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Turner Digital Fluorometer

OPERATING MANUAL

MODEL 450

45F00-01 • 2/18/97 Serial Number ________________________________________

BARNSTEAD|THERMOLYNE CORPORATION

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Table of Contents

Introduction ........................................................................................................................................... 3Safety Information ............................................................................................................................. 4Specifications .................................................................................................................................... 5

Installation ............................................................................................................................................. 6Standard Equipment ......................................................................................................................... 6Instrument Checkout ......................................................................................................................... 7Installation ......................................................................................................................................... 8

Operation ............................................................................................................................................ 10Description ...................................................................................................................................... 10Operation ........................................................................................................................................ 13 Models 450-000 and 450-003 ..................................................................................................... 13 Model 450-005 ............................................................................................................................ 14Cuvette Selection ............................................................................................................................ 16 Filters .......................................................................................................................................... 17 Selection ..................................................................................................................................... 19 NB versus SC Filter Use ............................................................................................................. 25 Care and Maintenance ............................................................................................................... 27Standard Curves and Calibration .................................................................................................... 29Fluorometric Performance Kit ......................................................................................................... 37Analog output .................................................................................................................................. 37

Preventive Maintenance...................................................................................................................... 38Fuse Replacement .......................................................................................................................... 38Sample Chamber Cleaning ............................................................................................................. 39Cover Removal ............................................................................................................................... 40Lamp Replacement ......................................................................................................................... 40Gain Switch ..................................................................................................................................... 41Filter Disassembly .......................................................................................................................... 41Troubleshooting .............................................................................................................................. 42

Appendix ............................................................................................................................................. 43Theory of Fluorescence .................................................................................................................. 44Considerations ................................................................................................................................ 46Frequently Asked Questions ........................................................................................................... 49Spectral Distribution of Mercury Line Lamp .................................................................................... 54Operation as a Nephalomter ........................................................................................................... 54Operation as a Luminometer .......................................................................................................... 56Accessories .................................................................................................................................... 58

Warranty.............................................................................................................................................. 60

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Introduction

Congratulations on your purchase of a DigitalFluorometer.

Introduction

The Turner Model 450 Fluorometer is a highsensitivity instrument utilizing integrated circuitanalog and digital signal processing. Excitationenergy is provided by a quartz-halogen lamp anddetection is by a high gain, low noisephotomultiplier tube. Wavelength selection isaccomplished by interchangeable filters.

The unit is designed to do a number of jobswithin your laboratory. Please read theinstructions carefully to insure that you receivethe maximum benefit from it. Also, be sure to fillout and return the enclosed warranty registrationcard. We would like to receive the informationrequested, and it will help us assure you ofproper warranty coverage.

Important Information

This manual contains important operating andsafety information. You must carefully read andunderstand the contents of this manual prior tousing this equipment.

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Safety Information

Your Turner Digital Fluorometer has beendesigned with function, reliability, and safety inmind. It is the user's responsibility to install it inconformance with local electrical codes. For safeoperation, please pay attention to the alert boxesthroughout the manual.

Safety Information

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Specifications

Specifications

Readout Seven Segment, 3 1/2 digits, Light Emitting Diode DisplayWavelength Range Excitation 340-750 with Quartz-Halogen Lamp

(254, 313 nm with Mercury Lamp)Emission 340-650 (340-750 nm with a red sensitive PMT)

Wavelength Selection Interchangeable, externally inserted filtersSensitivity 10 picograms per ml (Fluorescein Isothiocyanate)

15 parts per trillion (Quinine Sulfate Dihydrate, QSO4)Concentration Range 0-1999Resolution 0.05 % of Full ScaleFluorometric Linearity Typically within 1%.Detector Photomultiplier TubeStandard Sample Holder Accommodates 12 x 75 mm round cuvettes, and accessory

options available.Temperature range Operating 10 to 38° C, Storage 0 to 75° C.Humidity <95%Outputs 0 - 200 mVDC, linear in fluorescencePower Requirements 100, 120, 220, 240 VAC, 50/60 Hz, switchable, 75 wattsDimensions Width 39 cm (15 3/8") x Depth 29 cm (11 1/2") x

Height 13 cm (4 1/2")Weight Model 450-000, 450-003: 7.3 kg (16 lb.),

Model 450-005: 8.2 kg (18 lb.)

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Installation

Installation

Standard Equipment

The Turner Model 450 Digital Fluorometerconsists of the following:

1 Model 450-000/003/005 DigitalFluorometer

1 Power Cord1 NB360 Excitation Filter1 SC415 Emission Filter1 Blanking Filter (included with the

Model 450-005 only, for UV use.)3 12 x 75 mm cuvettes1 1/16" Allen Wrench4 Aperture Slides, 1 each of 0.266, 0.176,

0.080, 0.025 in. Diameters1 Square Cuvette Holder (HR904X4)1 2.0 Amp Slow Blow Fuse (for 100 and 120

VAC use only)1 1.0 Amp Slow Blow Fuse (for 220 and 240

VAC use only)1 45F00-01 Operation Manual1 Turner Warranty Card

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INSTALLATION

Instrument Checkout

After the Model 450 is unpacked,inspect it for physical damage. Reportany observed damage immediately tothe Freight Carrier and your dealer.

At this time fill out theBarnstead|Thermolyne Warranty Card toregister your instrument. Upon receipt,this card will serve to update you withthe current developments andapplications for your Model 450Fluorometer.

The Serial Number for the instrument islocated on the black rear panel. Pleasehave this number recorded in yourManual for future reference.

Serial Number:___________________.

Purchase Date: __________________.

Vendor:_________________________.

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Figure 1Back Panel, Model 450

INSTALLATION

Installation

A. The Model 450 requires 100, 120,220, or 240 VAC at 50 Hz or 60 Hz from aproperly wired and grounded, power outlet. TheModel 450 is delivered preset to 120 VAC, 60Hz. Verify which voltage is present and set thevoltage selection board on the back panel (seeFigure 2) to the appropriate setting as follows:

Failure to observe the proper voltage will resultin damage to your instrument.

1. Locate power inlet connector on the backpanel.

2. Be sure the power cord is not installed andslide the plastic protective cover to the leftexposing the voltage selector board. If thevoltage shown is incorrect, pull back the blackfuse lever to remove the fuse and remove theboard by pulling it firmly with a set of smallpliers.

3. Turn the board until the proper voltage isfacing you and reinsert the board. The propervoltage is now visible from the rear of theinstrument. (Figure 1)

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INSTALLATION

4. Insert the proper fuse for the voltage selected.For 100 and 120 VAC use 2.0 Amp Slow BlowFuse. (installed)For 220 and 240 VAC use 1.0 Amp Slow BlowFuse (included in the accessory package).

5. Slide the cover to the right.

B. Locate the instrument on a vibration-free laboratory surface. Allow two inches behindthe back panel for air circulation and the powercord.

C. Connect the power cord to theinstrument and to the proper outlet after verifyingthat the voltage of the instrument has beenproperly set.

D. The switch is on the back of the unit.The instrument panel should illuminate. If thedisplay does not illuminate, immediately turn theinstrument off and consult the trouble shootingsection (see Preventative Maintenance Section).

E. The excitation lamp is prealigned at thefactory and requires no further alignment. Seepg. 40 for lamp replacement or cleaning.

F. Refer to the Operation for operationaldetails of the Model 450.

CautionTwo spare fuses are provided

with the instrument, 1.0 Amp and 2.0Amp. In addition, the instrumentcomes with a 2.0 Amp fuse installedfor 100 and 120 VAC operation. Inareas where 220 and 240 VAC areused, the installed fuse and the spare2.0 Amp fuse should be discardedand replaced with the 1.0 Amp fuse.

