Download our new issue 1/2014 (pdf)

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ASA

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tech

Greek wine

Determination of organic acids in wineto ensure proper fermentation

New series: The big breakfasttest part 1/3

How do you like your eggs? Hardness test using the TextureAnalyzer EZ-Test-X

Masterpiece in speed and sensitivity

The new LCMS-8050 triple quadrupole

Farmland or flame? – Analysis ofheavy metals in sewage sludges 2

An acidic challenge – Online TOC determination in concentratedhydrochloric acid 6

Changes in the US Pharmacopoeia<643> – What are the impacts onTOC determination in the pharma-ceutical industry? 8

N-terminal protein sequencing in drug development – PPSQ-30 proteinsequencer for peptide 10

How do you like your eggs? – The big breakfast test using the EZ-Test-X Texture Analyzer 11

Rare earth salts prevent counterfeit money – High reso-lution UV-VIS-NIR range 12

Identify different sources of contamination – Applications with IRTracer-100 software 14

Greek wine – Determination of organic acids in wine 18

Analysis of pesticide residues – Chromatographic analysis of organic products 20

Time savings in protein analysis –Online digestion of proteins withPerfinity iDP 22

Masterpiece in speed and sensiti-vity – The new LCMS-8050 targets clinical research, food analysis andother markets 4

The best technique for elemental analysis – Criteria for selection 16

Rapid Scan Software and IRTracer-100 24

APPLICATION

PRODUCTS

SHIMADZU NEWS 1/2014

CONTENT

Farmland orflame?

I n Germany, 3.6 million tons ofsewage sludge (dryed matter)arises each year. Due to high

nutrient levels of nitrogen andphosphate, this waste product ofsewage treatment plants is of par-ticular interest to agriculture. Butbefore it can be used as a fertilizer,certain conditions must be met.

The heavy metal content, for in-stance, must not exceed specifiedlimit values. Should this be thecase, other applications are stillpossible, such as the use as fueland subsequent landfilling. Alldisposal routes for sewage sludgesas well as their potential percent-ages are summarized in figure 1.

Analysis of heavy metals in sewagesludges

Table 1: Analysis results of unknown samples including the corresponding limit values accor-

ding to the German Sewage Sludge Ordinance (AbfKlärV, § 4, section 12). All data refer to the

dryed matter of the corresponding sewage sludge sample (mg/kg dryed matter).

Sample 1ASample 1BSample 2ASample 2BLimit value

2.02.11.51.510

Cd

32321313

900

Cr

490490130140800

Cu

31302829

200

Ni

5755

< NWG< NWG

900

Pb

1,1001,100730770

2,500

Zn

3SHIMADZU NEWS 1/2014

APPLICATION

Within a very short time, the con-centration of many elements pres-ent in sewage sludge is determined,including the phosphorus content– a criterion for selecting theICPE-9000 over atomic absorp-tion spectrometry. Other aspectsfor selecting appropriate elementalanalysis methods can be found onpage 16 of this issue.

In the framework of this investiga-tion the elements lead (Pb), cad-mium (Cd), chromium (Cr), cop-per (Cu), nickel (Ni) and zinc (Zn)are determined. For these ele-ments, limit values are defined inthe German Sewage Sludge Ordi-nance (AbfKlärV, § 4, section 12).When these limit values are ex-ceeded, the sludge may not beused as a fertilizer and is to be

Fast determination of heavymetals in sewage sludges

How can the determination ofheavy metal content in sewagesludges be carried out within ashort time? For this purpose asimultaneous measurement meth-od is the best suitable one, like itcan be performed with Shimadzu’sICPE-9000, an inductively cou-pled plasma with optical emissionspectroscopy (ICP-OES). After asingle measurement the informa-tion of all calibrated elements isavailable, whereat more than 70elements can be chosen, depend-ing on the problem at hand, likein this case the heavy metals. Thismulti-element measurement ispossible due to a large 1 inch2

CCD chip whereby the entirewavelength range of 167 nm up to800 nm is detected simultaneously.

used in an alternative way (see fig-ure 1).

Sample preparation and measurement

Sample preparation was carriedout in accordance to DIN ISO11446. Essentially, this means that2 g of the dried sample is boiledunder reflux in 28 mL of aquaregia for two hours at 150 °C. Theresulting extract is subsequentlyfiltered (0.45 μm membrane filter)and diluted to a volume of 100 mL.After another 1: 5 dilution thesamples are placed with the cali-bration standards to the ASC-6100 autosampler and the fullyautomated measurement can start.

A special feature of the ICPE-9000is that measurements can be car-ried out under both radial and

axial plasma observation, withoutthe need for any instrument con-version. This fully automatedswitching between the observationdirections becomes important es-pecially with respect to additionalelements, as radial plasma obser-vation is less sensitive, and thisway it is possible to determinehigh-concentrated elements with-out further sample dilution. Inaddition to these savings in time,the use of the Mini Torch contrib-utes significantly in minimizingthe consumption of argon gas re-sulting in low level running costs.

Results

For method development, a certi-fied European Reference MaterialERM®-CC136a (sewage sludge)

Figure 1: Disposal routes for sewage sludges from communal wastewater treatment plants

in Germany in 2010 (source: German Federal Statistical Office, 08/2013)

was investigated prior to the meas-urement of real samples. When thecertified values can be determined,the accuracy of the method andsuitability of the ICPE-9000 is en-sured.

All elements could be determinedwith good recoveries (see figure 2)and the accuracy of the results(relative standard deviation of theanalysis results) even clearly ex-ceeds the permitted tolerances ofthe reference material. The toler-ances of the reference material arespecified in the certificate of ana-lysis and based on a previouslyconducted multiple determinationof the material by several labora-tories (ring trial). That is whythere always is a scattering of thecertified results.

Additionally more unknown sam-ples are now also investigatedusing the developed method (seetable 1).

Summary

The good recoveries for the certi-fied reference material in accor-dance with the certificate of analy-sis, as well as the good recoveriesof the measurement results showthat the ICPE-9000 is excellentlysuited for the analysis of sewagesludges. Other elements (Ca, Co,Fe, K, Mn, Na, P) were also in-cluded in this investigation, andthe determined values are in ac-cordance with the certificate of

analysis. The simultaneous meas-uring technique ensures that re-sults can be obtained within avery short time.

All results in table 1 are in com-pliance with the correspondinglimit values. Provided that, in ad-dition to the investigated heavymetals, all other parameters alsocomply with certain specifications.For instance this further specifica-tions are the determination of theorganic fraction by loss on igni-tion (LOI) test. If all points arechecked and in the ranges, thesewage sludge can be safely usedas a valuable source of nutrients inagriculture.

The background data on the topicof sewage sludge are obtainedfrom:• The report ‘Abwasserbehand-

lung – Klärschlamm – Ergebnis-bericht 2010’ of the GermanFederal Statistical Office, 02-08-2013

• The website of the GermanFederal Ministry for the Envi-ronment, Nature Conservationand Nuclear Safety:www.bmu.de/P608/-1 andwww.bmu.de/N39804/

50

60

70

80

90

100

110

120

130

140

150

Reco

very

[%

]

Cu 213

.598 n

m

Cu 327

.396 n

m

*Cd 21

4.438

nm

Pb 22

0.353

nm

Ni 231

.604 n

m

Cr 206

.149 n

m

Cr 267

.716 n

m

Zn 20

2.548

nm *no uncer-tainty certified

Figure 2: Results of the sewage sludge analysis for the ERM®-CC136a reference material.

The inaccuracies of the analytical results of the certified material (red, green) and those

determined using the ICPE-9000 (black) including error bars (relative standard deviation

of a trip-licate determination) are indicated.

PRODUCTS

4 SHIMADZU NEWS 1/2014

T he latest member ofShimadzu’s UFMS family(Ultra-Fast Mass Spectro-

metry) continues the evolution ofthe company’s UF technology.The high-end LCMS-8050 triplequadrupole mass spectrometerfeatures the world’s fastest dataacquisition rates and best-in-classsensitivity allowing quantitativeand qualitative analysis simultane-ously.

UF-sensitivity

The LCMS-8050 has been devel-oped based on the groundbreak-ing LCMS-8030/8040 to meet thegrowing demand for trace-levelquantitation in clinical research,food analysis and other markets.Outstanding sensitivity combinedwith high-speed analysis areachieved through two improvedtechnologies, the newly designedheated ESI source and the en-hanced UFsweeper™ III collisioncell featuring a better CID effi-ciency. In order to increase desol-vation efficiency, the newly devel-oped ESI probe uses a high-tem-perature gas (heating gas) in com-bination with the nebulizer spray(figures 2 and 3). This facilitates

Masterpiece in speed and sensitivitThe new LCMS-8050 targets clinical research, food analysis and other markets

Figure 1: The new triple quadrupole mass spectrometer LCMS-8050

Figure 2: Illustration of the newly developed heated ESI source

ionization of a wide range of com-pounds and expands the LC-MS/MS application range.

Durable high-sensitivity performance

Shimadzu continuously enhancesthe proprietary UFsweeper™technology by optimizing gaspressure within the collision cell,resulting in a 6-fold improvementin sensitivity compared to theUFsweeper™ II of LCMS-8040.The UFsweeper™ III cell acceler-ates ions out of the collision cellwithout loss of momentum. Fastcollision cell sweeping on succes-sive events is achieved while main-taining signal intensity and sup-pressing cross-talk even for high-speed multi-component analysis ata dwell time of 0.8 sec. High-speed MRM transitions up to 555channels per second acceleratelaboratory throughput for simul-taneous multi-component analy-ses.

