Home

Agilent 8453 UV-visible Spectroscopy System

1. VY 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 power cord designed for your region VY 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 36 Agilent 8453 UV visible Spectroscopy System Operator s Manual Installation and Start Up 2 Connection and Configuration in your Installing Your UV visible Spectroscopy System manual PC VY Make sure that all of your PC components are connected to line power VY Make sure that your general purpose software for UV visible spectroscopy is installed VY 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 8453 UV visible Spectroscopy System Operator s Manual 37 2 Installation and Start Up
2. Starting a Measurement Session 38 With a network connection to your spectrophotometer it is important that your spectrophotometer is recognized by the software This requires the assignment of a unique IP address at power on time to your spectrophotometer The assignment is either done by the Agilent BootP Service application running on your PC with a direct connection to the spectrophotometer or in a LAN 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 VY Switch on your PC and boot your PC operating system If a printer is connected to your system switch the printer on VY 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 Installing Your UV visible Spectroscopy System manual VY Launch your measurement session by pressing your operating system s Start button and select Programs UV Visible ChemStations Instrument 1 online v You are ready to use your system if the blue busy status display on the system s bottom message line turns off VY The first measurement you have to perform is a reference measur
3. andard Selected spectra As Export Selected Spectrum As L Agilent 8453 UV visible Spectroscopy System Operator s Manual 67 4 Using your Agilent 8453 UV visible Spectroscopy System 2 You select one of your already existing data files in the File name selection box of the Save Spectra As dialog or you type a valid file name into the File name edit box Sawe Spectra As File name Folders CAFFEINE SAMPLE SD cAchems2 1 data ADVEMAM SD fee cy ADVES4AM3 5D CHEM 32 ADVEMAM4 SD ADYEKAMS SO ADVEASM SD ADVE 8M6 SD BEN ENE SD NIST _HOLMIUM OSIDE 2034 50 Save file as type Drives Spectral SO S5TD B c Programe A valid file name consist of eight alphanumeric characters and the file extension sd or std Usually the extension std is used for standards only 4 If the file name exists already 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 3 Click OK to start the operation Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Saving a Selected Spectrum 1 Select the spectrum of interest in the graphic window Overlaid Sarniple Spectra e 0 8 0 7 0 6 0 5 EJ sarmnple Result Table Show Sample Into Last Spectrum H Name Dilut Factor Caffeine mg L Abs lt 2 3nm g
4. background correction 26 27 Beer s law 86 benzene 47 blank 38 40 51 blank push button 15 bootp service 56 bubble formation 48 busy 38 C caffeine 59 calculate 33 calculation results 80 calibration 28 32 86 89 coefficients 32 curve 2 carbon disulfide 47 Agilent 8453 UV visible Spectroscopy System Operator s Manual carbon tetrachloride 47 cell path length 80 81 83 cell changer 83 chloroform 47 cleaning cells 45 collimated beam 10 colloidal dispersions 48 command 20 common solvents 47 compartment for sample 12 concentration 33 concentration range 28 connection network 9 connector GPIB 17 GPIO 16 multicell transport 16 remote 16 RS232 16 correction for stray light 10 12 current context 20 current task 22 cut off filter 49 cut off wavelength 49 cyclohexane 4 cyclopentane 47 Index D data 29 absorbance 86 access 30 31 archieve 67 clear 72 clear math results 72 clear standards 72 evaluation 31 file extension 68 file information 72 file name 68 file selector box 72 format 67 load 67 local storage 67 network transfer 67 removing 72 replace 68 retrieve 67 71 save 67 save samples as 67 saving selected data 69 storage 67 degassed 48 derivative 26 derivative spectroscopy 96 description of instrument 14 determining purity 96 deuterium lamp 10 development of an analytical meth
5. 25 measure standard 89 standard information 90 measure push buttons 15 Agilent 8453 UV visible Spectroscopy System Operator s Manual measurement blank 57 noise 5 reference 5 sample 57 91 sample information 91 menu 20 message line 38 methanol 47 method 24 29 32 33 62 calibrated 34 86 current 65 information 65 last used 58 load 64 load method 64 modified 64 name 63 options amp information 63 parameter 29 parameters 62 print preview 66 printing 64 66 report 64 retrieve 64 save method as 62 store 62 methyl formate 47 minimum 25 MIO board slot 17 mode 23 advanced 24 color calculations 24 combined reports 24 dissolution testing 24 execute advanced method 24 kinetics 24 last used 58 multibath dissolution testing 24 standard 24 59 76 87 switch 24 thermal denaturation 24 verification and diagnostics 24 93 mouse cursor 21 multicell transport 83 7 cell 85 8 cell 83 connector 16 m xylene 47 n butyl alcohol 47 network administrator 36 37 connection 38 local 56 n hexane 47 nominal spectral slit width 12 0 offline 23 online 23 open sample area 40 operating system 38 operation 20 operation levels 23 operator level 23 operator name 29 optical filter 49 optical specifications of cells 42 optical surfaces 45 optical system 10 optimization 33 outliers 33 P paper orientation 37 size 37 parallelism 42
6. 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 Indicator Pushbuttons 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 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 On the front 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
7. 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 8453 UV visible Spectroscopy System Operator s Manual In This Guide To be able to use your new Agilent 8453 UV visible spectroscopy system quickly this book gives you step wise procedures and examples for basic operations and tasks This book shall not replace the detailed manuals available for installation Installing Your UV visible Spectroscopy System and operation of your software Understanding Your UV visible Spectroscopy System nor your Agilent 5453 Service Manual 1 introduction to Your System In this chapter you will find an introduction to your Agilent 8453 spectrophotmeter a
8. not touched or scratched by the pipette Agilent 8453 UV visible Spectroscopy System Operator s Manual Solvents Good Measurement Practices 3 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 8453 UV visible Spectroscopy System Operator s Manual 47 3 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
9. one of the position buttons to move the multicell transport 84 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Sampling q Multicell 8 cell Automation Multicell Transport Control Current Cell 1 Move To Close In an automated sequence the multicell transport can be used for automatic sample introduction A maximum of 8 samples can be introduced If you specify more than 8 measurements you get the warning Automation A Number of measurements is out of range 1 8 You may also control an existing 7 position multicell transport The major differences are that you have no separate reference position and one cell position less Agilent 8453 UV visible Spectroscopy System Operator s Manual 85 4 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 86 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 setup your method calibrated samples can be analyzed Several views of both your standards and the calibration as well as on your samples and results are available As a quick introduction a calibration using Beer s law with a single standard and the
10. the menu Your Instrument Panel shows you the current state of the spectrophotometer and the Blank button is enabled Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 6 The first task you have to perform is to measure a reference Typically the cell containing the solvent used with your samples is put in the measurement position and a 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 your cell see Inserting a