Figure 2Voltage Selector Board

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Operation

Description Refer to Figure 3 for location ofthe components.

A. Controls and Indicators:

1. Main Power Switch: The Main PowerSwitch is located on the rear panel of the Model450 Fluorometer, just above the Fuse andPower cord receptacle.

2. GAIN Switch: Controls the amount of voltagesupplied to the photomultiplier (light detector).The photomultiplier converts the lightfluorescing from the sample into an electricsignal and amplifies it. Higher gain settingsincrease the instrument's sensitivity and areused to read at low concentrations.

3. ZERO Knob: Controls the display value andis more sensitive than the SPAN control. Usedto set the display to zero with the blanking rodor blank solution (solvent) in the cuvette holder.

4. SPAN Knob: Controls the display value. Thisknob sets the display to any value you want toread for a sample with a known concentrationex. A 1000 mg/liter standard sample is set toread 1000.

Figure 3TURNER Model 450 Fluorometer

Operation

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5. DISPLAY: 3 1/2" digit display to indicaterelative fluorescence.

6. Mercury Lamp Power Switch: This switch islocated on the front panel, to the right of theSPAN control. This option is only available onthe Model 450-005. See Section 3-2 for furtherinformation.

B. Cuvette Holder:

The standard cuvette holder supplied with theinstrument accepts a 12 mm x 75 mm tube. Toinsert tube, lift the cover from the front and insertthe cuvette until it reaches the bottom. Close thecover. Optional Cuvette Holders for differentstyles of cuvettes are available from B/T.

C. Interchangeable Filter Holder

The Filters are designated Excitation andEmission. The Filter Holders areinterchangeable. The Excitation Filter is placedin the Holder on the left side of the samplechamber, and the Emission filter is placed in theHolder toward the front of the instrument. TheExcitation filter is always a Narrow Bandpass,

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NB, Filter, and the Emission filter is generally aSharp-Cut, SC, Filter. See pg. 17 for help withthe proper choice of filters for your applications.

To change the filters, lift the samplecompartment lid and slide out filter holder.Replace with the proper filter.

D. Aperture Slides:

The aperture slides are used to attenuate thelight when high sample concentrations are used.The aperture slides restrict the amount ofexcitation light reaching the sample through theexcitation filter. It may also be used to restrictthe amount of emitted light that passes throughthe emission filter to the photomultiplier tube.The aperture slides effectively reduce thesensitivity of the instrument.

Generally only the excitation side is attenuated.Too much excitation of concentrated samplestypically gives a non-linear fluorescentresponse. Attenuation of the emission light onlyreduces the sensitivity of the unit and will nothelp with non-linear readings.

To use an aperture slide, insert the appropriatefilter into the Filter Holder next to the SampleChamber. The aperture slide inserts betweenthe filter and the sample chamber. Make sure

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the hole end of the aperture slide is inserted first.If aperture slides are used with both theexcitation and emission filter holders, place thelarger aperture slide with the excitation filterholder

Operation

Models 450-000 and 450-003

Perform the proper installation steps (See theInstallation Section) before the instrument isoperated for the first time.

A. Make sure the proper voltage has beenselected. (see Section 2-3)

B. Switch on the power (on the backpanel). Allow a 15 minute warm-up.

C. Place the appropriate filters in thesample compartment (See Figure 4).

D. Insert the appropriate aperture slide ifrequired.

E. Place cuvette with blank solution, intothe cuvette holder.

F. Set SPAN fully Clockwise (CW) andadjust ZERO knob until the display indicates 000.

OPERATION

Note Dilutions of the most concentratedstandard solution should be tested toensure linearity.

Figure 4Sample compartment forthe Models 450-000/003

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G. Replace blank solution with the highestconcentration of standard sample to bemeasured.

H. Adjust the GAIN and SPAN controlsuntil desired concentration reading is obtainedon the display. i.e. 1000.

I. If the display goes blank reduce theGAIN until a number is displayed. If displayremains blank on the lowest Gain setting, installthe aperture slide, or dilute your standard. (SeeOperation Section)

J. Replace the Standard solution with anunknown sample. Read and record the relativefluorescence value.

Model 450-005

The operation of the Model 450-005 is identicalto that for the Models 450-000/003 with theaddition of the Mercury Vapor Line Lamp.

Perform the proper installation steps (See theInstallation Section) before the instrument isoperated for the first time.

A. Make sure the proper voltage has beenselected. (see Installation Section)

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B. Switch on the power (on the backpanel). Switch on the power for the Mercury LineLamp which is located on the front of theinstrument when using wavelengths below 340nm. Allow the instrument 15 minutes to warm-up.

C. For UV excitation place the blockingfilter in the excitation filter well corresponding tothe quartz-halogen lamp. (See Figure 4) Forwavelengths above 340 nm see step K.

D. Place the appropriate filters in theremaining sample compartment holders.

E. Place cuvette with blank solution, intothe cuvette holder.

F. Set SPAN fully Clockwise (CW) andadjust ZERO knob until the display indicates 000.

G. Replace blank solution with the mostconcentrated standard sample.

H. Adjust the GAIN and SPAN controlsuntil desired concentration reading is obtained onthe display. i.e. 1000.

I. If the display goes blank reduce GAINuntil a number is displayed. If display remainsblank on the lowest Gain setting, install theaperture slide. (See the Operation Section)

J. Replace the Standard solution with anunknown sample. Read and record the relativefluorescence value.

OPERATION

Note Dilutions of the most concentratedstandrd solution should be tested toensure linearity.

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K. To use the instrument over 340 nm,place the blocking filter in the mercury excitationfilter well, and switch off the mercury lamp.Refer to steps D-J for visible light operation (seepage 15).

Cuvette Matching Procedure

Cuvettes can affect the results and are importantto the performance characteristics of theinstrument. The cuvettes for any givendetermination should be matched so that theirfluorometric response is identical in theinstrument.

When using the Mercury Vapor Line Lamp forthe UV excitation source below 340 nm, youmust use quartz cuvettes.

Cuvettes may be matched as follows:

1. Install the excitation and emission filters forthe appropriate analytical procedure. (Usuallycuvettes are matched for use with a specificanalysis.)

2. Fill each cuvette to be checked with thefluorescent substance to be analyzed.

3. Set zero fluorescence with the blank referencesolution cuvette, and then observe thefluorescence of each individual cuvettes, whichhave been inserted into the cuvette holder andaligned according to any orientation indicators.

Note The quartz halogen lamp will stayilluminated when the Mercury Lamp ison.

Note When using excitation wavelengthsbelow 340 nm use quartz cuvettes.

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Note For the best results use quartzcuvettes for all your visible andultraviolet applications.

Note When using round cuvettes, youmust align them the same way in theinstrument every time. Variations inthe glues thickness & properties canaffect your results.

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4. Observe the fluorescence for each cuvette andselect those whose fluorescence agree closely.

Filters

The filters in your Turner Filter Fluorometer areused to perform two functions. The first is toallow only light of a specific wavelength to passinto the sample cell and excite a specificmolecule. The excitation filters are alwaysNarrow Band (NB) filters and are specific for thecompound of interest. The emission filters aregenerally Sharp-Cut (SC) filters that allow onlyemitted light above a specific wavelength to passinto the photomultiplier tube. Since thephotomultiplier tube is sensitive to a wide rangeof wavelengths, a filter allows the user to choosethe wavelength range detected by thephotomultiplier tube, to reduce background noiseand increase sensitivity.

