6850 Spectrophotometer Operating Manual 685 030 REV B/06-14
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.
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.
. 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.
Contents SECTION 1 – Introduction.......................................................................................... 8 1.1 1.2 INSTRUMENT DESCRIPTION............................................................................................. 8 INSTRUMENT SPECIFICATION.......................................................................................... 8 SECTION 2 – Installation ........................................................................................... 9 2.1 2.2 2.3 2.4 2.5 2.
5.4 5.4.1 5.4.2 5.4.3 SAMPLE MEASUREMENT ................................................................................................ 25 Photometric Measurements ................................................................................................ 25 Measuring a Sample after Entering a Concentration Factor .............................................. 25 Measuring a Sample after Calibrating to a Standard .........................................................
9.8 9.9 STORING METHODS/RESULTS ....................................................................................... 43 RECALLING STORED RESULTS ...................................................................................... 44 SECTION 10 – MULTI-WAVELENGTH ................................................................... 45 10.1 10.2 10.3 10.4 10.5 MULTI-WAVELENGTH MODE OPTIONS ......................................................................... 45 WAVELENGTH SELECTION .................
SECTION 1 – Introduction 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.
Protein Measurement modes Other Beam height Light source Outputs Power Size (w x d x h) Weight Direct UV 15mm Tungsten and Deuterium lamps USB, Parallel (Printer) 110/220V AC 50/60Hz 600 x 450 x 260mm 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.
The instrument will initially perform several power-on tests before displaying the warm up screen: 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.
2.4 CONTROLS 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 Function Keys: Allows on-screen options to be selected … Numeric Keys: Enter numbers and letters. Select corresponding menu options.
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).
2.5 REAR PANEL The image below shows the rear panel of the instrument: Fig. 2.5 – Rear Panel 2.6 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.
SECTION 3 – THEORY AND PRACTICE OF SPECTROSCOPY MEASUREMENTS 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.
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.
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.
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.
SECTION 4 – INSTRUMENT SETUP 4.1 INSTRUMENT NAVIGATION To navigate around the spectrophotometer screen press the arrow keys on the main keypad and use the Enter key to select the highlighted option. Alternatively, some options and actions require the user to press the function and numeric keys on the instrument keypad. The Esc key can be used to return to the previous menu without saving any changes. 4.
4.3 SYSTEM UTILITY MENU The system utility menu option allows the user to perform a variety of setup and service functions. System Utility Menu 4.3.1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.
4.3.3 Lamp Setup In the lamp setup menu the user can perform the following tasks. 1. Switch on/off the deuterium lamp 2. Reset the deuterium lamp usage timer 3. Switch on/off the tungsten lamp 4. Reset the tungsten lamp usage timer 5. Change the lamp switch point. Highlight the required option using the up and down arrow keys. Press the Enter key to confirm. 4.3.3.1 Change the Lamp Switch Point Highlight the Change the lamp switch point option and press the Enter key to confirm.
4.3.4.2 Set Date Highlight the set date option and press the Enter key to confirm. This will allow the user to enter the current date in the format DD.MM.YY using the numerical keypad and the decimal point keys. Press the Enter key to confirm. Once complete the instrument will return to the Clock Setup menu. 4.3.4.3 Display Time / Date Highlight the display time or display date option and press the Enter key to confirm. This will set the top right hand corner to show the selected option. 4.3.
4.3.10 Delete Entire Saved Files Highlight the delete entire saved files option and press the Enter key to confirm. A warning will be shown on screen and the user will need to confirm this command again by selecting the Yes option with the up and down arrow keys. This action will delete all saved results and methods from the instrument. 4.3.11 Restore Default Settings Highlight the restore default settings option and press the Enter key to confirm.
SECTION 5 – PHOTOMETRICS The photometrics measurement mode enables simple measurements of absorbance, % transmittance and concentration to be performed. In this measurement mode it is possible to calibrate against a standard of a known concentration or use a known factor. The sample is measured at one wavelength at one point in time. There are no post measurement calculations available in this measurement mode. 5.