11. 27 thermostattable cell holder 50 three point drop line 26 30 toluene 47 tool bar 20 transmittance 26 trimethylpentane 2 2 4 trimethylpentane 47 tungsten lamp 10 tutorial basic principles 96 basics of UV visible spectroscopy 96 derivative spectroscopy 96 U understanding Agilent ChemStation processing 29 used wavelength 30 useful wavelength range of solvents 47 Agilent 8453 UV visible Spectroscopy System Operator s Manual user definable equation 27 user interface elements 19 UV grade water 48 UV visible spectroscopy basics 96 V valley find 77 view 22 25 calibration 86 results 86 91 samples 61 79 standards 86 viscous solutions 50 volatile solvents 48 volume 27 W warning no results present 74 water 47 wavelength 30 wavelength reproducibility 34 wedge shaped cells 42 weight 27 width of slit 12 window 22 graphical 22 sample results table 30 tabular 22 Agilent 8453 UV visible Spectroscopy System Operator s Manual Index 103 Index 104 Agilent 8453 UV visible Spectroscopy System Operator s Manual www agilent com In This Book To be able to use your new Agilent 8453 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 Printed in Australia 08 11 90 042 ee Agilent Technologies
12. 3 Lock the sample cell in place by pushing the locking Utmost care should be taken when using small lever back down volume flow cells and particularly any cells with less oa 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 8453 UV visible Spectroscopy System Operator s Manual 53 3 Good Measurement Practices Inserting a Cell 54 Agilent 8453 UV visible Spectroscopy System Operator s Manual Agilent 8453 UV visible Spectroscopy System Operator s Manual 4 Using your Agilent 8453 UV visible Spectroscopy System Starting Your First Measurement Session 56 Starting Your UV visible Software 58 Measuring Caffeine Absorbance at 273 nm 59 Saving Your Parameters asa Method 62 Retrieving and Printing a Method 64 Saving and Retrieving Data 67 Print Preview of Reports 73 Finding the Caffeine Absorbance Maximum 76 Entering your Cell s Path Length 80 Controlling your Sipper System 81 Using your Multicell Transport 83 Quantitative Analysis using a Calibration with Standards 86 How Can I Be Sure That My Agilent 8453 Works Properly 93 How Can Get a Deeper Understanding of UV visible Spectroscopy 96 When Do I Have to Measure a Blank 98 Bee Agilent Technologies 55 4 Using your
13. OQ PV OQ PV kits for the 8453 are available as part numbers 5063 6503 and 5063 6521 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 Agilent 8453 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 H ry Hode a Verification and Diagnostics r 2 Select the Self Test task in the analysis panel s selection box Task Agilent 8453 UV visible Spectroscopy System Operator s Manual 93 4 Using your Agilent 8453 UV visible Spectroscopy System How Can Be Sure That My Agilent 8453 Works Properly 3 Choose Self Test Start from the Task menu or click Start to start the self test 9 845x UV Visible System 1 Online TK File Instrument View Mode ECM Config Hi Instrument Self test Self Test 3 Setup E Verification b Start Diagnostics b Ta Flow Test b s Information b S Maintenance b J f Dissolution Tests b 4 NOTE 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 The spectrophotometer should be allowed to warm up for 45 minutes P after switching the lamp on Do you want to proceed anyway 94 Agilent 8453 UV visible
14. Password Load Results Save Results As Print gt Current View Print To File b Selected Window ie Preview gt Method Printer Setup Results Exit Chem Station Automation Method 1 CACHEM32 METHODS CAFFEINE ANALYSIS 2 M MCUs 2 Dilut Factor Caf 3 e 10m NOTE To be able to print your method report your printer must be properly configured and online An alternative if your printer is currently not online is to view the print preview on the screen 66 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Saving and Retrieving Data This section explains how you can 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 has the ability to 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 Save and load 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 W atx UV visible System 2 Online TK Edit Method Measure Instrument Math View Mode ECM Config Help Load dave b Samples Asu Standards Asu aa
15. Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 The self test results are displayed with pass fail criteria 8453 Instrument Self Test Results El e Save to Instrument More INFO Specification Measured Result Self test results can be stored 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 Agilent 8453 UV visible Spectroscopy System Operator s Manual 4 95 4 Using your Agilent 8453 UV visible Spectroscopy System How Can I Get a Deeper Understanding of UV visible Spectroscopy The basic principles of UV visible spectroscopy are available with your help system The information contained in the Tutorial s Basics of UV Visible Spectroscopy covers Basic Principles through to details of Derivative Spectroscopy UV Vis ChemStation Help Amma e gt Hide Back Forward Basic Operation Good Measurement Practice J Working with the Spectrophat General Features Working with Files Working with Spectra Printing the Results ECM in the ChemStation Getting started Good Measurement Practice Working with the Spectrophotoreter and its Accessories General Features Working with Files Working with Spectra Printing the Results ECM inthe Chemstation hermal Denaturation Mode Multibath Dissolution
16. Wor Annotate Standards Math Results ah Te a eee RRS rT AEA ANG Mirame 8e Clear Standards and Clear Math Results can be used to delete the current standards respectively the current mathematical results spectra from Agilent ChemStation memory 72 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Print Preview of Reports Print preview allows you to look at 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 gg 845x UV Visible Systern 1 Online TK Edit Method Measure Instrument Math View Mode ECM Config Help Load Save lt untitled gt Import Export Selected Spectrum As gt imple Spectra New Method Load Method Save Method As Set Method Password Load Results Save Results As Print Print To File ae Print Preview Current View Printer Setup Selected Window Exit Chem tation Method Results Automation Method 1 CACHEM3ATNMETHODS CAFFEINE ANALYS
17. analysis of a sample are described Further 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 as sample For the calibration we use absorbance data at 273 nm Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Setup 1 Make sure that you are in the Standard mode The mode is indicated on the tool bar of your Agilent ChemStation session Clear a Method Mode lt untitled gt Standard 7 2 Select the Quantification task in the analysis panel s selection box iy Task Fixed Wavelengths Fined Wavelengths Spectrum Peaks R atio E quation Quantification 3 A new task panel is displayed and the Quantification Parameters dialog is opened automatically Task kai 8 amp aga me K If you are already in the Quantification task use the Setup on the analysis panel to open the parameter dialog Agilent 8453 UV visible Spectroscopy System Operator s Manual 87 4 Using your Agilent 8453 UV visible Spectroscopy System 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
18. 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 WA 8 A500 UV Misible Systern 1 Offline TK H Edit Method Measure Instrument Matl S A Load d Sawe e p pe Import e Export Selected Spectrum 45 Mew Method Load Method Sawe Method 445 Set Method Password Load Results Sawe Results As Print p Print To File Print Previews zz Printer Setup Exit Chemtation 62 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 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 Save Method As File name New Caffeine Analysis M c chem3z T methods CAFFEINE ANALYSIS EQUATION M CAFFEINE ANALYSIS M E CORTISONE CORTISOLE ANALYSIS M gt chem32 DNA MELTING M 5 1 P_PHENYL ACETATE HYDROLYSIS M 5 methods STDEFMET M ad CAVE AN YINSTALIMENT 1 M Li Save file as type Drives Method M E c Programs If you generate many methods you can use Options amp Infos from the Method menu to add text for documentation purposes and to simplify me