A. Selection

For nearly all fluorescent determinations, therequired filters are recommended by B/T. In theevent that a new procedure is being developed,the following considerations should be applied inselecting the proper filters.

NoteThe mercury lamp has a limitedlifespan. Turn off when not in use.

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1. Some compounds have the ability todischarge some of the energy obtained whenthey absorb light by emitting light of a longerwavelength. These compounds are fluorescent.The efficiency of this process may be anywherefrom a fraction of a percent to almost 100percent. A portion of the absorbed energy isreleased as heat.

2. The intensity of the light emitted isproportional to the amount of exciting lightabsorbed. At low concentrations, the emittedlight may be considered proportional to theconcentration. In practice, the meter reading ofthe fluorometer is linear with concentration of thefluorescent molecule involved. The rangebetween the lowest detectable sample and thepoint where significant non-linearity occurs isnormally a factor of 10

4 to 10

5 in concentration of

the fluorescent molecule.

3. If a compound is fluorescent, a wide range ofenergy which it absorbs causes it to fluoresce.The spectrum of the emitted light is normallyquite broad and its shape and peak areindependent of the wavelength of the excitinglight. The only variation is in intensity; whereless light (energy) is absorbed, less light will beemitted. Proportionality of fluorescence andconcentration are maintained even when theexciting light and measured fluorescence are farremoved from the peaks.

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Before outlining the specific steps involved infilter selection, several terms should beexplained.

Narrow Bandpass Filter (NB)

This filter is used when the excitation andemission wavelengths are close together.Typical bandwidths are in the 8 to 10 nm range.They require precision manufacturing techniquesand cost more than the other types of filters. Thefilter consists of a miniature interferometer inconjunction with blocking glasses. For the filterdesignated NB390 the range of wavelengthpassed by the filter is 385-395 nm. These filtersare constructed with a thin coating of materialand are susceptible to scratching. Extreme careshould be taken when handling these filters. Seethe Preventative Maintenance Section formaintenance.

Some Narrow Bandpass Filters are comprised ofspecial optical glasses, or a combination of glassor gelatin materials, and have bandwidths of 50to 100 nm. Examples of these excitation filtersare the NB360 (bandpass 40 nm) and the NB430(bandpass 90 nm). These filters are particularlyuseful where high sensitivity is required, andlower resolution is not a factor in distinguishing

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between different fluorescent moieties present inthe solution. By allowing more light to pass tothe sample, a greater number of thefluoroscence molecules are excited and moreemission light is produced per sample volume.

Narrow Band Filters can also be used in theemission side filter holder when specific emittedwavelengths need to be distinguished amongmany emitting fluoroscence present in solution.

Sharp-Cut Filters SC

This type of filter passes all light of a longerwavelength than a given value and blocks alllight of shorter wavelengths. The transition fromzero to full transmittance (approximately 80-90percent) usually takes place over a region ofabout 40 nm. The characteristic wavelength of aSharp-Cut Filter is defined as the wavelength atwhich the filter has a transmittance of 37percent. With most filter manufacturers, there isabout a 10 nm tolerance in the location of thelongest wavelengths of emitted light which canbe measured.

Photomultiplier Tube (PMT)

The photomultiplier tube in the Model 450-000 issensitive to wavelengths of 200-600 nm but fallsoff very rapidly above 650 nm. The

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photomultiplier tube in the Model 450-003 and -005 are red-sensitive to wavelengths of 200-750nm.

The PMT detector in all fluorometers measuresall the light reaching it from the cuvette. By usinga NB or SC filter, certain wavelengths of light arepermitted to be passed to the PMT andsubsequently detected. Unwanted wavelengthsare not allowed to pass to the PMT, and do notaffect the fluorescence measurement. The NBand SC filters select a part of the PMT responsecurve to measure.

Note: It is important that the pass bands of theexcitation and emission filters does not overlapand affect the measurements of the PMT.

Compatibility of Filters

Two filters are compatible and may be used asexcitation and emission filters if the wavelengthsof the light they transmit do not overlapsignificantly. If such an overlap is present,scattered light from an even slightly turbidsample, or from an optical defect in the cuvette,will reach the photomultiplier and be registeredas fluorescence.

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The steps involved in selecting the bestcombination of excitation and emission filters fora given compound are as follows:

1. Ideally the fluorescence spectra of thecompound will be available. Look for theexcitation and emission filters required in Table I.If the name of the compound is known, the usermay consult the manufacturer of the fluorescentcompound to obtain the absorbance maxima forthe compound and the fluorescent spectrummaxima for the compound. Direct the inquiriesto the technical service department of thechemical manufacturer.

2. If the fluorescence spectra are not available,then proceed as follows:

Refer to the sample spectra below for thisdiscussion.

a) Determine the maximum absorptionwavelength of the compound with aspectrometer. Choose a filter which is closest tothe peak from Table I and use this for excitation.

b) Having chosen a tentative excitationfilter, it requires but a few minutes to insert aseries of Sharp-Cut (SC) emission filterscompatible with the excitation filter, comparing

OPERATION

NB 360 NT 405 NT 440 NB 490 NB 540 SC 415 SC 430 SC 515 SC 535 SC 585 SC 665

Figure 5ABandpass Filter Transmittance Curves

Figure 5BSharp Cut Filter Curves

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the ratio of sample reading to reagent blank. Ifthe data obtained indicates a fairly sharp-peakedemission curve, the possibility of using a NarrowPass Emission Filter may be considered. Usuallythere is little advantage to a Narrow-PassEmission filter, though they are occasionally ofvalue where the emission peak falls close to theexcitation bandpass.

Table I Filter Selection Chart

All filters are mounted in filter holders specificallydesigned for simple wavelength selection in theModel 450. With the exception of the NB 360 (40nm) and the NB 430 (90 nm), all narrow bandfilters are designated by the center wavelength innanometers. See the graph below for a typicaltransmittance curve. Sharp-Cut filters,designated SC, are long pass filters. The numberfollowing the SC is wavelength, in nanometers, atwhich they exhibit a transmission of 37%. Theytypically have a transmission of less than 1% at awavelength 10 nm less than the ratedwavelength. The NB excitation filters may beused as emission filters where wavelengths areclose together but sensitivity will be reduced.

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OPERATION

45F01-01* Filter NB360 45F01-21* Filter SC41545F01-02 Filter NB390 45F01-22* Filter SC43045F01-03* Filter NB405 45F01-23 Filter SC45045F01-04 Filter NB420 45F01-24 Filter SC47545F01-05* Filter NB440 45F01-25 Filter SC50045F01-06 Filter NB460 45F01-26* Filter SC51545F01-07* Filter NB490 45F01-27* Filter SC53545F01-08 Filter NB520 45F01-28 Filter SC55045F01-09* Filter NB540 45F01-29* Filter SC58545F01-10 Filter NB450 45F01-30 Filter SC60545F01-11 Filter NB254 45F01-31 Filter SC63545F01-12 Filter NB430 45F01-32* Filter SC66545F01-13 Filter NB313* Filters marked with * are included in filter Set 45F01-50.

