Jenway 6850 User Manual
6850
Spectrophotometer
Operating Manual
685 030 REV B/06-14
2
Safety
Please read this information carefully prior to installing or using this equipment.
1. The unit described in this manual is designed to be operated only by trained personnel. Any
adjustments, maintenance and repair must be carried out as defined in this manual, by a
person qualified to be aware of the hazards involved.
2. It is essential that both operating and service personnel employ a safe system of work, in
addition to the detailed instructions specified in this manual.
3. Other than for those items defined in the maintenance procedures herein there are no user
serviceable items in this instrument. Removal of covers and attempted adjustment or service
by unqualified personnel will invalidate the warranty and may incur additional charges for
repair.
4. References should always be made to the Health and Safety data supplied with any
chemicals used. Generally accepted laboratory procedures for safe handling of chemicals
should be employed.
5. If it is suspected that safety protection has been impaired in any way, the unit must be made
inoperative and secured against any intended operation. The fault condition should
immediately be reported to the appropriate servicing authority.
Merci de lire attentivement ces informations avant d'installer ou d'utiliser cet appareil.
1. L'appareil décrit dans ce manuel est conçu pour être utilisé uniquement par des personnes
formées. Tout réglage, maintenance ou réparation doit être effectué comme décrit dans ce
manuel, par une personne qualifiée consciente des risques encourus.
2. Il est essentiel que les personnes utilisant et intervenant sur cet appareil respectent les
règles de sécurité de travail, en plus des instructions détaillées précisées dans ce manuel.
3. En-dehors des éléments décrits dans les procédures de maintenance ci-incluses, cet
appareil ne contient aucun élément réparable par l'utilisateur. L'enlèvement des capots et les
tentatives de réglage ou de réparation par des personnes non qualifiées invalide toute
garantie et entraîne un risque de frais de réparation supplémentaires.
4. Toujours se référer aux fiches techniques de santé et de sécurité accompagnant tout produit
chimique utilisé. Respecter les procédures de laboratoire généralement acceptées pour la
manipulation en toute sécurité des produits chimiques.
5. Si l'utilisateur suspecte qu'un problème quelconque puisse mettre en cause la sécurité,
l’appareil doit être rendu inopérant en empêchant son utilisation. Communiquer la défaillance
constatée au service de maintenance compétent.
3
Bitte lesen Sie diese Hinweise vor Installation oder Gebrauch dieser Ausrüstung sorgfältig
durch.
1. Das in diesem Handbuch beschriebene Gerät darf nur von geschultem Personal bedient
werden. Alle Anpassungen, Wartungsarbeiten und Reparaturen müssen entsprechend der
Vorgaben in diesem Handbuch und von einer kompetenten Person, die mit den damit
verbundenen Gefahren vertraut ist, durchgeführt werden.
2. Es ist wichtig, dass sowohl das Bedienungs- als auch das Service-Personal zusätzlich zu den
detaillierten Anweisungen in diesem Handbuch ein sicheres Arbeitssystem einsetzen.
3. Mit Ausnahme der Teile, deren Wartungsverfahren in diesem Handbuch beschrieben sind,
enthält dieses Gerät keine weiteren Teile, die vom Benutzer gewartet werden können. Das
Entfernen von Abdeckungen und Versuche von hierfür unqualifiziertem Personal,
Anpassungen oder Wartungsarbeiten durchzuführen, haben zur Folge, dass die Garantie
verfällt und können zusätzliche Reparaturkosten auslösen.
4. Es ist jederzeit auf die sicherheitsrelevanten Daten sämtlicher verwendeter Chemikalien
Bezug zu nehmen. Allgemein anerkannte Labormethoden zum sicheren Umgang mit
Chemikalien sollten eingesetzt werden.
5. Besteht der Verdacht, dass die Sicherheitsvorrichtungen in irgendeiner Weise beschädigt
wurden, muss das Gerät außer Betrieb genommen und gegen weiteren Gebrauch gesichert
werden. Die Störung sollte der zuständigen Serviceeinrichtung unverzüglich gemeldet
werden.
Leggere attentamente queste istruzioni prima di installare o utilizzare il dispositivo.
