry Forum” discusses some basic issues relevant to pressure measurement. 


In the ideal (off) world, we would assemble and operate 

our mass spectrometers in geosynchronous orbit. With 

ultralow pressure and infinite pumping capacity right 

outside the laboratory window, a few solar panels providing 

the power needed to operate the instrument (which is really 

minimal outside the power needed to operate vacuum 

pumps), and no terrestrial distractions, the only remaining 

impediment would be the wait that may be needed for appearance 

of a service engineer. The constrained transport 

chain for getting samples from Earth to the orbiting instrument 

might prove to be a blessing in disguise, serving to 

discourage submission of casual or repetitive samples, and 

focusing attention on properly prepared and validated samples. 

Should the return visit to Earth for the annual ASMS 

meeting become problematic, ASMS webcasts could prove 

useful. 

Such a scenario may be premature but it is not unrealistic. 

The location of mass spectrometers off-planet is not limited 

to the Viking instrument resident on the surface of Mars. 

Mass spectrometers have sampled the composition of other 

planetary atmospheres, comets, and interplanetary space 

itself. The need to design such instruments to meet the 

constraints of space and power, and to ensure robustness, 

has informed the development of the newest generation of 

mobile mass spectrometers for the rest of us. As with all 

design that pushes to extremes, it is a clear understanding of 

the basics that catalyzes progress. Note that the basic studies 

in extraterrestrial mass spectrometry (MS) extend back 

to the 1950s and 1960s. The designers of such instruments 

also had unique approaches to creating and maintaining a 

vacuum in the instrument. Creating a vacuum may be as 

simple as opening a port to space in transit. Maintaining 

a vacuum during descent through a planetary atmosphere 

requires careful consideration of the pressures that may 

be encountered, the composition of the gases encountered 

(these were in fact what was to be measured), and gas conductances 

within the system and through its ports. Perhaps 

the design would consider a control parameter involving

18 Spectroscopy 24(11) November 2009 


pressure measurements taken on-site, 

with the readings fed into a system 

that makes decisions on the fly. These 

complex topics deserve a more detailed 

exposition, which will appear in this 

column eventually. Until then, and to 

return to Earth orbit, interested readers 

might learn about the Wake Shield 

Facility (described at http://www.svec. 

uh.edu/wsfp.html), which takes advantage 

of the excellent vacuum available 

in the wake of the space shuttle. 

Earlier columns in “Mass Spectrometry 

Forum” covered general topics 

of vacuum systems (as well as our 

sometimes confusing uses of the terms 

vacuum and pressure), and the operation 

of high vacuum pumps (1,2). Here, 

to keep things simple, we will call any 

pressure below 1 atmosphere (760 

Torr) a vacuum. There are subsidiary 

terms of rough vacuum, low vacuum, 

high vacuum, and ultrahigh vacuum, 

with such terms corresponding, in 

order, to lower and lower pressures. 

The pressure in interstellar space, by 

the way, is about 10 -16 Torr, and the 

pressure within interplanetary space 

higher (depending upon where you 

are). These are isotropic gas pressures, 

and the fact that the interstellar pressure 

is so low is one reason why radiation 

pressure can be used to exert a 

force upon solar sails. 

The focus of this column is the 

measurement of pressure in a mass 

spectrometer, located somewhere on 

the surface of planet Earth (+/- 5 km) 

(3). The continued growth and diversification 

of MS should refocus our 

attention on the attainment of vacuum 

and the accurate measurement of pressures. 

At the heart of MS is the ability 

to create ions, to move them around, 

to differentiate them by their mass-tocharge 

ratios, and to detect them. For 

years, and certainly through the dominant 

era for electron ionization (EI) 

and chemical ionization (CI) sources, 

we thought of the MS instrument 

as under high vacuum from source 

through to the detector. We also came 

to know the vacuum pumping system 

as a high-cost, high-maintenance part 

of the instrument. Certainly, pumping 

systems evolved from the crude apparatus 

first used by Aston in his 

Source 

Inlet 

Ionization gauge 

Analyzer 

Diffusion pump 

Thermocouple gauge 

Rough 

pump 

Detector 

vent 

Figure 1: Simple diagram of placement of a thermocouple gauge (bottom) and an ionization 

gauge (top) on a generic mass spectrometer. 

Table I: Usual operational range for some common pressure gauges used 

in mass spectrometers. 

Device name 

Usual operational range (Torr) 

(760 Torr is 1 atmosphere of pressure) 

Mechanical gauge (various sorts) 1000 to 100 

Piezo or quartz sensor 760 to 1 

Capacitance manometer 1000 to 10 -3 

Thermocouple gauge 760 to 10 -3 

Ionization gauge 10 -4 to 10 -8 

parabola mass spectrographs, but the 

consistent general model was a highvacuum 

diffusion pump (or two) for 

the main system to achieve a high vacuum, 

with associated backing pumps 

(the rough pumps) that also could be 

plumbed into source interfaces for separators 

or direct insertion probes. Most 

pumps (including diffusion pumps 

and rotary vane backing pumps) transport 

gas molecules against a pressure 

gradient, so the ultimate exhaust for 

the backing pumps should be a hood 

vented to outside. The advent of reliable 

turbomolecular pumps did not 

shift the basic design of the pumping 

systems in our instruments, but added 

certain advantages of speed and pump 

placement. The basic goal still was to 

move the gas molecules from inside the 

system to outside of it. 

Within this general scheme, much of 

the plumbing intricacy of the vacuum 

pumping system was designed for the 

ionization source of the mass spectrometer, 

and the need to transport


samples from the outside world (760 

Torr) into the mass spectrometer 

(where the analyzer is at 10 -6 Torr). 

For example, a direct insertion probe 

would be fitted with a staged series of 

chambers, valves, and locks so that discrete 

samples at the laboratory pressure 

could be introduced safely into the 10 -5 

Torr EI source, or the 1 Torr CI source. 

Jet separators and membrane interfaces, 

and various other devices, were 

early interface devices that transported 

a stream of sample molecules from 

the exit of a gas chromatography (GC) 

system to those same under-vacuum 

sources, while pumping away most of 

the carrier gas and thereby enriching 

the concentration of sample. For liquid 

chromatography (LC), an even wider 

diversity of interface systems was devised 

because the evaporation of the 

LC solvent placed an even greater load 

on the vacuum-pumping system, and 

in the beginning at least, the ionization 

source was still operating at a pressure 

far below atmospheric pressure. Now, 

ionization sources operate at pressures 

above atmospheric pressure, and commonly 

at ambient pressure, such as 

electrospray ionization (ESI) sources, 

along with ambient sampling methods, 

and the problems of designing a successful 

interface are somewhat eased. 

At the other extreme, sources in surface 

science experiments may operate 

at pressures as low as 10 -9 Torr, and the 

idea of a chemical ionization reagent 

gas at 1 Torr pressure is anathema. 

Mass analyzers, however, still usually 

operate within the pressure range of 

10 -5 to 10 -7 Torr, with the notable exception 

of the ion trap. 

Instrument operators have to be 

aware of what pressure regime is 

proper for each part of a more complicated 

instrument, and measure and 

monitor those pressures for optimum 

and stable instrument operation. A 

rule-of-thumb in practical troubleshooting 

for erratic or deteriorating 

instrument operation is to “check the 

vacuum first.” The reason is explained 

directly in a short commercial publication 

about the need for accuracy in low 

pressure measurements (4): “But when 

it comes to high vacuum measurement, 

accuracy seems not to matter. 

We speak of being in “the 6 range” or 

in “the low part of the 8 range.” We 

seem to ignore the fact that the 6 range, 

that is, the pressure difference between 

1 X 10 -6 and 1 X 10 -5 Torr, involves 

a change in gas density of 10 times. 

We seem to think it is unimportant 

whether we are at 2 X 10 -8 or 1 X 10 -8 

Torr: “Not to worry. It’s okay. We’re in 

the 8 range.” Why does it matter if we 

pump to 2 X 10 -8 rather than to 1 X 

10 -8 Torr? At the higher pressure, we 

will have twice the density of molecules 


present as at the lower pressure. If this 

presents no problem, why then are we 

spending time and money pumping 

to a lower pressure than required? If 

a pressure of 2 X 10 -8 Torr is satisfactory, 

could we use 3 X 10 -8 Torr and 

save some pumping time? After all, 

nature abhors a vacuum, and reducing 

the pressure by a factor of two or more 

does not come free.” If the apparent 

sensitivity of an MS instrument is off 

10–15%, or seems to vary erratically, 

a “small” change in pressure in the


mass analyzer may be the cause. The 

author of this quote addresses the need 

for accurate pressure measurement in 

a vacuum chamber used for materials 

processing, but the lesson is valuable. 

For the mass spectrometer, scattering 

collisions reduce ion transmission, 

and reduce instrument sensitivity. A 

“small” change in pressure can lead 

to an amplified degradation in performance. 

Pumps “burp” for various 

reasons, and the short pressure surge 

takes time to dissipate. Erratic instrument 

performance may be your only 

clue if you are not carefully monitoring 

the pressure. The author may be the 

only mass spectrometrist in the world 

who would become concerned with a 

shift in measured base pressure from 

2 X 10 -6 to 3 X 10 -6 Torr, but now you 

know why. 

Performance of the vacuum pumping 

system of a mass spectrometer 

is monitored through measurement 

of the pressure at various points in 

the system. These measured pressures 

may or may not be logged into 

the automated control system of the 

mass spectrometer. In process vacuum 

chambers, system pressures certainly 

are recorded so that the conditions of 

materials processing are known precisely. 

But in analytical mass spectrometers, 

pressure measurement is often 

manually monitored. Consider Figure 

1, which is a schematic of a diffusion 

pump attached to the main vacuum 

chamber, supported by the backing 

pump (also known as the rough pump), 

with the system exhaust routed to a 

hood. Pressures within the backing 

pump lines are monitored (using the 

thermocouple gauge) as an indicator of 

the total gas load on the system, and to 

ensure that the main diffusion pump is 

properly supported. The main chamber 

pressure is monitored with the 

ionization gauge. The diffusion pump 

can operate for a short time at higher 

backing pressures, but prolonged operation 

leads inevitably to a rise in main 

system pressure and a degradation of 

the pumping fluid. In a simple EI mass 

spectrometer, monitoring the two pressures 

(the backing pump line thermocouple 

gauge and the main chamber 

ionization gauge) usually is sufficient 

for reassurance that the instrument 

was properly pumped. 

Details of how each measurement 

device works are explored in specialized 

texts (5–8) and commercial manufacturer’s 

applications notes, reflecting 

the broad application of vacuum 

science outside of MS. We concentrate 

here on the thermocouple gauge and 

the ionization gauge, because these are 

the most common devices found on 

mass spectrometers, usually configured 

approximately as shown in Figure 

1. Table I shows the operational pressure 

range for the thermocouple gauge 

and for the ionization gauge, and a few 

other devices included for comparison. 

