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Safety Information - Agilent Technologies

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1. CAUTION Organic and aqueous solvents can be hazardous if handled improperly Always follow the precautions listed below to protect both operator and instrument Flow Cells Avoid the use of alkaline solutions pH gt 9 5 which can attack quartz and thus impair the optical properties of flow cells Solvents Always filter solvents small particles can permanently block capillaries Avoid the use of the following steel corrosive solvents e Solutions of alkali halides and their respective acids for example lithium iodide potassium chloride and so on e High concentrations of inorganic acids like nitric acid sulfuric acid especially at higher temperatures replace if your analysis method allows by phosphoric acid or phosphate buffer which are less corrosive against stainless steel e Halogenated solvents or mixtures which form radicals and or acids for example 2CHCl O gt 2COCl 2HCl This reaction in which stainless steel probably acts as a catalyst occurs quickly with dried chloroform if the drying process removes the stabilizing alcohol e Analysis grade ethers which can contain peroxides for example THF dioxane di isopropylether such ethers should be filtered through dry aluminium oxide which adsorbs the peroxides Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide 13 1 Safety Information e Solutions of organic acids acetic acid formic acid and so on in organic solvents
2. transport can lead to measurements with poor reproducibility Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 57 4 Good Measurement Practices lt gt Quartz cells with transparent apertures fluorescence cells plastic cells Figure 6 Cells you should not use with the instrument Flow Cells We recommend a sipper system with a flow cell for obtaining high precision measurements Using a flow cell eliminates the necessity of moving the cell between blank measurement and sample measurement Also the cell can be rinsed thoroughly with the solution to be measured The design of the flow cell should minimize entrapment of bubbles and flow channeling to provide the most reliable results 58 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Good Measurement Practices 4 Handling and Maintaining Cells Passivating New Cells When filling a non passivated new cell with your sample you will observe that air bubbles stick on the windows of your cell To prevent the formation of sticky bubbles rinse the cell with cleaning and passivating fluid part number 5062 8529 The cleaning procedure is described on the label of the cleaning fluid container Cleaning Cells The fats in fingerprints are significant absorbers in the UV region and if left on optical surfaces can cause erroneous results Wipe off all fingerprints and contaminants before using a sample cell Use onl
3. wavelengths Use wavelength 273 nm Background correction pone Calibration Analyte name Caffeine Calibration curve type Enter Concentration Concentration ma L Unit Weight Volume Weight amp Volume ma aiL Unit V Prompt for standard information v Prompt for sample information Data type Display spectrum Absorbance x Erom 190 To 340 5 Click OK to set your parameters Now you are ready to run your measurements NOTE 4 y y 102 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Calibration 1 Fill your 1 cm path length quartz cell with distilled water Lift the lever at the left side of your cell holder Put the cell in the cell holder and make sure the transparent windows face towards the front and the back of the spectrophotometer Push the lever down to secure your cell in the cell holder 2 Press the Blank button on the front of the spectrophotometer or click Blank on the Instrument Panel to start the measurement Sampling Automation fl Standard Sample 3 Remember the orientation of the cell in the cell holder Lift the level to release the cell remove it and flush it three times with about 1 ml of your caffeine sample Then fill the cell with about 3 ml of the caffeine sample Make sure that the cell windows are clean and reposition the cell in the same orientation
4. CMChan ND Overlaid Sample Spectra ab Browie Analysis panel Data files Navigation bar DX AA Poaki Maleyt Modes Instrument panel Tabular results Spectra Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 33 2 Introduction to Your System Menu Eile Edit Method Measure Instrument Math View Mode ECM Config Help The more traditional menu interface at the top of the Agilent ChemStation window provides access to all operations When you choose an item from the menu bar a list of commands and submenus is displayed An operation is performed by choosing a command mouse click or ENTER key Toolbar Clear eS W Method Mode EN Ta Ss lhe WAVELENGTH ACCURACY TEST M Mod Standard gt Q 34 The tool bar below the menu bar provides direct access to basic operations such as printing result reports loading methods and saving methods and data Navigation Bar The navigation bar consists of mode switching bars and a file browsing tree view for saved files The navigation bar is dockable and can be hidden or resized The navigation bar window provides fast direct access to file system and mode switch functionality The size and position of the analysis and instrument panels are fixed but are a function of your display s resolution Files can be loaded from the navigation bar tree view by double clicking Clicking on the Browse section allows access to any
5. holder Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 6 Press the Blank button on the front of the spectrophotometer or click Blank on the Instrument Panel to start the measurement Sampling r 7 Remember the orientation of the cell in the cell holder Lift the level to release the cell remove it and flush it three times with about 1 ml of your caffeine sample Then fill the cell with about 3 ml of the caffeine sample Make sure that the cell windows are clean and reposition the cell in the same orientation as for the reference measurement Close the cell holder s level 8 Press the Sample button on the front of your spectrophotometer or click Sample on the Instrument Panel to start the measurement 9 The view shows you the spectrum of your caffeine sample with a vertical line indicating your wavelength of interest Below the spectrum is the Sample Result Table which shows the absorbance reading at 273 nm Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 75 5 Using your UV Visible Spectroscopy System Saving Your Parameters as a Method 76 This section describes how to save your settings for a future Agilent ChemStation session Simply by loading your method you adjust your Agilent ChemStation to repeat your measurement A library of methods facilitates routine laboratory work Let s assume that your current settings are
6. 24 layout 24 mechanical assemblies 24 warm up 71 instrument panel 35 instrument session 37 internal reference 40 43 IP address 50 51 52 70 isopropyl alcohol 61 L lamps 25 access through door 31 deuterium 24 tungsten 24 LAN crossover cable 50 LAN interface Jet Direct 50 Talk2Lab 50 lens 24 lens tissues 59 evels of operation 37 ight beam 56 59 ine power 51 input socket 31 switch 28 iquid samples 54 ogged 52 M main application window 37 maintenance 109 making measurements 54 manager level 38 72 maximum 39 measure standard 103 standard information 104 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual measure push buttons 29 measurement blank 70 noise 71 reference 70 sample 71 105 sample information 105 menu 34 message line 52 methanol 61 method 38 42 45 46 76 calibrated 47 100 current 79 information 79 last used 72 load 78 load method 78 modified 78 name 77 options amp information 77 parameter 42 parameters 76 print preview 80 printing 78 80 report 78 retrieve 78 save method as 76 store 76 methyl formate 61 minimum 39 MIO board slot 31 mode 37 advanced 38 color calculations 38 combined reports 38 dissolution testing 38 execute advanced method 38 kinetics 38 last used 72 multibath dissolution testing 38 standard 38 73 90 switch 38 thermal denaturation 38 verification and diagn
7. Figure 10 on page 66 show a good and a poor baseline Y Cell orientation of blank and sample measurements is the same V Ideally the cell is not removed between the measurements which means the cell is filled rinsed using a pipette or a flow cell is used V Time between blank and sample measurement should be short 0 0015 0 0010 0 0005 0 0000 0 0005 0 0010 200 400 600 800 1000 Figure 9 Example of a blank on water showing a good baseline Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 65 4 66 Good Measurement Practices 0 01 0 00 0 01 0 02 0 03 0 04 200 400 600 800 1000 Figure 10 Example of a blank on water with bubbles causing a poor baseline If your blank or spectra shows artifacts similar to those in Figure 10 see Solvents on page 61 to optimize the measurement procedure Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Inserting a Cell Good Measurement Practices 4 Your spectrophotometer is shipped with the standard single cell holder which you must install in the sample compartment This cell holder accommodates standard cells or flow cells To insert a sample cell in the cell holder 1 Move the locking lever to its up position 2 Insert the sample cell making sure you orient it correctly The frosted non clear sides of the sample cell should not be in the path of the light beam 3 Lock the sample cell in place by pushing the locki
8. For example a 1 solution of acetic acid in methanol will attack steel e Solutions containing strong complexing agents for example EDTA ethylene diamine tetra acetic acid e Mixtures of carbon tetrachloride with 2 propanol or THF 14 Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Safety Information 1 Other Precautions Use of the spectrometer system and accessories may involve materials solvents and solutions which are flammable corrosive toxic or otherwise hazardous Careless improper or unskilled use of such materials solvents and solutions can create explosion hazards chemical burn hazards fire hazards toxicity and other hazards that can result in death serious personal injury or damage to equipment Apply all necessary precautions including use of lab coats safety goggles and other appropriate forms of personal protection All wastes should be disposed of in accordance with local regulatory requirements Protective Earth Shock Hazard Connecting the Agilent Cary 8454 UV Vis to a power source that is not equipped with a protective earth contact creates s shock hazard for the operator and can damage the instrument Always operate your instrument from a power outlet which has a ground connection Disconnecting Device Shock Hazard To disconnect the instrument from line power disconnect the power cord from the supply The power supply still uses some power and a shock hazard remains insi
9. Peaks Pararneters Peak V alley find 2 peaks Find and annotate up to 3 valleys Prompt for sample information Data type Display spectrum Absorbance v Erom 190 nm To 400 nm 5 Fill your 1 cm path length quartz cell with distilled water Lift the lever at the left side of your cell holder Put the cell in the cell holder and make sure the transparent windows face towards the front and the back of the spectrophotometer Push the lever down to secure your cell in the cell holder Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 91 5 Using your UV Visible Spectroscopy System 6 Press the Blank button on the front of the spectrophotometer or click Blank on the Instrument Panel to start the measurement Sampling j 7 Remember the orientation of the cell in the cell holder Lift the level to release the cell remove it and flush it three times with about 1 ml of your caffeine sample Then fill the cell with about 3 ml of the caffeine sample Make sure that the cell windows are clean and reposition the cell in the same orientation as for the reference measurement Close the cell holder s level 8 Press the Sample button on the front of your spectrophotometer or click Sample on the Instrument Panel to start the measurement 92 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Finding the Caffeine Absorbance Maximu
10. System Operator s Manual Solvents Good Measurement Practices 4 Your choice of solvents should be based primarily on the solvent s absorbance characteristics over the wavelengths of interest its suitability as a solvent for the analyte and on experimental conditions Table 1 lists common solvents and the lower limit of their useful wavelength range Table 1 Lower limit of UV transmission for some common solvents Lower Limit Solvent 180 195 nm Sulfuric acid 96 200 210 nm 210 220 nm 245 260 nm 265 275 nm 280 290 nm Above 300 nm Water Acetonitrile Cyclopentane n Hexane Glycerol 2 2 4 Trimethylpentane Methanol n Butyl alcohol Isopropyl alcohol Cyclohexane Ethyl ether Chloroform Ethyl acetate Methyl formate Carbon tetrachloride Dimethyl sulfoxide Dimethyl formamide Acetic acid Benzene Toluene m Xylene Pyridine Acetone Carbon disulfide Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 61 4 Good Measurement Practices Many of the solvents in Table 1 are hazardous Be sure you fully understand their properties before using them When using volatile solvents such as acetone or methylene chloride make sure that the sample cell is stoppered Evaporation of a solvent does change the solute concentration and can cause solution noise due to solute convection currents Both of these will affect the accuracy of your measurements We also recommend stirring and tem
11. UV Visible Spectroscopy System Operator s Manual 87 5 Using your UV Visible Spectroscopy System Print Preview of Reports Printing a results report requires you to have set all parameters properly and have data available for evaluation If you do not have data you may get the message No results present on the message line No results present fa Fixed Wavelength Report 2 The report generated will be displayed in the preview window Method file Information Data File lt untitled gt Default Method i C HPCHEM 15 DATA ADVE XAN2 SD Created 02 06 1992 11 16 43 Overlaid Spectra Sample 1 Sample 2 Sample 3 ee i ee D c o 2 m 0 2 at Name 4bs lt 414nm gt Name 4bs lt 414nm gt 1 Sample 1 0 62494 3 Sample 3 0 10637 2 Sample 2 0 21198 Report generated by TK Signat pssssssssipsitas 88 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 This window allows you to look at your report page by page Scroll bars are available if a page does not fit into the actual preview window In addition you may use a different size for your preview display Three sizes are available with the size selection box Depending on your display resolution select the one which best fits your needs normal normal q The following functions are available with the print preview window Browse through the report pages with
