September 2012 · SIM mode permits identiﬁ cation of unknown contaminants in combination with...

LC TROUBLESHOOTING

Problem solvingGC–MS

Quantitative analysis of catalyst

poisoners

THE ESSENTIALS

Multidimensional GC

September 2012

Volume 15 Number 3

www.chromatographyonline.com

Detecting herbicides in tap water

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LC•GC Asia Pacifi c September 20124

Editorial Policy:

All articles submitted to LC•GC Asia Pacific

are subject to a peer-review process in association

with the magazine’s Editorial Advisory Board.

Cover:

Original materials: Don Farrall/Getty Images

Art Direction: Paul Davis

Article 8 Ultratrace Quantitative Analysis of Catalyst Poisoners

Using a Dedicated GC–MS Analyser

Kevin M. Van Geem, Jeroen Ongenae, Jean-Louis Brix, Joeri

Vercammen and Guy B. Marin

A GC–MS analyser is described that is reported to substantially

expand the workable application range of a classic catalyst

contaminants analyser. The use of mass spectrometry in FS/

SIM mode permits identifi cation of unknown contaminants in

combination with reliable quantifi cation at trace and ultratrace

amounts.

Columns 21 LC TROUBLESHOOTING

Readers’ Questions

John W. Dolan

Questions from the e-mail bag are considered in this month’s

instalment.

24 THE ESSENTIALS

A Short Introduction to Multidimensional GC

We present the governing principles and equipment required to

undertake comprehensive 2D GC techniques and highlight some of

the important applications areas.

Departments26 Application Notes

31 Products

34 Event Preview — FoodLytica 2012

COVER STORY15 Determination of Phenylurea

Herbicides in Tap Water and

Soft Drink Samples by HPLC–UV

and Solid-Phase Extraction

Manpreet Kaur, Ashok Kumar Malik

and Baldev Singh

A simple and sensitive high

performance liquid chromatography

(HPLC) method with ultraviolet (UV)

detection to analyse phenylurea

herbicides: — monuron, diuron,

linuron, metazachlor and metoxuron

— in three soft drinks brands and tap

water.

September | 2012

Volume 15 Number 3

4

ES119335_LCA0912_004.pgs 08.28.2012 05:15 ADV blackyellowmagentacyan

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LC•GC Asia Pacific September 20126

The Publishers of LC•GC Asia Pacific would like to thank the members of the Editorial Advisory Board

for their continuing support and expert advice. The high standards and editorial quality associated with

LC•GC Asia Pacific are maintained largely through the tireless efforts of these individuals.

LCGC Asia Pacific provides troubleshooting information and application solutions on all aspects

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knowledge to gain competitive advantage. Our scientific quality and commercial objectivity provide

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Editorial Advisory Board

Kevin AltriaGlaxoSmithKline, Harlow, Essex, UK

Daniel W. ArmstrongUniversity of Texas, Arlington, Texas, USA

Michael P. BaloghWaters Corp., Milford, Massachusetts, USA

Coral BarbasFaculty of Pharmacy, University of San

Pablo – CEU, Madrid, Spain

Brian A. BidlingmeyerAgilent Technologies, Wilmington,

Delaware, USA

Günther K. BonnInstitute of Analytical Chemistry and

Radiochemistry, University of Innsbruck,

Austria

Peter CarrDepartment of Chemistry, University

of Minnesota, Minneapolis, Minnesota, USA

Jean-Pierre ChervetAntec Leyden, Zoeterwoude, The

Netherlands

Danilo CorradiniIstituto di Cromatografia del CNR, Rome,

Italy

Hernan J. CortesH.J. Cortes Consulting,

Midland, Michigan, USA

Gert DesmetTransport Modelling and Analytical

Separation Science, Vrije Universiteit,

Brussels, Belgium

John W. DolanLC Resources, Walnut Creek, California,

USA

Roy EksteenTosoh Bioscience LLC, Montgomeryville,

Pennsylvania, USA

Anthony F. FellPharmaceutical Chemistry,

University of Bradford, Bradford, UK

Attila FelingerProfessor of Chemistry, Department of

Analytical and Environmental Chemistry,

University of Pécs, Pécs, Hungary

Francesco GasparriniDipartimento di Studi di Chimica e

Tecnologia delle Sostanze Biologica-

mente Attive, Università “La Sapienza”,

Rome, Italy

Joseph L. GlajchMomenta Pharmaceuticals, Cambridge,

Massachusetts, USA

Jun HaginakaSchool of Pharmacy and Pharmaceutical

Sciences, Mukogawa Women’s

University, Nishinomiya, Japan

John V. HinshawServeron Corp., Hillsboro, Oregon, USA

Tuulia HyötyläinenVVT Technical Research of Finland,

Finland

Hans-Gerd JanssenVan’t Hoff Institute for the Molecular

Sciences, Amsterdam, The Netherlands

Kiyokatsu JinnoSchool of Materials Sciences, Toyohasi

University of Technology, Japan

Huba KalászSemmelweis University of Medicine,

Budapest, Hungary

Hian Kee LeeNational University of Singapore,

Singapore

Wolfgang LindnerInstitute of Analytical Chemistry,

University of Vienna, Austria

Henk LingemanFaculteit der Scheikunde, Free University,

Amsterdam, The Netherlands

Tom LynchBP Technology Centre, Pangbourne, UK

Ronald E. MajorsAgilent Technologies,

Wilmington, Delaware, USA

Phillip MarriotMonash University, School of Chemistry,

Victoria, Australia

David McCalleyDepartment of Applied Sciences,

University of West of England, Bristol, UK

Robert D. McDowallMcDowall Consulting, Bromley, Kent, UK

Mary Ellen McNallyDuPont Crop Protection,Newark,

Delaware, USA

Imre MolnárMolnar Research Institute, Berlin, Germany

Luigi MondelloDipartimento Farmaco-chimico, Facoltà

di Farmacia, Università di Messina,

Messina, Italy

Peter MyersDepartment of Chemistry,

University of Liverpool, Liverpool, UK

Janusz PawliszynDepartment of Chemistry, University of

Waterloo, Ontario, Canada

Colin PooleWayne State University, Detroit,

Michigan, USA

Hans PoppeAnalytical Chemistry Laboratory,

University of Amsterdam, Amsterdam,

The Netherlands

Fred E. RegnierDepartment of Biochemistry, Purdue

University, West Lafayette, Indiana, USA

Harold RitchieThermo Fisher Scientific, Cheshire, UK

Pat SandraResearch Institute for Chromatography,

Kortrijk, Belgium

Peter SchoenmakersDepartment of Chemical Engineering,

Universiteit van Amsterdam, Amsterdam,

The Netherlands

Robert ShellieAustralian Centre for Research on

Separation Science (ACROSS), University

of Tasmania, Hobart, Australia

Robert SmitsRoyal Flemish Chemical Society, Belgium

Klaus K. UngerInstitute for Inorganic Chemistry, and

Analytical Chemistry, Johannes

Gutenberg University, Mainz, Germany

Yvan Vander HeydenVrije Universiteit Brussel,

Brussels, Belgium

Patricia M. YoungWaters Corporation, Milford,

Massachusetts, USA

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KEY POINTS

• High-yield metallocene catalysts are much more

susceptible to poisoning by feedstock impurities.

• MS is a powerful technique to quantify ultratrace levels of

target components and identify unknown contaminants,

simultaneously.

• A classic 2D GC column set-up with backflush and

vacuum restriction is capable of separating up to C12

hydrocarbons.

• A substantial increase in sensitivity is observed

compared to GC/FID analysers.

The use of high-yield metallocene catalysts has dramatically

increased both efficiency and selectivity of polymerization

processes (1). Unfortunately, these catalysts are extremely

prone to poisoning by feedstock impurities, such as

arsine (AsH3), phosphine (PH3), oxygenates (for example,

dimethylether) and sulphur-containing compounds

(mercaptanes, sulphides, etc) (2,3). Minute amounts of

these compounds are sufficient to impose undesirable

effects and induce immediate loss of catalytic activity and

reaction yield. At the same time, trace contaminants at the

part-per-billion (ppb) concentration levels can end up in

the polymers and alter subsequent polymer properties and

characteristics.

For decades, process chemical and petrochemical

analysts used to address their analytical challenges mainly

by relying on superior chromatography and smart tools

such as valve switching, backflush and Dean’s heart-cut.

In combination with relatively cheap, robust and selective

detectors, they were capable of providing all information

necessary to control and tweak petrochemical processes.

Ultratrace Quantitative Analysis of Catalyst Poisoners Using a Dedicated GC–MS AnalyserKevin M. Van Geem1, Jeroen Ongenae1, Jean-Louis Brix2, Joeri Vercammen2 and Guy B. Marin1,1 Ghent University, Laboratory for Chemical Technology, Zwijnaarde, Belgium,2 IS-X, Louvain-la-Neuve, Belgium.

A dedicated GC–MS analyser was developed to address the increasing need for more sensitive catalyst poisoner analysis. The system combines the separation power and robustness of a classic backfl ush confi guration with the selectivity and sensitivity of mass spectrometry.

Organic catalyst poisoners are usually determined using

dedicated chromatographic analysers. These systems

are, typically, equipped with a dual capillary column

configuration with backflush and fitted with a flame ionization



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detector (FID). Under these conditions, limits-of-detection

are usually situated around 100 ppb, depending on the

compound investigated and the complexity of the matrix that

is introduced (4). Unfortunately, this is far from sufficient to

protect the latest catalysts, which start to deteriorate as soon

as fed with low ppb amounts (5,6). An overview of some

typical specifications for catalyst poisoners in polymer-grade

hydrocarbons is given in Table 1.

Mass spectrometry (MS) is hardly used in petrochemical

QC laboratories, which is primarily because of its apparent

complexity and higher cost-of-ownership. Nonetheless, MS

detection has several distinct advantages over classic analogue

detectors. In full scan acquisition mode, for example, it allows

tracking and identification of unknown components using

spectral deconvolution and subsequent library matching.

In selective ion monitoring (SIM) mode, on the contrary,

MS permits trace and ultratrace quantification of target

analytes which is often superior to classic selective detectors.

Furthermore, MS permits the use of mass labelled internal

standards (ISs) that behave identically to their native analogues,

which has a positive effect on overall method precision and

accuracy. It is no surprise that instrument manufacturers have

invested substantially in solutions aimed at reducing overall

MS complexity and total cost-of-ownership in the last couple

of years. Easy tune and calibration functionalities, increased

sensitivity and speed, new acquisition modes and elegant

solutions that eliminate downtime, such as vacuum lock

technology, have contributed largely in this respect.

This article gives an overview of the main characteristics

and performance of a new gas chromatography mass

spectrometry (GC–MS) analyser that has been recently

developed. The system combines the chromatographic

separation power and backflush/Dean’s heart-cut capabilities

of a classic oxygenate analyser with the orthogonal separation

power, sensitivity, selectivity and overall robustness of the

latest generation single quadrupole mass spectrometers.

Experimental Standards: Standard oxygenate reference mixture from

Spectrum (Sugarland, Texas, US) at 10 ppm in hexane. For

more details with respect to the composition of the test

mixture, please consult Table 3. Calibration standards

were prepared by gradual dilution in hexane at 0.01 ppm,

0.05 ppm, 1 ppm and 5 ppm.

Gas chromatography: The GC analyser consisted of

a Thermo Trace GC (Austin, Texas, US) refurbished by

Global Analyser Solutions (GAS, Breda, The Netherlands).

