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8453 UV-visible system

1. Copyright Agilent Technologies 2002 All rights reserved Reproduction adaption or translation without prior written permission is prohibited except as allowed under the copyright laws Part No G1115 90019 Edition 04 02 Printed in Germany This handbook is for A 09 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 handbook Warranty The information contained in this document is subject to change without notice Agilent Technologies makes no warranty of any kind with regard to this material including but not limited to the implied warranties or merchantability and fitness for a particular purpose Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Agilent Technologies Deutschland GmbH Hewlett Packard Strasse 8 76337 Waldbronn Germany Agilent 8453 UV visible Spectroscopy System Operator s Manual 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 This book shall not replace the detailed manuals available for installation Installing Your UV visible Spectroscopy System and operation
2. Number of Samples 1 Date 10 18 99 Time 16 15 19 NOTE 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 71 NOTE Using your Agilent 8453 UV visible Spectroscopy System Saving and Retrieving Data Deleting Current Spectra Choose Clear Samples from the Edit menu or click the toolbar icon 845x UY Visible System 1 Offline TK Clear Eile Method Measure Instrument Math View Mc Uy Ch eat Bite apy Gilt I Paste HAY baste Append SAAE Fy Select All Fi Copy to Clipboard Clear dards Math Results Annotate TTT amp 2 T_ 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 Using your Agilent 8453 UV visible Spectroscopy System Print Preview of Reports 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 d
3. Processed Spectra Processed Spectra Data Evaluation Used Used Wavelength Wavelength Calibration Calibration Results Results 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 31 Introduction to Your System Standard Mode Data Processing become a function of a measured set of standards and the standard concentrations specified Processed Standard Spectra Ioj x 14 125 15 os o6 04 02 a l j F j i 200 250 Wavelength nm Calibration Curve os 04 03 02 01 DE 0 25 0 75 Analyte TA Path Absorbance AU Wavelength Result I Calibration Table olx Show Standard Info Show Coefficients Delete gelectedistandard Calibrate Standard Name Caffeine mg L Abs lt 273nm gt Eor f 1 Caffeine 10ma L 10 00000 0 53053 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 practic
4. 11 12 side panel 20 signal to noise ratio 50 single beam instrument 43 sipper 81 flow test 82 parameter 82 sipper system 45 sipper sampler system 49 slit 11 13 slit width 13 slots for MIO and accessory boards 17 software general purpose 8 18 solute convection 49 solution 51 solution noise 49 solutions 95 determining purity 95 enhancing sensitivity 95 solvent 43 57 solvent suitability 47 solvents 47 source lens 11 12 source of radiation 11 spectral acquisition 27 spectral operation 29 spectral processing 30 spectral raw data 33 spectrograph 13 101 Index lens 11 slit 11 spectrophotometer 32 37 front view 14 rear view 16 spectroscopy system 8 spectrum 25 standard 27 30 external 92 standard cells 53 standard push button 15 standard single cell cell holder 53 standards 85 current 31 minimum required number 32 number of 32 status 39 status indicator 15 stirring 49 stirring module 50 stop push button 15 stoppered sample cell 48 stray light 14 stray light correction 11 12 sulfuric acid 48 symbol 20 T task fixed wavelength 25 26 60 orientation 25 quantification 25 27 85 quantitative analysis 85 ratio equation 25 27 30 spectrum peaks 25 26 TCP IP protocol 38 temperature control 49 test kit 27 thermostattable cell holder 50 three point drop line 26 29 toluene 48 tool bar 20 transmittance 26 tungsten l
5. 80 PC 39 peak find 77 performance verification 92 photochemical reactions 49 photodegradation 50 photodiode array 13 photosensitive substances 49 pipette 47 plasma discharge 11 12 plastic door 17 plastic sample cells 43 pointer symbol 21 poor linearity 44 poor photometric accuracy 44 printer 38 39 configured 73 processed spectra 29 processing 28 spectral 29 standards 30 pump peristaltic 81 purity 27 push buttons 15 blank 15 sample 15 standard 15 stop 15 pyridine 48 Q quantification 25 85 quantitative analysis of samples 85 ready for analysis 89 quartz sample cells 43 R radiation source 11 rapid absorbance changes 50 ratio 25 raw data 28 rear view of spectrophotometer 16 recalculation 23 recommended cells 45 reference 39 remote connector 16 report results 73 result 32 results 29 precise 58 routine work 63 RS232C connector 16 S sample 43 51 compartment 12 push button 15 sample cell 43 49 sample data 30 sample information 27 sample result table 62 69 79 sampling device 20 sampling interval 13 sampling system 80 manual 80 sipper 81 security lever 17 self test 92 histories 94 results 94 start 93 working conditions 93 sensitivity 44 session data analysis only 23 instrument control 23 set of parameters 24 settings 63 setup dialog 20 sheet metal door 17 shine through aperture 11 12 shutter
6. 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 er Multicell 7 cell Multicell 8 cell Autosampler XY Autosampler Gilson 221 222 2 Click Setup on the Instrument Panel Type the path length of your flow cell in cm and click OK Setup Sipper Path Length fl cm 81 NOTE Using your Agilent 8453 UV visible Spectroscopy System Controlling your Sipper System 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 Sipper Parameter Pump jx s Pump Direction CW M Wait Time 3 s Sample Return 0 x Wash Time jo s Air Segment jo s 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 Instrument Panel measurement buttons or by pressing the s
7. Data type Absorbance M Absorbance 1st Derivative 2nd Derivative 3rd Derivative 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 Wavelengths Use wavelength 260 nm Background three point drop line 300 360 E 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 Name Equation WL1 WL6 Wt Y Unit Caffeine w1 0 09 mg71 Weight Volume l Use Weight Wt Volume Unit 29 Introduction to Your System St
8. N x Quartz cells Quartz cells with black apertures Y Quartz cells with black apertures smaller than 2 mm when used with a multicell transport can lead to measurements of poor reproducibility Cells You Should Not Use With the Instrument f e SS LN Quartz cells with transparent apertures uorescence cells plastic cells 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 45 Figure 7 Good Measurement Practices General Considerations The design of the flow cell should minimize entrapment of bubbles and flow channeling to provide the most reliable results 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 surfaces can cause erroneous results Wipe off all fingerpr
9. your network administrator has integrated your Agilent 8453 spectrophotometer into your local network A Start E CAG Bootp Server Switch on your Agilent 8453 spectrophotometer A running DHPC server will now assign the configured IP address to your spectrophotometer In the standard installation the CAG Bootp Server performs this task Start your measurement session by selecting Instrument 1 online from the menu Your Instrument Panel shows you the current state of the spectrophotometer and the Blank button is enabled The first task 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 57 NOTE NOTE Using your Agilent 8453 UV visible Spectroscopy System Starting Your First Measurement Session 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 rea