12. as for the reference measurement Close the cell holder s level 4 Press the Standard button on the front of your spectrophotometer or click Standard on the Instrument Panel to start the measurement Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 103 5 Using your UV Visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 104 r Standard Information Name Standard 1 Solvent Water Comment Value Caffeine 10 2 mg L i 5 Enter your standard s information in the Standard Information dialog box and click OK Your Agilent ChemStation software automatically calibrates and displays the calibration results After a successful calibration the task panel s calibration curve shows green This indicates that your method is ready for analysis Task Quantification Ed Doi The Task panel s Samples and Standards buttons can be used to switch your current view to the samples or standards view At this point you can also save your method for future use See Saving Your Parameters as a Method on page 76 for more information Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Analysis Using your UV Visible Spectroscopy System 5 1 Remember the orientation of the cell in the cell holder Lift the level to release the cell remove it and flush it three times with about 1 ml of your caffeine sample 1 1 dilution with distilled wat
13. as sample For the calibration we use absorbance data at 273 nm Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Quantitative Analysis using a Calibration with Standards Setup 1 Make sure that you are in Standard mode The mode is indicated on the tool bar of your Agilent ChemStation session Clear z Method Mode lt untitled gt Standard X 2 Select the Quantification task in the analysis panel s selection box Task Fixed Wavelengths vy Fixed Wavelengths Spectrum Peaks Ratio E quation Quantification 3 A new task panel is displayed and the Quantification Parameters dialog is opened automatically Task Quantification v pas g g Cees If you are already in the Quantification task use the Setup button on the analysis panel to NOTE open the parameter dialog Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 101 5 Using your UV Visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 4 Setup your analysis wavelength at 273 nm Use wavelength type Caffeine for the Analyte name set the Calibration curve type to Linear select Concentration entry and use mg l as Unit Check the Prompt for standard information and the Prompt for sample information Select Absorbance as Data type and set Display spectrum From 190 nm To 340 nm r Quantification Parameters A
14. available data directory Analysis Panel The analysis panel gives you a graphical visualization of the current working context In addition it provides access to the setup dialog of your actual task by means of the Setup button Task Fixed Wavelengths Fixed Wavelenaths S Ratio E quation Quantification Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 General Purpose Agilent ChemStation Software for UV Visible Spectro ny Overview Instrument Panel The instrument panel is below the analysis panel It displays and controls your sampling devices and spectrophotometer Some of the graphical elements on this panel are active items which are used for example to switch lamps on or off or run a peristaltic pump Sampling Sipper v Sample Automation When you move the mouse across an active area the cursor changes Click in an active area to bring up a small menu with selections or to perform an operation In addition you can select a sampling system from the list of available systems Its parameters can be adjusted by means of the Setup button Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 35 2 introduction to Your System General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview View The area on the right side of the side panels provides a view on a certain aspect of y
15. defining your method to analyze a caffeine sample To be able to repeat such an analysis all set parameters can be stored permanently to disk This allows you to load such a method on your system or even transfer such a method to your colleague with a Agilent ChemStation system 1 Choose Save Method As from the File menu or click the icon g 845x UV Visible System 1 Offline TK Edit Method Measure Instrument Matl Load gt 7 ain Save lly Import gt Export Selected Spectrum As gt New Method Load Method Save Method As Set Method Password Load Results Save Results As Print gt Print To File gt Print Preview gt Printer Setup Exit ChemStation Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Saving Your Parameters as a Method 2 This displays the Save Method As dialog box Type the method name in the File name field for example Caffeine m Click OK to save your method File name New Caffeine Analysis M CAFFEINE ANALYSIS EQUATION M CAFFEINE ANALYSIS M CORTISONE CORTISOLE ANALYSIS M DNA MELTING M P PHENYL ACETATE HYDROLYSIS M STDEFMET M TEST SAVF ANI Y INSTRIIMENT 1 M gm Save file as type NOTE If you generate many methods you can use Options amp Infos from the Method menu to add text for documentation purposes and to simplify method access This
16. displays the Method Options amp Information dialog box In the Method Information section you can enter a descriptive text which becomes part of your method Method Options amp Information E Autosave F Autosave Spectra to File Method Information Caffeine Analysis absorbance at 273 nm Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 77 5 Using your UV Visible Spectroscopy System Retrieving and Printing a Method Retrieving and Printing a Method This section describes how to access methods and print a method report 1 Choose Load Method from the File menu or click the icon in the toolbar g 845x UV Visible Systen 1 Online TK Edit Method Measure Instrument Math View Mode Load gt Save gt Import Export Selected Spectrum As gt New Method Load Method Save Method As Set Method Password Load Results Save Results As Print gt Print To File gt Print Preview Printer Setup Exit Chem Station 1 CACHEM32 METHODS CAFFEINE ANALYSIS 2 M 2 cj NOTE If your current method has been modified a dialog box will ask you whether you want to save or ignore these changes Save Method The curent method has been changed Do you want to save the changes Cancel 78 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System Retrieving and Printing
17. has the suffix Online and can be started as the first instance only on your PC In addition a data analysis session which has the suffix Offline can be launched The offline session allows recalculations based on stored data and is useful in the development of analytical methods Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 37 2 Introduction to Your System Operation Levels The operation levels manager level and operator level apply to all modes and allow managing an application and running an application only In the managing level of an application methods can be developed and stored permanently to disk The manager level of operation is password protected This assures the integrity of predefined methods and operation sequences In the operator level a reduced set of functions is available Functions which may affect the integrity of an analytical procedure are not available However an operator may use their own settings This is flagged on the tool bar and is indicated on printed reports Agilent ChemStation Modes The Agilent ChemStation modes are application oriented Each mode has its own mode specific menu panels operations and set of views Your general purpose software for UV Visible spectroscopy is the platform for all modes It is split into a Standard mode an Execute Advanced Method mode and a Verification and Diagnostics mode Mode Standard x Standard Thermal Dena
18. measurement session by selecting Instrument 1 online from the menu Your Instrument Panel shows you the current state of the spectrophotometer and the Blank button is enabled The first task is to measure a reference Typically the cell containing the solvent used with your samples is put in the measurement position and a Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 blank measurement performed To start this measurement click the Blank button on your Instrument Panel or press the spectrophotometer s Blank button A blank measurement is a reference measurement combined with the measurement of a baseline spectrum A baseline spectrum gives you additional hints on the absorbance of the cell windows and the solvent Areas with high noise indirectly indicate high absorbance For high precision measurements wait until the spectrophotometer and the lamps have reached thermal equilibrium The time required is a function of the environmental conditions Your spectrophotometer should be ready after 45 minutes 7 The next measurement is your sample measurement To get the most precise results use the same cell in the same orientation to the measurement beam Flush your cell about three times with your sample solution and start the measurement by clicking the Instrument Panel s Sample button or by pressing the spectrophotometer s Sample button For details on how to mount
19. sampling interval is about 0 9 nm Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 27 2 Introduction to Your System Spectrophotometer Description Line power switch with green light 28 Your spectrophotometer is very easy to use It has a line power indicator a status indicator and some push buttons All electrical connections are made at the rear of the spectrophotometer Front View The front view of the spectrophotometer is shown in Figure 2 Notice that the sample compartment is open Unlike conventional spectrophotometers the Agilent Cary 8454 UV Visible spectrophotometer does not suffer from ambient false light The open sample area makes it easier to access for cuvette handling and to connect tubing to a flow cell or thermostatable cell holder The spectrophotometer is shipped with the standard single cell holder Standard and accessory cell holders can be removed and replaced in seconds with few or no tools Pushbuttons Indicator Figure 2 Front view of spectrophotometer The line power switch is located at the lower left part of the spectrophotometer Pressing it in turns on the spectrophotometer It stays pressed in and shows a green light when the spectrophotometer is turned on When the line power switch stands out and the green light is off the spectrophotometer is turned off Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 On the fro
20. the solvent also influence the reference data In wavelength ranges where your solvent or cell is absorbing the noise of the baseline spectrum is high Reliable sample data cannot be expected in these areas Therefore new reference or blank measurements are required when e you change your measurement cell or its orientation relative to the measurement position e you use a different solvent or event a different lot of the same solvent e the time between your reference measurement and the sample measurement get too long e the time conditions are depending on the aging rate of your lamps and possible changes of your environmental conditions typically the last blank measurement should not be older than half an hour To ensure the highest accuracy of sample measurement the blank measurement should be taken immediately prior to the actual sample measurement 112 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Index A absorbance 40 high 71 absorbance maximum 90 accessing lamps 31 accessory slot 31 accurate results 54 acetic acid 61 acetone 61 acetonitrile 61 acquisition date 42 time 42 active item 35 active position 35 Agilent Bootp Service 50 52 Agilent Cary 8454 UV Visible 50 Agilent ChemStation analysis panel 73 90 family 37 graphical window 83 logon 72 measurement session 70 method 76 mode 38 offline session 72 online session 72 password 72 preview sizes 89
21. the analysis light beam have to be very parallel The parallelism is measured in terms of the angle between the two opposite cell walls We recommend that you use 10 mm path length cells with an angle that is below 0 1 degrees of an arc Light Beam In Parallel Cell Walls Light Beam Out Light Beam In Non parallel Cell Walls rd Figure 4 Shift of the spectrophotometer light beam due to non parallel cell walls Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Good Measurement Practices 4 Apertured Cells In applications where sample volume is limited apertwred or microcells are used The width of these cells is reduced to reduce the volume and the blank part of the cell must be blackened to avoid unwanted transmission and reflection through the side walls If the side walls are not blackened the result will be poor photometric accuracy and if different concentrations are measured poor linearity The disadvantage of apertured and microcells is that part of the light beam is blocked Not all the light passes through the sample and there can be some loss of sensitivity See Figure 5 for recommended and Figure 6 for cells you should not use with the instrument lt D Quartz cells Quartz cells with black Y Figure 5 Recommended cells CAUTION Quartz cells with black apertures smaller than 2 mm when used with a multicell
