Scanning Specification

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ISO 9001: 2000CERTIFIED COMPANY

CECIL INSTRUMENTS LIMITED MILTON TECHNICAL CENTRE CAMBRIDGE CB4 6AZ ENGLANDTELEPHONE: 01223 420821 FAX: 01223 420475

E-MAIL: [email protected] WEB SITE: www.cecilinstruments.com

Cecil Instruments policy is one of continuous development. We therefore reserve the right to change specification without notice.

CE4300 Dynamic Absorbance DetectorWavelength range 190-700nm, 4nm constant opticalbandpass, ultra fast wavelength scan (on-the-fly)with auto peak triggering.Variable wavelength monitoring, dual wavelengthoperation and programmable monitoring for up to6 wavelengths.Complete with instruction manual, power cable,signal cable and air test cell.Requires flowcell to be ordered.

CE4051Column mounting panel with column cover.

CE4050Column mounting panel with column cover andRheodyne injection valve fitted with 20L loop.

Flowcells1200 07 70 8L x 10mm pathlength1200 07 71 30L x 10mm pathlength1200 07 72 4L x 5mm pathlength1200 07 73 1mm pathlength preparative flowcell1200 07 74 12L x 7mm pathlength

Other flowcells available on request

Printers8000 70 01 Dot matrix printer with signal cable8000 72 01 Colour inkjet printer with signal cable8000 73 01 Laser printer with signal cable

CE4900PowerStream software provides PC workstationcontrol of Cecil HPLC systems and data processingof chromatograms.

9000 20 00DataStream Software - required for operation of theCE4300 in conjunction with a PC. No dataprocessing of chromatograms.

System Requirements

Computer with Pentium class processor Pentium 200 MHz or better Microsoft Windows XP Microsoft Windows 2000 128 MB of RAM or more Hard-disk space required: 15 MB CD-ROM drive XGA or higher resolution monitor Monitor resolution 1024 x 768 or better Microsoft Mouse or compatible pointer

Scanning Specification

Wavelength Range 190-700nm

Optical Bandwidth 4nm constantDigital Resolution 1nmWavelength Accuracy 1nmWavelength Reproducibility 0.1nmAbsorbance Range 0.001 to 3AScan Speeds Up to 1000nm/s UV scan in 200msBaseline Flatness 0.002A over most of rangeSecond Order Filter Fast automatic insertion at 370nmNoise < 0.5 x 10 5 A, 230nm, 2 s T/C, using air test cellDrift < 0.3 x 10 4 A/h, 230nm, at equilibrium using air test cellOverlayed Scans Up to 8 scans may be overlayedScan Trigger Start of peak, set Abs value, point of inflection or at peakNumber of Peak Scans Up to 100 peaks for a single elutionScan Storage Up to 100 scans password protectedMultiple Scans of a Peak Up to 8 scans of any peakUsed with DataStream Data to PC, PC control, scan triggering, Lambda max.Software scans, peak purity and purity calculation, graphical

presentation, overlay, normalisation, disk storage, librarystore and search, export, and many other functions.

Variable Wavelength Detection Specification

Wavelength Range 190-700nmWavelength Accuracy 1nmWavelength Reproducibility 0.1nmAbsorbance Range 0.001 to 3AAbsorbance Linearity Better than 1% 0.001 2AOptical Bandwidth 4nm constantDual Wavelength Operation Simultaneous measurement at 2 wavelengths and ratioWavelength Programming Measurements at up to 6 different wavelengths with a

time program and selectable sensitivityNoise 0.5 x 10 5 A at 230nm, 2s T/C, using air test cellDrift 0.3 x 10 4 A/h at 230nm at equilibrium, using test cellNoise Dual Wavelength 3 x 10 5 A at 250/280nmDrift Dual Wavelength 3 x 10 4 A/h at 250/280nm, constant temperature

General Specification

Optical System Double BeamMonochromator Littrow with 1200 l/mm holographic gratingStraylight < 0.02% at 220nm and 340nmDetectors Two Silicon DiodesLamp Deuterium lamp with elapsed time indicatorTaper Beam Opt ics All energy collected to minimise RI effectsSelf Test Automatic at switch-on; continuous monitoring of key

functionsAutozero By control panel or external triggerTime Constant 0.1 second 10 seconds, selectableWavelength Calibration Automatic at switch-on using deuterium emission line.Display Four-line display of parameters, menus, prompts,

wavelength, absorbance etc.Fast Number Entry By use of special number keysEvent Marking From keypad or by external triggerMethod Storage For up to 30 methods, security protectedAutomatic Power-Up/Off Pre-programmed to save warm up timeOutputs Two absorbance outputs, or absorbance and absorbance

ratio, parallel printer, data available to PC.Inputs Event, autozeroSize and Weight 365 x 290 x 140mm, 17kgPower Requirements 115V or 230V, 50/60Hz, 50VA

Ordering


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ULTRA FAST SCANNINGUV-VISIBLE DETECTOR


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WaveQuest the CE4300 Dynamic Absorbance Detector(DAD), is an ultra high speed scanning powerfulalternative to Photo Diode Array (PDA) detectors. HPLCmethod development, purity determination, R & D andVariable Wavelength Monitoring are all possible to thehighest quality. Dual wavelength monitoring andwavelength programming are also provided.