10. 23 B background correction 26 27 Beer s law 85 benzene 48 blank 48 51 blank push button 15 bootp server 57 bubble formation 49 busy 39 caffeine 60 CAG Bootp Server 37 39 calculate 32 calculation results 80 calibration 27 31 85 87 coefficients 31 curve 27 CAN connector 17 carbon disulfide 48 carbon tetrachloride 48 cell path length 80 81 83 cell changer 83 chloroform 48 cleaning cells 46 collimated beam 11 colloidal dispersions 49 command 20 common solvents 48 compartment for sample 12 concentration 32 concentration range 27 concept 7 connection network 8 connector CAN 17 GPIB 17 GPIO 16 multicell transport 16 remote 16 RS232 16 correction for stray light 11 12 current context 20 current task 22 cut off filter 49 cut off wavelength 49 cyclohexane 48 cyclopentane 48 D data 28 absorbance 85 access 29 30 archieve 68 clear 72 clear math results 72 clear standards 72 evaluation 30 file extension 69 file information 71 file name 69 file selector box 71 format 68 load 68 local storage 68 network transfer 68 removing 72 replace 69 retrieve 68 71 save 68 save samples as 68 saving selected data 69 storage 68 degassed 49 derivative 26 derivative spectroscopy 95 description of instrument 14 determining purity 95 deuterium lamp 11 development of an analytical method 23 dialog fixed wavelength
11. 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 64 Using your Agilent 8453 UV visible Spectroscopy System Retrieving and Printing a Method Retrieving and Printing a Method This section describes how to access methods and print a method report 1 Choose Load Method from the File menu or click the icon in the toolbar 845x UV Visible System 1 Online TK File 27 Load Method Ne NOTE If your current method has been modified a dialog box will ask you whether you want to save or ignore these changes Save Method 65 Using your Agilent 8453 UV visible Spectroscopy System Retrieving and Printing a Method 2 The Load Method dialog is displayed The selected method s information is shown in the File Information section of the dialog box File Name Directories CAFFEINE M c hpchem 1 methods a E eh E gt hpchem amp 1 gt methods Network Ix 5 List Files of Type Drives Method M 7 J c HARDDISK x File Information c hpchem 1 methods caffeine m METHOD Method Type Standard Fixed WaveLength Created by TK LastUpdate Date 18 10 99 Time 14 52 29 Comment My first Caffeine method absorbance at 274 nm 4 gt 3 If you want to load this method click OK
12. UV visible Spectroscopy System A few examples to demonstrate how typical standard applica tions can be performed 55 Starting Your First Measurement Session 57 Starting Your UV visible Software 59 Measuring Caffeine Absorbance at 273 nm_ 60 Saving Your Parameters asa Method 63 Retrieving and Printing a Method 65 Saving and Retrieving Data 68 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 85 How Can I Be Sure That My Agilent 8453 Works Properly 92 How Can I Get a Deeper Understanding of UV visible Spectroscopy 95 When Do I Have to Measure a Blank 97 Introduction to Your System Concepts behind your Agilent 8453 UV visible spectroscopy system and its components Introduction to Your System Operation of the system 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 Microsoft NT operating system 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 betwee
13. mode is indicated on the tool bar of your Agilent ChemStation session Mode Verification and Diagnostics M 2 Select the Self Test task in the analysis panel s selection box Task 92 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 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 93 Using your Agilent 8453 UV visible Spectroscopy System How Can I Be Sure That My Agilent 8453 Works Properly 4 The self test results are displayed with pass fail criteria E 8453 Instrument Self Test Results Iof x Save to Instrument More INFO Specification Measured Result Min intensity 190nm 220nm gt 2000cts 51301 cts Passed NOTE 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 94 Using your Agilent 8453 UV visible Spectroscopy System How Can Get a Deeper Understanding of UV visible Spectroscopy How Can I Get a Deeper Understanding of UV visible Spectroscopy The basic principles of UV visible s
14. of your software Understanding Your UV visible Spectroscopy System nor your Agilent 8453 Service Manual In Chapter 1 Introduction to Your System you will find an introduction to your Agilent 8453 spectrophotmeter and the concept of your Agilent ChemStation software In Chapter 2 Installation and Start Up you will find a summary of system installation and start up of a measurement session Good measurement practices are discussed in Chapter 3 Good Measurement Practices Stepwise examples for basic measurements and related tasks are given in Chapter 4 Using your Agilent 8453 UV visible Spectroscopy System Contents Introduction to Your System Concepts behind your Agilent 8453 UV visible spectroscopy system and its components 7 Agilent 8453 Spectrophotometer Overview 10 Optical System Overview 11 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 28 Installation and Start Up Summary of installation and startup of your system 35 Installation Summary for Your Agilent 8453 General Purpose UV visible System 37 Starting a Measurement Session 39 Good Measurement Practices General operating conditions of the spectrophotometer 41 General Considerations 43 Inserting a Cell 53 Contents Using your Agilent 8453
15. or thermostattable cell holder Making Measurements Blank Reference and Sample Measurement Your spectrophotometer is a single beam instrument so you must measure a blank before you measure a sample For the high accuracy measurements the blank and the sample measurement should closely follow each other In general a blank measurement should be repeated as often as is practical Even in a thermally stable environment a blank measurement should be taken 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 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 43 Figure 4 Parallel Cell Walls Non parallel Cell Walls Good Measurement Practices General Considerations Optical Specifications of Cells The accuracy of the readings of a diode array spectrophotometer is very sensitive
16. 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 Using your Agilent 8453 UV visible Spectroscopy System 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 Overlaid Sample Spectra 79 Using your Agilent 8453 UV visible Spectroscopy System Entering your Cell s Path Length 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 Manual M 3 Click OK to set the specified path length 80 Using your Agilent 8453 UV visible Spectroscopy System Controlling your Sipper System
17. 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 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 Photosensitive Samples A few substances are very photosensitive They degrade or undergo photochemical reactions if exposed to light This can be easily seen by a decrease of sample absorbance over time Use of Filters The shorter wavelength higher energy UV light is most likely to degrade photosensitive samples If 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 th