These technical improvementscombined with Shimadzu’s patent-ed ion optics system deliver du-rable high-sensitivity perform-ance, enabling excellent repro-

ducibility even at attogram levels.Table 1 shows the high-precision

quantitative results obtained withthe LCMS-8050 in the analysis of


PRODUCTS

y

Figures 5: Cable- and tubeless: newly designed sources for easy mounting and dismounting

Table 1: Quantification results of Verapamil

Verapamil in blood plasma at lev-els between 500 ag and 50 pg.

UF-switching and UF-scanning

Fast polarity switching is essentialwhen measuring positive and neg-ative ions simultaneously. Withthe world’s shortest switchingtime of only 5 msec, the LCMS-8050 is able to collect sufficientdata points even for the narrowpeaks obtained by UHPLC. Inaddition, the world’s fastest scan-ning rate of 30,000 u/sec allowstrue high-speed analysis by ob-taining quantitative and qualitativeinformation in a single run with-out compromise in sensitivity and

mass accuracy. A scan step of 0.1 ueven at full speed ensures quanti-tative accuracy even when per-forming MS/MS scans and MRMmeasurements concurrently.

%

Minutes

0

100

0.50 1.501.00

Figure 3: Effect of heating gas –

MRM chromatogram of testosterone

Easy maintenance

The new “plugin and play” design(figure 5) of the ionization unithas another big advantage: thesource housing is cable-free andtubeless, allowing quick removaland easy maintenance. Changingthe ionization probe is simple:only one lever has to be releasedand the probe can be lifted out.No tools are necessary to removethe corona needle in APCI orDUIS probes.

As known from Shimadzu’s othertriple quad systems, replacing thedesolvation line (DL) and ESIcapillary is quick and easy. It ispossible to replace the DL with-out breaking the vacuum, result-ing in longer uptime and usability.

User friendly operation

LabSolutions LCMS Version 5.60software provides intuitive, easy-to-use operation. It seamlesslyintegrates the operation ofShimadzu’s LC product lines(Nexera and prominence) as wellas the LCMS-8030, LCMS-8040and LCMS-8050 triple quad sys-tems. MRM transitions already

optimized with LCMS-8030/8040can also be used for LCMS-8050since parameters for MRM such asQ1 and Q3 pre-rod bias or colli-sion energy are identical in alltriple quad systems. Accordingly,the existing method packages canbe used, e.g. for Rapid ToxicologyScreening System, Primary Meta-bolites or Lipid Mediators.

Interface setting support software

The new optional interface settingsupport software is a tool forautomizing optimization of source

Figure 4: MRM chromatogram of

Verapamil

%

Minutes

0

100

0.8 1.0 1.1 1.20.9

0.00050.0050.050.55.0

50.0

0.0005010.004960.05060.4934.89

51.6

2.773.981.211.311.810.65

100.299.2

101.298.697.8

103.2

Concentration (ng/mL)

Calculated Conc.(ng/mL)

% RSD(n = 6)

Accuracy %(n = 6)

parameters. It automatically cre-ates a batch file to perform analy-sis and acquire data while chang-ing the heater temperature and gasflow rates in the interface. In thisway, optimal conditions for thetarget compound can be deter-mined, enabling higher sensitivityof analysis. This software can alsobe used with LCMS-8030 andLCMS-8040.

Summary

The new LCMS-8050 is a mile-stone in triple quad technology,enabling quantitative and qualita-tive analysis simultaneously. Itcombines the world’s best speedparameters with high sensitivity,enabling higher data quality. Thesystem meets the growing demandfor trace-level quantitation in clin-ical research, food analysis andother markets.

APPLICATION

C leaning and disinfectionagents, pesticides, pharma-ceuticals or plastics such as

PVC – they all consist of chlorinecompounds and have meanwhilebecome indispensible. Chlorine isone of the most important basicchemicals in the chemical indus-try.

Various processes for the produc-tion of chlorine, such as chlor-

electrolysis according to the ODC(Oxygen Depolarized Cathode)process has become established.

Hydrochloric acid is generated asa by-product in some processesand is, therefore, present in highamounts. The membranes used aresensitive to certain contaminationsin the hydrochloric acid, such asorganic compounds. This is why it is important to determine if hy-drochloric acid contains organiccontaminations prior to its use.Quality assurance also plays anincreasingly important role in thesale of hydrochloric acid.

TOC – the sum para-meter

The TOC (Total Organic Carbon)sum parameter is a measure ofcontamination by organic com-pounds. It provides an indicationof how much of the carbon pres-ent in the sample originates fromorganic compounds.

Specialized TOC analyzers areused for this purpose. The sampleis acidified in the analyzer inorder to destroy inorganic carboncompounds present, such as car-bonates and hydrogen carbonates.The resulting carbon dioxide issubsequently removed using asparging gas.

An aliquot of the pretreated sam-ple is injected onto a heated cata-lyst (680 °C). The organic com-

alkali electrolysis, have becomeestablished. The membrane elec-trolysis process is used about in2/3 of commercially run plants,since the end-products chlorine,hydrogen and sodium hydroxide(NaOH) occur with almost thesame purity as when using theamalgam electrolysis process – yet a clearly lower energy input isrequired. In addition to the mem-brane process, hydrochloric acid


An acidic challengeOnline TOC determination in concentrated hydrochloric acid

0

5

10

15

S#1 TOC/TNb

Figure 3: Trend graph: TOC and TNb -results measured over a period of four months


APPLICATION

pounds are converted to CO2 anddetected via an NDIR detector.

In addition to conventional labo-ratory analysis, TOC determina-tion can be simply and securelyimplemented online. Such processanalyzers operate autonomouslyin close proximity to the samplestream to be analyzed. Dependingon the parameter setting, theytake a sample automatically everyfew minutes, prepare and analyzeit and send the results to a controlstation or issue a warning when alimit value is exceeded.

TOC in concentrated hydrochloric acid

These types of process analyzersare used routinely in many appli-cation areas, such as wastewatermonitoring. Online TOC moni-toring of concentrated hydrochlo-ric acid, however, remains a majorchallenge.

As the extremely acidic sampledoes not contain any hydrogencarbonates, the sample prepara-tion step (acidification and sparg-ing) can be omitted. Reliable TOCdetermination in concentratedhydrochloric acid is, nevertheless,anything but easy.

Figure 1: Concentrated hydrochloric acid is diluted 1: 3 Figure 2: TOC-4200 in a protective cabinet

In close cooperation with the engineering department of aShimadzu costumer, a project wasrealized determining TOC in hy-drochloric acid.

Simultaneous TN determination

A major advantage of catalyticcombustion oxidation for the de-termination of TOC is the possi-bility to simultaneously detect theamount of nitrogen compounds.During combustion, the nitrogencompounds are converted tonitrogen monoxide (NO). A che-moluminescence detector (CLM)is connected in series followingthe CO2 selective NDIR detector.The incoming NO is oxidized tonitrogen dioxide (NO2) usingozone, and the photons emittedduring this reaction are detectedby the CLM detector. This combi-nation of detectors enables simul-taneous detection of TOC andnitrogen compounds.

Protection and safety

To protect the analyzer from ag-gressive hydrochloric acid fumes,various gas washers are needed.Shimadzu’s TOC-4200 is equip-

ped with several such gas washersthat chemically bind the aggres-sive chlorine gas.

Also needing to be considered inthis analysis, is that hydrochloricacid is a gas being released from aconcentrated solution. The con-centrated hydrochloric acid musttherefore be diluted prior toreaching the analyzer. This re-quires a dilution apparatus locatedoutside of the analyzer (figure 1),which dilutes concentrated hydro-chloric acid 1: 3 with ultrapurewater.

Another safety issue is occupa-tional safety. Employees shouldnot come into contact with ag-gressive vapors. This is why thesetypes of analyzers must be sur-rounded by a protective cabinetequipped with hydrochloric acidsensors. An alarm signal is issuedwhen HCl (hydrogen chloride)gas is detected. This protects em-ployees as well as materials.

Taking these issues into account, areliable system for the determina-tion of organic load in hydrochlo-ric acid is designed. And what haslong been a routine laboratory ap-plication can now also be ana-lyzed online.

In a test phase, concentrated hy-drochloric acid was monitoredover a period of four months. TheTOC as well as the TNb was cali-brated over a range of 1-10 mg/L.The TOC-4200 features an auto-matic dilution function enablingcreation of a multi-point calibra-tion from a standard solution. Asthe acids need no further acidi-fication, sample preparation (acid-ification and sparging) is not nec-essary. The injection volume is 50 μL.

I n 1996, the US Pharmacopoeiahas introduced the TOC pa-rameter for the determination

of impurities in purified water andwater for injections. For otherwaters used in the pharmaceuticalindustry, the wet-chemical potas-sium permanganate test continuedto be used. Meanwhile however,TOC determination has proven tobe so effective that it now replacesthe wet-chemical test.

In the current version of the UPS<643> (USP 36-NF 31) a distinc-tion is made between ‘bulk water’and ‘sterile water.’ The chapter‘Bulk Water’ includes purifiedwaters that are to be used rightaway as purified water, water forinjection, water for hemodialysis

and as condensate of pure steam.The following known conditionsapply to TOC determinations:

• Limit of detection of the TOCsystem used: < 0.05 mg/L C

• Blank water for the preparationof standards, rw: max. 0.1 mg/L C

• Standard solution (sucrose), rs: 0.5 mg/L C

• System suitability solution (ben-zoquinone), rss: 0.5 mg/L C

• Permitted response: 85 - 115 %• Limit response for the waters,

ru: < (rs-rw)

The chapter ‘Sterile Water’ is new.It includes sterile purified water,sterile water for injections, sterilewater for irrigation and sterilewater for inhalation. Sterile watercan be stored in various packagingconfigurations.