19. degrade photosensitive samples If you have a problem you can selectively 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 D5 Lamp off The short wavelength radiation leading to photodegradation comes from the light of the Do lamp For application where readings are taken at wavelengths above 400 nm the Do 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 conditions of your application Agilent 8453 UV visible Spectroscopy System Operator s Manual 49 3 50 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 integratio
20. e Quantification A task is selected and activated from the analysis panel s selection box Task Fixed Wavelengths Br Fired Wavelengths Spectrum Peaks Aatios Equation Quantification This 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 do offer only different views to your data acquired 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 8453 UV visible Spectroscopy System Operator s Manual 25 1 26 Introduction to Your System Fixed Wavelength Task Fired Wavelengths 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 Sp
21. for sample information Select Absorbance as Data type and set Display spectrum From 190 nm To 340 nm Quantification Parameters wavelengths Use wavelength ofa nm a correction mans Calibration Analyte name Catene Calibration curve type Enter Concentration Concentration mgL Unit weight Volume D Weight amp Volume mg fal Prompt for standard information Prompt for sample information Data type Display spectrum From 180 To a 5 Click OK to set your parameters Now you are ready to run your measurements 88 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Calibration 1 Fill your l 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 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 orie
22. parameter dialog 76 particulate matter 48 passivating new cells 45 path length setup 80 Agilent 8453 UV visible Spectroscopy System Operator s Manual Index PC 38 peak find 77 performance verification 93 photochemical reactions 49 photodegradation 49 photodiode array 13 photosensitive substances 49 pipette 46 plasma discharge 11 plastic door 17 plastic sample cells 41 pointer symbol 21 poor linearity 43 poor photometric accuracy 43 printer 37 38 configured 73 processed spectra 30 processing 29 spectral 30 standards 32 pump peristaltic 81 purity 27 push buttons 15 blank 15 sample 15 standard 15 stop 15 pyridine 47 Q quantification 25 87 quantitative analysis of samples 86 ready for analysis 90 quartz sample cells 41 radiation source 10 rapid absorbance changes 50 ratio 25 101 Index raw data 29 rear view of spectrophotometer 16 recalculation 23 recommended cells 43 reference 38 remote connector 16 report results 73 result 33 results 30 precise 5 routine work 62 RS232C connector 16 S sample 40 51 compartment 12 push button 15 sample cell 40 48 sample data 32 sample information 27 sample result table 61 69 79 sampling device 21 sampling interval 12 sampling system 80 manual 80 sipper 81 security lever 17 self test 93 histories 95 results 95 start 94 working conditions 94 sensitiv
23. this data analysis concept you can measure your standards before or after your samples and besides the minimum required number of standards you can use as many standards you like to use 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 and re calibrating your system And the elimination of outliers in your calibration is possible by simply removing this standard from you standard data set Agilent 8453 UV visible Spectroscopy System Operator s Manual 33 1 Introduction to Your System 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 In addition you may 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 e 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 The data analysis co
24. to Your System 1 In the Ratio Equation task for example this used wavelength data is processed by an evaluation of the equation specified This additional operation generates the calculation results These calculated result values are filled into the results container The results are available with the Sample Results Table Sample Result Table co tel es Show Sample Info Last Spectrum Analyze Ht Mame Bilut Factor Caffenne mg fL Abs lt 27 3nm gt 1 00000 0 28903 0 57807 Summary 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 8453 UV visible Spectroscopy System Operator s Manual 31 1 Introduction to Your System General Purpose Agilent ChemStation Software for UV visible Spectroscopy Overview Processing with Standards In the forth task in addition to your sample data standards are used This requires an extension to the above concept to handle standards Two independent sets of containers one for standards and another for samples have been implemented All processing containers are doubled as well As with the sample processing only all evaluation steps are done in
25. 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Finding the Caffeine Absorbance Maximum 9 The view shows you the spectrum of your caffeine sample Two peaks were found marked and these 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 Spectrum Sample Result Table sl Gos Show Sample Into Overlay Spectra Delete Selected Sample All Peaks Valleys Agilent 8453 UV visible Spectroscopy System Operator s Manual 79 4 Using your Agilent 8453 UV visible Spectroscopy System Entering your Cell s Path Length The cells used for your measurements are specified with the sampling system parameters In quantitative calculations these parameters are used in results calculation Due to the freedom of choices for the cell path length you must provide the correct value with the path length setting Usually you get this information from the supplier of your cells You so set the path length in manual mode cell handling as follows 1 Click Setup on the Instrument Panel Sampling 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 80 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy Syst
26. Agilent 8453 UV visible Spectroscopy System iy Agilent Technologies Notices Agilent Technologies Inc 2002 2003 2008 2011 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 90042 Edition 07 11 Printed in Australia Agilent Technologies 679 Springvale Road Mulgrave Victoria 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 ts for B 04 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 or implied with regard to this manual a
27. Agilent 8453 UV visible Spectroscopy System Starting Your First Measurement Session 56 1 Make sure that your Agilent 8453 UV visible system has been installed correctly For details of installation see your Installing Your UV visible Spectroscopy System manual 2 Switch on your PC monitor and printer 3 Log on to your PC operating system Check 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 Agilent 8453 spectrophotometer into your local network File Action View Help H Go is B bl gt gt Sh Services Local Name Description Status Startup Type Log On As Agilent Bootp Service Started Automatic Local Systern Sab Ag p y Agilent IO Libraries Service Provides syste Started Automatic Local System ag y yst Application Experience Processes appli Started Automatic Local System Ks Application Host Helper Ser Provides admin Started Automatic Local System A Application Information Facilitates the r Manual Local System W Application Layer Gateway Provides suppe Manual Local Service 4 Switch on your Agilent 8453 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 5 Start your measurement session by selecting Instrument 1 online from
28. Cell on page 53 Agilent 8453 UV visible Spectroscopy System Operator s Manual 57 4 Using your Agilent 8453 UV visible Spectroscopy System Starting Your UV visible Software This section describes how you 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 In addition 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 1 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 58 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System Measuring Caffeine Absorbance at 273 nm This section describes how you measure your caffeine sample that was shipped with your spectrophotometer Measurement of this caf