The system was fitted with a gas sampling valve (GSV),

a liquid sampling valve (LSV), a vapourizer, a standard

split/splitless injector and a FID. Inside the GC oven, a

universal pressure balanced Deans assembly was installed

to carry out heart-cut and/or backflush. Auxiliary pressure

for balancing was provided and controlled by a separate

Trace GC DCC unit.

The first dimension column was a Restek Rtx-1

(Bellefonte, Pennsylvania, US) with the following dimensions:

15 mL × 530 µm i.d., 5 µm df. The second dimension column

was an Agilent CP-Lowox (Middelburg, The Netherlands)

Table 1: Typical specifications for catalyst poisoners in polymer

grade hydrocarbons (6).

Table 2: Overview of the GC settings.

Impurity Typical Specification

Arsine Less than 20 ppb

Phosphine Less than 20 ppb

Ammonia Less than 100 ppb

Hydrogen sulphide Less than 20 ppb

Carbonyl sulphide Less than 20 ppb

Nitrogen dioxide Less than 50 ppb

HCN Less than 100 ppb

HCl, HF Less than 200 ppb

Phosgene Less than 50 ppb

Sulphur dioxide Less than 50 ppb

Chlorine Less than 30 ppb

Oven Setting Remarks

Initial temp., (°C) 50 –

Initial time, (min) 5.00 –

Final temp., (°C) 240 –

Final time, (min) 10.00 –

Rate, (°C/min) 5 Slow heating to maintain resolution

Inlet – –

Type Direct –

Mode Splitless –

Temp., (°C) 200 –

Carrier – –

Gas Helium –

Mode Constant pressure –

Setting, (kPa) 50 –

Detector – –

Type FID 1.2

Temp., (°C) 200 1.2

Table 3: Peak identification and typical SIM ions.

tR, min Name SIM ions

24.52 Diethyl ether 59, 74

25.05 Acetaldehyde 44

26.48 ETBE 59, 87

26.79 MTBE 57, 73

26.92 Di-isopropylether (DIPE) 59, 87

27.93 Propanal 57, 58

28.92 t-Amyl methyl ether (TAME) 73, 87

29.42 Propyl ether 73, 102

30.50 iso-Butanal 72

31.78 Butyraldehyde 57, 72

32.82 Methanol 29, 31

33.45 Acetone 58

35.26 Valeraldehyde 57, 58

36.13 MEK 57, 72

36.50 Ethanol 31, 45

39.28 iso-Propanol 45

39.45 Propanol 59, 60

40.21 Allyl alcohol 57, 58

41.54 iso-Butanol 41, 74

41.64 t-Butanol 57, 59

42.51 n-Butanol 55, 56


Vercammen et al.

ES117673_LCA0912_010.pgs 08.24.2012 08:03 ADV blackmagentacyan

EVENT OVERVIEW:

One of the most popular and useful techniques for polyolefins (PE,PP) character-

ization is Gel Permeation Chromatography or Size Exclusion Chromatography

(GPC/SEC). Traditionally it has been considered quite an involved technique

plagued with many practical difficulties, since it must be carried out at elevated

temperatures, handling chlorinated solvents.

Recently, instrumentation specifically designed for safe and reliable operation

under those hard conditions has become available and, for the first time, full

automation of all the analytical workflow, from sample preparation to data eval-

uation is possible.

Engineering advances implemented in most modern high temperature GPC/

SEC instruments will be described, showing how updated technology can help

to improving laboratory productivity, raising the safety standards and minimiz-

ing the downtime possibilities.

In this seminar, the great value of infrared detection as concentration detec-

tor for characterization of polyolefins and the synergy as online simultaneous

detection of chemical composition will be demonstrated. Real life examples of

several GPC/SEC methods with different detection schemes are discussed, from

routine to more challenging samples.

This session will be very valuable for those scientists who are considering the

acquisition of new gel permeation chromatograph equipment for polyolefin

application and want to learn more about the recent advances in instrumenta-

tion engineering as well as most appropriate detection technologies.

Who Should Attend:

n Anyone involved in molar mass distribution analysis of polyolefins.

n Scientists and engineers who need to understand what HT-GPC based on IR

detection can offer for a better understanding of copolymers and complex

resins.

n Laboratory managers and decision makers who need to gather information

on current status and future trends in high temperature instrumentation for

future purchase planning.

Key Learning Objectives:

n Learn how new developments in high

temperature GPC/SEC instrumentation

help in raising the laboratory safety and

health levels, while ensuring proper

sample care.

n Learn how to obtain chemical composition

and molar mass distribution for

copolymers in an efficient way thanks to

integrated high performance IR detector.

n Discover why GPC with infrared detector

is becoming the standard technique in

PO, given the excellent sensitivity and

stability of the IR detector and increasing

interest in the analysis of multi-reactor

resins and polypropylene / polyethylene

combinations.

n Keep updated with trends in polyolefin

industry in terms of molar mass and

chemical composition characterization.

Presenters:

Alberto Ortín

Scientist

Polymer Char

Juan Sancho-Tello

Senior Engineer

Polymer Char

Moderator:

Alasdair Matheson

Managing Editor

LCGC Europe

For more information, contact Kristen Farrell at [email protected]

Analytical and Engineering Advances in High Temperature GPC/SEC forPolyolefin Characterization

Sponsored by Presented by

ON-DEMAND WEBCAST

Register Free at http://www.chromatographyonline.com/polyole�n_gpc


with the following dimensions: 10 mL × 530 µm i.d., 10 µm

df. Restrictions were 250 µm i.d. uncoated Siltek-deactived

fused-silica capillary tubing (Restek) cut to the appropriate

length. All connections were made using micro Siltite unions

(SGE Analytical Science Victoria, Australia). Other relevant

parameters are summarized in Table 2.

Mass spectrometry: The GC analyser was hyphenated to

a Thermo ISQ single quadrupole mass spectrometer. The

system was applied in both full scan (range: 15–250 amu)

and SIM (dwell time: 0.2 s) as full scan/SIM mode. All relevant

MS settings are summarized in Table 3.

All data were acquired using Thermo QuanLab Forms.

The MS was applied after running a full EI tune. System

performance was verified using a daily tune check.

Results and DiscussionSystem set-up: The capillary column set comprises the true core

of any classic catalyst poisoner analyser. The second dimension

column is particularly important. Ultimately, it is here that

separation of the analytes, from each other as well as from the

aliphatic matrix in which they reside, occurs. A CP-Lowox column

(Agilent) was used for this purpose. This column, which is based

on a multilayer PLOT concept, is very polar and characterized

by a high MAOT with virtually no bleed at temperatures as high

as 350 °C (7). In combination with a backflushed apolar-coated

capillary column in the first dimension, matrix separations up until

C12 hydrocarbons are well within range.

Unfortunately, the CP-Lowox column is not available

in an MS-friendly narrow bore dimensions. To avoid the

MS vacuum from protruding the system, it is necessary to

incorporate an adequate restriction at the back of the column.

A schematic representation of system set-up for Lowox/MS

applications is depicted in Figure 1.

BF

MS

Rxi-1,

15m × 530 µm

Lowox,

10m × 530 µm

Restriction,

Oxygenates

HC/oxygenates

5m × 250 µm

Figure 1: Schematic representation of the GC–MS analyser.

BF = backflush; HCs = hydrocarbons.

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

5

00 5

0423.94

20 25 30

Time (min)

35 40 45

46.47 47.24

36.50

35.26

31.78

33.45

29.40

26.92

26.46

24.52

19.31

Rela

tive A

bu

nd

an

ce

39.28 41.56

41.51

Figure 2: Chromatogram of the oxygenates standard

at 10 ppm. The MS was applied in full scan mode. Peak

identification is referred to in Table 2.

(a)

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

25.0 25.1

25.08 25.14 25.19 25.23

25.68

25.38

Time (min)

Re

lati

ve

Ab

un

da

nce

25.77

25.88 25.98

25.2 25.3 25.4 25.5 25.6 25.7 25.8 25.9 26.

Figure 3: SIM traces at 0.01 ppm. (a) acetaldehyde;

(b) ethanol; and (c) propyl ether.

(b)

100

95

90

85

80

75

70

65

60

55

50

45

40

35

Re

lati

ve

Ab

un

da

nce

35.6 35.8 36.0 36.2 36.4 36.6 36.8 37.0 37.2 37.4 37.6 37.8

37.7237.4837.4437.3037.1637.02

36.64

Time (min)

(c)

100

95

90

85

80

75

70

65

60

55

50

Re

lati

ve

Ab

un

da

nce

29.35 29.40 29.45

29.48 29.50

29.50 29.55 29.60 29.65 29.70

29.74

29.60

29.75 29.80 29.85 29.90

Time (min)


Vercammen et al.


True backflush, as well as Dean’s heart-cut, is achieved by

increasing the auxiliary pressure at the column joint just above

the first dimension residual head pressure at a certain moment

in time. Debalancing induces full flow reversal, whilst maintaining

a small flow over the Lowox column for chromatography. It is

crucial to know the exact pressure at the column joint for this to

be successful. When set too low, standard flow direction will be

maintained and the first column is not backflushed. Conversely,

when set too high, none of the target analytes will be able to

reach the second dimension column and the detector. The

easiest way to determine the pressure at the column joint

involves setting the head pressure at regular and then reading

the residual pressure at the auxiliary DCC, which is kept off at

this stage. Five kPa differences are sufficient to induce flow

reversal. Although less straightforward because of the vacuum

conditions, a similar approach is applied in combination with

MS. It also permits the user to determine the minimal length of

the restriction capillary (Figure 1).

System suitability: System suitability was evaluated by

direct injection of the 10 ppm oxygenate standard. In order

to compare with a classic analyser set-up, experiments

were performed using both FID and MS as a detector. The

MS was applied in both full scan and SIM modes. A typical

chromatogram with the MS in full scan mode is depicted in

Figure 2. Peak identification is referred to in Table 3.

A comparative overview of the results is given in Table 4. For

each peak, the signal-to-noise ratio (S/N) was calculated (RMS)

in full scan, extracted ion and SIM mode. These results were

subsequently expressed relative to the S/N with FID detection.

The results in Table 4 clearly illustrate the significant gains in

sensitivity that can be reached when using MS compared to

FID. Minimal gain is 3.7 for ethanol. Unsurprisingly, sensitivity

gains are particularly significant when the MS was used in SIM

mode. Straight full scan mode proved to be less appropriate

for target analysis, which is predominately due to the low

molecular weight of the target compounds, and means

having to include highly interfering masses such as m/z =18

(water), 28 (nitrogen) and 32 (oxygen) in the scan range. More

convenient in this respect is the combined full scan/SIM mode,

which is available on all major instrument brands nowadays.

Afterwards, calibration curves were constructed for each

of the oxygenates in the standard mixture from 0.05 ppm to

5 ppm. Correlation coefficients were ≥ 0.995. Some typical

SIM traces at the 0.10 ppm level are depicted in Figure 3.

Method repeatability was determined as well. Results at the

10 ppm level are included in Table 4 (six consecutive analyses).

ApplicationsNaptha feed: Naphtha is a complex mixture of hydrocarbons

(C5–C12) in petroleum boiling between 30 °C and 200 °C.

Oxygenates are routinely determined in these samples

according to reference procedures such as ASTM D7423 (4)

as their cracking product cause problems in the downstream

separation processes (8). Naphthas are very complex and

fully require the chromatographic separation power of the

Lowox column. A typical chromatogram of a naphtha sample

100

95

90

85

80

75

70

65

60

55

50

45

40

35

30

25

20

15

10

20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56

5

0

Rela

tive A

bu

nd

an

ce

Time (min)

19.70

20.60

20.74

28.20

31.10

28.58

30.0531.8234.05

34.45

33.48 36.1937.60

39.36

39.80

40.5643.39

46.07

32.6

32.5232.5332.74

33.12

33.19

32.92

32.8 33.0 33.2Time (min)

41.41 45.08

46.60 47.3049.7650.8152.66 54.4656.64

21.5422.3624.45

26.43

27.53

Acetaldehyde

TAME

Methanol

Figure 4: Typical SIM trace of a naphtha feed.