1. L'unità descritta nel presente manuale è stata realizzata per essere utilizzata solo da
personale che ha ricevuto l'apposita formazione. Qualsiasi operazione di regolazione,
manutenzione e riparazione deve essere effettuata sulla base di quanto indicato nel presente
manuale da personale qualificato consapevole dei rischi connessi.
2. È fondamentale che il personale operativo e il personale addetto alla manutenzione utilizzino
un sistema di lavoro sicuro, oltre a seguire le istruzioni specificate nel presente manuale.
3. Oltre a quelli indicati nelle procedure di manutenzione, all'interno di questo dispositivo non
sono presenti altri elementi sui quali è possibile effettuare interventi. La rimozione delle
protezioni e qualsiasi tentativo di regolazione o di manutenzione posto in essere da
personale non qualificato invaliderà la garanzia. In questi casi, sarà necessario pagare un
importo per le riparazioni effettuate.
4
4. È sempre necessario fare riferimento ai dati sulla salute e sulla sicurezza forniti con le
sostanze chimiche utilizzate. Adottare le procedure di laboratorio generalmente accettate per
la gestione delle sostanze chimiche.
5. Nel caso in cui si sospetti che la salute possa essere pregiudicata in qualsiasi modo,
disattivare l'unità per renderla inutilizzabile. Qualsiasi condizione di errore deve essere
immediatamente segnalata al responsabile per la manutenzione.
Lea esta información atentamente antes de instalar o utilizar este equipo.
1. La unidad descrita en este manual está diseñada para que solamente la utilice personal con
formación. Cualquier operación de ajuste, mantenimiento y reparación debe llevarse a cabo
del modo indicado en este manual y debe realizarla una persona cualificada que sea
consciente de los peligros que implica.
2. Es fundamental que tanto los operarios como el personal de servicio utilicen un sistema de
trabajo seguro, así como las instrucciones detalladas que se especifican en este manual.
3. Cualquier elemento que no se encuentre entre los definidos en los procedimientos de
mantenimiento aquí descritos no podrá utilizarse en este instrumento. La extracción de las
tapas y los intentos de ajuste o reparación por parte de personal no cualificado invalidarán la
garantía y pueden incurrir en cargos adicionales por reparación.
4. Siempre deberían consultarse los datos sobre Salud y Seguridad que se suministran con
cualquier producto químico que se utilice. Es necesario llevar a cabo los procedimientos de
laboratorio de aceptación generalizada para la manipulación segura de productos químicos.
5. Si existe la sospecha de que las medidas protectoras de seguridad han quedado dañadas en
cualquier modo, la unidad debe inutilizarse y protegerse contra toda operación que se intente
llevar a cabo. El estado de fallo debe comunicarse inmediatamente a la autoridad de servicio
de mantenimiento y reparación pertinente.
5
Contents
SECTION 1 – Introduction .......................................................................................... 8
1.1 INSTRUMENT DESCRIPTION............................................................................................. 8
1.2 INSTRUMENT SPECIFICATION.......................................................................................... 8
SECTION 2 – Installation ........................................................................................... 9
2.1 UNPACKING......................................................................................................................... 9
2.2 INSTALLATION .................................................................................................................... 9
2.3 DISPLAY ............................................................................................................................. 10
2.4 CONTROLS ........................................................................................................................ 11
2.5 REAR PANEL ..................................................................................................................... 13
2.6 FRONT VIEW ..................................................................................................................... 13
SECTION 3 – THEORY AND PRACTICE OF SPECTROSCOPY MEASUREMENTS
................................................................................................................................ . 14
3.1 THEORY OF SPECTROSCOPY MEASUREMENT .......................................................... 14
3.2 NUCLEIC ACID DETERMINATION ................................................................................... 15
3.3 SPECTROSCOPY MEASUREMENT ................................................................................. 15
3.4 GOOD PRACTICE GUIDELINES ....................................................................................... 16
SECTION 4 – INSTRUMENT SETUP ...................................................................... 18
4.1 INSTRUMENT NAVIGATION ............................................................................................. 18
4.2 MAIN MENU SCREEN ....................................................................................................... 18
4.2.1 Slit Width Setting ................................................................................................................ 18
4.3 SYSTEM UTILITY MENU ................................................................................................... 19
4.3.1 Wavelength Reset .............................................................................................................. 19