18. the front of your spectrophotometer or click Standard on the Instrument Panel to start the measurement Standard Information Name Standard 1 Solvent water Comment m Value Caffeine filo mg L Enter your standard s information in the Standard Information dialog box and click OK Your Agilent ChemStation software automatically calibrates and displays the calibration results After a successful calibration the task panel s calibration curve shows green This indicates that your method is ready for analysis Task oro 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 63 for more information 89 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards Analysis 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 Make sure that the cell windows are clean and reposition the cell in the same orientation as for the standard measurement Close the cell holder s level Press the Sample button on the front of your spectrophotometer or click Sample on the Instru
19. to spatial shifts of the analysis light beam Cells having non parallel opposite faces or so called wedge shaped cells lead to a spatial shift of the light beam see Figure 4 Therefore the opposite cell walls illuminated by 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 Shift of the Spectrophotometer Light Beam due to non Parallel Cell Walls Light Beam In Light Beam Out Light Beam In Light Beam Out Apertured Cells In applications where sample volume is limited apertuwred 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 44 Figure 5 CAUTION Figure 6 Good Measurement Practices General Considerations Recommended Cells c
20. your spectrophotometer using a LAN make sure that your network administrator assigns the IP address to your spectrophotometer 38 NOTE Installation and Start Up Starting a Measurement Session Starting a Measurement Session 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 CAG Bootp Server 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 Switch on your PC and boot your PC operating system If a printer is connected to your system switch the printer on Make sure that the CAG Bootp Server is running or you are logged onto your LAN 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 Launch your measurement session by pressing your operating system s Start button and select Programs UV Visible ChemStations spectrophotometer 1 onl
21. 010 0 0005 0 0000 0 0005 0 0010 200 400 600 800 1000 51 Good Measurement Practices General Considerations Figure 10 Example of a Blank on Water with Bubbles Causing a Poor Baseline 0 01 0 00 0 01 0 02 0 03 0 04 200 400 600 800 1000 NOTE If your blank or spectra shows artifacts similar to the one in Figure 10 see Solvents on page 47 to optimize the measurement procedure 52 Good Measurement Practices Inserting a Cell 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 3 Lock the sample cell in place by pushing the locking lever back down Small volume flow cells and particularly any cells with less than a 2 mm aperture may require use of the optional adjustable cell holder The adjustable cell holder helps you ensure the cells are properly centered in the light path 53 Good Measurement Practices Inserting a Cell 54 Using your Agilent 8453 UV visible Spectroscopy System A few examples to demonstrate how typical standard app
22. 53 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 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 Before you switch on your spectrophotometer make sure that the CAG Bootp Server is running on your PC or your network administrator has assigned an IP address to your spectrophotometer For details see the chapter LAN Communication Installation Connection and Configuration in your Installing Your UV visible Spectroscopy System manual 37 Installation and Start Up Installation Summary for Your Agilent 8453 General Purpose UV visible System PC Make sure that all of your PC components are connected to line power Make sure that your general purpose software for UV visible spectroscopy is installed A printer must be configured on your PC e Adjust paper size for example Letter A4 e Set Orientation to Portrait The TCP IP protocol must be installed and configured on your PC Your spectrophotometer must be configured with its IP address If you connected your spectrophotometer directly to your PC be sure that your CAG Bootp Server application is installed and automatically launched when you start your PC If you connect to
23. NOTE Whenever you change a parameter of the current method you get an indication in the toolbar s modification field eto AER This triggers the reminder dialog mentioned above 66 Using your Agilent 8453 UV visible Spectroscopy System Retrieving and Printing a Method 4 To print a method choose Print Method from File menu 845x UV Visible System 1 Online TK File Print Automaton hesuit 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 67 Using your Agilent 8453 UV visible Spectroscopy System Saving and Retrieving Data 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 845x UY Visible System 1 Offline TK AEE Edit Method Meas
24. a set of standards Due to the spectral acquisition in addition background corrections can be applied to your data The calibration can be optimized for your concentration range of interest by changing the wavelength used for calibration A new calibration and a new analysis is automatically performed based upon your current standard data 27 Introduction to Your System Standard Mode Data Processing 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 Raw Data Data Access Processed Spectra Data Evaluation Used Wavelength 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 28 Introduction to Your System Standard Mode Data Processing Spectral Processing
25. al point of view are briefly descibed below Virtually Unlimited Number Of Standards Due to 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 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 32 Introduction to Your System Standard Mode Data Processing 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 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 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 n
26. amp 11 tutorial basic principles 95 basics of UV visible spectroscopy 95 derivative spectroscopy 95 U understanding Agilent ChemStation processing 28 used wavelength 29 useful wavelength range of solvents 48 user definable equation 27 user interface basic elements 19 UV grade water 49 UV visible spectroscopy basics 95 vV valley find 77 view 22 25 calibration 85 results 85 91 samples 62 79 standards 85 viscous solutions 50 volatile solvents 48 volume 27 WwW warning no results present 74 water 48 wavelength 29 wavelength reproducibility 33 wedge shaped cells 44 weight 27 width of slit 13 window 22 graphical 22 sample results table 29 tabular 22 102 ope Agilent Technologies 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 G1115 90019
27. andard Mode Data Processing In the Ratio Equation task for example this wsed 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 I Sample Result Table Show Sample Info Last Spectrum lolx Analyze Name Dilut Factor Caffeine mg L Abs lt 273nm gt 1 00000 0 26527 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 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 30 Introduction to Your System Standard Mode Data Processing with the sample processing only all evaluation steps are done in parallel on both the samples and the standards Samples Standards Spectral Processing Data Access