Filters

45F01-51 Application Filter Set, Fluorescein, NB490 & SC51545F01-52 Application Filter Set, Quinine Sulfate, Petroleum (heavy), NB360 & SC41545F01-53 Application Filter Set, Chlorophyll, NB440 & SC66545F01-54 Application Filter Set, Thiamine (Vitamin B1) & Ascorbic Acid (Vitamin C),

NB360 & SC43045F01-55 Application Filter Set, Riboflavin (Vitamin B2), NB440 & SC53545F01-56 Application Filter Set, Tocopherols (Vitamin E), NB360 & SC50045F01-57 Application Filter Set, DNA (DABA method), NB405 & SC51545F01-58 Application Filter Set, Catecholamines, NB405 & SC51545F01-59 Application Filter Set, Rhodamine, NB540 & SC58545F01-60 Application Filter Set, Ames TDA, NB405 & NB45045F01-61 Application Filter Set, Protoporphyrin, (Anemia & Lead Poisoning)

NB405 & SC58545F01-61 Application Filter Set, Petroleum, NB254, NB360 & SC415

Application Filter Sets

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B. NB versus SC Filter Use

The previous discussions detail which filters touse for an individual application, however thereare times when the results of the fluorescentmeasurements are not linear, or are affected byundesired fluorescent moieties. In theseinstances using a Narrow Bandpass Filter (NB) inthe emission side of the filter holder may givemore accurate results.

In some instances more than one compound ispresent which can absorb the excitation light. Ifthe undesired molecule subsequently does notfluoresce, then the undesired molecule will haveno effect on the reading of the fluorometer. Ifhowever the undesired molecule does fluorescethen the proper choice of emission filter isimportant. The SC filter allows light of a specificwavelength and longer to be passed through tothe photomultiplier tube. In the case when thislight is the result of emissions from two or moredifferent compounds in the sample then a choiceof using a Narrow Bandpass versus Sharp-Cutfilter must be made. This is where fluorometryhas a distinct advantage.

If the fluorescence maximum of the desirecompound is greater than the undesired moiety,choose a Sharp-Cut (SC) filter with a longerwavelength cut-off, above the fluorescencemaximum of the moiety. In the case where the

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undesired moiety fluoresces at a wavelengthlonger than the compound of interest, choose aNarrow Bandpass (NB) filter closest to thefluorescence maximum of the compound ofinterest.

If the fluorescent wavelengths of two compoundsoverlap, then careful choice of the excitationbandpass filter can decrease the contribution tothe fluorescent light emitted by the undesiredmoiety. Choose the excitation range for thedesired compound using an NB filter at awavelength where there is significant absorptionof light by the desired compound while there islittle or no absorbance of light by the undesiredcompound.

Compromises Between NB and SC Filters

Use of a NB filter in the Excitation side of thefilter holder allows light of a specific wavelengthto excite a molecule. The intensity of the lightpassed through to the sample depends on thelight source and the bandpass filter. Choosing aNB filter with a larger bandpass, like for theNB360 and NB 430 filters, will allow a largerspectral distribution of light to pass to the sampleand excite more molecules. This may increasesensitivity of the test. Care should be takenwhen using these filters.

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Use of a NB filter on the emission side of the filterholder passes less of the fluorescent lightproduced by the sample to the photomultipliertube. While being more specific for a wavelengthrange, the sensitivity of the test may be reduced.

Care and Maintenance

Three different types of filters are used in yourTurner Model 450 Fluorometer:

Solid Glass FiltersGelatin FiltersInterference Coated Filters

The Gelatin and Interference Coated Filters arethe most susceptible to environmental conditionsand handling. Care should be taken whencleaning, handling and storing of the filters toprevent damage, or scratching. Scratching of thefilter may result in the transmittance of light of anundesired wavelength to the sample ortransmittance of undesired light to thephotomultiplier tube. Carefully inspect the filtersfor cracks, scratches, or other damage. Replaceif necessary.

Handling of Gelatin filters:

1) Do not touch the filter surfaces, handle thefilter holders carefully.

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2) Avoid exposing the gelatin filters totemperatures above 50 °C (122 °F), thebandpass may change.

3) Under tropical or humid conditions, protect thefilters from damage from fungus by storing thefilters in a cool dry atmosphere, or suitably sealeddessicator.

4) Remove dust particles by brushing lightly witha clean, dry, camel’s hair brush, or by gentlyblowing clean, dry air across the surface.

5) Do not use a soft cloth or abrasive, i. e. lensepaper, on the filter directly.

6) Occasionally examine filters for excessivescratches or other damage. In some instanceserratic result may ensue, and these result may bedue to improper storage and handling of the filterand filter holder.

7) Do not allow water or other lense cleaningsolution to come into contact with the gelatin filter

8) Upon receipt of your fluorometer, perform astandard fluorescent measurement and record allfactors used for the standard test. Periodicallyrefer to these values to confirm the stability andresponse of the filters and fluorometer.Replacement of the filters to ensure accurateresults for critical tests may be recommended.

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Handling of Interference NB Filter

Interference filters have a thin coating of materialon the filter surface. Care must be taken notdamage the surface coating. Interference filtersshould be treated as gelatin filters in mostpurposes. The interference barrier is comprisedof a coating using microplasma filter techniques.They are sensitive to extremes of humidity, whichmay destroy the film.

Handling of Glass Filters

Glass filters are the most durable and may becleaned with lense paper or soft cloth makingsure the surfaces are not scratched in theprocess.

Standard Curves and Calibration

The Turner Model 450 Fluorometers measure therelative fluorescence of a sample. To accuratelydetermine the concentration of a fluorescentmaterial during an analysis, one must prepare astandard curve which correlates the fluorometricresponse of the instrument with knownconcentrations of sample. The Turner Model 450Fluorometer allows you to set the scale of theinstrument so that you can read the concentrationdirectly, facilitating the fluorescencemeasurement.

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Concentration Calculations:Parts per Million and Parts per Billion

Composition, concentration and related moleculeor particle composition is often expressed asparts per million (ppm, parts per billion (ppb), andsometimes parts per trillion (ppt). The term partsper million corresponds to one gram of thecompound of interest present in 1 million gramsof total solution. For an aqueous solution whosedensity is close to 1.00 g/mL, 1 ppm correlates to1 µg/ml or 1 mg/L. For dilute aqueous solutionsthe density of a mixture for all intended purposesis 1.00 g/mL.

Parts per Million, ppm = (grams of substance/grams of solution)x 10

6

Parts per Billion, ppb = (grams of substance/grams of solution)x 10

9

Parts per Trillion, ppt = (grams of substance/grams of solution)x 10

12

Preconditioning

Your Turner Fluorometer is capable ofmeasuring fluorescence of compounds atextremely low concentrations, e.g. in parts perbillion or parts per trillion. At theseconcentrations there are relatively few moleculesin solution. As a result, if molecules are removed

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from the solution, a large change in thefluorescence reading will occur. Depending onthe acid base chemistry of the solution andfluoroscence, the fluoroscence may adhere to thesides of the sample container, and be effectivelyremoved from the solution. This has been knownto occur between a basic moiety and a glasssurface.

To avoid this problem the acid-base chemistry ofthe glass surface must be equilibrated with thesolvent prior to the addition of the fluorescentmoiety. Add your solvent and allow the system tocome to equilibrium, then replace the solvent witha solution containing the fluorescent compoundof interest.

Preparation of a Stock Solution (Example)

Prepare a stock solution using thefluorescent compound you will be analyzing.You will use this stock solution for both theFluorescence Curve Determination and theInstrument Gain Check.

For example, if the user will be determining thefluorescence of Quinine Sulfate in a set ofsamples, the standard curve should bedetermined using known concentrations of

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NoteWeights and volumes may beadjusted accordingly to accommodateyour procedure.