4.3.2 Printer Setup ....................................................................................................................... 19
4.3.3 Lamp Setup......................................................................................................................... 20
4.3.3.1 Change the Lamp Switch Point .......................................................................................... 20
4.3.4 Clock Setup......................................................................................................................... 20
4.3.4.1 Set Time .............................................................................................................................. 20
4.3.4.2 Set Date .............................................................................................................................. 21
4.3.4.3 Display Time / Date ............................................................................................................ 21
4.3.5 Refresh Dark Current .......................................................................................................... 21
4.3.6 Connect to PC .................................................................................................................... 21
4.3.7 Beeper On/Off ..................................................................................................................... 21
4.3.8 Language Selection ............................................................................................................ 21
4.3.9 Refresh System Baseline ................................................................................................... 21
4.3.10 Delete Entire Saved Files ................................................................................................... 22
4.3.11 Restore Default Settings ..................................................................................................... 22
SECTION 5 – PHOTOMETRICS ............................................................................. 23
5.1 PHOTOMETRICS MENU SCREEN ................................................................................... 23
5.2 METHOD SET UP .............................................................................................................. 23
5.2.1 Selecting a Wavelength ...................................................................................................... 23
5.2.2 Selecting the Measurement Mode ...................................................................................... 23
5.2.3 Selecting the Unit of Measurement .................................................................................... 24
5.2.4 Entering a Concentration Factor ......................................................................................... 24
5.3 CALIBRATION .................................................................................................................... 24
5.3.1 Zero Calibration .................................................................................................................. 24
5.3.2 Calibrating to a Standard .................................................................................................... 25
6
5.4 SAMPLE MEASUREMENT ................................................................................................ 25
5.4.1 Photometric Measurements ................................................................................................ 25
5.4.2 Measuring a Sample after Entering a Concentration Factor .............................................. 25
5.4.3 Measuring a Sample after Calibrating to a Standard ......................................................... 26
SECTION 6 – QUANTITATION ................................................................................ 27
6.1 QUANTITATION MODE SCREEN ..................................................................................... 27
6.2 WAVELENGTH SELECTION ............................................................................................. 27
6.3 METHOD SETUP ............................................................................................................... 27
6.3.1 Selecting the Unit of Measurement .................................................................................... 28
6.3.2 Quantitation Table Settings ................................................................................................ 28
6.3.2.1 Select Curve Fit .................................................................................................................. 28
6.3.2.2 Manually Enter Curve Constants ........................................................................................ 29
6.3.2.3 Edit Calibration Table ......................................................................................................... 29
6.3.2.4 Display Calibration Curve ................................................................................................... 29
6.4 CREATING A NEW STANDARD CURVE .......................................................................... 30
6.5 STORING A STANDARD CURVE ..................................................................................... 31
6.6 RECALLING A STORED STANDARD CURVE ................................................................. 31
6.7 SAMPLE MEASUREMENTS .............................................................................................. 31
6.7.1 Quantitation Measurements................................................................................................ 32