Table 4: Average sensitivity gains in different MS detection modes. FS = full scan; EIC = extracted ion chromatogram; SIM = selected

ion monitoring. %RSD at the 10 ppm level (six analyses).

Name MS (FS), S/N MS (EIC), S/N MS (SIM), S/N %RSD

Diethyl ether 1.2 2.6 81 9.27

Acetaldehyde 0.2 4.1 10 18.9

ETBE 0.6 9.9 66 7.56

MTBE 0.6 3.1 53 6.84

Diisopropylether 0.7 6.5 70 10.1

Propanal 0.3 6.6 27 11.9

t-Amyl ether 1.1 4.4 64 4.60

Propyl ether 1.3 17 70 6.56

iso-Butanal 0.4 5.0 38 5.35

Butyraldehyde 0.8 4.4 23 2.31

Methanol 1.5 1.8 6.5 12.2

Acetone 0.8 18 100 7.21

Valeraldehyde 1.5 8.4 177 5.09

MEK 1.3 2.0 4.4 10.9

Ethanol 0.3 1.2 3.7 13.4

iso-Propanol 0.6 2.9 5.6 18.7

Propanol 0.5 1.6 5.5 12.4

Allyl alcohol 0.3 1.1 14 11.0

iso-Butanol 1.1 8.1 45 11.7

t-Butanol 0.9 2.1 49 9.34

n-Butanol 0.7 1.9 25 11.7

13www.chromatographyonline.com

Vercammen et al.


in SIM mode is depicted in Figure 4. Individual samples were

introduced using the LSV of the GC–MS analyser. The insert

shows the methanol trace (ion 29, 0.18 ppm); concentrations of

acetaldehyde and TAME were 8.3 and 4.9 ppm, respectively.

When idle, the GC oven was kept at 200 °C with the

backflush activated. This was necessary to prevent the

accumulation of siloxane bleed from the precolumn.

ConclusionsA GC–MS analyser is described that substantially expands the

workable application range of a classic catalyst contaminants

analyser. The use of mass spectrometry in FS/SIM mode

permits identification of unknown contaminants in combination

with reliable quantification at trace and ultratrace amounts.

AcknowledgementThe authors acknowledge the financial support from the

Long Term Structural Methusalem Funding by the Flemish

Government – grant number BOF09/01M00409. KVG holds

a Postdoctoral Fellowship of the Fund for Scientific Research

Flanders and a BOF tenure track position at Ghent University.

References(1) L. Resconi, L. Cavallo, A. Fait and F. Piemontesi, Chem. Rev., 100,

1253–1345 (2000).

(2) Almatis AC Inc., Application for Selective Adsorbents in Polymer

Production Processes, Technical Bulletin USA/6040-R00/0504.

(3) M.A. Graham, Selected Ethylene Feedstock Impurities: Survey Data.

Ethylene Producers Conference, Houston, Texas, USA (1993).

(4) J.G. Speight, Handbook of Petroleum Product Analysis, John Wiley

& Sons (2002).

(5) B. Biela, R. Moore, R. Benesch, B. Talbert and T. Jacksier, Gulf

Coast Conference, Galveston, Texas, USA (2003).

(6) S.S. Thind, Petro Industry News, 1–2, June/July, (2003).

(7) J. de Zeeuw and J. Luong, Trends Anal. Chem., 21, 594–607,

(2002).

(8) S.P. Pyl, C.M. Schietekat, M.-F. Reyniers, R. Abhari, G.B. Marin and

K.M. Van Geem, Chem. Eng. J., 176–177, 178–187 (2011).

Kevin M. Van Geem is a Fulbright alumnus of the

Massachusetts Institute of Technology, Massachusetts,

USA, and assistant professor in thermochemical reaction

engineering in the department of chemical engineering

and technical chemistry at Ghent University, Belgium.

Jeroen Ongenae is a masters student in civil engineering

in the department of chemical engineering and technical

chemistry at Ghent University, Belgium. He is currently

finishing his thesis on trace oxygenate analysis using mass

spectrometric techniques.

Jean-Louis Brix is an analytical support engineer at

IS-X. He specializes in instrument design and method

development for petrochemical applications.

Joeri Vercammen is managing expert of IS-X. He

specializes in method development and rationalization

from prep-to-rep, method validation and quality

assurance.

Guy B. Marin is chair of the department of chemical

engineering and technical chemistry at Ghent University,

Belgium. Chemical reaction engineering, catalysis in general

and reaction kinetics are the main topics in his research

programme.


Vercammen et al.

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KEY POINTS• Phenylurea herbicide residues can be found in water

sources, processed products and on the crops where

they are applied. Some types have also been detected

in soft drinks.

• The method described here can be used to

successfully detect monuron, diuron, linuron,

metazachlor and metoxuron in tap water and soft

drinks at concentrations of 5 ng/mL. Good recovery

rates were obtained for each analyte.

Phenylurea herbicides are used widely in a broad range of

herbicide formulations as well as for nonagricultural use.

Consequently, their residues are frequently detected as

major water contaminants in areas where these are used

extensively (1). Diuron and linuron are substituted urea

compounds that are soluble in water and can migrate in

soil and enter the food chain (2). These herbicides are

of significant toxicological risk to humans and wildlife.

Diuron, which is used in cotton‑growing areas and with fruit

crops, is rated as the third most hazardous pesticide for

groundwater resources. These herbicides are also applied

on railway tracks to maintain quality and provide a safer

working environment (3) but this may lead to groundwater

contamination as their leaching potential is significant.

Phenylureas enter the environment through pathways such

Determination of Phenylurea Herbicides in Tap Water and Soft Drink Samples by HPLC–UV and Solid-Phase ExtractionManpreet Kaur, Ashok Kumar Malik and Baldev Singh, Department of Chemistry, Punjabi University, Punjab, India.

A simple and sensitive high performance liquid chromatography (HPLC) method with ultra-violet (UV) detection has been developed for the analysis of phenylurea herbicides — namely, monuron, diuron, linuron, metazachlor and metoxuron — that involves a preconcentration step using solid-phase extraction (SPE). The mobile phase used was acetonitrile–water at a flow-rate of 1 mL/min with direct UV absorbance detection at 210 nm. Separation of analytes was studied on a C18 column. The method was applied successfully to the analysis of the herbicides in three soft drink brands and tap water. Good linearity and repeatability were observed for all the pesticides studied.



as spray drift, runoff from treated fields and leaching into

groundwater. Most of the excess material penetrates into the

soil where it is subjected to the action of microorganisms (4)

and degradation as studied by Canonica and colleagues

(5). Phenylureas are unstable photochemically, as

discussed by Khodja and colleagues (6) but these can

persist in water for several days or weeks depending on the

temperature and pH. Cases of incidental pesticide pollution

of water reservoirs (2–4,7–13) have become more numerous

in recent years.

Phenylurea residues can be found in water sources,

processed products and on the crops where these are

applied. In India, most of the soft drink bottling plants use

surface water from canals and rivers, which have a high risk

of pesticide contamination. The water treatment measures

used are insufficient for complete removal of these pesticide

residues, which have been found to be above permissible

limits. The evidence for the above stated facts was provided

in a 2003 Centre for Science and Environment (CSE, New

Delhi, India) report that found several pesticide residues

in many soft drink samples of leading international brands

procured from all over India. The CSE findings were

confirmed further by a Joint Parliamentary Committee (JPC)

created to verify the facts. In 2006, CSE conducted another

round of tests and found pesticides yet again in soft drink

samples. Keeping this in mind, the present work has great

importance, as it involves the determination of phenyl urea

herbicides in soft drink samples and tap water.

Therefore, it is imperative that sensitive, selective and

efficient methods for herbicide analysis be designed. The

common analytical methods used are high performance

liquid chromatography (HPLC)–UV (2–4,7–9), solid‑phase

microextraction (SPME)–HPLC (10), diode array (11),

immunosorbent trace enrichment and HPLC (12,14),

LC–mass spectrometry (MS) (15,16), gas chromatography

(GC)–MS (13), capillary electrophoresis (17,18,19),

photochemically induced fluorescence (20,21) and derivative

spectrophotometry (22). A useful review is presented by

Sherma (23) on the use of thin‑layer chromatography (TLC)

and its modified versions for the analysis of these herbicides.

Solid‑phase extraction (SPE) of phenylurea herbicides has

been reported in literature by several workers (24–29). The

SPE of soft drinks has been reported extensively (30–36).

As the use of polar and degradable pesticides becomes

widespread, it is urgent that more sensitive analytical

methods be developed for their residual analysis in various

matrices. HPLC has several advantages over GC because it

DiuronLinuron

Metazachlor

Monuron

Metoxuron

CH3

O

CH3

H

C

N

CI

CIN CH3

N

H

N

O

O

C

CI

CI

CH3

N

NN

CI C

CH3

O

CH2

CH2

H3C

CH3

N N

H

CI

O

C

CH3

CI

CH3O

CH3

CH3

NNH

O

C

Figure 1: Structures of phenylurea herbicides.A

bso

rban

ce (

mA

U)

Retention time (min)

1

0.8

0.6

0.4

0.2

0

-0.2-1 4

12

3

4 5

9 14

-0.4

Figure 2: HPLC–UV chromatogram of mixture containing

5 ppb each of the phenylurea herbicides: 1 = metoxuron,

2 = monuron, 3 = diuron, 4 = metazachlor and 5 = linuron.

Table 1: Analytical figures of merit obtained under optimum conditions.

Characteristic Metoxuron Monuron Diuron Metazachlor Linuron

Regression equation 0.0016x + 0.0712 0.0014x + 0.0308 0.0035x + 0.128 0.0017x + 0.083 0.002x + 0.1664

R2 0.992 0.994 0.992 0.992 0.993

Retention time (min) 4.3 4.9 7.25 8.68 12.4

Linear range (ng/mL) 5–500 5–500 5–500 5–500 5–500

LOD = 3.3 × S/m

(ng/mL)0.92 0.82 0.93 1.28 1.29

LOQ = 10 × S/m (ng/mL) 2.76 2.46 2.79 3.84 3.87

Recovery % (RSD) 81.0 (2.4) 85.4 (3) 91.1 (3) 88.2 (3.2) 92.3 (5)

* Amount of phenylurea herbicides taken 5 ng/mL each (n = 5)


Kaur et al


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can be used for simultaneous analysis of thermally unstable,

nonvolatile, polar and neutral species without a derivative

step. Because of the thermally unstable nature of phenylurea

herbicides, the direct application of GC to these compounds

is not possible and derivatization prior to the detection

is needed. For this reason, HPLC with UV absorption or

fluorescence detection (7–10) is preferred over GC. As

a result, HPLC is gaining popularity and preference as a

pesticide analysing technique.

The present work describes a simple and sensitive

HPLC–UV method for the analysis of phenyl urea herbicides

(namely, monuron, diuron, linuron, metazachlor and metoxuron)

and it involves a single‑step preconcentration by SPE.