28. are in the Standard mode The mode is indicated on the tool bar of your Agilent ChemStation session 845x UV Visible System 1 Offline Thomas Klink olx File Edit Method Measure Instrument Math View Mode Config Help ee Al SIL P o oe ER e Simin r 2 Select the Quantification task in the analysis panel s selection box Task Ratio E quation Quantification 85 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 3 Anew task panel is displayed and the Quantification Parameters dialog is opened automatically Task v x DP render NOTE If you are already in the Quantification task use the Setup on the analysis panel to open the parameter dialog 86 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 4 Setup your analysis wavelength at 273 nm Use wavelength type Caffeine for the Analyte name set the Calibration curve type to Linear select Concentration entry and use mg l as Unit Check the Prompt for standard information and the Prompt for sample information Select Absorbance as Data type and set Display spectrum From 190 nm To 340 nm Quantification Parameters Wavelengths Use wavelength nm Background correction none E Le Calibration Analyte name Caffeine Calibration curve type Linear x Enter Concentration Concentrat
29. arge in a low pressure deuterium gas the lamp emits light over the 190 nm to approximately 800 nm wavelength range The light source for the visible and SWNIR wavelength range is a low noise tungsten lamp This lamp emits light over the 370 nm to 1100 nm wavelength range Source Lens The source lens receives the light from both lamps and collimates it The collimated beam passes through the sample if one is present in the sample compartment Shutter The shutter is electromechanically actuated It opens and allows light to pass through the sample for measurements Between sample measurements it closes to limit exposure of sample to light If the measurement rate is very fast you can command the shutter to remain open Agilent ChemStation software or it stays open automatically handheld controller software 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 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 s
30. as Klink ox gt Ny Standard zi o 2 Select the Spectrum Peaks task in the analysis panel s selection box Spectrum Peaks Fixed Wavelengths Spectrum Peaks Quantification 3 Use the Setup button of the analysis panel to open the parameter dialog 76 Using your Agilent 8453 UV visible Spectroscopy System Finding the Caffeine Absorbance Maximum Type 2 for the number of peaks to find and uncheck the valley find option Set your data type to absorbance and adjust your spectral display to a wavelength range from 190 nm to 400 nm in the dialog s Display spectrum section Click OK to set your parameters Spectrum Peaks Parameters m 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 77 Using your Agilent 8453 UV visible Spectroscopy System Finding the Caffeine Absorbance Maximum 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
31. d reports Agilent ChemStation Modes The Agilent ChemStation modes are application oriented Each mode has its own mode specific menu panels operations and set of views Your general purpose software for UV visible spectroscopy is the platform for all modes It is split into a Standard mode an Execute Advanced Method mode and a Verification and Diagnostics mode Mode Standard Standard Advanced Kinetics Thermal Denaturation Dissolution Testing Multibath Dissolution Testing Combined Report Color Calculations Verification 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 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 24 Introduction to Your System Standard Mode Tasks Standard Mode Tasks The Agilent ChemStation modes also offer the ability to f
32. dentity of a Sample by Peak Comparison and Correcting for Background Absorbance Using an Internal Reference to Improve Reproducibility Using an Internal Reference to Improve the Sensitivity of a Q A Using Derivative to Emphasize Differences between Spectra Menus Aa Execute Advanced Method Mode 96 Using your Agilent 8453 UV visible Spectroscopy System When Do I Have to Measure a Blank When Do I 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 orientati
33. dy after 45 minutes 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 Cell on page 53 58 Using your Agilent 8453 UV visible Spectroscopy System Starting Your UV visible Software 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 A single online session can be started on your PC but multiple offline sessions can be started in parallel This allows you to optimize your method settings by direct comparison based on identical sets of data Switch on your PC monitor and printer Log on to your PC s operating system Start your Agilent ChemStation session by selecting Instrument 1 online for a measurement session or Instrument 1 offline for method optimization and data evaluation Enter your name to log on to your Agilent ChemStation session If you protected your manager level by password you must enter the correc
34. e 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 49 Good Measurement Practices General Considerations Turning the Dy 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 Stirring and Temperature Control Solution homogeneity can be a problem especially for viscous solutions There are cases where due to convection induced gradients rapid absorbance changes may give irreproducible data These changes can be observed spectroscopically by taking measurements with short integration times To minimize convection effects keep the 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 wa
35. easurement Practices General Considerations 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 Water Acetonitrile 200 210 nm Cyclopentane n Hexane Glycerol 2 2 4 Trimethylpentane Methanol 210 220 nm n Butyl alcohol Isopropyl alcohol Cyclohexane Ethyl ether 245 260 nm Chloroform Ethyl acetate Methyl formate 265 275 nm Carbon tetrachloride Dimethyl sulfoxide Dimethyl formamide Acetic acid 280 290 nm Benzene Toluene m Xylene Above 300 nm Pyridine Acetone Carbon disulfide WARNING 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 can change 48 Good Measurement Practices General Considerations the solute concentration or cause solution noise due to solute convection currents Both of these will affect 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
36. emStation 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 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 845x UY Visible System 1 Online TK File Edit Method Measure Instrument Math Vier Sz Load Save Import Export Selected Spectrum As New Method Set Method Password Print Print To File Print Preview Printer Setup Exit ChemStation oo Ina I 63 Using your Agilent 8453 UV visible Spectroscopy System Saving Your Parameters as a Method 2 This displays the Save Method As dialog box Type the method name in the File name field for example Caffeine m Click OK to save your method File name Folders Catfeinelm c hpchem 1 methods a Eck E gt HPCHEM Lewer _ E gt methods H A Save file as type Drives Methot M J c HARDDISK 7 NOTE If you generate many methods you can use Options amp Infos from the Method menu to add text for documentation purposes and to simplify method access
37. ent2 UV Vis offline offline Configuration Editor 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 an 845x UV Visible System 1 Offline TK File Edit Method Measure Instrument Math View Mode 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 only started as a single instance on your PC But multiple data analysis sessions which have the appendix Offline can be launched The offline sessions allow recalculations based on stored data and are 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 permanently to disk The manager level of operation is password protected This assures the integrity of predefined methods and operation sequences 23 Introduction to Your System Software Structure 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 printe