22. the required rated current and of the specified type normal blow time delay and so on are used for replacement The use of repaired fuses and the short circuiting of fuseholders must be avoided Some adjustments described in the manual are made with power supplied to the instrument and protective covers removed Energy available at many points may if contacted result in personal injury Any adjustment maintenance and repair of the opened instrument under voltage should be avoided as much as possible When inevitable this should be carried out by a skilled person who is aware of the hazard involved Do not attempt internal service or adjustment unless another person capable of rendering first aid and resuscitation is present Do not replace components with power cable connected Do not operate the instrument in the presence of flammable gases or fumes Operation of any electrical instrument in such an environment constitutes a definite safety hazard Do not install substitute parts or make any unauthorized modification to the instrument Capacitors inside the instrument may still be charged even though the instrument has been disconnected from its source of supply Dangerous voltages capable of causing serious personal injury are present in this instrument Use extreme caution when handling testing and adjusting Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Safety Information 1 Solvent Information
23. 4 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Using your Multicell Transport The multicell transport is a cell changer which allows you to position up to 8 cells in the measurement position automatically You may use different cells in each measurement position The path length can be specified individually for each of the cell positions For details of your multicell transport see the nstalling and Operating Your Multicell Transport manual 1 Select Multicell 8 cell in the Instrument Panel selection box Sampling Manual Sipper Multicell 8 cell XY Autosampler 2 Click Setup on the Instrument Panel Type the path lengths of all cells used in cm and click OK Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 97 5 ai yor UV vone oe System Usil our Multic ransport Setup Multicell Transport Cell Path Length cm cm cm cm cm cm cm loo I Im In lse lw Im le a or a a a a cm 3 To move your cell for the next measurement into the measurement position click the cell on the Instrument Panel or choose Multicell Transport Position from the Spectrophotometer menu to access the Multicell transport Control dialog In the Multicell Transport Control press one of the position buttons to move the multicell transport 98 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Vis
24. Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual oe Agilent Technologies Notices Agilent Technologies Inc 2002 2003 2008 2011 2013 2014 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 61115 90054 Edition 08 14 Agilent Technologies Australia M Pty Ltd 679 Springvale Road Mulgrave Victoria Australia Research Use Only This product may be used as a component of an in vitro diagnostic system if the sys tem is registered with the appropriate authorities and complies with the relevant regulations Otherwise it is intended only for general laboratory use Microsoft is a U S registered trademark of Microsoft Corporation Software Revision This handbook is for B 05 xx revisions of the Agilent ChemStation software where xx is a number from 00 through 99 and refers to minor revisions of the software that do not affect the technical accuracy of this hand book Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express
25. Config Help I Load Save Samples As Standards 4 Selected Spectra As Import Export Selected Spectrum As Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 81 5 Using your UV Visible Spectroscopy System Saving and Retrieving Data 2 Select a data files in the File name selection box of the Save Spectra As dialog or type a valid file name into the File name edit box Save Spectra As File name Folders a OK CAFFEINE SAMPLE SD e chem32 1 data a Cancel Al lea p E gt CHEM32 E G1 BENZENE S NIST_HOLMIUM OXIDE 2034 5D z Save file as type Drives Spectra SD STD v c Programs X A valid file name consist of eight alphanumeric characters and the file extension sd or std NOTE i Usually the extension std is used for standards only If the file name exists a message box is displayed allowing you to abort or continue with the operation Confirm Save As C chem32 1 data CAFFEINE SAMPLE SD already exists Do you want to replace it ys J u 3 Click OK to start the operation 82 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Saving a Selected Spectrum 1 Select the spectrum of interest in the graphic window r Overlaid Sample Spectra olez 08 07 0 6 05 0 4 0 3 0 2 0 1 o MSS 200 250 300 350 400 450 nm Or in the tabular Sample Results
26. Table window Sample Result Table Show Sample Info Last Spectrum Name Dilut Factor Caffeine mg L Abs lt 273nm gt 1 00000 0 28903 0 57807 2 Choose Save Selected Spectra As from the File menu ig 845x UV Visible System t Online TK File Edit Method Measure Instrument Math View Mode ECM Config Help Load Save Samples As Standards As Selected Spectra As Import Export Selected Spectrum As If you did not select either the Overlaid Sample Spectra window or the Sample Results Table window you may get the warning Select activate a window on the message line Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 83 5 84 Using your UV Visible Spectroscopy System Saving and Retrieving Data 3 Select a data files in the File name selector box of the Save Spectra As dialog or enter a valid file name into the File name edit box File name Folders OK CAFFEINE SSMPLE SD e chem32 1 data g J CHEM32 ADVEXAMS SD q E 4 47 50 a SD BENZENE SD NIST_HOLMIUM OXIDE 2034 SD z Save file as type Spectra SD STD v c Programs X Drives 4 Press OK to start the operation Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System Saving and Retrieving Data Retrieving Spectra 1 Choose Load Samples from the File menu Boo Ba ose i vrethls Get
27. This allows you to examine the results of this processing step by viewing the processed spectra container s content If for example you specified the first derivative data type the first derivative spectra of all your raw data are available in the processed spectra container after analysis The type of spectral operation is defined by your method settings Used Wavelength Wavelengths Use wavelength 273 nm poll three point drop line X 300 360 nm The next step in the data analysis process is the provision of data for further evaluation specified in terms of wavelength and background correction operations such as an internal reference calculation or three point drop line calculation This data is stored in the used wavelength container In the Fixed Wavelength task for example a tabular view of this data is available with the Sample Results Table window Results An additional step is the evaluation of the accessed data Calculation Name Equation WL LB WE Y Unit Caffeine WL1 0 05 mg L Weight Volume E Use Weight wt Volume Unit Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 43 2 Introduction to Your System Summary In the Ratio Equation task for example this used wavelength data is processed by an evaluation of the equation specified which generates the calculation results These calculated result values are stored in the results container The results are available w
28. Visible spectrophotometer are available as spare parts A quick check without the need of standards can be performed by the Verification and Diagnostics mode s self test This test can be performed always after starting the spectrophotometer Spectrophotometer Self test 1 Make sure that you are in the Verification and Diagnostics mode The mode is indicated on the tool bar of your Agilent ChemStation session Mode Verification and Diagnostics Sf 2 Select the Self Test task in the analysis panel s selection box Task Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 107 5 Using your UV Visible Spectroscopy System 3 Choose Self Test gt Start from the Task menu or click Start to start the self test t 845x UV Visible System 1 Online TK File Instrument View Mode ECM Config Hi Instrument Self test Self Test Setup E Verification Diagnostics Ta Flow Test S Information Maintenance Dissolution Tests The spectrophotometer should be in stable working conditions before you initiate the test If these conditions are not met you may get a warning message Lamp on time too short Bd The spectrophotometer should be allowed to warm up for 45 minutes after switching the lamp on Do you want to proceed anyway 4 The self test results are displayed with pass fail criteria Self test results can be stored
29. Warning Symbols 10 warning symbols 10 water 61 wavelength 43 wavelength reproducibility 47 wedge shaped cells 56 weight 41 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Index 117 Index 118 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual www agilent com In This Book To be able to use your new Agilent Cary 8454 UV Visible spectroscopy system quickly this book gives you step wise procedures and examples for basic operations and tasks Agilent Technologies 2002 2003 2008 2011 2013 2014 08 14 Agilent Technologies
30. a Method 2 The Load Method dialog is displayed The selected method information is shown in the File Information section of the dialog box EE Load Method METHOD Method Type Standard Quantification Created by TK LastUpdate Date 06 06 2008 Time 04 3 Calibrated No Number of Standards 0 3 If you want to load this method click OK NOTE Whenever you change a parameter of the current method an indication is displayed in the toolbar s modification field Look in a METHODS ef By n Name Date modif Type Size Tags CAFFEINE ANALYSIS M _ CORTISONE CORTISOLE ANALYSIS M DNA MELTING M Desktop P PHENYL ACETATE HYDROLYSIS M T C STDEFMET M C TEST SAVE ONLY INSTRUMENT 1 M Administrator WAVELENGTH ACCURACY TEST M H w C YELLOW DYE 3 WAVELENGTHS M Computer A c Network Fle name CAFFEINE ANALYSIS M Files oftype Method M 7 Cancel e i File Information c chem3251 methods caffeine analysis m This triggers the reminder dialog mentioned above WAVELENGTH ACCURACY TEST M Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 79 5 Using your UV Visible Spectroscopy System Retrieving and Printing a Method 4 To print a method choose Print Method from the File menu Edit Method Measure Instrument Math View Mode ECM Config Help Load saa lt untitled gt Impo
31. al is ceramic to reduce thermal effects to a minimum It main components of the spectrograph are the lens the slit the grating and the photo diode array with front end electronics The mean sampling interval of the diode array is about 0 9 nm over the wavelength range 190 nm to 1100 nm The nominal spectral slit width is 1 nm Spectrograph Lens The spectrograph lens is the first of the parts which are collectively known as the spectrograph It is mounted on the housing of the spectrograph The spectrograph lens refocuses the collimated light beam after it has passed through the sample Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 e Slit The slit is a narrow aperture in a plate located at the focus of the spectrograph lens It is exactly the size of one of the photo diodes in the photo diode array By limiting the size of the incoming light beam the slit makes sure that each band of wavelengths is projected onto only the appropriate photodiode e Grating The combination of dispersion and spectral imaging is accomplished by using a concave holographic grating The grating disperses the light onto the diode array at an angle linear proportional to the wavelength e Diode Array The photodiode array is the heart of the spectrograph It is a series of 1024 individual photodiodes and control circuits etched onto a semiconductor chip With a wavelength range from 190 nm to 1100 nm the
32. and re calibrating your system Also by simply removing this standard from your standard data set you can eliminate outliers in your calibration 46 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 e Calibrated method When you save your method the standards currently in memory are always saved with the method After loading a method you can directly analyze your samples e Optimization for a particular sample You can optimize your wavelength settings for a sample outside the linear range of your actual calibration Due to the excellent wavelength reproducibility of your spectrophotometer you can switch to a wavelength with a lower extinction coefficient for precise analysis of such a sample In summary the availability of spectral raw data offers you many additional opportunities to optimize your calibration and analysis for best results This optimization can be done quickly just by setting new method parameters You get new answers almost instantaneously with consistent and reliable results Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 47 2 Introduction to Your System General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview 48 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Agilent Cary 8454 UV Visible Spectroscopy System A e Operator s Manual ee 3 Installation and Start Up e a e
33. ar Load Save 2 Select the data file in the File name selector box of the Load Spectra to SAMPLES dialog box Look in J DATA a P ADVEXAM2 SD Recent Places L ADVEXAM3 SD L ADVEXAM4 SD Desktop _ ADVEXAMS SD L ADVEXAM SD _ ADVEXAMS SD Administrator Computer Ae L STDEXAM1SD r L STDEXAM2 SD Network STNEVARAA CN File name CAFFEINE SAMPLE SD bd Files of type Spectra SD STD File Information c chem32 1 data caffeine sample sd SPECTRA Sample Name Caffeine Number of Samples 1 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Name Date modified Type Size Tags e m Cancel Help 85 5 86 Using your UV Visible Spectroscopy System Select a file in the file selector box to view file information for that file 3 Click OK to start the operation The spectra available with your data file are added to the files currently in the Agilent ChemStation sample container Deleting Current Spectra 1 Choose Clear Samples from the Edit menu or click the toolbar icon A 845x UV Visible System 1 Online TK Clear File Method Measure Instrument Math View Mode ECM Co y Cle Cut Ctrl Method i Copy Ctrl C lt unt Paste Ctrl V Tas Paste Append Shift Ctrl v Overlaid Sample Spectra Ra Select All os l Copy to Clipboard ma N A l Clear Samples q Standard Np Annotate Sandard a SEPT TITTY Fi