5.2.3 Selecting the Unit of Measurement The unit of measurement can be selected by pressing the function key below the Unit option. The required unit of measurement can then be selected from the available options of IU, mM/L, M/L, μg/mL, mg/mL, mg/L, mEq, ppb, ppm, % and other. Press the Enter key to confirm. 5.2.4 Entering a Concentration Factor The concentration of a sample can be determined by using an appropriate multiplication factor with the measured photometric reading.
5.3.2 Calibrating to a Standard A calibration standard can be used to determine the concentration factor. Insert the cuvette containing the blank solution into the reference position in the sample chamber and insert the cuvette containing the standard solution into the sample position. Close the instrument lid. The concentration of the calibration standard is entered by pressing the function button below the Standard option.
5.4.3 Measuring a Sample after Calibrating to a Standard Remove the cuvette containing the standard sample and place a cuvette containing the sample to be measured in the sample chamber. Close the instrument lid and the calculated concentration value will be shown on the screen.
SECTION 6 – QUANTITATION The quantitation measurement mode enables sample concentrations to be calculated using a standard curve. In this mode a number of standard solutions covering a range of known concentrations are measured. The absorbance or % transmittance of these solutions is plotted to create a standard curve. Once the standard curve has been created a sample of unknown concentration can be measured and the concentration calculated using the standard curve. 6.
6.3.1 Selecting the Unit of Measurement The unit of measurement can be selected by pressing the function key below the Unit option. The required unit of measurement can then be selected from the available options of IU, mM/L, M/L, μg/mL, mg/mL, mg/L, mEq, ppb, ppm, % and other. Press the Enter key to confirm. 6.3.2 Quantitation Table Settings The quantitation table settings are accessed by pressing the function key below the Curve Fit option in the quantitation mode screen.
6.3.2.2 Manually Enter Curve Constants The user can manually enter the calibration curve constants by pressing the function key below the Params option. The user will be prompted to enter the constants that are required for the selected curve fit algorithm. 1. Linear Fit = K0 + (K1 x Abs) 2 2. Square Fit = K0 + (K1 x Abs) + (K2 x Abs ) 2 3. Cubic = K0 + (K1 x Abs) + (K2 x Abs ) + (K3 x 3 Abs ) 4. Linear Fit Through Zero = K1 x Abs The values can be entered using the numerical keypad.
6.4 CREATING A NEW STANDARD CURVE The user must firstly edit the calibration table before attempting to create a new standard curve. Insert the cuvette containing the blank solution into the reference position in the sample chamber and insert the cuvette containing the first standard solution into the sample position. Close the instrument lid and press the start/stop button. Once the measurement is complete the instrument will ask for the second standard solution to be inserted.
6.5 STORING A STANDARD CURVE A standard curve can be saved to the instrument’s internal memory to allow it to be recalled for future use. In the calibration table screen press the Save key on the instrument keypad. The instrument will ask the user to enter a filename (max. 8 characters) using the alphanumeric keypad.
6.7.1 Quantitation Measurements Ensure that a cuvette containing the blank solution is in the reference position in the sample chamber and insert the cuvette containing the sample solution into the sample position. Close the instrument lid and pres the Start/Stop key on the keypad. Once the measurement is complete the determined result will be displayed in the quantitation mode screen. Further samples can be measured in a similar manner. 6.
SECTION 7 – SPECTRUM The spectrum measurement mode enables measurements of absorbance or % transmittance over a range of wavelengths to be performed. The absorbance or % transmittance at each wavelength is plotted graphically. Post measurement tools such as peaks and spectral points analysis can be performed. This operating mode can be used to partially characterise a sample. 7.
