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Perkin Elmer Lambda 12-14-14P Operation manual

1. eee 3 1 3 2 Setting the Wavelength Manually Vere eaim e eei ran 3 2 3 3 Manual Background Correction 3 3 24 Quick Sample Measurement 3 4 35 4 2 2 3 5 Chapter 4 How to use Methods 41 What are Methods 4 1 42 Selecting a Method 4 2 421 Default Methods 4 3 43 Editing Methods 4 4 431 Modifying a Method 4 5 Changing a Parameter 4 6 Tagging a Parameter EEEE 432 Deleting a 4 8 4 3 3 Creating a New Method 4 9 Creating an Empty Method 4 0 Overwriting an Existing Method 4 10 4 3 4 New Method Name 43 5 Checking Method 4 12 43 6 Copying Method Parameters into a New Method File 4 13 Marking for Copy 4 13 Copying the Method into Another Method File 2 4 14 43
2. 2 0 AG 1 Selecting a Branch A3 2 A3 2 Selecting a Branch 1 Switch on the spectrometer in SuperUser mode 2 Press STOP unti a branch header is 3 4 displayed Use the arrow keys to select the required branch Press PARAMETER The branch methods can then be selected see Section 4 2 page 4 2 Can only be set in SuperUser mode APPLICATION PARAM lt gt 4 CONFIGURATION PARAM gt t 500 0 NM 0 000 ABS INPUT gt 2 B2187 20 Aalease 2 0 About Branches A3 3 About the Various Branches With branches that do not contain analytical methods you must always press START after making the change to make the changes effective A3 4 APPLICATION the Application Branch This branch contains the analytical methods A3 5 COMMUNICATION the Communication Branch This branch contains a method for controlling the spectrometer via an external computer Method Function 29 FREERUN Control of the spectrometer via an external computer precondition is that the computer should be equipped with a program compatible with the method in use e g UV WinLab or PECSS version 3 1 or later B2161 20 felease 2 0 A3 3 Calibration Branch 19 20 21 4 A3 6 CALIBRATION the Calibration Branch Only for service personnel or very experienced users This branch contains methods for wavelength calibr
3. 2 1 6 B2761 20 Asasa 2 0 Safety information This section contains important safety information Please read this information carefully before you install the instrument and observe the recommendations at all times Safety Information Contents Section Page Safety Conventions in the Handbook eee heh tesa rr 9 2 IEC 1010 Safety Requirements ena era e re enr Electricity 5 4 Radio Frequency PEE ee hh hs 22 5 5 eer ehm nn 6 Chemicals 22 222 2 2 4 2 2 2 6 6 Waste Disposal eee ehh a aram S 6 UV 25 25 Toxic Fumes TED 2 8 7 Compressed 48 k E 4 BOR OR X amp FM E 9 7 5 1 Safety Information Safety Conventions in the Handbook 5 2 Possible hazards that could harm the user or result in damage to the instrument are clearly stated at appropriate places throughout this handbook The following safety conventions are used throughout this handbook 1 01 Warnin We use the term WARNING to inform you about situations WARNING that could result in personal injury to yourself or other pers
4. Protect cells from scratches and never permit them to rub against one another or against other hard surfaces Avoid abrasive corrosive or stain producing cleaning agents and make sure that the exposed surfaces of cells are optically clean When measuring cold solutions always bear in mind that condensation can form on the optical surfaces Make sure no bubbles cling to the inner surfaces of the cell particularly when handling cold solutions For maximum precision and accuracy calibrate and test with celis of the same type and always insert cells into the holders with the same orientation 2 2181 20 Release 2 0 Use and Care of Cells 7 2 2 Pressure Buildup in Cells e Only fill the cell so full that the liquid meniscus is just above the radiation beam The remaining air space in the cell is then adequate to compensate for any slight increase in pressure in the cell during routine operation e If for analytical reasons it is necessary to fill the cell completely insert the stopper only lightly so that the liquid in the cell has a chance to expand Do not insert a stopper forcefully into a completely filled since this is likely to cause the cell to burst e When working at higher temperatures use a drilled stopper 0 4 mm hole to allow for expansion in the cell 7 3 Sample Compartment Windows Generally the windows should be installed at all times The windows are opti
5. TOTAL AREA PEAK AREA DERIV 2 FIX DERIV 2 PEAK TOTAL AREA the total peak area between wavelength maximum and wavelength minimum is calculated a WAVMIN WAVMAX PEAK AREA the peak area between wavelength maximum and wavelength minimum 15 calculated with a correction for the baseline drift WAV MIN WAV MAX A 82167 20 Release 2 0 10 15 Parameters Parameter No Description MODE 1 continued MMASS dA 66 MMASS dC 67 10 16 DERIV 2 Height of the 2nd order derivative curve at wavelengths CALC WAV2 and CALC WAV1 is measured CALC Wave CALC WAV1 DERIV 2 PEAK Height of the 2nd order derivative peak The software locates the extreme values around wave lengths CALC WAV2 and CALC WAV 1 and calculates the difference in height between these two points Only values that have exceeded the set THRESHOLD are taken into account This function can avoid errors that may occur through displacement of the spectrum CALC WAV2 CALC WAV1 Sets the relative molecular mass of the Adenine base Range 0 to 99999 Used with the Oligoquant methods Sets the relative molecular mass of the Cytosine base Range 0 to 99999 Used with the Oligoquant methods 82161 20 Release 2 0 Parameter MMASS dG 55 dT MMASS NUMBER OF NUMBER OF dC NUMBER OF dG NUMBER OF dT NUMBER OF N 2181 20 Melase 2 0 68 69 70 60 61 63 Parameters
6. gt P E is governed by the Michaelis Menten equation y V Ks Ky Os dS i with v dr dr Reaction Rate and ky Ke Ks Michaelis Constant 1 Due to the large excess of substrate there is limiting case Og gt V Ka Car 15 valid A4 2 82181 20 Release 2 0 Enzyme Kinetics Thus the reaction rate is constant no consumption of enzyme and directly proportional to enzyme concentration or enzyme activity Hence the enzyme activity of a solution can be directly determined by measuring the reaction rate In practice the course of the curve obtained deviate from the ideal form it becomes linear only after a certain lag time and flattens out towards the end In such a case only the linear region of the curve is used for calculating the reaction rate Lag Time Linear Region Time 8216120 Release 2 0 A4 3 Substrate Kinetics 4 3 Substrate Kinetics In substrate kinetics the substrate concentration of a sample solu tion is determined via enzyme controlled reactions The advan tages of such a process are High specificity 1 e only one substrate is converted This avoids the necessity of complex sample preparation A quicker reaction with measuring times of only 3 30 minutes In the course of the reaction the substrate is converted to product and the reaction can be followed photometr
7. 1 Select the WAKEUP method 901 901 WAKEUP lt gt IPARAM START 2 Press PARAMETER PARAMETER MODIFY METHOD PARAM gt EE 3 Press PARAMETER again PARAMETER 4 Type in the wakeup date using the DATE 000000 numeric keys year month day ENTER gt z e g 940429 n 5 Press ENTER to confirm the entry ENTER 6 Press PARAMETER again PARAMETER f 7 Type in the wakeup time using the TIME 0000 numeric keys hours minutes 0655 ENTER 8 Press ENTER to confirm the entry ENTER 9 Press START to activate wakeup START When this method is activated the lamps are switched off and are then switched on again at the preselected WAKEUP time To exit the WAKEUP method press STOP and the display returns to standby Both lamps go on 82161 20 2 0 5 13 2161 20 Fiefeasa 2 0 Self Test Self Test The spectrometer tests the signals from the optics electronically and prints a report at the end of the test We recommend you do this test after installing a new lamp 1 Select the SELF TEST method 999 999 SELF TEST START 2 Press START to activate the self test START ww Press STOP to cancel If there are any FAIL results in the self test report then repeat test If you have any further enquiries contact your Perkin Elmer office 5 43 5 44 B2161 20 Release 2
8. Not 39 PRINT DATA YES NO 36 OPER ID KH Only available with Lambda 14 and Lambda 14P 1Do not change the value for this parameter Once the OLIGOQUANT 1 method is created the method parameters can be edited The base sequence must be entered from base 5 to 3 Use the numeric keys according to the table below to enter the base sequence Base Description N Any base A Adenine C Cytosine G Guanine Key b T Thymine B2161 20 Aelease 2 0 B2161 20 Heleaso 2 0 Methods The following table lists typical OLIGOQUANT 2 parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter Parameter Value No Parameter Value No 17 2 11 Only available with Lambda 14 and Lambda 14 t Do not change the value for this parameter SLIT WAVELENGTH FACTOR 1 NUMBER OF dA NUMBER OF dG NUMBER OF N TM CALCULATION LAMP SAMPLES BATCH CYCLES GRAPHICS PLOT ALITO METHOD SAMPLE iD 2 0 nm 11 1 0 0 0 0 NO UV Vis 0 1 1 WAV 1 59 60 60 65 14 18 18 ORDINATE MODE ABS 260 0 1 0 cm NUMBER OF dC 0 NUMBER OF dT 0 CHNGE CONSTANTS NO 058 YES FIRST SAMPLE 1 0 1 min 5 39 Date Time 5 11 Date Time 1 2 3 4 5 6 7 Select the DATE TIME method 900 Press PARAMETER Press PARAMETER a
9. PERKIN ELMER Lambda 12 Lambda 14 Lambda 14P UV Vis Spectrometers Operation Part Number 0993 5011 Publication 82161 20 Release 2 0 Feb 95 UV Vis NIR Spectroscopy Bodenseewerk Perkin Elmer GmbH is registered for the design and manufacture of laboratory analytical equipment in respect to the quality system according to Certificate No FM 22178 ISO 9001 1987 EN 29001 1987 BS 5759 Part 1 1987 Perkin Elmer and UV WinLab are trademarks of the Perkin Elmer Corporation Registered names trademarks etc used in this document even when not specifically marked as such are not to be considered unprotected by law Release History Release Publication Date June 1994 september 1994 February 1995 The information contained in this document is subject to change without notice This document contains proprietary information that is protected by copyright rights are reserved No part of this document may be reproduced in any form whatsoever or translated into any language without the prior written permission of the Perkin Elmer Corporation or one of its subsidiaries Copyright 1994 Bodenseewerk Perkin Elmer GmbH Printed in the Federal Republic of Germany Technical Documentation Bodenseewerk Perkin Elmer GmbH D 88647 Ueberlingen Federal Republic of Germany Part Numbar 0993 5011 Publication 2161 20 Release 2 0 February 1995 Table of Contents Safety Information mE Safety Conventions i
10. Warning Warnung Bedautat daB es bai Nichtbeachten der genannten Anweisung zu einer Verletzung des Benutzers kommen kann Warning Advarsel Betyder at brugeren kan kv stet hvis anvisningen ikke overholdes Warning Peligro Utilizamos el t rmino WARNING PELIGRO para informarle sobre situaciones que pueden provocar da os personales usted otras personas En los recuadros como ste se proporciona informaci n sobre este tipo de circunstancias Warning Danger Nous utilisons la tormule WARNING DANGER pour avertir des situations pouvant occasionner des dommages corporels l utilisateur ou d autres personnes Los d tails sur ces circonstances sont donn es dans un encadr sembiable celui ci Warning Pericolo Con il termine WARNING PERICOLO vengono segnalate situazioni che potrebbero provocare incidenti alle persone Troverete informazioni su tali circostanze in un riquadro come questo Warning Waarschuwing Betekent dat wanneer de genoemde aanwijzing niet in acht wordt genomen dit kan leiden tot varwondingen van de gebruiker Warning Aviso Significa que a n o observancia da instru o referida poder causar um ferimento ao usuario 12 82181 20 Release 2 0 Translations of Warnings 11 01 Caution We use the term CAUTION to inform you about situations that could result in serious damage to the instrument or other equipment Details about these c
11. Analytical Notes 81 Background 8 1 82 Unusual Samples 8 2 83 Solvent Properties 8 4 Chapter 9 Error Messages 91 Error Messages Shown on the Display 9 1 92 Error Reports on the Printer 9 6 Chapter 10 Parameter Numbers and Descriptions 10 1 Parameter Numbers and Descriptions ehh rh 10 1 Appendix I SuperUser Activating SuperUser 1 1 Al 2 Deactivating SuperUser 1 1 Appendix 2 Protect Functions A2 1 General information about Protect Functions 224444 2 1 2 2 Setting Protect Functions enne 2 2 Setting Method Protect A2 3 Appendix 3 Instrument Branches A3 1 Overview Of the Instrument Branches 1 A3 2 Selecting a Branch 2 A3 3 About the Various Branches A3 3 34 APPLICATION the Application Branch A3 3 A3 5 COMMUNICATION the Communication Branch A3 3 A3 6 CALIBRATION the Calibration Branch A3 4 A3 7 CONFIGURAT
12. Changer _ CELL 1 7 CELL 8 13 STIRRER The locations at which measurements are to take place When CELL 1 7 shows the numbers 1 to 7 represent sample locations 1 to 7 When CELL 8 13 shows the numbers 1 to 6 represent sample locations 8 to 13 If none of the locations is to be used enter 0 Press ENTER to confirm the location numbers If e g measurement is to be carried out at locations 2 and 5 only enter 25 and then press ENTER CELL 1 7 and enter 0 and then press ENTER CELL 8 13 If BACK CORR YES has been selected location 1 is used for background correction measurement independent of the locations selected as described above Thus If BACK CORR YES has been selected place the blank solution at location 1 and the sample solutions from location 2 If BACK CORR NO has been selected ail locations can be used for sample measurement Example 1 CELL 1 7 246 Measurement will take place at CELL 8 13 20 locations 2 4 and 6 the rest will not be used Example 2 CELL 1 7 567 Measurement will take place at CELL 8 13 123456 locations 5 to 13 Switches the magnetic stirrer on and off Option YES Select with arrow key If the magnetic stirrer has been switched on place a small magnetic stirring bar in each of the cells The arrangement is such that whilst measurement is taking place in the one cell the following cell will be stirred 2161 20 Aefessa 2 0 Para
13. Description Sets the relative molecular mass of the Guanine base Range 0 to 99999 Used with the Ollgoquant methods Sets the relative molecular mass of the Thymine base Range 0 to 99999 Used with the Oligoquant methods sets the relative molecular mass of the base N is used to represent any base Range 0 to 99999 Used with the Ol goquant methods Sets the number of Adenine bases Range 0 to 999 Used with the Oligoquant methods Sets the number of Cytosine bases Range 0 to 999 Used with the Oligoquant methods Sets the number of Guanine bases Range 0 to 999 Used with the Oligoquant methods Sets the number of Thymine bases Range 0 to 999 Used with the Oligoquant methods r rr Sets the number of bases N is used to represent any base Range 0 to 999 Used with the Oligoquant methods 10 1 Parameters Parameter No Description OLIGO 78 the concentration of primer in the solution This is not necessarily the concentration in the cell Used for the melting point calculation Shows only when theoretical melting point is YES Refer to the Biochemical Application manual OPER ID 36 User identification up to a maximum 8 characters User identification appears on each printout p ORD MAX 26 Maximum ordinate scale range for graphics printout valid only if GRAPHICS PLOT YES Range 9999 9 to 9999 0 If th
14. spectrometer Sipper operation is not possible when using SUBSTRATE methods If ENZYME methods are used neither sipper nor autosampler can be used Prints method parameters prior to each method start Option YES NO Switches the autopurge function on and off Option YES 10 1 Parameters Parameter BACK CORR 10 2 No Description 16 Background correction Option YES Without tagging background correction should be carried out at the start of the method If tagging has been carried out background correction is offered At the start of the method CALL tagging Prior to each sample batch BATCH tagging Prior to each sample START tagging Time Drive Wavelength Prog Background correction 6 carried out at every wavelength Le the basic signal is set to zero Scan Background correction is carried out within the range WAV MAX and WAV MIN i e the baseline is set to zero Concentration 1 Concentration 2 If BACK CORR YES has been selected background correction must be carried out prior to measurement of the first reference solution Background correction should be carried out at wavelengths WAVELENGTH 1 3 i e the baseline signal is set to at these points Substrate Enzyme Background correction is carried out at the wavelength selected 82161 20 Release 2 0 Parameter BLANK No Parameters Description Blank value Enzyme Blank valu
15. 0 Operating with Accessories Operating with Accessories 6 6 1 6 2 82161 20 Release 20 General Accessories are components or instruments that are installed or connected in the sample compartment or otherwise connected to the spectrometer For some of these accessories parameters have to be taken into account in the methods The accessories described below have parameters in the various methods Accessories Samples can be applied either manually or with the help of a number of accessories The following accessories are currently available Changers Sippers Autosamplers 5Cell Changer 6Cell Changer 8Cell Changer 9Cell Changer 13Cell Changer Vacuum Sipper or Peristaltic Sipper 5 90 91 Operation with Accessories 6 3 6 4 Requirements for Operation with Accessories The following preconditions must be fulfilled in order to operate with accessories The accessory board and connector panel for the accessory in question must be installed in the spectrometer The various circuit boards and connector panels are described in the Installation Maintenance and System Description handbook e The accessory in use must be properly selected in the appropriate method Using Methods with Accessories When an accessory board is installed an additional parameter ACCESSORY appears in the method When the ACCESSORY parameter appears select the appropriate accessory type s
16. 1 Start of the Method 10 4 TOTAL TIME No Description 9 Slope calculation mode REGRESSION The slope i is calculated using all data points by means of linear regression INTERVAL The slope is calculated for each interval The mean of all the slopes is then used for the calculation of enzyme activity Note When using CALCULATE INTERVAL please bear in mind Instead of the parameter LAG TIME parameter DELAY TIME equilibration time appears Measurement begins only when the DELAY TIME has elapsed The TOTAL TIME must be a whole multiple of the INTERVAL time CALCULATESREGRESSION CALCULATE z INTERVAL TOTAL TIME 2 Start of Measurement 3 Start of Calculation 82181 20 Release 2 0 Parameter CELL 5 CELL 6 CELL 8 CELL 9 CELL 1 7 CELL 8 13 CHNG CONST B2151 20 2 0 39 43 41 42 65 Parameters Description To enter the location of the cell or cells to be measured in the 5cell changer To enter the location of the cell or cells to be measured in the cell changer To enter the location of the cell or cells to be measured in the Scell changer To enter the location of the cell or cells to be measured in the 9cell changer To enter the location 1 to 7 of the cell cells to be measured in the 13cell changer Example Cel 1 7 246 Measurement will take place at Cell 8 13 0 locations 2 4 and 6 the rest will not be used enter