38. ew answers almost instantaneously The data analysis concept applied assures consistent and reliable results 33 Introduction to Your System Standard Mode Data Processing 34 Installation and Start Up Summary of installation and startup of your system Installation and Start Up 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 The following topics are covered e Installation Summary for Your Agilent 8453 General Purpose UV visible System on page 37 e Starting a Measurement Session on page 39 36 WARNING WARNING Installation and Start Up Installation Summary for Your Agilent 8453 General Purpose UV visible System 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 Make sure that your spectrophotometer has the JetDirect card installed Check that your spectrophotometer is either connected to your PC directly using a twisted LAN cable or to your LAN using a direct connection Do not connect the LAN adapter of your PC to the CAN interface of the Agilent 84
39. ew of Spectrophotometer GPIB connector and switches MIO slot for LAN interface fo a E Line connector Two CAN connectors The multicell connector allows you to connect the cable which comes from the multicell transport The GPIO general purpose input output connector allow you to control a sipper and autosampler or other accessories depending on the software you are using The remote connector may be used in combination with other analytical spectrophotometers from Agilent Technologies if you want to use features such as common shut down and so on The RS232C connector may be used to control the spectrophotometer from a computer through RS232 connection using appropriate software for future use This connector needs to be defined by the configuration switch module next to the GPIB connector The software needs the appropriate drivers to support this communication which is intended for future use The RS232C port is used as printer interface to connect the printer using a serial parallel cable of the Agilent 8453E UV visible spectroscopy system 16 Introduction to Your System Spectrophotometer Description e The right CAN bus is used to connect the handheld controller of the Agilent 8453E UV visible spectroscopy system to the spectrophotometer e The GPIB connector is used to connect the spectrophotometer with a com
40. ight beam after it has passed through the sample Slit The slit is a narrow aperture in a plate located at the focus of the spectrograph lens It is exactly the size of one of the photo diodes in the photo diode array By limiting the size of the incoming light beam the slit makes sure that each band of wavelengths is projected onto only the appropriate photoed 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 proportional to the wavelength Diode Array The photoed array is the heart of the spectrograph It is a series of 1024 individual photocopies and control circuits etched onto a semiconductor chip With a wavelength range from 190 nm to 1100 nm the sampling interval is nominal 0 9 nm 13 Introduction to Your System Spectrophotometer Description Spectrophotometer Description 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 stray light The open sample area makes it easier to access for cuvette handling and to connect tubing to a flow cell or therm
41. ine You are ready to use your system if the blue busy status display on the system s bottom message line turns off The first measurement you have to perform is a reference measurement After this alignment your are ready to measure absorbance data and spectra It takes about 15 minutes for the lamps to reach stable state conditions For best results do not perform measurements before this period of time has elapsed 39 Installation and Start Up Starting a Measurement Session 40 Good Measurement Practices General operating conditions of the spectrophotometer Good Measurement Practices 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 42 Good Measurement Practices General Considerations General Considerations There are many factors that can affect the results of your measurements This section provides brief discussions of some of the more important ones Spectrophotometer Design The sample compartment of the Agilent 8453 spectrophotometer is open Unlike conventional instruments the Agilent 8453 does not suffer from ambient stray light The open sample area makes it easier to access it generally and to connect tubing to a flow cell
42. ints 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 Floating Particles in a Cell lt P Floating particles will deflect and scatter the light beam p O Good Measurement Practices General Considerations Figure 8 Spectrum Taken With Floating Particles in the Light Path 0 01 0 00 0 01 0 02 0 03 0 04 200 400 600 800 1000 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 Solvents Your choice of solvents should be based primarily on the solvent s absorbance characteristics over the wavelengths of interest its suitability as 47 Good M
43. ion mg L Unit Volume Weight amp Yolume a jL Unit X Prompt for standard information IX Prompt for sample information Display spectrum From fiso nm To 340 nm 5 Click OK to set your parameters Data type Absorbance M NOTE Now you are ready to run your measurements Calibration 1 Fill your 1 cm path length quartz cell with distilled water Lift the lever at the left side of your cell holder Put the cell in the cell holder and make sure the transparent windows face towards the front and the back of the spectrophotometer Push the lever down to secure your cell in the cell holder 87 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 2 Press the Blank button on the front of the spectrophotometer or click Blank on the Instrument Panel to start the measurement 3 Remember the orientation of the cell in the cell holder Lift the level to release the cell remove it and flush it three times with about 1 ml of your caffeine sample Then fill the cell with about 3 ml of the caffeine sample Make sure that the cell windows are clean and reposition the cell in the same orientation as for the reference measurement Close the cell holder s level 88 NOTE Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards Press the Standard button on
44. isplayed 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 am 845x UV Visible System 1 Offline TK Edit Method Measure Instrument Math View W Load Save Import Export Selected SpectrumAs gt New Method Setup Load Method Save Method As Set Method Password Print gt Print To Eile gt Print Preview Current View Printer Setup Selected Window Exit ChemS tation Method 1 Results A Automation Method f j 3 Autorration Results Print Preview Results Report 73 Using your Agilent 8453 UV visible Spectroscopy System Print Preview of Reports NOTE Printing a results report requires you to have set all parameters properly and have data available for evaluation If you do not have data you may get the message No results present on the message line No results present 2 The report generated will be displayed in the preview window Fixed Wavelength Report Iof x Fixed Wavelength Report Date 10 26 99 Time 10 36 42 Page 1 of 1 Method file lt untitled gt Information Default Method Data File C HPCHEM 1 DATA ADVE XAMZ SD Created 02 06 1992 11 18 43 Overlaid Spectra Sample 1 Sample 2 Sample 3 Absorbancef4U 300 Name Abe
