The Column #25/(’/)%()5−−9−)’!ˆ - UBMimages2.advanstar.com/PixelMags/lctc/pdf/2015-03-30-uk.pdf · “perfect” SOP for balance use. It should be reasonably straightforward

A Matter of BalanceHave you mastered the analytical balance?

2 Incognito A Matter of Balance

Incognito continues with his “back to basics” focus. This time is the turn of the analytical balance.

Cover Story

Features

18 Extending the Compatible Analyte Volatility Range for Indoor Air Quality and Material Emissions Testing Using Multi-Bed Thermal Desorption Tubes Caroline Widdowson and David Barden, Markes International This article describes sampling methodology for thermal desorption–gas

chromatography (TD–GC) that can extend the compatible analyte range of tests used to determine chemicals released from materials, as well as associated indoor air quality measurements.

9 Emerging Trends in Pharmaceutical Analysis Benedetto Natalini of the University of Perugia, Italy, spoke to Bethany Degg of The Column about the driving forces in pharmaceutical analysis, including the importance of regulation, chirality, and miniaturization.

Regulars7 News

Prof ling PBDEs in Baltic Sea f sh, detecting bromethalin poisoning in animals, and ensuring the safety of New Zealand shellf sh are featured this week.

13 GC Troubleshooting in Petrochemical Analysis Stephen Harrison, Linde Gases A guide to simple troubleshooting steps in gas chromatography (GC) with an emphasis on petrochemical analysis.

22 CHROMacademy

Find out what’s new on the professional learning site for chromatographers.

23 Training Courses and Events

24 Staff

30 March 2015 Volume 11 Issue 5

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ES586847_LCTC033015_cvtp1.pgs 03.20.2015 20:57 ADV blackyellowmagentacyan

A Matter of Balance

Have you mastered the analytical balance?

2 Incognito A Matter of Balance

Incognito continues with his “back to basics” focus. This time is the turn of the analytical balance.

Cover Story

Features

18 Extending the Compatible Analyte Volatility Range for Indoor Air Quality and Material Emissions Testing Using Multi-Bed Thermal Desorption Tubes Caroline Widdowson and David Barden, Markes International This article describes sampling methodology for thermal desorption–gas

chromatography (TD–GC) that can extend the compatible analyte range of tests used to determine chemicals released from materials, as well as associated indoor air quality measurements.

9 Emerging Trends in Pharmaceutical Analysis Benedetto Natalini of the University of Perugia, Italy, spoke to Bethany Degg of The Column about the driving forces in pharmaceutical analysis, including the importance of regulation, chirality, and miniaturization.

Regulars7 News

Prof ling PBDEs in Baltic Sea f sh, detecting bromethalin poisoning in animals, and ensuring the safety of New Zealand shellf sh are featured this week.

13 GC Troubleshooting in Petrochemical Analysis Stephen Harrison, Linde Gases A guide to simple troubleshooting steps in gas chromatography (GC) with an emphasis on petrochemical analysis.

22 CHROMacademy

Find out what’s new on the professional learning site for chromatographers.

23 Training Courses and Events

24 Staff

30 March 2015 Volume 11 Issue 5

ES586848_LCTC033015_001.pgs 03.20.2015 20:57 ADV blackyellowmagentacyan

2

Incognito2 News7 Q&A: Natalini9 Harrison137 99Widdowson and Barden18 CHROMacademy22 Training & Events23 Staff242222 2323

A Matter of BalanceIncognito continues with his “back to basics” focus. This time is the turn of the analytical balance.

It’s happened again. Following hot on

the heels of “Pipette-gate” at the end of

last year,1 I’ve been involved in another

dispute regarding the validity of a basic

laboratory skill. This time it’s the turn of

measurement of mass. Because my previous

article on the use of pipettes sparked so

much debate and interest, I thought this a

good opportunity to start another global

difference of opinion on the use of balances

within the analytical laboratory.

Starting with the same question as last

time — “Who legislates on the correct use of

balances in the analytical laboratory?” — ask

yourself who wrote your standard operating

procedure (SOP) on the use of analytical

balances to determine mass (not weight!)?

Which source was used to inform and guide

the author to ensure best practice was

taken into account as well as any regulatory

guidelines? Is the SOP always followed?

There are several regulations or regulatory

bodies that advise on the correct use of

balances; however, those which I use as

the de facto standards are USP Chapter 41

<Balances>,2 USP Chapter 1251 <Weighing

on an Analytical Balance>,3 and ISO

17025 Lab14 Calibration of Weighing

Machines (Edition 4 November 2006).4

Those of you who follow these things will

know that USP Chapter 41 changed in

December 2013 (after some 20 years in its

previous version), and that it is mandatory

in the pharmaceutical industry when

testing to USP standards. USP <1251> is a

guideline, mainly dealing with installation

qualif cation/operational qualif cation/

performance qualif cation (IQ/OQ/PQ)

matters and outside the scope of our

discussion here.

Let me highlight some of the issues that I

have seen in the determination of mass, one

of which was the cause of this latest dispute

on the correct use of an analytical balance.

Unlike the previous article on pipetting

I’m not going to formulate an imaginary

“perfect” SOP for balance use. It should

be reasonably straightforward to assemble

a reasonable SOP from a reference source,

even if this is the manufacturer’s instruction

manual. Instead I’m going to highlight

issues that are often not covered in SOPs,

common mistakes, and bad practice in the

measurement of mass.

General Weighing Practices

Location: Balances should be located

away from sources of heat (out of direct

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The Column www.chromatographyonline.com

sunlight and away from radiators or air

conditioning units) and in a position

where temperature does not f uctuate

considerably (typical temperature drift is

1–2 ppm/ºC). You should know how to

calculate the temperature coeff cient for

sensitivity of your balances should you

ever need to apply a correction factor! The

balance should be placed in the corner of

a room to ensure the least vibration, and

should be away from doors, windows, or

air conditioning units that can cause air

draughts. Ideally, balance tables should be

made of stone (should at least not suffer

from sag or vibration); be anti-magnetic (no

steel in the construction); and anti-static

(no plastic or glass). The balance should

be level (check bubble indicator before

each weighing session), and, if adjustment

is necessary, the balance should be

recalibrated (internal sensitivity adjustment)

before use.

Standby versus Power Off: Note that

if a balance has been powered off (rather

than put into standby mode), it may

take between one hour and one day to

“acclimatize” and you should consult your

balance literature for the recommended

time. If in standby mode, the balance may

be used once the internal calibration routine

is complete (where appropriate), or once a

stable reading is achieved with nothing on

the balance pan. It is good practice when

beginning a series of weighings to load

the balance pan, unload it again, and then

tare before beginning to avoid an “initial

weighing effect”.

Humidity: The humidity of the balance

location should be between 40% and

60% relative humidity (RH). High humidity

can lead to condensation and adsorption

of water; low humidity risks increased

electrostatic interference. If the balance

reading constantly drifts in one direction

or is not repeatable between weighings of

the same sample, use an anti-static gun,

metal receiver, or a balance with antistatic

ionizing blowers. Also note that changes in

humidity can affect air buoyancy (see later).

Sample Handling: The balance pan and its

surroundings should be free from powders

and liquids and should be brushed clean or

dried and recalibrated if found in a dirty state.

Use the smallest weighing vessel possible

to reduce the effect of f ow forces, don’t

touch with bare f ngers (skin oil residues

can reduce weighing accuracy), and use a

receiver with a smaller rather than a wider

neck to help prevent evaporation. Avoid

using plastic weighing vessels or receivers

because they are more susceptible to

electrostatic effects.

Ensure the sample is temperature

equilibrated with the weighing

Incognito

3


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environment. Samples that are too cold

will register a higher mass and vice versa

for warm samples. The readout may be

unstable if samples are not thermally

equilibrated and air buoyancy effects will

be markedly increased. Warm samples may

also suffer from evaporation.

If weighing hygroscopic materials, ensure

that the container closure is in place and that

the receiving vessel is as narrow as possible

(for example, stoppered volumetric f ask).