29. I0 connector RS 232 connector 16 Rear View All connections are made at the rear of the spectrophotometer see Figure 3 GPIB connector e aa aidew ches Z Fe F B O i sy O LAN interface k 4 Line connector Two CAN connectors Figure 3 Rear View of Spectrophotometer e The multicell connector allows you to connect a multicell transport e 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 e The remote connector is currently not used by Agilent instrument control software It can be used e g to synchronize instruments e The RS232C connector may be used to 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 Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 e 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 Only a single controller must be connected to the spectrophotometer at a time e Th
30. IS 2 M 2 3 Automation Results Agilent 8453 UV visible Spectroscopy System Operator s Manual 73 4 Using your Agilent 8453 UV visible Spectroscopy System 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 Wo results present on the message line Mo results present 2 The report generated will be displayed in the preview window A Fixed Wavelength Report Mil Ei a Fixed Wavelength Report Date 10 26 99 Time 10 36 42 Page 1 of 1 Method file cunt it led Information Default Method Data File Ci HPCHEM 15 DATA ADVE SANG SD Created 02 06 1992 11 18 45 Overlaid Spectra Sample 1 sample 2 Sample 3 ai m T a C L m ii E Hame 4be lt 41 4nm Name 4be lt 4 4nm gt 1 Sample 1 0 82494 3 Sample 3 0 10637 2 Sample 2 0 21156 Report Generated by TE SS he ease eda ee 74 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 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 The following functions are available w
31. 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 2 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 Solent Wi abed Dilution Factor 1 Comment rok diluted Cancel 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 8453 UV visible Spectroscopy System Operator s Manual 91 4 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards E Overlaid Sample Spectra Show Sample Info Last Spectrum Delete Selected Sample Analyze NOTE To save your data for future use or for documentation purposes see Saving and Retrieving Data on page 67 for more information 92 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 How Can I Be Sure That My Agilent 8453 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
32. 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 measurement 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 STop the spectrophotometer and or software aborts any ongoing activity and returns to a to ready state Agilent 8453 UV visible Spectroscopy System Operator s Manual 15 1 Introduction to Your System Remote connector Multicell transport connector GP
33. Testing Mod Combined Reports enfication and Diagnostics Mode Report Style Editor Maintenance troubleshooting Replacement Parts E FI An Introduction to Uy Visible Spec 9 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 96 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 How Can 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 OVVVO VHCmriolatiOn FT A Z x Ka Hide Back Forward Online Help Installation 5 Basic Operation Sq Standard Mode User Interface Reference within the views and dialog boxes of the user interface elect How to for information to help you make the choices presented How to use Standard Mode S Sy Standard Mode Examples B Correcting for Backgrounc fz Using an Internal Referen B Using an Internal Referen 2 Confirming the Identity of 2 Checking the Purity of a S Using Derivative to Emph B Calibrating and Quantifyirn Execute Advanced Method Mode Advanced Mode Kinetics Mode Thermal Denaturation Mode Dissolution Testing Mode Multibath Dissolution Te
34. al or a tabular representation You may see a graph showing your measured sample spectra and 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 are interested in 22 Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 Software Structure To reduce complexity of operation the software is divided into specific applications called modes In addition levels of operation are available and 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 vis instruments based 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 Agilent 845x UV visible System 1 845x UV Visible System 1 Onli File Edit Method Measure Instrument Math View Mode ECM Config Help Each instrument session is available for data analysis only and for instrument control An instrument control session has the appendix Online and can be started as the first instance only on your PC In addition a data analysis sessi
35. 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 sampling interval is about 0 9 nm Agilent 8453 UV visible Spectroscopy System Operator s Manual 13 1 Introduction to Your System Spectrophotometer Description Line power switch with green light 14 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 8453 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
36. ction 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 allows to control 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 H Waa m Method Mode D N y fla W5 IS WAVELENGTH ACCURACY TEST M Mod Standard z 20 The tool bar below the menu bar shows buttons with symbols icons which allow 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 treeview for saved files The navigation bar is dockable and can be hidden or resized The navigation bar window provides faster direct access to file system and mode switch functionality that also continues to be available in its traditional location in the menu bar 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 treeview by double clicking Clicking on the Browse section allows access to any available data directory Analysis Panel The analysis panel gives you a graphical visualization of the current context in which you are working In addition it pr
37. e Absorbance Tat Denvative 2nd Denvative ard Derivative AL Mee ob a 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 This concept allows you to have a look at the results of this processing step by viewing the processed spectra container s content If you for example specified 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 30 Wavelengths Use wavelength of nm ee thee point drop tine 300 380 mm A next step in the data analysis process is the access to 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 on these data is available with the Sample Results Table window Results An additional step is the evaluation of the accessed data Calculation Hame Equation ML MLE Wath W t Unit Caffeine WLT 0 05 rg L VW eight Volume Use Weight part Volume Unit Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction
38. e MIO board slot is reserved for a LAN interface board e 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 door 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 8453 UV visible Spectroscopy System Operator s Manual 17 1 Introduction to Your System General Purpose Agilent ChemStation Software for UV visible Spectroscopy Overview 18 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 on a practical point of view Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 General Purpose Agilent ChemStation Softwar
39. e for UV visible Spectroscopy Overview User Interface Elements Method name Tool bar Menu bar Analysis panel Data files Navigation bar Modes Instrument panel Tabular results Spectra 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 845x UV Visible System 1 Online x File Edit Method Measure Instrument Math Yiew Modd Config Help Clear S amp S HW Method 7 a TT m Task a i STDEFMET M SE Samples C Chem3241 D Overlaid Sample Spectra f olx sp Browse u ADVEXAM2 SD Spectrum Peaks z iy ADVEXAM3 SD u ADVEXAM4 SD 4 AD A D ADVEXAM7 SD a ADVEXAM8 SD 4 u BENZENE SD a SAVEFILE SD u STDEXAM1 SD a STDEXAM2 SD M Sooawsso PA liu STDEXAME SD Manual z a STDEXAM8 SD lt gt wW aaa feces Sone All Peaks Valleys X Advanced S Thermal Denaturation A Kinetics 4 Dissolution Testing Multibath Dissolution Testing Combined Report A erification and amp K Diagnostics i al Agilent 8453 UV visible Spectroscopy System Operator s Manual 19 1 Introdu