45. ks Fixed Wavelength Task Fixed Wavelengths M NA The Fixed Wavelength task is used to look at measured sample data at up to six different wavelength This data is available as absorbance transmittance and first to fourth derivative Due to the spectral acquisition additional techniques such as internal reference or three point drop line background corrections can be applied Spectrum Peaks Task Spectrum Peaks M L 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 26 Introduction to Your System Standard Mode Tasks Ratio Equation Task Ratio E quation x TEM ea Plik M SMA Meant Vea id bel ivy 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 v i g Z O inda The Quantification task allows you to do single component analysis based on four different types of calibration curves and
46. lications can be performed Using your Agilent 8453 UV visible Spectroscopy System This chapter includes the following Starting Your First Measurement Session on page 57 Starting Your UV visible Software on page 59 Measuring Caffeine Absorbance at 273 nm on page 60 Saving Your Parameters as a Method on page 63 Retrieving and Printing a Method on page 65 Saving and Retrieving Data on page 68 Print Preview of Reports on page 73 Finding the Caffeine Absorbance Maximum on page 76 Entering your Cell s Path Length on page 80 Controlling your Sipper System on page 81 Using your Multicell Transport on page 83 Quantitative Analysis using a Calibration with Standards on page 85 How Can I Be Sure That My Agilent 8453 Works Properly on page 92 How Can I Get a Deeper Understanding of UV visible Spectroscopy on page 95 When Do I Have to Measure a Blank on page 97 56 Using your Agilent 8453 UV visible Spectroscopy System Starting Your First Measurement Session Starting Your First Measurement Session 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 Bootp server is running on your system taskbar or make sure that
47. lt 414nm gt Name Abe lt 414mm gt 1 Sample 1 0 62494 3 Sample 3 0 10637 2 Sample 2 0 21198 Report generated by TK SIGHACULESS genenana 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 74 Using your Agilent 8453 UV visible Spectroscopy System Print Preview of Reports 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 with the print preview window e The Prev and Next buttons allow you to browse through the report pages e A selection box allows to jump to a page directly e The Print button sends the report to the printer which is displayed in the lower left corner of the print preview window HP LaserJet 5L PCL on LPT A4 210 x 297 mm Portrait e The Close button closes your print preview window and discards the report shown 75 Using your Agilent 8453 UV visible Spectroscopy System Finding the Caffeine Absorbance Maximum 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 se 845x UV Visible System 1 Offline Thom
48. ment Panel to start the measurement Sample Information Solvent Dilution Factor 1 Comment fist dilution of standard solution 90 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards 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 1 Sample 1 00000 4 99900 0 26521 NOTE To save your data for future use or for documentation purposes see Saving and Retrieving Data on page 68 for more information 91 Using your Agilent 8453 UV visible Spectroscopy System How Can Be Sure That My Agilent 8453 Works Properly 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 These procedures are described with your Operational Qualification Performance Verification for Agilent 8453 UV visible Spectroscopy Systems manual 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 Make sure that you are in the Verification and Diagnostics mode The
49. n 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 This chapter has two sections e Agilent 8453 Spectrophotometer Overview on page 10 e General Purpose Agilent ChemStation Software for UV visible Spectroscopy Overview on page 18 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 This overview contains e Optical System Overview on page 11 e Spectrophotometer Description on page 14 10 Introduction to Your System Optical System Overview Optical System Overview Optical System The optical system of the spectrophotometer is shown in Figure 1 Its radiation source is a combination of a deuterium discharge lamp for the ultraviolet UV wavelength range and a tungsten lamp for the visible and short wave near infrared SWNIR wavelength range The image
50. ocus 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 e Quantification A task is selected and activated from the analysis panel s selection box Task Fixed Wavelengths Fixed Wavelenaths Spectrum Peaks Ratio E quation 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 for 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 25 Introduction to Your System Standard Mode Tas
51. 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 Shutter stray light filter ey Deuterium f lamp Source lens Figure 1 Optical System of Spectrophotometer gt Tungsten lamp Source lens Slit J Grating Photo diode array Lamps The light source for the UV wavelength range is a deuterium lamp with a shine through aperture As a result of plasma discharge in a low pressure deuterium gas the lamp emits light over the 190 nm to approximately 800 nm wavelength range The light source for the visible and SWNIR wavelength range is a low noise tungsten lamp This lamp emits light over the 370 nm to 1100 nm wavelength range 11 Introduction to Your System Optical System Overview Lamps The light source for the UV wavelength range is a deuterium lamp with a shine through aperture As a result of plasma disch
52. 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 This overview contains e User Interface Elements on page 19 e Software Structure on page 23 e Standard Mode Tasks on page 25 e Standard Mode Data Processing on page 28 18 Mode switch Method name Menu bar Tool bar Analysis panel Instrument panel Tabular results Spectra Introduction to Your System User Interface Elements User Interface Elements Basic elements Your general purpose Agilent ChemStation software for UV visible spectroscopy facilitates operation of your diode array based UV visible spectrophotometer in daily routine operation The focus of this software is on ease of use and ease of learning A graphical user interface visualizes the spectrophotometer operation and usage This user interface consists of a number of elements as described in the following sections UV Visible ChemStation Online Soecwm Peaks I seu 19 Introduction to Your System User Interface Elements Menu File Edit Method Measure Instrument Math View Mode 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 s
53. oftware in case you want to do very fast repetitive scans or it is switched off automatically handheld controller software 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 adjustable cell holder the long path cell holder or the multicell transport All of these optional cell holders mount in the sample compartment using the 12 Introduction to Your System Optical System Overview 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 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 l