34. avelengths v Spectrum Peaks Ratio E quation Quantification The task orientation lets you quickly adjust the software to give you the right view and answers based on your data These tasks have been derived from a survey of the most common UV Visible spectroscopic tasks done on a routine basis in analytical laboratories Historically these tasks have been developed on filter photometers or scanning spectrophotometers With your spectrophotometer you have the advantage that by default the entire UV Visible spectrum of your samples is available So these four tasks offer different views of your acquired data A task switch within the Standard mode is much faster than an entire mode switch An additional benefit of these tasks is that the definition of a method is done within a single dialog Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 39 2 40 Introdu eral Purpose Ag ction to Your System ilent ChemStation Fixed Wavelength Task Fixed Wavelengths Y The Fixed Wavelength task is used to look at measured sample data at up to six different wavelength This data is available as absorbance transmittance and first to fourth derivative Due to the spectral acquisition additional techniques such as internal reference or three point drop line background corrections can be applied Spectrum Peaks Task Spectrum Peaks v 4 In the Spectrum Peaks task you can vi
35. blank measurement should be repeated as often as is practical Even in a thermally stable environment a blank measurement should be taken at least every half hour to ensure accurate results Chemically the only difference between the blank and the sample should be the presence of the analyte s For measurements with liquid samples the blank should be a sample cell filled with the solvent you plan to use 54 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Good Measurement Practices 4 Sample Cell Material Quartz sample cells or sample cells with quartz face plates are required if you want to use the full 190 to 1100 nm wavelength range of your spectrophotometer If you plan on working only in the visible and or short wave near infrared range of 350 to 1100 nm you can use good quality glass cells Disposable plastic sample cells for measurements in the range 400 1100 nm are also available The quality of these cells varies and they are generally not recommended Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 55 4 56 Good Measurement Practices Optical Specifications of Cells The accuracy of the readings of a diode array spectrophotometer is very sensitive to spatial shifts of the measurement light beam Cells having non parallel opposite faces or so called wedge shaped cells lead to a spatial shift of the light beam see Figure 4 Therefore the opposite cell walls illuminated by
36. ctively block portions of the UV spectrum with a UV cut off filter An optical filter wheel assembly with three cut off filters is available for the spectrophotometer The cut off wavelength of the filter you choose should be low enough that it does not eliminate important spectral information but high enough that it blocks the light that could degrade your sample If you use a filter with your samples you must use the same filter when you make your blank measurement Turning the Dz Lamp off The short wavelength radiation leading to photodegradation comes from the light of the Dy lamp For application where readings are taken at wavelengths above 400 nm the Dyg lamp can be turned off The light intensity supplied by the Tungsten lamp is sufficient for a good signal to noise ratio over the wavelength range 400 1100 nm When using cells with small apertures you should check the signal to noise ratio by making sample measurements under the conditions of your application Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 63 4 64 Good Measurement Practices Stirring and Temperature Control Solution homogeneity can be a problem especially for viscous solutions There are cases where due to convection induced gradients rapid absorbance changes may give irreproducible data These changes can be observed spectroscopically by taking measurements with short integration times To minimize convection effects keep the temperatu
37. d Installation Summary for Your General Purpose UV Visible System 50 Starting a Measurement Session 52 This chapter does not replace the information available with the Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide It is intended as a reminder of the key steps of the installation and system startup ig Agilent Technologies 49 3 Installation and Start Up Installation Summary for Your General Purpose UV Visible System General A detailed description of your UV Visible system is given with the manual Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide This summary reminds you of the key points of installation Spectrophotometer CAUTION V Make sure that your spectrophotometer has the LAN interface card installed V Check that your spectrophotometer is either connected to your PC directly using a crossover LAN cable or to your LAN using a direct connection The Agilent Cary 8454 UV Visible spectrophotometer does not have a CAN interface Do not connect the LAN adapter of your PC to the CAN interface of the Agilent 8453 spectrophotometer otherwise the LAN adapter of the PC will be seriously damaged because the operating voltage of the CAN interface 12 V is higher than the operating voltage of the LAN adapter 5 V V Check that your spectrophotometer is connected to a power outlet Always operate your instrument from a power outlet which has a ground connection Always use the p
38. dards in the Agilent ChemStation memory This means that results now become a function of a measured set of standards and of the standard concentrations specified Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 45 2 Introduction to Your System J See gt 5 alpom l lt ul E Processed Standard Spectra fo e js Sok es b Lon Lx o7 0 25 SE 0 2 0 5 3 i 0 4 i 0 15 3 3 0 1 0 2 A 0 05 o o 200 250 300 o 20 Wavelenethnm Analyte value Calibration Table Co Jie jes Show Standard Info Show Coefficients Calibrate Standard Name Caffeine mg L Abs lt 273nm gt Enor 54 89000 0 29195 0 00 These coefficients are then used to calculate concentration results for the samples currently in memory The same processing steps applied to sample and standard data lead to the most precise results Advantages A diode array based spectrophotometer in combination with powerful data evaluation based on Agilent ChemStation offers many advantages over traditional spectrophotometer systems Some of these advantages are e Virtually unlimited number of standards Measure your standards before or after your samples and as well as the minimum required number of standards use as many standards as you wish with your calibration e Easy optimization The availability of all raw data samples and standards means you can easily optimize your method s settings by choosing a different calibration wavelength
39. data 42 rear view of spectrophotometer 30 recalculation 37 recommended cells 57 reference 52 Regulatory Information 19 remote connector 30 report results 87 result 46 results 43 precise 71 routine work 76 RS232C connector 30 S sample 54 65 compartment 26 push button 29 sample cell 54 62 sample data 45 sample information 41 sample result table 75 83 93 sampling device 35 sampling interval 26 sampling system 94 manual 94 security lever 31 116 self test 107 histories 108 start 108 working conditions 108 sensitivity 57 session data analysis only 37 instrument control 37 set of parameters 38 settings 76 setup dialog 34 sheet metal door 31 shine through aperture 25 shutter 24 25 signal to noise ratio 63 single beam instrument 54 sipper flow test 95 parameter 95 sipper system 58 sipper sampler system 62 slit 24 27 slit width 26 slots for MIO and accessory boards 31 software general purpose 23 32 solute convection 62 solution 64 solution noise 62 solutions 110 determining purity 110 enhancing sensitivity 110 solvent 54 70 Solvent Information 13 solvent suitability 61 Solvents 13 solvents 61 source lens 24 25 source of radiation 24 spare parts 109 spectral acquisition 41 spectral operation 43 spectral processing 44 spectral raw data 47 spectrograph 26 lens 24 slit 24 spectrophotometer 46 50 front
40. de the instrument even if the power switch on the front panel is turned off For safety reason do not position the equipment so that it is difficult to operate the disconnecting device Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide 15 1 Safety Information Allowing Hot Instrument to Cool Hot Surface The lamps and its surroundings remain hot for up to five minutes after the lamp is turned off To avoid burns allow the lamp compartment to cool before carrying our any work in this area UV Light Source Eye Hazard Eye damage may result from directly viewing light produced by deuterium lamps Always turn off the deuterium lamps before opening the lamp door on the instrument When operating the instrument always wear safety glasses prescribed for viewing ultraviolet rays Direct viewing of ultraviolet rays without protection causes permanent blindness or vision impairment Power Cord Lithium Do not replace the mains power cord with one that is inadequately rated When replacing the mains power cord use only a mains power cord with a rating equivalent to the one supplied with the instrument Batteries Information Explosion Hazard The Lithium battery can be found on the Spectrometer Processor Main Board SPM Danger of explosion if battery is incorrectly replaced Replace only with the same or equivalent type recommended by the equipment manufacturer Do not dispose of lithium batteries in do
41. djust your current sampling system for sipper introduction In addition due to the length of tubing the dead volume of your flow cell and the flow rate of your pump you must adjust your sipper system parameters For details see the Installing and Operating Your Sipper System manual 1 Select the Sipper in the Instrument Panel selection box Sampling Manual Sipper Multicell 8 cell Autosampler XY Autosampler 2 Click Setup on the Instrument Panel Type the path length of your flow cell in cm and click OK Setup Sipper Path Length cm Cancel l Parameter 3 Click Setup again and access the Sipper Parameter dialog by clicking Parameter The parameters required can be determined using the Flow Test task of your Verification and Diagnostics mode Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 95 5 Using your UV Visible Spectroscopy System Controlling your Sipper System gt g Sipper Parameter Pump Time s Pump Direction Wait Time 3 s Sample Return Wash Time 0 s Air Segment 4 Click OK in the Sipper Parameter dialog and click OK in the Sipper dialog to set the parameters NOTE Every measurement you start by clicking one of the Instrument Panel measurement buttons or by pressing the spectrophotometer buttons uses the sipper for sample introduction Sipper introduction is also used by an automated sequence 96 Agilent Cary 845
42. ectrophotometer Measurement of this caffeine sample is also used for the IQ installation qualification of your spectrophotometer 1 Make sure that you are in Standard mode The mode is indicated on the toolbar of your Agilent ChemStation session 2 Select the Fixed Wavelength task in the analysis panel s selection box Task Fixed Wavelengths Fixed Wavyelenaths Spectrum Peaks Ratio E quation Quantification 3 Click Setup in the analysis panel to open the parameter dialog Task Fixed Wavelengths KA Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 73 5 74 Using y A our UV Visible Spectroscopy System 4 Type the wavelength of interest in the Wavelengths section of the Fixed Wavelength s Parameters dialog Adjust your spectral display to a wavelength range from 190 nm to 400 nm in the Display spectrum section Click OK to set your parameters Fixed Wavelength s Parameters Wavelengths Use wavelength s 273 in Background ii correction none E Prompt for sample information Data type Display spectrum ee From 190 nm To 340 nm Fill your 1 cm path length quartz cell with distilled water Lift the lever at the left side of your cell holder Put the cell in the cell holder and make sure the transparent windows face towards the font and the back of the spectrophotometer Push the lever down to secure your cell in the cell
43. ed suitable under Equipment class I category Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide 17 1 18 Safety Information Installation category The installation category is II based on IKC61010 1 The installation category implies the regulation for impulse withstand voltage It is also called the Over voltage category II applies to electrical equipment with a nominal supply voltage up to 300 V Pollution level The pollution level is 2 based on IEC61010 1 Pollution level describes the degree to which a solid liquid or gas that deteriorates dielectric strength is adhering 2 applies to a normal indoor atmosphere where only non conductive pollution occurs Electrical Power Your spectrophotometer can operate on a line voltage of 100 240 VAC witha line frequency of 50 or 60 Hz Maximum power consumption is 220 VA The instrument power supply accepts any line voltage supplied in the above mentioned range Consequently there is no voltage selector in the rear of the spectrophotometer There are no externally accessible fuses because automatic electronic fuses are implemented in the power supply Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Safety Information 1 Regulatory Information CE Compliance Your Agilent instrument has been designed to comply with the requirements of the Electromagnetic Compatibility EMC Directive and the Low Voltage electrical safety Direct
44. er Then fill the cell with about 3 ml of the caffeine sample Make sure that the cell windows are clean and reposition the cell in the same orientation as for the standard measurement Close the cell holder s level Press the Sample button on the front of your spectrophotometer or click Sample on the Instrument Panel to start the measurement Sample Information Name Sample Solvent wate Dilution Factor 1 Comment not diluted 3 Enter your sample s information in the Sample Information dialog box and click OK The view switches to the samples and your quantitative results will be displayed with the Sample Result table Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 105 5 Using your UV Visible Spectroscopy System Quantitative Analysis using a Calibration with Standards NOTE To save your data for future use or for documentation purposes see Saving and Retrieving Data on page 81 for more information 106 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 How Can Be Sure That My Spectrophotometer Works Properly The quality of your measurement data is dependent on the performance of your spectrophotometer For a full performance verification external standards are required Standard kits are available for operational qualification and performance verification OQ PV OQ PV kits for the Agilent Cary 8454 UV