In comparison to bulk water,however, other conditions forTOC determination apply:

• Limit of detection of the TOCsystem used: < 0.1 mg/L C

• Blank water for the preparationof standards, rw: max. 0.1 mg/L C

• Standard solution (sucrose), rs: 8 mg/L C

• System suitability solution (benzoquinone), rss: 8 mg/L C

• Permitted response: 85 - 115 %• Limit response for the waters,

ru: < (rs-rw)

Impact of the new determination

The present requirements of theUPS <643> (bulk water) are con-sistent with the requirements ofthe European Pharmacopoeia(limit of detection, concentration

Changes in the US Phar-macopoeia <643>

of the standard solution (sucrose)and system suitability solution(benzoquinone and response).Validation of the TOC system forboth determinations is thereforesufficient. In accordance with thenew USP <643>, the implementa-tion of a system suitability testusing higher concentrations isrequired. For users of Shimadzu’sTOC systems, this just means thecreation of an additional calibra-tion curve (sucrose, 8 mg/mL, seefigure 1) and control sample (ben-zoquinone, 8 mg/mL, see figure 2)as well as extension of the currentvalidation process with these data.Additional modifications of theTOC system are not necessary.

TOC determination in ultrapure water

Two oxidation techniques are nowcommonly used in TOC analysis:catalytic combustion and wet-chemical oxidation. In catalyticcombustion, carbon compoundsare converted to CO2 using hightemperatures and a catalyst, withsubsequent detection of the result-ing CO2 using an NDIR detector.Wet-chemical oxidation uses acombination of UV irradiationand persulfate oxidation. Bothmethods are suitable for TOCdetermination in ultrapure water.

Shimadzu TOC system

Shimadzu offers two systems thatare ideally suitable for TOC de-termination in ultrapure water.The TOC-VWP/WS uses wet-che-mical oxidation, whereas theTOC-LCPH uses catalytic com-bustion at 680 °C. With their widemeasuring range of 0.5 mg/L upto 30,000 mg/L, the instrumentssupport any application – from

Figure 1: Calibration curve of sucrose, 8 mg/L

What are the impacts on TOC determination in the pharmaceutical industry?

APPLICATION


ultrapure water (for instance incleaning validation) to highly polluted waters (such as waste-waters).

TOC-L series – catalytic oxida-tion at 680 °C

The ISP (Integrated Sample Pre-treatment) module for the TOC-Lseries significantly reduces theuser’s workload as the instrumentcarries out dilution, acidificationand sparging. The fully automateddilution from 4 μg/L up to 30,000mg/L extends the measuringrange.

In addition, the combustion meth-od can be used in combinationwith the TNM-L module, where-by a single injection is sufficientfor simultaneous determination ofthe total bound nitrogen (simulta-neous TOC/TNb determination).The EN-compliant determinationvia chemiluminescence detectionis applied. Catalytic combustion iscarried out at 720 °C. Simulta-neous TOC/TNb determination ishighly suitable for cleaning valida-tion, as it enables a differentialdetermination between the clean-ing agent and the product.

Figure 2: Graphical representation of benzoquinone peaks, 8 mg/L

TOC-LCSH


APPLICATION

TOC-V series – wet-chemicaloxidation

The key technique of the TOC-VWPseries is the powerful oxidationvia the combination of sodiumpersulfate and UV oxidation at 80°C. As a persulfate solution isused for the determination, it isimportant that it does not containany contaminations that coulddistort the actual measuring value.

The TOC-VWP features an auto-matic reagent preparation functionthat eliminates possible contami-nations of the persulfate solution.This ensures that the averageTOC value truly originates fromthe sample – and not from thereagent solution used.

The large injection volume (up to20.4 mL) in combination with thehighly sensitive NDIR detector,leads to an extremely low limit ofdetection and excellent reproduci-bility in the lower ppb range. TheTOC-VWP/WS is therefore highlysuitable for TOC determination inthe ultra-trace range.

Conclusions

Both types of instrument withtheir different oxidation methodscan be used for TOC determina-tion in accordance with the newUnited States Pharmacopoeia(USP <643>) and the EuropeanPharmacopoeia (EP 2.2.44). Theadvantage of the combustionmethod is its high oxidationpotential, especially for samplescontaining particulate matter.Moreover, simultaneousTOC/TNb measurements can becarried out, leading to a higherinformation content of the analy-sis. The advantage of wet-chemicaloxidation is its very high injectionvolume, which leads to highersensitivity and therefore enableshigh precision measurements inthe lower ppb range.

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Shimadzu NEWS, Customer Magazine of Shimadzu Europa GmbH, Duisburg

PublisherShimadzu Europa GmbHAlbert-Hahn-Str. 6 -10 · D-47269 DuisburgPhone: +49 - 203 - 76 87-0 Fax: +49 - 203 - 76 66 [email protected]

Editorial TeamUta SteegerPhone: +49 - 203 - 76 87-410 Ralf Weber, Tobias Ohme

Design and Productionm/e brand communication GmbH GWADüsseldorf

Circulation German: 6,270 · English: 15,920

CopyrightShimadzu Europa GmbH, Duisburg,Germany – January 2014.

Windows is a trademark of Microsoft Corporation. ©2014

Apple Inc. All rights reserved. Apple, theApple logo, Mac, Mac OS and Macintoshare trademarks of Apple Inc.

P rotein sequencing is a simpleand robust method based onEdman degradation. As me-

dical drugs are increasingly pro-tein-based, it is necessary for qua-lity control to determine the N-terminus. N-terminal amino acidsfrom immobilized proteins or pep-tides are cleaved, derivatized andchromatographically identifiedaccording to their retention times.

The Edman reaction starts in thereaction chamber under basic con-ditions with the coupling of phe-nylisothiocyanate (PITC) to theN-terminal amino acid of the pep-tide/protein. This amino acid iscleaved from the peptide/proteinvia a conversion reaction underacidic conditions and is subse-quently transferred to the conver-sion flask. It is then converted to a phenylhydantoin amino acid(PTH-AA) and injected onto areversed phase chromatographiccolumn. The isocratic separationenables PTH-AA identificationbased on retention time related to aPTH-AA standard.

While the conversion is still in pro-gress, the next cycle starts in thereactor by coupling of PITC to the

properties. The library beads aresynthesized in such a way that apart of the peptide can be liberatedfrom the bead by irradiation withUV light. Beads are spread in abacterial culture, and beads con-taining bactericidal peptides areidentified by the presence of anarea of killed bacteria around thebead (a halo). Again, the sequenceof such an antibacterial peptide isunknown in this stage and needs tobe determined by Edman sequenc-ing of the peptide-containing bead.

As the peptides are randomly syn-thesized using mix-and-split tech-nology, the exact sequence of thepeptides of interest is unknown

sands of peptide-containing beadsare incubated simultaneously witha molecule of interest, such as anantibody, in solution. Antibody-binding beads are visualized tostain for the presence of antibodyon particular beads. Such beadscontain possibly interesting pep-tides of which the sequence is notknown at this stage. The sequenceof the peptide is determined bysequencing the peptide-containingbead.

Testing peptides for antibioticproperties

Another applicationis the testing of pep-tides for antibiotic

next amino acid of the peptide/protein. The exact order of aminoacids, i.e. the protein or peptidesequence, can then be determinedin this way. Even isobaric aminoacids like isoleucin and leucinwhich have the same mass, can beseparated and identified.

The samples are usually alreadyimmobilized, e.g. by western blot-ting or are in solution and immobi-lized onto a membrane beforeanalysis.

The research group of Jan WouterDrijfhout from the Leiden Univer-sity Medical Center (LUMC) inHolland uses PPSQ for analysis ofrather unusual samples. They syn-thesize peptide libraries using amix-and-split protocol. This gener-ates millions of different peptidesstill attached to small polymericbeads. Each bead has a size ofabout 100 μm and is loaded with50 - 100 pmol of peptide. Everybead in the library contains a pep-tide with a unique sequence lead-ing to a peptide library with largediversity/complexity.

This library can be used to screenfor new active substances. Thou-

N-terminal protein sequencing indrug developmentPPSQ-30 protein sequencer for peptide-containing beads isolated from peptide libraries

APPLICATION


Figure 2: The protein sequencer PPSQ-30 consists of the Edman reaction unit and a HPLC unit.

Both units are controlled by one unified software.

Figure 1: The main user of the PPSQ Robert Cordfunke (left) and Huybert van de Stadt (right)

both from LUMC Leiden, who developed the reaction chamber design for bead analysis

and needs to be disclosed. In orderto sequence the peptides while stillcoupled to the bead, the reactor ofthe PPSQ had to be rebuilt. Thisnow enables sequencing of pep-tide-containing beads.

Data evaluation is supported bythe analysis software. Easy-to-usehandling, such as reprocessing ofchromatograms, individual reportsand automatic estimation of aminoacid sequence simplify analysis. An own software window allows

control of hardware, combining thesequencing unit with the HPLC.

The PPSQ-30 protein sequencerhas the following features:• stable base line and reproducible

retention times due to isocraticseparation

• high sensitivity• easy-to-use software and hard-

ware• suitability for low throughput

due to easy start-up• reduced costs due to low solvent

consumption based on recyclingof mobile phase.

F oods as a basic need of hu-man existence are subject toconstant inspections. The

Shimadzu News regularly reportson new analytical capabilities.

In addition to taste and the inspec-tion of ingredients, questions arealways raised on the physical pro-perties of our food: how quicklydoes our bread get stale? Howcrisp are our sausages? What arethe differences between eggshellsoriginating from different egg far-ming methods? ... These are thequestions that will be addressed inthis and in subsequent issues ofthe Shimadzu News – using thefoods that make a continentalbreakfast.