The design is based on a double beamspectrophotometer concept, with ultra fast wavelengthscanning over the range 190-700nm.

This dynamic scanning provides UV spectra in only200ms, previously only possible using the static opticalsystem of a PDA detector.

Powerful signal processing software has been developedwhich enables very high quality on-the-fly scans to betriggered by eluting peaks without peak degradation.

Spectral data is transferred by Cecils DataStreamsoftware to a PC workstation, where the same softwareoffers spectral manipulation and processing, libraryfacilities and the export of data to applications such asExcel, Word etc. All data may be stored on disk.

Eluting peaks may be integrated at the same time aspurity and peak identification is carried out. Quantitative

evaluation and reporting is carried out by Cecils CE4900PowerStream software, which is combined withDataStream software.

One Detector Covers All UV Applications

A single beam Photodiode Array Detector (PDA), used formethod development and R & D, will often be replaced bya dual beam UV variable wavelength detector (VWD), forroutine monitoring, when the developed method isadopted, because of the poor drift performance of singlebeam PDA detectors. Their performance can be as much

as 20 times worse. WaveQuest has a double beam opticalsystem with very high all round performance and thelowest drift available.

The CE4300 is used as a high performance UV detector forvariable wavelength monitoring. WaveQuest, which isless expensive than a PDA detector, therefore has adouble cost advantage.

Universal UV/Visible Detector

WaveQuest the ultra fast scanning Dynamic AbsorbanceDetector (DAD) is a major advance in UV-Visible detectordesign, offering the capabilities of a Photo Diode Array(PDA) detector with greatly improved performance whenused for chromatogram monitoring.

Very high speed scanning of eluting peaks, on-the-fly,makes purity checks, the determination of optimummonitoring wavelengths for unfamiliar peaks (lambdamax scans), and identification of peaks using libraryspectra all possible, with considerable cost saving.

CE4300 Dynamic Absorbance Detector

WaveQuest the new CE4300 Dynamic Absorbance

Detector for ultra fast scanning, part of the Adept HPLCrange from Cecil Instruments, offers all the spectralfacilities of Photo Diode Array detectors, with betternoise and drift performance, particularly important whenused to monitor and quantify chromatograms.

The CE4300 has a double beam optical system in whichthe dispersed radiation is dynamically scanned acrossthe detectors, in the manner of a UV-Visible double beamspectrophotometer, at ultra fast speeds up to1000nm/second.

All components of the detector monochromator, opticalsystem, lamp optics, detector optics and signalprocessing software have been carefully optimised toprovide unprecedented performance in both wavelengthscanning and single or multi-wavelength monitoring.

The lower noise level when monitoring a chromatogrammakes accurate measurements at the highestsensitivities possible when measuring very lowconcentrations.

The lower drift, as much as 20 times lower, gives flatchromatogram baselines making integration of peaks

more accurate, particularly at high sensitivities.

Taper Beam Optics

Taper beam optical imaging at the flowcell, with alltransmitted energy reaching the detector, providesmaximum energy collection. The result is minimalbaseline wander due to changes in the solvent refractiveindex during gradient formation.

Performance Validation

Validation of detector performance is carried outconveniently, using the instruments standard software.A Holmium Oxide filter is automatically inserted in theoptical path when the calibration routine is selected.

Bandwidth validation is automatic, using the Deuteriumemission line at 656.1nm. Absorbance accuracy ischecked using filters traceable to NPL standards.

New Super Fast Performance


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PowerStream Software CE4900

PowerStream is a chromatography control and dataprocessing software and interface developed by Cecil

Instruments to be comprehensive and convenient to usefrom a single PC workstation. It provides powerful datahandling and system control for isocratic, binary andternary HPLC systems.

DataStream software provides sophisticated spectralprocessing. Spectral data is transferred from the detectorto the PC where it is graphically displayed, manipulatedand exported as required.