Materials and MethodsThe HPLC system used included a P680 HPLC pump

(Thermo Scientific Dionex, Sunnyvale, California, USA),

a 250 mm × 4.6 mm, 5‑µm Acclaim C18 RP analytical

column (Thermo Scientific Dionex) and a UVD 170U

detector operated at a wavelength of 210 nm coupled to a

Chromeleon computer program for the acquisition of data

(Thermo Scientific Dionex).

Monuron, diuron, linuron, metoxuron and metazachlor

(Figure 1) pesticide standards were obtained from

Riedel‑de‑Haen (Seelze, Germany). HPLC‑grade acetonitrile

and methanol were obtained from J.T. Baker (Phillipsburg,

New Jersey, USA). All the solvents were filtered through nylon

6.6 membrane filters (Rankem, New Delhi, India) using a

filtration assembly (Perfit, India) and sonicated before use.

Triple‑distilled water was used for all purposes.

Standard PreparationStock solutions were prepared in a mixture of 50:50

methanol–water. All the solutions were stored under

refrigeration below 4 °C.

Sample PreparationThe SPE of the tap water and soft drink samples was

performed using a Visiprep SPE vacuum manifold (Supelco,

Bellefonte, Pennsylvania, USA) and C18 cartridges from

J.T. Baker. The SPE cartridges were attached to the

solvent‑recovery assembly and connected to a vacuum pump.

The conditioning was done with 1 mL each of acetonitrile,

methanol and triple‑distilled water.

Soft drink samples: The presence of phenylurea herbicides

was studied in three different types of locally purchased soft

drinks (Coke, Mirinda and Limca). These were filtered with

nylon 6.6 membrane filters and degassed by sonicating

for 30 min. The samples were spiked with the metoxuron,

monuron, diuron, metazachlor and linuron at a concentration

of 5 ng/mL. A 20 mL volume of these samples was passed

through the C18 SPE cartridges under vacuum and the

analytes were eluted with 1.5 mL of acetonitrile. The eluants

were further used for the HPLC–UV analysis. The sample

blanks were also prepared similarly.

Tap water sample: The tap water sample was taken from the

laboratory. It was filtered and then degassed with an ultrasonic

bath. The sample was spiked with metoxuron, monuron,

diuron, metazachlor and linuron at a concentration of 5 ng/mL

each. A 50 mL sample of the tap water containing the mixture

of herbicides was preconcentrated using C18 SPE cartridges.

A 1.5 mL volume of acetonitrile was used for the elution and

the eluant was subjected to HPLC–UV analysis. The sample

blanks were prepared by the same method.

ProcedureAliquots of the mixture of five herbicides were taken, having

concentrations of 5–500 ppb. These mixtures were analysed

at an optimum wavelength of 210 nm. The mobile phase is an

important factor in HPLC analysis, as it interacts with solute

species of the sample. Hence, the composition of the mobile

phase was selected carefully as 60:40 acetonitrile–water, and

the flow‑rate was set at 1 mL/min. All measurements were

(a)

3

2.5

2

1.5

Ab

sorb

an

ce (

mA

U)

Retention time (min)

1

0.5

0

3 5 7 9 11 13

(c)

(d)

(b)

Figure 3: HPLC–UV chromatograms of (a) tap water,

(b) Coke, (c) Limca and (d) Mirinda spiked with a mixture of

phenylurea herbicides containing 5 ppb of each, obtained

after preconcentration by SPE.

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Kaur et al


taken at ambient temperature. The calibration curves for all

five herbicides were prepared and the curves were linear in

the range studied.

Results and DiscussionHPLC–UV studies: The separation of these herbicides

was studied using direct injection of samples and

parameters such as the effect of flow‑rate, selection of

suitable wavelength and composition of mobile phase

were optimized. The composition of the mobile phase was

60:40 acetonitrile–water. At higher flow‑rates than 1.0 mL/

min, the separations were not up to the baseline, and with

lower flow‑rates, peak tailing was observed, so the flow‑rate

was optimized to 1.0 mL/min. The wavelength for detection

was selected from the UV absorption spectra of the five

herbicides as 210 nm.

Preparation of calibration curves: The calibration

curves were constructed for the detection of monuron,

linuron, diuron, metoxuron and metazachlor in the range

of 5–500 ppb under the optimized conditions using the

HPLC with UV detection. The calibration curves were

linear over this range. Various characteristics of HPLC–UV,

including regression equation, working range and RSD,

are summarized in Table 1. The LODs of the phenylurea

herbicides were calculated using 3.3 × S/m (S = standard

deviation, m = slope of calibration curve), and they were

found to be in the range 0.82–1.29 ng/mL. Characteristic

chromatograms with HPLC–UV detection at 210 nm are

shown in Figures 2 and 3 for the separation of these

herbicides.

Recoveries, repeatability and LODs: The method detection

limits were calculated for these herbicides per the ICH

Harmonized Tripartite Guidelines (www.ich.org/LOB/media/

MEDIA417.pdf). The method LOQs can be calculated by

using 10 × S/m. The accuracy (% recovery) and precision

(%RSD) of the HPLC–UV method were evaluated for each

analyte by analysing a standard of known concentration

(5 ng/mL) five times and quantifying it using the calibration

curves. Method optimization and validation parameters are

presented in Tables 1 and 2. Good linearity and repeatability

were observed for all the compounds studied (with

correlation coefficient > 0.99). The method gives satisfactory

results when used to quantify these herbicides in soft drink

and tap water samples (Table 2) with percentage recoveries

ranging from 75% to 90.1%.

ApplicationsThe phenylurea herbicides were studied in various soft drink

and tap water samples and no interfering peaks appeared

at the retention times of these herbicides in the spiked

samples. The tap water, Coke, Mirinda and Limca (Figure 3)

samples were spiked with metoxuron, monuron, diuron,

Table 2: Analytical figures of merit obtained using various samples.

Samples testedPhenylurea Herbicide

Metoxuron Monuron Diuron Metazachlor Linuron

Tap water

Linear range

(ng/mL)5–500 5–500 5–500 5–500 5–500

LOD (ng/mL) 0.92 0.84 0.91 1.30 1.35

LOQ (ng/mL) 2.76 2.52 2.73 3.90 4.05

Recovery* % (RSD) 80.6 (4) 84.2 (3.1) 90.1 (4) 87.1 (4) 76.3 (5)

Limca

Linear range

(ng/mL)5–500 5–500 5–500 5–500 5–500

LOD (ng/mL) 0.95 0.89 0.99 1.39 1.41

LOQ (ng/mL) 2.85 2.67 2.97 4.17 4.23

Recovery* % (RSD) 79.4 (4) 83.1 (4) 87.8 (4.2) 87.8 (4.5) 75.4 (5.1)

Coke

Linear range

(ng/mL)5–500 5–500 5–500 5–500 5–500

LOD (ng/mL) 0.95 0.90 1.0 1.37 1.40

LOQ (ng/mL) 2.85 2.70 3.0 4.11 4.20

Recovery* % (RSD) 77.5 (4.6) 80.2 (4.7) 88.6 (5) 85.3 (5) 77.3 (4.8)

Mirinda

Linear range

(ng/mL)5–500 5–500 5–500 5–500 5–500

LOD (ng/mL) 0.96 0.89 0.99 1.37 1.42

LOQ (ng/mL) 2.88 2.67 2.97 4.11 4.26

Recovery* % (RSD) 81.1 (3.4) 83.4 (3.2) 87.6 (4) 85.1 (4.3) 77.3 (5.3)

* Samples spiked at 5 ng/mL, n = 5


Kaur et al

ES117846_LCA0912_019.pgs 08.24.2012 08:17 ADV blackmagentacyan

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Mughari and I.M. Ortiz‑Rodríguez, Journal of Fluorescence, 18,

365–373 (2008).(22) I. Baranowiska and C. Pieszko, Anal. Letters, 35, 413–486 (2002).(23) J. Sherma, Acta. Chromatographia, 15, 5–30 (2005).(24) M.M.C. de la Peña and A. Bautista‑Sánchez, Talanta, 13, 279–285

(2003).(25) I. Ferrer, V. Pichon, M.C. Hennion and D. Barceló, Journal of

Chromatography A, 1, 91–98 (1997).(26) F. Li, D. Martens and A. Kettrup, Se Pu, 19, 534–537 (2001).(27) T. Cserhati, E. Forgács, Z. Deyl, I. Miksik and A. Eckhardt,

Biomedical Chromatography, 18, 350–359 (2004).(28) M.J.I. Mattina, Journal of Chromatography A, 549, 237–245 (1991).(29) M. Hamada and R. Wintersteiger, Journal of Planar

Chromatography-Modern TLC, 15, 11–18 (2002).(30) J.F. García‑Reyes, B. Gilbert‑López and A. Molina‑Díaz, Anal.

Chem., 30, 8966–8974 (2002).(31) M.A. Mumin, K.F. Akhter and M.Z. Abedin, Malaysian Journal of

Chemistry, 8, 45–51 (2008).(32) X.L. Cao, J. Corriveau and S. Popovic, J. Agric. Food Chem., 57,

1307–1311 (2009). (33) Z. Pan, L. Wang, W. Mo, C. Wang, W. Hu and J. Zhang, Anal. Chim.

Acta., 545, 218–223 (2005).(34) R. Lucena, S. Cardenas, M. Gallego and M. Valcarcel, Anal. Chim.

Acta., 530, 283–289 (2005).(35) E. Papadopoulou‑Mourkidou, J. Patsias, E. Papadakis and A.

Koukourikou, Fresenius J. Anal. Chem., 371, 491–496 (2001).(36) N. Yoshioka and K. Ichihashi, Talanta, 74, 1408–1413 (2008).(37) J. Patsias and E. Papadopoulou‑Mourkidou, JAOAC International,

82, 968–981 (1999).(38) A. C. Gerecke, C. Tixier, T. Bartels, R.P. Schwarzenbach and S.R.

Müller, J. Chromatography A., 930, 9–19 (2001).(39) A.R. Mughari, P. Parrilla Vázquez and M. M. Galera, Anal. Chimica

Acta., 593, 157–163 (2007).

Manpreet Kaur, Ashok Kumar Malik and Baldev Singh are

with the Department of Chemistry, Punjabi University, Punjab, India.

metazachlor and linuron at a concentration of 5 ng/mL. The analytical validation for the simultaneous quantification of metoxuron, monuron, diuron, metazachlor and linuron has been performed with good recovery. The recoveries obtained are very good in all cases. Thus, this method can be used to detect the presence of these harmful herbicides in the soft drink and water samples.

ConclusionsThe objective of the current study is to develop a simple, isocratic, reproducible, specific and highly sensitive method for quantitative and qualitative determination of phenylurea herbicides. In the present method the analysis time is 13 min (linuron tR 12.4 min), which is rapid in comparison to some of the other reported methods, such as Patsias and colleagues (37) (linuron tR = 18.88 min.), Gerecke and colleagues (38) (linuron tR = 17.58 min) and Mughari and colleagues (39) (linuron tR = 15 min). The proposed method can determine phenylurea herbicides at very low concentrations. The present paper describes the application of HPLC to the separation and quantitative determination of five phenylurea herbicides, and the feasibility of the method developed was tested by simultaneous determination of these herbicides in different brands of soft drinks and in tap water samples. Good linearity and repeatability were observed for all the compounds studied (with correlation coefficient > 0.99). It is hoped that the results of the present study contribute to increased scientific knowledge in the field of pesticide residue analysis in various food and environmental samples.

Please browse the Forum Web site for program updates: www.casss.org

December 3-4, 2012

Royal Park Hotel, Tokyo, Japan

JAPAN 2012

www.casss.org

Organized by


Kaur et al



LC TROUBLESHOOTING

One aspect I enjoy about being the

“LC Troubleshooting” editor is getting

to interact with readers through a wide

variety of liquid chromatography (LC)

questions that I get via e-mail. This

month I’ll share some of the more

interesting ones I’ve received recently.