Note that f ngerprints are hygroscopic!

Place the weighing vessel in the centre of

the balance pan, otherwise the result will

be skewed because of a phenomenon that

is known as “eccentricity” or “off-centre

loading error”.

Samples containing magnetic materials

(nickel, iron, steel, magnetic stirrer bars!!)

can affect the measurement of mass. In this

case, you may need to use antimagnetic

balance pans or increase the distance from

the pan to the magnetic sample by using an

upside down beaker. You will typically note

that balance readings are stable but not

repeatable for the same sample or when

positioning the sample at different positions

on the balance pan.

Balance Calibration

You should adjust the sensitivity of the

balance daily (usually via the balance

internal calibration algorithm) or when you

operate the balance for the f rst time; when

you change the location of the balance;

after levelling the balance; and after major

changes in temperature, humidity, or air

pressure.

Sensitivity is def ned as “change in the

output variable of a measuring instrument

divided by the associated change in the

input variable”. For a balance, this is

the change in the weighing value (mass

displayed) ∆W divided by the load variation

∆m (mass measured). Sensitivity is one

of the most important specif cations of a

balance and is typically determined from

the slope of the curve of mass measured

versus mass displayed over the nominal

range of the balance. Most analytical

balances have a built in calibration function

that can assess the sensitivity and linearity

of the instrument.

Adjustment for Gravity: The further a

mass is from the centre of the earth, the

lower the gravitational force action upon it.

A mass of 200 g measured on the ground

f oor will be 199.99937 g on the 10th

f oor — an error of 3.15 ppm. The further

a mass is from the equator, the lower the

centrifugal acceleration because of the

rotation of the earth that counteracts

gravitational force (around 92 ppm

per degree of latitude!). It is therefore

Incognito

4




important to calibrate balance sensitivity

in the balance location — especially after

moving the balance from one location to

another.

For highly accurate weighing, or when

comparative weighing is done on different

days, changes in environmental factors

must be considered. The sensitivity of the

balance is calibrated using weights

of 8.0 g/cm3 and when measuring the

mass of substances with different

densities, air buoyancy errors can arise.

A full treatment of this subject is outside

our scope here; however, changes in

atmospheric pressure, atmospheric

humidity, and temperature all require an

adjustment to be made for air buoyancy.

You can f nd a good reference source at

this link: http://www.npl.co.uk/upload/pdf/

buoycornote.pdf

Complying with USP General

Chapter <41> Requirements

USP Chapter 41 “repeatability” def nes the

starting point of a balance operating range:

• Perform 10 measurements with the exact

same weight. Use a single certif ed weight

(see ISO 17025 below) below 5% of the

balances nominal range.

• Calculate 2× standard deviation (SD)/

nominal value — the result should be

<0.10%.

• Calculate the starting point of the

operating range: 2 × SD × 1000.

• If SD <0.41 d, replace it by 0.41 d.

The coeff cient “d” is the measurement

interval or “readability”. Examples include:

• Microbalances: 1 d = 1 μg = 0.000001 g

(6-digit) (0.41 d = 0.41 μg)

• Semi-microbalances: 1 d = 0.01 mg

= 0.00001 g (5-digit) (0.41 d = 0.041 mg)

• Analytical balances: 1 d = 0.1 mg =

0.0001 g (4-digit) (0.41 d = 0. 41 mg)

So, if the standard deviation of the

determination of 10 measurements on your

analytical balance is <0.41 × d, then the

minimum weighable amount is 2 × 0.41 ×

0.1 × 1000 = 82 mg

This minimum weight should be

periodically assessed and is a unique feature

of each balance — not the balance type,

model, or manufacturer.

Accuracy: Balance accuracy must be

assessed using a test weight between 5%

and 100% of the balance capacity, and the

indicated reading should be less than or

equal to 0.1% of the test weight nominal

value. The test weight should have a

maximum permissible error of 0.03%.

One really important provision in USP <41>

states that: Unless otherwise specif ed, when

substances must be “accurately weighed” the

Incognito

5


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Contact author: IncognitoE-mail: [email protected]

weighing shall be performed using a balance

that is calibrated over the operating range

and meets the requirements def ned for

repeatability and accuracy.

How do you calibrate your balance? Is

it done by your external service provider

or metrology department? Note that here

we don’t mean the daily adjustment of

the balance that most folks carry out

(internal instrument calibration) but a full

assessment of repeatability, sensitivity,

linearity, departure of indication from

nominal value, and eccentric loading. Most

folks will use the ISO17025 guidelines for

balance calibration and the United Kingdom

Accreditation Service (UKAS) document

Lab 14 (http://www.ukas.com/library/

Technical-Information/Pubs-Technical-

Articles/Pubs-List/LAB14.pdf) that outlines

the requirements for test weights and

calibration procedures nicely.

I think that will probably do for this

instalment, except to say that I haven’t

touched upon the estimation of measurement

uncertainty when using an analytical balance

or indeed the frequency with which checks

should be carried out. I will leave this to

your own further study, but would highlight

that under the new USP <41> regulations,

daily balance sensitivity calibration is not

required and that you should be familiar with

the “risk-based approach” that is described

therein.

The argument that prompted this

article was regarding the proper (simple)

specif cation in our in-house balance SOP

of a “safety factor”, which should be

considered when specifying the smallest net

weight measured using a balance to avoid

out-of-specif cation measurements as a result

of f uctuations in the minimum weighable

amount. Any suggestions?

References

1. Incognito, The Column 10(21), 2–5 (2014).

2. General Chapter <41> “Balances” in Second

Supplement to USP36 – NF31 (United States

Pharmacopeial Convention, Rockville, MD, USA, 1

June 2013).

3. General Chapter <1251>“Weighing on an

Analytical Balance” in Second Supplement to

USP36 – NF31 (United States Pharmacopeial

Convention, Rockville, MD, USA, 1 June 2013).

4. http://www.ukas.com/library/Technical-

Information/Pubs-Technical-Articles/Pubs-List/

LAB14.pdf

Incognito

6


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Researchers from the California Animal Health and Food Safety Laboratory at the University of California Davis in the USA have

developed an ultrahigh-performance liquid chromatography–mass spectrometry (UHPLC–MS) method for the detection of

desmethylbromethalin (DMB) residues in animal tissues.1 DMB is the toxic metabolite of bromethalin, a neurotoxic rodenticide

that has increased in usage because of new regulations introduced by the U.S. Environmental Protection Agency (EPA) to phase

out the use of other “second-generation” or long-acting anticoagulents in rodenticides intended for use in residential areas,

leading consumers to look for alternatives.

The U.S. EPA introduced regulations in 2008 to phase out the use of anticoagulents in rodent poisons because of risks to

children, pets, and wildlife. However, lead author Michael S. Filigenzi told The Column that consumers have now increased

their use of an alternative chemical, bromethalin, increasing the danger of poisoning non-target animals. Toxicologists therefore

needed a method to detect bromethalin in brain, liver, and fat tissue to conf rm bromethalin exposure in animals post-mortem.

The team initially developed the UHPLC–MS–MS method with electrospray ionization using a bromethalin standard, but found

the primary ion signal was not as expected. Filigenzi told The Column: “The results were consistent on different instrumentation

and over time, so we developed HPLC conditions and an extraction method which appeared to give us a reasonable detection

limit (in the low to mid part per billion range) and we detected it in a tissue sample from an animal suspected to have been

exposed to bromethalin.” He added: “We felt that we had a useful method but our inability to determine exactly what we

were detecting was worrisome, to say the least. Then, a desmethylbromethalin standard became commercially available.

When we analyzed that standard, it became evident that the compound we had been detecting all along was actually the

desmethylbromethalin — we weren’t detecting bromethalin at all.”

Simultaneous analysis by HPLC with UV and MS of the bromethalin standard detected a low level impurity of

desmethylbromethalin (<1%), which Filigenzi attributes to the poor ionization of bromethalin by electrospray LC–MS; DMB ionizes

exceedingly well. Feligenzi concluded: “Fortunately, bromethalin is metabolized

to desmethylbromethalin, which is the toxic form of the compound

and which will be present in animals exposed to bromethalin.