17. 1 4 initialization 2 2 standby 2 2 symbols 4 6 Divisor 10 9 10 10 Double Index of Refraction 8 3 Editing Methods 4 4 Electricity safety information 8 4 End Time 10 10 Enz factor 10 10 unit 10 11 Enzyme Kinetics 5 26 A4 2 activity 5 26 A4 2 international unit 4 2 blank value 5 28 enzymatic analytical proce dures A4 1 F Factor 10 11 10 12 concentration 10 6 dilution 10 9 enzyme 10 10 Features 1 1 Filter change points A3 7 First Sample 10 12 Form Feed 1 3 G Gases safety information S 7 Graphics Plot 10 12 Grid 10 12 Help A3 5 key 4 17 key combinations 4 17 text 4 17 Installing cell holder 2 5 Instrument care of the 7 1 Interface Failure A3 5 Interference Pattern 8 3 Interrupting the Measurement 82161 20 Release 2 0 Interval 10 12 10 13 Interval Time 5 34 K Key arrow 1 2 back corr 1 2 ce 1 2 combinations 1 3 enter 1 2 goto L 1 2 1 3 help 1 2 1 3 method 1 2 1 3 minus 1 2 parameter 1 2 1 3 point 1 2 start 1 2 stop 1 2 Keyboard 1 1 L Lag Time 5 34 10 13 Lamp 10 13 Lamp Compartment 1 1 Lifter 2 4 Line Feed 1 3 Line Type 10 13 Lower Wavelength Lirnits of solvents 8 5 Mark for Copy 4 13 Measurement Wavelengths 5 19 2nd derivative 5 19 peak areas 5 19 peak heights 5 12 5 13 82161 20 Release 2 0 Method checking 4 12 concentration 5 12 copying 4 14 copy
18. 5 42 Startup 2 2 Stirrer 6 8 10 24 Substrate Kinetics 5 30 A4 4 biank value 5 33 procedure with cell changer 5 32 Super User 1 1 activating 1 1 deactivating Al 1 mode 1 3 T Tagged Parameters 5 2 Tagging 10 2 parameters 4 7 symbols 4 7 type of 4 7 Temp Check 10 24 Temperature 8 3 10 25 Peltier cell holder 10 25 sensor 10 24 Test branch A3 7 Thin film Samples 8 3 Threshold 5 20 10 25 Time Drive 5 6 Time Unit 10 25 Calculation 10 25 Total Area 10 15 Total Time 5 34 10 26 82161 20 2 0 Toxic fumes safety information S 7 Tray 10 26 Tube ports 2 5 U Unit concentration 10 6 enzyme 10 11 time 10 25 Unusual Samples 8 2 User configuration A3 6 UV radiation 5 7 Validation branch 3 7 Value n 10 26 Volatile Samples 8 2 2161 20 Release 2 0 Index Wakeup 5 42 Warnings translations of T 1 Waste disposing of 5 6 Wav Max 5 19 10 27 Wav Min 5 19 10 27 Wave n 10 27 Wavelength 10 27 manual setting 3 2 Wavelength Program 5 10 Symbois of Refs 10 1 Wavelengths 10 1 Co T 10 19 Numbers 3WL Analys 10 14 Index 5 index 6 2161 20 Ralpase 2 0
19. Protect Functions Protect functions define the right of access to methods and branches They can only be defined in the SuperUser mode You can prevent access to branches and methods using the protect function You can set the protection so that for routine sample checking only the method dedicated to that task is acces sible Table A2 1 lists the protect functions available in order of priority WRITE has lowest priority ALL full has highest priority Table A2 1 Protect Functions Protect Function Effect Designation Write protection Prevents method parameters WRITE from being written over Read Write protection Prevents methods from being RDAVR read and from being over written Execute protection Prevents methods from being EXECUTE used Full protection Prevents access to a branch ALL and all of its methods B2161 20 Release 2 0 2 7 Setting Protect Functions A2 2 Setting Protect Functions Please observe the following when setting protect functions Protect functions set for a method are valid for this partic ular method only Protect functions set for a branch are automatically valid for all the methods contained in the branch If a particular method has a higher protect function priority than the branch the method protection is valid If the method has a lower priority than the branch the branch protection is valid Example When a branch has read and write protection all the methods in
20. SAMPLEID Only available with Lambda 14 and Lambda 14 2 Modify the method as required 52167 20 Release 2 0 62161 20 2 0 Enzyme Kinetics 3 if necessary determine the blank value of the reaction see 5 28 and enter the value in the parameter BLANK 4 Press START to start the measurement 5 Depending on the display shown SAMPLE ID Change the displayed parameter values if ENTER gt required and press START Nou n BACK CORR Insert the cell containing a blank solution PRESS START distilled water and press START SEO UR n SAMPLE 1 Place solutions in the cell according to PRESS START the procedure and mix Place the cell in the sample cell holder Press START to start the measurement or to continue the method Example of the Display Shown During the Measurement xxx nm nm Wavelength xxx min xxx ABS xxx min Time xxx ABS Measured value The display shown when using a cell changer EN n 001 Type of method xxx min Xx ABS Temperature n Cell location C 001 Cycle number xxx min Interval time xxx ABS Measured value Printout The results are printed out at the end of the analysis 5 27 Enzyme Kinetics Determining the Blank Value Determine the blank value of the reaction as follows 1 Select the appropriate ENZYME method 2 Set parameter BLANK 0 0 in the method 3 Carry out a
21. been started independent of whether the parameter has been tagged or not Insert the reference solutions in sequence e g when asked Always start at location 1 REFS IN CELL 1 5 PRESS START 6 6 82181 20 Release 2 0 Accessory Parameters 6 8 Accessory Parameters Parameter Description General ACCESSORY Select the accessory MANUAL operation with standard cell holder i no accessories CELL for cell changers e g 5Cell Changer 6Cell Changer 8Cell Changer 9Cell Changer 13Cell Changer SIPPER for Vacuum Sipper or Peristaltic Sipper AS 90 91 for autosamplers AS 90 and AS 91 Note Sipper operation is not possible when using SUBSTRATE methods If ENZYME methods are used neither Sipper nor Autosampler can be used Cell changer 5cell 6cell CELL 1 n The locations at which measurements are to take place Enter the number of the locations and press ENTER If e g measurement is to be carried out at locations 2 and 5 only enter 25 If BACK CORR YES has been selected location 1 is used for background correction independent of the locations selected as described above Thus If BACK CORR YES has been selected place the blank at location 1 and the sample solutions from location 2 If BACK CORR NO has been selected all locations can be used for sample measurement B2161 20 Release 2 0 6 7 Accessory Parameters Parameter Description Cell changer
22. creation of the new method i OF nnn SCAN Press STOP to cancel lt gt PARAM START 6 Modify the new method as required Creating a New Method Overwriting an Existing Method 1 Select a method that can be overwritten m 13 WAVELENGTHPROG lt gt PARAM START 2 Press PARAMETER PARAMETER MODIFY METHOD gt 3 Use the arrow keys to select Y NEW METHOD NEW METHOD gt 4 Press PARAMETER again PARAMETER 5 Use the arrow keys to select the method 4 b type t NEW TIME DRIVE 6 Press PARAMETER PARAMETER The existing method is written over 18 TIME DRIVE Press STOP to cancel lt PARAM START 7 Modify the new method as required 4 10 82161 20 Release 2 0 New Method Name 43 4 New Method Name You can rename methods using the NEW METHOD NAME parameter The method number remains the same when you rename method 1 Select the method to be renamed 13 TIME DRIVE lt 2 PARAM START T qa 2 Press PARAMETER PARAMETER MODIFY METHOD PARAM gt 3 Use the arrow keys to select v NEW METHOD NAME NEW METHOD NAME PARAM gt 4 Press PARAMETER again PARAMETER 5 Use the arrow keys to select letters 4 gt Confirm each letter by pressing ENTER OF NEW METHODNAME Use the numeric keys to enter numbers gt TIME DRI Confirm each number by
23. does not appear when using the cell changer 28 61 17 Length of the oligonucleotide base sequence If batch procedure is not used enter 0 Abscissa scale in nm cm or mm min for graphics printout valid only when GRAPHICS PLOT YES Options 0 5 1 2 5 10 20 50 100 nm cm 1 2 5 10 20 50 100 mm min Sequence of the oligonucleotide bases The sequence must be entered in order from base 5 to 3 Use the numeric keys according to the table below to enter the base sequence Option Base Kev is Adenine 7 isCytosine 4 i Guanine 1 is Thymine 0 is any base 8 Range 2 to 50 Required slit width in nm Options 0 5 1 2 4 Note Only shows with Lambda 14 and Lambda 14P spectrometers 82161 20 Release 2 0 Parameter SMOOTH 82167 20 He sasa 2 0 Parameters No Description 14 Smoothing according to Savitzky Golay in nm The acceptable level of smoothing is dependent on the scan speed SPEED up to 960 nm min 1920 nm min 2880 nm min SMOOTH 0 2 3 4 0 4 6 0 6 6 8 10 nm 8 10 nm 8 10 nm Guideline 0 5 x peak width of lowest peak Smooth 2 nm corresponds to 14 Peak width Spectrum quality net compromised Smooth 6 nm Spectrum quality poor Smooth value is 10 nm Benzene spectrum not recognizable Smoothing influences resolution and noise an increase smoothing decreases the noise but resolution suffers A too high degree of smoothing tends to ave
24. equilibration time has elapsed Substrate Once the equilibration time has elapsed add the enzyme solution to the cell and mix Operating with a Sipper Delay between the end of the aspiration process and the start of the measurement Range 0 1 to 99 9 Dilution factor Range 0 00001 to 9999 9 Divisor Range 0 00001 to 9999 9 For example the molar absorption coefficient value can be entered a5 divisor Values can be obtained from the literature Note if the absorption coefficient is already included in the ENZ FACTOR enzyme factor enter DIVISOR 1 Enzyme Enzyme activity is automatically calculated as follows pg enzyme factor x dilution factor x L divisor x d4 dr Substrate Substrate concentration c is calculated as follows C Sub concentration factor x dilution factor x 1 divisor x with absorbance difference 10 9 Parameters Parameter DIVISOR continued END TIME ENZ FACTOR TO 10 No Description 12 11 Concentration 1 Concentration 2 Divisor The measured value is multiplied by the factor or divided by the divisor and the resultant value displayed Thus dilution procedures or differing masses can be taken into account If a dedicated correction factor is to be used for each sample select FACTOR or DIVISOR as a START tag parameter see page 4 7 The factor or divisor can be entered immediately prior to each analysis Example p
25. equivalent of substrate per minute The unit may appear in capitals on the display Factor Range 0 000001 to 9999 The measured value is multiplied by the factor and the result displayed Thus concentration can be read off directly or a dilution taken into account If only the true measured value is to be shown then choose FACTOR 1 If concentration units are to be read off directly calculate the factor according to the Beer Lambert law c where is the absorbance is the molar absorption coefficient c is the concentration of the sample d is the pathlength of the celi Thus the concentration c A ed and the factor f 1 ed 10 11 Parameters Parameter FACTOR n FIRST SAMPLE GRAPHICS PLOT GRID INTERVAL 10 12 No Description 11 29 21 Factor for each wavelength n Range 9999 0 to 9999 9 The measured value is multiplied by the factor and the result displayed Thus concentration can be read off directly or a dilution taken into account If only the true measured value is to be shown then choose FACTOR n 1 If concentration units are to be read off directly calculate the factor according to the Beer Lambert law where is the absorbance the molar absorption coefficient c is the concentration of the sample d is the pathlength of the cell Thus the concentration c A ed and the factor f 1 ed Number of the first sample in th
26. peak 82161 20 Release 2 0 No 5 B2187 20 Configuration Branch A3 7 CONFIGURATION the Configuration Branch This branch contains methods for configuration of the spectrom eter to the individual requirements of the user Method HELP CONFIG AS90 91 CONFIG COMM CONFIG PORT USAGE CONF Ralease 2 0 Function Level and language of help messages Parameters LEVEL Extent LANGUAGE Language Setting for the AS 90 91 sample locations upper and lower limits for the sampler arm etc Details are contained in the AS 90 91 User s Manual Protocol for the RS 232 interface for use with a The default parameter values are for use with PECSS and Epson PC Default values Port Enable No 015 Respond Prompt Prompt Character 021 Baud Rate 4800 Break Character 043 Bits Character 8 Erase Character 010 Stopbit 1 Kill Character 010 Parity none Range 2 Terminator CRLF Usage of the RS 232 interface The parameter MAIN PORT determines whether a printer or a PC can be connected to the RS 232 interface PORT 2nd RS 232 PRINTER printer PC COMPUTER PC printer After a full reset the value is set to PRINTER 7o facilitate adminis tration set MAIN PORT PRINTER and connect the printer to the Ist RS 232 interface the PC to the 2nd RS 232 in
27. service personnel Default 0 0 nm ABS FACT Only for service personnel Default 1 0 D2 OFFSET Only for service personnel Default 0 0 nm FILTER n Wavelengths for filter change filter 2 7 Default filter 2 830 0 nm filter 3 683 0 nm filter 4 558 0 nm filter 5 2 420 0 nm filter 6 2 383 0 nm filter 7 326 0 nm lamp change point A3 8 VALIDATION the Validation Branch The methods in this branch are reserved for customer service The methods are used to test that the spectrometer s perfor mance lies within specification A3 9 TEST the Test Branch These methods are for use by Perkin Elmer Service only 2161 20 Release 2 0 A3 7 BU caa a F M METHOD ENTER P PARAMETER S STOP Arrow keys B2T161 20 Release 2 0 Enzyme Kinetics Appendix 4 A4 1 52161 20 Aleasa 2 0 Enzymatic Analytical Procedures Enzyme analysis makes use of enzyme controlled reactions to determine a substance These procedures are used especially in clinical chemistry and food chemistry There are two methods of enzymatic analysis The determination of enzyme concentration or enzyme activity enzyme kinetic measurement For example the determination of enzymes in blood serum The determination of the concentration of a substrate substrate kinetic measurement For example the determina tion of components in food e g alcohol in wine The basis of both methods is the con
28. solution containing 1 g substance in 100 mL of solution in a cell of 1 cm pathlength is color number The units appear all capitals on the display 2161 20 Release 2 0 Parameters Parameter No Description CREEP CYCLE 21 Number of cycles after the reaction end time Range 0 no further measurements after end point 2 to 99 This parameter is used to compensate for creeping reactions The spectrometer calculates the slope for each interval If it remains constant the substrate reaction is complete the spectrometer will then determine the difference in absorbance for the substrate reaction If the slope does not remain constant two further measuring intervals are added CREEP TIME 24 Duration of the measuring interval units as selected for TIME UNIT Appears only if CREEP CYCLES is 2 or more Range 0 1 to 999 9 CREEP CYCLES 1 rm _ r CREEF TIME Creeping Reaction J Absorbance Difference ENO TIME Time D 2 CD Start of the Method 2 Enzyme Added 3 Start of Measurement 82161 20 Relaasa 2 0 10 7 Parameters Parameter CUR FIT CYCLE TIME CYCLES 10 8 No Description 22 21 240 Different types of calibration Curve Fit can be calculated the spectrometer software LINEAR used when the measured values vary linearly with the concentration the curve passes through the origin LINEAR INT
29. then press START SAMPLESIN9 CELL Insert samples in the cell changer and then PRESS START press START BACKCORR siPPER Press START on the Sipper background ACCESSORY START correction SAMPLE1 SIPPER Press START on the Sipper sample ACCESSORY START Measurement 62187 20 Release 2 0 6 6 Procedure Locations Blank solution Sample solution BACK CORRz YES BACK CORRZNO H2161 20 2 0 Background Correction with Cell Changers Background Correction with Cell Changers The locations used for sample measurement depend on the tagging and option chosen for the BACK CORR parameter Not all locations can be used for sample measurement see Table 6 1 Table 6 1 BACK CORR Tagging BACK CORR tagging FIX CALL START BATCH Insert solutions When selecting a back If no background according to ground correction correction is selected display First insert all solu BACK CORR is only tions E selectable in the Then select YES or appropriate method NO and press Thus on demand to START insert the sample First insert all solu tions Then start the first measurement 1 2 n 1 0 6 5 CONCENTRATION Methods with Cell Changers 6 7 CONCENTRATION Methods with Cell Changers Please note the following when using CONCENTRATION methods When BACK CORR YES has been selected background correction is carried out once the method has
30. they absorb radiation With such samples which are mostly bio chemical lack of reproducibility will characterize the resultant data 5 Other Sample Properties Samples that are polarizing in nature or have a double index of refraction are often difficult to measure accurately The emerg ing monochromatic radiation is slightly polarized due to having been refracted Thin film samples also pose a problem since optical interferences may develop causing a regular interference pattern to be super imposed on the spectral curve 8 3 Solvent Properties 8 3 Solvent Properties The soivent should meet the following requirements e It should dissolve the sample without reacting with it e The radiation absorption in the scanning region should be low High absorption by the blank reduces the reference energy thus increasing noise Evaporation should be fairly low at ambient temperature In general aromatic compounds exhibit high absorption in the UV region and hence are not suitable as solvents for measure ments in this region Water is virtually the only useful solvent below 195 nm but it must be freed from oxygen to attain best transmission Whenever you are going to use a solvent with unknown absorp tion characteristics scan its spectrum first to determine whether it is suitable The lower wavelength limits of a number of commonly used sol vents are presented in the following table The lower limit h
31. using the numeric keys e g 325 5 2 Then press GOTO The monochromator slews to the selected wavelength 3 2 82761 20 Release 2 0 Manual Background Correction 3 3 Manual Background Correction In this operation the BACK CORR key is used to set the measured absorbance value to 0 or transmittance value to 100 for the actual wavelength shown on the display 1 Open sample compartment cover 2 Place cells with blank solution in the reference and sample cell holders OF Place empty cells in the reference and sample cell holders 3 Close the sample compartment cover 4 Set the desired wavelength using GOTO 5 Press BACK CORR Background correction is complete once the standby display reappears The ordinate mode of the last used method always appears on the display To convert absorbance to transmittance or vice versa select a TIME DRIVE method and then select the desired ordinate mode B2161 20 Ralesse 2 0 3 3 Quick Sample Measurement 3 4 Quick Sample Measurement You can make quick sample measurements as follows 1 2 3 4 3 6 7 8 9 10 11 12 13 Prepare the sample Press GOTO Select your desired wavelength Press ENTER The monochromator slews to the selected wavelength Open the sample compartment cover Insert cells with blank solutions in the reference and sample cell holders Or use air as blank Close sa