Egg farming methods andeggshell strength

Table 1 shows inconsistent values.While there were hardly any dif-ferences between the values forboiled and raw barn-raised eggsand free-range eggs, the valuesbetween raw and boiled organiceggs and for grain-fed eggs in-creased significantly.

Also the strength profiles betweenthe individual egg farming meth-ods were not as expected. Whileeggs produced according to in-creasingly more ecological farmingmethods, have harder shells whenboiled, this was not the case forraw eggs. Surprisingly, organiceggs exhibited significantly lowervalues than free-range eggs.

Consequently, it cannot be con-cluded that the egg farming meth-od has a significant influence onthe eggshell. However, it must benoted here that organic eggs exhi-bited the lowest measured valuefluctuations and the most uniformbreak patterns.

To be continued ... *

*Those of you who cannot wait for the next

breakfast test, the entire text can be down-

loaded in advance.

www.shimadzu.eu/breakfast-test

Eggs are part of a rich, healthybreakfast – whether boiled, friedor as an omelet. On average, theegg consumption per capita peryear is 218 in the European Union.Whereas the Spanish eat 285 eggsper person per year, each Britonconsumes only 176. Germany near-ly meets the average of 217 eggsper person per year.

This high level of consumption requires a high production rate,which can only be attained bykeeping high populations of layinghens in egg farming systems. Eversince the ban on battery cages, achange in mindset has been trig-gered with respect to egg produc-tion, and the number of produc-

How do you like your eggs?The big breakfast test using the EZ-Test-X Texture Analyzer

Figure 1: Texture Analyzer EZ-Test-LX with customized compression jig

Table 1: Test measurements of raw and boiled eggs


APPLICATION

Barn-raised, Grade AFree-range, Grade AGrain-fed free-range,Grade AOrganic eggs, Grade A

142.74181.27162.79

109.41

135.39172.26193.30

161.39

Egg farming method Raw (in N) Boiled for 10 min (in N)

tion facilities for organic eggs orfree-range eggs is increasing. Whatis the influence of egg farmingmethods on the product itself?

The eggshell could be used as anindex for natural egg farming meth-ods, since it can be assumed thatdue to better animal welfare con-ditions, eggs may grow at a slowerrate, and in turn the eggshell canabsorb more calcium and thus maybe stronger.

Hard center,hard shell?

The rigidity of eggs from variousegg farming methods have beencompared. Eggs were obtainedfrom a random supermarket andwere subjected to a pressure testusing the EZ-Test-X Texture Ana-lyzer. The eggs were from barn-raised, free-range and organicallyraised laying hens as well as fromfree-range laying hens that re-ceived special grain feeds. All eggswere clamped in the testing ma-chine and compressed at a con-stant crosshead movement untilthe break. To level possible naturalunevenness of the shell, the bot-tom support surface as well as thebottom of the pressure plate wascovered with a 40 mm thicknessfoam board.

All test measurements were carriedout on raw eggs as well as eggsthat had been hard-boiled for tenminutes.

APPLICATION

R are earth elements are locat-ed everywhere, but are sel-dom concentrated in signifi-

cant amounts at one point (rareearth); their extraction is time-consuming and expensive. Rareearths include 17 elements belong-ing mainly to the group of Lan-thanides. They are used in variousindustries such as illuminants,magnets, glasses, catalytic con-verters, polishes and in metallur-gy. A short approximation is givenin an article from BGR in 2009 [1].The pie chart shows the distribu-tion of rare earth elements overvarious industries. In many indus-trial applications the earth elementused to color a target object spe-cifically or even make it unique.

Figure 1 does not include nichemarkets or applications. One suchuse can be the coloring of paper,

achieved using rare earth salts.Their structures bear a uniquecolor. Small changes in the crystalconfiguration will change the ap-pearance. This application shows

e.g. banknotes. Paper money iscopied very often. One attempt tosolve this problem applies the useof colors which are unique anddifficult to prepare. This can be


Rare earth salts prevent counterfeit moneyHigh resolution spectra in diffuse reflectance for powder samples measuredwith integrating sphere in the UV-VIS-NIR range

Figure 1: Usage of rare earth metals in t (REO) and by commercial use 2012 (Total consumption

189,000 t approximately) [1], source Kingsnorth (2007)

54,534

60,000

70,000

80,000

83,496

524.4 530.0 535.0 540.0 543.9

R %

Figure 2: UV-VIS spectra from NdVO4 powder under conditions of 1 nm (green) and 5 nm

(red) color. The identification and uniqueness of such substances is of interest. A target

application is to prevent copying of the printed graphics on currency.

Figure 3: UV-VIS Reflectance spectrum of Neodymium vanadate, 1 nm slit width,

ISR-3100 with three detectors, range 220 to 860 nm

30,971

40,000

60,000

80,000

91,498

220.0 400.0 600.0 800.0 860.0

R %

the effect of different Neodymiumand Samarium salts in UV-VISspectroscopy.

Rare earth salts are a product’s finger prints

The salts from the rare earth ele-ments form UV-VIS spectra whichhave similarities to the so called“finger print” range of the midinfrared spectra. Normally, theUV spectrum of liquids formsbroad signals with low resolution.High resolution in a UV-VIS spec-trum is expected with gas phasespectra such as the benzene gasphase or for the well-known mer-cury, deuterium or halogen iodidelamps. In this case, the sample wasa solid in powder form pressedbehind a quartz window and di-luted with BaSO4. Even under thiscondition, the salts form a finger-


APPLICATION

Table 1: Parameter settings for the UV-3600

46,454

50,000

55,000

58,172

330.0 350.0 400.0 450.0 490.0

R %

nm

Figure 4: UV-VIS spectrum from Samarium vanadate measured under conditions of diffuse

reflectance (SmVO4)

59,778

70,000

80,000

90,000

100,000

107,079

240.0 500.0 1,000.0 1,500.0 2,000.0 2,600.0

R %

nm

Figure 5: UV-VIS NIR spectrum from Neodymium phosphate NdPO4, 5 nm measurement in

the range from 240 to 2,600 nm

Wavelength Range (nm.)Scan SpeedSampling IntervalInstrument PropertiesInstrument TypeMeasuring ModeSlit WidthLight Source Change WavelengthDetector UnitDetector Change WavelengthGrating Change Wavelength

220 to 2,600Medium0.01

UV-3600 SeriesReflectance1.0 nm310.00 nmExternal (3 Detectors)870 nm 1,650 nm720 nm

Measurement Properties

print spectrum in the UV-VISrange. The signals reflect not onlycolors but also energy transfertransitions from the salt pattern.The energy modes shown arecharacteristic for the lattice orcrystal form. They are probablyso unique that a specific identifi-cation is possible.

Due to the rough surface of apowder, the ISR-3100 integratingsphere was used for the analysis.The integrating sphere collectsdiffuse reflected light from thepowder surface. The sphere has asize of 60 mm, and the diffusereflectance can be measured.

The sample is placed for this pur-pose in the 0 degree position atthe sphere. Under this conditionspecular reflected light is excluded

from the measurement, and onlydiffuse reflectance is measured.

In this case, the possibility ofobtaining a 1 nm resolved spec-trum depends on a good responsefrom the material. Looking at therange of 520 to 550 nm showssubstructures in high resolution.

Figure 2 includes two spectra indifferent resolutions. The red andgreen colored lines are the 5 nmand 1 nm spectra respectively. Inthe case of a signal group, the res-olution can be shown.

The Neodymium salt yielded suc-cessful spectra with many signalgroups. For reference, NdPO4 and(for the Lanthanide Group) a Sa-marium vanadate spectra are pre-sented.

Homogeneous sample in short time

Sample preparation was complet-ed in short time. The salts were ina ratio of 1 : 10 volume percentdiluted with BaSO4. The powdermixture was transferred into a cupwith quartz window. The cup wasclosed using manual pressure ontothe sample mixture to generate ahomogeneous layer, pressed witheven distribution of the sampleover the quartz window.

Objective was to create a homoge-neous surface of the sample. Thecup was placed into the sampleposition at the integrating sphere.As reference a cup filled withBaSO4 only was used. The ISR-3100 integrating sphere (withthree detectors) was equippedwith PM, InGaAs, and PbS detec-tors for the complete range ofUV-VIS-NIR.

The benefits of such rare earthsalts are the uniqueness of thecrystal lattice which forms a spec-trum in the visible range depend-ent on the lattice. Charge transfertransitions are mainly measured.The color of the material dependson all of these characteristics,thereby being unique. Identifi-cation can be done using UV-VIS-NIR spectroscopy.

Literature

[1] Seltene Erden; Maren Liedtke, Harald

Elsner; http://www.bgr.bund.de/DE/

Gemeinsames/Produkte/Downloads/Comm

odity_Top_News/Rohstoffwirtschaft/31_er

den.pdf?__blob=publicationFile&v=2

AcknowledgmentMany thanks to Prof. Dr. Robert Glaum

(Inorganic Chemistry Department at the

University of Bonn) for the neodymium

and samarium salts and the discussions

regarding this topic.

APPLICATION

I nfrared spectroscopy as classi-cal analysis has been a majortechnique for the identification

of solid materials with KBr pelletand cell techniques for liquid sam-ples. Nowadays, other reflectionstyle measurement methods areincreasingly being used.

Users need a new view on spectra,because the measurement resultsnot only include air from theatmosphere, but also extra signalsfrom the accessory, e.g. a diamondprofile. Finally, the spectrum iscontaminated by accessory absor-bance or reflection.

A different class of contaminantscan be particles in materials whichas impurities restrict the quality ofuse (uneven, broken etc.). Conta-mination due to organic and inor-

ganic compounds can be present indifferent media, such as tap water.In this application, methods areshown which are designed to assistin the identification of contami-nants.