This makes it a good indicator of exposure. The published method

detects less than 1 ppb of desmethylbromethalin in tissue samples,

which has proven to be suff cient to demonstrate exposure to the

rodenticide in a number of cases.” — B.D.

Reference1. M.S. Filingenzi, A.C. Bautista, L.S. Aston, and R.H. Poppenga, Journal of

Agricultral and Food Chemistry DOI: 10.1021/jf5052706 (2015).

Detecting Off-Target Bromethalin Poisoning

Prof ling PBDE Levels In Baltic Sea Fish

Fish from the Baltic Sea are a major source of lipophilic environmental

pollutants for consumers in Finland. Surrounded by land, the Baltic

Sea is one of the most threatened marine environments, making

f sh from the Baltic Sea a major source of lipophilic environmental

pollutants including polybrominated diphenyl ethers (PBDEs).

PBDEs were used as f ame retardents on many products before

the EU introduced regulations in 2004 to restrict their use. To

determine the exposure of consumers in Finland to PBDEs as the

result of eating Baltic Sea f sh, researchers sampled f sh from the

Baltic Sea, freshwater lakes, and f sh farms. Corresponding author

Hannu Kiviranta, from the National Institute for Health and Welfare in

Kuopio, Finland, told The Column: “The motivation behind the study

was that unfortunately the Baltic Sea is polluted by many organic

pollutants and for protection of the population it is necessary to know

current levels of various pollutants. There is also already EU maximum

limit values for dioxins and PCBs in food and feed and maybe in the

future also for brominated f ame retardants. For legislation work we

need to know the levels of these pollutants in f sh.”

Over 200 samples of 17 edible f sh were prepared and subsequently

analyzed using gas chromatography–mass spectrometry (GC–MS) to

screen for 15 PBDE congeners including BDE-28, -47, -66, -71, -75,

-77, -85, -99, -100, -119, -138, -153, -154, -183, and -209. According

to the paper, analyses showed high levels of BDE-209 in Baltic herring

sampled near the city of Pori (Finland), and in farmed whitef sh. — B.D.

Reference1. R. Airaksinen et al., Environmental Science & Technology DOI: 10.1021/

es505266p (2015).

7



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Analyzing PSTs in Shellfish AquacultureParalytic shellf sh toxins (PSTs) are naturally

occurring toxins produced by some species of

microscopic algae that can accumulate in f lter

feeding shellf sh. These toxins are a threat to

shellf sh aquaculture and pose a serious hazard

to public health when ingested. A group

of scientists has developed a method using

hydrophilic interaction liquid chromatography

coupled to ultrahigh-performance LC tandem

mass spectrometry (HILIC UHPLC–MS–MS) to

determine PSTs in a variety of shellf sh species

as part of the Safe New Zealand Seafood

Research programme.1

Historically, shellf sh toxins have been

analyzed using mouse bioassay methods,

but over recent years alternatives have been

sought for ethical and technical reasons.

Corresponding author Michael J. Boundy

told The Column: “In 2001, the Cawthron

Institute (New Zealand) began implementing

routine monitoring of the lipophilic shellf sh

toxins using high performance liquid

chromatography (HPLC)–mass spectrometry.

For many years, these lipophilic shellf sh toxins

were routinely monitored by LC–MS with

export clearance required to be combined

with a mouse bioassay screen whereas

paralytic shellf sh toxins were determined

solely by mouse bioassay.” He said: “At the

time, the only available off cial method of

analysis was the AOAC 2005.06 pre-column

oxidation LC f uorescence (FL) method, which

was decided to be a suitable interim method

for regulatory monitoring in New Zealand.

Appraisal of more recent off cial methods

of analysis AOAC 2011.02 (post-column

oxidation LC-FL) and AOAC 2001.27 (receptor

binding assay) indicated that none of the

methods of analysis met all of our laboratories

requirements for both regulatory monitoring

and research projects (fast analysis, short

turn-around-time, high throughput, sensitive,

quantitation of toxin prof le, low cost).

Therefore, we sought to develop a new

method that would meet our requirements.”

PSTs are a broad range of small, structurally

very similar compounds that have a wide

range of toxicities meaning that mass

spectrometry analyses can be challenging,

but HILIC separation was initially shown

to be very promising. However, it was

evident very early on in the research that

chromatographic reproducibility was poor.

Boundy told The Column: “Most of the effort

spent in the development of the method

was developing a set of mobile phases and

chromatographic conditions that could be easily

and consistently prepared, and would allow

robust chromatography that would meet the

requirements of a routine testing lab.”

There were signif cant problems when

identifying and quantifying toxins in shellf sh

using HILIC that were caused by the high

concentrations of salt solutions found in

the marine environment. Boundy said: “In

the presence of high concentrations of salt

solutions, retention on the analytical column

is signif cantly impacted, and retention times

would greatly differ between standards

and samples. Due to the wide range of

compounds sharing mass-spectrometric

MRM transitions, mitigating this retention

shift in samples was very important to ensure

specif city and correct identif cation of the

compounds of interest.”

To overcome this issue, sample clean-up

was therefore necessary. The study authors

chose graphitized carbon for this purpose as

it had been previously shown to be successful

in retaining highly polar compounds. Samples

were then analyzed using a rapid, selective,

and sensitive HILIC UHPLC–MS–MS method

for identif cation of PSTs in shellf sh. A

validation study on 12 commercially produced

shellf sh species has been performed and

will be published shortly. Michael concluded:

“The improved sensitivity and specif city of the

developed HILIC UHPLC–MS–MS method over

the previous methods of analysis has been

extremely benef cial with a range of research

projects. The improved sensitivity will provide

industry with an earlier warning during a

harmful algal bloom, and the improved

turn-around time will reduce the delays before

harvested product can be sold.” — K.M.

Reference

1. M.J. Boundy, Andrew I. Selwood, D.T. Harwood,

et al., Journal of Chromatography A 1387, 1–12

(2015).


Emerging Trends in Pharmaceutical AnalysisBenedetto Natalini of the University of Perugia, Italy, spoke to Bethany Degg of The Column about the driving forces in pharmaceutical

analysis, including the importance of regulation, chirality, and miniaturization. Natalini also discussed his recent research in chiral

chromatography, as well as developments in diagnostics using novel orthogonal liquid chromatography (LC) methods to quantify

neurotransmitters involved in Parkinson’s disease.

Q. Why is pharmaceutical analysis

important and what areas are you

focusing on at the moment?

A: Regulators worldwide require increasingly

high quality and safety standards from

the pharmaceutical industry. To ensure

these standards are met, reliable analysis

tools and methods are constantly required

and developed by analytical scientists.

Pharmaceutical analysis therefore plays a

pivotal role in advancing the concepts and

theories of analytical science, as well as

providing important information on practical

aspects of drug design, quality control, and

quality assurance of industrial manufacturing.

Chiral analysis also plays an important role in

the pharmaceutical analysis.

The current trend in drug discovery

towards enantiomerically pure drugs is

the result of the need to reduce the dose

of a drug, simplify the dose—response

relationship, and reduce the toxicity caused

by the therapeutically less active (or inactive)

enantiomer.

There is, therefore, a great demand for

chiral separations to determine enantiomeric

purity at the early stages of the drug

development process; for enantioselective

bioanalysis in clinical trial studies; to analyze

drugs in the environment; and for the quality

control of medical products and their raw

materials. All these areas have played a part

in my research activities, which involves the

study of the main principles governing the

molecular recognition mechanism in the

various domains of chiral chromatography.

Another area of my research is aimed

at devising chromatographic strategies to

determine the physico-chemical properties of

pharmaceutically relevant compounds. I have

also recently been involved in nutraceutical

analytical chemistry in relation to the study

of the enantiomeric composition of amino

acids and other functional compounds in

fresh and treated foodstuffs, as well as the

development of new high performance

liquid chromatography (HPLC) methods for

bioanalytical studies.Ph

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Q. How has pharmaceutical analysis

evolved and what are the most

interesting recent trends?