32. 1 all the results of a particular batch are printed out sequen tially This enables spectra to be more easily compared than if they are printed out separately Prints out the calibration curve Option YES NO Allows you to recalculate the results Option YES Enzyme When POSTRUN YES is used the results for a changed TOTAL TIME and LAG TIME can be recalculated When using a cell changer the values for TOTAL TIME and LAG TIME must be a whole multiple of the set INTERVAL time Substrate When POSTRUN YES is used the results for a changed reaction END TIME can be recalculated When using a cell changer no recalculation is possible Prints out a table containing analytical data when activated Option YES 2101 20 Release 2 0 Parameters Parameter No Description PAINT REFS 26 Prints out data from the references when activated Option YES REFS 9 Reference solutions Choice as to whether a calibration curve should be established or not at the start of a method Optio OLD NEW Old means you wish to use the OLD stored calibration curve New means you wish to make a NEW calibration curve If a calibration curve is to be used again or if the values of the curve are to be entered directly select REFERENCES OLD see also parameter VALUE It is often useful to tag REFERENCES e g as CALL parameters select REFERENCES NEW when selecting the first method and generate a calib
33. 181 20 Release 2 0 How to use Methods lt lt lt SURES Re ESE Methods 4 1 B2161 20 Aelease 2 0 4 What are Methods Methods are a collection of those parameters necessary for a particular analysis using the spectrometer and are stored as method files You can process large numbers of samples efficiently using the methods The parameter values necessary for the analysis in question need only be set once and are then available on request Up to 200 methods can be stored in the spectrometer each method can be allocated a number between 1 and 999 On delivery 10 basic methods are programmed in the spectrometer these are immediately available for use 4 1 Selecting a Method 4 2 Selecting a Method 1 Switch to the standby display use STOP or PARAMETER 500 0nm 0 000 ABS INPUT gt lt 3 Enter the method number 4 Press ENTER 500 0 0 000 ABS E SELECT METHOD gt lt The method is loaded onto the operational memory Entry Field The method header then appears on the display 1 In the standby display enter the method number 2 Press METHOD Method Number The method is loaded onto the Method Name operational memory 2 SCAN The method header then appears on PARAM START the display Method header The method can now be used for measurement If an unused method number is entered the function NEW METHOD appea
34. 7 Printing Out a Method 4 15 44 Printing Out a Directory 4 16 441 Spectrometer Directory 14 4 4 tarea 4 16 4 4 2 Branch Directory 4 16 45 4 17 Chapter 5 Measuring using methods 51 2 2 4 5 1 5 2 5 2 53 Analysis Procedure 8 3 2 B2181 20 2 0 5 4 5 5 5 6 5 7 3 8 5 9 5 10 5 11 5 12 5 13 Table of Contents continued Single Wavelength Measurements 3 6 Scanning a Spectrum ehe nhan 5 8 Measurement at Several Wavelengths 5 10 Concentration Determination 2222224 5 12 5 7 1 CONCENTRATION 1 Method Peak heights 3 12 5 7 2 CONCENTRATION 2 Method Peak Areas 2nd Derivative 5 18 5 7 3 Processing the Calibration Curve CONCENTRATION Methods 5 24 Enzyme Kinetics NONU 5 26 Substrate Kinetics 5 30 5 9 1 Recalculation of Results with POSTRUN 5 34 Metho
35. ABS d 1 0 pg mL 3 0 pg mL 0 1 0 3 1 0 15 YES 1 0 1 min Modify the parameters as required using the wavelengths determined above Once a method is created you can save it and use it for the same analysis when required without having to redetermine the wavelengths 52161 20 Release 2 0 Concentration 1 Establishing the Calibration Curve 1 Press START to start the measurement 2 Depending on the display SAMPLE ID Change the displayed parameter values if ENTER gt lt required and press START BACK CORR Insert cell s containing a blank solution PRESS START and press START REF n xx Inserta cell containing a reference PRESS START solution and press START 3 Insert the references in sequence when asked When they have all been measured the instrument prints out the calibration curve and results You can now amend the calibration curve see Section 5 23 page 5 24 if required You can use previously established calibration curves or reference values see REFS page 10 21 B2161 20 Release 2 0 5 15 Concentration 1 5 16 Measuring the Sample 1 Press START to start the measurement 2 Depending on the display SAMPLE ID Change the displayed parameter values if ENTER required and press START 2 BACK CORR Insert cell s containing blank solution PRESS START and press START OL sMPL1 Insert a cell cont
36. E n n is the sample number Press START to start the measurement Tagged parameter amp is the CALL tag if desired enter a new value and press ENTER OL Select a new value using the arrow keys Press START to proceed with the analysis 82181 20 Release 2 0 Analysis Procedure 5 3 Analysis Procedure 1 Select the appropriate method see Section 4 2 page 4 2 2 Ifnecessary modify the method parameters 3 Press START 4 Depending on the display SAMPLE ID Change the displayed parameter values if ENTER gt required and press START OF Insert cell s containing a blank solution PRESS START and press START var sMPL1 Insert a cell containing the sample PRESS START solution and press START is any method type 5 Continue to insert samples when asked until they have all been measured interrupting the Measurement Press STOP to interrupt a method in progress 82161 20 Release 20 5 3 Method Procedure Diagram 8 4 n METHOD lt gt GALL Parameter 4 START BATCH Parameter 1 G STAAT Parameter Measurement Next Sample Measurement Repeated in the Group measured Figure 5 1 Flow diagram of a typical method procedure 82181 20 Release 2 0 B2T61 20 Release 2 0 5 5 Time Drive 5 6 5 4 Single W
37. ERC used when the measured values vary linearly with the concentration the curve has an intercept on the measured value axis to compensate for background interferences QUADRATIC used when the measured values do not vary linearly with the concentration the curve passes through the origin QUAD INTERAC used when the measured values do not vary linearly with the concentration the curve has an intercept on the measured value axis to compensate for background interferences The time in minutes between the start of one sample measurement to the start of the next sample measurement Range 0 002 to 999 99 CYCLE mar CYCLE Measurement Measurement2 Duration of the analysis CYCLES x CYCLE TIME Note When using accessories set the CYCLE TIME longer than required for scanning the spectrum This avoids time problems The number of t mes one sample is scanned or measured Range 1 to 99 Note If CYCLES is set to 0 the sample is scanned or measured continu ously until you stop the method B21801 20 Release 2 0 Parameter DELAY TIME DiL FACTOR DivVISOR 52161 20 Flalcsso 2 0 Parameters No Description 23 47 10 12 Equilibration time Only in the case of use with the cell holder or in manual operation with CALCULATE INTERVAL This is the time from the start of the method to the start of measurement units as selected for TIME UNIT Range 0 0 to 999 9 Note Measurement begins after
38. G Documentation Only a Perkin Elmer service representative or similarly trained and authorized person should be permitted to service the instrument 7 1 Daily Care Do not leave samples particularly those given to fuming or evaporation in the sample compartment for longer than necessary e If any type of sample handling system is installed and portions of it are left in the sample compartment such as a sipper and flowcell make certain that the system is cleaned at the end of the working Generally such systems should be filled with deionized water when left overnight Immediately clean all spilled materials from affected area and wipe it dry with lintless paper or cloth If you have to wipe sample compartment windows make sure you do not introduce scratches Sample windows are optical components and you should handle them in the same way as high quality cells 82167 20 2 0 Use and Care of Cells Expensive damage can result to the optics or electronics if liguids are spilled and run inside the instrument or onto the keyboard Take care not to spill liquids onto the spectrometer CAUTION 7 2 Use and Care of Cells 7 2 7 Handling Only hold cells by non optical surfaces such as the matt finish surfaces e Always wipe the optical surfaces of cells dry and free of fingermarks using a soft lint free cloth or cleaning tissue just before placing them in the cell holder
39. HOD YES 36 OPERID 2 o 87 SAMPLEID Only available with Lambda 14 and Lambda 14P When a particular parameter is set to YES the extra parameters required automatically appear in their correct order eg When GRAPHICS PLOT is YES the extra parameters ORD ORD MIN SCALE and GRID appear When GRAPHICS PLOT is NO the extra parameters ORD MAX ORD MIN SCALE and GRID no longer appear 2 If necessary modify the method parameters 3 Press START to start the method 2181 20 Haelesso 2 0 B2157 20 Release 20 Scan 4 Depending on the display shown SAMPLE ID Change the displayed parameter values if ENTER gt required and press START EOM BACK CORR Insert containing a blank solution PRESS START and press START Of SCAN sMPL1 Insert a cell containing the sample PRESS START solution and press START 5 Continue to insert samples when n asked until they have been measured Example of the Display Shown During the Measurement SCAN 1 nm Wavelength XXX ABS ABS Measured value ordinate as selected CYC xx Repeat measurement cycles still to be performed This appears on the top right when cycles gt L Printout Graphics are printed out during the measurement process numerical data follow at the end of the analysis Wavelength Program 5 6 Measurement at Several Wavelengths Select a wavelength p
40. ION the Configuration Branch 42444 A3 5 A3 8 VALIDATION the Validation Branch A3 7 A3 9 TEST the Test A3 7 C 4 B21601 20 Release 2 6 Table of Contents continued Appendix 4 Enzyme Kinetics 41 Enzymatic Analytical Procedures 4 1 A42 Enzyme Kinetics 4 2 A43 Substrate Kinetics 4 4 Annex Translations of Warnings 82101 20 Release 20 C 5 Table of Contents continued illustrations 1 1 Features common to Lambda 12 14 and 1 1 2 1 Single Cell Holder B050 5071 hehehe rures essere 2 4 2 2 Correct Alignment of the Radiation Beam in the Cell Sample Area 2 7 5 1 Flow diagram of a typical method procedure 5 4 Tables 24 Minimum Volume 2 9 41 Text and symbols on the lower line of the display 4 6 4 2 Typeoflags TONNEN 4 7 453 Help Key Combinations 4 17 61 BACK CORR Tagging 6 5 8 1 Lower Wavelength Limits of Solvents 8 5 2 1 Protect Functions
41. MARK A method has been tagged MARK FOR COPY although the method in question is no longer available This error is shown when the instrument gets no response from an attached accessory during startup Check that the accessory is correctly connected Switched on functioning normally range Enter number within the range 1 to 999 An attempt was made to delete all methods Retain at least one method in the memory otherwise the spectrometer cannot work An attempt was made to select a fully protected method The protection has to be modified if the method 15 to be used An attempt was made to ALL protect all the methods Retain at least one method otherwise the spectrometer cannot work An attempt was made to ALL protect all branches Retain at least one branch otherwise the spectrometer cannot work An attempt was made to modify methods in a write protected branch modify the method alter the branch protection 82181 20 Felease 2 0 Error PROBLEM DIRECTORY FULL PROBLEM MEMORY FULL ERROR NO ENERGY ERROR NO ENERGY UV LAMP PROBLEM NO ENERGY VIS LAMP 82157 20 Halcase 2 0 Error Messages Meaning An attempt was made to store more than 200 methods To create space for the new method delete a method that is no longer required The available memory is insufficient to cope with the new method To create space for the new method delete a method that i
42. ME UNIT 20 Units for all subsequent time parameters min minutes seconds TM CALCULATION 76 Calculates the theoretical melting point Ty Refer to the Biochemical Application manual B2161 20 Re onse 2 0 10 25 Parameters Parameter TOTAL TIME TRAY VALUE n 10 26 No Description 22 50 Total time from the start of the method or end of DELAY TIME to the end of the measurement units as selected for TIME UNIT Range 0 to 999 9 Select the measuring time so that ihe end of the measurement ts stil within the linear portion of the curve Manual operation For CALCULATE REGRESSION the time is that between the stant of the method and end of the measurement For CALCULATE INTERVAL the time is that between the end of the DELAY TIME and end of the measurement Operation with Cell Changer The time from the end of the DELAY TIME io the end of the measurement Within this measuring period all samples are measured n times consecu tively The number of cycles is determined by the measuring time and the INTERVAL time Number of cycles TOTAL TIME INTERVAL 1 If the result proves to be a decimal fraction the next higher whole number is taken e g 100 30 I 433 5 cycles The cycle time is identical to the INTERVAL time The final cycle begins at the end of the measuring time i e measurement ends only on completion of the final cycle Enter the number of the tray used with the AS90 91 auto
43. Measurement at several wavelengths 5 6 differential and ratio analysis at several wavelengths 4 CONCENTRATION 1 Determination of concentration using peak 571 height S CONCENTRATION 2 Determination of concentration using peak 5 7 2 area or 2nd derivative 6 ENZYME KINETICS Enzyme kinetics 5 8 7 SUBSTRATE KIN Substrate kinetics 5 9 OLIGOQUANT 1 Quantitative analysis of oligonucleotides up 5 10 to 50 bases long OLIGOQUANT 2 Quantitative analysis of oligonucleotides 5 10 longer than 50 bases 900 DATE TIME To enter and change date and time 5 11 901 WAKEUP To switch on the lamps and allow them to 5 12 warm up before the start of the working day 990 SELF TEST Instrument internal test to check the optics 5 43 B2161 20 Rebase 2 0 5 1 5 2 Method Procedure When a method is selected it can be used for measurements When starting the method the system automatically makes requests via the display eg BACK CORR PRESS START SCAN SMPLn PRESS START Background correction Place a cell containing a blank solution in cach of the sample and reference cell holders OL Place an empty cell in each of the sample and reference cell holders m asurement against air Press START to start the background correction Sample measurement Place the cell containing the sample solu tion in the sample cell holder n ENTER can be used to switch directly to SAMPL
44. PARAMETER tion 0 Press STOP to cancel 6 Exit SuperUser mode to activate the protect function A2 4 B2161 20 Roloste 2 0 H2161 20 Release 2 0 Setting Protect Functions Preventing Access to the Methods and Branches You can prevent access to branches and methods using the protect functions Example If only one particular method is to be used without access to the parameters set the protect functions as follows Set full protection for all branches except the application branch Set read write protection for the application branch Set full protection for all methods in the application branch not to be used For the method to be used the read write protection of the branch is valid This method can be used outside of the SuperUser mode i e started but it cannot be read or altered Tagged parameters continue to appear when methods are being run and may then be modified 2 5 A2 6 lt 8016120 Aelsass 20 Instrument Branches Appendix 3 A3 1 Overview Of the Instrument Branches The basic spectrometer has 6 branches Branch Content Application Analysis methods Communication Interface methods Calibration Methods to calibrate the spectrometer Configuration set up basic spectrometer settings Test Test methods Validation Validation methods Can only be accessed as SuperUser Methods can only be modified you cannot create any new methods 52181 20
45. R ACCESSORY START H REF 1 xxx PRESS START 82781 20 2 0 Description Request to start background correction Display during background correction Request to start sample measurement Display during sample measurement SCAN method rn ee ee Display of a tagged parameter If necessary the parameter can be changed Cell Changet request to insert blank solutions and start background correction Cell Changer request to insert sample solutions and start the measurement Sipper request to press start key on the Sipper back ground correction Sipper request to press start key on the Sipper sample measurement rr c a aua Request to measure reference solution 1 5 1 6 82187 20 Ralease 2 0 Switching On and Off ay MOX ON uus H EE ee Pa ea ao Startup and Shutdown 2 Please take your time when operating spectrometer wait until the BUSY display has switched off before pressing the next key in the sequence This allows the software to complete the calculations and the motors to move the optics to their required setting Before starting analysis leave the spectrometer switched on for approximately 10 minutes to allow the lamps to warm up and stabilize B2161 20 Ralease 2 0 2 T Startup 2 2 2 1 Star
46. T as required 2 Press START The new curve fit is calculated on already available data Remeasuring the Reference Solution Should a measuring point lie outside the calibration curve and need to be remeasured proceed as follows 1 Modify the method select REFS 2 NEW 2 Press START 3 When asked for first reference solution REF 1 enter the number of the reference to be remeasured and press ENTER Xxx PRESS START 4 Place the reference solution in the sample cell holder and press START 5 Wait until measurement is complete and then press STOP 6 Set REFS OLD 7 Press START The new calibration curve is calculated with the new value If the new cutve is satisfactory measure the sample OF if it is not acceptable repeat the procedure 5 24 82761 20 Release 2 0 Calibration Curve Deleting a Point from the Calibration Curve To delete such a point proceed as follows Linear curve through zero In the method parameters 1 Set REF n 0 000 and VALUE n 0 000 n is the number of reference solutions 2 Press START to confirm the changes 2 04 Press STOP to cancel Non linear curves and curves with intercept In the method parameters 1 Note the ordinate value and the concentration value of the last point 2 Replace the ordinate value and the concentration value of the point to be deleted REF n with the values noted in step 1 above 2 Reduce the v
47. a Record the spectrum of the sample see SCAN method page 5 8 b Select a strong peak and note wavelength at its start WAV MAX and end WAV MIN c Record the spectrum of the same sample using the 2nd derivative D2 mode over the wavelength range determined in step b above d From the 2nd derivative spectrum determine the wavelength at the peak maximum and peak minimum Use these values for CALC WAV 1 and CALC WAV 2 D2 WAV MIN CALC WAV 2 CALOC WAV 1 WAV MAX Peak minimum Peak maximum B2161 20 Bolease 20 5 19 Concentration 2 Determining the Threshold Value 2nd derivative 1 To determine the threshold value a Record the spectrum of the most dilute reference solution using the 2nd derivative D2 mode over the wavelength range determined in step 1b of Determining the measurement wavelengths 2nd derivative above b Determine the value D2 of this spectrum i e the height from peak maximum to peak minimum D2 WAVMIN CALC WAV 2 CALC WAV 1 WAVMAX Peak minimum Peak maximum c Select a value for the threshold parameter somewhat lower than this 02 value 5 20 82767 20 Release 2 0 Creating a Method Concentration 2 1 Create a new CONCENTRATION 2 method see Section 4 3 3 page 4 9 The following table lists typical CONCENTRATION 2 parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter Mo Param