Contaminant analysis software for complex challenges

The new IRTracer-100 system withits new LabSolutions IR softwareplatform enables complete applica-tion analysis, e.g. a contaminantanalysis. In such cases, it oftenhappens that the contaminant con-sists of a mixture of several sub-stances. A conventional searchresult covers just one of the maincomponents and needs furtherexpertise to identify the remainingunknown substances.


Identify different sourcesof contaminationApplications with IRTracer-100 and »ContaminantAnalysis« software

cm-1

Abs

.

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.54

0.50

4,000 3,600 3,200 2,800 2,400 2,000 1,800 1,600 1,400 1,200 1,000 800 600

Figure 1: ATR spectrum from PVC resin measured with a diamond reflection unit

IRTracer-100


APPLICATION

Several infrared spectra in vinyl chloride resin

Several types of additives are ad-ded to resin materials to enhancetheir material properties. Each onehas its own infrared spectrumoverlaying the main material spec-trum.

In the following article, results ofa vinyl chloride resin are present-ed. The sample was prepared witha diamond-based single reflectionunit, the DuraSamplIR, and ana-lyzed by Contaminant analysisprogram using ATR measurementtechnique. Result of the analysisindicates phthalic ester present inPVC and calcium carbonate (seefigures 2 and 3). These additivesare commonly used as plasticizerand non-reinforcing filler respec-tively in PVC products.

Contaminants in tap water

A different approach in identifyingunknown materials is the analysisof inorganic and organic contami-

The Contaminant analysis soft-ware sorts out such complex iden-tification.

The LabSolutions IR program of-fers two main application fields,i.e. Transmission and ATR (At-tenuated Total Reflection) mode.533 spectra (typical contaminants)are contained in specific librarieswhich are used for sophisticatedsearching. The search algorithmhas been improved to also enableprecise re-identification of inor-ganic material. As an example, aresin was selected.

cm-1

Abs.

Figure 3: Spectra output window for direct comparison of search results from

the library

Figure 2: Analysis results using the contaminant analysis software, listing with

judgment levels

0.0

0.2

0.4

0.6

0.8

1.0

4,000 3,600 3,200 2,800 2,400 2,000 1,800 1,600 1,400 1,200 1,000 800 600

cm-1

Abs

.

Figure 4: Combination with the EDX database allows judgment of the contaminant’s

color or other characteristics

4,000 3,600 3,200 2,800 2,400 2,000 1,800 1,600 1,400 1,200 1,000 800

Details of infrared spectrum database search result:“Discharge pipe 32 packing_Gray_Inside”Materials: styrene butadiene styrene (SBS),calcium carbonate (CaCO3),magnesium silicate (TALC, 3Mg4SiO2H2O)Major elements: Cl, Ca, Si, Mg, S, Zn, TiColor: Gray Shape: Resin/Ring-shaped Hardness: Softmetallic luster: No technique: ATR (Diamond)

nants in tap water. They can origi-nate from different sources, suchas tap water system materials,minerals and microorganisms.Major contaminant sources in-clude rubber and metallic compo-nents resulting from degradationof the water supply system.

The tap water analysis programincludes two databases for FTIRand EDX analysis. As soon as arun is started, the system identifiesthe IR-Spectrum and gives addi-tional information based on theEDX analysis of various materials.

An example of analysis is shownon the right (figure 4).

Conclusion

With dedicated knowledge basessuch as libraries or databases, it iseasy to analyze contaminants.Mixtures, e.g. polymers, are easyto identify. The software can beadapted for contaminant analysisin environmental, petrochemicaland food fields.

PRODUCTS

A tomic spectroscopy in-cludes a variety of techno-logies that are designed pri-

marily for the determination ofthe elemental composition of sam-ples from many industries, e.g. en-vironmental, geochemical, metal-lurgical, pharmaceutical, food,agriculture and more. Amongthese techniques, the most popu-lar ones in routine applications areatomic absorption- (AAS), induc-tively coupled plasma- (ICP-OES),and X-ray fluorescence- (XRF)spectroscopy.

Which of these techniques forqualitative and quantitative deter-mination of element concentra-tions is best suited for solving aparticular analytical problem?Due to the techniques’ specificstrengths and advantages, but alsolimitations and disadvantages theoptimum solution for a particularproblem may not always be clear.Selecting the right technique re-quires consideration of a varietyof important criteria such as de-tection limits, analytical workingrange, sample throughput, inter-ferences, ease-of-use and the in-vestment budget available. Thisarticle gives an overview of themost important techniques andprovides the information required

GFA-7000, where the sample isintroduced directly into a graphitetube before it is heated in a pro-grammed sequence to remove thesolvent and matrix componentsand to atomize the element of in-terest. All of the analyte is atom-ized, and the residence time of theatoms within the tube is extendedresulting in a high sensitivity andsignificantly improved detectionlimits in comparison to flameatomization.

The key differentiator betweenelectrothermal atomization andflame sampling is the analysistime, which is much longer whenusing the graphite furnace system.On the other hand, the enhancedsensitivity of the graphite furnacewhich is typically 100 to 1,000times better than flame makes thisan ideal solution for the analysisof ultra low concentrations. Fig-ure 1 shows the AA-7000 which isa fully automatic double beam dualatomizer system in combinationwith the graphite furnace GFA-7000 with digital control and theASC-7000 sample preparation sta-tion.

The AA-7000 in the dual atomizerversion includes both flame and

as well as reporting of the results.The light source is a hollow cath-ode lamp containing the elementto be determined in the cathode.The atomization unit is either aburner head for flame atomizationor a graphite furnace for electro-thermal atomization.

The typical flame system operatesa titanium burner head with anair/acetylene or nitrous-oxide/acetylene flame. The sample isintroduced as an aerosol into theflame by the sample-introductionsystem consisting of a nebulizerwith Pt/Ir capillary and a spraychamber which is inert to aqueousand organic solvents. The burnerhead is aligned so that the lightbeam passes through the flame,where the light is absorbed. Un-fortunately, sample introductionusing a nebulizer and spray cham-ber is not very efficient, so thatonly a small amount of sample isdelivered to the flame, therebylimiting the sensitivity of suchsystem.

In order to improve the sensitivityof atomic absorption spectrome-ters, another atomization unit isneeded consisting of a high sensi-tivity graphite furnace such as the

to help in selecting the best solu-tion to a specific analytical prob-lem.

Atomic AbsorptionSpectroscopy

AAS quantitates concentrations ofelements in a vapor, when a groundstate atom absorbs light energy ofa specific wavelength and is elevat-ed to an excited state. The amountof light energy absorbed at thiswavelength is increased when thenumber of atoms of the selectedelement in the light path increases.The relationship between theamount of light absorbed and theconcentration of the element pres-ent in known standard solutionscan be used to determine un-known sample concentrations bymeasuring the amount of lightthey are absorbing.

Quantitative analysis of elementsis typically carried out using atom-ic absorption spectrometers suchas the Shimadzu AA-7000, con-sisting of a primary light sourcefor the target element, an atomiza-tion unit, a monochromator to setthe specific wavelength of light tobe measured, a photomultiplierdetector to measure the light, andelectronics for the data processing


The best technique for elementalanalysis Criteria for selection

Figure 1: Fully automatic atomic absorption spectrometer AA-7000

Figure 2: Guide to selecting the right technique


PRODUCTS

graphite furnace atomization unit,allowing fully automatic change-over from flame to graphite fur-nace mode and element-specificoptimization of the atomizer posi-tion. The system includes twomethods of background correc-tion for the determination of ele-ment concentrations in sampleswith complex matrices. The Deu-terium background correction isuseful for compensation of spec-tral interferences generated bymolecular absorption and particu-late caused scattering. In addition,the high speed self-reversal tech-nique (high current pulse tech-nique) is useful for compensationof interferences caused by over-lapping absorption lines andstructured background.

ICP-OES: Inductively CoupledPlasma Optical Emission Spec-troscopy

Inductively coupled plasma opti-cal emission spectroscopy (ICP-OES) is the measurement of lightemitted by all elements present ina sample introduced into an ICPsource. The emission intensitiesmeasured are then compared withthe intensities of standard samplesof known concentration to obtainthe elemental concentrations inthe unknown samples. The argon

plasma is generated by an RF fieldand ionized argon gas. The advan-tage of the plasma in comparisonto other energy sources is the hightemperature of 10,000 K, enablingcomplete atomization of the ele-ments in a sample while minimiz-ing interferences.

The ICPE-9000 applies a mini-torch with reduced argon con-sumption which is mounted in avertical position. This setup al-lows two ways of viewing thelight emitted from the torch. Inthe typical ICP-OES configura-tion, the light across the plasma isviewed axially from the top, re-sulting in the highest sensitivity.In radial view from the side, thesensitivity is reduced by a factorof 5 to 10. The most effective wayof operation allows the plasma tobe viewed in either orientation ina single analysis, providing thebest detection capabilities and thewidest working range, the so cal-led dual view mode.

The vacuum optical system ofICPE-9000 consists of a spectro-meter including a diffraction grat-ing and a prism for separation ofthe individual wavelengths of lightand focusing onto the surface ofthe large CCD detector (Charge-Coupled Device). This setup

Table 1: Overview of strengths and limitations

allows true simultaneous multielement analysis with highest sam-ple throughput. In addition toquantitative measurements againstcalibration curves of standardsamples, the system also enablesqualitative analysis.

X-ray Fluorescence Spectros-copy (XRF)

XRF allows analysis of elementcomposition of samples in a widevariety of applications. This tech-nique provides non-destructiveand fast measurements of liquidand solid samples and is best suit-ed for analyzing the elementalrange from sodium/carbon to ura-nium, which covers the majorityof the metallic elements.