6.8 STORING RESULTS .......................................................................................................... 32
6.9 RECALLING STORED RESULTS ...................................................................................... 32
SECTION 7 – SPECTRUM ...................................................................................... 33
7.1 SPECTRUM MODE SCREEN ............................................................................................ 33
7.2 METHOD SETUP ............................................................................................................... 33
7.3 SELECTING THE MEASUREMENT MODE ...................................................................... 34
7.4 SAMPLE MEASUREMENTS .............................................................................................. 34
7.5 ADJUSTING THE DISPLAYED SCAN RANGE ................................................................. 34
7.6 SPECTRUM SEARCH ........................................................................................................ 35
7.7 SPECTRUM SMOOTHING ................................................................................................ 35
7.8 STORING RESULTS .......................................................................................................... 35
7.9 RECALLING STORED RESULTS ...................................................................................... 36
SECTION 8 – KINETICS ................................................................ .......................... 37
8.1 KINETICS MODE SCREEN ............................................................................................... 37
8.2 WAVELENGTH SELECTION ............................................................................................. 37
8.3 METHOD SETUP ............................................................................................................... 37
8.3.1 Selecting the Measurement Mode ...................................................................................... 38
8.4 SAMPLE MEASUREMENTS .............................................................................................. 38
8.5 ADJUSTING THE DISPLAYED SCAN RANGE ................................................................. 38
8.6 CALCULATING I/U CONCENTRATION ............................................................................ 39
8.7 SPECTRUM SMOOTHING ................................................................................................ 39
8.8 STORING RESULTS .......................................................................................................... 40
8.9 RECALLING STORED RESULTS ...................................................................................... 40
SECTION 9 – DNA/PROTEIN .................................................................................. 41
9.1 DNA MENU OPTIONS ....................................................................................................... 41
9.2 ADJUSTING THE MEASUREMENT MODE ...................................................................... 41
9.3 WAVELENGTH SELECTION ............................................................................................. 42
9.4 ADJUSTING THE CONCENTRATION CALCULATION FACTORS .................................. 42
9.5 SELECTING THE UNIT OF MEASUREMENT ................................................................... 42
9.6 RESET MODE SETTINGS ................................................................................................. 43
9.7 DNA/PROTEIN MEASUREMENTS .................................................................................... 43
7
9.8 STORING METHODS/RESULTS ....................................................................................... 43
9.9 RECALLING STORED RESULTS ...................................................................................... 44
SECTION 10 – MULTI-WAVELENGTH ................................................................... 45
10.1 MULTI-WAVELENGTH MODE OPTIONS ......................................................................... 45
10.2 WAVELENGTH SELECTION ............................................................................................. 45
10.3 SAMPLE MEASUREMENT ................................................................................................ 46
10.4 STORING METHODS/RESULTS ....................................................................................... 46
10.5 RECALLING STORED RESULTS ...................................................................................... 46
SECTION 11 – SAVING/RECALLING RESULTS USING A USB MEMORY STICK 47
SECTION 12 – ACCESSORIES AND SPARE PARTS ............................................ 48
11.1 OPTIONAL ACCESSORIES............................................................................................... 48
11.2 INSTALLING THE PASSIVE AND ACTIVE ACCESSORIES ............................................ 48
11.2.1 Passive Accessories ........................................................................................................... 48
11.2.2 Active Accessories .............................................................................................................. 49
11.2.2.1 8 Position Automatic Cell Changer ................................................................................ 49
11.3 USING THE 8 POSITON AUTOMATIC CELL CHANGER ................................................ 50
11.4 SPARES ............................................................................................................................. 51