48. a is defect Make a note of the steps you made leading up to this message Press PARAMETER to continue If you cannot continue call your Perkin Elmer office and inform them of the error message and the steps you made leading up to the etror Note After full reset all methods are erased This error is shown after changing the instrument software or after a full reset or when the method memory is defect Make a note of the steps you made leading up to this message Press PARAMETER to continue If you cannot continue call your Perkin Elmer office and inform them of the error and the steps you made leading up to the error Note After a full reset all methods are erased 82761 20 Release 2 0 Error SPECTROMETER 4 DIALOG FULL RESET DONE BUS ERROR a message 82161 20 Aelease 2 0 Error Messages Meaning This error is shown after changing the instrument software or after a full reset Make a note of the steps you made leading up to this message Press PARAMETER to continue If you cannot continue call your Perkin Elmer office and inform them of the error and the steps you made leading up to the error Note After a full reset all methods are erased This error plus a message is shown when the instrument has an address error Make a note of this message and the steps you made leading up to the error Press PARAMETER to continue If you cannot continue call your Perkin Elm
49. absorbance against concentration will be nonlin ear for a number of reasons The absorption characteristics of a sample can be changed during sample preparation depending on the amount of reagent added for color development and so on For details refer to reference books covering these subjects 8 2 82167 20 Release 2 0 82161 20 Aeieasa 2 0 Unusual Samples Temperature has an influence to a greater or lesser degree on the absorption characteristics of a sample You should check this effect if non repeatable results are obtained If you are measuring temperature dependent samples either wait until temperature equilibrium has been attained or use a thermo statted cell or cell holder 3 Chemically Reactive Samples If a reaction takes place in the cell between the sample material and the solvent spectral data based that sample cannot always be expected to have sufficient reliability or repeatability For samples of this type use a quantitative method that takes advantage of the change in transmittance with time at a fixed wavelength For details refer to reference books covering this specific subject 4 Photoactive Samples Some samples are known to be photoactive in that they fluoresce upon absorbing radiation Since a small portion of the fluorescent radiation will be measured by the detector a higher apparent transmittance will often result Samples are also known that undergo photochemical reactions as
50. aining the sample PRESS START solution and press START 3 Continue to insert samples when asked until they have all been measured Example of the Display Shown During the Measurement CONC1 SMPL1 200 m Wavelength nm 96 Result units as selected CYCLES xx CYCLES XXRepeat measurement cycles still to be performed This appears on the top right when cycles 21 XXX X Printout If PLOT REFERENCES and PRINT DATA are set to YES the calibration curve and results are printed out 82167 20 Ragase 2 0 5 17 B2181 20 Aelease 2 0 Concentration 2 5 7 2 CONCENTRATION 2 Method Peak Areas 2nd Derivative Summary of procedure for creating a CONCENTRATION 2 method Determine the measurement wavelengths see page 5 19 Determine the threshold value 2nd derivative see page 5 20 Create a CONCENTRATION 2 method see page 5 21 e Fstablish a calibration curve using references see page 5 22 Measure the sample see page 5 23 5 78 82191 20 Release 2 0 Concentration 2 Determining the Measurement Wavelengths Peak areas 1 To determine the wavelengths a Record the spectrum of the sample see SCAN method page 5 8 b Select a strong peak and note the wavelength at its start WAV MAX and end WAV MIN o WAV MIN WAV MAX A WAV MIN WAV MAX Determining the Measurement Wavelengths 2nd derivative 1 To determine the wavelengths
51. alue for OF REFS by 1 3 Press START to confirm the changes Press STOP to cancel 5 25 B2161 20 Release 20 Enzyme Kinetics 9 8 5 26 Enzyme Kinetics Select an ENZYME method for enzyme kinetic measurements Note Enzyme activity is strongly dependent on temperature Thus the following should be taken into account All measurements should be carried out at a constant temperature You can use the temperature sensor Part Number B018 5227 for monitoring the temperature All solutions and essential instrument accessories especially cells and cell holders should be thermostatted prior to use Procedure 1 Select the appropriate ENZYME method The following table lists typical ENZYME parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter No Parameter _ No Parameter SLIT 2 0 nm 3 WAVELENGTH 340 0 nm RESPONSE 05s 15 LAMP UN Vis 20 TIME UNIT min 9 CALCULATE REGRESSION 22 TOTAL TIME 1 0 min 21 INTERVAL 0 2 min 24 LAGTIME 0 0 min 11 ENZ FACTOR 1 0 10 DiL FACTOR 1 0 12 BIVISOR 1 0 7 BLANK 0 0 6 ENZ UNITS U L 16 BACK CORA YES 14 SAMPLES BATCH 0 19 FIRST SAMPLE 1 25 GRAPHICS PLOT YES 27 ORD MIN 0 000 ABS 29 GRID YES 34 POSTRUN KIN YES 36 OPER ID 26 ORDMAX 1 000 ABS 2B SCALE 20 mm min 42 PRINT DATA ALL 35 AUTO METHOD YES
52. ample compartment window T4 7 3 Clock internal 5 41 realtime 5 41 Cold Solutions 7 2 Communication branch 43 3 configuration A3 5 1 index Compressed gases safety information S 7 Conc 10 19 factor 10 6 unit 10 6 Concentration Method 5 12 concentration 1 calibration curve 5 15 create 5 14 measurement wave length s 5 13 sample measurement 5 16 summary 5 12 concentration 2 2nd derivative 5 19 calibration curve 5 22 create 5 21 measurement wavelengths 5 19 peak areas 5 19 sample measurement 5 23 summary 5 18 threshold 5 20 with cell changers 6 6 Configuration accessory 43 6 5 90 91 A3 5 branch A3 5 communication 43 5 factory A3 7 help 3 5 port usage 3 5 printer A3 6 user 3 Connector panel 1 1 Copying method 4 14 method parameters 4 13 Cor 10 1 10 19 10 20 cycle 10 7 time 10 7 Creeping reactions 44 5 Cur Fit 10 8 Index 2 Curve calibration 5 12 5 15 5 22 5 24 10 26 changing the type of fit 5 24 type of fit 10 8 Cycle Time 10 8 Cycles 10 8 D1 10 19 122 10 19 23 10 19 D4 10 19 Daily Care 7 1 Date Time 5 40 Default Methods 4 3 Delay Time 6 9 10 9 Deleting a method 4 8 Delta ABS 10 14 Deriv 2 Fix 10 15 10 16 Deriv 2 Peak 10 15 10 16 Derivative second 5 19 Dif 10 1 10 19 10 20 Dil Factor 10 9 Directory branch 4 16 printing 4 16 spectrometer 4 16 Display 1 1
53. and continue a method Numerical keys Decimal point Minus used to enter negative values Confirms parameter Clears unconfirmed parameter entry B2151 20 Helaase 2 0 Key Combinations 1 1 1 Key Combinations Key Combinations Description nnn METHOD nnn n GOTO 4 Selects method nnn To change to wavelength nnn n nn PARAMETER Selects parameter nn when you are in the parameter list level PARAMETER PARAMETER e HELP 0 HELP 1 HELP 2 HELP 3 HELP 4 HELP 5 HELP 6 HELP 7 HELP 7 9 1 5 7 2181 20 Release 2 0 Selects parameter tagging Selects default methods from Application level Selects previous parameter Prints out a method or branch directory Prints out current values shown on the display Prints out method parameters Prints out a directory of the methods available in the branch Prints out the additional method information shown on the display Line feed Form feed Prints out the Peltier cell holder temperature shown on the display Functions only when the Peltier accessory is installed Prints out the spectrometer status Full reset see page 3 5 Caution all methods are deleted Switches to Super User mode see page A1 1 1 3 Displays 1 1 2 Displays This section contains a summary of the most common displays Display Description 500 0 NM 0 000 ABS Standby display with wavelength and m
54. articular component of a powder is to be determined and displayed in mg g The calibration curve is compiled using pure substance in solution made up in mg L In order that the results can be displayed independently of the actual mass of powder used the mass of the powder should be entered as the divisor the powder were dissolved in 0 25 L instead of in 1 L an additional dilution factor of 0 25 should be entered The results are then automatically calculated as follows 0 25 mass of powder in g x concentration in mg L concentration in mg g Time from the start of the reaction ie addition of enzyme to the end of the reaction units as selected for TIME UNIT Range 0 1 to 999 9 Enzyme factor Range 0 00001 to 9999 9 Calculate the enzyme factor as follows Enzyme factor Vidv where Vis the volume of the total solution in the cell mL d is the pathlength in cm v is the volume of sample in mL Note Depending on the procedure used the molar absorption coefficient may need to be taken into account B2161 20 Release 2 0 Parameter ENZ UNIT FACTOR 62161 20 Aelease 2 0 Parameters No Description 6 11 Enzyme Unit U L is units per liter U mL is units per milliliter mU L is milliunits per liter U is units mU X ismilliunits mg mL is milligrams per milliliter is unit Enzyme Unit U is the amount of enzyme which catalyzes the conversion of I nmol or micro
55. as been defined as that wavelength at which 10 mm of pure solvent has a transmission of 10 8 4 2161 20 Release 2 0 Solvent Properties Table 8 1 Lower Wavelength Limits of Solvents etone 1 rac h oO r 0 et h n ge Ere E Errare Re e 1 TENERE Totu ana HRAT HR Benzen aCe an nn nna b a B n Lr N Di m et h y fo m id e n NE PK ra s sp prec LAA Ba Gian natn atta t h V ro i 0 n at e EEE SPB alg RE Ga ri Tetrach ride Ser TE ee ae Glycerol E e a Mee aaa LL er eR meee a a acer A E oaae e a ie e e e A a E A Aa a A e A A ANa a AAA N Di l oxan AI RECS II Ene lA LIII m L ma II II mamm mpa m mm mmm LI E mmmn Cyclohexane Methanol Ethanol Methylcyclohexane Propanol Water EEE 190 210 230 250 270 am 29 310 330 350 370 82161 20 Release 20 8 5 8 6 2161 20 Raleasa 2 0 Error Messages Error Messages 9 If an error occurs during the operation of the spectrometer an error message 15 shown on the display or is printed out if a printer is connected 9 1 Error Messages Shown on the Display Errors remain displayed until they are deleted To delete
56. ate methods that have been inadvertently erased or written over regularly print out all important methods 4 4 82161 20 Release 2 0 4 3 1 Modifying a Method 1 Select the method to be modified 2 Press PARAMETER 3 Press PARAMETER again 4 Change the displayed parameter values if required OF Press PARAMETER to select the next parameter Press PARAMETER to recall the previous parametet OF Enter the appropriate parameter number and press PARAMETER to select a partic ular parameter see Chapter 10 for parameter description and parameter numbers OT Press STOP to cancel B2161 20 Helease 2 0 Modifying a Method e g 2 SCAN lt gt 5 PARAMETER MODIFY METHOD PARAM gt PARAMETER f SLIT 1 0 nm dx m oF PARAMETER 4 5 Modifying a Method 4 6 Changing a Parameter 1 Select the parameter to be changed 2 Depending on the parameter shown change as described in procedure table 4 1 Text Symbol lt gt ENTER gt ENTER fabie 4 1 and symbols on the lower line of the display Procedure Select option with the arrow keys Enter the desired value with the numeric keys Press ENTER to confirm the value entered Appears if several values have to be entered Use the arrow key to select the desired parameter Enter the required value Press ENTER to confirm the value ente
57. ation of the spectrometer Method 0 T CALIBRATION ONE WAVEL CALIB TWO WAVEL CALIB Function Switches the dark signal compensation on and off A residual current dark signal flows through the detector even when there 15 no beam This signal is taken into account when the dark signal function is switched on Compensation then takes place automatically either at the start of a method or every 10 minutes whichever occurs first Wavelength calibration with one peak To check the calibration record the spectrum of a wavelength standard and compare with that recorded by the spectrometer If they do not correlate the spectrometer should be recalibrated Parameters nm PEAK Internal calibration at 0 nm D2 PEAK Internal calibration at 656 1 nm SPEC PEAK Calibration using an external wavelength standard e g holmium oxide OLD PEAK Measured wavelength of the external standard NEW PEAK Actual wavelength of the external standard Wavelength calibration with two peaks To check the calibration record the spectrum of a wavelength standard and compare with that recorded by the spectrometer If they do not correlate the spectrometer should be recalibrated Parameters AUTO PEAK Internal calibration at 0 0 and 656 1 nm SPEC PEAK Calibration using an external wavelength standard e g holmium oxide OLD PEAKn Measured wavelength of the peak NEW PEAKn Actual wavelength of the
58. avelength Measurements Select a TIME DRIVE method to measure a sample at one wavelength over a defined period of time For Enzyme Activity methods see Section 5 8 page 5 26 Procedura 1 Select the desired TIME DRIVE method The following table lists typical TIME DRIVE parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter No Parameter Valus Mo Parameter SLIT 2 0 1 ORDINATE MODE ABS 23 WAVELENGTH 500 0 nm 11 FACTOR 1 0 14 RESPONSE 0 58 i5 LAMP UV Vis 16 BACK CORR YES 18 SAMPLES BATCH 0 19 FIRST SAMPLE 1 21 CYCLES 1 22 0 1 min 25 GRAPHICS PLOT YES 26 ORD MAX 0 000 ABS 27 ORD MIN 1 000 ABS 28 SCALE 20 nm min 29 GRID NO 32 PRINT DATA YES 35 AUTO METHOD NO 36 OPERID 387 SAMPLEID Only available with Lambda 14 Lambda 14 When a particular parameter is set to YES the extra parameters required automatically appear in their correct order e g When GRAPHICS PLOT is YES the extra parameters ORD MAX ORD MIN SCALE and GRID appear When GRAPHICS PLOT is NO the extra parameters ORD MAX ORD MIN SCALE and GRID no longer appear 2 Ifnecessary change the method parameters 3 Press START to start the measurement 82161 20 Release 2 0 Be151 80 Release 2 0 Time Drive 4 Depending on the display SAMPLE ID Change the displayed parameter values i
59. blish a calibration curve using references see page 5 15 Measure the sample see page 5 16 5 12 82161 20 Release 2 0 Concentration 1 Determining the Measurement Wavelength s 1 To determine the wavelengths a Record the spectrum of the sample see SCAN method page 5 8 b Select a strong peak and note the wavelength at its absorbance maximum WAV 1 1 WAV 2 Select the type of baseline correction required a With a straight but offset baseline Select a second wavelength at the baseline minimum WAV 2 WAVE WAV b With a sloping baseline Select a wavelength at the beginning and at the end of a peak WAV 2 and WAV 3 WAV WAV t WAV2 82161 20 Release 2 0 5 13 Concentration 1 Creating a Method 1 Create a new CONCENTRATION 1 method see Section lt 4 3 3 2 5 14 page 4 9 The following table lists typical CONCENTRATION 1 parameters in the order jeft to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter Parameter Value Mo Parameter Value SLIT WAV 1 CONC UNIT REF 2 REFS VALUE 2 CUR FIT DIVISOR LAMP SAMPLES BATCH CYCLES PRINT DATA PRINT AEFS OPER ID MODE OF REFS REF 1 REF 3 VALUE 1 VALUE 3 FACTOR RESPONSE BACK CORR FIRST SAMPLE PLOT REFS AUTO METHOD SAMPLE ID Only available with Lambda 14 and Lambda
60. cal components and require the same care and handling as cells Youcan remove the windows to clean them they are held in place a magnetic frame Windows are most suitably cleaned by wiping them with a soft lint free cloth moistened with ethanol B2161 20 2 0 7 3 7 4 52161 20 2 0 Analytical Notes lisi ie ER PEPPER Pee tee 23 OPE PED EE um TAE MOI eem T XQEINESUSESUSNEE I Hz ER Peo PEEL aA EC Analytical Notes 8 8 1 2161 20 Release 2 0 Background Correction The type of background correction depends on the method type selected In methods with a fixed wavelength e g TIME DRIVE WAVELENGTH PROGRAM CONCENTRATION 1 the displayed measurement value for absorbance is set to 0 for transmission to 100 at the measurement wavelength this is called an autozero You can use the BACK CORR key to perform a manual autozero in fixed wavelength methods see Section 3 3 page 3 3 In methods with measurement over a wavelength range e g SCAN CONCENTRATION 2 a background correction is performed over the selected wavelength range background correction can only be performed in a method The ordinate mode of the last used method always appears on the display To change from absorbance to transmittance or vice versa select a TIME DRIVE method and then select the desired ordinate mode A backgrou
61. d Or Press STOP to cancel You can also press 1 and then HELP to print out the method parameters 82161 20 Release 2 0 4 1 Printing Out a Director 4 16 4 4 4 4 1 4 4 2 Printing Out a Directory Printing out a directory provides a list of its methods A printer must be installed and configured refer to the installation handbook There are two directories the spectrometer directory and the branch directory Spectrometer Directory The spectrometer directory is a list of all methods for the spectrometer including the SuperUser methods Print out the directory as follows 1 Select a branch header e g 2 Press e and then HELP lt gt to print a directory of e HELP Branch Directory The branch directory is a list of all the methods in the selected branch Print out all the methods in the selected branch as follows To select SuperUser branches you must first enter as SuperUser see Appendix 1 1 Select a method in the branch tobe eg 2 SCAN 2 Press e and then HELP to print out the branch HELP directory You can also press 2 and then HELP to print out the branch directory B2181 20 HRaleasa 2 0 Help Key 4 5 Help Key 8218120 Release 2 0 The help key be used on its own to provide additional information about the parameters currently shown on the display or in combination with other keys to provide oth
62. d value Printout The results are printed out at the end of the analysis 2181 20 Release 2 0 Substrate Kinetics Delermining the Blank Value Determine the blank value of the reaction as follows 1 Select the appropriate SUBSTRATE method 2 Set parameter BLANK 0 0 in the method 3 Carry out a measurement according to your procedure using a cell filled with redistilled water in place of the sample 4 Enter the resuit of the measurement in the parameter BLANK 5 33 82161 20 Heofease 2 0 Postrun Kinetics 5 34 5 9 1 Recalculation of Results with POSTRUN KIN You can recalculate the results from ENZYME and SUBSTRATE methods using the POSTRUN KIN parameter When you set the POSTRUN KIN parameter to YES the following parameters in the corresponding method can be modified ENZYME method LAG TIME and TOTAL TIME SUBSTRATE method END TIME manual operation only Note Observe the following The time set for the LAG TIME must be smaller than the TOTAL TIME If a cell changer is being used make sure that both LAG TIME and TOTAL TIME are whole multiples of the INTERVAL time Proceed as follows to calculate the results D 2 3 4 EP Create a method with POSTRUN KIN YES Carry out a measurement see Section 5 8 or 5 9 At the end of the measurement POSTRUN KIN appears on the display Press START to recaiculate results GOL If the results are not to be recalculat