In a typical energy dispersiveXRF-spectrometer such as EDX-720P/800P, the tube directs X-raysonto the sample, ejecting electronsfrom the inner electron shells ofthe atoms. When outer electronsfill in the hole left by the ejectedelectron, energy is emitted as X-rays and measured on the detectorof the XRF spectrometer. Thestrength of the signal from eachelement reveals the relative con-centration in the sample. Energydispersive XRF spectrometerssuch as EDX-720P/800P are

multi-purpose instruments forquantitative analysis using stan-dard samples for calibration withhigh accuracy, as well as the fun-damental parameter method whenstandard samples are not available.Simultaneous screening in the ele-mental range from sodium/carbonto uranium is also possible.

Shimadzu is one of the worldwideleading manufacturers of analyti-cal instrumentation and has beensetting milestones in the field ofatomic spectroscopy for morethan 40 years. With a state of theart product line ranging fromflame atomic absorption and highperformance graphite furnaceatomic absorption to simultaneousICP-OES- and X-ray fluorescencesystems, Shimadzu offers a totalhardware and software solutionfor the determination of elementconcentrations in every type ofsample.

Flame AAS – Flame AtomicAbsorption Spectroscopy

GFAAS – Graphite FurnaceAtomic AbsorptionSpectroscopy

ICP-OES – InductivelyCoupled Plasma OpticalEmission Spectroscopy

ED-XRF – Energy DispersiveX-ray Fluorescence Spectro-scopy

Very easy-to-useWidely acceptedReference method in many fieldsWide application rangeInexpensiveLow detection limitsWide application rangeUnatteneded operation

Fast analytical speedSimultaneous multi-elementanalysisHighest sample throughputWidest analytical rangeGood documentationWide application rangeUnatteneded operation Qualitative and quantitative analysisEasy-to-useNon destructive analysisFast screeningNo need for sample pretreat-ment

Low sensitivitySequential analysisUse of flammable gas

Limited analytical working rangeSample throughput limited in comparison to flame AASor ICP-OESHigher initial investment

Lower precision at lowconcentration levels

Ideal for laboratories analyzingmany samples for up to six elements and for the determi-nation of concentrations in ppm levelIdeal for laboratories analyzingmany samples for up to six ele-ments at low detection limits with typical concentrations in ppb levelIdeal for laboratories doing multi-element analysis on a large number of samples inppm to ppb level

Ideal for laboratories doing fast analysis in the upper ppm to % level with no sample preparation

AA-7000F

AA-7000G + GFA-7000

ICPE-9000

EDX-720P/800P

Technique Strengths Limitations Applications System

APPLICATION

O rganic acids contributegreatly to the composition,stability and organoleptic

qualities of wine, especially whitewine. Their preserving propertiesenhance microbiological stability.So dry white wines not subjectedto malo-lactic fermentation aremore stable in terms of potassiumhydrogen tartrate and calcium tar-trate precipitation. Red wines arestable at lower acidity levels dueto the presence of phenols.

Since the composition of the or-ganic acids directly influences theflavor and color of wine, it mustbe controlled carefully to ensureproper fermentation and to pre-vent spoilage (table 1).

The Union of Agricultural Coop-eratives of Peza in Greece is locat-ed in the prefecture of Heraklion,Crete. Its main products are wineand olive oil. By collecting highquality raw materials from localproducers, the Union of Agricul-tural Cooperatives of Peza Creteensures excellent quality of thesetraditional products for its custom-ers. The red wines have P.D.O.Peza (Protected Designation ofOrigin Peza) status since 1971,and the white wines have had thisstatus since 1982.

for phosphoric buffer addition.Using water as a mobile phasemakes initial equilibration fasterand total time of everyday analy-sis shorter, as it eliminates the ad-ditional time needed to clean thesystem from unwanted salts. Inaddition, a mobile phase compati-ble with a LCMS detector and anESI probe was tested since thisinstrument is included in theUnion’s future plans. Formic acidwas selected because of its appro-priate properties. Two differentconcentrations (0.1 and 0.5 %formic acid) were tested and thebest separation was obtained at0.1 %. This is in agreement with aprevious study conducted byGamoh et al [2].

Polar analytes are not always re-tained and often do not separatewith a satisfactory capacity factoron conventional C18 columns.Synergi Hydro-RP is a C18 bond-ed phase endcapped with a polargroup to provide extreme reten-tion of both hydrophobic andpolar compounds under 100 %aqueous conditions. The high 4 μm silica surface area (475 m2/g)combined with a dense bondedphase coverage allows substantialinteraction between the sampleanalyte and the bonded phase. The result is a very retentive C18phase, well suited to separate or-ganic acids.

Running a 100 % aqueous mobilephase on a C18 column can pro-vide improved retention of polarcompounds. However, conven-

ditions are presented in the fol-lowing paragraph:

Analytical conditions

Analysis mode: IsocraticMobile phase: Formic acid 0.1 %in waterMobile phase flow rate: 0.5 mL/minColumn: Phenomenex SynergiHydro-RP, 250 mm x 4.6 mm, 4 μmColumn temperature: 35 °CDetector: PDA SPD-M20ADetection wavelength: 210 nmTotal run time: 20 minInjection volume: 20 μL

Column selection

The goal was to select a columnthat could operate at 100 % aque-ous conditions without the need

In collaboration with N. Asteria-dis S.A., the Peza Union Crete hasdeveloped in its quality controllaboratory a green, fast HPLCmethod for the determination oforganic acids in wine based onresearch of the University of Ca-labria [1]. This method requiresno hazardous organic solvents. In addition no buffers are used,thereby eliminating the need foradditional time to clean theHPLC system.

Experimental

The analyses were performed on aShimadzu prominence HPLC sys-tem equipped with a PDA detec-tor (SPD-M20A). Chromatogra-phic separation was achieved on a250 mm x 4.6 mm, 4 μm Pheno-menex Synergi Hydro-RP column(table 2). Detailed analytical con-


Greek wineDetermination of organic acids in wine

Minutes

mAU

0

500

1,000

1,500

2,000

2,500

3,000

5.0 7.5 10.0 12.5 15.0

Figure 1: Chromatogram of a mixed standard solution of D-malic acid 5 g/L, L-malic acid

5 g/L, lactic acid 5 g/L and tartaric acid 10 g/L

Figure 3: Typical chromatogram of a wine sample

Minutes

mAU

0

100

200

300

400

500

600

5.0 7.5 10.0 12.5 15.0

AceticCitricFumaric

Malic

Tartaric

Monitored to prevent spoilageMonitor for export limitsAdded to prevent malo-lactic fermentation and adjust acidityDetermined to measure the progress of malo-lactic fermentationConcentration is useful for deacidification and might be applied to cold stability testing

Organic acid Reasons of interest for wine

Table 1: Reasons of interest of organic acids in wine


APPLICATION

tional C18 phases are poorly wet-ted by highly aqueous mobilephases, causing the C18 ligands tomat down on the surface of thesilica with the consequence thatover time, retention is lost com-pletely. Organic acids are oftendifficult to separate as their polar-ity hinders interaction with con-ventional C18 ligands, but it wasobserved that this is accomplishedusing Synergi Hydro-RP under100 % aqueous conditions.

Procedure

Working standard solutions of D-malic, L-malic, lactic and tartaricacid were prepared in ultrapurewater. Standard solution concen-trations for D-malic, L-malic andlactic acid were 0.5, 1, 2 and 5 g/L.Standard solution concentrationsfor tartaric acid were 1, 2, 4 and10 g/L. In figure 1, a chromato-gram of a mixed standard solutionof D-malic acid 5 g/L, L-malicacid 5 g/L, lactic acid 5 g/L andtartaric acid 10 g/L is shown.

Calibration curve creation andquantitation were performed auto-matically using LC solution soft-ware.

Table 2: Column technical specifications

Table 3: Calibration curve slopes ([Organic acid] = a *Area), correlation coefficients and QC parameters for organic acids. Limits of detection

and quantitation are calculated by multiplying the residual standard deviations of the calibration curves (Sy/x) by 3.3 and 10 respectively.

Table 4: Quantitative results and QC parameters from some wine samples

Concentration Concentration

0.0

2.5

5.0

0.0

2.5

5.0

0 2,500,000 5,000,000 7,500,000 Area

0 2,500,000 5,000,000 Area

0.0

0.0

2.5

5.0

7.5

10.0

2.5

5.0

0 5,000,000 10,000,00015,000,000 Area

0 10,000,000 20,000,000 Area

Concentration Concentration

Figure 2: Calibration curves for D-malic, L-malic, lactic and tartaric acid

Synergy Hydro-RP 4 80 1.05 475 19 2.45 Hydrophilic

Packingmaterial

Particle size(μm)

Pore size(Å)

Pore volume(mL/g)

Surface area

(m2/g)

Carbon load (%)

Calculatedbonded

phase cove-rage

(μmol/m2)

End capping

D-malicL-malicLacticTartaric

Organic acid

4.6697 x 10-7

2.4624 x 10-7

7.8443 x 10-7

2.8536 x 10-7

Slope(a)

0.999970.999910.999990.999

R2

15.84.04.5––

Resolution

2.4100.6210.9640.333

k’

165007000

100006300

Theoreticalplates

0.050.080.030.55

LOD (g/L)

0.140.230.091.68

LOQ (g/L)

RetsinaTTV3 - 150

Average resolutionAverage k’

0.530.082.85

72.5

4.854.037.67

1.60.7

13.3520.88

8.34

1.01.0

21.5221.1556.64

0.90.35

Wine [D-malic acid] (g/L) [L-malic acid] (g/L) [Lactic acid] (g/L)

QC parameters

[Tartaric acid] (g/L)

Correlation coefficients and QCparameters for all organic acidsare shown in table 3. Correlationcoefficients for all calibrationcurves were excellent as can beseen in figure 2.