Performance of 

the vacuum 

pumping 

system of a mass 

spectrometer is 

monitored through 

measurement of 

the pressure at 

various points in 

the system. 

Note that in addition to monitoring 

the pressure in the backing lines, thermocouple 

gauges also can be used to 

monitor vacuum pressures in sample 

introduction interfaces. Note that for 

sources that operate at atmospheric 

pressure, the ambient pressure usually 

is not measured. We emphasize in this 

column three basic issues relevant to 

the vacuum pumping system in a mass 

spectrometer. First, the vacuum gauge 

produces an electrical output that can 

be related to pressure through a calibration, 

and not all residual gas mixtures 

follow the same calibration curve. 

Second, the pressure measured is the 

pressure at the gauge, not necessarily in 

the system, and so we have to consider 

conductance. Third and finally, proper 

vacuum-pumping system care 

maintains instrument performance. 

Calibration: Like any transducer, 

a vacuum gauge must be calibrated. 

In MS, that calibration entails some 

special issues. The composition of a gas 

mixture at Earth atmospheric pressure 

contains an expected mix of nitrogen, 

oxygen, water, carbon dioxide, argon, 

and some trace components. Does this 

mixture change in its relative composition 

as a pump lowers the pressure in 

a vacuum chamber? It most certainly 

does, because various pumps may act 

to pump one gas component more effectively 

than another. Thus the composition 

of that starting gas mixture 

at 10 -3 Torr will be slightly different 

from the composition at atmosphere, 

even more different at 10 -6 Torr, and 

vastly different at 10 -9 Torr. For the 

pressure-measuring devices, a calibration 

should be completed so that the 

electrical output can be related to the 

actual pressure, and the calibration 

must factor in the composition of the 

gas. Any device reacts to different gases 

with a different sensitivity, and thus 

exhibits a different calibration curve. 

A thermocouple gauge that measures 

1 Torr of pressure when the composition 

is residual atmospheric gases will 

be slightly different in response from 

a thermocouple gauge reading 1 Torr 

of methane in a CI source. Many commercial 

companies offer calibration 

services for vacuum gauges. In MS, 

traceability to a NIST or other national 

standard is not usually necessary. NIST 

calibration is described in detail in 

publications available on the web (9,10). 

Conductance: The connection of 

a vacuum gauge to the mass spectrometer 

deserves some discussion. 

Consider the two situations depicted 

in Figure 1 for a thermocouple gauge 

and for an ionization gauge. The thermocouple 

gauge is connected directly 

into the vacuum line, and many gauges 

are built into threaded assemblies for 

such a purpose. We can be assured 

that the pressure measured by the 

thermocouple gauge is the pressure in 

the line because of this direct connection. 

On the other hand, consider the 

connection of an ionization gauge to 

the main vacuum chamber of a mass 

spectrometer. Sometimes the glass-en-



closed ionization gauge is found in a T 

configuration equipped with a bolted 

vacuum machine flange. This vacuum 

flange is then bolted to the receiving 

flange teed off the main chamber. 

We want to know the pressure in the 

main chamber, but what we actually 

measure is the pressure at the gauge, 

which is related to the pressure in the 

chamber through the conductance of 

the connection. As a rule, instrument 

design should maximize conductance 

between the vacuum gauge and the 

chamber. For example, large diameter 

connections have better conductance 

than do smaller-diameter connections, 

and curved 90° bends have better conductance 

than do right-angle bends. 

To minimize possible issues of conductance, 

a direct mounting of the gauge 

is available with the use of a “nude” ion 

gauge. Here the analyst must be aware 

of the ionic and photonic emission of 

the gauge itself, as well as its fragility. 

Care: A vacuum pumping system 

does not thrive on neglect, but this 

author’s experience is that few currently 

practicing mass spectrometrists 

receive basic education in vacuum 

science. For example, a proper pumpdown 

technique (11,12) is part of the 

necessary care of a vacuum system 

and requires more attention than 

usually documented. Additionally, a 

good maintenance schedule includes, 

for example, attention to air filters, 

water filters, exhaust filters, flushing of 

water lines, pump oil replacement and 

proper disposal, seals testing, leak testing, 

replacements for degraded seals 

and hoses, and general system cleanliness. 

Pumps sited away from the mass 

spectrometer for purposes of noise 

abatement or vibration isolation are 

especially likely to fall off the maintenance 

schedule. “Bake-out” is a term 

that is not much used anymore, and it 

is doubtful that the reasons for completing 

one are still known. Finally, 

while source ionization cleanliness 

usually is acknowledged to be vital for 

good instrument performance, the 

cleanliness of the rest of the system 

(comprising the preponderance of 

surface area for the vacuum system) 

rarely is considered. Maintenance is 

not the most pleasant work of the MS 

laboratory, but it is one of the more 

important ones. Let the author know if 

you have assembled a workable reward 

structure that results in proper maintenance 

— “beer and pizza” will not be 

considered an adequate response. 

Good vacuum system design is a 

crucial underpinning for high performance 

instrumentation. In “Mass 

Spectrometry Forum,” our range of 

topics is extraordinarily broad, ranging 

from the basics of electronic and 

vacuum technology through to the 

ethical use of MS data. The important 

aspects of pressure and vacuum need 

regular teaching, and we will return 

to additional topics in these areas in 

future columns. 

References 

(1) K.L. Busch, Spectroscopy 15(9), 22 

(2000). 

(2) K.L. Busch, Spectroscopy 16(5), 14, 

(2001). 

(3) There are mass spectrometers that 

operate in the undersea environment,


and also instruments that operate in 

the lower reaches of the atmosphere. 

Interfaces will differ, of course, but 

the basic pumping needs within the 

instrument itself remain similar. Outside 

that range, design considerations 

diverge. 

(4) Granville-Phillips, Issues in Vacuum 

Measurement, “It’s a Myth that 

Less Accuracy is Needed for Low- 

Pressure Measurement.” Found at 

http://www.brooks.com/documents. 

cfm?documentID=4879. 

(5) K. Jousten, Handbook of Vacuum 

Technology (Wiley-VCH, New York, 

New York, 2008). 

(6) J.F. O’Hanlon, A User’s Guide to 

Vacuum Technology (John Wiley and 

Sons, Hoboken, New Jersey, 2003). 

(7) D.J. Hucknall and A. Morris, Vacuum 

Technology: Calculations in Chemistry, 

(Royal Society of Chemistry, UK, 

2003). 

(8) A very useful compendium of vacuum 

technology references is found at: 

http://www.atomwave. 

org/rmparticle/ao%20refs/ 

aifm%20refs%20sorted%20by 

%20topic/beam%20detectors/ 

Vacuum%20References.doc. 

(9) S. Dittmann, “NIST Measurement 

Services: High Vacuum Standard and 

Use, 1989.” Found at http://ts.nist. 

gov/MeasurementServices/Calibrations/upload/SP250-34.pdf 

(10) J.H. Hendricks , P.J. Abbott, J.E. 

Ricker, J.H. Chow, and J.D. Kelley, 

“Development of a New NIST Calibration 

Service Using the Comparison 

Method for Vacuum Gauges Spanning 

the Range 0.65 Pa to 133 kPa,” found 

at www.cstl.nist.gov/projects/fy06/ 

indst0683608.pdf. 

(11) See http://www.vacuumlab.com/Articles/VacLab32.pdf. 

(12) See http://www.plasma.org/activity/ 

groups/subject/vac/Events_Archive/ 

file_8392.ppt. 


grew up in the era of 

belt-driven rough pumps. 

KLB always remembers to 

put a leak tray under his 

rough pumps, he knows 

what a sight glass is, and 

he knows the chemical molecular composition 

of many diffusion pump oils. In his 

youth, with a steadier hand, he could replace 

filaments in nude ionization gauges. 

Extra bonus points with no redeemable 

cash value are sent via email to those who 

deduce the pun not quite hidden in this 

column. No hints provided. Responsibility 

for this column resides solely with the author, 

who can be reached at: 

wyvernassoc@yahoo.com 

For more information on 

this topic, please visit: 

www.spectroscopyonline.com/busch


Focus on Quality 

The Tiger Has Sharp New Teeth 

The new FDA Commissioner wants a strong FDA and is backing her words with action by initiating 

a program that cuts the time that firms must respond to 483 observations from 30 to 

15 business days. Not only is the time halved but the response must be complete. Therefore 

it is better and cheaper to be compliant than not. 

R.D. McDowall 

The Food and Drug Administration (FDA) has not had 

good press over the last few years, as they have been on 

the receiving end of two critical reports from the Government 

Audit Office (GAO). The last report covered the 

FDA foreign inspection program and was damning in the 

fact that the FDA does not know how many establishments 

it has to inspect, relies upon volunteers to inspect overseas, 

and when they do conduct inspections, the frequency is 

lower than in the U.S. (1). 

FDA Modernization Act 2009 

As a result of this report and also the increased threat of 

supply chain contamination, the FDA Modernization Act 

2009 (2) is being passed into law and covers the following 

areas: 

• Creates an up-to-date registry of all drug and device facilities 

serving American consumers. 

• Increases funding for more GMP inspections for ethical 

and generic drugs as well as introduces preapproval inspections 

for generic drugs. 

• Requires parity between foreign and domestic inspections, 

and to meet this, the FDA is setting up offices in 

China, India, Europe, and Latin America. 

• Denies entry to drugs coming from facilities that limit, 

delay, or deny FDA inspections. 

• Requires manufacturers to know their supply chain including 

the identification and mitigation of risk throughout 

their supply chain. 

• Requires country-of-origin labeling for components. 

Concomitantly, there also has been an increase in budget 

to fund this and increase the inspection program. So what 

does that mean for me in the laboratory? This stuff appears 

to be too high a level to even contemplate doing anything 

about. What’s this about a tiger and new teeth? It may seem 

that the tiger has been stuffed but the good bits start in the 

next section. 

New FDA Commissioner Acts 

In addition to the FDA Modernization Act 2009, there also 

has been a change at the top with the appointment of Dr. 

Margaret Hamburg as the new FDA commissioner in May 

2009. On August 6, 2009, the new commissioner made a 

speech that emphasized the need for a “strong FDA,” which 

highlighted the benefits of this new approach as having 

credibility with the public, being transparent in explaining 

its decisions, being able to enforce the law, and being creative 

in promoting health. In this speech, with an accompanying 

video (3), were such quotations as 

• “Through regular inspections and follow-up on signals 

indicating problems, the FDA must work to identify and resolve 

problems early.” 