45. eter Self test 107 What Maintenance Does My Spectrophotometer Require 109 Cleaning 109 Spare Parts 109 How Can Get a Deeper Understanding of UV Visible Spectroscopy 110 When Do Have to Measure a Blank 112 Index 113 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 7 Contents 8 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 1 Safety Information Safety Information 10 Warning Symbols 10 Precautions 12 Solvent Information 13 Other Precautions 15 Environmental Conditions and Power Requirements 17 Regulatory Information 19 Agilent Technologies on the Internet 21 ee Agilent Technologies 1 Safety Information Safety Information 10 Agilent products must only be used in the manner described in the Agilent product user guides Any other use may result in damage to the product or personal injury Agilent is not responsible for any damages caused in whole or in part by improper use of the products unauthorized alterations adjustments or modifications to the products failure to comply with procedures in Agilent product user guides or use of the products in violation of applicable laws rules or regulations Warning Symbols To avoid personal injury death or damage to the instrument the operator must refer to an explanation in the manual This symbol is placed on the product where it is necessar
46. ew absorbance minima and maxima The focus here is more on the wavelength scale but the relevant absorbance readings are also provided Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 cihla Snarctrn lt Ratio Equation Task Ratio E quation w WE x Li i Wi eit je a P g rni AM Ac A WEE Viani ite lalli The Ratio Equation task is used to evaluate a user definable equation based on measured data and sample information An equation can be set up using sample data at up to six wavelengths Weight and volume data is entered with the samples measured By means of an equation for example analysis results based on chemical test kits can be automatically calculated and reported Another application for this task is to use a ratio of data values to check for the identity or the purity of a sample Quantification Task Quantification x The Quantification task allows you to do single component analysis based on four different types of calibration curves and a set of standards Due to the spectral acquisition background corrections can also be applied to your data The calibration can be optimized for your concentration range of interest by changing the wavelength used for calibration A new calibration and a new analysis is automatically performed based upon your current standard data Agilent Cary 8454 UV Visible Spectroscopy System Operator
47. fined in FAR 52 227 19 c 1 2 June 1987 U S Government users will receive no greater than Limited Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are fully understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual In This Guide This book is an introduction to your Agilent Cary 8454 UV Visible spectroscopy system It includes step wise procedures and examples for basic operations and tasks For more detailed information see the following manuals e Agilent Cary 5454 UV Visible Spectroscopy System Installation Guide e Understanding Your UV Visible Spectroscopy System e Agilent Cary 8454 Service Manual 1 Safety Information Introduces your UV Visible spectrophotometer and the Agilent ChemStation soft
48. he software This requires the assignment of a unique IP address to your spectrophotometer when it is powered on The assignment is done either by the Agilent BootP Service application running on your PC with a direct connection to the spectrophotometer or by a server application on your LAN Therefore it is important that either one of these applications is up and running before you switch your spectrophotometer on V Switch on your PC and boot your PC operating system If a printer is connected to your system switch the printer on V Make sure that the Agilent BootP Service is running or you are logged onto your LAN Y Switch your spectrophotometer on and wait until the spectrophotometer s indicator light turns to green This process includes the spectrophotometer s self test and takes about one minute For details on the startup sequence see chapter Installation and Start Up in your Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide V Launch your measurement session by pressing Start gt Programs gt UV Visible ChemStations gt Instrument 1 online Y You are ready to use your system if the blue busy status display on the system s bottom message line turns off V The first measurement you have to perform is a reference measurement After this alignment you are ready to measure absorbance data and spectra It takes about 15 minutes for the lamps to reach stable state conditions For best results do n
49. ible Spectroscopy System 5 Using your Multicell Transport Sampling ranspor Multicell 8 cell z Current Cell 1 Move To 2 ba sD ore mia ma mis emf enti Automation In an automated sequence the multicell transport can be used for automatic sample NOTE introduction A maximum of 8 samples can be introduced If you specify more than 8 measurements this warning is displayed Automation H A Number of measurements is out of range 1 8 a Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 99 5 Using your UV Visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 100 Your quantitative analysis task is based on a calibration with standards After a successful calibration the measured standards can become part of your method Such a method can be used directly for quantitative analysis of samples After having set up your method calibrated samples can be analyzed Several views of both your standards and the calibration as well as of your samples and results are available As a quick introduction a calibration using Beer s law with a single standard and the analysis of a sample is described The only limitation on the number of samples and standards is the memory capacity of your Agilent ChemStation For the practical experiment we use the IQ caffeine sample as standard and a 1 1 dilution with distilled water
50. ilent Cary 8454 UV Visible Spectroscopy System Installation Guide 19 1 20 Safety Information Operation is subject to the following two conditions 1 This device may not cause harmful interference 2 This device must accept any interference received including interference that may cause undesired operation If this equipment does cause harmful interference to radio or television reception which can be determined by turning the equipment off and on the user is encouraged to try one or more of the following measures 1 Relocate the radio or antenna 2 Move the device away from the radio or television 3 Plug the device into a different electrical outlet so that the device and the radio or television are on different electrical circuits 4 Make sure that all peripheral devices are also certified 5 Make sure that appropriate cables are used to connect the device to peripheral equipment 6 Consult your equipment dealer Agilent Technologies or an experienced technician for assistance Changes or modifications not expressly approved by Agilent Technologies could void the user s authority to operate the equipment ICES NMB 001 This ISM device complies with Canadian ICES 001 Cet appareil ISM est conforme la norme NMB 001 du Canada South Korean Class A EMC declaration Aw 7 71 G73 34 5417 214 This equipment is Class A suitable for professional use and is for use in electromagnetic environments outside of the h
51. information The result is a fundamental increase in the rate at which spectra can be acquired 24 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Spectrograph lens Introduction to Your System 2 ophotometer Overview Shutter stray light filter Tungsten lamp Slit Grating Source lens Photo diode array Figure 1 Optical system of spectrophotometer e Lamps The light source for the UV wavelength range is a deuterium lamp with a shine through aperture As a result of plasma discharge in a low pressure deuterium gas the lamp emits light over the 190 nm to approximately 800 nm wavelength range The light source for the visible and SWNIR wavelength range is a low noise tungsten lamp This lamp emits light over the 370 nm to 1100 nm wavelength range Source Lens The source lens receives the light from both lamps and collimates it The collimated beam passes through the sample if one is present in the sample compartment Shutter The shutter is electromechanically actuated It opens and allows light to pass through the sample for measurements Between sample measurements it closes to limit exposure of sample to light If the measurement rate is very fast you can force the shutter to remain open using Agilent ChemStation software or it stays open automatically handheld controller software Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 25 2 26 Introductio
52. ith the Sample Results Table Sample Result Table Cs ip EE Show Sample Info Last Spectrum Analyze Name Dilut_ Factor Caffeine mg L Abs lt 273nm gt 1 00000 0 28903 0 57807 The generalized basic data processing is divided into three steps 1 spectral processing 2 data access 3 data evaluation These steps are always performed in the above order identically for all spectra in the raw data container The results are placed in the results container The previous content of the processed spectra used wavelength and results container is replaced Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview Processing with Standards In the fourth task standards are used in addition to your sample data This requires an extension to the above concept to handle standards Two independent sets of containers are implemented one for standards and another for samples All processing containers are also duplicated As with the sample only processing all evaluation steps are done in parallel on both the samples and the standards Samples Standards s s Spectral Processing e Data Access i a i S Data Evaluation a P gt Calibration The evaluation in quantification is now a calibration using standards The coefficients are calculated based on the method s settings and the current stan
53. ive commonly referred to as the LVD of the European Union Agilent has confirmed that each product complies with the relevant Directives by testing a prototype against the prescribed EN European Norm standards Proof that a product complies with these directives is indicated by e the CE Marking appearing on the rear of the product and the documentation package that accompanies the product containing a copy of the Declaration of Conformity The Declaration of Conformity is the legal declaration by Agilent that the product complies with the directives listed above and shows the EN standards to which the product was tested to demonstrate compliance Electromagnetic Compatibility EN55011 CISPR11 Group 1 ISM equipment group 1 contains all Industrial Scientific and Medical ISM equipment in which there is intentionally generated and or used conductively coupled radio frequency energy which is necessary for the internal functioning of the equipment itself Class A equipment is equipment suitable for use in all establishments other than domestic and those directly connected to a low voltage power supply network which supplies buildings used for domestic purposes This device complies with the requirements of CISPR11 Group 1 Class A as radiation professional equipment Therefore there may be potential difficulties in ensuring electromagnetic compatibility in other environments due to conducted as well as radiated disturbances Ag
54. lear 86 clear math results 86 clear standards 86 evaluation 44 file extension 82 filename 82 file selector box 86 format 81 load 81 local storage 81 network transfer 81 removing 86 replace 82 retrieve 81 85 save 81 save samples as 81 saving selected data 83 storage 81 degassed 62 derivative 40 derivative spectroscopy 110 description of instrument 28 determining purity 110 deuterium lamp 24 development of an analytical method 37 dialog fixed wavelength s parameters 74 method options amp information 77 dimethyl formamide 61 dimethyl sulfoxide 61 diode array 27 46 door to access lamps 31 dust 62 E enhancing sensitivity 110 114 entrapment of bubbles 58 Environmental Conditions and Power Requirements 17 equation 39 44 ethyl acetate 61 ethyl ether 61 examples 111 extinction coefficient 47 F false light 28 54 filter for stray light correction 24 26 fixed wavelength 39 40 73 flow cell 58 flow cells 13 67 flow test 95 front view of spectrophotometer 28 G glass cells 55 glycerol 61 GPIB connector 30 GPIO connector 30 grating 24 27 handling cells 60 highest precision measurements 58 holographic grating 24 27 homogeneity 64 HPLC grade water 62 ICES NMB 001 20 identity 41 indicator 29 installation 50 installation qualification 73 instrument construction 24 description 28 electronic assemblies
55. log boxe How to use Standard Mode amp Standard Mode Examples B Correcting for Backgrounc Using an Internal Referen 5 Using an Internal Referen Confirming the Identity of on at bottom right on B Checking the Purity of a S B Using Derivative to Emph B Calibrating and Quantifyiru Execute Advanced Method Mode Advanced Mode Online Help ut Solutions for troubleshooting information an lt Thermal Denaturation Mode Dissolution Testing Mode Multibath Dissolution Testing Mod Combined Reports Verification and Diagnostics Mode Report Style Editor Maintenance A Troubleshooting Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 111 5 Using your UV Visible Spectroscopy System When Do Have to Measure a Blank The measurement data acquired by your spectrophotometer is instrument independent To achieve this independence a reference measurement must be performed All consecutive measurements refer to the last measured reference In the Agilent ChemStation software the reference measurement is combined with a baseline measurement The baseline displayed is information about the quality of the current reference In absorbance mode the data should be close to 0 AU and in transmission mode the data should be close to 100 Typically reference measurements for dissolved samples are made on the cell filled with the solvent used The absorbance properties of the cell and