If you have a question for me, feel free

to contact me at the e-mail address

listed at the end of this article.

Acceptable RetentionReader: I’ve heard you say that the

retention factor, k, should not be less

than 2 for an isocratic method. Is this a

hard-and-fast rule? I’m having trouble

getting the first peak retained and

would like to have a fast run.

JWD: As a general rule, a retention

factor in the range 2 < k < 10 will

give you the “best” chromatography,

but this is no guarantee of the best

separation. Also, some samples

have such a wide polarity range that

you can’t fit them in this retention

window. In such cases, 1 < k < 20

certainly is acceptable. When even

this extended range of k-values is

not possible, you should seriously

consider gradient elution instead of

an isocratic method.

Let’s review why we set these k-value

guidelines. First, recall that the retention

factor is calculated as

k = (tR − t

0)/t

0 [1]

where tR is the retention time and

t0 is the column dead time, usually

determined by the first rise in the

baseline at the “solvent front.”

Resolution is a function of k/(1 + k),

so if we plot retention as the retention

factor on the x-axis and resolution

as k/(1 + k) on the y-axis, we see a

plot like that of Figure 1. You can see

that the resolution line starts out at a

very low value and rises to a plateau

as k increases. This relationship is

the basis of the recommendations for

k-ranges for isocratic methods. When

2 < k < 10, you can see that the plot

begins to flatten out, but run times

aren’t excessive. In this region small

changes in k will result in very small

changes in resolution. Or another way

of looking at this is that the method is

robust to small changes in variables

that might change retention, such as

the percentage of organic solvent in

the mobile phase, temperature or pH.

On the other hand, if we extend the

acceptable k-range to 1 < k < 20, the

early peaks lie on a much steeper

portion of the curve. This means that

the same change in k that caused

little concern with longer retention

times will cause larger changes

in resolution. Thus, methods with

k < 2 tend to be less stable. Another

problem with peaks with k < 2, and

certainly k < 1, is that there is more

likelihood of interferences from

unretained material at t0. I’ve also

plotted the run time and peak height

in Figure 1. As k increases, run time

increases and peaks broaden and are

shorter; both of these are undesirable,

so smaller k-values for the last peak

are desirable.

However, it must be acknowledged

that the recommendations of

k-ranges shown in Figure 1 are just

that, recommendations, not hard-

and-fast rules; there will always be

exceptions. For example, sometimes

it is not possible to get sufficient

retention of a very polar peak so

that k > 1 can be obtained. Or for

very clean samples, the baseline

disturbance at t0 may be small

enough that k = 0.5 provides

acceptable separation for adequate

quantification. But when we make

a decision to develop and validate

a method with such small retention,

we should go into it with our eyes

open and recognize the potential

problems.

What are some alternatives? If run

time is your major concern, it may

be possible to increase k-values so

that the first peak has k > 2, then to

increase the flow rate and reduce

the retention time, because k is not

affected by flow rate. Or if retention

on a conventional C18 column is

too short for a polar compound,

maybe an embedded polar phase

column will provide an acceptable

alternative. Another alternative

might be to use hydrophobic

interaction chromatography (HILIC),

which is a form of normal-phase

chromatography. With HILIC, retention

orders typically are the opposite of

those obtained using reversed-phase

chromatography, so polar compounds

are well retained and nonpolar ones

come out early.

New or Used Column?Reader: Should I start my method

validation experiments with a new

column? I’ve heard some people say

this is mandatory, but I don’t see why

I can’t continue with the column I have

been using — after all, it still works.

JWD: I don’t think it is necessary

to always put a new column in

when validation commences, but

I do strongly recommend that the

influence of the column should be

investigated as part of the validation

experiments. One of the checks

of precision that is often made is

called intermediate precision, which

refers to changes in conditions

that are not easy to quantitatively

Readers’ Questions John W. Dolan, LC Resources, Walnut Creek, California, USA.

Questions from the e-mail bag are considered this month.



LC TROUBLESHOOTING

control. For example, changes

to the mobile-phase composition

or column temperature can be

quantitative, and fall in the category

of repeatability. Changes resulting

from different operators, different

equipment and different columns

are things that we can identify as

changes, but are more qualitative

than quantitative changes — these

are the intermediate precision items

that are tested.

Traditionally, intermediate precision

includes checking the results for

three different columns — two from

one batch of packing material and

one from another batch. I think

such checks are of less importance

today with the high degree of

reproducibility achieved by column

manufacturers for modern columns.

Perhaps a more important check is

to compare results between a new

column and a well-used column.

So in your case, it would be smart

to check both your used column

and a new one to be sure the same

analytical results could be obtained.

One caution is appropriate when

a used column is included in your

validation experiments: You want

to be sure that the used column

accurately reflects the chemistry

of a used column under normal

application of the method. If the used

column has been operated under

a wide variety of conditions during

method development or used with

another method, and especially if

any of the experiments were outside

the 2 < pH < 8 range where most

silica-based columns are stable,

you may have inadvertently changed

the column chemistry. However, if a

new column was installed at the time

you began your final prevalidation

experiments for the method, where

you perform a mini-validation to be

sure the method is sufficiently stable

to pass validation, the column ageing

process is more likely to reflect what

a column would experience in real

life.

So the bottom line here is that

you should check the performance

of your method with more than

one column, and selecting an

appropriately used column as one of

the test columns seems reasonable

to me. However, interpretation of

regulatory guidelines differs widely,

so rather than take my word as

gospel, I’d suggest you seek advice

from your quality unit, as well.

Setting Limits for Herbal ProductsReader: I work for a company that

“manufactures” herbal materials that

are sold to clients who formulate

these into products that are sold to

the public. As a supplier, we need

to ensure that the material has the

appropriate potency, so I need to set

acceptance limits for my LC methods.

How do I go about that?

JWD: As you know, herbals are not

regulated as strictly as traditional

pharmaceutical products, and the

performance criteria for pharmaceutical

methods may not be appropriate to

apply to your raw materials. However,

the general principles that are used

for pharmaceuticals can be used as

guidelines.

First, you need to start with the

product specifications your company

quotes to clients. For example, herbal

material X contains 50–100 mg/kg of

active ingredient Y. This means that

you need to show with some degree

of confidence that X contains 75 ±

25 mg/kg of Y. Next, you need to

decide how often it is acceptable

to ship product outside this range

— for example, if 95% of the time

you want to comply with the target

range, this would correspond to 4

standard deviations (SD), so your

passing material would have to be

75 ± 12.5 mg/kg (1 SD). This would

correspond to a relative standard

deviation (RSD) of 12.5/75 = 17%

RSD. To have confidence reporting

17% RSD, you probably want your

method to perform at half this level of

imprecision or better. So developing

a method that has an imprecision

of 5–10% may be adequate. The

regulations for this are pretty

vague, but you need to develop a

test process that is scientifically

sound and defensible. Finally, your

decisions should be influenced by

any safety risk, such as toxicity, that

might be involved if analytical errors

are made.

Peak PurityReader: One of the peaks in my

samples tails a bit, and I think it may be

an impurity that is not separated from

the peak. Will the diode-array detector’s

peak-purity output show me if the peak

is pure or not?

JWD: This is one of those questions

that gets answered with a “maybe.”

The peak-purity determination is

made by comparing UV spectra

taken at different points across a

Ideal: 2 < k < 10

1.4

1.2

1.0

0.8

RS

0.6

0.4

Retention (k)

0.2

0.00 2 4 6 8 10 12 14 16 18 20

Run time

Resolution

Peak height

Acceptable: 1 < k < 20

Figure 1: A plot of resolution, expressed as k/(1 + k) vs. retention (k). The effect of

retention on peak height and run time are also shown.



LC TROUBLESHOOTING

the past. However, there are many

LC systems, such as yours, that

are still in use and do not have an

automatic degasser installed. For

years, sparging the mobile phase

with helium was the gold standard

for degassing, and this is still the

most effective way to remove air from

the mobile phase. Another popular

technique that has been used for

years is vacuum degassing of the

bulk solution. Vacuum degassing

while simultaneously sonicating the

solution is considered by many users

to be superior to vacuum degassing

alone, but I have never seen a

well-executed study comparing the

two techniques. In one study I read,

helium sparging removed about 80%

of the dissolved air and vacuum

degassing about 60%. However,

sonication alone was only about 30%

effective, so it is not very promising.

Another consideration is that

different pumping system designs

have different levels of tolerance for

dissolved gas in the mobile phase.

At the extremes of systems that

I have used, I remember one LC

system that required simultaneous

helium sparging and a positive

head pressure on the mobile-phase

reservoir to avoid bubble problems

in the pump. In the same laboratory

we had another brand of pump that

was so tolerant of air that it would

prime itself if a dry inlet tube was

dropped into a reservoir. In general,

high-pressure-mixing systems are

more tolerant of dissolved gas than

LC systems that use low-pressure

mixing. My conclusion is that if you

are using a high-pressure-mixing,

bubble-tolerant system, then

sonication may be adequate, but for

other systems, sonication is unlikely

to provide sufficient degassing for

reliable operation. Try it and see —

you may be lucky!

John W. Dolan is vice president

of LC Resources, Walnut Creek,

California, USA. He is also a member

of LC•GC Asia Pacific’s editorial

advisory board. Direct correspondence

about this column should go to “LC

Troubleshooting”, LC•GC Asia Pacific,

4A Bridgegate Pavilion, Chester

Business Park, Wrexham Road,

Chester, CH4 9QH, UK, or e-mail

the editor, Alasdair Matheson, at

[email protected]

peak. If the spectra are the same,

the peak is considered pure,

whereas if the spectra are different,

the presence of an impurity is

indicated. This is all well and good in

principle, and I have read convincing

articles showing the utility of this

measurement. However, in actual

practice, I have found that most

users don’t provide such glowing

praise. I think this has to do with

the challenge of the measurement.

Often if the two peaks have similar

retention times, the structures are

similar, which means that the UV

spectra are also likely to be similar.

Many compounds do not show

much UV absorbance other than

the end-absorbance characteristic

of most organic compounds in the

< 210 nm region, so this may further

compromise spectral comparison.

Finally, if a small peak is eluted on

the tail of a large one, there may

be sufficient difference in peak

size that even if there are small

differences in the spectra, they

will not be of sufficient magnitude

to definitively show up in the

peak-purity calculations. These

problems are likely the source of

the rather mediocre endorsement of

peak-purity measurements by most

users.

On the other hand, there is plenty

of literature supporting peak purity

measurements, and if the spectra of

the two compounds are sufficiently

different and there is enough of

the minor component present, the

peak-purity calculations may indeed

indicate the presence of a second

compound. My advice is to try the

peak-purity measurement and see what

happens. Just remember that it may be

possible to show that a peak is impure

by using peak-purity or mass-spectral

measurements, but it is not possible to

prove that a peak is pure.

DegassingReader: I have been using sonication

to degas my mobile phase, but recently

I was told that this is not effective. Can

you clarify this?

JWD: Today most LC systems

include an in-line vacuum degasser,

so issues with mobile-phase

degassing, which once were at

the top of the list of common LC

problems, are largely a thing of

w

2 Dreaming of highpurity AND yield?

It will probably remain a dream –at least for batch chromatography!But it‘s different with continuousLC processes. Repeated column switching utilizes the stationary phase much better. Thus separation tasks that are intractable with batch LC can be mastered and the solvent consumption is greatly reduced.

www.knauer.net/purifi cation

2 Modular SMB chromatography for high-effi ciency separations of binary mixtures. Over 99 % yield and purity are feasible.