• “Companies must have a realistic expectation that if 

they are crossing the line, they will be caught, and that if 

they fail to act, we will.” 

• “The agency must show industry and consumers that we 

are on the job. We must publicize our enforcement actions 

— and the rationale for those actions — widely and 

effectively.”


• “The agency must place greater 

emphasis on significant risks and violations, 

and use meaningful penalties 

to send a strong message to discourage 

future offenses.” 

The strong message coming through 

loud and clear is be compliant and 

remain compliant with the regulations 

— or else. You’ll now see that the tiger, 

far from being ready for the taxidermist, 

is starting to sharpen its teeth. 

Not only does this apply to the 

here-and-now but also, as noted in the 

final bullet point, the commissioner 

wants to discourage future offences. 

So why change the FDA approach to 

enforcement? One of the rationales for 

this was mentioned in the speech as 

the pathways for enforcement action 

can be too long and arduous when the 

public’s health is in jeopardy. So from 

this and the FDA, we must infer that 

the FDA will be increasing inspections 

while tightening up and speeding up 

enforcement actions. 

Thus. it was not surprising that the 

FDA announced in the Federal Register 

of August 11 (4) the trial of a new 

program entitled “Review of Post-Inspection 

Responses.” The purpose of 

this program is to facilitate the timely 

issuance of warning letters that started 

on September 15, 2009 and will run for 

18 months (the tiger has just come back 

from the dentist and is feeling hungry 

— are you getting the message?). 

Issues with the Current 

Inspection Process 

Before we look in detail at the inspection 

goodies coming your way, I would 

just like to step back and review what 

happened before implementation of 

this new program. FDA inspections 

come in three flavors and will continue 

to do so: 

• Facility inspection: routine inspection 

of the facility and the six areas 

quality — however, we will just focus 

on the quality system and the laboratory 

in this column. 

• Preapproval inspection (PAI): inspection 

of the manufacturing facility 

for a new drug or a generic drug before 

licensing. 

• For cause inspection: either there 

is a public health issue that must be 

Late 

response 

Establishment 

Inspection 

Report (EIR) 

Regulatory 

action 

stopped 

Adequate 

response 

rapidly investigated or a whistleblower 

has alerted the agency. In either case, 

the first thing you’ll probably notice is 

the knock of the inspection team at the 

front door of your site. 

Regardless of the inspection type, 

the inspection follows the same format 

and begins with the inspectors 

FDA inspection 

483 

observations 

15 days 

Response 

letter 

Inadequate response 

Warning 

letter 

15 days 

Response 

letter 

Inspection 

follow-up 

Closeout 

letter 

Serious 

violations 

Figure 1: Process flow of the new FDA postinspection response program. 

Serious 

violations 

Further 

enforcement 

action 

presenting their credentials and a copy 

of FDA Form 482, which is the notice 

of inspection, at the opening meeting. 

During the inspection, they may notice 

noncompliances and these are documented 

on FDA Form 483 entitled 

“Inspectional Observations” (this is 

the dreaded 483!), which is given to the



company at the closing meeting of the 

inspection. Written on each page of 

Form 483 is the following wording: 

“This document lists observations 

made by the FDA representative during 

the inspection of your facility. 

They are inspectional observations, 

and do not represent a final agency 

determination regarding your compliance.” 

The company currently has 30 

working days to respond to the 483 but 

what happens in practice is that a first 

response is presented within the time 

limit and then further letters are fed in 

over a few months for the more serious 

issues. If you read some of the warning 

letters on the FDA website, you’ll see if 

the inspection occurred in January, the 

warning letter finally emerges into the 

light 6–9 months later. The reason for 

this is that there has been protracted 

correspondence between the company 

and the FDA. This will stop under the 

new program. 

Warning letters are only issued for 

significant violations from the regulations. 

After the warning letter has been 

sent out to the company, a copy is hung 

on the “Wall of Shame” in the warning 

letter section of the FDA’s website. 

Here anyone can have a good laugh 

about the problems of another company 

while secretly hoping that they 

will not have a starring role in a future 

warning letter. The response time for 

the company to respond to a warning 

letter is 15 working days; in the new 

program, this also will be the response 

time for a 483. 

Regardless of whether you get a 

483 or not, the FDA inspectors will 

write a detailed Establishment Inspection 

Report (EIR) of the inspection 

that details the people they had talks 

with and the procedures, systems, 

and records that were inspected; this 

document provides the agency with 

its elephant’s memory. Failure to correct 

the noncompliances outlined in 

a warning letter may lead to further 

enforcement action by the FDA, and 

ignoring a warning letter is not advisable. 

However, the agency has often 

issued further warning letters to companies 

in the past, a practice that will 

apparently stop with the new program. 

Further regulatory enforcement by the 

FDA can include a consent decree of 

permanent injunction, which will bind 

the company in perpetuity to comply 

with the regulations; this can cost a 

company millions of dollars or even its 

existence or withdrawing a product licence. 

For foreign companies, the FDA 

can ban import of its products. 

483 Complaints Procedure 

Under the “GMP for the 21st Century” 

initiative, the FDA issued a guidance 

for industry with a delightful title of 

“Formal Dispute Resolution: Scientific 

and Technical Issues Related to Pharmaceutical 

CGMP” (5). In essence, this 

document encourages the company 

and the inspector to discuss and resolve 

everything while the inspection 

is still ongoing. However, if this is not 

possible, only with regard to observations 

where it is down to interpretation 

is a company allowed to appeal to the 

FDA in a formal, two-tiered response. 

For example, if you don’t keep 

complete records of a spectroscopic 

analysis, you will not have a leg to 

stand on as the regulations explicitly 

require you to do this. Here you are 

noncompliant and you have been 

caught — no discussion and no argument. 

In contrast, if the regulations are 

vague and it is down to differences of 

interpretation between the company 

and the inspector, then this is where 

the dispute procedure can be used. 

Postinspection Response 

Program 

So what has changed with this new 

program? Two changes that will impact 

laboratories is that a very short 

timeframe is being put around how 

quickly firms must respond to 483 

observations, and the second is that 

there will be a formal close out of 

warning letters to demonstrate that 

the noncompliances listed there have 

been resolved. So let’s look in detail at 

what you have to do and you will now 

see that the tiger has sharp new teeth 

and that they will be sinking into your 

flesh. Figure 1 shows the process as a 

diagram and readers should look at 

this to put the text below into context.


• FDA has now set postinspection 

deadlines for responses by organizations. 

The response time for firms to 

respond has been halved to 15 working 

days. This is not just to send a letter 

to begin a cozy correspondence with 

the agency but to have a complete 

response to all the 483 observations. 

If the compliance problems are significant, 

the timeframe for the response is 

critical and the response must be full, 

adequate, and complete. The letter will 

not be able to show that the problems 

have been resolved for all but the simplest 

observations but will demonstrate 

how they will be resolved and in what 

timeframe. If a response is not received 

in this time or the response is inadequate, 

then the agency can start work 

on a warning letter or other enforcement 

action. We’ll take a look at ways 

to respond to a 483 or warning letter in 

a later section of this column. 

An important point to understand 

here is that the FDA now takes the 

systems-based approach to inspections 

and coupled with ICH Q10 on 

pharmaceutical quality systems (6) 

you will need a different approach to 

respond to 483 observations. If there 

is an observation that has an impact 

outside of the area inspected, you will 

be expected to fix it as part of the systems-based 

approach. Remember, the 

wording at the end of virtually every 

warning letter on the FDA website: 

“this letter is not intended to be an 

all-inclusive list of the violations at 

your facility. It is your responsibility 

to ensure compliance with applicable 

laws and regulations administered by 

FDA.” However, there is an onus on 

the inspector to discuss findings with 

management to minimize surprises 

and errors when issuing the 483 at the 

inspection closing meeting. 

• The warning letter issuing process 

is being streamlined and speeded 

up by the FDA. Instead of the FDA 

lawyers reviewing all warning letters, 

only those where there are significant 

legal issues will now get the legal eagle 

eye. The agency will not delay issuing 

a warning letter if you are late in 

responding to the 483. Even if your 

response to the 483 is perfect, the FDA 

does “not plan to routinely include a 

response on the apparent adequacy 

of the firm’s corrective actions in the 

warning letter (4).” Instead, the agency 

will respond to evaluate both your 

response to the 483 and the warning 

letter together. Therefore, the message 

is clear to all: Your 483 response must 

be timely and complete, or else you 

could end up with a warning letter on 

the front doormat pretty quickly afterwards. 

• Next, the FDA will prioritize 

enforcement follow-up. After a warning 

letter is issued or a major product 

recall occurs, the FDA will make it a 

priority to follow up promptly with 

appropriate action, such as an inspection 

or investigation to assess whether 

or not a company has made required 

changes in its compliance practices. 

• In the past, it was not uncommon 

for a company to have a number 

of warning letters after several inspections; 

it may be the companies



involved thought that they could 

manage the warning letters, the FDA, 

and still do business. However, under 

this new program, the agency will 

not issue multiple warning letters 

like confetti to noncompliant firms 

before taking greater enforcement action. 

The agency will move quickly 

and aggressively to address significant 

health concerns or serious violations 

by immediate action — even before 

they have issued a formal warning letter. 

This message is crystal clear — be 

compliant or there will be regulatory 

trouble and the FDA will move to 

protect public health. 

• In a novel approach to noncompliance, 

the FDA will now issue a 

close-out letter to companies issued 

warning letters to document that they 

have made necessary corrections. As a 

copy of this close-out will be posted on 

the FDA website alongside the warning 

letter, readers will be able to see 

how quickly companies respond to the 

regulator. It is hoped that this will be 

an important motivator for companies 

to fix the problem quickly. 

So does the FDA have sharp new 

teeth or are they toothless? It is your 

call to decide, but as a word of advice, 

be compliant and inspection-ready in 

your laboratory. 

Are You Inspection-Ready? 

Remember, the cost of compliance is 

always, always, cheaper than the cost 

of noncompliance. You can decide how 

to do things rather than rush through 

something with a regulator breathing 

your neck. Therefore, the easiest way 

to respond to a 483 observation is to be 

compliant and don’t get one. However, 

we have to manage risk in the industry 

and this is probably unrealistic. So, 

don’t get a serious 483 observation. 

You’ll need to have a quality system 

that is defendable, as this is always 

better than arguing with the inspector 

on the day of the inspection. Rather 

than just sit there and wait for the next 

inspector to drop, all staff should be 

vigilant and ensure that they work in a 

compliant manner and — this point is 

very important — improve the current 

processes and the associated compliance. 