40. ectrum Peaks Task Spectrum Peaks z 3 In the Spectrum Peaks task you are looking at absorbance minima and maxima The focus here is more on the wavelength scale but you get the according absorbance readings in addition Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 Ratio Equation i EGINI A Ua eli oy i AAS Il ii Liat The Ratio Equation task is used to perform a user definable equation based on measured data and sample information An equation can be setup using sample data at up to six wavelengths and weight and volume data 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 is to use a ratio of data values to check for the identity or the purity of a sample Quantification Task K A Pd egoin 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 in addition background corrections can be applied to your data Agilent 8453 UV visible Spectroscopy System Operator s Manual 27 1 28 Introduction to Your System 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 automat
41. em 4 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 have to adjust 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 adjust your sipper system parameters For details see your Installing and Operating Your Sipper System manual 1 Select the Sipper in the Instrument Panel selection box Sampling HMulticell 7 cell HMulticell 8 cell Autosampler AY Autosampler Gilson 2217222 2 Click Setup on the Instrument Panel Type the path length of your flow cell in cm and click OK Setup Sipper Path Length crm 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 8453 UV visible Spectroscopy System Operator s Manual 81 4 82 Using your Agilent 8453 UV visible Spectroscopy System Sipper Parameter Pump Time Ei z Pump Direction wait Time 3 Z Sample Aeturn 0 zE wash Time O amp Ar Segment 0 4 Click OK in the Sipper Parameter dialog and click OK in the Sipper dialog to set the parameters Every measurement you start by clicking one of the Instru
42. ement After this alignment your are ready to measure absorbance data and spectra lt takes about 15 minutes for the lamps to reach stable state conditions For best results do not perform measurements before this period of time has elapsed Agilent 8453 UV visible Spectroscopy System Operator s Manual Agilent 8453 UV visible Spectroscopy System Operator s Manual ee 3 Good Measurement Practices General Considerations 40 Inserting a Cell 53 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 Bee Agilent Technologies 39 3 Good Measurement Practices General Considerations 40 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 Agilent 8453 spectrophotometer is open Unlike conventional instruments the Agilent 8453 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
43. ertures Figure 5 Recommended Cells CAUTION Quartz cells with black apertures smaller than 2 mm when used with a multicell transport can lead to measurements of poor reproducibility Agilent 8453 UV visible Spectroscopy System Operator s Manual 43 3 Good Measurement Practices A E 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 the 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 44 Agilent 8453 UV visible Spectroscopy System Operator s Manual Good Measurement Practices 3 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 you should apply 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 surface
44. feine sample is also used for the IQ installation qualification of your Agilent 8453 spectrophotometer 1 Make sure that you are in Standard mode The mode is indicated on the toolbar of your Agilent ChemStation session Ue Clear lt 4 Method Mode lt untitled gt Standard X 2 Select the Fixed Wavelength task in the analysis panel s selection box Task Fired Wavelengths Fixed Wavelengths Spectrum Peaks Aatio E quation Quantification 3 Click Setup in the analysis panel to open the parameter dialog Task Fired Wavelengths Agilent 8453 UV visible Spectroscopy System Operator s Manual 59 4 4 Using your Agilent 8453 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 Wravelength s Parameters Wavelengths Use wavelengths 273 nim Sate none oD comection Bone C Prompt for sample information Data type Display spectrum 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 font and the back of the spectrophotometer Push the lever down to secure your cell in the cell holder 60 Agile
45. ically performed based upon your current standard data Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 Panars Disyrnneo Agnilan Cham Ctat ag a Cattharara tar IV waeesthin nari f varun a a F iyi 1 Ve a LNA Io AAS F r ay as a SNA d amp ff nw wart a UECNCral FUPPOSE AGHENT GNCMolalUon SOIVWale TOT VISIDIE VDDECLIOSCOD VeVi Standard Mode Data Processing General Data Processing Although knowledge of the internal design of the data flow and processing in not required to use Agilent ChemStation software it helps to understanding how Agilent ChemStation processes your data and how this processing is controlled by your method s parameters The data processing can be easily 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 will be 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 as well as the acquisition operator s name Agilent 8453 UV visible Spectroscopy System Operator s Manual 29 1 Introduction to Your System Spectral Processing Data type Transmittanc
46. ion is shown in the File Information section of the dialog box Look in J METHODS Date modif Type Fy TATA TRAD AA TIDE LATA LTR TL LILES LITE T LATTE TALE RE LEELA ETRE EE EELS _ CORTISONE CORTISOLE ANALYSIS L DNA MELTING M _ P PHENYL ACETATE HYDROLYSIS M STOEFMET M TEST SAVE ONLY INSTRUMENT 1 M _ WAVELENGTH ACCURACY TEST M _ YELLOW DYE 3 WAVELENGTHS M File name CAFFEINE ANALYSIS M Files of type Method M ki Cancel Help File Information c chem3241 methods caffeine analysis m METHOD Method Type Standard Quantification Created by TK LastUpdate Date 06 06 2008 Time 04 3 Calibrated No Number of Standards O I 3 If you want to load this method click OK Whenever you change a parameter of the current method you get an indication in the toolbar s modification field Clear H t H Method ll A ER An WAVELENGTH ACCURACY TEST H Hod This triggers the reminder dialog mentioned above Agilent 8453 UV visible Spectroscopy System Operator s Manual 65 4 Using your Agilent 8453 UV visible Spectroscopy System Retrieving and Printing a Method 4 To print a method choose Print Method from File menu te 845x UV Visible Systern 1 Online TK File Edit Method Measure Instrument Math View Mode ECM Config Help Load Save lt untitled gt Export Selected Spectrum As gt imple Spectra New Method Load Method Save Method As Set Method
47. ith the print preview window The Prev and Next buttons allow you to browse through the report pages A selection box allows to jump to a page directly The Print button sends the report to the printer which is displayed in the lower left corner of the print preview window HP Laserlet 5L PCL on LPT1 Ad 210 8 297 mm Portrait The Close button closes your print preview window and discards the report shown Agilent 8453 UV visible Spectroscopy System Operator s Manual 75 4 Using your Agilent 8453 UV visible Spectroscopy System 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 Clear H H Method Mode lt untitled gt Standard 2 Select the Spectrum Peaks task in the analysis panel s selection box Task Fixed Wavelengths El Fixed Wavelengths Spectrum Peaks Hatio E quation Quantification 3 Use the Setup button of the analysis panel to open the parameter dialog Task Spectrum Peaks L 76 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 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 fro