63. ds for Quantitative Analysis of Oligonucleotides enne 5 36 2 22424244241 5 40 2 5 42 5 43 Chapter 6 Operating with accessories 6 1 6 2 6 3 6 4 6 5 6 6 6 7 6 8 PP 6 1 ACCESSOTIES 2 hs herren 6 1 Requirements for Operation with Accessories V 62 Using Methods with Accessories 6 2 6 4 1 Spectrometer without Accessory Board 6 3 Running Methods with an 6 4 Background Correction with Cell Changers 6 5 CONCENTRATION Methods with Cell 6 6 Accessory Parameters v 0 7 Chapter 7 Care 71 72 Daily 2222 2 2 2 2 7 1 Use and Care of Cells 7 2 7 21 Cell Handling 7 2 72 2 Pressure Buildup in Cells 7 3 72 3 Sample Compartment Windows sese 7 3 B2181 20 Release 2 0 C 3 Table of Contents continued Chapter 8
64. e units as selected for ENZ UNT Range 0 00001 to 9999 9 The enzyme activity is calculated as follows 4Enz Blank Substrate Blank value units as selected for CONC UNIT Range 9999 9 to 9999 9 The substrate concentration 1s calculated as follows C Sub Total 7 Blank CALC WAV 1 CALC WAV 2 2761 20 2 0 Wavelength in nm used in CONCENTRATION 2 methods for calculations using the 2nd order derivative only Range 190 0 to 1100 0 in steps of 0 1 The values must lie within the WAV MAX and WAV MIN values set Enter the value at peak maximum for CALC WAV 1 see page 5 20 In MODE DERIV 2 FIX the height of the derivative curve 15 measured at these wavelengths In MODE DERIV 2 PEAK a derivative maximum or minimum is located around these wavelengths and the nearest one evaluated see also parameter MODE Wavelength in nm used in CONCENTRATION 2 methods for calculations using the 2nd order derivative only Range 190 0 to 1100 0 in steps of 0 1 The values must lie within the WAV MAX and WAV MIN values set Enter the value at peak minimum for CALC WAV 2 see page 5 20 MODE DERIV 2 FIX the height of the derivative curve is measured at these wavelengths MODE DERIV 2 PEAK a derivative maximum minimum is located around these wavelengths and the nearest one evaluated see also parameter MODE 10 3 Parameters Parameter CALCULATE
65. e analytical value obtained is outside the set values the latter should be changed Note Derivative spectra can also have negative values 27 Minimum ordinate scale range for graphics printout valid only if GRAPHICS PLOT YES Range 9999 9 to 9999 9 If the analytical value obtained is outside the set values the latter should be changed Note Derivative spectra can also have negative values 10 18 82161 20 Refaase 2 0 Parameters Parameter No Description ORDINATE MODE 1 Ordinate Mode transmittance in percent ABS Absorbance Dito D4 1st to 4th derivatives of the spectrum derivative spectra RAT Absorbance ratio DIF Absorbance difference COR Corrected Absorbance ratio conc Concentration see also FACTOR Derivative modes D1 to D4 can be used to resolve overlapping peaks to reduce interference and to enhance the fine structure of a particular peak This facilitates the qualitative evaluation of spectra with overlap ping peaks and the quantitative evaluation of spectra with unde sired background absorption The derived values obtained are multiplied by 10 for every degree of derivation in order to produce graphics that are easier to inter pret Resolution and noise increase with the degree of derivation In general the 2nd derivative is more helpful in this respect than the Ist the resohution is better and the characteristic maximum of the signal is easy to recognize as a derivative m
66. e batch All subsequent samples are automatically numbered consecutively Graphics printout Option YES NO Graphics printout with grid valid only when GRAPHICS PLOT YES Option YES NO Interval time units as selected for TIME UNIT Range 0 1 to 999 9 When using CALCULATE REGRESSION the change in absorbance dA dt is printed out When using CALCULATE INTERVAL the slope is calculated for each interval The mean of all slopes is then used for the calculation of the enzyme activity see also parameter CALCULATE 82167 20 Release 2 0 Parameter INTERVAL continued No 21 Parameters Description Operating with the Changer During the interval time the spectrometer measures once at each location To do this you need to know the minimum measuring time t min f min X 3 x RESPONSE x 0 1 seconds where N the number of cells 3 x RESPONSE measuring time per cell X relocation time from cell to cell The interval time should always be greater than the required minimum time re ERR ee ee eee eee ee eee ee eee ee ee eee LAG TIME LAMP LINE TYPE 82161 20 Aelease 2 0 15 31 Lag time This is the time from the start of the method to the start of calculation units as selected for TIME UNIT After this time a constant reaction rate should have been reached Range 0 0 to 999 9 Measurement begins with the start of the method However
67. e to center the radiation beam Then retighten the two milled posts and the two locking SCrews 82181 00 Release 2 0 Single Cell Holder 6 Now visually examine the vertical alignment of the radiation beam in the cell sample area Alignment is correct when the radiation beam is just above the floor of th cell sample a area 2 mm or covers the cell window min 2 mm Figure 2 2 Correct Alignment of the Radiation Beam in the Sample Area 7 If alignment is required turn the vertical alignment screw on the lifter either clockwise to raise the cell or counterclockwise to lower the cell 8 Recheck the horizontal alignment of the radiation beam through the cell and correct if necessary 9 Using the GOTO key slew the monochromator to any value above 200 nm 10 Remove the card blocking the sample and reference beam windows and close the sample compartment cover This completes coarse alignment of the cell holders If necessary proceed with fine alignment as described on the next page 1 The center of the window for micro flowcells should be ideally approximately 15 mm above the base of the cell 8215120 Release 2 0 2 Single Cell Holder 2 8 Fine Alignment If fine alignment is necessary proceed as follows 1 2 3 4 5 6 7 Using the GOTO 3 key slew the monochromator to the requested measurement wavelength or to 460 nm Call up a method that uses a
68. easured value INPUT gt Starting point appears after switch on following initialization routine Standby display Set up absorbance manually set wavelength manually select method print out method directory of the relevant branch return to branch header select another processing function return to method header APPLICATION Branch header with branch name PARAM Select the branch method print out the spectrometer directory change to another branch select default method of branch return to standby display 2 SCAN Method header with method number and type lt gt PARAM START gt Start method select method processing select another method return to standby display MODIFY METHOD Method processing with selected processing function PARAM Start processing function ORDINATE MODE ABS Parameter directory with parameter names and value gt Change parameter select next previous parameter start method select help messages for current parameter return to method header 4 82161 20 Release 2 0 Displays Displays shown during the measurement Display BACK CORR PRESS START BACK CORR XXX xxx ABS n SAMPLE n PRESS START SCAN CYCLES xxnm xxx ABS ORDINATE amp ABS lt gt BACK CORR IN CELL1 PRESS START SAMPLES IN 9 CELL PRESS START BACK CORR SIPPER ACCESSORY START SAMPLE 1 SIPPE
69. ed select NO using the arrow keys and press START Enter new values as required for the parameter displayed and press ENTER Press START to continue e g POSTRUN YES lt gt START t LAG TIME 0 0 MIN 82161 20 Aslease 2 0 82167 20 Release 2 0 5 35 Oligo Methods 5 10 Methods for Quantitative Analysis of Oligonucleotides Select an OLIGOQUANT 1 method for quantitative analysis of oligonucleotides up to 50 bases long and to calculate the theoretical melting point Select an OLIGOQUANT 2 method for quantitative analysis of oligonucleotides longer than 50 bases and to calculate the theoretical melting point You create an OLIGOQUANT method as follows 1 Press METHOD METHOD 0 000 ABS 500 0 SELECT METHOD gt lt t 2 Enter a method number nnn not nnn previously used 3 Press ENTER The first method of the NEW METHOD level appears i 4 Use the arrow keys to select the required 4 gt method type Oligoquant 1 or Oligoquant 2 NEW OLIGO gt f 5 Press PARAMETER this confirms the PARAMETER creation of the new method t nnn OLIGOQUANT 1 Press STOP to cancel lt gt PARAM START 6 Modify the new method as required 2 5 36 82181 20 Release 2 0 82151 20 2 0 Methods Procedure 1 Select the appropriate OLIGOQUANT method see page 5 38 2 if necessary modi
70. ee Section 10 1 page 10 1 Note The method can still be used even if the accessory in question is not connected the spectrometer simulates operation with the accessory However with cell changers only one sample can be measured B2161 20 Ral amp esa 2 0 Spectrometer without Accessory Board 6 4 1 Spectrometer without Accessory Board Spectrometers without an accessory bo rd cannot be operated with accessories but the method parameters for accessories can be selected The methods can be stored and used on spectrometers with the necessary equipment The procedure for setting up a method for an accessory although an accessory board is not installed is as follows 1 Switch on the spectrometer in Super User mode see Appendix 1 2 Select the CONFIGURATION branch 3 Select method 7 ACCESSORY CONFIG 4 Modify the method by selecting ACCESSORY YES 5 Press START to store the changes and to activate the accessory mode 6 deactivate the Super User mode switch off the spectrometer 7 Wait about two minutes to allow the lamps to cool down 8 Switch on the spectrometer 9 Set up the method as required 6 3 82161 20 Releasa 20 Running Methods with an Accessory 6 4 6 5 Running Methods with an Accessory When using methods with accessories the spectrometer automatically presents the necessary actions e g BACK CORR IN GELLI Insert blank at location 1 of the cell PRESS START changer and
71. eligro el ctrico Cualquier interrupci n del conductor de protecci n deniro o fuera aparato o la desconexi n del terminal del mismo toma de tierra podr an ocasionar serios peligros usar ef equipo Prohibida fa interrupci n intencionada Risque d lectrocution Toute interruption du conducteur de protection l int rieur ou l ext rieur de l instrument ou d connexion du raccord du conducteur de protection terre peut rendre l instrument dangereux H est interdit d interrompre volontairement ce conducteur Pericolo elettricit Quals asi interruzione della protezione del conduttore all interno o all esterno dello strumento o fo scollegamento del terminale di terrajmassa del conduttore di protezione possono rendere pericoloso lo strumento vietato provocare volontariamente queste interruzioni Risico s door elektriciteit Het instrument moet voor de werking geaard zijn geen geval mag de aarding van het instrument of de netvoeding worden onderbroken of worden verwijderd Perigo por electricidade Para a opera o o aparelho tem de estar sempre ligado terra De forma alguma separar ou retirar os condutores de protecc o terra no aparelho ou no cabo de alimenta o da rede 90 OO 0 8 9 82161 20 Release 2 0 r5 Translations of Warnings WO2 02 e Even with the power switch OFF line power voltages can still be present Within the instrument e Whe
72. enzyme activity is only calculated from the end of the lag time Only when using POSTRUN KIN YES or manual operation together with CALCULATE REGRESSION Switched on lamps Uv 190 nm to 326 nm VIS 326 nm to 1100 nm UV VIS 190 nm to 1100 nm Note In order to preserve the UV lamp Switch off the lamp only at the end of the working day Allow the lamp to cool off for at least 2 minutes before switching on again Type of line used for the graphics printout valid only when GRAPHICS PLOT z YES DASHT AUTO For each curve a different type of line is used in the sequence DASH1 DASH2 DASHS3 DASH4 DASHI etc 10 73 Parameters Parameter MODE 1 10 14 No Description Concentration 1 Sets the mode in which the measurements are made ABS is absorbance DELTA ABS is delta absorbance Analysis is 3 wavelength analysis ABS Measuring absorbance at one wavelength Measured value A4 Wy DELTA ABS Correction for an offset baseline by measuring at two wavelengths Measured value Wi Wa 3 WL ANLYS Correction for a sloping baseline by measuring at three wavelengths wa _ Measured value A Aus Be161 20 2 0 Parameters Parameter No Description MODE 1 Concentration 2 continued Sets the mode in which the measurements are made There are four possibilities
73. equired and press START OF n BACK CORR Insert the cell containing a blank solution PRESS START distilled water and press START OF n SAMPLE 1 Place solutions with the exception of PRESS START enzyme solution ina cell and mix Place the cell in the sample cell holder and press START 6 Allow equilibrium time delay time to DELAY TIME xxx elapse MIN xx ABS 7 Add the enzyme solution mix WAIT SAMPLE 1 and press START PRESS START B2167 20 Release 2 0 5 31 Substrate Kinetics 5 32 Procedure with Cell Changer Analysis is performed analogous to manua operation the essential difference being that instead of one cell several can be used for measurement in the one operation The procedure is as follows 1 Once the equilibrium time has elapsed prior to addition of enzyme the absorbance in each cell is measured automatically 2 After adding enzyme only location 1 is measured to follow the course of the reaction 3 Once the reaction is complete the absorbance in all the remaining cells is measured Example of the Display Shown During the Measurement SUBSTRATE xxx nm SUBSTRATE of method xxx min ABS xxx nm Wavelength min Time xxx ABS Measured value The display shown when using a cell changer SUBST xC n SUBST Type of method xxx min ABS XC Temperature n Cell location Xxx min Time ABS Measure
74. er functions see table 4 3 fable 4 3 Help Key Combinations Key Description HELP Provides additional parameter information on the display HELP Prints out a method or branch directory 0 HELP Prints out the current values shown on the display 1 HELP Prints out the method parameters 2 HELP Prints out a directory of methods available in the branch 3 IHELP Prints out the additional method information 4 HELP Line feed 5 HELP Form feed 6 HELP Prints out the Peltier cell hoider temperature shown on the display Functions only when Peltier accessory is installed 7 HELP Prints out spectrometer status To yiew the help text for a particular parameter proceed as follows 1 Select the desired parameter 2 Press HELP 3 Continue to press HELP to view all the text 2 04 Press PARAMETER to interrupt the help function Help text is available in German refer to the help configuration in SuperUSer 4 18 82757 20 Release 2 0 Measuring using Methods SEWIBSUSSSEEESSENEL mer Ez EIE II Using Methods 5 5 1 Overview The spectrometer incorporates the basic types of methods shown in the table below No of Method Use Section 1 TIME DRIVE Measurement over a certain period at one 54 gt wavelength 2 SCAN scanning spectra and derivative spectra 5 5 3 WAVELENGTH PROG
75. er office and inform them of the error the error message and the steps you made leading up to the error Note After a full reset all methods are erased 9 5 Error Massages 9 2 Error Reports on the Printer Error Baseline correction data do not fit Start baseline correction Cannot approximate calibration curve Check references or change curve fit algorithm Cannot calculate delta absorbance Because too few points read Cannot calculate slope Because too few points read End time out of limits Change end time Lag or total time is not divisible by interval time 0 0 Lag or total time out of limits Change lag and or total time Lag tima greater than total time Change lag time Meaning The background correction last carried out did not correlate to the method used Carry out a new background correction with the proper method A The calibration data deviate strongly from the curve form selected or not enough points were measured for the curve form selected Check the references select another curve form or carry out more measurements kinetic method was interrupted with STOP Too few points available to calculate delta absor bance kmetic method was interrupted with STOP Too few points available to calculate the slope In recalculation an out of limits value for END TIME was entered Enter a lower END TIME value and
76. eter Value No Parameter Value 17 SLIT WAV MAX OF REFS REF 1 REF 3 VALUE 1 VALUE 3 FACTOR SPEED LAMP SAMPLES BATCH CYCLES PRINT DATA PRINT REFS OPER ID 2 0 nm 600 0 nm 3 1 9 30C 0 1 0 3 1 0 960 mm min UV Vis 0 1 YES YES 4 6 f g 8 10 12 14 16 19 22 25 35 37 MODE WAV MIN CONC UNIT REF 2 REFS VALUE 2 CUR FIT DIVISOR SMOOTH BACK CORR FIRST SAMPLE CYCLE TIME PLOT REFS AUTO METHOD SAMPLE ID Only available with Lambda 14 and Lambda 14P PEAK AREA 500 0 nim C 20C NEW 0 2 m d oh d od boa 2 Modify the parameters as required using the values determined above Once a method is created you can save it and use it for the same analysis when required without having to redetermine the values 02751 20 Heloaso 2 0 5 21 Concentration 2 Establishing the Calibration Curve 1 Press START to start the measurement 2 Depending on the display SAMPLE ID Change the displayed parameter values if ENTER gt lt required and press START s BACK CORR Insert cell s containing a blank solution PRESS START and press START REF n xxx Insert a cell containing the sample PRESS START solution and press START 3 Insert references in sequence when asked When they have all been measured the instrument prints out the calibration curve and resuits You can
77. f ENTER gt required and press START ww BACK CORR Insert cell s containing a blank solution PRESS START 200 press START TIMEDRIVE sMPL1 Inserta cell containing the sample PRESS START solution and press START 5 Continue to insert samples when asked until they have all been measured Example of the Display Shown During the Measurement xxx nm xxx nm Wavelength xxx min ABS ABS Measured value ordinate as selected min Time units as selected Cx Repeat measurement cycles still to be performed This appears on the top right when cycles gt 1 Printout The result is printed out at the end of the analysis 5 7 Scan 5 5 Scanning a Spectrum Select a SCAN method to scan and record a spectrum of the sample Procedure 1 Select the desired SCAN method The following table lists typical SCAN parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter Mo Parameter Value Parameter Value 17 Sut 2 0 nm 1 ORDINATE MODE ABS 3 WAV MAX 1100 0 nm 4 WAV MIN 190 0 nm 13 SPEED 960 nm min 14 SMOOTH 2 nmi 15 LAMP UV e Vis 16 BACK CORR YES 18 SAMPLES BATCH 0 19 FIRST SAMPLE E 21 CYCLES 1 22 CYCLE TIME 0 1 min 25 GRAPHICS PLOT YES 26 ORD MAX 1 000 ABS 27 ORD MIN 0 000 ABS 28 SCALE 50 0 nm cm 29 GRID YES 30 OVERLAY NO 32 PRINT DATA YES 33 THRESHOLD 0 1 ABS 35 AUTO MET