56. m 9 The view shows you the spectrum of your caffeine sample Two peaks were found and are annotated with the wavelength Below the spectrum graph the Sample Result Table shows the wavelength of the peaks found and the measured absorbance values Last Sample Spectrurn Show Sample Info Overlay Spectra Delete Selected Sample All Peaks Valleys Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 93 5 Using your UV Visible Spectroscopy System Entering your Cell s Path Length 94 The cells used for your measurements are specified with the sampling system parameters In quantitative calculations these parameters are used in results calculations You must provide the correct value for the path length setting Usually you get this information from the supplier of your cells You set the path length in manual mode cell handling as follows 1 Click Setup on the Instrument Panel Sampling 40 Automation 2 Type the path length in cm in the Setup Manual dialog box Setup Manual Path Length cm 3 Click OK to set the specified path length Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Controlling your Sipper System A sipper system transfers your sample by means of a peristaltic pump into a flow cell for the measurement To control your sipper system through the Agilent ChemStation software you must a
57. mestic waste 16 Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Safety Information 1 Environmental Conditions and Power Requirements Your spectrophotometer will operate within specifications at ambient temperatures of 0 to 40 C 82 to 104 F Hot Surface If you use the instrument at environmental temperatures higher than 50 C 122 F the rear of the instrument may get hot CAUTION The spectrophotometer can be shipped or stored where the ambient temperature is between 40 and 70 C 4 and 158 F Do not store ship or use your instrument under conditions where temperature fluctuations could cause condensation within the instrument Condensation will damage the electronics If your instrument was shipped in cold weather leave it in its box and allow it to warm up slowly to room temperature to avoid condensation You can operate your spectrophotometer in environments with relative humidity up to 95 non condensing provided the temperature is between 25 40 C 77 to 104 F Avoid air circulation of varying temperatures like from refrigerators or air conditioners and exposure to direct sun light to ensure the instrument operates within performance specifications Your spectrophotometer can be stored at altitudes up to 4 600 m 14 950 ft and operated at altitudes up to 2 000 m 6 500 ft The Agilent Cary 8454 UV Vis instrument is suitable for indoor use only and is classifi
58. n to Your System e Stray Light Correction Filter In a standard measurement sequence reference or sample intensity spectra are measured without and then with the stray light filter in the light beam Without the filter the intensity spectrum over the whole wavelength range from 190 1100 nm is measured The stray light filter is a blocking filter with 50 blocking at 420 nm With this filter in place any light measured below 400 nm is stray light only This stray light intensity is then subtracted from the first spectrum to give a stray light corrected spectrum Depending on the software you can switch off the stray light correction using Agilent ChemStation software if you want to do very fast repetitive scans or it is switched off automatically Sample Compartment The spectrophotometer has an open sample compartment for easier access to sample cells Because of the optical design a cover for the sample area is not required The spectrophotometer is supplied with a single cell holder already installed in the sample compartment This can be replaced with the Peltier temperature control accessory the thermostattable cell holder the long path cell holder or the multicell transport All of these optional cell holders mount in the sample compartment using the same simple mounting system An optical filter wheel is also available for use with the spectrophotometer and most of the accessories Spectrograph The spectrograph housing materi
59. ng lever back down Utmost care should be taken when using small volume flow cells and particularly any cells with less than a 2 mm aperture Here it is important that the cells are properly centered in the light path The cell holder is designed for cells with 15 mm center height If applicable these cells should not be removed between the respective reference blank measurement and the sample measurement Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 67 4 Good Measurement Practices Inserting a Cell 68 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 5 Using your UV Visible Spectroscopy System Starting Your First Measurement Session 70 Starting Your UV Visible Software 72 Measuring Caffeine Absorbance at 273 nm 73 Saving Your Parameters asa Method 76 Retrieving and Printing a Method 78 Saving and Retrieving Data 81 Print Preview of Reports 87 Finding the Caffeine Absorbance Maximum 90 Entering your Cell s Path Length 94 Controlling your Sipper System 95 Using your Multicell Transport 97 Quantitative Analysis using a Calibration with Standards 100 How Can I Be Sure That My Spectrophotometer Works Properly 107 What Maintenance Does My Spectrophotometer Require 109 How Can Get a Deeper Understanding of UV Visible Spectroscopy 110 When Do Have to Measure a Blank 112 ot Agilent Techn
60. ng with Files E Working with Spectra Printing the Results E ECM in the ChemStation Standard Mode Execute Advanced Method Mode Advanced Mode Gi Kinetics Mode Thermal Denaturation Mode E Dissolution Testing Mode 4 Multibath Dissolution Testing Mod Combined Reports G Verification and Diagnostics Mode Report Style Editor Maintenance E Troubleshooting E E Replacement Parts F An Introduction to UV Visible Spec e Getting Started e Good Measurement Practice e Working with the Spectrophotometer and its Accessories e General Features e Working with Files e Working with Spectra e Printing the Results e ECM in the ChemStation In addition solutions to selected topics in UV Visible analysis are described There you can find help on topics such as Enhancing Sensitivity or Determining Purity 110 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 How Can I Get a Deeper Understanding of UV Visible Spectroscopy More specific examples including data are available in the Examples section of the Standard Mode help These can be used to run your Agilent ChemStation software for the special purpose described UV Vis ChemStation Help tobe es gt Hide Back Forward Contents Index Search E Installation Basic Operation 4 amp Standard Mode How to for inf a E User Interface Reference zithin the views and dia
61. nt panel of the spectrophotometer is a status indicator which will display different colors depending of the actual condition of the spectrophotometer e Green the spectrophotometer is ready to measure e Green blinking the spectrophotometer is measuring e Yellow the spectrophotometer is in not ready state for example turning one of the lamps on or if both lamps are switched off e Red error condition that is the spectrophotometer does not pass one of the self tests which are run when the spectrophotometer is turned on or an error occurred during operation In this case the UV Visible operating software gives a detailed error message and possible explanations are in the online help system and in your Service Manual Chapter 3 Diagnostics and Troubleshooting e Red blinking error condition of the spectrophotometer processor system Because in this case there is no communication with the computer there will be no error message The online help system and your Service Manual Chapter 3 Diagnostics and Troubleshooting give more information about troubleshooting The four measure push buttons on the front panel cause the following actions to be performed and the resulting data being sent to the computer The push button functionality is controller by the UV Visible ChemStation software and adjusted to the current measurement task e BLANK the spectrophotometer takes a blank measurement This comprises a reference meas
62. o control the spectrophotometer from a computer through RS232 connection using appropriate software The Multiple Instrument Firmware Tool provided can be used with RS232 communication The UV Visible ChemStation software currently supports LAN and GPIB communication only The GPIB connector is used to connect the spectrophotometer with a computer The 8 bit configuration switch module next to the GPIB connector determines the GPIB address of your spectrophotometer The switches are preset to a default address recognized by the operating software from Agilent Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 Only a single controller must be connected to the spectrophotometer at a time e The MIO board slot is reserved for a LAN interface board The accessory board slot is reserved for future use e The power input socket does not have a voltage selector because the power supply has wide ranging capability for more information see your Service Manual Chapter 1 Specifications There are no externally accessible fuses because automatic electronic fuses are implemented in the power supply The security lever at the power input socket prevents that the spectrophotometer cover is taken off when line power is still connected Side of the Spectrophotometer On the right side of the spectrophotometer is a double door for access to the lamps For exchanging lamps both doors the plastic d
63. ologies 69 5 Using your UV Visible Spectroscopy System Starting Your First Measurement Session Starting Your First Measurement Session 70 1 Make sure that your spectrophotometer has been installed correctly For details of installation see your Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Switch on your PC monitor and printer Log on to your PC operating system Confirm that your Agilent BootP Service is running using the Control Panel s Administrative Tools Services dialog or make sure that your network administrator has integrated your spectrophotometer into your local network File Action View Help es oblHm gt un gt Sh Services Local Name Description Status Startup Type Log On As OA Agilent Bootp Service Started Automatic Local System Gh Agilent 10 Libraries Service Provides syste Started Automatic Local System KoA Application Experience Processes appli Started Automatic Local System KoA Application Host Helper Ser Provides admin Started Automatic Local System KoA Application Information Facilitates the r Manual Local System Application Layer Gateway Provides suppo Manual Local Service Extended A Standard 4 Switch on your spectrophotometer A running BootP service will now assign the configured IP address to your spectrophotometer In the standard installation the Agilent BootP Service performs this task Start your
64. ome o 71713 ATS A Se AA REA H a EL ALBA JIA ME A AAAA AANA ALE AS Bao w Bey clr x lo Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Safety Information 1 Agilent Technologies on the Internet For the latest information on products and services visit our website at http www agilent com Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide 21 1 Safety Information Agilent Technologies on the Internet 22 Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual ee 2 Introduction to Your System Agilent Cary 8454 UV Visible Spectrophotometer Overview 24 General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview 32 This chapter introduces the principles of data acquisition data evaluation and data handling to help you to run your system successfully Your spectroscopy system is based on a UV Visible spectrophotometer with general purpose Agilent ChemStation software for UV Visible spectroscopy running on a PC with a supported Microsoft Windows operating system The spectrophotometer and PC are linked by a network connection This type of link is very flexible It can be used for a direct connection between the spectrophotometer and the PC as well for integration in a enterprise network with network access from the PC to the spectrophotometer The spectrophotomete
65. oor and the sheet metal door must be opened Safety light switches are automatically turning off the lamps when the inner sheet metal door is opened Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 31 2 Introduction to Your System General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview This section gives an overview of the elements of the user interface implemented with your Agilent ChemStation software and the data analysis concept behind It explains how data are processed and what the advantages of this processing are from a practical point of view 32 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview User Interface Elements Your general purpose Agilent ChemStation software for UV Visible spectroscopy facilitates operation of your diode array based UV Visible spectrophotometer in daily routine operation The focus of this software is on ease of use and ease of learning A graphical user interface visualizes the spectrophotometer operation and usage This user interface consists of a number of elements as described in the following sections Method name Tool bar B4hx UV Visible System 1 Online x TET ee Ede Method Measure Instrument Math View Mod Menu bar ops Methods C ChemJ2 1 M ab Brown STDEFMET M OJ STOEFMET M iy Sarg
66. or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms in the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend If software is for use in the performance of a U S Government prime contract or subcon tract Software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 or asa commercial item as defined in FAR 2 101 a or as Restricted computer soft ware as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or disclo sure of Software is subject to Agilent Tech nologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as de