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NoteFor concentrations in the ppb rangedilute 1 mL of Solution A into another1 liter volumetric flask. (Alternatively,you may dilute 0.1 mL int a 100 mLvolumetric flask.) Fill to the mark with0.1N acetic acid. This gives astandard solution of 0.00000001grams per liter (10.0 ppb). Using thissolution, proceed with steps 2 and 3above to arrive at solutions of 2.50ppb and 5.0 ppb respectively. Usethese three ppb solutions in the samemanner as their ppm counterparts inFluorescence Curve Determinationan Instrument Gain Check.

When working with concentrations inthe ppb range, you may need toadjust the Gain control. See theInstrument Gain Check section fordetails.

Quinine Sulfate. The Quinine Sulfate StandardReference Material (SRM) 936a used in thefollowing example was obtained from theNational Institute for Standards and Technology(NIST), Standard Reference Material Program,Room 204, Building 202, Gaithersburg, MD20899-001. (301) 975-6776. StandardFluorescence Grade Chemicals are alsoavailable from the commercial laboratory supplycompanies.

1) Add approximately 500 ml of 0.1N acetic acidto a 1 liter volumetric flask. Weigh 0.1000 grams(10.0 mg) of quinine sulfate and transfer it to theflask. Swirl to mix. Fill the flask to the mark with0.1N acetic acid. This gives a stock solution of10.0 mg/L (10.0 ppm) of quinine sulfate. Label asSolution A (10.0 ppm).

2) Accurately measure 250 ml of Solution A intoanother 1 liter volumetric flask. (Alternatively,you may dilute 25 mL into a 100 mL volumetricflask.) Fill to the mark with 0.1N acetic acid. Thisgives a standard solution of 2.50 mg/L (2.50ppm). Label as Solution B (2.50 ppm).

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3) Accurately measure 500 ml of Solution A intoanother 1 liter volumetric flask. (Alternatively, youmay dilute 50 mL into a 100 mL volumetric flask.)Fill to the mark with 0.1N acetic acid. This gives astandard solution of 5.0 mg/L (5.0 ppm). Label asSolution C (5.0 ppm).

4) Use 0.1N acetic acid as the "blank" standard(0.0 ppm). Label as Solution D (0.0 ppm).

Following the above general dilution procedure,concentration ranges of parts per million (ppm),parts per billion (ppb) and parts per trillion (ppt,etc., can be prepared. You may reduce theamount of compound initially weighed out, orincrease the number of dilutions made forstandard preparation, i.e. 0.001 g, 0.01 g orlower, as fluorescence is very sensitive!

Instrument Gain Check

When you are working with very lowconcentrations of a fluorescent compound, theGain setting may need to be adjusted upward toprovide sufficient sensitivity for your analysis. Toset the fluorometer Gain control:

NoteBecause of the sensitivity offluorescence, minor variations in thewalls of cuvettes may be reflected inyour results. To maintain consistencyand accuracy, it is important tocarefully orient the markings on yourcuvettes to the same point on theinstrument for each reading. For thebest possible results, use the samecuvette for each and everymeasurement.

OPERATION

34

1) Turn on the fluorometer and allow the systemto warm up for 15 minutes. Insert filtersappropriate for your application into the left andfront holders of the instrument. For QuinineSulfate, the appropriate filters are the NB360Excitation filter (left holder) and SC415 Emissionfilter (front holder). The Model 450-005 alsorequires the blank filter, supplied with theinstrument, to be inserted into the right holder.

2) Set the fluorometer Gain control to 1 and theSpan control fully clockwise.

3) Insert a cuvette containing Solution D, theBlank. Using the Zero control, set the display toread 000.

4) Remove the cuvette containing Solution Dfrom the instrument and insert a cuvettecontaining Solution A. The display should read avalue above 1000. If it does, proceed withFluorecence Curve Determination. If it does not,continue with step 4.

Fluorescence Curve Determination

1) Turn on the fluorometer and allow the systemto warm up for 15 minutes. Insert filtersappropriate for your application into the left andfront holders of the instrument. For QuinineSulfate, the appropriate filters are the NB360Excitation filter (left holder) and SC415 Emission

OPERATION

35

filter (front holder). The Model 450-005 alsorequires the blank filter, supplied with theinstrument, to be inserted into the right holder.

2) Place Solution D, the Blank, into a cleancuvette and place the cuvette into the cuvetteholder in the fluorometer, then close the cover.

3) Turn the SPAN control fully clockwise. Usingthe ZERO control set the display to read zero(000).

4) Place Solution A into a clean cuvette andinsert the Solution A cuvette into the fluorometer.Using the SPAN control, adjust the display toread 1000, or some chosen value. This value willbe equivalent to 10.0 ppm. (When you first insertthe 10.0 ppm standard, the instrument displaymay indicate an over range reading. Adjust theSPAN control until a value appears, then adjustto your chosen value.)

5) Place Solution B into a clean cuvette andinsert the Solution B cuvette into the fluorometer.The Display should read 250±10 (2.50 ppm) ifyou set the SPAN control to 1000 in step 4. If youset the SPAN to some other number in step 4,the Display should display a value one-fourth ofthe value of the SPAN value.

OPERATION

NoteThe fluorometer display will over-range when the cover is not closed.

NoteAfter the adjustment of the SPAN andZERO controls, these controls shouldnot be adjusted.

36

6) Place Solution C into a clean cuvette andinsert the Solution C cuvette into the fluorometer.The Display should read 500±10 (5.0 ppm) if youset the SPAN control to 1000 in step 4. If you setthe SPAN to some other number in step 4, theDisplay should display a value one-fourth of thevalue of the SPAN value.

7) Place your unknown solution into a cleancuvette and insert the unknown cuvette into thefluorometer. The Display will read XXX andcorrespond to 0.XXX g of compound per liter ofsolution (XX.X ppm) if you set the SPAN controlto 1000 in step 4. If you set the SPAN to someother number in step 4, determine the solution'sconcentration using the following formula:(Display value/SPAN vlaue chosen) X 10 ppm =solution's ppm.

Following the above general dilution procedure,concentration ranges of parts per million (ppm),Parts per billion (ppb) and parts per trillion (ppt),etc., can be prepared.

The user should reduce the amount compoundinitially weighed out, or increase the number ofdilutions made for standard preparation, i. e.0.001g, 0.01 g or lower, as Fluorescence is verysensitive!

Fluorometric Performance Verification

GAIN1) Install filters. Front SC415, Left NB360 andblanking rod.2) Set span FCW.

OPERATION

NoteAssuming that the stock solutionswere prepared properly, steps 5 and 6will demonstrate that the fluorometeris linear. If the tests in steps 5 and 6do not return the expected values,contact Barnstead|ThermolyneCustomer Service for assistance at(800) 553-0039.

NoteFor best results, stock solutions mustbe prepared carefully in cleanglassware to ensure accuracy.Positive displacement pipettes -available from Barnstead|Thermolyne- and volumetric flasks should beused whenever possible. For low levelanalysis, it is suggested that cleanglassware be covered with aluminumfoil and backed in an oven at 250°Cfor at least one hour to destroyorganic contaminants.

Refer to the cuvette matching andmarking procedures outlined in theinstrument manual.

37

3) Set gain to 1.4) Set "zero" to 0.5) Insert the sample gain solution QS04 (1 ppm)(see pg. 29)6) Display will read 520 ±50.

NOISE1) Install filters. Front SC415, Left NB360,blanking rod and set to zero.2) Remove rod and insert the sample gainsolution QS04 (1 ppm).3) Set gain at 1000.4) 0.1% apertures at left position.5) 1% apertures at front position.6) Observe the display and ensure display flickerwill remain within ± -004 over a 30 second period.