A: Pharmaceutical analysis methods are

traditionally and commonly applied to

the chemical analysis of drug molecules.

However, in the last two decades, modern

pharmaceutical analysis has evolved

enormously, capitalizing on combination

techniques, high-throughput technologies,

chemometrics, and most recently

miniaturization and nanotechnology.

The combination of various techniques

allows the modern pharmaceutical analyst

to exploit the virtues of each technique

and, in turn, to improve the overall quality

of analysis. Indeed, modern analytical

techniques and methods offer the possibility

of increasing the amount of information

received from individual analysis, with

reduced cost, analysis time, and sample

volumes.

High-throughput technologies are having

an increasingly important role in early-stage

drug development, providing a fast

qualitative and quantitative characterization

of thousands of compounds evaluated in

the frame of preclinical and clinical ADME

(Absorption, Distribution, Metabolism,

Excretion) studies.

The principal component analysis (PCA)

and projections to latent structures (PLS) are

two chemometric methods applied in the

domain of computer-aided drug discovery,

and which prove particularly successful

in early stage preclinical research as a

fast computational and analytical tool for

screening the increasing numbers of potential

drug candidates.

The interest in miniaturization technology

has grown rapidly, particularly in the

pharmaceutical industry where it has been

fuelled by the need to speed-up the analysis

in high-throughput screening applications.

Research in this area is particularly focused

on “lab-on-a-chip” nanotechnology because

of the potential to identify, study, and

evaluate new drug entities. Nanotechnology

will have an increasingly important role in the

development of commercial analytical and

preparative tools.

Q. Why is there so much emphasis on

chiral analysis and the detection of

enantiomers?

A: Confronted with the disastrous

consequences of the “Thalidomide Drama”,

scientists in academia, pharmaceutical

industry, and drug regulatory authorities

started recognizing the crucial role of drug

stereochemistry in the development of

new pharmaceutically active ingredients.

In response to the enhanced knowledge

on enantioselective drug action, in the

early 1990s a pragmatic approach to the

regulation of chiral drugs was imposed.

In most countries, regulatory authorities

require separate (or distinct) pharmacological

and toxicological tests of the racemate and

the individual enantiomers. This means that

both racemate and enantiomers must be

tested separately. The decision to develop

the racemate or single isomer is then mainly

based on (a) achievement and exhibition

of safety and effi cacy of the new drug and

(b) optimal use of time and money: In this

frame, the development of the racemate is

sometimes the appropriate decision, with a

number of issues favouring the development

of the only enantiomer endowed with

activity.

The recognition of chirality as a new asset

in drug development has had an enormous

effect on the product pipelines of the major

players in the pharmaceutical industry,

unavoidably leading to the development

of numerous enantiorecognition systems,

mostly in the various domains of

chromatography, as well as of increasingly

improved methods of analysis.

Enantiomers of specifi c compounds have

been established to act as neuromodulators

or neurotransmitters in the central nervous

system, while others were found to play

an important role in endocrine tissues.

Interestingly, the occurrence of racemization

Q&A: Natalini

10


Use of Novel Zirconia-Based Cleanup Sorbents for the

Analysis of Contaminants in Fatty Food Samples

LIVE WEBINAR: Tuesday, March 31, 2015 8 am PDT/ 11 am EDT/ 4 pm BST/ 5 pm CEST

Register Free at www.chromatographyonline.com/novel

EVENT OVERVIEW:High fat content of foods has been a problem when analyzing samples for nonpolar contaminants such as pesticides, PCBs, and PAHs. Extraction methods for these compounds tend to generate samples that are highly contaminated with fatty matrix, causing a number of problems with the subsequent chromatographic anal-ysis. Traditional cleanup techniques, such as gel permeation chro-matography (GPC) and normal phase column chromatography can be very efective, but are often expensive and time consuming.

In this seminar, we will present an alternative approach for the

cleanup of fatty matrices using a novel zirconia-based family

of sorbents, for both QuEChERS (dispersive SPE) and traditional

cartridge SPE.

Key Learning Objectives:

n The use of a zirconia-based sorbent, (Supel™ QuE Z-Sep) will be demonstrated for the QuEChERS cleanup of various fatty foods, in the analysis of pesticides, PCBs, and PAHs.

n Learn about a new dual-layer SPE cartridge, Supelclean™ EZ-POP NP, designed specifcally for the extraction of POPs from oily food samples.

n Broaden your knowledge on advances in QuEChERS technology.

Sponsored by

Presented by

For questions, contact Kristen Moore

at [email protected]

Presenters

Katherine StenersonPrincipal Applications ChemistSupelco/Sigma-Aldrich

Moderator

Laura BushEditorial DirectorLCGC & Spectroscopy

Who Should Attend:

Chromatography and sample prep method develop-

ers, especially those interested in learning about advances and

innovations in QuEChERS cleanup technology.



of certain compounds in biological fl uids

and tissues can be associated to various

pathophysiological conditions. All these

relatively new acquisitions have expanded

the interest towards chirality issues as a

whole.

Q. In a recent study by your research

group, novel orthogonal liquid

chromatography methods were applied

to quantify neurotransmitters involved

in Parkinson’s disease.1 What were the

main objectives of this study?

A: Parkinson’s disease is a multifactorial

neurodegenerative disorder, characterized

by severe motor and non-motor symptoms

resulting from a selective loss of the

dopaminergic neurons in the nigrostriatal

pathway, with consequent reduction of

dopamine (DA) levels. A relatively long

preclinical phase precedes classical motor

symptoms that appear when more than 80%

of dopaminergic neurons are lost. Indeed,

non-motor symptoms, primarily cognitive

dysfunction, often occur many years before

the appearance of movement disorders,

thus limiting a prompt medical diagnosis.

Moreover, the molecular pathways involved

in the pathogenesis of disease have not yet

been fully disclosed. In this scenario, we

were motivated to develop new diagnostic

tools to evaluate early stage depletion

of DA and of some of its metabolites

(3,4-dihydroxyphenylacetic acid, DOPAC;

homovanillic acid, HVA; 3-methoxytyramine,

3-MT), thus facilitating biochemical

evaluations and clinical applications at once.

A recent study showed that the neurons

responsible for DA release also produce the

neurotransmitter gamma-aminobutyric acid

(GABA), which, in contrast to DA, contributes

to limit the neuronal hyperactivity.

Accordingly, we deemed it interesting to

develop an HPLC method for the quantitative

measurements of GABA levels in specifi c

biological samples, to apply for diagnostic

purposes.

The information derived from the

chromatographic study, when combined

to those from imaging approaches, could

facilitate the diagnosis at different stages.

Q. Why did you choose reversed-phase

ion-pairing chromatography (IPC)

and hydrophilic liquid interaction

chromatography (HILIC) for this study?

A: To take advantage of MS-compatible

chromatographic methods providing distinct

selectivity profi les, which enhances the

chance of correct species quantifi cation,

and allows the compensation of the intrinsic

limits characterizing all (single dimension)

chromatographic methods. To the best of my

knowledge, this represents the fi rst study in

Q&A: Natalini

11


Triumphs and Challenges of High-Resolution Mass

Spectrometry in Comprehensive Pesticide Residue ScreensON-DEMAND WEBCAST Originally aired February 25, 2015

Register for free: http://www.chromatographyonline.com/triumphs

Triumphs and Challenges of High-Resolution Mass

Spectrometry in Comprehensive Pesticide Residue Screens

EVENT OVERVIEW:If we want to continually expand our analytical capabilities and allow for

post-acquisition data mining, we can no longer rely solely on triple-quad-

rupole instruments for rapid, single-injection, easy-to-manage analysis. The

Florida Department of Agriculture and Consumer Services pesticide regula-

tory program now has several single stage high resolution mass spectrometers

(ST-HRMS), which have a mass resolving power of ~100,000 and continually

scan over a large mass-to-charge range. ST-HRMS allows for the mass resolu-

tion of, among other analytes, phosmet from azinphos methyl, two commonly

applied pesticides with very close masses and similar chromatographic reten-

tion times, a scenario that arises frequently in pesticide analysis. After a year of

analyzing ~800 samples for 226 pesticides using ST-HRMS and systematically

comparing the results to those acquired using a QTRAP instrumentwe fnd a

high degree of agreement and thus conclude that ST-HRMS is valid analytical

technique when analyzing for many compounds in complex food matrices.