SECTION 12 – MAINTENANCE AND SETVICE ..................................................... 52
12.1 ROUTINE MAINTENANCE ................................................................................................ 52
12.2 LAMP REPLACEMENT ...................................................................................................... 52
12.2.1 Tungsten Lamp Replacement............................................................................................. 52
12.2.2 Deuterium Lamp Replacement ........................................................................................... 53
12.3 SERVICE ............................................................................................................................ 54
SECTION 13 – TROUBLESHOOTING .................................................................... 55
13.1 TROUBLESHOOTING GUIDE ........................................................................................... 55
13.2 TECHNICAL SUPPORT ..................................................................................................... 55
SECTION 14 – DECLARATION OF CONFORMITY ................................................ 56
8
SECTION 1 – Introduction
6850
Wavelength
Range
190 to 1100nm
Resolution
0.1nm
Accuracy
± 0.3nm (at 0.5 and 1nm bandwidth) ± 0.5nm (at 2, 4 and 5nm
bandwidth)
Repeatability
± 0.2nm
Spectral bandwidth
Variable 0.5 / 1 / 2 / 4 and 5nm
Photometrics
Transmittance
0 to 200%
Absorbance
-0.3 to 3.0A
Accuracy
±0.3%T (0 – 100%T), ±0.002A (0 – 0.5A)
Reproducibility
±0.001 Abs (0 to 0.5 Abs) ±0.002 Abs (0.5 to 1.0 Abs)
Resolution
0.1%T, 0.001A
Stray light
<0.05% at 360nm and 220nm
Noise
0.0005A
Stability
±0.001A at 500nm after 15min warm up
Quantitation
Range
0 to 99999
Data points
Up to 3 wavelengths
Calibration
Blank with up to 10 standards
Units
IU, mM/L, M/L, μg/mL, mg/mL, mg/L, mEq, ppb, ppm, % and other
Curve fit algorithms
Linear, Linear through zero, Quadratic and Cubic
Multi-wavelength
Range
0 to 99999
Data points
Up to 10 wavelengths
Kinetics
Measurement Time
Up to 12 hours
Scan Interval
0.1, 0.2, 0.5, 1, 2, 5, 10 or 30 seconds
Calibration
Blank with a single standard or factor
Display
Graphical and concentration
Analysis
Slope and line of best fit between any two points
Spectrum
Range
Any range between 190 to 1100nm
Scan speed
100 to 2000nm/min
Scan interval
Selectable 0.1, 0.2, 0.5, 1, 2 or 5nm
DNA
Measurement
modes
DNA/RNA Ratio, concentration, A320 correction
1.1 INSTRUMENT DESCRIPTION
The 6850 is a variable bandwidth, double beam UV/visible spectrophotometer with an integrated user
interface for local control. This instrument has highly stable optics and two detectors that measure the
sample and reference solutions simultaneously to optimise the accuracy of the measurement. The
6850 has measurement modes for photometrics, concentration, multi-wavelength, spectrum
scanning, quantitation, kinetics and DNA and Protein analysis. The instrument is supplied with
Jenway Prism PC software to allow full PC control of the spectrophotometer.
1.2 INSTRUMENT SPECIFICATION
9
Protein
Measurement
modes
Direct UV
Other
Beam height
15mm
Light source
Tungsten and Deuterium lamps
Outputs
USB, Parallel (Printer)
Power
110/220V AC 50/60Hz
Size (w x d x h)
600 x 450 x 260mm
Weight
22kg
SECTION 2 – Installation
2.1 UNPACKING
Remove the 6850 from the packaging and ensure the following items are included:
1. Model 6850 spectrophotometer fitted with 10 x 10mm cuvette holder (685-SC)
2. Power supply cables HH179(S) – UK lead
HH180(S) – EU lead
CABLEUS – US lead
3. Instruction manual (685 030)
4. 10mm Glass Cuvettes x 4 (035 027)
5. 10mm Quartz Cuvettes x 2 (035 028)
6. Dust Cover (685 040)
7. PC software CD and USB security dongle (685 035)
2.2 INSTALLATION
The 6850 is supplied ready to use.
The working temperature and humidity range of the 6850 spectrophotometer is 15 – 35˚C, 15 – 70%
relative humidity. The storage temperature and humidity range of the 6850 spectrophotometer is -10
– 50˚C, 15 – 70% relative humidity.
The unit should be placed on a clean flat surface which is free from drafts and vibrations. The units
are designed for operation on 110V±11V / 60Hz±1Hz and 220V±22V / 50Hz±1Hz AC.
Connect the power supply unit to the power inlet socket on the rear panel of the instrument and
connect to the mains socket. Turn the power on at the mains and switch the instrument on using the
power switch on the rear of the instrument.
10
The instrument will initially perform several power-on tests before displaying the warm up screen:
Main menu options
1. Photometrics measurement mode
5. DNA/Protein measurement mode
2. Quantitation measurement mode
6. Multi-wavelength measurement mode
3. Spectrum measurement mode
7. Instrument settings menu
4. Kinetics measurement mode
8. Slit width
Fig 2.2 – Instrument power-on tests complete
The power-on tests comprise
1. Lamp test
2. Detector test
3. A to D converter test
4. Grating position check
5. Dark current check
2.3 DISPLAY
The instrument has an inclusive display which enables text and graphs to be displayed clearly.
Following successful completion of the power-on tests and the instrument warm up time, the main
menu screen will be displayed:
Fig. 2.3 – Display
11
2.4 CONTROLS
Function Keys: Allows on-screen options to be selected
…
Numeric Keys: Enter numbers and letters. Select
corresponding menu options.
CLEAR Key: Delete the entered value or stored data
BACK Key: Delete a single character
The keypad used for this model enables an easy and effective way of navigating the different
measurement modes, entering numbers, saving and analysing results. The keypad is
displayed below.