This is not just a job for quality 

assurance personnel who conduct 

internal audits and document checks, 

it is everybody’s job. This internal effort 

must be coupled with an increased 

program of laboratory audits aimed at 

verifying what the real level of compliance 

is in your organization. It is 

all well and good knowing that in six 

months time an inspection is due and 

that you can prepare for it. When the 

inspector comes around the laboratory 

is clean and tidy and all the staff are in 

their Sunday best. However, what is the 

reality of the day-to-day operations? 

Here, audits can be a very effective 

means to determining the real level of 

compliance. Not just planned audits 

that analytical scientists know will 

occur and can prepare for but unannounced 

audits that give the real level 

of compliance and inspection readiness 

of a laboratory. This latter type 

of audit can be disruptive but is a vital


means of avoiding the 15-day postinspection 

panic that will become a 

feature of noncompliant organizations 

in the coming months. Also, from personal 

audit experience, it is very useful 

to use a digital camera to record some 

types of noncompliance such as untidy 

laboratories or missing calibration 

stickers on equipment as the auditee 

has less wiggle room when the evidence 

is in the report. To be effective, 

audits need to be conducted by trained 

staff external to the laboratory under 

audit to remove bias from the results. 

Responding Effectively to a 483 

Observation 

Ok, so you ignored the advice above 

and now you have one or more 483 

laboratory observations — so what are 

you going to do? Remember, the clock 

starts ticking 15 days from the closing 

meeting so if you want to avoid the FDA 

tiger taking a bite out of various portions 

of your anatomy, you had better 

start thinking and quickly. A good option 

is to talk through the observation 

with the inspector while he or she is still 

on site to clarify the issue and to inform 

the inspector what action will be taken 

to resolve the problem. This should help 

you understand the problem and to get 

feedback if this is a way to bring you 

back into compliance. 

The response to the agency must be 

formal and that is typically with a covering 

letter written at a minimum by 

the site head or better still by the chief 

executive officer. This is intended to 

demonstrate to the FDA how seriously 

the firm takes the situation and that it 

also provides a written commitment to 

comply voluntarily. 

This will be coupled with the attachments 

discussing how the company will 

resolve the inspection observations. 

The documents may be sent initially 

by fax or e-mail but it always should be 

followed up with a hard copy couriered 

to the field office. To write the attachments, 

you will need to gather the experts 

in the organization who will help 

draft the response to specific observations. 

Also, you should know when you 

have to obtain external expertise to help 

you. This is especially important with 

the new program, as you only have a 

maximum of 15 days before the time 

limit is up and external resources may 

need time to mobilize. The attachments 

will detail how you will resolve each of 

the observations or noncompliances. It 

is extremely important that these are 

laid out clearly: therefore, address each 

observation at the beginning of a new 

page. Ensure also that the response 

numbering is the same as the observation 

numbering in the 483. This makes 

it easier for the official at the FDA to 

correlate the material (remember your 

reader!). 

For each observation: 

• Do you agree or disagree with the 

inspector’s observation? 

Acknowledging that this is correct 

means you understand the noncompliance. 

Coupled with this needs to be 

an understanding of the underlying 

regulation that has caused the observation: 

Is your interpretation correct and 

how has this been documented in your 

SOPs? 

• A root cause analysis may be important 

for some observations as the 

underlying cause of the observation 

may not be immediately apparent in 

many cases. 

• Either provide: 

A description of a completed corrective 

action with evidence of the work 

carried out. 

A corrective action plan with evidence 

of work carried out to date. 

A corrective action plan with sufficient 

detail and a reasonable timescale 

outlining what will be done and by 

when. Will you be working with external 

experts who will help and guide you 

in your corrective action plan? 

• Make sure that the work package to 

resolve the observation is specific and 

complete: for example, if an SOP is to be 

updated as there is an omission, provide 

a copy of the new SOP but also outline 

what training was provided to the impacted 

staff with copies of the material 

and updated training records. 

• Realism is important as the FDA 

officials reviewing your response are 

not idiots. As you cannot validate a 

laboratory information management 

system (LIMS) in one month, don’t 

expect the FDA to believe you if you put



this in your response letter. Credibility 

is a vital commodity here — do not lose 

it. Therefore, be able to ensure that you 

can deliver on the plan and have the 

resourcing to do this. Strangely, at times 

like this, words such as “budget” and 

“constraint” are never found together in 

the same sentence. 

• Addressing just the observation 

is like putting a band-aid on a broken 

leg: you need to understand what the 

impact is on other parts of the organization, 

other products, methods, instruments, 

systems, and so forth. This is 

the systems-based approach. For larger 

organizations, it may not be at just one 

site but globally. Remember, it is the 

EIR that will provide the FDA with ammunition 

to look at other sites and if 

they find the same problems — you are 

in trouble. 

• It’s all well and good providing 

plans and schedules for doing the work. 

How do you know the work will resolve 

the problem? More importantly, how 

do you know if the problem will not 

reoccur in the future? Therefore, you 

must anticipate these questions from 

the agency: How will you verify that the 

work has been done, what documentation 

will be available to demonstrate 

this, and how will you monitor the area 

to ensure that it does not happen again? 

A well-constructed response letter 

delivered within the new 15-day timeframe 

will go a long way toward restoring 

your regulatory credibility. Still, 

you could look on the bright side: The 

program is an 18-month trial, so will 

the FDA drop this approach after the 

trial ends? I don’t think so, look at the 

resume of the new commissioner, read 

the speech and watch the video. I think 

this new program is here to stay. We 

will see an initial increase in warning 

letters but these will tail off as industry 

ensures it stays in compliance but it will 

be a rough time initially, as the tiger 

builds up momentum with its sharp 

new teeth. 

and you had better beware — complete 

responses to the 483 observations are 

required by 15 working days after the 

inspection or a warning letter or increased 

enforcement action may ensue. 

I have also outlined a practical way for 

audits to check you are in compliance 

but if that fails, a means to respond to 

483 observations. But remember, time is 

not on your side here. 

References 

(1) “Better Data Management and More 

Inspections are Needed to Strengthen 

FDA’s Foreign Drug Inspection Program,” 

U.S. Government Accountability 

Office, Report GAO-08-970, 

September 2008. 

(2) HR 759 FDA Modernization Act 2009. 

(3) “Law Enforcement and Benefits to 

Public Health,” Margaret Hamburg, 

6th August 2009. 

A copy of the speech is available at 

http://www.fda.gov/NewsEvents/ 

Speeches/ucm175983.htm and the 

video of the speech can be accessed 

on the same page. 

(4) Review of Post-Inspection Responses 

Federal Register, August 11, 2009, 74 

(153) 40211–40212. 

(5) “Guidance for Industry, Formal Dispute 

Resolution: Scientific and Technical 

Issues Related to Pharmaceutical 

CGMP,” FDA January 2006. 

(6) International Conference on 

Harmonisation (ICH) Q10: Pharmaceutical 

Quality Systems 

step 4 (see www.ich.org). 

R.D. McDowall 

is principal of McDowall 

Consulting and director 

of R.D. McDowall 

Limited, and “Questions 

of Quality” column editor 

for LCGC Europe, 

Spectroscopy’s sister magazine. Address 

correspondence to him at 73 Murray 

Avenue, Bromley, Kent, BR1 3DJ, UK. 


Summary 

In this column, I have looked at the 

new postinspection response program, 

which was introduced on an 18-month 

trial by the FDA on the September 15, 

2009. The tiger’s new teeth are sharp 

Visit Us at MRS Fall Booth 916

30 Spectroscopy 24(11) November 2009 


Atomic Perspectives 

Advantages and Disadvantages 

of Different Column Types for 

Speciation Analysis by 

LC–ICP-MS 

With the growing popularity of speciation analysis, there are many options to consider on 

the chromatography side. Given that many speciation users have spectroscopy backgrounds, 

it is important to understand some fundamental concepts of liquid chromatography (LC) 

before deciding upon methodology to pursue. This article focuses on three aspects of LC 

that should be considered before embarking on speciation analysis: ion exchange versus 

reversed-phase ion pairing separations, narrow bore versus standard bore LC columns, and 

ultrahigh-pressure LC versus traditional high performance liquid chromatography (HPLC). 


Speciation analysis by liquid chromatography–inductively 

coupled plasma-mass spectrometry (LC–ICP- 

MS) has been growing rapidly in popularity and application 

over the past several years. Not only have people 

begun looking at different elements and species, but there 

has been an increase in the variety of matrices that speciation 

analysis is being performed on: various types of environmental, 

waste, and process waters; both solid and liquid 

foods; and biological fluids. With this increased interest, 

more laboratories are offering speciation analysis as service. 

When deciding upon a speciation method, many factors 

must be considered. 

The chromatographic separation of components is based 

upon competition between the column, mobile phase components, 

species, and the sample matrix. With the variety 

of matrices now being analyzed, there are a variety of chromatographic 

options available. This column will discuss 

three column types and the relative advantages and disadvantages 

of each. 

Ion-Exhange Versus Reversed-Phase Ion-Pairing 

Chromatography 

Two common types of separation schemes used in chromatography 

are ion-exchange and reversed-phase ion-pairing 

chromatography. These schemes differ primarily in the 

column stationary phase, which affects the way components 

compete for active sites on the column. 

Ion-exchange chromatography involves the interaction of 

ionic (that is, charged) components in the mobile phase with 

charged stationary groups on the column packing material. 

Charged species in the sample compete with mobile phase 

components for sites on the column. Species with a stronger 

attraction to column sites than mobile phase components 

will be retained on the column longer and have longer



Anion 

exchanger 

A _ 

elution times. Species with a weaker attraction 

to the column than the mobile 

phase components will have shorter 

elution times. This is the mechanism 

by which separation is achieved and is 

represented in Figure 1. 

Reversed-phase ion-pairing chromatography 

involves the use of reversedphase 

columns that are characterized 

by nonpolar stationary phases composed 

of carbon chains, typically 18 

carbons (C18), although other columns 

exist with different length carbon 

chains (for example, four and eight 

carbons). Because most inorganic species 

exist in charged states in solution, 

it appears that reversed-phase columns 

A _ 

A _ 

NH 4 

+ 

CI- 

CI- 

=Analyte - CI 

A _ 

CI- 

+ 

NH 4 

CI- 

A _ 

= Counterion 

Figure 1: Schematic of the ion-exchange separation mechanism. 

Support O-C18 Organic - NH 3 

(+) 

Bonded reversedphase 

packing 

Ion pairing 

reagent 

(in mobile phase) 

Flow 

Figure 2: Diagram illustrating the reversed-phase ion-pairing mechanism. 

(-) O-R 

Ionic 

compound 

would not be able to perform the 

separation. 