We fnd that this approach has the potential for transformational improve-

ments in our analytical capabilities by allowing a broad scope single-injec-

tion analysis to be paired with a broad scope analytical extraction, allowing

for more efcient analysis. In this presentation, we will also address some of

the fundamental limitations of ST-HRMS analysis, the lack of parent isolation

capabilities and the inability to distinguish between two analytes with the

same exact mass. Lastly, we will demonstrate strategies that successfully mit-

igate these potential impediments, including the use of structurally relevant

fragments for, among other pesticides, terbumeton, prometon and secbu-

meton, and the use of trusted fragments that have been observed for years

on triple quadrupole instruments, but the structures of which are unknown,

as is the case for azoxystrobin and others.

Key Learning Objectives:

nHow HRMS may be used to screen and quantify 100s-1000s pesticides,

toxins, and other residues in food products

nHow very large analytical screens are enabled by full scan HRMS

experiments

n Learn approaches that mitigate potential challenges to HRMS analyses for

compounds with same exact mass

For questions, contact Kristen Moore at [email protected]

Presenter:

MARK CROSSWHITE, Ph.D.

Florida Department

of Agriculture and

Consumer Services

Division of Food Safety

Chemical Residue Laboratory

Moderator:

LAURA BUSH

Editorial Director

LCGC & Spectroscopy

Who Should Attend:

n Food safety chemists and

researchers interested in pesticide

analysis

n Food scientists interested in the

application of HRMS technology

for screening and quantitation of

contaminants

Sponsored by

Presented by



which the above fairly orthogonal methods

have been complementarily and successfully

applied to real samples with this aim.

Q. What were your main fi ndings?

What are the advantages of your

novel approach compared to existing

methods? Are other applications

possible?

A: The two fairly orthogonal HPLC methods

were directly applied to the biological

samples without preliminary derivatization

of the compounds of interest. A high level

of selectivity was obtained for dopamine

metabolites and GABA by running the

gradient reversed-phase IPC method with

a volatile ion-pairing reagent, which makes

it suitable for the quantitative assay of four

out of fi ve compounds. Matrix-deriving

interferences enabled the baseline separation

of dopamine, which was instead successfully

achieved with the HILIC-based method.

As previously reported, taking advantage

of HPLC methods providing distinct

selectivity profi les makes correct species

quantifi cation possible and allows analysts

to compensate the intrinsic limits

characterizing all chromatographic methods.

The two HPLC methods offer a valid

contribution to neuroscience research

and are potentially applicable to several

human biological samples. This approach

E-mail: [email protected]: http://www.unipg.it/it/pagina-personale?matricola=000818

will make it possible to diagnose the

early-stage of pathologies or to monitor

the progression of some neurodegenerative

disorders characterized by depletion of vital

neurotransmitters.

Q. Where do you see your research

taking you in the future? Do you have

anything further to add?

A: My future research activity will focus on

(a) studying the variation of the enantiomeric

composition of chiral biomarkers in biological

samples from healthy to pathological

conditions with 2D HPLC applications; (b)

developing new HPLC methods to identify

and quantify chiral biomarkers of bacterial

contamination in foodstuffs; and (c) gaining

a deeper understanding into the basic

principles governing the enantiorecognition

mechanism with both low- and

high-molecular-weight chiral selectors used

in LC applications, through the development

and application of spectroscopic and

molecular dynamic-based computational

protocols.

Reference

1. R. Sardella, S. Scorzoni, C. Conte, A. Lisanti,

F. Ianni, and B. Natalini, Journal of Pharmaceutical

and Biomedical Analysis 98, 253–259 (2014).

Benedetto Natalini

graduated in chemistry

at the Institute of

Organic Chemistry,

Faculty of Science,

University of Perugia

(Italy) in 1973. After

military service, he

began his research activity in 1976 at

the Institute of Medicinal Chemistry and

Pharmaceutical Technology, Faculty of

Pharmacy, University of Perugia (Italy). In

2003 he was designated an expert for the

ECM National Programme by the Ministry

of Health. In 2010 he was an Editor of the

International Journal of Medicinal Chemistry.

In 2011 he became coordinator of the

International Doctorate in Chemistry and

Technology of Drugs. From November 2011

to December 2013 he was Director of the

Department of Chemistry and Technology

of Drugs at the University of Perugia (Italy).

Since January 2014 he has been Director of

the Department of Pharmaceutical Sciences

at the University of Perugia.

Q&A: Natalini

12


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HPLC systems


GC Troubleshooting in Petrochemical AnalysisStephen Harrison, Linde Gases, Munich, Germany.

Gas chromatography (GC) is a core analytical

technique in the petrochemical sector, primarily

used to analyze the main process stream

components in fuel production but also to

detect trace impurities that can impact the

production process and f nal product. Against

this background, GC technology has advanced

towards higher sensitivity (or lower detection

limits), and detection of a greater number of

chemical components within a sample. Multiple

detectors can be combined for the analysis of

complex mixtures, resulting in instruments with

multifaceted and highly involved conf gurations

that can analyze 30 or more components from

a single sample injection. Another key trend is

the miniaturization of GC instruments, allowing

on-site analysis and a reduction in ref nery

running costs because they require very low

f ow rates of carrier gas.

Troubleshooting GC Analysis

As in other industries, chromatographers

in the petrochemical sector face the same

issues as all GC users. However, knowing

where to start troubleshooting can be

diff cult because there are so many potential

impurities, a broad range of analytes, and

wide concentration ranges encountered

during analysis. This is further complicated

where instrumentation is contained within

one “black box”. Merging and packaging

different technologies within one GC

unit can simplify analysis, but this creates

a greater number of issues because it is

impossible to visualize each step of the

analysis.

Calibration: A common cause of GC

problems is a lack of precision in the

calibration of the instrument and detector:

• The certif cate supplied with the

calibration gas mixture must be read and

clearly understood to ensure that the

component concentrations are similar to

the concentrations that will be measured.

• The accuracy of the reported values in the

calibration mixture should be appropriate

A guide to simple troubleshooting steps in gas chromatography (GC) with an emphasis on petrochemical analysis.

Ph

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for the measurement being undertaken

and all required components must be

present in the calibration gas mixture.

• The certif cate should be checked to

ensure that the gas mixture is within its

shelf life or expiry date.

Beyond these fundamentals, the use of

appropriate cylinder connection techniques is

vital and this may involve purging the system

with an inert gas to remove atmospheric

air after calibration cylinder connection, but

prior to calibration sample introduction.

Troubleshooting Unexpected Peak

Shapes in Gas Chromatograms

A fundamental error in process or

malfunction of the equipment can be

diagnosed if a chromatogram displays

results far removed from expectations. If

the result is not what was anticipated, or

the result indicates only a small number

of components in a complicated chemical

mixture, it is possible the operator has

chosen a set-up for the separator column

and detector which are simply not suitable

for the sample being measured. Below are

key hints and tips on how to approach

troubleshooting unexpected chromatograms.

“Fuzzy” Chromatograms: A problem

occasionally encountered is that peaks may

become smeared to the point where there

is no apparent difference between peaks,

referred to as a “fuzzy” chromatogram.

This can be caused by using a damaged GC

column, or using a GC column that is not

capable of reaching the level of separation

required. The column should be replaced or

exchanged for a different column that will

achieve a better separation.

Another possible cause is that the carrier

gas is not appropriate for the application.