Fig. 2.4 – Keypad
12
ESC Key: Return to previous menu screen
100%T/0Abs Key: Zero / Blank instrument
OPEN Key: Open results stored in internal memory
SAVE Key: Save results to internal memory
START/STOP Key: Start/Stop measurement
GOTO Key: Set the instrument wavelength using the numeric
keys. Press the Enter key to confirm.
PRINT Key: Print result
ENTER Key: Confirm operation
CELL Key: Select/Deselect Auto-cell Holder position (If fitted).
Use the numeric keys (1-8) to move to cell holder to the
corresponding cell position
,
RIGHT, LEFT Keys: Search peak/valley and set X axis scale
,
UP, DOWN Keys: Scroll menu/data and set Y axis scale
13
2.5 REAR PANEL
The image below shows the rear panel of the instrument:
Fig. 2.5 – Rear Panel
1. Fan Cover Allows access to lamp when replacement is necessary
2. Power switch On/off switch for the unit
3. Power in socket Connection socket for power supply unit
4. LCD contrast adjustment Connection to a PC or external serial printer
5. Parallel port Allows the accessory printer to be connected
6. USB port Allows the instrument to be connected to a PC
2.6 FRONT VIEW
The image below shows the front view of the instrument:
Fig. 2.6 – Front Panel
1. LCD display
2. Keypad
3. Sample chamber lid
4. Sample chamber
14
SECTION 3 – THEORY AND PRACTICE OF SPECTROSCOPY MEASUREMENTS
l
I
o
I
t
Where:
Io is the incident light
lt is the transmitted light
l is the path length
3.1 THEORY OF SPECTROSCOPY MEASUREMENT
UV-visible spectroscopy is the measurement of the absorbance of light at a specific wavelength in a
sample. This is used to identify the presence and concentration of molecular entities within the
sample. The Beer-Lambert law is used to relate the absorption of light to the properties of the sample
through which the light is travelling through. The Beer-Lambert law states that:
A is the absorbance
is the molar absorption coefficient (l mol-1cm-1)
c is the concentration (mol l-1)
l is the path length (cm)
This law shows that absorbance is linear to concentration but this is only true for low concentrations.
For absorbance levels above 3 the concentration starts to move away from the linear relationship.
Transmittance is the proportion of the light which passes through the sample:
Therefore:
I
Absorbance is inversely related to transmittance:
T
T = It
A = log 1
o
15
3.2 NUCLEIC ACID DETERMINATION
DNA, RNA and oligonucleotides can be measured directly in aqueous solutions in a diluted or
undiluted form. Aqueous buffers with low ion concentrations (e.g. TE buffer) are ideal for this method.
The concentration is commonly determined by measuring at 260nm against a blank and then
evaluating against a factor.
The 6850 has pre-defined methods installed which assume that absorption of 1 OD (A) is equivalent
to, approximately: 50μg/ml dsDNA, 37μg/ml ssDNA, 40μg/ml RNA and 30μg/ml for oligonucleotides.
DNA interference by contaminants can be assessed by the calculation of an absorption ratio. The
ratios A260/A280 and A260/A230 are used to estimate the purity of nucleic acids, since proteins
absorb at 280nm and substances such as peptides, phenols, aromatic compounds or carbohydrates
absorb at 230nm. Pure DNA should have an A260/A280 ratio of approximately 1.8 and pure RNA 2.0.
In pure nucleic acid samples the A260/A230 ratio should be approximately 2.2.
Nucleic acid concentration can also be estimated with the following calculations:
Conc (μg/ml) = (Abs@260nm x 62.9) – (Abs@280nm x 36.0)
Conc (μg/ml) = (Abs@260nm x 49.1) – (Abs@230nm x 3.48)
Referring to a blank value where no absorption should occur is commonly required. On the 6850 the
default reference wavelength is 320nm and the user can include the measured absorbance value in
all nucleic acid calculations. The default wavelength can be modified from 320nm if required.
3.3 SPECTROSCOPY MEASUREMENT
There are four main components of a spectrophotometer. These are a light source to emit a high and
constant amount of energy over the full wavelength range; a method for separating the light into
discreet wavelengths; a sample holder and a light detector.