Analog Output

External devices, such as data processinginstruments or strip chart recorders (SeeAccessories), may be interfaced to the Model450. Fluorometric analog output is on the rearpanel (see Figure 2). The zero to 200 millivoltDC output is equivalent to the fluorescencedisplayed. If the Computer/Printer Interface isbeing used (PN: 46F00-01), set the voltagesetting on the Interface to 0-200 mDC.

Connection is made to the Model 450 withstandard banana plugs. Observe the polarity asmarked on the panel, red is positive (+) and blackis negative (-)or ground.

OEPRATION

38

Preventive Maintenance

The Model 450 has been designed with the samereliability inherent in all TURNER Fluorometers.It requires minimal preventive maintenance andcare given any quality instrument. It should bekept clean and out of dusty areas or those withtemperature and humidity extremes. There arefew user serviceable parts in the Fluorometer.

Fuse Replacement

The fuse is mounted in a holder which preventsaccess unless the power cable is removed. Firstturn off the instrument and then remove the cablefrom the power outlet.

A. Remove the power cable from the backof the instrument.

B. Slide the plastic cover to the leftexposing the fuse holder.

C. Locate the black lever marked FUSEPULL. Swing the lever out and to the left.

D. The Fuse can now be removed.E. Replace the fuse only with the proper

Slow Blow type. If the voltage is changed refer tothe installation instructions (Section InstallationSection).

2.0 Amp for 100 and 120 VAC applications.1.0 Amp for 220 and 240 VAC applications.

CautionService must only be attempted bytrained and qualified personnel.

Preventative Maintenance

39

F. Insert the fuse and push the lever backinto place.

G. Slide the cover to the right and replacethe power cord in the instrument and then intothe power outlet.

Sample Chamber Cleaning

A. Lift the sample chamber cover and viewthe chamber from the top.

B. Remove the Excitation and Emissionfilters and store them in a clean dry place asrecommended.

C. Remove two phillips screws holding thesmall black plastic alignment adaptor.

D. Insert a finger into the sample cuvetteholder, loosen and lift the cuvette adaptor up andout of the sample holder housing. Do notscratch, damage or distort the cuvette holder.

E. Clean the cuvette adaptor, and samplecompartment carefully with water and allow theparts to dry thoroughly

F. Reassemble.

Other than the lamp and fuse, the Model 450 hasno user replaceable or adjustable componentsinside the cover. Do not remove the cover unlessspecifically authorized by the dealer or thefactory. If steps below do not solve the problemcontact your Barnstead|Thermolyne Dealer’sService Representative.

PREVENTATIVE MAINTENANCE

40

Cover Removal

A. Turn off the instrument and remove thepower cable.

B. Remove the Excitation and EmissionFilters and store them in a clean dry place asrecommended.

C. Using the Allen Wrench, remove twoscrews in the lid of the cuvette holder andremove the lid.

D. Turn the instrument over and removethe five recessed screws holding the cover inplace.

E. Turn the instrument right side up and liftoff the cover.

F. Reverse the above steps to replace thecover.

Lamp Replacement/Cleaning

A. Turn the instrument off.B. Follow the instructions for cover

removal (see Cover Removal Section, pg. 39).C. Locate the lamp in the black lamp

housing. Using a phillips screwdriver, remove tosmall screws holding the lamp in place.

D. Remove the lamp and disconnect twowires from the board.

E. Clean the new lamp (Turner partnumber 45F02-01) with lens tissue and acetone.

PREVENTATIVE MAINTENANCE

41

F. Install the new lamp, replace the crewsand connect the two leads to the board. Thelamp is pre-aligned and requires no furtheralignment.

G. Replace the cover.

Gain Switch Cleaning

A) Follow the direction for cover removal.B) Spray an electronics switch cleaner on

the GAIN contacts and switch the GAIN switchthrough its positions a number of times. Repeatthe process if necessary.

C) Reassemble the cover, sample coverhinge, and replace the cover hold down screws.

Filter Disassembly

Disassembly of the filters in the filter holder isnot recommended! In the event disassemblyneeds to be done, observe all warnings andhandling procedures as outlined in Section 3-5.Damage of the filters by the user is very likely,and is not covered by the warranty, i. e. thewarranty is voided.

A) Inspect the filters for damage. Thereshould be no cracks or scratches on the filters.

PREVENTATIVE MAINTENANCE

42

B) If the filters need to be cleanedbetween the filters, remove the spring ringholding the filters in place and record the order ofthe small 1/2” filter components which areremoved. Clean the filter disks carefully andreplace them in the filter holder in the properorder, then replace the spring ring.

Troubleshooting

A. Front panel does not illuminate:

1. Turn the power off.2. Remove the power cord from the

instrument and the main power line.3. Remove and inspect Fuse (See pg. 36).

Replace if necessary.4. Re-insert the power cord firmly to

insure proper contact.5. Turn the instrument on and verify proper

operation.

B. Front panel illuminates, source lampdoes not.

1. Replace lamp (See Lamp ReplacementSection, pg. 39.

C. Front panel and source illuminate,display will not change.

1. Perform installation procedure (See theInstallation Section) and operation procedure(See the Operation Section).

PREVENTATIVE MAINTENANCE

43

Appendix

Theory of Fluorescence

When a molecule absorbs radiation, an electronis promoted from one energy level in themolecule to a higher energy level within themolecule, absorbing the energy. The moleculemay release the absorbed energy and return theelectron to its ground state by converting thisenergy to vibrational energy or releasing theenergy in the form of light emitted by themolecule. If part of this energy is converted tovibrational energy , the remainder, if radiatedwithin 10

-8 seconds, is emitted as light of lower

energy (longer wavelength) than the absorbedenergy. This property is called fluorescence.Atoms or molecules which fluoresce have well-defined excitation and emission spectra whichallow both qualitative and quantitative analysis ofthe material.

The shape of the excitation spectrum is that ofthe absorbance curve of the molecule and isindependent of the wavelength at whichfluorescence is measured. Each molecule also

Appendix

44

APPENDIX

has a characteristic number called the quantumefficiency, which is the ratio of total number ofemitted photons to the total number of absorbedphotons. A non-fluorescent molecule is onewhose quantum efficiency is zero or so close tozero that the fluorescence is not measurable.Both the quantum efficiency and the shape of theemission spectrum are independent of thewavelength of the exciting light. If the excitinglight used is of a wavelength which is differentfrom that of the absorption peak, a smallerportion of the light will be absorbed andproportionately less light will be emitted,illustrating the constancy of the quantumefficiency. However, the shape and location ofthe emission spectrum will not change.

Fluorescence/Concentration Relationships

Fluorescence is related to and dependent onconcentration according to the following equation:

F = KIφC

Where F = Fluorescence reading observed onthe instrument.K = A constant which accounts for instrumentalfactors, pH, T, electronics.

l

45

APPENDIX

I = The intensity of the exciting light at awavelength.C = The concentration of the fluorescingmolecule. = The path length of the solution or solid beingmeasured.φ = The quantum efficiency of the molecule.

An examination of this equation shows that for agiven molecule, at a constant concentration, theamount of fluorescence observed can be affectedby:

I, the intensity of the exciting wavelength, andD, the path length of the molecules.

As the concentration of a molecule decreases,the observed fluorescence can be kept constantby increasing the intensity of the excitingwavelength and/or the pathlength.