78. f the sample compartment allow you io lead tubes from flowcells water thermostatted cell holders etc in and out of the sample compartment When not in use you should always insert the caps into the ports 82161 20 Heiease 2 0 2 5 Single Cell Holder 2 6 2 9 3 Aligning the Single Cell Holder Coarse Alignment 1 2 3 4 5 Open the sample compartment cover Fill ceils with a low absorbing solvent e g deionized water or ethanol Insert cell into the sample celi holder and one into the reference cell holder Make certain that the cells are pushed down fully Note The alignment procedure is for a given cell in a given _ holder After alignment the cell should always be used in the same holder Using the GOTO key slew the monochromator to 0 nm to obtain a beam of visible zero order radiation in the sample compartment Block the sample and reference beam windows on the right hand side of the sample compartment with a card to prevent white light from saturating the detector By holding a piece of matt white paper behind each cell holder visually examine the light spot to see that the radiation beam is passing through the cell sample area Diffraction patterns become apparent if the radiation beam impinges on the cell wall If the radiation beam is not centered exactly loosen the two locking screws and the two milled posts on the relevant cell holder and shift the cell holder plat
79. fy the method parameters 3 Press START to start the measurement 4 Depending on the display SAMPLE ID Change the displayed parameter values if ENTER gt required and press START OF BACK CORR Insert cell s containing a blank solution PRESS START and press START ws OLIGOn sMPL1 Inserta cell containing the sample PRESS START solution and press START 5 Continue to insert samples when asked until they have all been measured Example of the Display Shown During the Measurement OlGOn SMPLn nm Wavelength xxx nm xx ABS XXX ABS Measured value ordinate as selected Printout Graphics are printed out during the measurement process numerical data follow at the end of the analysis 5 37 Oligo Methods 5 38 Oligoquant Parameter Tables The following table lists typical OLIGOQUANT 1 parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter No Parameter 17 2 WAVELENGTHS 11 FACTOR 1 80 SEQUENCE LENGTH 61 SEQ 76 TM CALCULATION 18 LAMP 18 SAMPLES BATCH 21 CYCLES 25 GRAPHICS PLOT 35 AUTO METHOD 37 SAMPLE 10 Lane NEN Value No Parameter _ Value 20nm 1 ORDINATE MODE ABST 11 1 260 0 1 0 59 PATHLENGTH 1 0 cm 20 61 SEQ 1 2 65 CONSTANTS 14 RESPONSE 15 UV Vis 16 BACK NO 0 19 FIRST SAMPLE 1 1 22 CYCLE TIME 0 01 min
80. g with the BACK CORR arrow keys PARAM gt 4 Press ENTER Every parameter can be tagged For parameters where tagging is less meaningful e g LAMP GRAPHICS PLOT tagging is accepted but not carried out B2161 20 2 0 4 7 Deleting a Method 43 2 Deleting a Method 1 Select a method that can be deleted 2 Press PARAMETER 3 Use the arrow keys to select DELETE METHOD 4 Press PARAMETER again to delete the method The method is deleted as soon as PARAMETER is pressed and the display returns to the next method header in the list SUE Press STOP to cancel e g 2 SCAN lt gt PARAM START PARAMETER MODIFY METHOD gt 4 gt DELETE METHOD PARAM gt PARAMETER tt B2161 20 Release 2 0 20 4 3 3 Heiessa 2 0 Creating a New Method Creating a New Method You can create a new method in one of the following ways 1 Create a new empty method file 2 Overwrite an existing method file Crealing an Empty Method File 1 Press METHOD METHOD 0 000 ABS 500 0 SELECT METHOD gt lt 2 Enter a method number nnn not nnn previously used 3 Press ENTER The first method of the NEW METHOD NEW TIMEDRIVE level appears gt 4 Use the arrow keys to select the required 4 gt method type NEW SCAN PARAM gt 5 Press PARAMETER this confirms PARAMETER
81. gain Use the arrow keys to select the realtime or the internal clock Press PARAMETER again Use the arrow keys to select the day Press PARAMETER again 900DATE TIME lt PARAM START PARAMETER MODIFY METHOD PARAM gt PARAMETER f CLOCK INTERNAL j 4 gt E CLOCK REALTIME lt gt f pana ETER BAY MONDAY lt gt 4 gt DAY FRIDAY PARAMETER 82161 20 Aefease 2 0 Date Time 8 Type in the date using the numeric Keys DATE 000000 year month day e g 940429 ENTER gt 9 Press ENTER to confirm the entry ENTER 4 10 Press PARAMETER again PARAMETER mE 11 Type in the time using the numeric TIME 0000 keys hours minutes e g 1430 ENTER z 12 Press START to activate the clock START OF Press STOP to cancel The realtime clock need only be set once and has the following functions day Monday Tuesday etc date yymmdd and time hhmm and continues working when the instrument is switched off The internal clock is limited to the following functions date and time hhmm and counts from the time the spectrometer is switched on The internal clock must be reset to actual time after each switch on 82161 20 Release 2 0 5 41 Wakeup 5 42 5 12 Wakeup You can use WAKEUP to set the spectrometer to switch on the lamps to warm up before the start of the working day
82. ically l he reaction is started by the addition of enzyme and proceeds rela tively quickly until a state of equilibrium is attained substrate has been converted by this time and the absorbance does not alter any more The measured difference in absorbance AA is directly propor tional to the substrate concentration CSub FAA where Sub 15 the substrate concentration is the concentration factor is the measured difference in absorbance e Time 4 4 2161 20 Release 2 0 Substrate Kinetics The course of the reaction can deviate from the ideal described above creeping reactions take place and the absorbance can hence increase even after the substrate reaction has been completed The end point Of such a reaction is reached when the slope of the curve remains constant The actual end point can then be deter mined by extrapolation Enzyme added Creeping reaction 82167 20 Release 2 0 A4 5 A4 6 2181 20 BRolsase 2 0 Translations of Warnings UU SO nee Translations of Warnings This annex contains translations of the numbered warnings used in this handbook 82781 20 Helease 20 1 Translations of Warnings WD1 D1 Warning We use the term WARNING to inform you about situations that could result in personal injury to yourself or other persons Details about these circumstances are in a box like this one
83. ing parameters 4 13 creating a file 4 9 creating a new 4 9 deleting 4 8 editing 4 4 modity 4 5 name 4 11 overview 5 1 overwriting 4 10 printing 4 15 procedure 5 2 protect function A2 3 types 5 1 Michaelis constant A4 2 Michaelis Menten Equation 4 2 Microcell 2 9 alignment 2 9 liquid height 2 9 minimum volume application 2 0 MMass dA 10 16 dC 10 16 dG 10 17 dN 10 17 dT 16 17 Mode 10 14 10 15 16 16 ordinate 10 19 10 20 N New from Mark 4 14 New Method creating a 4 9 Number of dA 10 17 dC 10 17 dG 10 17 dT 10 17 N 10 17 index O Oligo 10 18 Oligo Method oligoquant 1 5 36 oligoquant 2 5 36 Oper ID 10 18 Optimization 6 9 Ord Max 10 18 Ord Min 10 18 Ordinate Mode 10 19 10 20 Overlay 10 20 Overview branches A3 1 methods 5 1 P Parameter changing 4 6 descriptions I0 1 10 28 numbers 10 1 10 28 tagging 4 7 Part numbers cell 2 9 Pathlength of cells 2 9 Peak areas 5 19 10 15 deriv 2 10 15 10 16 heights 5 12 5 13 Peristaltic Sipper 6 9 Personal Computer operation with 3 5 RS 232 interface A3 5 Photoactive Samples 8 3 Flot Refs 10 20 Polarizing Samples 8 3 Port usage A3 5 Postrun Kinetics 5 34 10 20 Power switch 1 1 Pressure buildup in cells 7 3 Index 3 Index Print branch directory 1 3 current values 1 3 data 10 20 directory 4 16 method 4 15 directory 1 3 inf
84. inimum Should the ist or 2nd derivatives prove insufficient the 3rd or 4th derivatives can be used providing the noise level remains within acceptable limits E The parameters SPEED and SMOOTH influence the quality of derived spectra In choosing parameter values take the following into account SPEED Guideline value peak width in nm x 10 High scan speeds decrease resolution low scan speeds increase noise SMOOTH In the case of derivation spectra smoothing exerts a greater influence than in absorbance measurement Smoothing should thus be kept to a minimum Guideline value peak width in nm x 0 5 Derivation is not possible with a degree of smoothing of 0 inde pendent of the ordinate mode selected 52161 20 Release 2 0 10 19 Parameters Parameter ORDINATE MODE continued OVERLAY PLOT REFS POSTRUN KIN PRINT DATA 10 20 No Description 1 25 32 Ratio difference and corrected ratio RAT DIF COR modes are determined according to the equations below A 2 23 or A AS an A4 As As COR Ihe subscript 1 stands for the first wavelength 2 for the second eic Prints spectra from the same batch onto the same graphics printout valid only when GRAPHICS PLOT YES Option YES Overlaying graphics printouts functions only if the printer used has an automatic paper reverse function If CYCLES
85. ircumstances are in a box like this one Caution Achtung Bedeutet daf die genannte Anleitung genau befolgt werden um einen Ger teschaden zu vermeiden Caution Bem rk Dette betyder at den n vnte vejledning skal overholdes noje for at undg en beskadigelse af apparatet Caution Advertencia Utilizamos el t rmino CAUTION ADVERTENCIA para advertir sobre situaciones que pueden provocar averias graves en este equipo o en otros En recuadros ste se proporciona informaci n sobre este tipo de circunstancias Caution Attention Nous utilisons le terme CAUTION ATTENTION pour signaler les situations susceptibles de provoquer de graves d t riorations de l instrument ou d autre mat riel Les d tails sur ces circonstances figurent dans un encadr semblable celui ci Caution Attenzione Con il termine CAUTION ATTENZIONE vengono segnalate situazioni che potrebbero arrecare gravi danni allo strumento o ad apparecchiatura Troverete informazioni su tali circostanze in un riquadro come questo Caution Opgelet Betekent dat de genoemde handleiding nauwkeurig moet worden opgevolgd om beschadiging van het instrument te voorkomen Caution Atenc o Significa que a instru o referida tem de ser respeitada para evitar a danificagao do aparelho 90 0 9 9086 02161 20 Helease 2 0 Transiations of Warnings WOA Explosive Aimosphere This instrument is not designed for
86. measurement according to your procedure using a celi filled with redistilled water in place of the sample 4 Enter the result of the measurement in the parameter BLANK 5 28 B2181 20 Release 20 5 29 0 Aeeese 2 4 Substrate Kinetics 5 9 5 30 Substrate Kinetics Select a SUBSTRATE KIN method for substrate kinetic measurements Manual Procedure 1 Select the appropriate SUBSTRATE KIN method The ollowing table lists typical SUBSTRATE KIN parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter Parameter SLIT RESPONSE TIME UNIT END TIME CONC FACTOR DIVISOR CONC UNIT SAMPLES BATCH GRAPHICS PLOT ORD MIN GRID POSTRUN KIN OPER ID 0 000 ABS Value No Para YES Parameter Value WAVELENGTH 3940 0 LAMP UV t Vis DELAY TIME 0 0 min CREEPING CYCLE 0 DIL FACTOR 1 0 BLANK 0 0 BACK CORR YES FIRST SAMPLE 1 ORD MAX 1 000 ABS SCALE 21 mm min PRINT DATA ALL AUTO METHOD YES SAMPLEID Only available with Lambda 14 and Lambda 14 2 Modify the method as required 3 If necessary determine the blank value of the reaction see page 5 33 and enter the value in the parameter BLANK 4 Press START to start the measurement B2161 20 Release 2 0 Substrate Kinetics 5 Depending on the display shown SAMPLE iD Change the displayed parameter values if ENTER gt lt r
87. meter SAMPLING TIME DELAY TIME AUTO PURGE RETURN Peristaltic Sipper OPTIMIZATION Peristaltic Sipper HEV TIME Peristaltic Sipper only SIPPERS 5 90 91 SIPPER 2167 20 Release 20 Accessory Parameters Description Sample aspiration time in seconds for the sipper Range 0 1 to 99 9 Enter value and confirm by pressing ENTER Delay between the end of the aspiration process and the start of the measurement Range 0 0 to 99 9 Enter value and confirm by pressing ENTER Switches the autopurge function on and off Option YES NO Select with arrow keys Pump in reverse direction after sample measurement Option YES Select with arrow keys To optimize the RETURN function Option YES NO Select with arrow keys enter the reversed flow time for the optimization Range 0 0 to 99 9 Enter value and confirm by pressing ENTER Parameters for control of the sipper must be entered via the sipper see sippers user documentation Parameters and operation with the AS 90 91 are described in the AS 90 91 user documentation 6 10 82181 20 Release 2 0 meme 5 Si a IE MIU MT HEHEHE Han Care WOH Unauthorized Adjustments and Servicing A Do not attempt to make adjustments replacements or repairs to this instrument except as described in the accompanying User WARNIN
88. more pair than height of beam 4 mm 1cm 007 9402 pair Cell Pathlength Part Number Volume Cell window com m liquid 25 05cm B051 0077 SuL 01cm B051 0078 3 1 0 cm B050 5823 30 uL 1 0 cm B019 0608 82101 20 Release 2 0 nmr ara a You should align microcells very carefully in the radiation beam by following the procedures in Section 2 3 3 above page 2 6 When aligning microcells fill each cell with the minimum volume of liquid specified i in the above table to make sure that the liquid meniscus is not in the radiation beam 2 10 B2161 20 Release 2 0 uiii iu 5 TUTUTUTETWTUNNT GT EET Operating without Methods cose f Operating without Methods 3 3 1 Overview Measurements are usually carried out using methods containing all the necessary parameters see Chapter 5 The following functions can be carried out via the keyboard Setting the wavelength Manual background correction Quick sample measurement Reset B2181 20 Relesse 2 0 3 1 Setting the Wavelength Manually 3 2 Setting the Wavelength Manually The wavelength can be set manually using the GOTO key as follows either 1 Press GOTO 4 2 Enter the desired wavelength e g 325 5 3 Press ENTER The monochromator slews to the selected wavelength OF 1 Enter a wavelength
89. mple compartment cover Press BACK CORR and wait until the background correction is completed Open the sample compartment cover Remove the blank and insert the with sample solution in the sample cell holder Close the sample compartment cover The absorbance or transmittance reading and wavelength are shown on the display The ordinate mode of the last used method always appears on the display Use a TIME DRIVE method to change from absorbance Ate to transmittance WT Press 0 and then HELP to print out the reading Bg161 20 2 0 Reset 3 5 Reset By a full reset the spectrometer and its program are returned to the default condition You can carry out a full reset at any time Note In carrying out a full reset all methods will be erased Before carrying out a full reset make sure that all important methods are printed out To carry out a full reset 1 Switch off the spectrometer 2 Press 7 9 e seven nine point simultaneously 3 Keep the keys pressed and switch on the spectrometer 4 Keep the keys pressed until the display appears After the full reset is completed a status report is printed out when a pr nter is connected Note There are default methods stored in the internal memory of the spectrometer These methods are not deleted after a full reset and can be copied and amended see Section 4 2 1 page 4 3 82161 20 Release 2 0 8 5 2
90. n steps of 0 1 When using MODE ABS only 1 wavelength can be entered When using MODE DELTA ABS 2 wavelengths must be entered When using MODE 3 WL ANALYS 3 wavelengths must be entered WAVELENGTH 1 measuring wavelength WAVELENGTH 2 and 3 wavelengths for baseline correction See page 10 14 WAVELENGTH 3 The wavelength nm at which measurements are made Range 0 for adjustment of cell holder 190 0 to 1100 0 in steps of 0 1 2161 20 feleass 20 10 27 10 28 82161 20 Release 2 0 Appendix EE FETEH e ES EEE E see H TIUDMEEMIMEUNBEzLnIN ae SuperUser Appendix 1 A1 1 A1 2 Bz161 20 Aeleasa 2 0 Activating SuperUser Mode SuperUser mode gives you access to all the branches in the instrument You activate SuperUser mode as follows 1 Switch off the spectrometer 2 Wait about two minutes to allow the lamps to cool down 3 Press 1 5 one five minus simultaneously 4 Keep the keys pressed and switch on the spectrometer 3 Release the keys when the display appears The SuperUser mode is now active Deactivating SuperUser Mode 1 Switch off the spectrometer 2 Wait about two minutes to allow the lamps to cool down 3 Switch on the spectrometer The SuperUser mode is now deactivated 1 2 82161 20 Release 2 0 Protect Functions 2 A2 1 General Information about
91. n the Handbook TUNE TP 5 2 1010 Safety Requirements 8 3 Electricity 24 64 2 MM 5 4 Radio Frequency tem ehh mh hem mh ra DII 25 5 Environment 124 Me ehm ree ha haerere 5 6 TED TD 5 6 Waste Disposal 1 9 6 UV Radiation rsen 5 7 Toxic Fumes Lederer hs hn 5 7 Compressed Gases ee e hahaha hn 8 7 Chapter 1 Introduction 11 Keys TC 1 2 1 1 1 Key Combinations 1 3 1 1 2 1 4 Chapter 2 Switching On and Off 21 Startup SNMP seserra 2 2 2 2 Shutdown 2 3 23 Single Cell Holder 1242 4 1 6 3 2 4 2 31 2 4 2 3 2 Installing the Single Cell Holder 255 233 Aligning the Single Cell Holder 2 6 2 34 Minimum Volume Applications 2 9 2161 20 Release 2 0 1 Table of Contents continued Chapter 3 Operating without Methods 3l Overview Sete
92. n the instrument is connected to line powar terminals may be live and opening covers or removing paris except ihose to which access be gained by hand is likely to expose live parts e Capacitors inside the instrument may still be charged even if the instrument has been disconnected from all voltage sources Lethal voltages are present in the instrument WARNING Gef hrliche Spannung im Ger t e Auch in ausgeschaltetem Zustand kann an einigen Stellan im Ger t Netzspannung anliegen wenn das Gerat am Stromnetz angeschlossen ist e Auch bei ausgeschaltetem Ger t und getrennter Netzverbindung k nnen Kondensatoren im Ger t noch mit gef hrlicher Spannung geladen sein Farlig sp nding i apparatet fare for kv stelser Ogs slukket tilstand kan der v re netspesnding nogle steder f apparatet hvis apparatet er tilsluttet til stramnetiet e Selv nar apparatet er Slukket og stremforbindelsen er afbrudt kan kondensatorerne i apparatet v re ladet med farlig sp nding En el aparato existen voltajes letales e incluso con al interruptor desconectado puede haber voltaje dentro del equipo e Cuando instrumento se encuentre conectado red el ctrica los terminales pueden estar bajo corriente y stos quedar expuestos al abrir las cubiertas o al extraer componentes exceptuando aquellos a los cuales se puede acceder con la mano e Los condensadores internos del aparato pueden permanecer cargado
93. nd correction or autozero must be performed e atthe start of a new method e when the wavelength is changed whenthe wavelength range is extended each time the solvent is changed To perform a background correction or autozero place cells with a blank solution or reference solution in the sample cell holder and reference cell holder Unusual Samples 8 2 Unusual Samples If a sample is chemically stable and undergoes no physical or chemical change other than to absorb incident radiation errors in photometric values should not be caused by the sample Many samples are not this stable and special consideration must be given to them 1 Volatile Samples Some liquid samples are so volatile that their concentration change while recording is in progress If this occurs the resulting data will lack reproducibility If you are analyzing volatile samples use stoppered cells to prevent this problem 2 Samples not Governed by the Beer Lambert Law Quantitative analyses utilizing the absorption of spectral radi ation are based on the Beer Lambert law which states that the absorption is proportional to the concentration of the analyte The law can be expressed in the form where A is absorbance is molar absorption coefficient c is molar concentration d is thickness through which the radiation is transmitted This law is mostly true for dilute solutions but at higher concen trations a plot of