48. ity 43 session data analysis only 23 instrument control 23 set of parameters 24 settings 62 setup dialog 20 sheet metal door 17 102 shine through aperture 11 shutter 10 11 signal to noise ratio 49 single beam instrument 40 sipper 81 flow test 81 parameter 81 sipper system 44 sipper sampler system 48 slit 10 13 slit width 12 slots for MIO and accessory boards 17 software general purpose 9 18 solute convection 48 solution 50 solution noise 48 solutions 96 determining purity 96 enhancing sensitivity 96 solvent 40 57 solvent suitability 47 solvents 47 source lens 10 11 source of radiation 10 spectral acquisition 27 spectral operation 30 spectral processing 31 spectral raw data 34 spectrograph 12 lens 10 slit 10 spectrophotometer 33 36 front view 14 rear view 16 spectroscopy system 9 spectrum 25 standard 27 32 external 93 standard cells 53 standard push button 15 standard single cell cell holder 53 standards 86 current 32 minimum required number 33 number of 33 Status 38 Status indicator 15 stirring 48 Stirring module 50 stop push button 15 stoppered sample cell 48 stray light correction 10 12 sulfuric acid 47 symbol 20 T task fixed wavelength 25 26 59 orientation 25 quantification 25 27 86 quantitative analysis 86 ratio equation 25 27 31 spectrum peaks 25 26 TCP IP protocol 37 temperature control 48 test kit
49. le VY Blank measurement shows a flat baseline Figure 9 and Figure 10 on page 52 show a good and a poor baseline v Cell orientation of blank and sample measurements is the same VY Ideally the cell is not removed between the measurement which means the cell is filled rinsed using a pipette or a flow cell is used VY 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 8453 UV visible Spectroscopy System Operator s Manual 51 3 Good Measurement Practices 0 01 0 00 0 01 0 02 0 03 0 04 200 400 600 S00 1000 Figure 10 Example of a Blank on Water with Bubbles Causing a Poor Baseline If your blank or spectra shows artifacts similar to the one in Figure 10 see Solvents on page 4 to optimize the measurement procedure 52 Agilent 8453 UV visible Spectroscopy System Operator s Manual Good Measurement Practices 3 Inserting a Cell Your spectrophotometer is shipped with the standard single cell holder you first have to 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
50. lel 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 the analysis light beam have to very parallel The parallelism is measured in terms of the angle between the two opposite cell walls We recommend to use 10 mm path length cells with an angle which is below 0 1 degrees of an arc Light Beam In Parallel Cell Walls Light Beam Out Light Beam In Non parallel Cell Walls __ Light Beam Out Figure 4 Shift of the Spectrophotometer Light Beam due to non Parallel Cell Walls 42 Agilent 8453 UV visible Spectroscopy System Operator s Manual Good Measurement Practices 3 Apertured Cells In applications where sample volume is limited apertured 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 in sensitivity See Figure 5 for recommended and Figure 6 for cells you should not use with the instrument IE Quartz cells Quartz cells with black ap
51. ly 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 quick simple mounting system An optical filter wheel is also available for use with the spectrophotometer and most of the accessories Spectrograph The spectrograph housing material 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 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 e Slit The slit is a narrow aperture in a plate located
52. m 190 nm to 400 nm in the dialog s Display spectrum section Click OK to set your parameters Spectrum Feaks Parameters Peak alley find IM Find and annotate up t z peaks Find and annotate up to a Walley Prompt for sample information Data type Display spectrum Absorbance From 190 nm To 400 nm Cancel 5 Fill your l 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 holder Agilent 8453 UV visible Spectroscopy System Operator s Manual 77 4 Using your Agilent 8453 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 Automation 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 78 Agilent
53. ment Panel measurement buttons or by pressing the spectrophotometer buttons uses the sipper for sample introduction Sipper introduction is also used by an automated sequence Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 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 about your multicell transport see your nstalling and Operating Your Multicell Transport manual 1 Select Multicell 8 cell in the Instrument Panel selection box Sampling Autosampler YT Autosampler Gilson 2217222 2 Click Setup on the Instrument Panel Type the path lengths of all cells used in cm and click OK Agilent 8453 UV visible Spectroscopy System Operator s Manual 83 4 Using your Agilent 8453 UV visible Spectroscopy System setup Multicell Transport Cell Path Length 1 crm Ean cm 3 cm 4 7 cm 5 Jl cm G 1 cm Fy od cm 11 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 Multicell transport Control dialog In the Multicell Transport Control you press
54. mits light over the 370 nm to 1100 nm wavelength range e 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 e 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 command the shutter to remain open Agilent ChemStation software or it stays open automatically handheld controller software Agilent 8453 UV visible Spectroscopy System Operator s Manual 11 1 12 Introduction 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 Agilent ChemStation software in case you want to do very fast repetitive scans or it is switched off automatical
55. n times To minimize convection effects keep the temperature 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 VY Apertured cells has black sides VY Apertured cells has an aperture greater than or equal to 3 mm v Cell windows are free of fingerprints and other contamination Y Flow cell used instead of an apertured standard cell Measurements Y Solution in cell is free of floating particles Y Solution in cell and cell walls are free of bubbles vY Solution in cell is mixed homogeneously Agilent 8453 UV visible Spectroscopy System Operator s Manual Good Measurement Practices 3 VY Blank measured on same solvent as samp
56. ncept applied assures consistent and reliable results 34 Agilent 8453 UV visible Spectroscopy System Operator s Manual Agilent 8453 UV visible Spectroscopy System Operator s Manual 00 ee 2 Installation and Start Up Installation Summary for Your Agilent 8453 General Purpose UV visible j System 36 Starting a Measurement Session 38 This chapter does not replace the information available with the Installing Your UV visible Spectroscopy System manual It is meant as a reminder of the key steps of the installation and the system startup Bee Agilent Technologies 35 2 Installation and Start Up Installation Summary for Your Agilent 8453 General Purpose UV visible System General A detailed description of your Agilent 8453 general purpose UV visible system is given with the manual Installing Your UV visible Spectroscopy System The summary reminds you of the key points of installation Spectrophotometer VY Make sure that your spectrophotometer has the LAN interface card installed VY Check that your spectrophotometer is either connected to your PC directly using a crossover LAN cable or to your LAN using a direct connection CAUTION 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
57. nd 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 as a 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 defined in FAR 52 227 19 c 1 2 June 1987
58. nd the concept of your Agilent ChemStation software 2 Installation and Start Up In this chapter you will find a summary of system installation and start up of a measurement session 3 Good Measurement Practices Good measurement practices are discussed in this chapter 4 Using your Agilent 8453 UV visible Spectroscopy System Stepwise examples for basic measurements and related tasks are given in this chapter Agilent 8453 UV visible Spectroscopy System Operator s Manual 3 Agilent 8453 UV visible Spectroscopy System Operator s Manual Contents Introduction to Your System 9 Agilent 8453 Spectrophotometer Overview 10 Optical System Overview 10 Spectrophotometer Description 14 General Purpose Agilent ChemStation Software for UV visible Spectroscopy Overview 18 User Interface Elements 19 Software Structure 23 Standard Mode Tasks 25 Standard Mode Data Processing 29 Installation and Start Up 35 Installation Summary for Your Agilent 8453 General Purpose UV visible System 36 General 36 Spectrophotometer 36 PC 3 7 Starting a Measurement Session 38 Good Measurement Practices 39 General Considerations 40 Spectrophotometer Design 40 Making Measurements 40 Sample Cell Material 41 Optical Specifications of Cells 42 Apertured Cells 43 Flow Cells 44 Agilent 8453 UV visible Spectroscopy System Operator s Manual Contents Handling and Maintaining Cells 45 Solvents 47 Sample Preparation 48 Photosensitive Sam