54. on relative to the measurement position e you use a different solvent or event a different lot of the same solvent e the time between your reference measurement and the sample measurement get too long e the time conditions are depending on the aging rate of your lamps and possible changes of your environmental conditions typically the last blank measurement should not be older than half an hour 97 Using your Agilent 8453 UV visible Spectroscopy System When Do I Have to Measure a Blank 98 Index Numerics 2 2 4 trimethylpentane 48 A absorbance 26 high 57 absorbance maximum 76 accessing lamps 17 accessory slot 17 accurate results 43 acetic acid 48 acetone 48 acetonitrile 48 acquisition date 28 time 28 active area 21 active item 21 active position 21 Agilent 8453 general purpose UV visi ble system 57 Agilent ChemStation analysis panel 60 76 family 23 graphical window 69 log on 59 measurement session 57 method 63 mode 24 offline session 59 online session 59 password 59 preview sizes 75 print preview 73 printer display 75 samples 71 session 23 session online 58 workstation 32 analysis 90 analysis panel 20 92 analysis setup calibration 87 concentration unit 87 data type 87 display 87 prompt for sample information 87 wavelength 87 analytes 43 analytical task 24 analyze 63 apertured cells or cuvettes 44 apertures 50 application specific
55. ormation 89 measure push buttons 15 measurement blank 57 noise 57 reference 57 sample 58 90 sample information 91 menu 20 message line 39 methanol 48 method 24 28 31 32 63 calibrated 33 85 current 66 information 66 last used 59 load 65 load method 65 modified 65 name 64 options amp information 64 parameter 28 parameters 63 print preview 67 printing 65 67 report 65 retrieve 65 save method as 63 store 63 methyl formate 48 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 59 multibath dissolution testing 24 standard 24 60 76 85 switch 24 thermal denaturation 24 verification and diagnostics 24 92 mouse cursor 21 multicell transport 83 7 cell 84 8 cell 83 connector 16 m xylene 48 N n butyl alcohol 48 network administrator 37 38 connection 39 local 57 100 Index n hexane 48 nominal spectral slit width 13 o offline 23 online 23 operating system 39 operation 20 operation levels 23 operator level 23 operator name 28 optical filter 49 optical specifications of cells 44 optical surfaces 46 optical system 10 11 optimization 32 outliers 32 P paper orientation 38 size 38 parallelism 44 parameter dialog 76 particulate matter 49 passivating new cells 46 path length setup
56. ostattable cell holder The spectrophotometer is shipped with the standard single cell holder Standard and accessory cell holders can be removed and replaced in seconds with few or no tools Figure 2 Front View of Spectrophotometer Indicato Pushbuttons Line power switch with green light 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 14 Introduction to Your System Spectrophotometer Description When the line power switch stands out and the green light is off the spectrophotometer is turned off On the front panel of the spectrophotometer is a status indicator which will display different colors depending of the actual condition of the spectrophotometer Green the spectrophotometer is ready to measure Green blinking the spectrophotometer is measuring Yellow the spectrophotometer is in not ready state for example turning one of the lamps on or if both lamps are switched off 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 Tr
57. oubleshooting 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 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 the baseline spectrum is measured and displayed on the PC SAMPLE the spectrophotometer takes a sample measurement or starts a series of measurements This depends on the parameters set in your software 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 Stop the spectrophotometer and or software aborts any ongoing activity and returns to a to ready state 15 Figure 3 Remote connector Multicell transport ose connector GP IO connector RS 232 connector Introduction to Your System Spectrophotometer Description Rear View All connections are made at the rear of the spectrophotometer see Figure 3 Rear Vi
58. pectrophotometer buttons uses the sipper for sample introduction Sipper introduction is also used by an automated sequence 82 NOTE Using your Agilent 8453 UV visible Spectroscopy System Using your Multicell Transport 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 Installing and Operating Your Multicell Transport manual 1 Select Multicell 8 cell in the Instrument Panel selection box Sampling Manual Sipper Multicell 7 cell Multicell 8 cell Autosampler XY Autosampler Gilson 221 222 2 Click Setup on the Instrument Panel Type the path lengths of all cells used in cm and click OK Setup Multicell Transport Cell Path Length TTN 83 NOTE Using your Agilent 8453 UV visible Spectroscopy System Using your Multicell Transport 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 one of the position buttons to move the multicell transport Multicell Transpor
59. pectroscopy 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 Help Topics Basic Operation 24 xi Contents Index Find Click a topic and then click Display Or click another tab such as Index QL Tutorial E Basics of UV Visible Spectroscopy gt Basic Principles Instrumentation The Diode Array Advantages Instrumental Specifications e Accessories Making Measurements Quantitative Analysis Multicomponent Quantification e Kinetics Enzyme Kinetics Thermal Denaturation Derivative Spectroscopy Solving nalytical Problems Print Cancel 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 95 Using your Agilent 8453 UV visible Spectroscopy System 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 Help Topics Basic Operation gt Installation e Basic Operation VE Standard Mode e Using Tasks Calibration and Quantification of a Single Component Checking the Purity of a Sample by a Ratio Calculation Confirming the I
60. puter 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 The GPIB port is not used when the handheld controller of the Agilent 8453E UV visible spectroscopy system is connected to the spectrophotometer However the 8 bit configuration switch of the port must be set for GPIB communication e The 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 there is a door for exchanging the lamps Behind this plastic door there is another sheet metal door Two independent safety light switches are implemented They automatically turn off the lamps when the sheet metal door is opened 17 General Purpose Agilent ChemStation Software for UV visible Spectroscopy Overview This section gives an overview of the elements
61. re Instrument Math View Mod Load gt ar Samples As Standards As Import Export Selected Spectrum s gt Selected Spectra As NOTE 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 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 As File name Folders catteine sd c N 1 data E c E gt HPCHEM amp 1 CHROM SD x Save file as type Drives Spectra SD STD x J c HARDDISK 4 Press OK to start the operation 70 Using your Agilent 8453 UV visible Spectroscopy System Saving and Retrieving Data Retrieving Spectra 1 Choose Load Samples from the File menu 845x U Visible System 1 Offline TK AES Edit Method Measure Instrument Math W Save er Standards 2 You select the data file in File name selector box of the Load Spectra to SAMPLES dialog box File Name Directories CAFFEINESD se Apchem 1 data ADVEXAM2 SD Help ADVEXAM3 SD ADVEXAM4 SD ADVEXAM5 SD ADVEXAM7 SD ADVEXAM8 SD BENZENE SD CAFFEINE SD List Files of Type Drives Spectraf SD STD z J c HARDDISK File Information c hpchem 1 data caffeine sd SPECTRA Sample Name Caffeine 10mg L