7.3 SELECTING THE MEASUREMENT MODE The measurement mode can be selected by pressing the function key below the Mode option. The required measurement mode can then be selected from the available options of Abs and %T using the up and down arrow keys. Press the Enter key to confirm. 7.4 SAMPLE MEASUREMENTS Insert a cuvette containing the blank solution into the reference position in the sample chamber and insert the cuvette containing the sample solution into the sample position.
7.6 SPECTRUM SEARCH The photometric values of the measured spectrum can be viewed by pressing the function key below the Search option. The user can press the up and down arrow keys to move between the peaks in the scan that are above the threshold peak height. The user can alternatively press the left or right arrow keys to review each point in the scan in turn. The peak height threshold can be adjusted by pressing the function key below the PK Height option.
7.9 RECALLING STORED RESULTS A result file can be opened from the instrument’s internal memory. Press the Open key on the instrument keypad. The user can select from the list of stored files by pressing the up and down arrow keys. Once the required file has been located press the Enter key to confirm.
SECTION 8 – KINETICS The kinetics measurement mode enables the absorbance or % transmittance of an active molecule to be measured over a period of time; for example enzyme analysis of horseradish peroxidase. The absorbance or % transmittance is measured at regular time intervals at a set wavelength over a period of time. The results are plotted on a graph to show the change in absorbance or % transmittance over time.
8.3.1 Selecting the Measurement Mode The measurement mode can be selected by pressing the function key below the Mode option. The required measurement mode can then be selected from the available options of Abs and %T using the up and down arrow keys. Press the Enter key to confirm. 8.4 SAMPLE MEASUREMENTS Insert a cuvette containing the blank solution into the reference position in the sample chamber and insert the cuvette containing the sample solution into the sample position.
8.6 CALCULATING I/U CONCENTRATION The instrument software allows the user to calculate the concentration of a substance from the difference between the photometric values at two time points in the displayed kinetics scan. To enter the calculation parameters press the function key below the Process option and enter a Start Time, an End Time and a Factor, pressing the Enter key to confirm each value.
8.8 STORING RESULTS A result file can be saved to the instrument’s internal memory. Press the Save key on the instrument keypad. The instrument will ask the user to enter a filename (max. 8 characters) using the alpha-numeric keypad. The user can select the indicated letters, numbers and symbols by repeatedly pressing the key until the required value is displayed, after a short period the next character can be entered in the same manner. Once the filename has been entered, press the Enter key to confirm.
SECTION 9 – DNA/PROTEIN The DNA/Protein measurement mode allows the user to measure multi-wavelength absorbance ratios, such as 260nm/280nm and 260nm/230nm, which are commonly used to estimate a protein or nucleic acid sample’s purity. The mode also includes calculations that can be used to estimate the concentration of the protein or nucleic acid sample. 9.1 DNA MENU OPTIONS The DNA/Protein measurement mode enables measurement parameters to be changed.
Absorbance Difference 2 Purity Ratio = Abs@260nm / Abs@230nm RNA Conc (μg/ml) = (Abs@260 x 49.1) – (Abs@230 x 3.48) Protein Conc (μg/ml) = (Abs@230 x 183) – (Abs@260 x 75.8) The option to include the third wavelength as a reference is given after the measurement mode is selected. Use the up and down arrow keys to select Yes or No and press the Enter key to confirm.
9.6 RESET MODE SETTINGS The DNA/Protein mode settings can be reset to their default values by pressing the function key below the Default option. 9.7 DNA/PROTEIN MEASUREMENTS Ensure that a cuvette containing the blank solution is in the reference position in the sample chamber and insert the cuvette containing the sample solution into the sample position. Close the instrument lid and pres the Start/Stop key on the keypad.
9.9 RECALLING STORED RESULTS A method/result file can be opened from the instrument’s internal memory. In the DNA/Protein mode screen press the Open key on the instrument keypad. The user can select from the list of stored files by pressing the up and down arrow keys. Once the required file has been located press the Enter key to confirm. If more than one result is stored in a file the up and down arrow keys can be pressed to scroll though the results.