Chromatographic peaks are automatically scanned todetermine purity, identify components, and determineoptimum wavelengths for chromatogram monitoring

Lambda max scans. Multiple scans of peaks enablechecks to be made of peak homogeneity.

Wavelength scans are compared with your own library toestablish the identity of unknown components. A spectrallibrary search facility is provided.

Visual comparison of spectra is valuable for identifyingcomponents and for comparing scans made for puritydetermination. To further assist assessment , amathematical calculation is provided, which produces afigure indicating the degree of match or similarity.

New Advanced Design

The design of the WaveQuest, CE4300, DynamicAbsorbance Detector is based on a double beam UV/Visible

Wavelength Scanning Spectrophotometer concept, with anoptical system which also provides the highestperformance as a dual wavelength, programmable detector.

The 4nm optical bandwidth, constant over the 190-700nmrange, provides high quality spectral scans for easyidentification of unknown components. The digital signalprocessing and optical and mechanical systems provideultra fast scanning and the best noise and driftperformance available. Accurate measurements at highsensitivities are therefore possible.

Ultra Fast Scans of Narrow Peaks

The ultra high speed scanning of the WaveQuest isshown. A very fast binary separation was carried out, ona Cecil gradient elution system 4S, of sixteenpolyaromatic hydrocarbons, designated by the AmericanEnvironmental Protection Agency as priority pollutants.The time for the elution was less than 4 minutes, using a5cm column with a 3 micron packing.

Several of the peaks have widths at half height less than1.5 seconds. The spectra of all peaks were displayed inreal time and are shown here overlayed in groups of four.

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Dual High Performance

When a method has been developed using the CE4300Dynamic Absorbance Detector, the detector may beused as a sensitive and very stable monitor for theapplication, with performance much superior to that ofa Photo Diode Array detector.

Should the CE4300 be required for further methoddevelopment or R & D it may be replaced by theCE4200 Variable Wavelength Monitor. This is a topperformance detector with superb noise and drift andis therefore recommended for detection at the veryhighest sensitivities. Excellent linearity allowsaccurate quantification of samples at very low or high,concentrations.

WaveQuest offers better sensitivity and lowerdetection limits than a Photo Diode Array detector.

The WaveQuest represents two instruments in one, ascanning spectrophotometer and a variablewavelength detector, resulting in major cost saving.

Peak Impurities Revealed

The outstanding performance of the WaveQuest isillustrated by a number of chromatographicapplications described in this brochure.

The application shown here is a chromatogram of amixture, obtained under isocratic conditions. Itappears to be a simple three component mixture.

Peak purity checks were made for each of the peakswith ultra-fast on-the-fly scans on both theascending and descending sides of each peak.

Peaks 1 and 2 gave identical spectral scans but Peak 3gave totally different spectra between the up sideand down side scans indicating a major peak

impurity, giving rise to the scan shown in red.

Despite the presence of the impurity, peak 3 appearsvisually symmetric and indeed the asymmetry factorcalculated for the peak was close to 1.00, giving noindication of the presence of an impurity.


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Lambda Max Scans

The same trial mixture as before, which separated intothree peaks under isocratic conditions, separated into fourpeaks under binary gradient conditions using an AdeptSystem 4S.

Each peak was scanned at its maximum - Lambda max scan and the spectra displayed whilst the chromatogram wassimultaneously displayed and quantified by integration toprovide baseline corrected peak areas and peak heights.

Peaks 1, 2 and 4 are quantitatively similar and all have peakmaxima close to 222 and 273nm.

Peak 3 scan is clearly very different having maxima near234, 268, 316, and 331nm.

Optimum monitoring wavelengths for each peak may bechosen from these scans.

The photo shows an Adept Isocratic System 2S. TheWaveQuest detector, fitted with a column and injector valvepanel, is mounted on the CE4100 pump, with thePowerStream interface unit at the bottom of the stack.

An optional column oven is fitted on the right. Thesystem offers Diode Array performance and the very bestdual and variable wavelength monitoring, withexceptional compactness, as does the Adept GradientSystem 4.


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Normalised Overlays Confirm Purity

Automatic peak purity checks were made by the sameupside and downside, on-the-fly, scans for all fourpeaks.

The results are shown for peaks 3 and 4 only, overlayedbefore and after normalisation of the spectral scans. Peak4 is shown in the upper two plots and Peak 3 in the lowertwo.

From the overlays, particularly when normalised, it isseen that a near perfect match is obtained indicating thatthese peaks were homogenous and hence pure, as alsowere Peaks 1 and 2.

Eight Scans of Peaks

Several means of triggering the wavelength scan of apeak are user selectable.