62. s parameters 61 99 Index method options amp information 64 dimethyl formamide 48 dimethyl sulfoxide 48 diode array 13 32 door to access lamps 17 dust 49 E enhancing sensitivity 95 entrapment of bubbles 46 equation 25 30 ethyl acetate 48 ethyl ether 48 examples 96 extinction coefficient 33 F filter for stray light correction 11 12 fixed wavelength 25 26 60 flow cell 45 flow cells 53 flow test 82 front view of spectrophotometer 14 G glass cells 43 glycerol 48 GPIB connector 17 GPIO connector 16 grating 11 13 H handling cells 47 highest precision measurements 45 holographic grating 11 13 homogeneity 50 HPLC grade water 49 I identity 27 indicator 15 installation 37 installation qualification 60 instrument construction 10 description 14 electronic assemblies 10 layout 10 mechanical assemblies 10 warm up 58 instrument panel 20 instrument session 23 internal reference 26 29 introduction system 7 IP address 37 38 39 57 isopropyl alcohol 48 J Jet Direct 37 L lamps 11 12 access through door 17 deuterium 11 tungsten 11 LAN 37 lens 11 lens tissues 46 levels of operation 23 light beam 44 46 line power 38 input socket 17 switch 14 liquid samples 43 logged 39 M main application window 23 making measurements 43 manager level 23 59 maximum 25 measure standard 89 standard inf
63. select a sampling system from the list of available systems Its parameters can be adjusted by means of the Setup button 21 Introduction to Your System User Interface Elements View F4 Overlaid Sample Spectra Transmittance 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 graphical 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 Introduction to Your System Software Structure 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 is part of Agilent s ChemStation family of instrument control software An installation of Agilent ChemStation software can control up to four different instruments such as UV GC LC or CE based on a single PC Each of these instruments has its own session Instrument 1 Instrument 2 Installation online online Qualification mm a Z Instrument 1 nstrum
64. t password The system will then come up with the last used mode and method 59 Using your Agilent 8453 UV visible Spectroscopy System Measuring Caffeine Absorbance at 273 nm Measuring Caffeine Absorbance at 273 nm This section describes how you measure your caffeine sample that was shipped with your spectrophotometer Measurement of this caffeine 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 845x UYV Visible System 1 Offline Thomas Klink OF x File Edit Method Measure Instrument Math View Mode Config Help 2 Select the Fixed Wavelength task in the analysis panel s selection box Task Fixed Wavelengths x Fixed Wavelenaths Spectrum Peaks Ratio E quation Quantification 3 Click Setup in the analysis panel to open the parameter dialog Task Fixed Wavelengths M Setup Using your Agilent 8453 UV visible Spectroscopy System Measuring Caffeine Absorbance at 273 nm Type the wavelength of interest in the Wavelengths section of the Fixed Wavelength s Parameters dialog Adjust your spectral display to a wavelength range from 190 nm to 400 nm in the Display spectrum section Click OK to set your parameters Fixed Wavelength s Parameters Lo Eu se Bea Fill your 1 cm path length quartz cell with distilled wa
65. t Control 2 Jia a hone 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 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 84 Using your Agilent 8453 UV visible Spectroscopy System Quantitative Analysis using a Calibration with Standards Quantitative Analysis using a Calibration with Standards 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 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 Setup 1 Make sure that you
66. ter 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 61 Using your Agilent 8453 UV visible Spectroscopy System Measuring Caffeine Absorbance at 273 nm 6 Press the Blank button on the front of the spectrophotometer or click Blank on the Instrument Panel to start the measurement Sampling Manual gt 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 62 Using your Agilent 8453 UV visible Spectroscopy System Saving Your Parameters as a Method Saving Your Parameters as a Method This section describes how to save your settings for a future Agilent Ch
67. ubmenus is displayed An operation is performed by choosing a command mouse click or ENTER key Toolbar te Al S P veto ME BE ooe E E 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 Side Panels The panels on the left side are the analysis panel and instrument panel The size and position of these panels are fixed but are a function of your display s resolution The minimum resolution is 600 x 800 pixels Analysis Panel The analysis panel gives you a graphical visualization of the current context in which you are working In addition it provides access to the setup dialog of your actual task by means of the Setup button Task Fixed Wavelengths Fixed Wavelenaths Spectrum Peaks Ratio E quation Quantification Instrument Panel The instrument panel is below the analysis panel It visualizes and controls your sampling devices and spectrophotometer Part of the graphical elements 20 Introduction to Your System User Interface Elements on this panel are active items for example to switch lamps on or off or run a peristaltic pump 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
68. ure Instrument Math View Mod lla Load ull m Save P Samples As Standards As Selected Spectra As Import gt Export Selected Spectrum s gt 2 Youselect 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 Save Spectra As 21x File name Folders caffeine sd c N 1 data Cancel E ct Cancel E E HPCHEM z 1 EX BENZENE SD CHROM SD z a Save file as type Drives Spectral SD STD c HARDDISK 7 68 Using your Agilent 8453 UV visible Spectroscopy System Saving and Retrieving Data NOTE 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 If the file name exists already a message box is displayed allowing you to abort or continue with the operation Save Spectra As 3 Click OK to start the operation Saving a Selected Spectrum 1 Select the spectrum of interest in the graphic window Overlaid Sample Spectra Or in the tabular Sample Results Table window E Sample Result Table __Show Sample Info Last Spectrum 69 Using your Agilent 8453 UV visible Spectroscopy System Saving and Retrieving Data 2 Choose Save Selected Spectra As from the File menu 845x UV Visible System 1 Offline TK Edit Method Measu
69. y 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 L Cell is made of quartz or glass Apertured cells has black sides Apertured cells has an aperture greater than or equal to 3 mm Cell windows are free of fingerprints and other contamination OU O wu Flow cell used instead of an apertured standard cell Figure 9 Good Measurement Practices General Considerations Measurements m m Ly m m m m m Solution in cell is free of floating particles Solution in cell and cell walls are free of bubbles Solution in cell is mixed homogeneously Blank measured on same solvent as sample Blank measurement shows a flat baseline Figure 9 and Figure 10 show a good and a poor baseline Cell orientation of blank and sample measurements is the same 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 Time between blank and sample measurement should be short Example of a Blank on Water Showing a Good Baseline 0 0015 0 0

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