As a practical matter, this means that the onlylimits on the sensitivity of fluorescencemeasurements are the electrical noise in theinstrument, competing radiations, physicallimitations, i. e. sample volume available,maximum energy available, and maximumacceptable cell size.

The accuracy of measurement is excellentbecause the technique is a direct measure of theradiant energy is being made.

l

46

Considerations

Blank Fluorescence

One of the most common limits on sensitivity isset by blank fluorescence. Blank fluorescencearises from the reagents or other sourcesseparate from the compound of interest. Ideally,the fluorescence of the “blank” should be 000,relative to the fluorescence of the material beinganalyzed.

Reagents

Usually, little difficulty will be encountered withcommercially available reagent-grade chemicals.This is particularly true in laboratories utilizingprocedures where the majority of assays aredone with visible light activating the fluorescenceof the sample.

If the samples are under ultraviolet excitation, orat high instrument sensitivity, then high “reagentblank” fluorescence may be encountered. Evenspectro-quality solvents sometimes containtraces of impurities which may fluoresce underultraviolet light. Simple distillation in an all-glassapparatus frequently remedies this problem.

APPENDIX

47

There are also several companies marketingfluorescent-grade reagents for those who mustwork at very low concentration.

A frequent source of contaminants are theplasticizers in some plastics and rubber. Fordispensing distilled water, an all polyethylene orglass system is recommended. Water standingin rubber tubing may develop high fluorescencefrom moieties contained in the tubing duringmanufacture.

Standards

Standards should always be stored asconcentrated solutions, out of the light, and at lowtemperature. Standard solutions of lowconcentrations may degrade with time. Dilutionsto working concentrations should always beprepared as required using appropriate dilutentsince fluorescence is usually pH dependent.

Glassware

Borosilicate glassware is recommended forgeneral storage of reagents. Test tube or flasksmay be capped with parafilm for mixing andstorage, however care should be taken whenusing this material. For work at very low levels of

APPENDIX

48

fluorescence, it may be necessary to rinse allglassware with an appropriate solvent. See theOperation Section).

Temperature

Fluorescence is usually more temperaturesensitive than absorption. Although not allfluorescent materials exhibit marked sensitivity,there are instances where as much as a 2percent reduction in fluorescence is observed forevery degree centigrade temperature rise.Standards, reagent blanks, and sample shouldbe at the same temperature prior tomeasurement of fluorescence. Sometimesdrifting of fluorometer readings may be observeddue to an change in the temperature of thesample while in the sample chamber.

pH

The pH of the solution may affect the fluorescentspecies being tested. Protonation changes theresonance structures of the molecule, theequilibrium of these species, and consequentlythe concentration of the fluorescent molecule.Check your applications carefully for thepossibility of this problem. The pH of standardsolutions may change over time due to the acidic

APPENDIX

49

nature of the storage container materials. e. g.glass. Similarly, the fluorescent compound maydecompose under a variety of pH ranges.

Miscellaneous

Some materials are photosensitive; that is, theydecompose when exposed to wavelengths theyabsorb. This property can be determined bysimply inserting a sample solution into theinstrument and observing its change influorescence over a period of time. Similarlydecomposition may occur while the sample isexposed to room light. Test your samples forsensitivity to room light if there is a question ofstability.

A few materials exhibit delayed fluorescence.This property can be verified by placing thesolution in the instrument and observing a rise inthe fluorescence reading after a period of time.

Frequently Asked Questions:

Can the Model 450-000 be upgraded to aModel 450-003, or 450-005?

The Model 450-000 can be upgraded to a Model450-003 for use in chlorophyll analyses by theinstallation of the extended range red sensitivephotomultiplier tube. Contact your dealer for

APPENDIX

50

availability, or Barnstead|Thermolyne for serviceinstallation. Upgrading the Model 450-000 orModel 450-003 to the Model 450-005 for use inthe UV range requires installation of a mercuryvapor light source, power supply, and new opticalblock.

My instrument displays a 1, and does notrespond to any changes made to the SPAN orZERO controls. What is the problem?

A 1 on the display represents an indication ofover-ranging of the instrument. Adjust the Gain,Zero and Span to give a display reading. Insome cases, use of the aperture slides may berequired, along with the Gain, to attenuate thelight entering or leaving the sample compartment.

The instrument does not have the samereading as a test performed 3 months ago.What is the problem?

Many factors may affect a fluorescent reading.All fluorometers measure relative fluorescence.A lower reading may be due to sampledecomposition, loss of fluorescent material due toadsorption on the surface of a container, or manyother factors.

How can I tell if the lamp is going bad?

Unless the quartz halogen lamp is burned out,the lamp will continue to provide the requiredexcitation light for an analysis. With the

APPENDIX

51

instrument turned on, remove the excitation filterand observe the light present in thecompartment. The light should be on.

When a sample is being read, the readingdrifts to lower or higher displayed values.What is the problem?

When a cuvette is inserted into the fluorometer,the fluorescent reading quickly stabilizes to avalue. When reading drifts lower, thefluorescence of the sample is decreasing due towarming of the sample, or decomposition of thefluorescent moiety. A higher reading may be dueto solvent evaporation or delayed fluorescence.

The display reading seems erratic. What isthe cause?

Check the sample solution for particulate matterlike dust that induces light scattering. Make surethe filters are properly inserted and there are noscratches on the cuvette. Check the filters toensure they have not been damaged. Replacethe filters if necessary.

How can I increase the sensitivity of theinstrument and the measurement?

The sensitivity of the instrument is dependent ona number of factors: the intensity of theexcitation light, the bandpass of the excitationlight, the concentration and quantum efficiency ofthe fluoroscence, the bandpass of the emission

APPENDIX

52

filter, the volume of the sample, and othersources. Careful examination of the testprocedure may indicate changes that can bemade. Choosing a wider bandpass filter willincrease the amount of excitation light enteringthe sample and subsequently excite moremolecules for fluorescent emission. Theconcentration of the sample can be increased.The quantum efficiency is dependent on themolecule and cannot be changed. The samplevolume which is exposed to the excitation lightcan be increased. For additional samplecuvettes and accessories contact your localTurner dealer, or Turner directly.

Are there flow cells available?

Currently Turner offers a 500 microliter flow cellfor sampling. The holder for the flow cell canaccept smaller fluorometer flow cells if they arethe standard 12.5 mm x 12.5 mm square varietywith four polished sides. Contact your Turnerdealer for recommendations for these cuvettes,and accessory items offered by Turner to fit yourneeds.

APPENDIX

53

My old Model 110, 111, or 112 TurnerFluorometer used a 47B blue cobalt filter butnow, for the same test, the recommendedNB490 Filter is Yellow/Silver? Do I have thecorrect filter?

The Narrow Band NB490 filter is specificallydesigned to meet the application. The NB onlyallows light of a small wavelength range to pass.By viewing a light source through the NB filteronly blue light will be observed to pass. Otherfilters will behave similarly.

Where can I get Technical Assistance?

Turner’s customer support continues with a largeapplication database covering fluorometer andspectrophotometer applications, accessed by ourtechnical service department. We are continuingto update our references to provide the customerwith the latest developments in fluorometry andspectrophotometry for eventual use by newcustomers and current instrument owners.Contact your B/T Dealer, orBarnstead|Thermolyne directly.