In the example shown here, WaveQuest wasprogrammed to make repetitive scans of pyrene as iteluted.

The use of the cursor is demonstrated by using it tomeasure the absorbance maximum at 326.7nm. The dropline shows that all the scans have the same wavelengthfor this absorption maxim.


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Spectral Overlay Confirms Identity

A different mixture of the same components was used toproduce the chromatogram shown here with its 4 peaks.

Spectral library searches were made in order to identifythe components, in conjunction with retention time data.

Peaks 1, 2 and 4 can be identified as alkyl phthalates, mostprobably dimethyl, dibutyl and di(ethylhexyl) phthalates.

Peak 3 is identified as pyrene. The library referencespectrum of pyrene overlayed on this impurity peakshows an almost perfect fit, and together with theretention time data for pyrene provides confirmation ofidentity.

ADVANCED DESIGN

190-700nm Ultra fast on-the-fly scans UV scan in 200 ms

Double beam 4nm optical bandpass 1nm digital resolution Superb stability Fast warmup


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Analysis of Sugar Free Soft Drinks

The new ultra fast scanning detector from CecilInstruments, the WaveQuest, was used as part of anAdept System 4S to separate, identify and quantify thecomponents of soft drinks.

Using the CE4300, high quality UV-Visible spectra wereobtained on-the-fly without the use of an expensivePhoto Diode Array detector.

The spectra indicate the most appropriate wavelength formonitoring any given material. The spectra together withretention time data are sufficient to provide positiveidentification of eluted peaks.

The chromatographic separation of commonly occurringcomponents of soft drinks and fruit juices is shown. Thegradient elution used a C18 column, with a phosphatebuffer: methanol binary gradient, monitored at 210nm assome of the components lack chromophores at longerwavelengths.

The peaks in the chromatogram were scanned on-the-fly during elution and the spectra displayed in adjacentwindows.

Four scans are shown here, overlayed. Sufficientinformation is available to identify a component peakwhen taken in conjunction with the retention time.

VERSATILITY Highest sensitivity VWD

Very low noise

Very low drift

Method storage

Dual wavelength operation

Programmed monitoring of 6 wavelengths

Simultaneous 4 wavelength monitoring


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Library Search Yields Identification

The use of the Spectral Library for peak identification isdemonstrated by the chromatogram shown, which wasobtained from a low cost lemonade style soft drink.

Comparisons were made between the UV spectra of theeluted peaks, with those of reference spectra which hadbeen previously scanned and stored in the SpectralLibrary.

Overlay of these scans after normalisation indicated thatthese peaks were identical with Citric Acid (Peak 3),Saccharin (Peak 5), Aspartame (Peak 7) and Benzoic Acid(Peak 9).

Retention times for the major components coincided with

those of the library spectra confirming the identifications.

The scan of Peak 9 is shown overlayed with the LibrarySpectrum of Benzoic Acid displayed in the upper window.The very high quality of the spectral match obtained, isclearly seen.

A statistical calculation is programmed in the DataStreamsoftware, enabling a match index to be calculatedbetween spectra, for identification and also purity.

BENEFITS Spectral Storage Simple intuitive menu operation

Stackable with Adept HPLC modules Flowcell replacement without adjustment Validation software Spectral library Library search


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Spectral Match Index

To confirm the identity of the sixteen separated peaksshown in the fast separation, known reference standardswere injected and eluted under the same conditions asthe sample mixture.

Comparison of spectra and retention times allowspositive identification of all the components.

The screen display shown here illustrates the process,showing a reference library spectrum, which is overlayedon peak 8 in the chromatogram of the sixteen componentmixture.

Applying the mathematical routine for spectral match, orsimilarity, to the data for the sample and library spectra

shown here, produced a match index of 996.5, which isdisplayed on the screen.

A figure greater than 990 indicates a good degree ofmatch, and a figure above about 900 indicates somedegree of match.

A figure less than 100 indicates very little match, orsimilarity between the scans.

The photograph shows the WaveQuest detectorfitted with the column mounting panel and cover. ARheodyne injection valve is also fitted to the panel

The arrangement provides close coupling betweenthe column and flow cell, for minimum deadvolume.


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Excellent AnalyticalPrecision

The excellent precision of theWaveQuest, and indeed thecomplete Adept Gradient System4S, is demonstrated by 7 repeatinjections and elutions of the softdrink shown here in overlay mode,each chromatogram being offsetby 50mA.

The phosphate buffer: methanolbaseline was first recorded and isshown.

Statistical analysis of some ofthese results is shown in summaryreport comment.

The report shows a % RSD for thepeak caffeine of 0.33.

Component Summary

Scanning Specification

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