94. nders of compressed gas with care in accordance with local regulations We recommend that gas cylinders be located outside the laboratory and the gases led to the laboratory through approved gas supply lines Use only approved tubing connectors and regulators for gas supply es 5 7 Safety information Introduction ELE SESE TAPE LTT reuse Introduction The Lambda 12 Lambda 14 and Lambda 14P are versatile spectrometers operating in the ultraviolet UV and visible Vis spectral ranges The spectrometers have some common features Display keyboard Connector Panel Sampie Compartment Figure 1 1 Features common io Lambda 12 14 and 14 82167 20 Release 20 1 1 Keys 1 1 Keys METHOD HELP GOTO X PARAMETER STOP BACK CORR 4 START 0 to 9 ENTER CE 1 2 T HERE ecc TIT wed nd Bn 5 MINIMSOGMISUDMIEI CHI nmn ete de ii nunn wet Description Selects methods Use with numerical keys see page 4 2 Provides additional parameter information on the display To change the wavelength setting see page 3 2 Selects next parameter Switches to next lower level Stops a method Switches to next higher level Starts background correction see page 3 3 Selects previous or next element in a particular level To start
95. now amend the calibration curve see Section 5 7 3 page 5 24 if required You can use previously established calibration curves or reference values see REFS page 10 21 5 22 62167 20 Release 2 0 2181 20 Release 2 0 Concentration 2 Measuring the Sample 1 Press START to start the measurement 2 Depending on the display SAMPLE ID Change the displayed parameter values if ENTER gt required and press START vw BACK CORR Insert cell s containing a blank solution PRESS START and press START 2 Of CONC2 Inserta cell containing the sample PRESS START solution and press START 3 Continue to insert samples when asked until they have all been measured Example of the Display Shown During the Measurement nm Wavelength Result units as selected CONC2 SMPL 1 XXx x nm xxx C CYCLES XXRepeat measurement cycles still to be performed CYCLES XX This appears on the top right when cycles gt 1 Xxx x nm xxx Printout If PLOT REFERENCES and PRINT DATA are set to YES the calibration curve and results are printed out 5 23 Calibration Curve 5 7 8 Processing the Calibration Curve CONCENTRATION Methods Changing the Type of Curve Fit The type of calibration curve fit linear or quadratic can be altered without having to carry out additional measurements The procedure is as follows 1 Modify the method Select REFS OLD CUR FI
96. ons Details about these circumstances are in a box like this one Coe Caution We use the term CAUTION to inform you about situations CAUTION that could result in serious damage to the instrument or other equipment Details about these circumstances are in a box like this a Translations of the warning messages used in this handbook are given in an Annex at the end of this handbook Safety Information IEC 1010 Safety Requirements This instrument has been designed and tested in accordance with Perkin Elmer specifications and IEC 1010 Safety requirements for electrical equipment for measurement control and laboratory use The instrument is protected in accordance with IEC Class 1 rating This handbook contains information and warnings that must be fol lowed by the user to ensure safe operation and to maintain the instru ment in a safe condition Before using this instrument it is essential to read the handbook care fully and to pay particular attention to any advice it contains concerning potential hazards that may arise from the use of the instrument The advice is intended to supplement not supersede the normal safety code of behavior prevailing in the user s country Safety information Electricity To ensure satisfactory and safe operation of the instrument it is essen tial that the green yellow lead of the line power cord is connected to true elec
97. operation an explosive atmosphere gt z 2 Explosionstahige Atmosph ren Das darf nicht in explosionsfahigen Atmospharen betrieben werden Apparatet m ikke anvendes i eksplosive omgivelser Eksplosive omgivelser Atm sfera explosiva Este aparato no ha sido dise ado para utilizarlo en atm sferas explosivas Atmosph re explosive Cet instrument n est pas concu pour fonctionner dans une atmosph re explosive Atmosfera esplosiva Questo strumento non adatto per l uso in atmosfera esplosiva Explosiegevaarlijke omgevingen Hat instrument mag niat in een explosiegevaariijke omgeving worden gebruikt Atmosferas explodiveis aparelho n o pode ser utilizado em atmosferas explodivels 9 60 0886 T4 82161 20 Aelease 2 0 Translations of Warnings Electrical Hazard A Any interruption of the protective conductor inside or outside the instrument or disconnection of the protective conductor earthiground WARNING terminal is likely to make the instrument dangerous intentional interruption is prohibited Gefahrdung durch Elektrizitat Das zum Betrieb immer geerdet sein Auf keinen Fall die Schutzieiter Gerat oder in der Neizzuleitung trennen oder entfernen Fare p grund af elektricitet Apparatet skal altid v ra jordet Man m under ingen omsteendigheder skille eller fjerne jordlederen inde i apparatet eller i str mledningen P
98. ormation 1 3 parameters 1 3 Peltier temperature 1 3 tefs 10 21 status 1 3 using help key combinations 4 17 Printer configuration A3 6 output configuring A3 6 Protect Function 42 1 execute 2 1 for branches 42 4 for methods A2 3 full A2 1 preventing access to methods and branches 42 5 read write 2 1 setting A2 2 write A2 1 Quick Sample Measurement 3 4 R Radiation beam 2 7 Rat 10 1 10 19 10 20 Ref n 10 21 Reference Solution remea suring 5 24 Refs 10 21 Reset 3 5 fuil 1 3 Response 10 21 index 4 Return 6 9 to standby 5 42 Rev Time 6 9 RS 232 interface use of A3 5 use with PC A3 5 S Safety Information 5 3 Salt 10 21 Sample Compartment 1 1 window 7 1 7 3 cleaning 7 1 7 3 Sample ID 10 22 Sample Measurement 5 2 sample Volume minimum in microcell 2 9 Samples Batch 10 22 chemically reactive 8 3 not governed by Beer Lam bert law 8 2 photoactive 8 3 polarizing 8 3 thin film 8 3 unusual 8 2 volatile 8 2 Sampling Time 6 9 10 22 Scale 10 22 Scan 5 8 Select branch A3 2 default method 1 3 4 3 method 4 2 previous parameter 1 3 tag 1 3 Self Test 5 43 Seq n 10 22 Sequence Length 10 22 Shutdown 2 3 Sipper 6 9 10 1 Slit 10 22 Smooth 10 23 Solvent lower wavelength limits of properties 8 4 Spectrometer directory 4 16 Speed 10 24 Spilled Materials 7 1 Standby display 2 2 return to
99. presente all interno dello strumento e Quando lo strumento collegato alla rete di alimentazione i terminali possono essere sotto tensione aprendo le calotte di protezione o rimuovendo alcune parti ad eccezione di quelle raggiungibili con mano 8 possibile esporre altre parti sotto tensione e Anche se strumento stato scollegato da tutte le fonti di tensione i condensatori suo interno possono essere ancora carichi Gevaarlijke spanningen het instrument gevaar van letsel NL e Ook in uitgeschakelde toestand kan wanneer het instrument op de netvoeding is aangesloten op sommige pleatsen in het instrument netspanning staan e Ook bij een uitgeschakeld instrument en een onderbroken netvoeding kunnen condensatoren in het instrument nog een gevaarlijke lading bevatten Tens o perigosa no aparelho perigo de ferimento e Mesmo desligado aparelho podera ainda ter tens o de rede em alguns pontos enquanto estiver ligado rede de corrente e Mesmo com o desli gado e a liga o rede de corrente interrompida os condensadores dentro do ainda poder o ter ums tens o perigosa aplicada B2161 20 Release 2 0 m T Translations of Warnings r8 B210120 Aletease 2 0 index BATUNSEUTETUDIEEN RRC ur CARR UR eRe ee a eee ca a ABS 10 14 10 19 Accessory 10 1 configu
100. press PARAMETER Error RANGE ERROR XXXK X XXXX X Meaning The value entered is outside the displayed range Press PARAMETER and enter a value within the range shown ee PARAMETER XX DOES NOT EXIST PARAMETER XX NOT USED ee rr ee ee HINT INT GREATER TOT TIM HINT SELECT INTERVAL TIME 82161 20 Release 2 0 The parameter XX does is not exist Press PARAMETER to continue eee eee ee ee The parameter XX is not used or is not active in this method Press PARAMETER to continue The Interval time is greater than the total time This message appears with kinetic measurements Select a shorter interval time Appears with enzyme methods with changers if the interval time does not correlate with the measurement time Select the INTERVAL time so that the TOTAL TIME can be divided by the INTERVAL time eveniy 9 1 Error Messages Error PROBLEM MARK NOT SET PROBLEM METHOD NOT FOUND PROBLEM ACCESSORY NOT INITIALIZED PROBLEM METHOD NO LIMITS 1 999 ERROR LAST METHOD PHOBLEM METHOD PROTECTED DON T PROTECT METHODS DON T PROTECT ALL BRANCHES PROBLEM BRANCH WRITE PROTECT The method number entered is outside the displayed Meaning COPY FROM MARK was selected without first initiating MARK FOR COP First use MARK FOR COPY and then select COPY FROM
101. pressing ENTER OF Press ENTER without entering a letter or number for empty space You can combine numbers and letters 6 Press PARAMETER to confirm the new PARAMETER name 82767 20 falasse 2 0 4 11 13 TIME DRIVE 2 lt gt PARAM START Checking a Method 4 3 5 Checking a Method When using the CHECK METHOD function the parameter values are displayed but cannot be changed 1 Select the method to be checked 2 Press PARAMETER 3 Use the arrow keys to select CHECK METHOD 4 Press PARAMETER to check each param eter in turn Press STOP to cancel 4 12 e g 2 SGAN lt gt PARAM START PARAMETER MODIFY METHOD gt CHECK METHOD PARAM gt _ Y PARAMETER SLIT 1 0 nm CHECK ONLY PARAMETER Copying Method 4 3 6 Copying Method Parameters into a New Method File This is useful when you wish to make a new method with only a few parameters different from the original Two steps are involved in this procedure 1 Marking the method for copy 2 Copying the method into another method file Marking for Copy 1 Select the method who s parametets are to be copied es 2 SCAN lt gt PARAM START t 2 Press PARAMETER PARAMETER MODIFY METHOD FARAM gt i 3 Use the arrow keys to select 4 MARK FOR COPY MARK FOR COPY PARAM gt 4 Press PARAMETER to mark
102. r the following conditions e Indoors Temperature 15 C to 35 e Relative humidity 20 to 80 Chemicals Use store and dispose of chemicals that you require for your analyses in accordance with the manufacturer s recommendations and local safety regulations Waste Disposal 5 6 Waste containers may contain corrosive or organic solutions and small amounts of the substances that were analyzed If these materials are toxic you may have to treat the collected effluent as hazardous waste Refer to your local safety regulations for proper disposal procedures Deuterium lamps are maintained under reduced pressure When you dispose of lamps that are defective or otherwise unusable handle them correctly to minimize the implosion risk Safety Information me BL e i I UV Radiation You should be aware of the health hazard presented by UV radiation When the deuterium UV lamp is switched on do not open spectrometer covers unless specifically instructed to do so in the handbook Always wear UV absorbing eye protection when the deuterium lamp is exposed Never gaze into the deuterium lamp Toxic Furnes If you are working with volatile solvents toxic substances etc you should provide an efficient laboratory ventilation sysiem to remove vapors that may be produced when you are performing analyses Compressed Gases Handle cyli
103. rage out the values and the fine structures of the spectrum disappear If smoothing is too low spectral fine structures may be hidden by noise If an unacceptable value is selected an appropriate indication is displayed If then no other value is substituted ihe spectrometer will automatically continue to operate at smoothing 9 Note Derivative spectra cannot be scanned at smoothing 0 Concentration methods Select a smoothing factor that is as low as possible so that the peak is smoothed as little as possible The value selected should depend on the level of noise 10 23 Parameters Parameter SPEED STIRRER TEMP CHECK 10 24 No 13 49 50 Description Scanning speed in nm min Select the scanning speed depending on the type of sample and the desired resolution The following can be used as a genera rule For narrow peaks select a low scanning speed and low degree of smoothing in order to limit the noise level For broader peaks higher scanning speeds be selected For derivative spectra observe the comments given under ORDINATE MODE Guideline values Overview spectra 2880 1920 nm min Broad peaks 960 480 240 nm min Solid and liquid samples 120 60 30 nm min Gaseous samples spectra with higher resolution expanded spectra 30 15 7 5 nm min Switches magnetic stirrer on and off Optio YES If magnetic stirring has been switched on place a small magnetic s
104. ration A3 6 operation with 6 1 parameters 6 7 requirements 6 2 spectrometer without board 263 type 6 1 with methods 6 2 Alignment cell holder 2 6 microcell 2 9 Analysis Procedure 5 3 Application branch A3 3 minimum volume 2 9 AS 90 91 6 9 10 1 configuration 43 5 Auto Method 10 1 Auto Purge 16 1 Autopurge 6 9 Back Corr 10 2 Background Correction 5 2 8 1 10 2 mantal setting 3 3 with cell changers 6 5 Baseplate 2 4 Baud rate for PC A3 5 82161 20 Release 2 0 Beer Lambert Law 10 11 10 12 Beer Lambert Law 8 2 Blank 10 3 Branch application A 3 3 calibration A3 4 communication 43 3 configuration A3 5 directory 4 16 protect function A2 4 selecting A3 2 test 3 7 validation 43 7 Branches overview A3 1 C Calc Wav 1 10 3 Calc Wav 2 10 3 Calculate 10 4 10 12 10 26 interval 10 4 regresston 10 4 Calibration branch A3 4 curve 10 5 10 26 deleting a point from 5 25 processing 5 24 Care daily 7 1 of the instrument 7 1 index Cell 2 9 10 1 centre height 2 7 changer 13cell 6 8 Scell 6 7 6 7 Scell 6 7 Ocell 6 7 holder 2 4 alignment 2 6 installation 2 5 lifter for short cells 2 4 part numbers 2 9 pathlength 2 9 use and care of 7 2 Cell 1 7 160 5 Cell 5 10 5 Cell 6 10 5 Cell 8 10 5 Cell 8 13 10 5 Ceil 9 10 5 Chemically Reactive Samples 3 Chng Const 10 5 Cleaning sample compartment 7 1 s
105. ration curve At the next call up select REFERENCES OLD The available calibration curve is used and measurement can start immediately REFn 7 Concentration of reference solution n RESPONSE 14 Response time Time constant in seconds s Option 0 1 0 2 0 5 1 2 5 10 large value for time constant gives good signal noise ratios but can cause undesirable smoothing of the curve especially with rapidly altering signals Thus the time constant should be kept as low as possible but maintaining an acceptable signal noise ratio Note in analyses involving creeping reactions the time constant set must be lower than the CREEP TIME set Note The time constant set must be lower than the INTERVAL in order to calculate see parameter INTERVAL SALT 77 Toenter the salt concentration monovalent cation concentration of the oligonucleotide sample Used for the melting point calculation Uni mmol L Shows only when theoretical melting point is YES Refer to the Biochemical Application manual 70 21 82767 20 Aelease 2 0 Parameters Parameter SAMPLING TIME SAMPLE ID SAMPLES BATCH SCALE SEQ n SEQUENCE LENGTH SUT 10 22 37 Description Aspiration time in seconds for sipper Range 0 0 to 99 9 Used with the sipper accessory Sample identification up to a maximum of 8 characters Sample identification appears on every printout Number of samples per batch
106. red e g Several reference values have to be entered Enter the value for REF 1 Confirm with ENTER Use the arrow key to move to REF 2 Continue until values have been entered for all the references Press CE to delete wrongly entered values 3 Press PARAMETER to proceed to the next parameter You can press HELP for additional information about a parameter When a particular parameter is set to YES the extra parameters required automatically appear in their correct order Eg When GRAPHICS PLOT YES the extra parameters ORD MAX ORD MIN SCALE and GRID appear When GRAPHICS PLOT is NO the extra parameters ORD MAX ORD MIN SCALE and GRID no longer appear 2761 80 2 0 Modifying a Method Tagging a Parameter You tag a parameter to change it at appropriate times during the analysis e g prior to the start of each sample measurement Untagged parameters can only be changed prior to the start of a method Table 4 2 shows the type of tagging and when it appears during the analysis Table 4 2 Type of Tags Tag Symbol Appears CALL amp Prior to the start of a method BATCH Prior to the start of each sample batch START Prior to the start of each sample FX No tag a parameter as follows 1 Select the parameter to be BACK CORR NO tagged for example gt 2 Press PARAMETER e PARAMETER j 3 Select the appropriate taggin
107. references or change the concentration range or change the curve fit algorithm The measured sample concentration is outside the calibration range Measure additional calibration solutions within the concentration range or change the curve fit algorithm Appears with TIME DRIVE methods if the wave length has been modified since the last back ground correction Perform a new background correction using the proper method 2161 20 Aplease 2 0 Parameter Numbers and Descriptions Parameter Numbers 1 0 and Descriptions 10 1 Parameter OF REFS Parameter Numbers and Descriptions Description Number of reference solutions used for the calibration Range 1 to 20 ee ee Un FEAT LLLA WAVELENGTHS ACCESSORY AUTO METHOD AUTO PURGE 2161 20 Release 2 0 38 35 48 Number of wavelengths at which measurements are made Range 0 to 20 If ordinate modes RAT and DIF are used the number must be divisible by 2 If ordinate mode COR is used the number must be divisible by 3 See also parameter Ordinate Mode Lets the spectrometer recognize the accessory type connected MANUAL 15 for operations without using an accessory or for manual cell changers CELL is for a cell changer SIPPER isforasipper _ AS 90 91 15 for the AS 90 91 autosampler Note This parameter only shows when an accessory board is installed in the
108. repeat the procedure In recalculation the LAG TIME or TOTAL TIME entered was not divisible by the INTERVAL time Enter a value for LAG TIME or TOTAL TIME that is fully divisible by the INTERVAL time In recalculation a greater value for LAG TIME or TOTAL TIME was entered than the actual measuring time Enter lower value for LAG TIME or TOTAL TIME In recalculation a value for LAG TIME was entered that was greater than the measuring time Enter a LAG TIME less than the TOTAL TIME 82181 20 Raleasa 2 0 Error More than one peak within wavelength limits Change threshold or measurement wavelengths No peak detected Change threshold No points stored Start measurement Two solutions for 1 ABS value Change curve fit algorithm Value not within valid limits Check references or change curve fit algorithm Wavelength data do not fit Start background correction 82161 20 Aeiease 20 Error Messages Meaning More than one peak identified within the selected measuring range Change WAV and WAV MAX or THRESHOLD so that only one peak is detected The THRESHOLD is too high to detect peaks Select a lower value for THRESHOLD The method was interrupted with STOP before data could be stored e g during the equilibrium time of a substrate method Restart the method n a non linear calibration curve two reference solutions exhibit the same absorbance value Check the