APPENDIX

54

APPENDIX

Spectral distribution of Mercury Line Lamp

Operation of the Model 450 as aNephalometer

A Nephalometer is an instrument for measuringthe amount of light scattered by extremely dilutesuspensions. It is the most useful where theappearance of the sample is of prime importance,as in the measurement of clarity of beverages,water, etc. In many cases light scattering is an

55

index of a harmful or potentially noxious propertyof a liquid, like silica which can damage turbineblades. Although light scattering is frequentlyused for quantitative analyses, particle size,shape, refractive index, wavelength of light,coefficient of reflection, and color are someproperties which must be considered as theyaffect the amount of light scattered.

For most cases the amount of scattered light islinear to respect to turbidity, and the displayreading can be directly related to turbidity.

Operation

General procedures use a square cuvette to holdthe samples. Barnstead|Thermolyne is pleasedto offer a square cuvette holder for the Model450. Contact your Turner Dealer, orBarnstead|Thermolyne directly for the squarecuvette accessory.

Any filter can be used to select the color of thelight used for excitation. Except when surfacechemistry is such that the molecules on thesurface can fluoresce, only a non-absorbingglass filter can be used to protect the detectorfrom dust. In some cases, it may beadvantageous to have identical filters in theexcitation and emission filter holders to reducethe interference from fluorescent materials.When the solution to be measured is extremelyclear and the particle size very small, a gain in

APPENDIX

56

sensitivity will result from the use of blue light.The wavelengths selected for the measurementshould not be absorbed by the solution.

Nephalometric measurements may be handled inthe same way as fluorescence measurements.For turbidities above 100 ppm, or forstandardization purposes, a standard curveshould be prepared. Colored solutions, wherethe transmittance is consistent from sample tosample, may be treated as if they were colorless,provided the reagent blank and standards arealso colored. Bubbles of gas in the solution or onthe cuvette walls, and cuvette scratches, must beavoided.

Operation of the Model 450 as a Luminometer

Light produced by a chemical reaction can bedetected and measured by the Model 450Fluorometer. These analyses are generallyknown as bioluminescence reactions, and aTurner Fluorometer used in this way is called aLuminometer. Bioluminescence reactions aremost commonly seen in the biological world infireflies. One common test is to measure the lightproduced in the luceferin-lucerferinase reactionmonitoring ATP, or other biological systems.

No filters are required on the excitation side ofthe fluorometer and all excitation light must beblocked by from entering the sample. Block theexcitation light by using a blanking filter holder,inserting an aperture slide with the aperture holeat the top, or by inverting a filter holder. The

APPENDIX

57

fluorometer should be operated in the proceduresoutlined in the Operation Section. A standardcurve should be prepared using theluminescence testing solutions as for a standardfluorometric procedure as detailed in theOperation Section.

Accessories

Model 450 Application Filter Sets

45F01-51 Fluorescein, NB490 & SC51545F01-52 Quinine Sulfate, Petroleum (heavy),

NB360, SC41545F01-53 Chlorophyll, NB440 &SC66545F01-54 Thiamine (Vitamin B1), Ascorbic

Acid (Vitamin C) NB360, SC43045F01-55 Riboflavin (Vitamin B2) NB440,

SC53545F01-56 Tocopherols, (Vitamin E) NB360,

SC51545F01-57 DNA (DABA method), NB405,

SC50045F01-58 Catecholamines, NB405, SC51545F01-59 Rhodamine, NB540, SC58545F01-60 Ames TDA, NB405, NB45045F01-61 Protoporphyrin, (Anemia & Lead

Poisoning) NB405, SC58545F01-62 Petroleum,NB254, NB360, &

SC415

APPENDIX

58

Model 450 Accessories

46F00-01 Computer/Printer InterfaceProgrammable Pocket Computer connects toModel 450. Performs curve fitting and samplecomputation with print-out. User programmed inBasic. 24 Character L.C. Display. Alpha-numericQUERTY Keyboard; 4K bytes RAM, 27K BytesBasic ROM. 24 Character /line printer. Analog toDigital Interface.

45F02-11 Model 450 Flow CellLow Fluorescence, 90 degree emission with 500µl view volume. Complete assembly includesAdapter, Flow Cell and Cover. Requires externalpump or vacuum source.

45F01-50 Model 450 Filter SetContains 11 most commonly used filters. NB360,NB405, NB440, NB490, NB540, SC415, SC430,SC515, SC535, SC585, SC665. (See Filterdescriptions).

Model 450 Supplies & Accessories

11F08-01 Cuvette, 12 x 75mm, RoundBorosilicate matched to 1%, 1-5 ml, Pkg of 511F06-07 Cuvette, 12 x 75mm, RoundBorosilicate selected for optical clarity, 1-5 ml,Pkg of 1245F00-01 Model 450 Operating Manual

(Additional Copies)45F02-01 Lamp, Quartz Halogen, High

Energy, pre-aligned.

APPENDIX

59

45F02-02 Cuvette, Blanking, 12 x 75mm(Replacement)

45F02-03 Aperture Slide Set, (Set of 4)45F02-05 Photomultiplier Tube, High Gain,

Low Noise for Models 450-000 and450-003 (94000-01 and 94003-01),300-600 nm.

45F02-06 Fuse, 2.0 Amp, 120 VAC SBPkg of 5

45F02-07 Fuse, 1.0 Amp, 220 VAC SBPkg of 5

45F02-08 Service Manual, all models.45F02-10 Photomultiplier Tube, High Gain,

Low Noise, R928 Extended Range.200-800 nm

45F02-12 Lamp, Mercury VaporLine source at 254, 313, 360, 405,436, 546, & 578 nm. For Model450-005 (45F00-05) only.

APPENDIX

60

Barnstead|Thermolyne Corporation warrants that if a product manufactured byBarnstead|Thermolyne and sold by it within the continental United States or Canadaproves to be defective in material or construction, it will provide you, without charge, for aperiod of ninety (90) days, the labor, and a period of one (1) year, the parts, necessary toremedy any such defect. Outside the continental United States and Canada, the warrantyprovides, for one (1) year, the parts necessary to remedy any such defect. The warrantyperiod shall commence either six (6) months following the date the product is sold byBarnstead|Thermolyne or on the date it is purchased by the original retail consumer,whichever date occurs first.

All warranty inspections and repairs must be performed by and parts obtained from anauthorized Barnstead|Thermolyne dealer or Barnstead|Thermolyne (at its owndiscretion). Heating elements, however, because of their susceptibility to overheating andcontamination, must be returned to our factory, and if, upon inspection, it is concluded thatfailure is not due to excessive high temperature or contamination, warranty replacement willbe provided by Barnstead|Thermolyne . The name of the authorizedBarnstead|Thermolyne dealer nearest you may be obtained by calling 1-800-446-6060 orwriting to:

Barnstead|ThermolyneP.O. Box 797

2555 Kerper BoulevardDubuque, IA 52004-0797

USAFAX: (319) 589-0516

E-MAIL ADDRESS: mkt@barnsteadthermolyne.com

Barnstead|Thermolyne’s sole obligation with respect to its product shall be to repair or (atits own discretion) replace the product. Under no circumstances shall it be liable forincidental or consequential damage.

THE WARRANTY STATED HEREIN IS THE SOLE WARRANTY APPLICABLE TOBarnstead|Thermolyne PRODUCTS. Barnstead|Thermolyne EXPRESSLY DISCLAIMSANY AND ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDINGWARRANTIES OF MERCHANTABILITY OR FITNESS FOR USE.

One Year Limited Warranty

9001ISOREGISTERED

Barnstead|Thermolyne2555 Kerper Blvd.P.O. Box 797Dubuque, IA 52004-2241Phone: 319-556-2241 800-553-0039Fax: 319-589-0516E-Mail Address: mkt@barnstead.com