This situation is rectified by incorporating 

an ion-pairing reagent in the 

mobile phase. Ion-pairing reagents 

consist of both nonionic and ionic 

parts: The nonionic sections interact 

with the carbon chain on the column, 

and the ionic sections interact with 

the charged species in solution by 

mechanisms similar to those in ion-exchange 

chromatography. In this way, a 

reversed-phase column can be made to 

perform like an ion-exchange column. 

The mechanism of reversed-phase ionpairing 

chromatography is shown in 

Figure 2. 

Both ion-exchange and reversedphase 

ion-pairing chromatography 

each have their advantages and disadvantages. 

One of the main advantages 

of ion exchange is that there is only 

one interaction involved in the separation: 

the analytical species interacting 

with the stationary phase. With reversed-phase 

ion pairing, two interactions 

are involved with the separation: 

the ion-pairing reagent interacting 

with stationary phase of the column 

and the species interacting with the 

ion-pairing reagent. As a result, ionexchange 

chromatography may have 

more matrix tolerance. 

The downside of ion-exchange chromatography 

is that these columns typically 

are much more expensive than 

reversed-phase columns. Also, because 

reversed-phase columns have been in 

use for many years, they are reliable 

and established, meaning that results 

will be reproducible from one column 

to the next. This may not always 

be true with ion-exchange columns 

because new columns are constantly 

being developed with new ionic groups 

bound to the stationary phase. Because 

of this constant evolution, some ionexchange 

columns may not always 

be well established, which could lead 

to irreproducibility from column to 

column. 

The decision to use either ion-exhange 

or reversed-phase ion-pairing 

chromatography depends upon the 

application: the elements and species 

of interest, the sample matrix, and the 

levels of the different species that need 

to be measured. 

Standard-Bore Versus 

Narrow-Bore Columns 

Regardless of which type of column 

is chosen, users are then faced with 

another choice: column diameter. For 

speciation analysis, the two most common 

column diameters used are 4.6 

mm (referred to as standard bore) and 

2.1 mm (narrow bore). Again, each diameter 

offers specific advantages and 

limitations. 

Standard-bore columns are available 

in a variety of particle sizes, ranging 

from 3 µm to 10 µm. Depending upon 

the size, the optimal high performance


HETP (µm) 

60 

40 

20 

0 

0 2 4 6 8 

Flow Rate (mL/min) 

liquid chromatography (HPLC) flow 

rate for achieving the best separation 

can vary from 0.7 mL/min to 2 mL/ 

min, as shown in the Van Deemter 

plot in Figure 3. In this figure, the 

HETP label on the y-axis refers to the 

height equivalent to a theoretical plate 

— the lower the HETP, the better 

the separation. As shown in the Van 

Deemter plot, the best separations are 

achieved with 3-µm packings and a 

flow rate of 1.5–2 mL/min. ICP-MS 

can handle 1.5-mL/min sample introduction 

rates without a problem, so 

the best separations are achieved with 

a 3-µm packing and an LC flow rate 

of 1.5 mL/min. These conditions are 

well suited for standard-bore 

columns. 

If a 3-µm packing is used in a narrow-bore 

column, a flow rate of 1.5 

mL/min will result in a very high back 

pressure, typically too high to be run 

safely. This is the result of the smaller 

internal diameter of the column. As a 

result, narrow-bore columns usually 

are run with LC flow rates of 0.2–0.5 

mL/min. As seen in the Van Deemter 

plot, these low flow rates do not provide 

the best separation, which means 

that using a narrow-bore column may 

involve sacrificing some separation 

capability. This compromise may 

or may not be a problem, depending 

upon the spacing between peaks. 

However, this usually can be 

10m 

5m 

3m 

Figure 3: Van Deemter plots for various LC stationary phase particle diameters. 

compensated for by changing the 

mobile phase conditions. 

Narrow-bore columns offer two advantages 

compared with standard-bore 

columns: sharper peaks and less reagent 

consumption. The sharper peaks 

are a direct result of the narrower column 

width — there is less radial dispersion, 

which leads to sharper, more 

intense peaks than those obtained 

using standard-bore columns. The increased 

peak sharpness may compensate 

for the loss of resolution resulting 

from the nonoptimal flow rates. The 

increased peak height may also mean 

that lower levels of the species can be 

seen. 

A main drawback of narrow-bore 

columns versus standard-bore columns 

is that their capacity is reduced. 

Because fewer stationary phase particles 

can physically fit into the narrower 

column, there is less surface area available 

for species to bind. As a result, 

these columns can become overloaded 

easily, which could be a problem when 

analyzing unknowns. 

Typically, this reduced capacity 

is compensated for by diluting the 

samples more or using smaller injection 

volumes. Because less sample is 

injected onto the column, this may 

offset the advantage of increased peak 

height. Therefore, it is not always possible 

to measure lower levels with a 

narrow-bore column. 

Another potential disadvantage of 

narrow-bore versus standard-bore columns 

is sample throughput. Because 

lower LC flow rates are used for narrow-bore 

columns, separations tend to 

take longer than with standard-bore 

columns, which will affect sample 

throughput. The advantage of a lower 

LC flow rate is that there is less consumption 

of reagents and less waste to 

dispose. 

When deciding whether to use a 

standard-bore or narrow-bore column, 

users must consider which combination 

of factors is best for a particular 

laboratory and application. There is no 

single correct answer — each situation 

is different. 

HPLC Versus UHPLC for 

Speciation 

Within the past several years, a new 

form of LC has emerged: ultrahighpressure 

liquid chromatography 

(UHPLC). The Van Deemter plot 

shows that the smaller the particle size 

in the column, the better the separation. 

UHPLC uses columns with 

particle sizes less than 3 µm, typically 

1.5–2.5 µm. These smaller particles 

provide more surface area for mobile 

phase and species to interact, which 

leads to better separation. Or, another 

way to think of it is that shorter 

columns with smaller packings can 

provide the same separation as longer 

columns with larger packings. The advantage 

of the shorter columns is that 

the time for separations are reduced 

greatly. 

The other signifcance of the smaller 

packings is a much higher back pressure, 

typically more than 10,000 psi. 

This increased pressure requires a special 

HPLC pump capable of handling 

it. Normal HPLC pumps cannot be 

used with columns containing packing 

material with a diameter of



analysis times required for the separation 

of biological molecules (such as 

proteins, peptides, and so forth), which 

can take up to an hour or more. Most 

typical speciation analyses with either 

standard- or narrow-bore columns 

can be accomplished in less than 10 

min. Therefore, there would not be a 

great time savings. 

Users of UHPLC also must be aware 

of other factors associated with these 

columns. First, because the particles 

are so small and packed so tightly, the 

columns can clog easily. To eliminate 

particulates, all mobile phases and 

samples must be filtered before analysis. 

Because ICP-MS is such a sensitive 

detection method, filtering can introduce 

conatmination, that could raise 

the baseline. Also, filtering samples 

may facilitate the interconversion of 

species in solution. Therefore, the effects 

of filtering on species conversion 

must be studied and understood. 

The additional active sites available 

on UHPLC columns provide the 

increased separation capability, but 

this also means that these columns 

take longer to equilibrate. While a 

typical 3-µm column may take 20 

min to equilibrate, a UHPLC column 

could take upwards of 45 min for 

proper equilibration. This increased 

time would have consequences when 

starting the system, changing mobile 

phases, running gradient methods, 

and performing method development. 

Another aspect to consider are the 

types of UHPLC columns available. 

Because UHPLC is relatively new and 

was developed primarily for separation 

of biological substances, columns 

that will separate inorganic species 

of interest may not be available. For 

example, there are few ion-exchange 

UHPLC columns available. 

Where UHPLC could be advantageous 

is in the emerging field of 

metallomics. This field involves the 

separation of biological substances 

using ICP-MS to detect inorganic molecules 

associated with proteins, peptides, 

and other biomolecules. UHPLC 

columns may be available to separate 

the substances of interest. 

Summary 

When performing speciation analysis, 

there are many aspects that must be 

considered: separation scheme, column 

type, column diameter, mobile 

phase, species of interest, required 

measurement levels of each species, 

sample throughput, and reagent usage 

and disposal. There is no single right 

answer for every situation. Each user 

must consider these factors when undertaking 

speciation analysis. 


is a Senior Scientist 

at PerkinElmer LAS, 

where he works with 

both ICP-MS and 

HPLC–ICP-MS. He received 

a B.A. in Chemistry 

from Colgate University, Hamilton, 

New York, and a Ph.D. in Analytical Chemistry 

from the University of Delaware, 

Newark. Ken joined PerkinElmer in 1997. 

For more information on 

this topic, please visit: 

www.spectroscopyonline.com


Interaction of Indigo Carmine 

with Nucleic Acids in the Presence 

of Cetyltrimethylammonium 

Bromide: Spectral Studies and the 

Confirmation of Combined Points 

The interaction of indigo carmine with nucleic acids in weak acid medium was studied in the 

presence of cetyltrimethylammonium bromide (CTMAB) by the measurements of resonance 

light scattering (RLS), UV–vis, and nuclear magnetic resonance (NMR) spectra. In trihydroxymethyl 

aminomethane (Tris) buffer (pH 5.47), indigo carmine and nucleic acids interact with 

CTMAB to form a ternary complex, which results in the enhanced RLS signals at 285 nm, 

336 nm, 405.5 nm, and 548 nm, respectively. The enhanced RLS intensity at 336 nm is proportional 

to the concentration of nucleic acids in a wide range with a detection limit of 3.1 

ng/mL. And the concentrations of nucleic acids in three synthetic samples were analyzed 

satisfactorily. Mechanistic studies show that the enhanced RLS stems from the aggregation 

of indigo carmine on nucleic acids through the bridged and synergistic effect of CTMAB. The 

combined points of the anionic dye indigo carmine and nucleic acids-CTMAB have been confirmed 

tentatively by using 1 H NMR spectroscopy. 

Changqun Cai, Xiaoming Chen, Hang Gong 

Some organic dyes have important environmental effects; 

for instance, dye dilution methods for determining 

blood flow are used in a wide variety of circumstances 

(1). Most of them have low-potential toxicity and can 

be detected easily by ordinary spectrophotometric methods 

(2). However, some azo dyes can be decomposed to produce 

aromatic amine changing the structure of DNA, which results 

in human pathological changes and induces cancer (3). 

Ever since its introduction in 1903 by Voelcher and Joseph, 

indigo carmine (IC) has been used to test renal function and 

it has been the subject of investigation for many years (4,5). 

IC usually is dispensed for intravenous injection in 0.8% of 

water solution in 5-cc ampules. The compound’s molecular 

structure is shown in Figure 1. 