2 Contichrom® for the purifi cation of substances in multi-component mixtures. 50 % higher yield and purity than batch LC, up to10 times higher throughput.

Two purifi cation solutions:



THE ESSENTIALS

Multidimensional gas chromatography

(GC) is now an established

technique for the analysis of complex

samples in application areas such

as petrochemistry, metabolomics,

environmental and flavour and

fragrance science.

The technique uses GC columns

connected in series to achieve a

complete separation of complex

samples using orthogonal column

chemistries. These separations

are either impossible or very time

consuming using a one-dimensional

(1D) technique (that is, using only one

GC column).

In theory, if each dimension is totally

orthogonal, then the maximum peak

capacity (Φ) can be calculated as

the product of the individual peak

capacities for each dimension:

Φmax

= Φ1 × Φ

2 [1]

In a situation where the first

dimension has a peak capacity of 1000

and the second dimension has 30, the

2D GC×GC system would offer a peak

capacity of 1000 × 30 = 30 000. To

achieve such peak capacity with a 1D

separation, a 2-km GC column would

be required (analysis time in the order

of 1.5 years)!

Second-dimension columns must

achieve separation much faster than

their first-dimension counterparts to

optimize the “sampling rate” from the

first dimension and, therefore, they

tend to be short. The length of the first

column might typically be 20–30 m,

the inner diameter 0.25 mm and the

film thickness 0.25 μm. The second

column is typically shorter (1–2 m), the

inner diameter is narrower (0.1 mm)

and the stationary phase is thinner (0.1

μm), to allow for faster separations. The

reduction in internal diameter is used

to counterbalance the decreases in

efficiency (plate numbers) obtained from

shorter columns. It is common to select

a nonpolar column for the first-dimension

separation and use a more highly polar

phase in the second dimension.

The major instrument challenge

in multidimensional GC is to achieve

efficient “injection” of the effluent of

the first dimension into the second.

Columns joined in series are the simplest

embodiments of multidimensional

chromatography; however, the

separations produced are limited by

carrier-gas velocities because all the

solutes transit both columns in a single

continuous stream. When working with

complex samples, peaks that are well

separated by elution from the first column

can come back together or might interfere

with other peaks as they pass through

the second column. Therefore, we need

to “trap” or “bunch” discrete fractions

from the first column before introduction

into the second dimension. This is

typically achieved using a “modulator”

that is used to transfer effluent from the

first-dimension column to the head of

the second-dimension column in short

repetitive pulses. Modern instruments

use two types of modulators: thermal

(cryogenic or heated) and valve (time

or pressure) modulators. Regardless of

the design or principle, the rapid and

efficient transfer of discrete fractions

from one, many, or all peaks in the first

dimension is absolutely critical to maintain

the separation quality. There are as

many subtle variations in the design and

implementation of modulator devices

as there are instrument manufacturers;

however, there is no doubt that the

modulator is the heart of the GC×GC

system.

In “heart-cutting” systems, one

or several discrete portions of a

separation are directed from the first

column to the second. Because only a

few selected peaks enter the second

column at a time, interference from

other nearby peaks that precede

or follow the heart cut is eliminated,

and the second column’s separation

becomes largely independent from the

first one.

In the much more complex technique

of comprehensive multidimensional GC,

all of the effluent from the first dimension

column is sampled into the second.

Correct sample modulation is essential

in the comprehensive technique to

successfully maintain resolution of

all components in both the first and

second dimensions. This technique

generates huge amounts of data, and

complex software is required to reduce

the data to a usable form, typically

represented via a 2D or 3D plot of the

type shown in Figure 1. This 2D contour

plot of a separation of light cycle oil uses

colours to represent the signal intensity,

the x-axis plots the separation in the

first dimension (in minutes) and the

second-dimension separation is plotted

on the y-axis (in seconds).

Multidimensional GC data are primarily

used for qualitative analysis. However,

quantitative multidimensional analysis is

possible.

While multidimensional GC brings

many separation benefits, achieving

efficient analyte transfer between

columns and the complexity of data

analysis are potential barriers to more

wholesale adoption as a routine analytical

technique.

A Short Introduction to Multidimensional GC An excerpt from LCGC’s e-learning tutorial on multidimensional GC at CHROMacademy.com

Get the full tutorial at www.CHROMacademy.com/Essentials

(free until 20 October).

More Online:

High

Low85

Time (min)0

0.0

4.0

2D

rete

nti

on

tim

e (

s)

Sig

nal in

ten

sity

key

Figure 1: Data generated in a

comprehensive multidimensional GC

separation of light cycle oil.


Ignition coil

Collector

electrode

Air

Fuel + Makeup gas

Cathode (-ve)

Anode (+ve)

Jet electrode

Capillary column

200 - 300V

Electron collector

β e -e -

e -

e -

e -e -

β

β

ββ

β

Radioactive foil

emits β particles

Inlet liner

Gassupply

Septum Septum purgegas outlet

Split linegas outlet

Split valve

Heated Injector body

Nut andferrule Capillary column

Propazine

Atrazine

SimazinePropyzamide

3000+ cups of coffee1000 +1000 1000

lots of takeaway food20x 21x

175 hours overtime

141 yrs GC experience> 20x

1 programmer

2 designers

5 chromatographers

* Data compiled from LCGC online surveys and the CHROMacademy GC Troubleshooter June - August 2012

http://www.chromacademy.com/gc_troubleshooting.asp

CHROMacademy’s New Interactive GC Troubleshooter

5 7 11

21

23

33

ECD Detector

MS Detector

FID Detector

Pressure / Flow

FPD DetectorOther

%Top Instrument

problems*

5% 20

% 36%

39

%

Retention

Baseline

Separation & Quantitation

Peak shape

%Top Chromatographic

problems*

OtherDecreasefilm

thickness

Use hydrogenas a carrier gas

Reducecolumnlength

Increase thecarrier gas

linear velocity

Increasetemperatureprogrammeramp rates

42%

25%

14%

12% 4

%3%

What is the first thing that you do

to reduce analysis time in GC?*

powered by

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26 LC•GC Asia Pacifi c September 2012

ADVERTISEMENT FEATURE

There is an increasing public interest in the analysis of aromatic

amines since this class of organic compounds includes many

carcinogenic substances.

In recent years other sources of aromatic amines apart from tobacco

smoke have gained more and more interest, for example, azo dyes (1).

Therefore a fast and reliable method for the determination of aromatic

amines in dyes like printer ink was developed. Five primary aromatic

amines (PAAs) (aniline, 2-anisidine, 3-chloro-4-methoxyanline,

2,4-dimethylaniline, o-toluidine) were chosen for this demonstration.

Method Parameters for Full Mass Scan Tests

The mass spectra of single compound standards are shown in

Figure 1. The resulting m/z values manifest the fragmentation

patterns of the PAAs. For every PAA the highest intensity was

detected for the single charged quasi molecule ion [M+H]+.

Therefore this mass was chosen for quantifi cation in all cases.

With the calibrated m/z values the extracts of two printer inks were

analysed in order to determine PAA composition and concentrations

of these five PAAs.

Sample Preparation

Samples were prepared as cold water extracts according to EN

645:1993 from printed paper.

Instrument Confi guration

This application was performed on a PLATINblue binary high

pressure gradient UHPLC system equipped with degasser,

autosampler, column thermostat and MSQ Plus mass detector.

UHPLC Parameters: Column: BlueOrchid 175–1.8 C18, 100 x 2 mm

i.d.; Eluent A: water + 0.1% formic acid; Eluent B: methanol + 0.1%

formic acid; Gradient: yes (details on request); Flow rate: 0.2 mL/min;

Injection volume: 50 μL; Column temperature: 40 °C

MS Detection Parameters: Ionization mode: ESI, positive mode;

Needle voltage: 1 kV; Cone voltage: 20 V; Probe temperature:

200 °C

Conclusion

The UHPLC-ESI-MS method presented in this application demonstrates

the fast and simultaneous separation, qualifi cation and quantifi cation of

fi ve PAAs usually found in printer ink. The limit of detection was in the

range between 1 to 5 μg/L (S/N = 3). Only 7 min are required for the

analysis of one sample, including a washing step and re-equilibration

of the column. Therefore the method is well-suited for routine analyses.

Due to the fast separation and low eluent fl ow rate of this method, only

about 1.5 mL of eluent and less than 1 mL of methanol are needed

for one run. Thus this method is both economical and environmentally

acceptable.

Reference

(1) M.J. Zeilmaker, H.J van Kranen, M.P. van Veen and J. Janus, Cancer risk

assessment of azo dyes and aromatic amines from tattoo bands, folders

of paper, toys, bed clothes, watch straps and ink. Rijksinstituut voor

Volksgezondheid en Milieu RIVM, 22-Feb-2000.

Determination and Quantifi cation of Primary Aromatic Amines in Printer Ink M. Margraf, Dr S. Marten and R. Borstel, Knauer GmbH

Figure 1: Mass spectra of single standard.

KNAUERWissenschaftliche Gerätebau Dr. Ing. Herbert Knauer GmbH,

Hegauer Weg 38, 14163 Berlin, Germany

tel: +49 30 809727 0 fax: +49 30 801501 0

E-mail: [email protected] Website:www.knauer.net

Figure 2: SIC scans of two printer inks (P1 + P2) after sample preparation.


LC•GC Asia Pacifi c September 2012 27


Introduction

Blockcopolymers such as SBS (styrene-butadiene-styrene) are an important product class and a typical example of complex polymers. In addition to the molar mass distribution, a chemical composition distribution may also be present in copolymers. While GPC/SEC is the established method for the determination of molar mass averages and distribution, gradient HPLC can be applied to separate based on chemical composition.

Gradient HPLC can be hyphenated with GPC/SEC in a fully automated setup to measure both distribution simultaneously with a high peak capacity and to detect differences in batches (cf. Figure 1).

Experimental

All experiments were performed on PSS SECcurity equipment using the following conditions:Eluent 1st dim.: n-Hexane/THF p.a. gradientColumn 1st dim.: PSS Si-60 5 μm Flow-rate 1st dim.: 0.1 mL/minInjection volume: 20 μLTransfer: PSS 2D tandem transfer valve with two 100 μL loopsEluent 2nd dim.: THF p.a.Column 2nd dim.: PSS HighSpeed SDV 5 μm, 10 000 ÅFlow-rate 2nd dim.: 6.25 mL/minDetection: SECcurity VWD 1260 UV at 254 nmCalibration: PSS Polystyrene ReadyCal Standards, PSS Polybutadiene standardsData system: PSS WinGPC Unity 7.5

Results

The 2D approach is the only way to determine two property distributions independently and unambiguously. The online combination of gradient HPLC and GPC/SEC increases the peak capacity of the separations and those peaks which cannot be separated by either method alone to be examined more closely. The HPLC conditions are selected to separate according to polybutadiene content.

Figure 2 shows the contour plot for a thermoplastic elastomer that shows one narrow main peak in GPC/SEC. However, 2D separation reveals that four different compositions are present that co-elute in the GPC/SEC experiment. The species differ in composition and polybutadiene content. The colour code indicates the concentration of each peak. Simultaneous molar mass results and composition results can be measured using the calibrated GPC/SEC and HPLC axis.

2D Analysis of Thermoplastic Elastomers TPE

Peter Kilz, PSS Polymer Standards Service GmbH

PSS Polymer Standards Service GmbH

In der Dalheimer Wiese 5, D-55120 Mainz, Germany

tel.+49 6131 96239 0 fax +49 6131 96239 11

E-mail: [email protected]

Website: www.pss-polymer.com

Figure 1: Scheme of two-dimensional chromatography.