Wine samples were diluted 1 : 10with ultrapure water and filteredusing 0.45 μm pore size prior toanalysis. Because of the dilution, adilution factor of 10 was used forquantitation. In figure 3, a typicalchromatogram of a wine sample isshown.

Quantitative results and QC pa-rameters for the most typical wineproducts are shown in table 4.

Conclusions

The method described above hasbeen developed and successfullyimplemented in the Union’s Qual-ity Control laboratory. It has prov-en to be fast and reliable while re-quiring no organic solvents andminimum time for equilibrationand cleaning. Linearity of the cali-bration curves for all organic acidswas excellent in the range of themeasurements, providing a solidbase for the determination of or-

ganic acids in wine. Using a Phe-nomenex Synergi Hydro-RP col-umn with particle size of 4 μmproved to be an excellent choicefor this type of analysis. Thismethod, although developed usingUV detection, is also suitable forLC/MS systems. Using such a sys-tem in the future, the Union ex-pects to achieve even better LODs*and LOQs.**

* LODs: Limits of detection** LOQs: Limits of quantification

References[1] RT-OR5-ALTRO-CHIM/2, Rapporto Technico:

Verifica Delle Metodiche Analitiche

E Loro Applicazione A Vini Calabresi DOC,

Progetto: LOGICA, Laboratorio Tecnologico

Della Logistica In Calabria, Laboratorio Di

Cosenza, R&D.LOG, Italy

[2] K. Gamoh, H. Saitoh, H.Wada, Rapid

Commun. Mass Spectrom. 17 (2003)

685-689

[3] A. Rodríguez-Bernaldo de Quirós, M.A.

Lage-Yusty, J. López-Hernández, Talanta 78

(2009) 643-646

Emmanouil G. Barbounis*, Konstantinos

Tampouris*, Kleanthi Garefalaki**,

Georgios Koumantakis**

* Application Department N. Asteriadis S.A

**Union of Agricultural Cooperatives of Peza,

Crete

APPLICATION


Figure 2: The relationship between matrix-matched and solvent calibration for

2-phenylphenol

Concentration (mg/kg)

2-phenylphenol calibration curves

0

20,000

40,000

60,000

80,000

100,000

120,000

140,000

160,000

0 0.02 0.04 0.06 0.08 0.10 0.12

Peak

are

a

Figure 3: The relationship between matrix-matched and solvent calibration for

chlorpyrifos

Concentration (mg/kg)

Chlorpyrifos calibration curves

0

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

0 0.02 0.04 0.06 0.08 0.10 0.12

Peak

are

a

O rganic products differ fromother conventional prod-ucts in the way that they

are produced and processed whileobserving the rules of organicagriculture. These rules, amongothers, forbid the use of pesti-cides, and as Regulation EC No889/2008 states: the quantifiablepresence of a pesticide residue inan organic product (positive analy-sis result) leads to “substantiatedsuspicion.“

Differing controlling authoritiesdon’t share the same opinion re-garding maximum allowable quan-tity in mg/kg of pesticide residuesin organic products, while the„quantifiable presence“ (probablycorresponding to the limit of quan-tification) also differs for pesti-cides. Since the LOQ for mostpesticides is currently around0.010 mg/kg, it is common for thecontrol authorities to take thisvalue as determinant when declar-ing a product to be BIO or not.One of the largest associations oforganic processors – Bundesver-band Naturkost Naturwaren

currently used for pesticide re-sidue analysis in foods were devel-oped based on the need to searchfor a large number of pesticides ata reasonable price. In addition, theuse of proper matrix-matched cal-ibration is not often seen in com-mon laboratory practice as thiscould lead to higher expenses, ifproper blank commodities were tobe purchased for each measure-ment. Nevertheless, DocumentNo. SANCO/12495/2011 strictlyprescribes the use of exact matrix-matched calibration for difficultmatrices.

Inaccurate quantification possible

Everything mentioned so far canlead to inaccurate quantificationof pesticide residues if multire-sidue methods and non matrix-matched, or even solvent calibra-tion curves are used for analysis of plants with difficult matrices,while on the other hand organiccertification authorities can in thecase of a minimal violation ofmaximum residue limit (MRL,

during pesticide residue chro-matographic analysis. As a result,a serious over/underestimation ofthe quantity of pesticide residuesfound in such plant samples canoccur in gas and/or liquid chro-matographic analysis if no propermatrix-matched calibration curvesare used.

This is shown in the report issuedby European Union ReferenceLaboratory for Pesticide Residues:Pesticide analysis in teas andchamomile by liquid chromatog-raphy and gas chromatographytandem mass spectrometry. Vali-dation data of 86 pesticides usinga multiresidue method by LC-MS/MS and GC-MS/MS in greentea, red tea, black tea and chamo-mile, have shown that the GCresponse for these pesticides wasup to nine times higher in matrixthan it was in the pure solvent,whereas the LC response was upto four times lower in matrix thanin the pure solvent.

Furthermore, it is well-knownthat the multiresidue methods

(BNN) e.V. from Germany hasintroduced the following guide-lines for decision-making regard-ing pesticide residue findings inagricultural products: „...The ori-entation value for each substance(active ingredient) is 0.010 mg/kg,and applies to the original un-processed product ... No morethan a total of two pesticides maybe present ...“

Plant species with very complexchemical composition

It is well-known that some plantspecies have very complex chemi-cal compositions, which result invery difficult sample matrices inQuEChERS sample preparation

New approach for analysis ofpesticide residues Scientific and economical chromatographic analysis of pesticide residues in organic products with difficult matrices

Figure 1: Vials with different vegetable and

fruit matrices


APPLICATION

Figure 5: The results of standard addition method for chlorpyrifos

Conc. of chlorpyrifos standard solution added (mg/kg)

Standard addition – chlorpyrifos

-10,000

10,000

20,000

30,000

40,000

50,000

60,000

70,000

80,000

0-0.005 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05

Peak

are

a

0

0.011 mg/kg instead of the al-lowed 0.010 mg/kg) stop the saleof the commodity in which exces-sive residue has been found, andorganize challenging inspections.

When considering that medicinalherbs and teas (commodities thatoften have very complex matrices)are mostly acquired by “wild col-lecting“ from areas where no pes-ticides are used, but which can be

present in high quantities in thecase of an unpredictable pollutionfrom the surroundings by air orwater or in case of unfavorableatmospheric conditions, it is clearthat there is a greater risk of find-ing high pesticide residues reach-ing MRL-s in these commodities.Methods more accurate than themultiresidue methods for determi-nation of quantity of pesticideresidues should be used in thesecases.

New approach in four steps

For analysis of pesticide residuesin organic products with difficultmatrices, the authors suggest thefollowing scientific and economi-cal approach – analysis in severalsteps with respect to the resultsobtained:

1. Use of multiresidue methodsfor the analysis of organic com-modities and possible identifi-cation of pesticide residues.

2. Rough estimation of whetherthe sample analyzed is „toobad“, or records quantities of

Figure 4: The results of standard addition method for 2-phenylphenol

Conc. of 2-phenylphenol standard solution added (mg/kg)

Standard addition – 2-phenylphenol

-50,000

50,000

100,000

150,000

200,000

250,000

300,000

350,000

400,000

450,000

0-0.005 0.005 0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05

Peak

are

a

0

pesticide residues which areclearly under the MRL-s (inwhich case further analysis isnot necessary).

3. Recalculating the quantities ofresidues found using multire-sidue method, by using the fac-tors showing the difference inresponse of the respective pesti-cides in matrix compared tothose in pure solvent (these fac-

tors can be obtained by in-house experiments or found inthe literature).

4. If the values recalculated showquantities for one or two pesti-cide residues around 0.010 mg/kg (BNN guidelines), performthe method of standard additionfor the most accurate determi-nation of the quantity of thepesticides found.

In order to perform the third stepof the proposed approach, theauthor’s laboratory obtained ma-trix-matched calibration curvesfor difficult matrices, particularyfor herbal teas. As a blank matrix,a mixture of 13 blank herbs, fruits,flowers and roots was used formatrix-matched calibration. Thesecommodities were confirmed tobe blank by own and external laboratories. Ratios of calculatedconcentrations with matrix-matched calibration curve, andconcentration calculated with sol-vent calibration curve were ob-tained for different concentrationsof standard solutions of pesticides,at 0.010, 0.025, 0.050 and 0.100 mg/

kg, and named „matrix effect“(ME).

New method in practice:analysis of daisy

To demonstrate the proposal, theauthors conducted analysis of oneBellis Perennis (daisy) sample.This analysis was conducted onShimadzu’s GCMS-TQ8030 withOptic 4 injector, using a multi-

residue method for determinationof 124 pesticides (including iso-mers). Sample was prepared usingthe European style QuEChERSsample preparation method.

Step 1Multiresidue analysis of the sam-ple investigated showed positiveidentification of two pesticides inconcentrations above 0.010 mg/kgas calculated by solvent calibra-tion, specifically 2-phenylphenolat 0.011 mg/kg, and chlorpyrifosat 0.012 mg/kg.

Step 2Elimination of further analysisdoes not apply.

Step 3The authors obtained the follow-ing ME factors for the identifiedpesticides at 0.010 mg/kg: 2-phenylphenol 1.22, and chlor-pyrifos 2.39. Both concentrationswould therefore be lower than0.010 mg/kg after dividing theresults by the ME factors.