Resonance light scattering (RLS) is a creative application 

of light signals to the measurement of RLS by simultaneously


NaO 3 S 

N 

H 

Figure 1: Molecular structure of indigo carmine. 

scanning the excitation and emission 

monochromators with a common fluorescence 

spectrophotometer. It has been 

proved that RLS has the advantages of 

simplicity and high sensitivity for detecting 

cation porphrins (6–8), triphenylmethane 

dyes (9), phenothiazinium 

dyes (10,11), xanthene dyes (12,13), metal 

complexes (14–16), cationic surfactant 

(17), zwitterionics (18), and nanoparticles 

(19,20). Up until now, only a few 

investigations have been carried out to 

determine nucleic acids as probe reagents 

for anionic dye (21–23), due to the 

electrostatic repulsion between nucleic 

acids and it. 

In this study, we first find that the 

ternary complex of IC-cetyltrimethylammonium 

bromide (CTMAB)- 

nucleic acids can cause a stronger RLS 

signal. The enhanced intensity of RLS 

is proportional to the concentration of 

nucleic acids in a wide range. The sensitivity, 

selectivity, and linear range for 

the determination of nucleic acids by 

RLS are reported. The combined points 

of the anionic dye IC and nucleic acids- 

CTMAB have been tentatively confirmed 

by using 1 H NMR spectroscopy. 

The reaction mechanism and the influential 

factors on the variation of RLS 

have been investigated. 

b 

a 

c 

O 

H 

N 

O 

c 

a 

b 

SO 3 Na 

Experimental 

Reagents 

Stock solutions of nucleic acids were 

prepared by dissolving commercially 

purchased yeast DNA (Shanghai Changyan 

Pharmaceutical Factory, Shanghai, 

China), calf thymus DNA (Shanghai 

Changyan Pharmaceutical Factory), 

fish sperm DNA (fsDNA, Shanghai 

Institute of Biochemistry, Academy of 

Science, Shanghai, China), and yeast 

RNA (Shanghai Changyan Pharmaceutical 

Factory) in doubly distilled water. 

These stocks needed to be stored at 0–4 

°C with only an occasional gentle shake 

if needed. The concentration of DNA 

was determined according to the absorbance 

values at 260 nm by using ε DNA 

= 6600 mol -1 L cm -1 (24). In this experiment, 

all working solutions of DNA were 

25.0 mg/L. 

A 1.0 X 10 -3 mol/L stock solution of 

IC (Shanghai Chemical Reagents Co. 

Shanghai, China), was prepared by dissolving 

0.4664 g of the crystal product 

in water in a 1000-mL volumetric flask. 

The working solution of IC was 5.0 X 10 -5 

mol/L. 

A 5.0 X 10 -4 mol/L stock solution 

of cation surfactant CTMAB (Merck, 

Germany) was prepared by dissolving 

Intensity 

450 

400 

350 

300 

250 

0.3645 g of the crystal product in water 

in a 1000-mL volumetric flask. 

A 0.10 mol/L Tris-HCl buffer solution 

was prepared by dissolving 12.1140 

g of Tris in 1000 mL volumetric flash in 

water and then adjusting the pH to 5.47 

with 1.0 mol/L HCl. 

All reagents were of analytical reagent 

grade without further purification and 

doubly distilled water was used throughout 

the process. 

Apparatus 

The RLS spectrum and the intensity 

of RLS were measured with an RF- 

5301PC (Shimadzu, Japan) fluorescence 

spectrometer by using a 1.00-cm quartz 

fluorescence cell. All absorption spectra 

were measured on a UV-265 spectrophotometer 

(Shimadzu, Japan). The surface 

tension (σ) was measured with a Krüss 

KizMK5 program surface tension instrument 

(Krüss GmbH, Hamburg, 

germany) by using the suspended plate 

method. 1 H NMR spectra were recorded 

on a BRUKER 400 spectrometer (Bruker, 

Billerica, Massachusetts) for solution in 

D 2 O with tetramethylsilane (TMS) as 

internal standard. Chemical shifts are 

reported downfield in parts per million 

(ppm) and J-values are in Hz. A pHs-3C 

digital pH meter (Leici, Shanghai) was 

200 

2 

150 

100 

3 

50 

4 

0 

250 300 350 400 450 500 550 600 650 700 

Wavelength (nm) 

Figure 2: RLS spectra of IC-CTMAB-DNA system. Conditions: CIC, 3.0 X 10 -6 mol/L; pH, 5.47; 

CDNA, 500 µg/L; CCTMAB, 3.0 X 10 -5 mol/L. 1 = IC-CTMAB-DNA, 2 = CTMAB-DNA, 3 = IC- 

CTMAB, 4 = DNA-IC. 

1



utilized to detect the pH values of the 

aqueous solutions. 

Standard procedure 

Into a 25-mL volumetric test tube were 

successively added appropriate volumes 

of nucleic acids solution according to the 

concentration needed: 1.50 mL 5.0 X 10 -4 

mol/L CTMAB, 1.50 mL 5.0 X 10 -5 mol/L 

IC, and 2.00 mL HMTA-HCl buffer. The 

mixture was diluted to the mark with 

water and mixed thoroughly before RLS 

was measured. All measurements were 

made at an ambient temperature of 25 

°C. 

All RLS spectra were obtained by scanning 

simultaneously the excitation and 

emission monochromators (namely Δλ 

= 0 nm) from 250 to 700 nm. The intensity 

of RLS was measured at λ = 336 nm in 

a quartz fluorescence cell with slit width 

at 3.0 nm for the excitation and emission. 

The enhanced intensity of the CTMAB- 

IC system by DNA was represented as 

ΔI RLS = I RLS – I0 RLS. Here, I RLS and I 0 RLS 

were the intensity of the system with and 

without nucleic acids. 

Absorbance 

0.55 

0.50 

0.45 

0.40 

0.35 

0.30 

0.25 

0.20 

0.15 

0.10 

0.05 

0.00 

300 

1 

2 

3 

4 

5 5 

1 

2 

3 

4 

400 500 600 700 


Figure 3: The absorption spectra. 1 = IC-CTMAB-DNA, 2 = IC-CTMAB, 3 = IC-DNA, 4 = IC, 5 = 

CTMAB-DNA. Conditions: CIC, 5.0 X 10 -5 mol/L; pH, 5.47; CCTMAB, 5.0 X 10 -4 mol/L; CDNA, 500 

µg/L. 

Results and Discussion 

Features of the resonance light scattering 

spectrum 

As can be seen from Figure 2 (curves 1– 

4), when yDNA is mixed with CTMAB, 

strong RLS signals could be found, because 

CTMAB could be aggregated on 

the molecular surface of nucleic acids (25) 

(curve 2). However, it is still much weaker 

than those of curve 1, which indicates that 

the RLS of DNA is enhanced with the 

help of both IC and CTMAB, and a ternary 

complex (26) of IC-CTMAB-DNA is 

produced. The CTMAB in the system acts 

as a bridge between IC and DNA, which 

results in the greatly enhanced RLS intensities. 

Similar RLS spectral characteristics 

can be surveyed when fsDNA, ctDNA, 

and yRNA are used. 

The absorption spectra of the IC- 

CTMAB-DNA system in the 220–700 

nm region are presented in Figure 3. By 

comparing the light-scattering and absorption 

spectra, it can be seen that the 

peaks of light scattering at 336 nm appear 

at the red side of the absorption bands at 

292 nm. According to the theory of RLS 

(1,27), the peaks of light scattering at 336 

nm are ascribed to the absorption bands 

of IC at 292 nm. Because the light-scat-


Intensity 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

250 

300 350 400 450 500 550 600 650 700 


Figure 4: Effect of surfactants on the I RLS value of IC-DNA-CTMAB system. Conditions: CIC, 3.0 X 

10 -6 mol/L; pH, 5.47; CDNA, 500 µg/L. 1 = CTMAB, 2 = CPB, 3 = TX-100, 4 = SDBS, 5 = SDS. 

Surface tension (mN/m) 

100 

80 

60 

40 

20 

0 

1 2 3 4 5 6 7 8 9 

C 

CTMAB (10-5 ) 

Figure 5: Effect of CTMAB concentration on surface tension. Conditions: CIC, 3.0 X 10 -6 mol/L; 

pH, 5.74; CDNA, 500 µg/L. 

I RLS 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

0 0.5 1 1.5 2 

Indigo carmine concentration 

Figure 6: Effect of IC concentration on the ΔI RLS intensity. Conditions: CCTMAB, 3.0 X 10 -5 mol/L; 

pH, 5.47; CDNA, 500 µg/L. Top: I RLS of the system with DNA; bottom: I RLS of the system without 

DNA). 

1 

2 

3 

4 

5 

tering spectrum depends on the nature 

of the system, also reflects the characteristics 

of the instrument (28), the RLS 

peak around 470 nm is considered to be 

possibly the emission of the xenon lamp 

in this region (29). The RLS intensity at 

336 nm is the maximum, so 336 nm was 

selected in the further study. 

Effect of buffer and surfactant 

The effects of buffer and surfactant are 

examined. Tris is the best buffer tested, 

HMTA-HCl (88.7); NH 4 Ac-HAc (19.2); 

NH 3 -NH 4 Cl (11.5). It can be seen from 

Figure 4 that CTMAB is the best surfactant. 

The effects of buffer and surfactant are 

examined (presented in Figure 4) and we 

know the enhanced RLS signals strongly 

depend upon the concentration of 

CTMAB. If the concentration of CTMAB 

is too small, the enhanced RLS intensity 

is not significant. However, when the 

concentration of CTMAB increases, the 

enhanced RLS signals are very strong. 

When the concentration of CTMAB is 

above 4.0 X 10 -5 mol/L, perhaps CTMAB 

reacts with IC directly and forms larger 

particles of IC-CTMAB which is not favorable 

for further reaction with DNA, so 

the ΔIRLS decreases. 

From the measurement of surface tension 

(shown in Figure 5), we can obtain 

the critical associate concentration (CAC) 

in this system, which is 3.0 X 10 -5 mol/L. 

So 3.0 X 10 -5 mol/L CTMAB is selected for 

further research. 

The effects of buffer are examined, and 

the dependence of light-scattering upon 

pH might be relevant to the form of IC 

and DNA. When the pH is below 4.50, 

both the free base form of IC and DNA 

are protonized, which is not favorable for 

positively charged CTMAB to be close 

to the DNA molecule. At the same time, 

the concentration of IC with two negative 

charges is low, which is not favorable for 

the reaction of IC and CTMAB. When 

the pH is above 6.50, the ΔI RLS of IC- 

CTMAB-DNA system begins to decline 

markedly, perhaps as a result of the direct 

reaction CTMAB with IC. So pH 5.47 is 

chosen for further research. 