67. ostics 38 107 mouse cursor 35 multicell transport 97 8 cell 97 connector 30 m xylene 61 N55011 CISPR11 19 n butyl alcohol 61 network administrator 50 51 connection 52 local 70 n hexane 61 nominal spectral slit width 26 0 offline 37 online 37 open sample area 54 operating system 52 operation 34 operation levels 38 operator level 38 operator name 42 optical filter 63 optical specifications of cells 56 optical surfaces 59 optical system 24 optimization 46 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Index outliers 46 P paper orientation 51 size 51 parallelism 56 parameter dialog 90 particulate matter 62 passivating new cells 59 path length setup 94 PC 52 peak find 91 performance verification 107 photochemical reactions 63 photodegradation 63 photodiode array 27 photosensitive substances 63 pipette 60 plasma discharge 25 plastic door 31 plastic sample cells 55 poor linearity 57 poor photometric accuracy 57 Precautions 12 printer 51 52 configured 87 processed spectra 43 processing 42 spectral 43 standards 45 pump peristaltic 95 purity 41 push buttons 29 blank 29 sample 29 standard 29 stop 29 pyridine 61 115 Index Q quantification 39 101 quantitative analysis of samples 100 ready for analysis 104 quartz sample cells 55 radiation source 24 rapid absorbance changes 64 ratio 39 raw
68. ot perform measurements before this period of time has elapsed Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual ee 4 Good Measurement Practices General Considerations 54 Inserting a Cell 67 This chapter describes making measurements selecting material optical specification and type of cell handling and maintaining cells checklist for good results solvents selection sample preparation use of filters stirring and temperature control of sample how to insert cells into the cell holder phe Agilent Technologies 53 4 Good Measurement Practices General Considerations There are many factors that can affect the results of your measurements This section provides brief discussions of some of the more important ones Spectrophotometer Design The sample compartment of the spectrophotometer is open Unlike conventional instruments the UV Visible spectrophotometer does not suffer from ambient false light The open sample area makes it easier to access it generally and to connect tubing to a flow cell or thermostattable cell holder Making Measurements Blank Reference and Sample Measurement Your spectrophotometer is a single beam instrument so you must measure a blank before you measure a sample For the high accuracy measurements the blank and the sample measurement should closely follow each other In general a
69. our current task A view consists of one or more separate windows These windows provide information such as a graph showing your measured sample spectra or a table with the calculated results Views are usually handled automatically by the operation you performed but you may also use the view menu s commands to select the view you prefer 36 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 D Anilant Cham Ctat Onftisraro fnr LIV lieth inartrr unn UIE erail Furpose Ag len cNemotation somware for UV V sible spectroscopy UVerview Software Structure The software is divided into specific applications called modes In addition levels of operation are available and there is support for data evaluation sessions without spectrophotometer control Agilent ChemStation Sessions Your Agilent ChemStation belongs to the Agilent s ChemStation family of instrument control software An installation of Agilent ChemStation software can control up to four different UV Visible instruments on a single PC Each of these instruments has its own session If you start a session its name is indicated in the main application s window title bar for example 845x UV Visible System 1 845x UV Visible S File Edit Method Measure Instrument Math View Mode ECM Config Help Each instrument session is available for data analysis only or for instrument control An instrument control session
70. ower cord designed for your region 50 V Before you switch on your spectrophotometer make sure that the Agilent Bootp Service is installed on your PC or your network administrator has assigned an IP address to your spectrophotometer For details see the chapter LAN Communication Installation Connection and Configuration in your Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Installation and Start Up 3 PC V Make sure that all of your PC components are connected to line power V Make sure that your general purpose software for UV Visible spectroscopy is installed V A printer must be configured on your PC Adjust paper size for example Letter A4 Set Orientation to Portrait V The TCP IP protocol must be installed and configured on your PC Y Your spectrophotometer must be configured with its IP address V If you connected your spectrophotometer directly to your PC be sure that the Agilent Bootp Service is installed and configured If you connect to your spectrophotometer using a LAN make sure that your network administrator assigns the IP address to your spectrophotometer Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 51 3 Installation and Start Up Starting a Measurement Session 52 With a network connection to your spectrophotometer it is important that your spectrophotometer is recognized by t
71. perature control when you use volatile solvents When using water as solvent we recommend using UV grade or HPLC grade water to reduce unwanted absorbance from impurities in the water If you are using the sipper sampler system the water should be degassed to avoid bubble formation in the flow cell especially if the water comes from a pressurized water supply Sample Preparation 62 The sample cell should be rinsed three to five times with your intended solvent before you fill it with the pure solvent that will be used in the measurement Turning the cell upside down on a small stack of absorbent tissues will help remove any residual solvent This treatment will minimize contamination from previous experiments Samples which contain colloidal dispersions dust or other particulate matter should be filtered centrifuged or allowed to settle If not the overall attenuation of transmittance spectrum due to light scattering and or reflection will hide the spectral information from the analyte Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Good Measurement Practices 4 Photosensitive Samples A few substances are very photosensitive They degrade or undergo photochemical reactions if exposed to light This can be easily seen by a decrease of sample absorbance over time Use of Filters The shorter wavelength higher energy UV light is most likely to degrade photosensitive samples If this is a problem you can sele
72. print preview 87 printer display 89 samples 86 session 37 session online 71 workstation 46 Agilent Technologies on the Internet 21 ambient light 28 analysis 105 analysis panel 34 107 analysis setup calibration 102 concentration unit 102 data type 102 display 102 prompt for sample information 102 wavelength 102 analytes 54 analytical task 38 analyze 76 apertured cells or cuvettes 57 apertures 63 application specific 37 background correction 40 41 Beer s law 100 benzene 61 blank 52 54 64 blank push button 29 bootp service 70 bubble formation 62 busy 52 C caffeine 73 calculate 46 calculation results 94 calibration 41 45 100 103 coefficients 45 curve 41 carbon disulfide 61 carbon tetrachloride 61 CE compliance 19 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual cell changer 97 cell path length 94 95 97 chloroform 61 cleaning 109 cleaning cells 59 collimated beam 24 colloidal dispersions 62 command 34 common solvents 61 compartment for sample 26 concentration 46 concentration range 41 connection network 23 connector GPIB 30 GPIO 30 multicell transport 30 remote 30 RS232 30 correction for stray light 24 26 current context 34 current task 36 cut off filter 63 cut off wavelength 63 cyclohexane 61 cyclopentane 61 113 Index D data 42 absorbance 100 access 43 44 archieve 81 c
73. r acquires and provides absorbance data which is handled by the PC s application software All of the data display evaluation and long term storage is done under software control on the PC Ee Agilent Technologies 23 2 Introduction to Your System Agilent Cary 8454 UV Visible Spectrophotometer Overview This section gives an overview of the optical system and explains the spectrophotometer front and back panels It also explains the layout and construction of the spectrophotometer including the electronic and mechanical assemblies inside the spectrophotometer Optical System Overview Optical System The optical system of the spectrophotometer is shown in Figure 1 Its radiation source is a combination of a deuterium discharge lamp for the ultraviolet UV wavelength range and a tungsten lamp for the visible and short wave near infrared SWNIR wavelength range The image of the filament of the tungsten lamp is focused on the discharge aperture of the deuterium lamp by means of a special rear access lamp design which allows both light sources to be optically combined and to share a common axis to the source lens The source lens forms a single collimated beam of light The beam passes through the shutter stray light correction filter area then through the sample to the spectrograph lens and slit In the spectrograph light is dispersed onto the diode array by a holographic grating This allows simultaneous access to all wavelength
74. re of your sample the same as the cell holder or environmental temperature Problems like these can also be minimized by using a thermostattable cell holder and or a stirring module A similar effect can occur in cases of incomplete mixing This is especially true where the specific gravities or miscibilities of the solvent and analyte are quite different Again stirring is a way to prevent this kind of problem In an unstirred cell it is sometimes possible to observe local photodegradation of sensitive analytes Because the actual volume of the sample in the light path is very small stirring the sample will reduce the time any given analyte molecule is in the light path This minimizes the photodegradation and increases homogeneity Using a flow cell with continuous flow can yield similar results Checklist for Best Results Cell V Cell is made of quartz or glass V Apertured cell has black sides Y Apertured cell has an aperture greater than or equal to 3 mm V Cell windows are free of fingerprints and other contamination V Flow cell used instead of an apertured standard cell Measurements V Solution in cell is free of floating particles Y Solution in cell and cell walls are free of bubbles Y Solution in cell is mixed homogeneously V Blank measured on same solvent as sample Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Good Measurement Practices 4 V Blank measurement shows a flat baseline Figure 9 and
75. rpo ETE Math Results AEA Aan ame 8e Clear Standards and Clear Math Results can be used to delete the current standards and the current mathematical results spectra from the Agilent ChemStation memory Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Print Preview of Reports Print preview allows you to view the report in a separate Agilent ChemStation window based on the currently configured printer All types of available reports can be checked page by page in the preview window The number of pages generated and the layout also can be checked In addition the currently displayed report can be printed Print Preview of a Results Report The print previews work similarly for all available types of reports The example below shows you how to preview your results report 1 Choose Print Preview Results from the File menu fail 845x UV Visible System 1 Online TK File Edit Method Measure Instrument Math View Mode ECM Config Help Load Se lt untitled gt Import Export Selected Spectrum As AEE New Method Load Method Save Method As Set Method Password Load Results Save Results As Print Print To File a Print Preview Current View Printer Setup Selected Window Exit ChemStation Method 1 CACHEM32 1 METHODS CAFFEINE ANALYSIS 2M fet 2 3 Automation Method Automation Result Agilent Cary 8454
76. rt Export Selected Spectrurn As mple Spectra New Method Load Method Save Method As Set Method Password Load Results Save Results As Print 3 Current View Print To File Selected Window pring Preview wena Printer Setup Results Exit ChemStation Automation Method Aute Results 1 CACHEM32 I METHODS CAFFEINE ANALYSIS 2 M Arradon masuk 2 Dilut Factor Caf 3 e 10 NOTE To print your method report your printer must be properly configured and online If your printer is currently not online you can view the print preview on the screen 80 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Saving and Retrieving Data This section explains how to save and retrieve measured data This data can be used for archiving for method development at a later stage or for exchange with other Agilent ChemStations Your Agilent ChemStation can store and retrieve your data using a binary checksum protected data format extension sd std All current spectra samples or standards can be saved to disk for permanent storage Saving and loading of data is possible using local and network drives In addition a single spectrum can be selected for storage Saving your Samples 1 Choose Save Samples As from the File menu or click the toolbar icon 9 45x LV Visible System Online TK H Edit Method Measure Instrument Math View Mode ECM
77. s Manual 41 2 42 Introduction to Your System General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview Standard Mode Data Processing General Data Processing Although knowledge of the internal design of the data flow and processing is not required to use Agilent ChemStation software it can be useful to understand how Agilent ChemStation processes your data and how this processing is controlled by your method parameters The data processing can be described using a model of data containers and operations visualized in a flow diagram Spectral Processing Data Access Data Evaluation All basic data go into a raw data container This container is empty when you start your Agilent ChemStation session and it is filled by measuring data or loading data from a file The raw data container held the originally acquired data as specified with your acquisition parameters and stamped with for example acquisition date and time and the acquisition operator s name Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 Spectral Processing Data type Transmittance Absorbance 1st Derivative 2nd Derivative J 3rd Derivative ih Marinhana Your method defines how this data is analyzed The first processing step is spectral processing The processed raw data spectra are transferred automatically into a second container for processed spectra