Hydrogen has a low viscosity and high

separation velocity and will often achieve

the fastest results, but helium will generally

achieve a better peak resolution despite a

slightly slower response time. A change in

carrier gas can sometimes address the issue.

Unexpected Peaks: The appearance of

unexpected peaks can sometimes be the

result of impurities in the carrier or detector

gas. Check the correct grade of gas has

been connected; for example, a purity gas of

99.8% has been connected to a GC system

that requires a purity of 99.999% or higher.

The next step is to check the system for

leaks that can let gases out of the system,

but also allow contaminant gases in. Leaks

are particularly problematic because they

lower method sensitivity and can result in a

loss of carrier gas, with associated costs and

potential safety issues. If leaks are found,

connections should be tightened, and the

system allowed to settle with gas f owing

Harrison

14




through to purge before resuming analysis.

It is also possible that there may be damage

to the GC column from the moisture in

incoming air.

If no leaks are found, the carrier, detector,

and gas cylinders should be replaced. It

is important when changing from an old

cylinder to a new cylinder to use appropriate

techniques such as purging and leak testing

to avoid the introduction of contaminants

during the cylinder change-over. If the

carrier gas or detector gas is sourced from

a gas generator, the gas could be replaced

by a high purity specialty gas cylinder to see

if any change in the results occurs. If so, it

could indicate that the generator produces

gas with non-favourable impurities for the

specif c analysis.

Masking Effect: Peaks in a chromatogram

can sometimes appear to be overlapping

creating a “masking effect”. Solutions on

how to address this are:

• Impurities in the carrier gas: Check for gas

purity and system leakages.

• The sample volume is too high: Typical

GC sample volumes are millilitres or micro

litres, so if too much volume is introduced

to the system, the detector or separator

could become overloaded and this leads

to masked peaks.

• Carrier gas: Analytes with a similar

separation coeff cient will elute at a

similar time and can be masked by the

carrier gas. If this is suspected, the best

troubleshooting idea is simply to switch to

a different type of carrier gas.

Peak Shifting: If the carrier gas f ow rate

is too high or too low, peaks will show

up in places where they are not expected,

effectively shifting the whole chromatogram

to the left or the right. The f rst step is to

check the carrier gas f ow rate. “Pressure

creep” is characteristic of single-stage gas

pressure regulators; as the cylinder empties,

carrier gas f ow rate can increase. Using

two-stage pressure regulation will maintain

a stable gas inlet pressure to the GC.

Inappropriate gas f ow rates can also

cause problems in the detector. The FID

f ame operates best when gas f ow rates

produce an even f ame with laminar f ow

and the correct stoichiometric mix of fuel

and oxidant gases. If the fuel gas (normally

hydrogen) or oxidant gas (normally synthetic

air) f ow rates are not matched, the f ame

will burn with an unstable characteristic

and can cause erratic sample detection.

The remedy here is simple: Gas f ow rates

should be checked and it should be ensured

that high quality gas regulators are used to

deliver the gases to the FID detector to avoid

pressure f uctuations that may cause the gas

f ow rates to change. In some modern

Harrison

15


Register for free at

www.chromatographyonline.com/comparison

EVENT OVERVIEW:

A wide range of pharmaceuticals and personal care products (PPCPs)

have been detected as contaminants in surface and wastewaters around

the globe, which could possibly be linked to adverse health efects. This

webinar will discuss a study that investigated multiple water samples

from various points of the Tar River and from water treatment plants

in eastern North Carolina (USA) to determine the types and levels of

PPCPs in surface waters, and their potential link to the high incidences

of disease in this area.

Samples were prepared by solid-phase extraction (SPE) or liquid–liquid

extraction and analyzed for parent PPCPs and their metabolites by liquid

chromatography–time-of-fight mass spectrometry (LC–TOF-MS) and

gas chromatography–mass spectrometry (GC–MS). Many parent PPCPs

and their metabolites were detected in municipally treated wastewaters

as well as in the Tar River including: carbamazepine, iminostillbene,

oxcarbazepine, epiandrosterone, loratidine, gabapentin, β-estradiol,

triclosan, and others. In this presentation, the presenters discuss and

compare the results obtained using diferent extraction techniques

combined with GC–MS and LC–TOF-MS detection systems.

Who Should Attend

■ Environmental scientists using LC–MS and GC–MS in water analysis.

■ Water research scientists involved in the analysis of PPCPs.

Key Learning Objectives

■ Introduction to emerging PPCPs as

water contaminants.

■ Comparison of current methods used

for the extraction and identifcation

of PPCPs in surface and treated

wastewaters.

■ Scope and advantage of diferent

extraction and detection systems used.

Presenters:

Mustafa I. Selim

Tenured Professor of

Pharmacology and Toxicology

The Brody School of Medicine

East Carolina University (ECU)

Blake Rushing

PhD Research Student

The Brody School of Medicine

East Carolina University (ECU)

Moderator:

Laura Bush

Editorial Director

Spectroscopy & LCGC

For questions, contact Kristen Moore at [email protected]

Comparison of LC–MS and GC–MS Analysis of Pharmaceuticals and Personal Care Products in Surface Water and Treated Wastewaters

ON-DEMAND WEBCAST (Originally aired March 17, 2015)



GC–FID setups the f ame will not ignite if the

fuel gas f ow rates are unsuitable. While a

good feature, if the sample is run through

the GC–FID without the f ame being ignited,

the results will clearly be wrong.

Off-the-Scale Peaks: Peaks on the

chromatogram scale can disappear off the

paper for the following reasons:

• High detector sensitivity: If it is possible,

the simplest troubleshooting solution

would be to reduce the detector sensitivity

level.

• Sample volume: Reduce the sample

volume or dilute the sample prior to, or

during, injection to the GC.

To be sure the GC works well and is f t for

purpose, good practice would be to run a

method specif c system suitability test. In

addition, to track any system drift over time,

known samples could be analyzed regularly

during the analytical run.

Sample Considerations

In extreme cases, a skewed result may have

nothing to do with the f ow rate, volume

of sample, purity of the carrier gas, or any

leakages. The wrong sample may have been

introduced to the instrument, or samples may

have become contaminated or decayed. If this

is suspected, the sampling technique, sample

preparation, and storage should be reviewed.

Sample Decay: Sample decay, or changes

in sample composition, can take place

during the chromatography process. For

example, if hydrogen is used as a carrier gas,

any unsaturated hydrocarbons or aromatic

hydrocarbons present in a sample are likely

to react. This will be vastly accelerated in

the GC column oven. In this case, the best

troubleshooting advice would be to change

the carrier gas.

Sample Collection: Samples can also be

inadvertently transformed prior to injection

into the column. Volatile components can

evaporate from the sample mixture, or

components within the sample can react

with each other or with air or moisture

from the ambient environment. Collecting

samples in evacuated sample containers

or using temperature control during

sample transportation can be effective

troubleshooting remedies.

The above risks can be signif cantly

mitigated by taking and analyzing multiple

samples that will signif cantly increase the

chance that a sample handling error will be

detected.

Petrochemical Analysis Focus

Gas Chromatography–Flame Ionization

Detection (GC–FID):

Overview: Perhaps the most common gas

chromatography technique used in ref ning

and petrochemical applications is gas

chromatography with a f ame ionization

detector (GC–FID). The FID detects analytes

by measuring an electrical current generated

by electrons from burning carbon particles

in the sample. FID harnesses a combination

of hydrogen and oxygen. The oxygen for

the f ame combustion is normally supplied

by the use of high purity synthetic air to

minimize the amount of impurities coming

into the detector.

Key Considerations: It is important when

changing over from one cylinder of

synthetic air to another to ensure that the

composition of the air in the new cylinder

is consistent with that of the previous

cylinders, in terms of blend tolerance. For

example, the target oxygen concentration

might be 20%, but that mixture might

have a blend tolerance of plus or minus

1% absolute (5% relative) meaning that

the actual oxygen concentration can be

between 19% and 21%. While a small

change in the consistency of the contents of

the new cylinder might be acceptable, more

pronounced differences will inf uence how

the FID f ame burns and could lead to a very

different analytical result, even though the

sample has not changed.