Effect of indigo carmine 

concentration 

The effect of IC concentration on 

ΔI RLS of the IC-CTMAB-DNA system is 

investigated and shown in Figure 6. As is



indicated by the figure, the IC concentration 

has an obvious effect on ΔI RLS of 

the IC-CTMAB-DNA system. It is found 

that when the concentration of IC is in 

the range 2.0 X 10 -6 to 4..0 X 10 -6 mol/L, 

the ΔI RLS of the IC-CTMAB-DNA system 

reaches a maximum. So we select 3.0 X 

10 -6 mol/L IC for further research. 

Effect of the ionic strength 

The ionic strength of the medium 

has an effect on the interaction of IC, 

CTMAB, and DNA. As Figure 7 shows, 

the ΔI RLS of the IC-CTMAB-DNA system 

decreased slightly when NaCl was at 

a low concentration, while it decreased 

markedly with the increasing ionic 

strength. The reason could be that Na+ 

shielded the phosphorus negative charge 

of DNA, which weakened the combination 

of DNA and CTMAB, so the ΔI RLS 

decreased, despite the strong dependence 

of the enhanced light scattering of IC by 

DNA upon ionic strength ranges from 0 

to 0.05. 

Reaction time and stability 

The binding reaction of DNA and IC 

with CTMAB occurs rapidly at room 

temperature after less than 2 min, and 

the ΔI RLS remains a constant for about 

4 h. It shows that the reaction does not 

require any crucial timing and has good 

stability. 

Table I: Interference of foreign substances 

Foreign 

substance 

Conc. 

Coexisting 

(µg/L) 

Relative 

error 

(%) 

Foreign 

substance 

Conc. 

Coexisting 

(µg/L) 

Relative 

error 

(%) 

Ca(II) 5.0 -0.66 Fe(III) 48 -0.98 

Al(III) 1.5 3.88 KH 2 PO 4 30.0 -2.76 

Mg(II) 2.0 7.63 Co(III) 0.10 9.01 

Ni(II) 0.90 -1.92 Pb(II) 0.10 -6.12 

EDTA 12.0 3.40 L-Leucine 0.08 0.97 

Adenine 16.0 -2.74 DL-Alanine 15.0 2.33 

20.0 -3.34 Sucrate 20.0 -4.65 

D-Galactose 40.0 -5.02 DL-Cystine 7.5 3.27 

L-Lysine 30.0 7.11 

L-Methionine 

DL-Tryptophan 

40.0 2.18 

DL-Histidine 20.0 2.16 Thymine 15.0 -5.23 

Tolerance of Foreign Coexisting 

Substances 

The effect of substances such as metal 

ions, amnion acids, galactose, and adenine 

were examined for interference. 

The results are summarized in Table I. It 

can be seen that most of the metal ions in 

biological systems, such as K + , Na + , and 

Ca 2+ , can be tolerated at high concentrations 

because they are hard ions (30) and 

tend to bind almost exclusively with the 

phosphate groups, stabilizing the Watson–Crick 

double helix of DNA, so their 

effect on the interaction of IC is related to 

the shielding effects of counter ions on the 

negative backbone of nucleic acids, which 

shows that this method is very useful and 

valuable. 

Calibration 

Under the optimum conditions, the dependence 

of ΔI RLS upon the concentration 

of DNA is determined. The analytical parameters 

of this method are listed in Table 

II, which shows that there was a good


Table II: Analytical parameters 

Nucleic acids 

Linear range 

(mg/L) 

Linear regression 

equation 

(mg/L) 

Limit of 

determination 

(ng/mL) 

Correlation 

coefficient (r) 

yDNA 0–2.50 

ΔI = 156.53C 

+ 81.43 

ctDNA 0.05–2.00 

ΔI = 139.28C 

+ 96.26 

fsDNA 0.12–2.20 

ΔI = 128.81C 

+ 107.68 

RNA 0.10–2.30 

ΔI = 

125.56C+80.27 

3.10 0.9997 

7.59 0.9993 

9. 97 0.9995 

14.86 0.9986 

Table III: Chemical shifts (ppm) of individual protons of IC in the systems 

a b c 

IC 8.10 7.55 6.52 

IC-CTMAB-yDNA 8.13 7.58 6.58 

I RLS 

450 

400 

350 

300 

250 

200 

150 

100 

50 

0 

0 0.01 0.02 0.03 0.04 0.05 0.06 

Sodium ion concentration 

Figure 7: Effect of ion strength on the ΔI RLS value of IC-DNA-CTMAB system. Conditions: 

CCTMAB, 3.0 X 10 -5 mol/L; pH, 5.47; CDNA, 500 µg/L; CIC, 3.0 X 10 -6 mol/L. Top: I RLS of the system 

with DNA; bottom: I RLS of the system without DNA. 

linear relationship between ΔI RLS and 

nucleic acids in a wide range. It is well 

known that no groove or helix structure 

exists in RNA, However, both DNA and 

RNA can increase the resonance light 

scattering intensity of IC-CTMAB in 

our experiment. So groove binding and 

intercalative binding are not the reason 

for the RLS enhancement. 

Nature of the interaction of IC, 

CTMAB, and nucleic acids 

Generally, organic dyes interact with 

DNA in the following models: intercalative, 

groove or electrostatic binding, 

and the long-range assembly on the 

molecular surface of DNA does not involve 

intercalative or groove binding. It 

can be seen from Figure 2 that with the 

addition of CTMAB, DNA can enhance 

the ΔI RLS without IC, which is due to the 

formation of a DNA-CTMAB association, 

although the ΔI RLS is smaller than 

that in the presence of IC. IC and DNA 

cannot react with each other due to the 

electrostatic between them. By introducing 

a bridge, namely, cationic surfactant 

CTMAB into DNA-anionic dyes system, 

IC, CTMAB, and DNA formed a ternary 

complex of IC-DNA-CTMAB, which 

shortened the distance between IC and 

DNA and enhanced the assembly of IC 

on the surface of DNA. Several other



(a) 

(b) 

8.0 7.5 7.0 6.5 

chemical shift is attributed to the reduction 

of the electron cloud density. It is 

considered that the decrease of electron 

cloud density of hydrogen atoms is attributed 

to the fact that IC combines 

with nucleic acid and CTMAB. The (c) 

hydrogen atoms are moved downfield, 

and this indicates the combined points 

are the three hydroxyl of IC through 

hydrogen bond and form IC-CTMABnucleic 

acids ternary complex, resulting 

in the RLS enhancement of this system. 

This is consistent with the earlier 

studies. 

8.0 7.5 7.0 6.5 

Figure 8: The NMR spectra of the system: (a) IC, (b)-CTMAB-yDNA. 

surfactants have been employed to examine 

further the surfactant effects 

functioning in RLS enhancement. The 

cationic surfactants revealed RLS enhancement 

effects much more prominently, 

which in turn disclosed that 

electrostatic force performs an important 

function in the combination 

of DNA with IC and surfactant. The 

relatively weak RLS enhancement effects 

of non-ion and anion surfactant 

confirms that hydrophobic force is 

also a factor in the reaction of DNA 

with IC and surfactant (31) 

It is recognized that viscosity is one 

of the important parameters in the 

reaction of DNA and small molecules 

(32). The relative viscosities of different 

mixtures were examined in the experiment 

and the relative viscosity of 

the ternary complex declining slightly 

indicated that there was no intercalative 

among CTMAB, DNA, and IC. 

So we can conclude that the interaction 

of nucleic acids, CTMAB, and 

IC mainly depend upon electrostatic 

binding. Hydrophobic force is also the 

factor in the reaction of nucleic acids 

and IC with surfactant. 

The NMR spectra of the system 

To further understand the interaction 

between IC, CTMAB, and 

DNA, NMR spectra at optimum experimental 

conditions were measured, 

as is shown in Figure 8 and Table 

III. Corresponding to IC, the chemical 

shift signals of all hydrogen atoms 

moved downfield significantly when IC 

was added into the CTMAB and yDNA 

solution. The motion to downfield of the 

Conclusion 

In this article, a new RLS assay of nucleic 

acids is proposed. IC, CTMAB, and nucleic 

acids can form a ternary complex 

mainly through electrostatic binding, 

hydrophobic force, and hydrogen bond, 

which results in the enhanced DI RLS of 

IC-CTMAB-nucleic acids system. The 

combined points of the anionic dye 

with nucleic acids have been tentatively 

confirmed through the measurement 

of 1 H NMR spectra, which establishes a


foundation for further investigation on 

the nucleic acids at the molecular level, 

and have enlarged the range of probe reagents 

of nucleic acids at the same time. 

This method enlarges the range of probe 

reagents of nucleic acids, which will be 

applied widely for quantitative determination 

of nucleic acids. The reaction 

of IC and DNA directly hope to benefit 

the further research on the interaction of 

pathological changes part of DNA and 

the antineoplastic. 

Acknowledgments 

This work was supported by Xiangtan 

University (08XZX11). 

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For more information on this topic, 

please visit our homepage at: 

www.spectroscopyonline.com



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includes Multimode, Single 

Mode, and Polarization Maintaining 

with core diameters ranging from 

50–1500 μm and connector interface 

selections of SMA (standard), 

ST, FC, and ferrule style. According 

to the company, the fibers are 

helium leak-tested and certified to 

10 −7 torr. Fiberguide Industries, 

Stirling, NJ; www.fiberguide.com 

Air-jet sieve 

The AS 200 Jet air-jet sieve is designed for separating light, fine 

particle sizes. The sieve is intended for use with 203-mm (8-in.) 

diameter sieves as small as 10 

μm (~1200 mesh). According 

to the company, an industrial 

vacuum generates a jet of air that 

evenly disperses the particles via 

a slotted nozzle. The material that 

passes through the sieve is either 

discarded through the vacuum or 

collected in an attached cyclone. 

The procedure reportedly 

requires 2–3 min. 

Retsch, Inc., Newtown, PA; 

www.retsch-us.com 

XRF inorganic elemental analyzer 

EDAX’s Orbis micro-XRF inorganic elemental analyzer is designed to 

perform nondestructive measurements that require minimal sample 

preparation and provide improved 

sensitivity over SEM and EDS 

methods. The analyzer includes a 

motorized turret for coaxial sample 

view and X-ray analysis. Primary 

beam filters can be used with 

X-ray optics for micrometer to millimeter 

spot elemental analyses. 

Applications include forensics, 

materials analysis, failure analysis, 

and elemental imaging. 

EDAX Inc., Mahwah, NJ; 

www.edax.com 

DPSS laser 

Cobolt’s Zouk CW DPSS laser is designed for UV-range applications. 

The 355-nm laser reportedly 

has an output power of 10 mW 

and a TEM00 beam. According 

to the company, the laser is an 

alternative to the Ar/Kr-ion UV 

lines, quasi-CW UV lasers, and 

diode lasers for applications 

such as fluorescence-based bioanalysis, 

semiconductor inspection, 

Raman spectroscopy, and 

microlithography. 