SECTION 10 – MULTI-WAVELENGTH The multi-wavelength measurement mode allows the user to measure a sample’s photometric absorbance or %transmittance at up to 10 wavelengths. Following sample measurement, the photometric readings are displayed on the multi-wavelength mode screen. 10.1 MULTI-WAVELENGTH MODE OPTIONS The multi-wavelength measurement mode enables measurement parameters to be changed.
10.3 SAMPLE MEASUREMENT Insert a cuvette containing the sample to be analysed into the sample chamber and press the key below the sample measurement icon. The instrument will take a reading at each of the specified wavelengths the operating menu screen will then display the result of the selected measurement calculation and the photometric readings for each of the measured wavelengths. 10.4 STORING METHODS/RESULTS A method/result file can be saved to the instrument’s internal memory.
SECTION 11 – SAVING/RECALLING RESULTS USING A USB MEMORY STICK There is a USB port on the side of the instrument for data storage. The instrument only recognises a USB memory stick being inserted or removed when it is operating in the home screen. A USB icon appears at the top of the home screen (circled in red). Insert the USB memory stick when the instrument is in the home screen and enter the required measurement mode.
SECTION 12 – ACCESSORIES AND SPARE PARTS 11.1 OPTIONAL ACCESSORIES Part Code 685 204 685 131 685 005 685 304 685 401 035 088 035 091 060 422 035 143 685 035 685 040 11.
11.2.2 Active Accessories 11.2.2.1 8 Position Automatic Cell Changer Unscrew the two fixing screws to undo the passive accessory. Lift out the passive accessory. Fixing screws On the underside of the 8 cell accessory, locate the accessories power supply and communication connector. Accessory connector Take the 8 cell accessory and align the accessories connector with the instrument connection socket.
Place the accessory in the correct orientation and re-tighten the two fixing screws to fix the 8 cell accessory in place. Fixing screws 11.3 USING THE 8 POSITON AUTOMATIC CELL CHANGER When the automatic 8 cell turret is installed the current cell position is indicated below the lamp status icons in the top right hand corner of the screen. To activate the accessory press the Cell key on the instrument keypad. The indicated cell position will be highlighted on screen.
11.
SECTION 12 – MAINTENANCE AND SETVICE 12.1 ROUTINE MAINTENANCE Ensure the external surfaces of the unit are clean and free from dust. The sample area should always be kept clean and any accidental spillage should be wiped away immediately. To give added protection when not in use, the unit should be disconnected from the mains supply and covered with the optional dust cover. 12.2 LAMP REPLACEMENT Warning! HOT.
3. Whilst wearing cotton gloves remove the defective tungsten lamp and replace with a Tungsten Lamp new one. Entrance Window 4. Switch on the power and move the Switch Mirror down into the position shown in the Switch Mirror Entrance Hole picture opposite. Observing the entrance window on the instrument’s monochromator, adjust the tungsten lamp until the light is centred on the entrance hole. 5. Disconnect the power and re-attach the lamp compartment cover and the top case of the spectrophotometer.
12.3 SERVICE Our dedicated service team are on hand to help in the unlikely event that your Jenway equipment develops a fault. Please contact them by one of the following means with a clear description of the problem: E-mail: service@bibby-scientific.com Tel: +44 (0) 1785 810475 Fax: +44 (0) 1785 810471 On occasion it may be necessary for your equipment to be sent back to our Service Department for repair.
SECTION 13 – TROUBLESHOOTING 13.1 TROUBLESHOOTING GUIDE Issue Unable to achieve a reading when measuring a sample Solution Ensure the correct cuvettes are being used so that light isn’t being absorbed by the cuvette. Ensure the sample isn’t too dense that light is not transmitted through the sample. Ensure the lamp is working. 13.
SECTION 14 – DECLARATION OF CONFORMITY 56