78. the Prev and Next buttons Jump to a page directly using the selection box The Print button sends the report to the printer which is displayed in the lower left corner of the print preview window HP LaserJet 5L PCL on LPT 1 44 210 x 297 mm Portrait The Close button closes your print preview window and discards the report Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 89 5 Using your UV Visible Spectroscopy System Fin bsorbance Maximum ding the Finding the Caffeine Absorbance Maximum This section describes how you find the absorbance maximum for your caffeine IQ sample 1 Make sure that you are in the Standard mode The mode is indicated on the tool bar of your Agilent ChemStation session 2 Select the Spectrum Peaks task in the analysis panel s selection box Task Fixed Wavelengths v Fixed Wavelengths Spectrum Peaks Ratio E quation Quantification 3 Use the Setup button of the analysis panel to open the parameter dialog Task Spectrum Peaks v 4 90 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 4 Type 2 for the number of peaks to find and uncheck the Valley find option Set your data type to absorbance and adjust your spectral display to a wavelength range from 190 nm to 400 nm in the dialog s Display spectrum section Click OK to set your parameters Spectrum
79. tructure 37 Standard Mode Tasks 39 Standard Mode Data Processing 42 Installation and StartUp 49 Installation Summary for Your General Purpose UV Visible System 50 General 50 Spectrophotometer 50 PC 51 Starting a Measurement Session 52 Good Measurement Practices 53 General Considerations 54 Spectrophotometer Design 54 Making Measurements 54 Sample Cell Material 55 Optical Specifications of Cells 56 Apertured Cells 57 Flow Cells 58 Handling and Maintaining Cells 59 Solvents 61 Sample Preparation 62 Photosensitive Samples 63 Stirring and Temperature Control 64 Checklist for Best Results 64 Inserting a Cell 67 Using your UV Visible Spectroscopy System 69 Starting Your First Measurement Session 70 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Contents Starting Your UV Visible Software 72 Measuring Caffeine Absorbance at 273 nm 73 Saving Your Parameters as a Method 76 Retrieving and Printing a Method 78 Saving and Retrieving Data 81 Saving your Samples 81 Saving a Selected Spectrum 83 Retrieving Spectra 85 Deleting Current Spectra 86 Print Preview of Reports 87 Finding the Caffeine Absorbance Maximum 90 Entering your Cell s Path Length 94 Controlling your Sipper System 95 Using your Multicell Transport 97 Quantitative Analysis using a Calibration with Standards 100 Setup 101 Calibration 103 Analysis 105 How Can Be Sure That My Spectrophotometer Works Properly 107 Spectrophotom
80. turation Kinetics Dissolution Testing Multibath Dissolution Testing Combined Report Verification and Diagnostics Depending on your needs modes are available for Advanced operation Dissolution Testing runs Multibath Dissolution Testing runs Combined Reports evaluation Kinetics measurements Thermal Denaturation studies and Color Calculations These modes of operation can be switched within a running Agilent ChemStation session All current raw data will be preserved during such a switch This allows you to view your data from different perspectives Most of the modes offer you the ability to define your analytical task by a set of parameters and if necessary data A set of parameters and data can be saved as a method This allows you to repeat your analysis task under defined conditions simply by loading a method and running your samples 38 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Introduction to Your System 2 Standard Mode Tasks The Agilent ChemStation modes also offer the ability to focus on specific tasks The standard mode of your general purpose software for UV Visible spectroscopy is oriented towards the most common tasks performed in a analytical laboratory that employs UV Visible spectroscopy Four tasks are available e Fixed Wavelength e Spectrum Peaks e Ratio Equation e Quantification A task is selected and activated from the analysis panel s selection box Task Fixed W
81. urement that is used in all subsequent sample measurements until a new blank measurement is taken Following the reference measurement an additional baseline spectrum is measured and displayed on the PC e SAMPLE the spectrophotometer takes a sample measurement or starts a series of measurements This depends on the parameters set in your software e STANDARD the spectrophotometer takes a measurement of a standard Additional information such as concentration and so on have to be entered in the operating software e Srop the spectrophotometer and or software aborts any ongoing activity and returns to a to ready state Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 29 2 Introduction to Your System Remote connector Multicell transport connector GP 10 connector Rear View All connections are made at the rear of the spectrophotometer see Figure 3 GPIB connector and switches MIO slot for LAN interface Line connector RS 232 connector 30 Figure 3 Rear view of spectrophotometer The multicell connector allows you to connect a multicell transport The GPIO general purpose input output connector allow you to control a sipper and autosampler or other accessories depending on the software you are using The remote connector is currently not used by Agilent instrument control software It can be used e g to synchronize instruments The RS232C connector may be used t
82. view 28 rear view 30 spectroscopy system 23 spectrum 39 standard 41 45 external 107 standard cells 67 standard push button 29 standard single cell cell holder 67 standards 100 current 45 minimum required number 46 number of 46 status 52 status indicator 29 stirring 62 stirring module 64 stop push button 29 stoppered sample cell 62 stray light correction 24 26 sulfuric acid 61 T task fixed wavelength 39 40 73 orientation 39 quantification 39 41 100 quantitative analysis 100 ratio equation 39 41 44 spectrum peaks 39 40 TCP IP protocol 51 temperature control 62 test kit 41 thermostattable cell holder 64 three point drop line 40 43 toluene 61 tool bar 34 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual transmittance 40 width of slit 26 trimethylpentane window 36 2 2 4 trimethylpentane 61 sample results table 43 tungsten lamp 24 tutorial basic principles 110 derivative spectroscopy 110 U understanding Agilent ChemStation processing 42 used wavelength 43 useful wavelength range of solvents 61 user definable equation 41 user interface elements 33 UV grade water 62 UV Visible spectroscopy basics 110 UV Visible system 70 V valley find 91 view 36 39 calibration 100 results 100 105 samples 75 93 standards 100 viscous solutions 64 volatile solvents 62 volume 41 W warning no results present 88
83. ware 2 Introduction to Your System Introduces your UV Visible spectrophotometer and the Agilent ChemStation software 3 installation and Start Up Summarizes the system installation process and explains how to start a measurement session 4 Good Measurement Practices Provides guidance for good measurement practices 5 Using your UV Visible Spectroscopy System Provides examples of basic measurements and related tasks Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 3 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Agilent Cary 8454 Contents 1 Safety Information 9 Safety Information 10 Warning Symbols 10 Precautions 12 Solvent Information 13 Flow Cells 13 Solvents 13 Other Precautions 15 Protective Earth 15 Disconnecting Device 15 Allowing Hot Instrument to Cool 16 Environmental Conditions and Power Requirements 17 Installation category 18 Pollution level 18 Electrical Power 18 Regulatory Information 19 CE Compliance 19 Electromagnetic Compatibility 19 Agilent Technologies on the Internet 21 Introduction to Your System 23 Agilent Cary 8454 UV Visible Spectrophotometer Overview Optical System Overview 24 Spectrophotometer Description 28 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 24 Contents General Purpose Agilent ChemStation Software for UV Visible Spectroscopy Overview 32 User Interface Elements 33 Software S
84. with the spectrophotometer Storing the self test results with the spectrophotometer allows you to monitor the performance over time Graphical representations of the self test histories can be generated 108 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 What Maintenance Does My Spectrophotometer Require Cleaning Any spills should be wiped up immediately The exterior surfaces of the spectrophotometer should be kept clean All cleaning should be done with a soft cloth If necessary this cloth can be dampened with water or with a mild detergent Do not use organic solvents or abrasive cleaning products Spare Parts For spare parts and consumables ordering information refer to the Agilent Technologies website at www agilent com Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 109 5 Using your UV Visible Spectroscopy System How Can I Get a Deeper Understanding of UV Visible Spectroscopy How Can I Get a Deeper Understanding of UV Visible Spectroscopy The basic principles of UV Visible spectroscopy are covered with your help system This covers Basic Principles through to details of Derivative Spectroscopy UV Vis ChemStation Help Hide Back Forward Contents Index Search E E Installation E Getting Started E Good Measurement Practice E Working with the Spectrophot E G General Features E E Worki
85. y for you to refer to the manual in order to understand a hazard Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide Safety Information 1 The following symbols appear on the instrument for your information The hazard they describe is also shown Single phase alternating current When attached to the rear of the instrument indicates that the product complies with the requirements of one or more EU directives Indicates a protective ground terminal Eye damage may result from directly viewing light produced by deuterium lamps used in detectors and spectrophotometers Always turn off the deuterium lamp before opening the lamp door on the instrument Agilent Cary 8454 UV Visible Spectroscopy System Installation Guide 11 1 Safety Information Precautions 12 Before applying power comply with the installation section Additionally the following must be observed Do not remove instrument covers when operating Before the instrument is switched on all protective earth terminals extension cords auto transformers and devices connected to it must be connected to a protective earth via a ground socket Any interruption of the protective earth grounding will cause a potential shock hazard that could result in serious personal injury Whenever it is likely that the protection has been impaired the instrument must be made inoperative and be secured against any intended operation Make sure that only fuses with
86. y high quality lens tissues part number 9300 0761 and never dry the inside of a cell with lens tissues Dry the inside of the cell with pressurized oil free air that prevents the cell from contamination with tissue particles or rinse the cell with blank or sample solution Floating particles in the cell will deflect the light beam and result in very poor quality of the measured spectrum lt gt Floating particles will deflect and scatter the light beam Figure 7 Floating particles in a cell Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 59 4 Good Measurement Practices CAUTION 0 01 0 00 0 01 0 02 0 03 0 04 200 400 600 800 1000 Figure 8 Spectrum taken with floating particles in the light path Lens tissues for glasses or other uses often contain detergents or lubricants which can affect your measurements If possible avoid cleaning the faces of your cell between blank and sample measurements Handling Cells Always install a cell so that it faces the same direction to minimize problems with cell non uniformity For best results with microcells leave your sample cell clamped in position throughout the measurement sequence Solutions should be removed and replaced by pipette or use flow cells If glass pasteur pipettes are used make sure that the optical windows of the cell are not touched or scratched by the pipette 60 Agilent Cary 8454 UV Visible Spectroscopy
87. your cell see Inserting a Cell on page 67 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual 71 5 Using your UV Visible Spectroscopy System Starting Your UV Visible Software This section describes how to start an Agilent ChemStation session on your PC If you want to perform measurements you can start an online session or you can start an offline session for optimizing the analytical parameters of a method recalculating results or printing reports An online session can be started only as the first instance on your PC An offline session can be started in parallel This allows you to optimize your method settings by direct comparison based on identical sets of data 1 Switch on your PC monitor and printer 2 Log on to your PC s operating system 3 Start your Agilent ChemStation session by selecting Instrument 1 online for a measurement session or Instrument offline for method optimization and data evaluation 4 Enter your name to log on to your Agilent ChemStation session If you protected your manager level by password you must enter the correct password The system will then come up with the last used mode and method 72 Agilent Cary 8454 UV Visible Spectroscopy System Operator s Manual Using your UV Visible Spectroscopy System 5 Measuring Caffeine Absorbance at 273 Measuring Caffeine Absorbance at 273 nm This section describes how to measure the caffeine sample that was shipped with your sp

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