Cobolt AB, Stockholm, Sweden; 

www.cobolt.se 

Custom standards 

Custom inorganic standards from Inorganic Ventures can be used 

in applications such as ICP, ICP-MS, 

atomic absorption, and ion chromatography. 

Each standard is supplied 

with a certificate of analysis that 

details NIST traceability, certified 

values, and trace impurities. The standards 

reportedly are manufactured in 

a “green” manufacturing facility. 

Inorganic Ventures, Lakewood, NJ; 

www.inorganicventures.com 

Confocal mapping FLIM system 

The DynaMac fluorescence lifetime imaging microscopy system 

from HORIBA Scientific is a 

confocal mapping system 

designed to provide information 

about molecular motion, 

sizes, local environment, 

interaction, and binding with 

respect to microscopic locations 

in a sample. The standalone 

system reportedly 

includes software control 

of light source, camera, pinholes, 

and filters. HORIBA 

Scientific, Edison, NJ; 

www.horiba.com/scientific



Calendar of Events 

January 2010 

Contact: Email: info@ifpacnet.org, 

Website: www.ifpac.com; 

November 2009 

16–19 Eastern Analytical Symposium 

and Exposition, Garden State Exhibit 

Center, Somerset, NJ; 

Contact: E-mail: askEAS@eas.org, 

Website: www.eas.org 

18–22 16th Annual Meeting SFRBM 

— The Society for Free Radical 

Biology and Medicine, 

San Francisco, CA 

Contact: Society for Free Radical Biology 

and Medicine, 8365 Keystone Crossing, 

Suite 107, Indianapolis, IN 46240; Tel. 

(317) 205-9482; 

E-mail: info@sfrbm.org, Website: www. 

sfrbm.org/annualMeetings.cfm 

19–23 30th SETAC Annual Meeting, 

New Orleans, LA 

Contact: Society of Environmental 

Toxicology and Chemistry, 1010 N. 12th 

Avenue, Pensacola, FL 32501-3367; 

E-mail: setac@setac.org, 

Website: www.setac.org 

24–27 LILS — Light in Life Sciences 

Conference, Melbourne, Australia; 

Contact: Website: http://www.physics. 

mq.edu.au/research/fluoronet/LILS09/ 

30 – 4 December, Materials Research 

Society, Boston, Massachusetts 

Contact: E-mail: info@mrs.org, Ph: 

(724)779-3003, Fax: (724)779-8313; Website: 

www.mrs.org 

December 2009 

2–5 10th European Meeting on 

Environmental Chemistry (EMEC10), 

Limoges, France 

Contact: EMEC 10 Secretary; 

E-mail: emec10@unilim.fr 

Website: www.unilim.fr 

3–9 2010 Winter Conference on 

Plasma Spectrochemistry, 

Fort Myers, FL 

Contact: Ramon Barnes, University Research 

Institute for Analytical Chemistry, 

P.O. Box 666, Hadley, MA 01035-0666; 

E-mail: wc2010@chem.umass.edu, 

Website: http://icpinformation.org 

22–25 Sanibel Conference on Mass 

Spectrometry, “From Structural Biology 

to Drug Discovery: New Roles 

for Mass Spectrometry of Nucleic 

Acids,” St. Petersburg Beach, Florida 

Contact: ASMS, 2019 Galisteo Street, 

Building I-1, Santa Fe, NM 87505, E-mail: 

asms@asms.org, Ph: (505)989-4517 or 

(800)825-3220, Fax: (412)825-3224; 

Website: www.asms.org 

23–28 SPIE Photonics West, 

San Francisco, CA 

Contact: Tel. (888) 504-8171; E-mail: 

customerservice@spie.org, Website: 

http://spie.org/x2584.xml 

24–27 LabAutomation2010, Palm 

Springs, CA 

Contact: Association for Laboratory Automation, 

330 West State Street, Geneva, 

IL 60134, E-mail: Brenda Dreier, bdreier@ 

labautomation.org, Ph: (888) 733-1252, 

Fax: (630) 578-0172; 

Website: www.labautomation.org 

31–February 3 Applications of Lasers 

for Sensing and Free Space 

Communications, San Diego, CA 

Contact: Kristin Mirabal, E-mail: kmirab@ 

osa.org 

February 2010 

2–4 IFPAC 2010 24th International 

Forum Process Analytical 

Technology, Baltimore Marriott Waterfront, 

Baltimore, MD 

6–9 SPIE Medical Imaging 

San Diego, CA 

Contact: Website: http://spie.org/ 

x12166.xml 

28–March 5 Pittcon 2010, Orlando, FL; 

Contact: Website: http://www.pittcon. 

org 

March 2010 

3–6 IRUG9 — Ninth Biennial International 

Conference of the Infrared 

and Raman Users Group, Buenos 

Aires, Argentina; Contact: E-mail: irug9@ 

qu.fcen.uba.ar, Website: www.irug9.com 

5–9 Defense, Security, and Sensing 

2010 

Orlando World Center Marriott Resort & 

Convention Center, Orlando, Florida. 

Contact:Website: http://spie.org/defense-security-sensing.xml 

21–25 239th ACS National Meeting & 

Exposition, San Francisco, CA; 

Contact: Website: http://portal.acs.org 

May 2010 

2–6 Joint Conference of the 14th In 

Vivo ESR/EPR Spectroscopy & Imaging 

and the 11th International EPR 

Spin Trapping/Spin Labeling (EPR 

2010), San Juan, Puerto Rico 

Contact: Antonio E. Alegria, Ph.D, Scientific 

Secretariat, Department of Chemistry, 

University of Puerto Rico at Humacao, 

Estacion Postal CUH, Humacao, P. R. 

00791, E-mail: antonio.alegria1@upr. 

edu, Ph: (787)309-6961, Fax: (787)850- 

9422. Website: www.epr2010.org


16–21 CLEO/QELS 2010, Laser Science 

to Photonic Applications, 

San Jose McEnery Convention Center, 

San Jose, CA 

Contact: E-mail: Angela Stark, astark@ 

osa.org; Website: www.cleoconference. 

org 

17–19 SPIE Scanning Microscopy 

2010, Monterey, CA; 

Contact: Website: www.spie.org/ 

x19036.xml 

23–27 58th ASMS Conference on 

Mass Spectrometry (ASMS 2010), 

Salt Lake City, UT 

Contact: American Society for Mass 

Spectrometry, 2019 Galisteo Street, 

Building I, Santa Fe, NM 87505; Tel. 

(505) 989-4517, Fax: (505) 989-1073; 

E-mail: office@asms.org 

June 2010 

6–9 5th Nordic Conference on 

Plasma Spectrochemistry, Loen, 

Norway 

Contact: Yngvar Thomassen, National 

Institute of Occupational Health, P.O. Box 

8149 DEP, N-0033 Oslo, Norway; E-mail: 

yngvar.thomassen@stami.no, 

Website: www.nordicplasma.com 

28–July 2 Laser Optics 2010, 

St. Petersburg, Russia 

Contact: E-mail: conf2010@laseroptics. 

ru, Website: www.laseroptics.ru 

July 2010 

13–16 International Symposium on 

Luminescence Spectrometry, Prague, 

Czech Republic 

Contact: Symposium Secretariat, 

CZECH-IN s.r.o., Professional Event & 

Congress Organiser, Prague Congress 

Centre, Prague, Czech Republic, E-mail: 

info@isls2010.org, Ph: 420 261 174 301, 

Fax: 420 261 174 307; 

Website: www.isls.org 

31–August 6 IDRC 2010 — 

International Diffuse Reflectance 

Conference, Chambersburg, PA 

Contact: Website: www.idrc-chambersburg.org/ 

August 2010 

1–5 SPIE Optics & Photonics 2010, 

San Diego, CA 

Contact: Website: www.spie.org 

2–6 Denver X-Ray Conference 2010, 

Denver Marriott Tech Center Hotel 

Denver, CO 

Contact: Website: http://www.dxcidd. 

com/



8–13 XXII International Conference 

on Raman Spectroscopy, Boston, MA 

Contact: ICORS 2010 Conference Office, 

2019 Galisteo Street, Building I-1, Santa 

Fe, NM 87505; Tel. (505) 989-4735; E- 

mail: office@icors2010.org 

September 2010 

13–17 Laser Induced Breakdown 

Spectroscopy 2010 (LIBS 2010), 

Memphis, TN 

Contact: Dr. Jagdish P Singh, E-mail: 

LIBS2010@icet.msstate.edu, Ph: 

(662)325-7375, Fax: (662)325-8465; 

19–22 European Symposium on 

Polymer Spectroscopy (ESOPS 18), 

Zadar, Croatia; 

Contact: Prof. Vesna Volovšek, ESOPS 

18 Secretariat, Faculty of Chemical Engineering 

and Technology, University of 

Zagreb, Zagreb, Croatia, E-mail: esops18@ 

fkit.hr, Ph: 385 1 4597135. 

Website: www.esops18.com 

21–24 SRI 2010 — 50th U.S. National 

Conference on Synchrotron Radiation 

Instrumentation, Argonne, IL; 

Contact: Website: www.lightsources.org 

October 2010 

17–21 Annual Conference of the Federation 

of Analytical Chemistry and 

Spectroscopy Societies (FACSS), 

Raleigh, NC 

Contact: FACSS, P.O. Box 24379, Santa 

Fe, New Mexico 87502, E-mail: facss@ 

facss.org, Ph: (505)820-1648, Fax: 

(505)989-1073; Website: www.facss.org 

18–21 26th Annual International 

Conference on Contaminated Soils, 

Sediments, and Water, Amherst, MA 

Contact: Denise Leonard, Environmental 

Health Sciences, N344 Morrill, University 

of Massachusetts, 639 North Pleasant 

Street, Amherst, MA 01003-9298; 

E-mail: dleonard@schoolph.umass.edu 

Website: www.umass-soils.com 

November 2010 

7–11 31st SETAC Annual Meeting, 

Portland, OR 

Contact: Society of Environmental Toxicology 

and Chemistry, 1010 N. 12th Avenue, 

Pensacola, FL 32501-3367; E-mail: 

setac@setac.org, Website: www.setac.org 

TBA Eastern Analytical Symposium 

and Exposition, Garden State Exhibit 

Center, Somerset, NJ; 

Contact: E-mail: askEAS@eas.org, 

Website: www.eas.org 

December 2010 

15–20 Pacifichem 2010, Honolulu, HI 

Contact: Pacifichem 2010 Congress 

Secretariat, c/o American Chemical 

Society, 1155 16th St. N.W., Washington, 

DC, 20036; E-mail: pacifichem@acs.org, 

Website: http://pacifichem.org/ 

Please visit our homepage at: 


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