The same principle applies to ordering

calibration gas mixtures. Using a calibration

gas mixture with an analytical accuracy of

plus or minus 10% could create an apparent

shift in process parameters when a process

analyzer is recalibrated and the instrument

then begins to respond differently.

Troubleshooting Steps: The f rst step is to

check that the fuel gas to the detector

has been switched on, that the f ame is

functioning, and has been successfully

ignited. Troubleshooting relies on checking

gas f ow rates and re-ignition of the f ame

prior to re-running the sample. It should also

be ensured that high quality gas regulators

are used to deliver the gases to the FID

detector to avoid pressure f uctuations that

may cause the gas f ow rates to change.

Gas Chromatography–Sulphur

Chemiluminescence Detection

(GC–SCD):

Overview: The sulphur chemiluminescence

detector (SCD) has emerged as a powerful

tool in ref nery GC, and is primarily used

for the quantitative determination of

various sulphur organic species (such as

hydrogen sulphide, mercaptans, thiophenes,

benzothiophenes, and sulphides in

hydrocarbon samples). It is a highly

sensitive and useful technique for the

characterization of crude oils of different

origin, because sulphur speciation is

essential during oil catalytic processing in a

ref nery.

Harrison

16




Key Considerations: A key consideration

when performing GC–SCD analyses are

the physical properties of the sample

delivery lines. It is essential that these

delivery lines are constructed from an inert

material, because using the wrong material

could result in sample components reacting

with the walls of the line. The most common

material used in general industry is 316

stainless steel, but it is not appropriate for

ref nery analysis. This is because certain

sulphur compounds in the sample line can

adhere to the walls and therefore not reach

the analyzer at the same time as the bulk of

the sample. The best alternatives are highly

chemically resistant non-metallic materials

such as Tef on and Kel-F or Hastelloy C-22,

a nickel-chromium-molybdenum-tungsten

alloy with high corrosion resistance.

Troubleshooting Steps: This problem can

go undetected because analysts do not

know that these compounds are present

until the line becomes saturated, resulting

in a sudden concentration of the substance

being released and detected as an anomaly.

If sulphur compound peaks appear in an

analysis result, but several hours after they

might have been expected, the reason

could be that the sulphur concentration is

actually several hours old. To validate or

rule out this issue, test injections of known

concentration calibration gas mixtures into

the sample delivery pipework upstream of

the analyzer could validate or rule out this

problem.

Summary

The analysis of chemical components for

petrochemical plant process control has

been elevated to unprecedented levels of

accuracy. As legislation becomes ever more

stringent, the importance of quantifying

and qualifying emission pollutants in an

accurate and transparent manner through

GC has become a priority. Emissions

measurement has serious f nancial

implications and compliance to measurement

is critical.

Stephen Harrison is a British Chartered

Engineer (MIChemE) with a career in

industrial gases spanning 26 years, over 12

of which have been focused in the area

of specialty gases. He has worked in an

international capacity for both Linde Gases

and previously BOC and now leads Linde’s

global Specialty Gases & Specialty Equipment

business from Munich, Germany. Stephen

has a Masters degree in chemical engineering

from Imperial College, London, UK.

E-mail: [email protected]

Website: http://hiq.linde-gas.com/

Harrison

17


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Extending the Compatible Analyte Volatility Range for Indoor Air Quality and Material Emissions Testing Using Multi-Bed Thermal Desorption Tubes

This article describes sampling methodology for thermal desorption–gas chromatography (TD–GC) that can extend the compatible analyte range of tests used to determine chemicals released from materials, as well as associated indoor air quality measurements. We summarize studies that demonstrate how packed multi-bed thermal desorption tubes, popular in environmental analysis, deliver improved recovery of very volatile compounds over single-bed tubes without compromising the recovery or stability of heavier target analytes.

Caroline Widdowson and David Barden, Markes International, Llantrisant, Wales, UK.

Thermal desorption (TD) is a versatile gas

chromatography (GC) pre-concentration

technology that is applicable to the

analysis of volatile and semi-volatile

organic compounds (VOCs and SVOCs)

in a wide range of sample matrices. As

well as boosting sensitivity, it allows full

automation of the processes of sample

pre-concentration, desorption and

extraction, and GC injection, greatly

improving sample throughput.

The typical two-stage TD process, used

for concentrating volatiles from relatively

large volumes of air or gas, involves the

gentle heating of sorbent-packed TD tubes

in a f ow of inert carrier gas, with the

released components being swept into an

electrically-cooled focusing (“cold”) trap Ph

oto

Cre

dit

: Tri

cia

Sh

ay P

ho

tog

rap

hy/G

ett

y I

ma

ge

s

18




within the TD system. The focusing trap is

then heated rapidly in a reverse f ow of carrier

gas to inject the organic compounds into

the GC column as a sharp band of vapour.

This two-stage desorption process optimizes

concentration enhancement and produces

narrow chromatographic peaks, thus

optimizing sensitivity.

The selection of sorbents for thermal

desorption sampling tubes and focusing

traps involves the consideration of a range of

factors, including the strength of the sorbent–

sorbate interaction, artefacts, hydrophobicity,

inertness, and mechanical strength. Packed

with appropriate sorbent(s), a TD sampling

tube can allow quantitative retention and

release of compounds ranging from C2

hydrocarbons and freons to semi-volatiles

such as PCBs, phthalates, and PAHs, without

exceeding optimized tube dimensions or

requiring liquid cryogen coolant.

Material emissions test methods have

traditionally been targeted at relatively narrow

analyte ranges — typically from n-hexane

to n-C16 or, in some cases, from n-hexane

to n-C22. Such methods typically use tubes

packed with Tenax TA,1,2 and although

this sorbent has many desirable qualities

(for example, hydrophobicity, low inherent

artefacts, good recovery of semi-volatiles,

and inertness), it is not suitable for some

very polar compounds, and is too weak for

quantitative retention of species more volatile

than n-hexane.

Therefore, with the growing demand to

measure very volatile and semi-volatile toxic

compounds, increasing attention has been

given to the use of sampling tubes packed

with additional sorbents.3,4 In these so-called

“multi-bed” tubes, up to four sorbents are

arranged in order of increasing strength from

the sampling end, so that the less volatile

“sticky” components only encounter the

weakest sorbent, and are easily released

when the gas f ow is reversed during the

subsequent thermal desorption process. This

allows a wider volatility range of components

to be quantitatively sampled and analyzed.

Although such multi-bed sorbent tubes

are commonly used in ambient (outdoor)

air monitoring,5 their deployment for

material emissions testing and indoor air

quality monitoring has historically been

limited. However, a recent revision of a key

international standard method1 includes

the option to use multi-bed sorbent tubes.

This article describes studies that assessed

the effectiveness of these tubes against the

single-bed tubes more widely used in this f eld.

Experimental

The protocols used are brief y summarized

here. Full details can be found in the cited

references.

Tenax TA tubes

(in series)(a)

(b)

Ab

un

da

nce

(x

10

6 c

ou

nts

)

5

2

1

0 5 10 15 20 25 30 35 40 45

IS 3

2

1

5

6

7 8

4

3IS

4

6

7

6

5

4

3

2

1

0

Ab

un

da

nce

(x

10

6 c

ou

nts

)

7

8

6

5

4

3

2

1

0

1

2

3

87

Multi-bed tubes

(in series)

First tube

1 n-Pentane2 Methylcyclobutane3 Butyl acetate4 m/p-Xylene5 Styrene6 o-Xylene7 2-Ethoxyethyl acetate8 Benzaldehyde

Second tube

Time (min)

0 5 10 15 20 25 30 35 40 45

Time (min)

First tube

Second tube

Figure 1: Analysis of emissions from PU foam (sampling volume ~4.6 L), sampled onto (a) two Tenax TA tubes connected in series, and (b) two multi-bed tubes connected in series. Internal standard (IS) = toluene-d8. Adapted and reproduced with permission from Analytical Methods 5, V.M Brown and D.R Crump, An investigation into the performance of a multi-sorbent sampling tube for the measurement of VVOC and VOC emissions from products used indoors, 2746–2756 (2013) © Royal Society of Chemistry.