The optical layout of the 6850 spectrophotometer is shown overleaf:
The light from the tungsten and deuterium lamps is focused onto the grating which separates the light
into discreet wavelengths. The diffracted spectrum of light then passes through a further slit and lens
arrangement before passing through a beam splitter which directs half of the light towards to sample
holder and half towards the reference sample holder. The light which is not absorbed by the two
solutions is transmitted through a collecting lens and onto the signal detector. The signal from each
photo-diode detector is used to calculate the % transmittance. The result is displayed either as %
transmittance or absorbance on the instrument display.
16
3.4 GOOD PRACTICE GUIDELINES
1. For optimum performance all spectrophotometers should be sited in a clean, dry, dust
free atmosphere. When in use ambient temperature and light levels should remain as
constant as possible.
2. If required adherence to Standard Operating Procedures (SOP) and Good Laboratory
Practice (GLP) should be monitored with regular calibration checks and a suitable Quality
Control (QC) programme.
3. The sample chamber lid must be fully closed during measurement and before any
readings are recorded or printed.
4. The correct selection of sample containers is imperative for accurate and reproducible
results:
a) Check that the material of the sample container is compatible with the wavelengths to
be used for measurement. In general glass can only be used down to 360nm or
320nm depending on quality. Standard plastic cuvettes can be used down to 320nm.
Special UV versions can be used down to 260nm. Below this level quartz cuvettes
must be used.
b) Plastic disposable cuvettes should only be used ONCE.
c) Glass cuvettes should be thoroughly cleaned after use. Discard when scratches
become evident on optical surfaces.
d) Care should be taken when selecting semi-micro or micro cuvettes. The cuvette
window on the inner chamber (the area filled with sample) must be wider than the
aperture in the sample holder or light will reach the detector without passing through
the sample. In this case, semi-micro or micro cuvettes with self-screening black
surrounds must be used or, alternative holders for these cuvettes should be used.
e) Glass test tubes and other sample tubes should be used with care. Where possible,
matched tubes should be used and any index mark set to the correct position before
measurements are made.
f) Ensure any sample containers used are compatible with the constituents of both the
samples and standards they are to hold. Plastic cuvettes are not compatible with
organic solvents.
g) All sample containers must be handled with care; by the top, bottom and non-optical
surfaces only. Any finger marks evident must be removed by a suitable cleaning
process.
17
h) Flow-through cuvettes must be selected with care and consideration for the sample
type, sample volume, pumping system, rinse, sample and waste handling to be used.
5. Samples and standards should not be stored in open cuvettes or sample containers as
evaporation will change the value and lead to staining of the walls which may be
irreversible. If stored in stoppered and sealed cuvettes, they should be filled with little or
no air space and the values regularly checked against a reference standard or quality
control material.
6. Samples should be allowed to equilibrate to ambient temperature before measurement
(unless a suitable temperature controlled sample holder is in use). Temperature change
during measurement may cause air bubbles to form on the walls of the sample holder.
This is a common cause of drift during measurement.
7. In the preparation of samples and standards high grade borosilicate glass and AR grade
chemicals and reagents must be used. Good quality deionised water or other suitable
solvents must be used for dissolving or diluting samples, chemicals and reagents.
8. All measurements require calibration to a blank, for maximum accuracy this should be
prepared with care using the same deionised water or solvent used for dissolving or
diluting the sample. Where reagents are added to the sample to produce a colour
proportional to its concentration a ‘sample based’ blank should be used. In this case the
blank should consist of all reagents or chemicals to be used, except the sample which
will produce the colour to be measured.
9. Deviations from the Beer-Lambert Law may occur at high and low concentrations giving
non-linear response during sample concentration measurements. For all new methods a
linear range should be defined by the preparation of a calibration curve. The quantitation
mode may be used to construct such a curve against which sample results are
automatically measured.
10. Cuvettes and sample holders must be filled to a minimum level which covers the light
path. All Jenway spectrophotometers have a beam height of 15mm.
11. The instrument must be calibrated to zero absorbance/100% transmittance prior to taking
readings. In the spectrum measurement mode a baseline scan must be performed before
performing a sample scan.
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