59. nt 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 6 Press the Blank button on the front of the spectrophotometer or click Blank on the Instrument Panel to start the measurement Sampling 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 8453 UV visible Spectroscopy System Operator s Manual 61 4 Using your Agilent 8453 UV visible Spectroscopy System Saving Your Parameters as a Method 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 defining your method to
60. ntation 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 8453 UV visible Spectroscopy System Operator s Manual 89 4 Using your Agilent 8453 UV visible Spectroscopy System Standard Information Name Standard 1 Solvent Wi aber Comment mgL Cancel 5 Enter your standard s information in the Standard Information dialog box and click OK 6 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 aC ee 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 62 for more information 90 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Analysis 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 water Then fill the cell with about 3 ml of the caffeine sample
61. od 23 dialog fixed wavelength s parameters 60 method options amp information 63 dimethyl formamide 47 dimethyl sulfoxide 47 diode array 13 33 door to access lamps 17 dust 48 100 E enhancing sensitivity 96 entrapment of bubbles 44 equation 25 31 ethyl acetate 47 ethyl ether 47 examples 97 extinction coefficient 34 F false light 14 40 filter for stray light correction 10 12 fixed wavelength 25 26 59 flow cell 44 flow cells 53 flow test 81 front view of spectrophotometer 14 G glass cells 41 glycerol 47 GPIB connector 17 GPIO connector 16 grating 10 13 handling cells 46 highest precision measurements 44 holographic grating 10 13 homogeneity 50 HPLC grade water 48 identity 27 indicator 15 installation 36 installation qualification 59 instrument construction 10 description 14 electronic assemblies 10 layout 10 mechanical assemblies 10 warm up 57 instrument panel 21 instrument session 23 internal reference 26 30 IP address 36 37 38 56 isopropyl alcohol 47 L lamps 11 access through door 1 deuterium 10 tungsten 10 LAN crossover cable 36 LAN interface Jet Direct 36 Talk2Lab 36 lens 10 lens tissues 45 levels of operation 23 light beam 42 45 line power 37 input socket 17 switch 14 liquid samples 40 logged 38 M main application window 23 making measurements 40 manager level 23 58 maximum
62. ombined Report Wenfhication and Diagnostics According to 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 look at your data with the focus on different aspects 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 to disk as a method This allows you to repeat your analysis task under defined conditions simply by loading a method and running your samples Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 Standard Mode Tasks The Agilent ChemStation modes also offer the ability to focus on a certain aspect many parameters have to be managed to customize a mode for a certain application To overcome this complexity your standard mode offers an additional focus on 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
63. on 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 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 information The result is a fundamental increase in the rate at which spectra can be acquired 10 Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 we a N gt Tungsten Spectrograph lens AA T lamp 7 ai UE Deuterium Slit lamp 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 e
64. ons which has the appendix Offline can be launched The offline session allow recalculations based on stored data and is useful in the development process of an analytical method 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 usually a method is developed and stored Agilent 8453 UV visible Spectroscopy System Operator s Manual 23 1 24 Introduction to Your System 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 only a reduced set of functions is available Especially functions which may affect the integrity of an analytical procedure are not available But on the other hand an operator may use their own settings This gets flagged on the tool bar and is be 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 Hode Standard Advanced Thermal Denaturation Kunetics Dissolution Testing Multibath Dissolution Testing C
65. ovides access to the setup dialog of your actual task by means of the Setup button Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 Duantification Instrument Panel The instrument panel is below the analysis panel It visualizes and controls your sampling devices and spectrophotometer Part of the graphical elements on this panel are active items for example to switch lamps on or off or run a peristaltic pump Sampling Sipper kd Automation You can recognize the active areas by a pointer change when you move the mouse across the area A mouse click in such an active position brings up a small menu with selections or simply performs 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 8453 UV visible Spectroscopy System Operator s Manual 21 1 Introduction to Your System General Purpose Agilent ChemStation Software for UV visible Spectroscopy Overview View Overlaid Sample Spectra Absorbance CALL Wavelength rm Fe eS ES Sample Result Table ME Es Show Sample Info Last Spectrum Delete Selected Sample All Peaks alleys The area on the right side of the side panels offers you a view on a certain aspect of your current task A view consists of one or more separate windows These windows provide information in mainly a graphic
66. parallel on both the samples and the standards Samples Standards Spectral Processing Data Access Data Evaluation Calibration The evaluation in quantification is now a calibration using standards The coefficients are calculated based on the method s settings and the current standards in the Agilent ChemStation memory This means results now become a function of a measured set of standards and the standard concentrations specified 32 Agilent 8453 UV visible Spectroscopy System Operator s Manual Introduction to Your System 1 E Processed Standard Spectra Ss fe lle dinaa 0 7 0 6 me 2 0 5 F a a 0 4 k 5 a wi 0 3 2 0 2 0 1 20 Analyte value 250 40 Wavelenethtnml Calibration Table oo es Show Standard Info Show Coefficients Calibrate H Standard Name Caffeine mgfL Abs lt 2 Error EHET 7 rm oe 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 The concept of 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 from a practical point of view are briefly described below e Virtually Unlimited Number Of Standards Due to
67. ples 49 Stirring and Temperature Control 50 Checklist for Best Results 50 Inserting a Cell 53 Using your Agilent 8453 UV visible Spectroscopy System 55 Starting Your First Measurement Session 56 Starting Your UV visible Software 58 Measuring Caffeine Absorbance at 2 3 nm _ 59 Saving Your Parameters asa Method 62 Retrieving and Printing a Method 64 Saving and Retrieving Data 6 Saving your Samples 6 7 Saving a Selected Spectrum 69 Retrieving Spectra 71 Deleting Current Spectra 72 Print Preview of Reports 73 Finding the Caffeine Absorbance Maximum 76 Entering your Cell s Path Length 80 Controlling your Sipper System 81 Using your Multicell Transport 83 Quantitative Analysis using a Calibration with Standards 86 Setup 87 Calibration 89 Analysis 91 Agilent 8453 UV visible Spectroscopy System Operator s Manual Contents How Can I Be Sure That My Agilent 8453 Works Properly 93 Agilent 8453 Self test 93 How Can I Get a Deeper Understanding of UV visible Spectroscopy 96 When Do I Have to Measure a Blank 98 Index 99 Agilent 8453 UV visible Spectroscopy System Operator s Manual 7 Contents 8 Agilent 8453 UV visible Spectroscopy System Operator s Manual Agilent 8453 UV visible Spectroscopy System Operator s Manual eece 1 Introduction to Your System Agilent 8453 Spectrophotometer Overview 10 General Purpose Agilent ChemStation Software for UV visible Spectroscopy Overview 18 Operation of the s