Figure 2: Contour plot of a thermoplastic elastomer.




This application note demonstrates the analysis of trypsin-digested bovine serum albumin (BSA) using a Thermo Scientifi c Accucore 150-C18 (150 Å pore diameter) nanoLC column.

Accucore™ HPLC columns use Core Enhanced Technology™ to facilitate fast and high effi ciency separations. Accucore 150-C18 has been further optimized for the analysis of biomolecules and protein digests by extending the pore size to 150 Å.

The increased pore diameter enables larger peptide fragments to diffuse into the particle and interact with the stationary phase more effectively, resulting in high resolution of these fragments.

Herein, we demonstrate the excellent performance of Accucore 150-C18 nanoLC columns for the separation of digested BSA.

Standard Preparation

A 50 fmol/μL solution of digested BSA was prepared.

Instrumentation, Column, Consumables and Method

Thermo Scientifi c Dionex UltiMate 3000 RSLCnano LC system, coupled to a Thermo Scientifi c LTQ-Orbitrap XL mass spectrometer fi tted with a Proxeon Nanospray Flex ion source.Accucore 150-C18 2.6 μm, 75 μm i.d. × 150 mm nanoLC column (P/N 16126-157569). Thermo Scientifi c National Vials and Closures (P/N MSCERT4000-34W).

The sample was loaded directly on the column (1 μL injection volume) through sample loop at gradient start.

Flow rate: 300 nL/min; A: 0.1 % formic acid in water B: 80:20 acetonitrile: water (4–40% B gradient over 30 min; ramp to 95% B over 2 min; hold for 2 min; drop to 4% B over 1 min; hold for 4 min).

Results

Elution of tryptic peptides using the conditions described above was achieved within 36 min (Figure 1). Triplicate analyses showed excellent retention time reproducibility for a set of 12 peptides, with % RSD values below 0.14%. Figure 2 shows the extracted ion chromatograms (EIC) of a subset of the peptides monitored. In all cases the peak shapes were found to be excellent, with minimal peak tailing. A peak capacity value of 200 was obtained (1), showing the high resolution power of Accucore 150-C18 nanoLC columns.

Conclusion

Accucore 150-C18 nanoLC columns are an ideal choice for complex proteomic samples, featuring excellent resolution power and run-to-run reproducibility.

References

(1) X. Wang, Anal. Chem. 78(10), 3406–3416 (2006).

Analysis of Bovine Serum Albumin (BSA) Digest on a Thermo Scientifi c Accucore 150-C18, NanoLC ColumnValeria Barattini1, Joanna Freeke1, Duncan Smith2 and John Griffi ths2, 1Thermo Fisher Scientifi c;2The Paterson Institute of Cancer Research, Manchester, UK.

Thermo Fisher Scientifi cTudor Road, Manor Park, Runcorn, Cheshire WA7 1TA, UK

tel. +44 (0) 1928 534110

Website: www.thermoscientifi c.com/chromatography

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m/z 507.81

m/z 768.04

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Figure 1: Base peak chromatogram of 50 fmol of digested BSA loaded on an Accucore 150-C18 nanoLC column, 75 μm i.d.×150 mm.

Figure 2: EIC of a set of eight peptides.


LC•GC Asia Pacifi c September 2012 29


This application note demonstrates the analysis of intact proteins using a Thermo Scientifi c Accucore 150-C4 (150 Å pore diameter) HPLC column. Analysis of six proteins ranging in mass from 6 to 45 kDa is carried out in 15 min with pressures compatible with conventional HPLC instrumentation.

Accucore™ HPLC columns use Core Enhanced Technology™ to facilitate fast and high effi ciency separations. The 2.6 μm diameter particles have a solid core and a porous outer layer. The optimized phase bonding creates a series of high-coverage, robust phases. The tightly controlled 2.6 μm diameter of Accucore particles results in much lower back pressures than typically seen with sub 2 μm materials. For the analysis of large biomolecules the Accucore pore size has been further optimized and a C4 phase with reducedhydrophobic retention has been prepared. This 150 Å pore size enables the effective analysis of molecules unable to penetrate into smaller diameter pores, whilst the low hydrophobicity C4 phase results in protein separation by hydrophobicity.

Chromatographic separation of proteins at the intact level prior to MS analysis is desirable for reducing sample complexity and maintaining global protein information. In this application note we demonstrate the excellent performance of an Accucore 150-C4 HPLC column for the chromatographic separation of six intact proteins (6–45 kDa).

Thermo Scientifi c Column and Consumables

Accucore 150-C4, 2.6 μm, 100 × 2.1 mmVials and closures (P/N MSCERT 4000-34W)

Thermo Scientifi c Accela HPLC System

Flow rate: 400 μL/min Run time: 15 min Column temperature: 40 °C Injection details: 2 μL (10 pmol/μL solution of each protein)UV detector wavelength: 214 nmBack pressure at starting conditions: 185 bar (c.f. 320 bar on sub 2 μm material)

Data Processing

Software: Thermo Scientifi c Xcalibur 2.0 SR2

Mobile Phase

Mobile phase A: 0.1 % TFA in 30:70 acetonitrile:water Mobile phase B: 0.1 % TFA in 98:2 acetonitrile:water Gradient: 0–30% B in 8 min, 30–95% B in 2 min, hold at 95% B for 1 min and re-equibrilate for 4 min

Results

Under these conditions, six proteins covering the mass range of 6 to 45 kDa can be separated on an Accucore 150-C4 HPLC column in less than 15 min with back pressures compatible with conventional HPLC equipment. The chromatography is shown in Figure 1 with all of the proteins eluting with sharp symmetrical peaks and being baseline resolved, with the exception of an impurity from carbonic anhydrase which co-elutes with lysozyme.

Conclusion

• Accucore 150-C4 HPLC columns show excellent separation of six

test proteins of differing mass (6–45 kDa) within 15 min. • Good peak shape is observed for all proteins.

• The back pressure is compatible with use on a conventional HPLC

system.

Analysis of Intact Proteins on a Thermo Scientifi cAccucore 150 C4 HPLC ColumnJoanna Freeke and Valeria Barattini, Thermo Fisher Scientifi c

Thermo Fisher Scientifi cTudor Road, Manor Park, Runcorn, Cheshire WA7 1TA, UK

tel. +44 (0) 1928 534110

Website: www.thermoscientifi c.com/chromatography

70000

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Figure 1: Chromatogram for six proteins separated on an Accucore 150-C4 HPLC column. 1. insulin 2. cytochrome c 3. lysozyme 4. myoblobin 5. carbonic anhydrase 6. ovalbumin * carbonic anhydrase impurity.




Poly(lactic-co-glycolic acid) (PLGA) is a copolymer based on glycolic

acid and lactic acid. The two monomer units are linked together

by ester linkages and form linear polyester chains. The obtained

product is biodegradable and biocompatible, and it is approved by

the Food and Drug Administration (FDA) for production of various

therapeutic devices as well as for drug delivery applications. The

properties of PLGA can be tuned by the ratio of the two monomers

and by its molar mass distribution.

The characterization of PLGA by means of conventional size

exclusion chromatography (SEC) is problematic because of the lack

of suitable calibration standards. In addition, the linear polyester

structure can be modifi ed by the addition of small amounts of

polyfunctional monomer to obtain branched chains of differing

degrees of branching. The degree of branching becomes an

additional parameter that can be used to adjust PLGA properties —

all of which renders conventional column calibration an inadequate

analytical technique.

In this application note, two commercially available samples were

analysed by SEC coupled to a multi-angle light scattering (MALS)

detector (HELEOS), a refractive index detector (Optilab rEX) and a

viscosity (VIS) detector (ViscoStar). The ViscoStar was used in order

to discover additional information about the molecular structure

of the analysed polymers. In addition to molar mass distributions,

the SEC–MALS-VIS system yields the relationship between intrinsic

viscosity and molar mass (Mark-Houwink plot) that can provide

deep insight into the molecular structure of the polymers being

analysed.

In Figure 1, the molar mass distributions are given as differential

distribution plots. As seen from the plots, the two samples span

markedly different molar mass ranges. The Mark-Houwink plots of

the two samples are shown in Figure 2 together with the plot of

linear polystyrene that is shown simply for the sake of comparison.

The slope of the Mark-Houwink plot of the linear polystyrene is

0.71, a typical value for linear random coils in thermodynamically

good solvents. The slope of the red sample roughly corresponds

to a linear structure as well. However, there is a slight indication of

deviation from linearity at the region of high molar masses that may

indicate the presence of branched molecules. The Mark-Houwink

plot of the blue sample is curved. Curvature of the Mark-Houwink

plot generally reveals branching. In addition, the slope of the higher

molar mass portion of the Mark-Houwink plot of 0.48 suggests

signifi cant branching.

SEC-MALS-VIS is an excellent method for the characterization of

PLGA polyesters as it has the ability to determine not only the molar

mass distribution, but also to reveal subtle differences in PLGAs

molecular structure.

Characterization of PLGA Using SEC–MALS-VISWyatt Technology Corporation

Wyatt Technology Corporation6300 Hollister Avenue, Santa Barbara, California 93117, USA

tel. (805) 681 9009 fax (805) 681 0123

Website: www.wyatt.com

LOGO GOES HERE

Figure 1: Differential molar mass distribution curves of two PLGA samples.

Figure 2: Mark-Houwink plots of two samples of PLGA (red and blue) and linear polystyrene (magenta). The lines are linear extrapolations of the data.



PRODUCTS

GPC system

Polymer Char has announced the

release of a 4D gel permeation

chromatography (GPC/SEC) system

to measure the composition and molar

mass of polyolefi ns. According to the

company, the GPC-IR instrument

incorporates features such as the full

integration of the new infrared detector IR5 MCT, with sensitivity

and stability to measure both concentration and composition

(SCB/1000C). The GPC-IR is said to automate all sample

preparation steps, including the initial vials fi lling as well as an

in-line fi ltration with blackfl ush cleaning. As a result, neither vials

transfer nor manual solvents handling is required during the

whole analytical process.

www.polymerchar.com

Polymer Char, Valencia, Spain.

UHPLC columns

Advanced Chromatography Technologies has

launched the ACE Excel Ultra Robust UHPLC

columns which, according to the company, have

been especially engineered to withstand the

demanding conditions of UHPLC. The columns are available

in a range of selectivities, including the ACE C18-AR and ACE

C18-PFP phases, enabling chromatographers to take advantage of

the benefi t of selectivity combined with efficiency. They are packed

with the same “ultra inert” ACE particles that are said to have have

set industry standards for purity and efficiency, resulting in a range

of UHPLC columns that claim to be truly scalable to larger particle

HPLC columns. ACE Excel UHPLC columns are compatible with

all UHPLC and HPLC systems and are robust up to 1,000 bar

(15 000 psi) and temperatures up to 100 °C.

www.ace-hplpc.com

ACT, Aberdeen, Scotland.

Water purifi cation

Merck Millipore has announced the

introduction of Millitrack Compliance,

an e-solution designed to enable

regulatory compliance for lab

water purifi cation system users. According to the company,

this fully-embedded water purifi cation system software has

been developed especially for the pharmaceutical, biotech

and contract labs that follow GxP regulations (GLP, GCP or

cGMP – U.S. Food and which are required to conform to FDA

Title 21 CFR Part 11 guidelines — or similar requirements set

by other global regulatory organizations. Millitrack Compliance

is reported to offer users four important key benefi ts, including

full system control, audit trail, electronic signature, and account

management.

www.millipore.com

Merck Millipore, Guyancourt, France.