Widdowson and Barden

19




A Micro-Chamber/Thermal Extractor

(Markes International) was used to sample

volatiles emitted by polyurethane (PU)

foam onto the sorbent tubes, followed

by analysis using a TD-100 automated

thermal desorber (Markes International)

with gas chromatography (GC) (Agilent

Technologies) coupled to MS (Agilent

Technologies).

Emissions from Polyurethane (PU)

Foam:6 PU foam was freshly cut from a

composite door known to release a mixture

of very volatile organic compounds (VVOCs),

VOCs, and SVOCs. Material samples were

placed into individual micro-chambers

equilibrated at 23 °C and the emissions

sampled onto the two types of sorbent tube.

Two identical tubes, in series, were used in

each case — a front “sampling” tube, and a

rear “back-up” tube to collect any analytes

that broke through. Sampling conditions

were (a) 60 min with an air f ow of ~80 mL/

min, to generate the chromatograms shown

in Figure 1, and (b) 15 min with an air f ow

of 50 mL/min, to generate the mean values

shown in Figure 2.

Stability Studies:7 To generate the results

shown in Figure 3, a mixture of chemical

standards relevant to material emissions

testing was loaded onto sorbent tubes in

the gas phase (with or without methanol,

as necessary) to give a nominal loading of

100 ng for each component, with a bubbler

used to generate humid atmospheres.

Results and Discussion

Figure 1 compares emissions from the

PU foam sample sampled using the two

types of tube. It is immediately apparent

that the multi-bed tubes show better

recovery of the lightest analytes, n-pentane

(b.p. 36 °C) and methylcyclobutane (b.p.

36 °C), with no detectable breakthrough

of either analyte on the respective back-up

tubes.

These results are confirmed by the

analysis shown in Figure 2, which used the

same setup except with a lower flow rate

and shorter sampling time, more typical of

a standard sampling protocol.

In the past, there have been concerns

that, during storage, less volatile analytes

might migrate from weaker sorbents to

stronger sorbents within multi-bed tubes — a

phenomenon that would result in compounds

being irreversibly bound to the stronger

sorbent. However, the 4-week stability

data presented in Figure 3 shows that the

performance of the multi-bed sorbent tubes

was at least equal to that of the Tenax TA

tubes. It is worth noting that, whatever the

sorbent combination used, it is still advisable

to analyze tubes as soon as possible after

sampling, and ideally within four weeks.

All experiments used stainless steel 3½”

× ¼” sorbent tubes (Markes International),

packed either with Tenax TA or with quartz

wool–Tenax TA–Carbograph 5TD. All tubes

were stringently conditioned before use using

a TC-20 off-line tube conditioner (Markes

International). The conditioned tubes were

capped prior to and after sampling with

two-piece brass long-term storage caps f tted

with PTFE ferrules.

500 Tenax TA

Multi-bed

Co

nce

ntr

ati

on

(µ

g/m

3) 400

300

200

100

n-Pen

tane

Met

hylcy

clobuta

nea,

b

Butyl a

ceta

te

m/p

-Xylen

e

Styr

enea

o-X

ylen

e

2-Et

hoxyet

hyl a

ceta

teb

Benza

ldeh

ydeb

0

Figure 2: Mean concentrations (n = 7–9) with standard deviations for eight dominant compounds in the analysis of emissions from PU foam (sampling volume 0.75 L), sampled onto two Tenax TA tubes connected in series (red), and two multi-bed tubes connected in series (blue). a) Values divided by 10 for ease of comparison; b) no pure standard was available, and so quantitation used toluene equivalents. Adapted and reproduced with permission from Analytical Methods 5, V.M Brown and D.R Crump, An investigation into the performance of a multi-sorbent sampling tube for the measurement of VVOC and VOC emissions from products used indoors, 2746–2756 (2013) © Royal Society of Chemistry.


20




Conclusions

These results demonstrate that multi-bed

sorbent tubes packed with quartz wool,

Tenax TA, and Carbograph 5TD are

compatible with an extended analyte

range compared to single-bed Tenax TA

tubes. As well as allowing simultaneous

active/pumped sampling of volatiles from

n-butane to n-C30, the stability of a

number of volatile analytes is found to be

very similar on both tube types,

confirming the suitability of these

multi-bed tubes for a range of TD

applications.

Acknowledgement

The kind assistance of Dr Derrick Crump

and Dr Veronica Brown (Cranf eld University,

UK) in supplying the data for this article,

and in reviewing a draft, is gratefully

acknowledged.

References

1. ISO 16000: Indoor air. Part 6: Determination

of volatile organic compounds in indoor and

test chamber air by active sampling on Tenax

TA sorbent, thermal desorption and gas

chromatography using MS or MS-FID. Annex

D specif es multi-bed sorbent tubes packed

(as in this study) with quartz wool–Tenax TA–

Carbograph 5TD, as an alternative to single-bed

Tenax TA tubes.

2. EN TS 16516: Construction products:

Assessment of emissions of regulated

dangerous substances from construction

products – Determination of emissions into

indoor air.

3. Application Note 005: Advice on sorbent

selection, tube conditioning, tube storage, and

air sampling, Markes International, April 2014,

http://www.markes.com/Resources/Application-

notes/default.aspx.

4. E. Woolfenden, Journal of Chromatography

A 1217, 2685–2694 (2010), http://dx.doi.

org/10.1016/j.chroma.2010.01.015

5. US EPA Method TO-17: Determination of volatile

organic compounds in ambient air using active

sampling onto sorbent tubes.

6. V.M. Brown and D.R. Crump, Analytical

Methods 5, 2746–2756 (2013), http://dx.doi.

org/10.1039/c3ay40224j

7. V.M. Brown, D.R. Crump, N.T. Plant, and

I. Pengelly, Journal of Chromatography A

1350, 1–9 (2014), http://dx.doi.org/10.1016/j.

chroma.2014.05.011

E-mail: [email protected]: www.markes.com

Caroline Widdowson is the product

marketing manager for thermal desorption at

Markes International, having completed her

chemistry degree at Cardiff University in 2004,

followed by a Ph.D. in organic chemistry. As

part of her current role, she works closely with

manufacturers, test laboratories, and research

institutes to advise analytical chemists on

sampling, analysis, and regulations surrounding

the release of chemicals from materials indoors

or in vehicles.

David Barden is a technical copywriter at

Markes International, having joined the company

in 2011. David studied natural sciences at the

University of Cambridge, UK, and remained

there for his Ph.D. in organic chemistry, which

he received in 2003. A placement at the

European Journals Department of Wiley-VCH,

Weinheim, Germany, was followed by seven

years as a technical editor for various scientif c

journals at the Royal Society of Chemistry,

Cambridge, UK.

Reco

very

(%

)

100

98

96

94

Storage time (weeks)

40% RH <3% RH

920 1 2 3 4

Reco

very

(%

)

100

98

96

94

Storage time (weeks)

920 1 2 3 4

Tenax TA

Tenax TA

Multi-bed

Multi-bed

Figure 3: Mean percentage recoveries for n-hexane, methyl isobutyl ketone, toluene, butyl acetate, cyclohexanone, 1,2,3-trimethylbenzene, and 4-phenylcyclohexene (nominally 100 ng each) from Tenax TA and multi-bed sorbent tubes (n = 5), stored for up to 4 weeks at room temperature, after loading using air of moderate and low relative humidity (40% RH and <3% RH). The individual 4-week recoveries across all seven compounds with a 40% RH loading were between 93% and 104%. Image generated from data in reference 7 with the permission of the authors.


21



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The Column #25/(’/)%()5−−9−)’!ˆ - UBMimages2.advanstar.com/PixelMags/lctc/pdf/2015-03-30-uk.pdf · “perfect” SOP for balance use. It should be reasonably straightforward