68. s can cause erroneous results Wipe off all fingerprints and contaminants before using a sample cell Use only 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 getting contaminated with tissue particles or rinse the cell with blank or sample solution Floating particles in the cell will deflect the light beam and so lead to a very poor quality of the measured spectrum SA Floating particles will deflect and scatter the light beam Figure 7 Floating Particles in a Cell Agilent 8453 UV visible Spectroscopy System Operator s Manual 45 3 Good Measurement Practices CAUTION Ab 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
69. 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 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 Agilent 8453 UV visible Spectroscopy System Operator s Manual Good Measurement Practices 3 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 8453 UV visible Spectroscopy System Operator s Manual 41 3 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 paral
70. sting Mod 5 Combined Reports Verification and Diagnostics Mode 9 Report Style Editor Maintenance Fa Troubleshooting lt uw r Agilent 8453 UV visible Spectroscopy System Operator s Manual 97 4 Using your Agilent 8453 UV visible Spectroscopy System When Do Have to Measure a Blank The measurement data acquired by your spectrophotometer is instrument independent To achieve this independency a reference measurement must be performed All consecutive measurements are referring 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 Here in addition the absorbance properties of the cell and the solvent are influencing 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
71. t 1 00000 0 28903 0 57807 2 Choose Save Selected Spectra As from the File menu pa 043x UN Visible System 1 Online TK Edit Method Measure Instrument Math View Mode ECM Config Help Load Save j Samples s Standards Asu Import Export Selected Spectrum As seein as You may get the warning Select activate a window on the message line if you did not select either the Overlaid Sample Spectra window or the Sample Results Table window Agilent 8453 UV visible Spectroscopy System Operator s Manual 69 4 Using your Agilent 8453 UV visible Spectroscopy System Saving and Retrieving Data 3 You select one of your already existing data files in the File name selector box of the Save Spectra As dialog or you enter a valid file name into the File name edit box Save spectra s File name Folders CAFFEINE SAMPLE SD cAchemse data ADVEXAM SD cs ADVEXAM3 SE T ADVEXAM SD CHEM32 ADVEXAMS SD ADVEXAM SE ADVEXAMS SE BENZENE SD NIST _HOLMIUM OXIDE 2034 50 Save file as type Drives Spectral 50STD E c Programs 4 Press OK to start the operation Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 Saving and Retrieving Data Retrieving Spectra 1 Choose Load Samples from the File menu samples Standardin i 2 You select the data file in File name selector box of the Load Spec
72. the accuracy of your measurements We also recommend stirring and temperature 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 48 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 8453 UV visible Spectroscopy System Operator s Manual Good Measurement Practices 3 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
73. thod access This displays the Method Options amp Information dialog box In the Method Information section you can enter a sort descriptive text which becomes part of your method Method Options amp Information Autosave Autosave Spectra to File Method Information Caffeine Analysis absorbance at 273 nm Agilent 8453 UV visible Spectroscopy System Operator s Manual 63 4 Using your Agilent 8453 UV visible Spectroscopy System 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 gg 845x UV Visible Systern 1 Online TK Edit Method Measure Instrument Math View Mode Load b Save gt Import gt Export Selected Spectrum As gt I New Method Load Method Save Method As Set Method Password Load Results Save Results As Print gt Print To File gt Print Previews gt Printer Setup Exit ChemStation 1 C CHEM32 1 METHODS CAFFEINE ANALYSIS 2 M 2 3 If your current method has been modified a dialog box will ask you whether you want to save or ignore these changes The current method has been changed Do you want to save the changes 7 64 Agilent 8453 UV visible Spectroscopy System Operator s Manual Using your Agilent 8453 UV visible Spectroscopy System 4 2 The Load Method dialog is displayed The selected method s informat
74. 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 sample measurement the blank measurement should be taken immediately prior to the actual sample measurement 98 Agilent 8453 UV visible Spectroscopy System Operator s Manual Index A absorbance 26 high 57 absorbance maximum 76 accessing lamps 17 accessory slot 17 accurate results 40 acetic acid 47 acetone 47 acetonitrile 47 acquisition date 29 time 29 active area 21 active item 21 active position 21 Agilent 8453 36 Agilent 8453 general purpose UV visible system 56 Agilent Bootp Service 36 38 Agilent ChemStation analysis panel 59 76 family 23 graphical window 69 log on 58 measurement session 56 method 62 mode 24 offline session 58 online session 58 password 58 preview sizes 75 print preview 73 printer display 75 samples 72 session 23 session online 57 workstation 33 ambient light 14 analysis 91 analysis panel 20 93 analysis setup calibration 88 concentration unit 88 data type 88 display 88 prompt for sample information 88 wavelength 88 analytes 40 analytical task 24 analyze 62 apertured cells or cuvettes 43 apertures 49 application specific 23
75. tra to SAMPLES dialog box g F cad Spect ta to TA ha PLES S Ea Loak in rr DATA e M g Egr A Name Date modified Type SIZE Tags ADVEXAMZ2 SD _ ADVEXAM3 SD Recent Places J ADVEXAM4 SD Desktop L SDVEXAMS 30 d J ADVEXAMT SD ADVEXAME SO Administrator _ BENZENE SD A CAFFEINE SAMPLE SD Ma __ NIST_HOLMIUM OXIDE 2034 30 Computer Ai L STOEXSM1 SO 2 L STOEXSM2 S0 Network ETARE ARAA Ch File name CAFFEINE SAMPLE SD Files of type Spectral SD STD Cancel Help File Information c chen3241 data cattein sample sd SPECTRA Samda Mame Agilent 8453 UV visible Spectroscopy System Operator s Manual 71 4 Using your Agilent 8453 UV visible Spectroscopy System You can look at the File Information of your files by moving the selection in the file selector box The content is always updated with the current selection 3 Click OK to start 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 gg 845x UV Visible Systern 1 Online TK Clear File Method Measure Instrument Math View Mode ECM Co ll 7 Cle Cut Ctrl X Method Paste Ctrl u Tas Paste Append Shift Ctrl i Overlaid Sample Spectra Ra Select All 08 We Copy to Clipboard A klear gt Sarnples Chap p
76. ystem is much easier if you understand the implementation models The mind models of data acquisition data evaluation and data handling will help you to run the system successfully Your spectroscopy system is based on an Agilent 8453 spectrophotometer and general purpose Agilent ChemStation software for UV visible spectroscopy running on a PC with the supported Microsoft operating system s These two components are linked together 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 as the integration in a enterprise network with network access from the PC to the spectrophotometer The tasks are split between these devices such that the spectrophotometer acquires and provides absorbance data which are handled by the PC s application software All of the data display evaluation and longterm storage is done under software control on the PC Agg Agilent Technologies 9 1 Introduction to Your System Agilent 8453 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 combinati

Agilent 8453 UV-visible Spectroscopy System

Contents

Download Pdf Manuals

Related Search

Related Contents