Absolute molecular weight

detector

Wyatt presents the Minidawn Treos,

a multi-angle light scattering detector

that can be coupled to any liquid chromatograph or used

off-line in a “micro-batch” mode for determining the absolute

molecular weights and sizes of polymers or biopolymers

directly, without resorting to column calibration or reference

standards. According to the company, the detector reveals

aggregation phenomenon that are not usually detected by

UV, RI or NMR instruments, and the light-scattering detector

can show how the molecules are behaving in solution.

Users are also able to study aggregation, kinetics and

reaction rates as they occur.

www.wyatt.com

Wyatt Technology Corp., California, USA.

LC system

Agilent Technologies has introduced the

Agilent 1290 Infi nity Quaternary LC system,

a quaternary UHPLC system that can

reportedly deliver the accuracy and precision

of binary systems. According to the company,

the pump is the foundation of the system and

offers a maximum pressure of 1200 bar. Features include

active damping and high-resolution pump drives, as well as

Agilent’s microfl uidic technology in the Inlet Weaver and Jet

Weaver mixer. The system features Agilent’s 1290 Infi nity

diode-array detector, which is said to deliver the highest

levels of ultraviolet sensitivity and baseline robustness.

Spectral acquisition is as fast as 160 Hz.

www.agilent.com

Agilent Technologies, Santa Clara, California, USA.

Advanced chemistries

AB Sciex has announced the commercial availability of

Amplifex Reagents, a family of advanced chemistries

designed to boost the performance of mass

spectrometers, improve data precision and increase

sensitivity beyond convential limits. According to the

company, the reagents boost ionization efficiency, improve

fragmentation and chromatographic properties and

overcome other workfl ow restraints that laboratories can

face. Improvements in sensitivity are said to range from

5× to 1000×, depending on the analyte. The Amplifex

Reagents expand the SCIEX iChemistry Solutions

portfolio of mass spec tagging chemistries.

www.absciex.com

AB Sciex, Framingham, Massachusetts, USA.



PRODUCTS

Antibody analysis

Tosoh Bioscience has introduced the TSKgel STAT columns. These were engineered to increase throughput for high-efficiency separations of recombinant proteins, monoclonal antibodies and other biocompounds. The columns are packed with mono-disperse nonporous particles of which the surface consists of an open access network of multi-layered ion-exchange groups. According to the company, the innovative bonding chemistry combined with a relatively large particle size results in a respectable loading capacity and a low operating pressure. The series encompasses a range of anion and cation exchange chromatography columns, suitable for various applications from research to quality control. TSKgel STAT columns can be applied in standard HPLC and in UHPLC systems. www.tosohbioscience.com

Tosoh Bioscience, Stuttgart, Germany.

Two-column system

Knauer has introduced Contichrom lab, a preparative two-column liquid chromatography system designed for discovery, process development and scale-up. The system is reported to offer great fl exibility for all process choices (batch LC, SMB, MCSGP, multi-column) using a single equipment and control software. The MCSGP process principle works with two columns instead of one. Through continuous column switching, impure side fractions containing product are recycled internally, thus maximizing extraction of product. This is reported to increase both yield and purity by 50% at a 10-fold throughput rise and 70% buffer reduction compared with batch LC. Process development is said to be easy, because it starts from a simple non-optimized batch process that is switched to a superior MCSGP process by the included control software.www.knauer.net/purifi cation

Knauer, Berlin, Germany.

Tube accessories

Phenomenex has introduced two tabless tube holder accessories for its Strata and Strata-X lines of silica-based and polymeric solid phase extraction (SPE) sorbents. These accessories hold SPE tubes in 96-well spacing to integrate with automated systems. All Strata and Strata-X products are now available in the tabless 1-mL tube format, reportedly enabling the user to arrange multiple sorbents within the holders, adding fl exibility for method development. According to the company, users can also remove and replace a single SPE tube should they make an error, which cannot be done with standard 96-well SPE plates. The holders are compatible with the Phenomenex vacuum manifold as well as positive pressure systems. Strata and Strata-X SPE sorbents are reported to simplify the method development process for fast and efficient sample preparation prior to chromatography. www.phenomenex.com

Phenomenex Inc., Torrance, California, USA.

UHPLC system

Thermo Fisher Scientifi c has expanded its range of the UltiMate 3000 UHPLC+ to include the UltiMate 3000 XRS UHPLC system. The system reportedly offers new UHPLC capabilities in solvent delivery and sample handling with a wide range of detector options for high-throughput laboratories. According to the company, the instrument has the lowest gradient delay volume and unmatched fl ow precision and accuracy amongst all leading quaternary UHPLC platforms, and is designed to support very robust chromatographic runs with column pressures up to 1,250 bar (18 130 psi). www.thermofi sher.com

Thermo Fisher, San Jose, California. USA.

Wine analysis

Gerstel has released the Gerstel

Solutions Magazine Wine Special which takes a closer look at modern wine analysis. “Corky” or “reductive notes” are terms that wine experts use to describe off-fl avours in wine. The causes and how these can be determined is explained. Articles are offered on modern wine analysis including how to determine pesticides on grapes, tasting wine like a professional and correlating the aftertaste of a wine with the presence of fl avour compounds in the mouth. The magazine is aimed at both experts and chemists just interested in wine.www.gerstel.com

Gerstel, Mülhein an der Ruhr, Germany.

High-temperature columns

PSS has released a range of high-temperature columns for the separations of poly(ethylene), poly(propylene) and other polyolefi ns in TCB, o-DCB or Decalin. The columns can be used with any detector including on-line light scattering detectors. Analytical columns available include 8 mm i.d., 300 mm length; ideal fl ow-rate 0.5–1 mL/min and HighSpeed columns (20 mm i.d., 50 mm length; ideal fl ow-rate 3–5 mL/min). Standard particle size is 10 µm, with other sizes available on request. Available porosities are in the range of 100 Å to 10 000 000 Å. In addition two linear columns are available. The maximum temperature is 200°C. www.polymer.de

PSS Polymer

Standards Servive

GmbH, Mainz,

Germany.


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LC TROUBLESHOOTING

Trouble appearing everywhere!

GC CONNECTIONS

Headspace sampling

COLUMN WATCH

HPLC column trends

March 2012

Volume 15 Number 1


using polymeric monolithic capillary columns

Protein Profiling

LC TROUBLESHOOTING

Retention time changes

GC CONNECTIONS

Pittcon Product Review 2012

SAMPLE PREPARATION

PERSPECTIVES

HF3LPME in practice

June 2012

Volume 15 Number 2


using solid-phase microextraction

Innovative in vivo Analysis

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10–12 September 2012CoSMoS 2012 - Conference on

Small Molecule Science

Rhode Island Convention Centre,

Providence, Rhode Island, USA

Website: http://www.cvent.com/events/

cosmos-annual-conference/event-

summary-7b8db8b280df403085ce-

7c539aa348ef.aspx

9–13 September 201229th International Symposium on

Chromatography

Nicolaus Copernicus University

Campus, Torun, Poland

Contact: Polish Chemical Society/

Commission for Chromatographic

Analysis

Tel: +48 56 6114308

Fax +48 56 6114837


Website: www.isc2012.pl

3–5 October 2012SFC 2012 – 6th International

Conference on Packed Column SFC

Brussels, Belgium

Organizers: The Green Chemistry

Group

Tel: +1 412 805 6296

Fax: +1 412 967 9446

E-mail: register@greenchemistrygroup.

org

Website: www.greenchemistrygroup.org

22–23 October 20122012 International Light Scattering

Colloquium (ISLC)

Four Seasons Biltmore Resort, Santa

Barbara, California, USA

Organizers: Wyatt Technology

Corporation

Tel: 805 681 9009


Website: http://www.wyatt.com/compo-

nent/option,com_jumi/Itemid,120/fileid,12/#

7–8 November 2012 Lab Innovations 2012

Organizers: easyFairs

Tel: (0)20 8843 8822

Fax: (0)20 8892 1929

E-mail: Richard.Thompson@easyFairs.

com

Website: www.easyFairs.com/

labinnovationsS

Send any event news to Kate Mosford

[email protected]

FoodLytica 2012

FoodLytica 2012 will take place

on 31 October–2 November

2012 in New Delhi, India.

India is one of the most

attractive markets globally for

food and beverages, and it is

vital to ensure the safety and

quality of these products by

domestic food processors.

This need can mainly

be attributed to the rapidly

changing dynamics of the Indian markets alongside stringent regulatory

implications, an increasingly aware consumer base and extensive penetration

of multinational corporations (MNCs).

Organized by Fi Conferences, FoodLytica 2012 is launching in New Delhi.

The conference will be led by renowned experts in food safety and quality

testing methodologies. The Food Safety and Standards Authority of India

(FSSAI) and the National Accreditation Board for Testing and Calibration

Laboratories (NABL) will be on hand to discuss regulatory standards and

solutions to complicated issues of traceability, SOPs and SSOPS, chemical

analysis, microbiological testing, nutrient profiling, validation of test methods,

calibration of instruments and many more.

Some of the industry experts speaking at this conference include: Dr

Srinivasa Bhat, Nestle; Dr Rahul Singh, Dabur; Mr K.V. Shashikumar, Unilever;

Mr Shailesh Ghodekar, Marico; and Dr N.K. Kansara, BIS-CL, alongside

experts from established research institutes like Dr Roy Betts, Campden

BRI-UK, Dr K.C. Gupta, Indian Institute of Toxicology Research (IITR); Dr

Lalitha Gowda, Central Food Technology Research Institute (CFTRI); Dr R.R.

Mallya, Institute of Food Technology and Management (IFTM); and Mr Udai

Saxena, UKSOLUTIONS & AFSTI.

In addition there will be an exclusive technology presentation and instrument

training on GC–MS–MS led by the Bruker Corporation, LC–MS–MS and

IC-PMS led by Agilent Technologies; a focused day on food safety training

led by Mr Tom Chestnut, Vice President, Food Safety Global Division, NSF

International and a must-attend workshop on rapid microbiological testing

led by Dr Roy Betts, HOD Microbiology, Campden BRI. There will also be

a workshop on sampling techniques led by Dr Deepa Bhajekar, Managing

Director, MicroChem Silliker with an additional site visit to TUV SUD South Asia.

FoodLytica 2012 will bring together the vice presidents, directors and heads

of quality assurance, quality control, quality management, quality systems

and operations, food safety, corporate quality assurance, food technology,

analytical development, laboratory operation and senior food scientists from

Indian food processing and beverage companies as well as international

organizations and institutes to share their expertise on the latest methodologies

for testing the safety and quality of food and beverage products.

Tel: +91 (022) 4046 1466

Website: http://www.foodlytica.com/default.aspx

EVENT NEWS



Enjoy performance in your conventional HPLC methods far beyond that of columns packed with 5 µm,

4 µm or even 3 µm fully porous particles using Thermo Scientific Accucore XL HPLC columns.

4 µm solid core particles provide very high separation efficiencies using standard HPLC instruments

and conditions, resulting in increased peak resolution and lower limits of detection. An ultra-stable

packed bed results in exceptionally robust columns that demonstrate excellent retention and response

reproducibility.

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• Find more information at thermoscientific.com/accucoreXL

perform better

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• 4 µm solid core particles for all users

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September 2012 · SIM mode permits identiﬁ cation of unknown contaminants in combination with...

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Transcript of September 2012 · SIM mode permits identiﬁ cation of unknown contaminants in combination with...