Step 4To confirm these findings, the

method of standard addition forthese two pesticides was per-formed later. The daisy sampleextract was divided into threeequal parts, after which increasingvolumes of pesticide standard so-lutions were added into the threeportions of an unknown sample,resulting in three different con-centrations of added pesticides,0.010, 0.025 and 0.050 mg/kgrespectively.

After analysis of the preparedsolutions was completed and cal-culations were made, the resultsconfirmed (as can be seen fromthe graphs 2 and 3) that the truequantities of the pesticide residuesfound in the daisy sample weresmaller than the maximum al-lowed quantity of 0.010 mg/kg.Corrected values were 0.0044 mg/kg for 2-phenylphenol and 0.0025mg/kg for chlorpyrifos.

The proposed approach showedthat the product which would nothave been marketable as organicwhen analyzed using multiresiduemethod and external calibration(based on positive identification of two pesticides with quantities >0.010 mg/kg), satisfies the organicregulation criterion after all. Thisknowledge was gained by invest-ing in moderately higher analyti-cal costs while acquiring muchmore precise results.

APPLICATION

P rotein analysis is complexand time-consuming. Mostof the proteins needing to

be characterized are relativelylarge molecules that are not easilyanalyzed without pretreatment.Enzymatic cleavage into smallerfragments is a common procedure.In addition to chemical and thusrelatively drastic methods, enzy-matic reactions are widely used inbiochemical laboratories, and haveproven to generate reproducibleresults. The problem is that enzy-

and then to carry them out in atest tube. Nor is it possible to pre-dict reproducibilities or even sta-tistical data for such reactions.

Special column for trypticonline protein digestion

Experiments to speed up enzy-matic digestion or to carry thisout on a stationary phase are notnew. But thanks to advanceddevelopments in HPLC stationaryphases, it is now possible to pro-duce a specialized column fortryptic online protein digestion.

From the initial idea of an immo-bilized trypsin column to the

The function of endopeptidases

Endopeptidases are important substances

in chemical analytical protein sequencing.

The cleaved (denatured) proteins are easily

hydrolyzed, and bind water molecules.

In the intestinal tract, trypsin selectively

cleaves peptide bonds into the basic amino

acids lysine, arginine as well as modified

cysteine.

Similar reactions occur in the hu-man intestinal tract during diges-tion of proteins. These are wellknown in terms of mechanism andfunctioning. By experience it is,however, much more difficult topredict exactly or to describe suchprocesses for unknown proteins

matic cleavage processes are oftentime consuming, as they requireincubation of the protein with aspecific enzyme. It can take 24hours or longer to selectivelycleave a protein without denatur-ing it.

Tryptic digestion is frequentlyused. The name is derived fromtrypsin, an endopeptidase that cleaves proteins at specific locations in the molecule.


Time savings in protein analysisOnline digestion of proteins with Perfinity iDP

Figure 2: Reaction equation of the trypsin activity test by BAPNA

Figure 1: Perfinity iDP system

Figure 3: Flow schematics of the Perfinity iDP system

Figure 4: Start screen of the Perfinity iDP software


APPLICATION

reproducibility refers equally toresults obtained on various non-consecutive days for the samesample. This illustrates the meritof the method.

What the UV detector does notshow, however, are the manysmaller digestion products whichcan subsequently be detectedusing a highly sensitive MS/MS.On the other hand, the limitedcomplexity was also advantageousduring the initial experimentsusing the system (and is certainlyalso the case elsewhere).

the user-created methods. Just likethe standard pre-built methods,these user-created methods aresubsequently available in thecombo box. Being able to adaptthe digestion times in particularcan provide interesting results inorder to be able to assess the truevalue of the system for proteinanalysis.

Fast results

Using the standard pre-builtmethod, it is possible to quicklyobtain results for various proteins.The following chromatogramshows the tryptic digestion of Cy-tochrome C. In addition to theconsiderable savings in time (8 mi-nutes digestion time ‘on-column’compared with 20 hours at 37 °Cusing the conventional approach),the reproducibility of the methodis remarkable. One can only won-der whether experienced proteinanalysts can obtain such resultsusing a manual approach and howoften this actually succeeds. The

phase are used to transfer thedigested protein fragments via anadditional valve and a downstreamdesalting column onto the actualseparation column. Separationtakes place in an acetonitrile/buffer mixture suitable for LCMS/MS. In addition to the UV detec-tor that is part of the system, amass spectrometer can easily beconnected. This is certainly thedetector of choice when as manyof the peptides as possible are tobe detected and identified.

Simple user interface for LabSolutions

Since most protein analysts findthe control and the complexitiesof a HPLC system only of limitedinterest, straightforward user in-terfaces and omitting of all non-essential parameters are an abso-lute requirement for routine useof the system.

The start screen of the PerfinityiDP software meets all these re-quirements (figure 4):

The software features a speciallyadapted user interface for theLabSolutions LC Workstationsoftware.

For everyone interested in HPLC,it goes without saying that all set-tings can also be completed in theLabSolutions software itself.Being able to start the analysisusing pre-built methods is anenormous help to novices in thisanalytical application area, andshould not be underestimated.In addition to the main menu, it ispossible to adapt individualmethod parameters and to save

Figure 5: Method details of the standard pre-built method

Minutes

75,000100,000125,000150,000175,000200,000225,000250,000275,000300,000325,000350,000375,000400,000425,000450,000475,000500,000

uV

11.0 11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0

Figure 6: Chromatograms obtained using Perfinity: Tryptic digestion of cytochrome C

and examples of their reproducibilities

The use of trypsin and trypticdigestion is just one possibleapproach in protein analysis tosomewhat simplify this complexapplication area. Of course inpractice, a variety of enzymes areused for protein digestion.

Acknowledgment

Shimadzu Europa sincerely thanks its col-

leagues at Shimadzu Scientific Instrument

(Maryland, USA) as well as Perfinity Bio-

systems (Purdue University, Indiana, USA)

for the development of the system.

Perfinity iDP™ is a trademark of Perfinity

Biosystems.

Perfinity iDP™ system (PerfinityiDP – integrated Digestion Plat-form) described here, it was andstill is a long way: on-column di-gestion times must be optimizedfor individual proteins. Questionsin this context include: How longshould the protein remain on thecolumn in order to be fully digest-ed? What is the optimal digestiontime, and what concentrations canbe handled? How many digestioncycles can be performed using onecolumn? How can the method betested and what is involved inquality control?

Hardware, software,application kit

We should not try to answer allthese questions ad hoc here, butthe Perfinity iDP system offers acombination of HPLC hardware,columns, buffer mixtures and auser-friendly software interfacethat simplifies an important partof protein analysis: sample prepa-ration for MS/MS analysis, orquality control for biotechnologi-cal production of proteins.

In addition to the hardware men-tioned here, the system also fea-tures an application kit. The kitincludes the trypsin column andthe appropriate buffer mixtures asa set for every 1-liter ready-madebuffer solution, as well as soft-ware and a series of prebuiltmethods that facilitate systemoperation. All that is required isto place the samples in the auto-sampler and start the analysis.

Detecting the enzyme activity

of trypsin

The BAPNA test is used as a functionality

test. The enzyme activity of trypsin is de-

tected via N-benzoyl-D,L,-alanine-p-nitro-

alanine (BAPNA). BAPNA is cleaved by

trypsin at arginine to form p-nitroaniline.

The p-nitroalanine concentration can be

detected spectroscopically at a wavelength

of 405 nm.

Briefly returning to the systemflow line: the previously cleanedprotein is loaded onto the trypsincolumn via a switching valve andis ‘parked’ on the column using alow flow rate suitable for diges-tion. In a subsequent step, a high-er flow and a different mobile

PRODUCTS

24

T he new IRTracer-100 Fou-rier Transform Infrared(FTIR) spectrophotometer

is a highly versatile instrument incombination with the newLabSolutions IR software. Com-bining high speed, sensitivity,and resolution with enhancedexpandability and easy-to-use

Using the additional ‘Rapid Scan’software, the IRTracer-100 de-monstrates its advantages in fastkinetics analyses. Up to 20 spec-tra per second are recorded andare evaluated in terms of signalintensity, area, ratio of two sig-nals according to intensity andarea, as well as the TAC (Total

Absorbance Curve).These values arebasic requirementsfor kinetics analy-ses.

The resolution, thenumber of measure-ments and the mir-ror velocity are pa-rameters that affectthe measurement

speed. A resolution of 16 cm-1

and a mirror velocity of 40 mm/sresults in an acquisition time of0.05 seconds per spectrum.

‘Rapid scan’ is suitable for far-infrared (FIR), middle-infrared

software, the IRTracer-100 quick-ly and easily obtains high-qualitydata for samples in such fields aspharmaceuticals, foods, chemicalsand electronics.

Rapid ScanSoftware andIRTracer-100

(MIR) and near-infrared (NIR)measurements. In the NIR, meas-uring range data points affect thespeed of measurement.

Figure 1 shows the fast kineticsof the changes in a resin underthe influence of UV light. Thespectra are recorded at 50 msintervals. Below is the correspon-ding signal analysis at 1,400 cm-1

and the absorption profile over

time – 30 seconds in the presentcase. The ‘Rapid Scan’ software istherefore recommended for fastkinetics analysis within a timeframe of less than one second.

IRTracer-100

Abs

Abs cm-1

Time[sec]

Time[sec]

Sample: UV light cure adhesiveResolution: 16 cm-1

No. of scan: 1

Interval: 50 msecMonitor: Peak at around 1,400 cm-1

Detector: MCT

0.0

0.200.220.240.260.280.300.320.34

0.10.20.30.40.50.60.70.80.91.01.11.21.3

1,600 1,500 1,400

10 15 20 25 30

1,300 1,200 1,100 1,000 900 800 510

1520

2530

Figure 1: Analysis of the curing reaction of a resin under the influence of UV light

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