109. rogram WAVELENGTH PROG method to measure a sample at several different wavelengths Procedure 1 Select the desired WAVELENGTH PROG method The following table lists typical WAVELENGTH PROG parameters in the order left to right in which they appear See Section 10 1 page 10 1 for a detailed description of each parameter No Parameter Value No Parameter Value 17 SLIT 2 0 nm 1 ORDINATE MODE ABS 2 WAVELENGTH 3 WAV 1 459 9 nm 3 WAV 2 418 5 nm 3 WAV 3 360 0 nm it FACTOR 1 1 0 11 FACTOR 2 1 0 11 FACTOR2 1 0 14 RESPONSE 058 15 LAMP UV Vis 16 BACK CORR YES 18 SAMPLES BATCH 0 19 FIRST SAMPLE i 21 CYCLES 1 22 CYCLE TIME 0 1 min 25 GRAPHICS PLOT YES 26 ORD MAX 1 000 ABS 27 ORD 0 000 ABS 28 SCALE 20 mm min 20 YES 32 PRINT DATA YES 35 AUTO METHOD NO 36 OOPERID 37 SAMPLE iD Only available with Lambda 14 and Lambda 14 When a particular parameter is set to YES the extra parameters required automatically appear in iheir correct order eg When GRAPHICS PLOT is YES the extra parameters ORD MAX ORD MIN SCALE and GRID appear When GRAPHICS PLOT is NO the extra parameters ORD MAX ORD MIN SCALE and GRID no longer appear 2 If necessary modify the method parameters 3 Press START to start the measurement 5 10 82161 20 Aelease 2 0 Wavelength Program 4 Depending on the display SAMPLE ID Change the displayed parameter
110. rs new method can now be created see Section 4 3 3 page 4 9 If you don t know the method number return to the standby display and use PARAMETER fo switch to the first method header Then use the arrow keys to view the available methods in turn 52161 20 Aelease 2 0 Default Methods 4 2 1 Default Methods Default methods are stored in the spectrometer The default methods can be read and copied but not modified The copied default methods can then be modified to suit your own requirements You access the default methods as follows 1 Switch on the spectrometer in SuperUser mode gt see Appendix 1 2 Press STOP repeatedly until the APPLICATION APPLICATION branch header is displayed 3 Press e PARAMETER to select the first of the default methods 4 Use the arrow key to select the required method type 5 Press PARAMETER and then use the arrow key to select COPY FROM MARK 6 Press STOP to return to the APPLICATION branch 7 Create an empty method file see page 4 9 8 Copy the default method into the empty method file using the NEW FROM MARK parameter see page 4 14 The default method can now be amended as required B2167 20 Release 2 0 4 3 Editing Methods 43 Editing Methods The following options are available MODIFY METHOD e DELETE METHOD NEW METHOD NEW METHOD NAME CHECK METHOD MARK FOR COPY NEW FROM MARK PRINT METHOD Ib recre
111. s incluso cuando aparato haya sido desconeciado dol voltaje de la l nea Pr sence de tensions mortelles dans l instrument e M me lorsque l interrupteur de puissance est sur ARRET des tensions de secteur peuvent encore tre pr sentes dans l instrumant e Lorsque l instrument est reli au secteur les raccords peuvent tre sous tension et des parties sous tension peuvent tre d couvertes en ouvrant des capots ou en retirant des pi ces l exception de celles auxquelles il est possible d acc der manuellement e Les condensateurs contenus dans l instrument peuvent encore tre charg s si l instrument a t d connect de toutes les sources de tension F6 82761 20 Release 2 0 Translations of Warnings rr LS WOo2 02 e Even with the power switch OFF line power voltages can stil be present within the instrument WARNING e When the instrument is connected to line power terminals may be liva and opening covers or removing parts except those to which access can be gained hand is likely to expose live parts e Capacitors inside the instrument may stili be charged even if the instrument has been disconnected from all vollage sources Lethal voltages are prasent the instrument CD strumento sono presenti tensioni mortali e Anche se di alimentazione in posizione OFF la tensione di linea essere ancora
112. s no longer required This error is shown when not enough energy is detected Possible causes Beam is blocked in the sample compartment Loose lamp connection Lamp burnt out Lamp s switched off Defective detector This error is shown when not enough energy is received from the UV lamp Possible causes Beam is blocked in sample compartment UV lamp loose connection UV lamp burnt out UV lamp switched off Defective detector This error is shown when not enough energy is received from the Vis lamp Possible causes Beam is blocked in the sample compartment Vis lamp loose connection Vis lamp burnt out Vis lamp switched off Defective detector 9 3 Error Messages 9 4 Error PROBLEM SYSTEM ERROR SPECTROMETER FULL RESET DONE DIALOG FULL RESET DONE Meaning This error is shown when the instrument operating software crashes full reset is then automatically carried out After the instrument is reset one of the following messages is shown on the second line of the display BATTERY LOW TIMER FAIL RS232 IROQ FAIL TIMER IRO FAIL Make a note of this message Press PARAMETER to continue If you cannot continue call your Perkin Elmer office and inform them of the error message Note After a full reset all methods are erased This error is shown after changing the instrument software or after a full reset or when the spectrometer dat
113. s the ordinate If necessary change the ordinate mode to Remove the reference cell from the sample compartment Make horizontal fine alignment to the sample cell holder locking screws and milled posts loosened to obtain the highest possible transmittance reading on the display close the sample compartment cover while measuring transmittance Make fine alignment to the vertical alignment screw again to obtain the highest possible reading close the sample compartment cover while measuring transmittance When you are satisfied with the alignment tighten the milled posts and the locking screws on the cell holder Reinsert the reference cell in the reference cell holder The sample cell remains in its holder Repeat steps 4 and 5 with the reference holder but this time obtain the lowest possible transmittance reading on the display This completes the fine alignment procedure When the cell holder has been aligned once you can take it out and reinstall it without aligning it again 2181 20 Raelaasa 2 0 Minimum Volume Applications 2 3 4 Minimum Volume Applications To measure minimum sample volumes use microcells offered by Perkin Elmer The minimum sample volume required is a function of the cell internal width or volume and is specified in Table 2 1 Table 2 1 Minimum Volume Requirements Cell Type Cell Pathlength Min Part Number Internal Width Height of liquid 2mm 1 cm B007 9404 slightly
114. sampler If the calibration curve is to be compiled from known values e g from the literature values or from previous measurements these values should be entered here Make sure that the entering sequence is the same as for the corresponding concentrations parameter REF If the calibration curve is to be recorded do not enter anything here After calibration the absorbance values for the reference solutions are automatically entered Ihe spectrometer calculates the calibration curve from these values 82161 20 Release 2 0 Parameters Parameter No Description WAV MAX 3 Upper limit of the wavelength range in nm Value range 190 0 to 1100 0 in steps of 0 1 WAV MIN 4 Lower limit of the wavelength range in nm Range 190 0 to 1100 0 in steps of 0 1 Ifpeak areas are used for the calculation see parameter MODE the area is cal culated between the upper and lower wavelength values If calculation 15 made via a 2nd order derivative the derivative spectrum i5 recorded between the upper and lower wavelength values WAVE n 3j Wavelength program Oligoquant 1 Measuring wavelength in nm Range 190 0 to 1100 0 in steps of 0 1 Note The wavelengths is run in the sequence in which they are entered when using the ORDINATE MODE take RAT and DIF into account Enter wavelengths decreasing sequence if possible highest first lowest last Concentration 1 Wavelength n in nm Range 190 0 to 1100 0 i
115. t with any covers parts removed Servicing should be carried out only by a Perkin Elmer Service Representative or similarly authorized and trained person Disconnect the instrument from all voltage sources before opening it for any adjustment replacement maintenance or repair If afterwards the opened instrument must be operated for further adjustment maintenance or repair this must only be done by skilled person who is aware of the hazard involved Use only fuses with the required current rating and of the specified type for replacement Do not use makeshift fuses or short circuit the fuse holders Whenever it is likely that the instrument is no longer electrically safe for use make the instrument inoperative and secure it against any unauthorized or unintentional operation The instrument is likely to be electrically unsafe when it Shows visible damage Fails to perform the intended measurement Has been subjected to prolonged storage under unfavorable conditions Has been subjected to severe transport stresses This equipment generates radio frequency energy and if not installed and used properly i e in strict accordance with the handbook may cause harmful interference to radio communications 5 5 Safety information Environment wWwo1o03 Explosive Atmosphere This instrumant is not designed for operation an WARNING explosive atmosphere The instrument will operate correctly unde
116. terface If one interface fails change the method to switch the interface usage A3 5 Configuration Branch No Method 6 PRINTER CONFIG 7 ACCESSORY CONFIG 8 USER CONFIG A3 6 Function Printer Output Parameters PRINTER ON Switches output to the printer on and off PRINTER Selects the printer EPSON LQ EPS EX FX BIDIRECTIONAL GAP Sets the gap between two pages COLOR ON Switches color printing on and off only for color printers PERFORATION Skip over Perforation PLOT HEADER Header printed at the start of each plot Accessory mode for spectrometers without accessory board Parameters ACCESSORY ON Switches accessory mode on and off CELL To select the type of cell changer NO is no cell changer 5 CELL6 CELLS CELL9 CELL13 is for cell changer type SIPPER To select the type of sipper VASI PESI Si sipper VA vacuum PE peristaltic AS 90 91 autosampler Switches from single to double beam mode switches back ground correction on and off Parameters BEAM DB Double beam BEAM SBH Single reference beam BEAMSBS Single sample beam BASELINE CORR YES Background correction on BASELINE CORR NO Background correction off Note When using single beam mode operate only with ordinate mode 2161 20 Reisasae 2 0 Configuration Branch No Method Function 9 FACTORY Calibration peaks offsets and filter change points CONFIG Parameters ONM OFFSET Only for
117. the location 8 to 13 of the cell or cells to be measured in the 13cell changer Example 1 7 567 Measurement will take place at Cell8 13 2123456 locations 5 to 13 To change the molecular mass and molar absorption coefficient values of oligonucleotide bases Option YES NO Shows only when theoretical melting point is YES Refer to the Biochemical Application manual 10 5 Parameters Parameter CONC FACTOR CONC UNIT 10 6 No Description 11 Concentration factor Range 0 00001 to 9999 9 Note The concentration factor is calculated as follows V x M d x v x 1000 where V is the volume of the total solution the cell in mL M is the molar mass of the substrate in g mol d is the pathlength in cm v is the volume of sample in mL 1000 is the conversion factor for volume units in liters Concentration Factor Depending on the procedure used the molar absorption coefficient may need to be taken into account Concentration Unit defines the concentration unit used for the analysis is any concentration unit 1 gram per liter mg L is milligram per liter mg mL is milligram per milliliter mg dt is milligram per deciliter ug is microgram is microgram per milliliter mol mole mmol is millimole nmol is nanomole pmol is picomole ppm isparts per million mg kg ppb js parts per billion pg kg Is percent At isthe absorbance of a
118. the method PARAMETER The method is now marked for copying in the next step Press STOP to cancel B2761 20 Fielease 2 0 Copying Method 4 14 Copying the Method into Another Method File 1 Create a method see Section 4 3 3 13 SCAN lt gt PARAM STAAT page 4 9 into which the parameters from the marked method can be copied 2 Press PARAMETER PARAMETER MODIFY METHOD PARAM gt 3 Use the arrow keys to select v NEW METHOD NEW METHOD PARAM gt 4 Press PARAMETER again PARAMETER 5 Use the arrow keys to select 4 gt NEW FROM MARK NEW FROM MARK PARAM gt 6 Press PARAMETER PARAMETER The parameters from the marked 13 SCAN method are copied into the newly created JpARAM START method i Or MEE Press STOP to cancel 7 Modify the new method as required 2761 20 Aatease 2 0 Printing Out a Method 4 3 7 Printing Out a Method Printing out a method provides a list of its parameters and their current values A printer must be installed and configured refer to the installation handbook 1 Select the method to be printed out e g 2 SCAN lt gt PARAMS TART 2 Press PARAMETER PARAMETER i MODIFY METHOD PARAM gt t 3 Use the arrow keys to select 4 PRINT METHOD PRINT METHOD gt 4 Press PARAMETER again to print out the PARAMETER metho
119. this branch have the same protection _ However full protection can be set for individual methods since full protection has a higher priority than read write protection Write protection is not possible for individual methods since the branch has the higher priority 82161 20 Release 2 0 B216120 Release 2 0 Setting Protect Functions Setting Method Protect Function Set the method protect function as follows 1 Switch on the spectrometer in SuperUser mode 2 Select appropriate method e g 2 SCAN lt PARAM START 3 Press PARAMETER PARAMETER MODIFY METHOD gt f 4 Use the arrow keys to select 4 gt CHANGE PROTECTION CHANGE PROTECTION gt t 5 Press PARAMETER s PARAMETER 6 Use the arrow keys to select the desired lt q protect function i NO PROTECT lt gt f 7 Press PARAMETER to confirm the PARAMETER protection OD Press STOP to cancel 8 Exit SuperUser mode to activate the protect function A2 3 Setting Protect Functions Setting Branch Protect Function Set the branch protect function as follows 1 Switch on the spectrometer in SuperUser mode 2 Select the appropriate branch e g APPLICATION PARAM gt 3 Press e METHOD e METHOD 4 Use the arrow keys to select the required 4 gt protect function 4 NO PROTECT lt gt j 5 Press PARAMETER to confirm the protec
120. tirring bar in each of the cells The arrangement is such that whilst measurement is taking place in one cell the following cell is stirred Used with the 13Cell changer accessory Temperature measurement Option YES If TEMP CHECK YES has been selected a temperature sensor Part Number B018 5227 must be in stalled T he temperature measured in the cell is included in the printout If a temperature sensor has not been installed select TEMP CHECK NO B2161 20 Release 20 Parameters Parameter No Description TEMPERATURE 51 To enter the required temperature only in combination with TEMP CHECK YES Range 0 0 to 150 0 Measurement begins as soon as the temperature in the cell has reached the required temperature 0 1 C For temperatures of 0 0 or less the temperature is only registered and printed out together with the analytical results Operation with a Peltier cell holder To enter the required temperature of the Peltier cell holder rr P n M M M Fu I FE I T THRESHOLD 33 Scan Threshold value for the printout of analytical data valid only when PRINT DATA YES Range 0 0 to 9999 Only data above the given threshold value is printed out Concentration Threshold value for MODE DERIV PEAK 2 Only values above the given threshold value will be recognized as peaks TI
121. trical earth ground If any part of the instrument is not installed by a Perkin Elmer service representative make sure that the line power plug is wired correctly Cord Lead Colors Terminal Protective Conductor earth ground Green Yellow Green A a B LL A WARNING Wo2 01 Electrical Hazard Any interruption of the protective conductor inside or outside the instrument or disconnection of the protective conductor earth ground terminal is likely to make the instrument dangerous intentional interruption is prohibited Lethal voltages are present in the instrument Even with the power switch OFF line power voltages can Stil be present within the instrument e When the instrument is connected to line power terminals may be live and opening covers or removing parts except those to which access can be gained by hand likely to expose live parts e Capacitors inside the instrument may still be charged even if the instrument has been disconnected from voltage sources WD2 02 Safety Information When working with the instrument Radio Frequency Connect the instrument to a correctly installed line power outlet that has a protective conductor earth ground Do not attempt to make internal adjustments or replacements except as directed in this handbook Do not operate the instrumen
122. trometer the single cell holder can be installed in two different positions in the sample compartment Always install the holder such that the arrow on the cell holder lines up with the center point on the baseplate see next page Inscription Use in Spectrometer on Holder LAMBDA this position the cell holder can be used with legible Lambda Series Spectrometers mE BIO LAMBDA 2 In this position the cell holder be used with legible Lambda 2 Series Spectrometers as Lambda 2 11 12 14 14 Bio etc baseplate with 4 threaded holes The smallest beam diameter is exactly in the middle of the cell This is useful especially for operation with micro and semi micro cells X IM 11 1 Am mL LL m 82167 20 Hslaase 20 Single Cell Holder 2 3 2 Installing the Single Cell Holder There are two single cell holders provided with the instrument one for the sample beam and one for the reference beam Install the single cell holder in the sample compartment as follows 1 Lower the cell holder so that the two alignment holes slip onto the two studs on the baseplate at the bottom of the sample compartment The arrow on the cell holder must line up with the center point of the baseplate and BIO LAMBDA 2 must be legible Center point 2 Move the milled posts a little to locate the threaded holes in the baseplate and then tighten the milled posts The tube ports located at the front o
123. tup 1 Open the sample compartment cover 2 Make sure that the beam paths are free i e No objects cables etc project into the beam paths Nosamples are in the sample compartment Accessories are properly installed Note If the sample compartment is obstructed during the startup procedure the spectrometer will not initialize correctly 3 Close the sample compartment cover 4 Switch on at the power switch 5 Wait for the standby display LAMBDA 12 to appear BUSY Lambda 14 or Lambda 14P shows initialization display on those spectrometers standby spicy 500 0 0 000 ABS Other values be shown INPUT gt 6 Switch the accessories Standby display B218120 Helsase 2 0 Shutdown 2 2 Shutdo wn 1 Return the spectrometer to standby use STOP or PARAMETER 2 Switch off the accessories 3 Open the sample compartment cover 4 Remove samples and cells from the sample compartment 5 If accessories e g flowcell are installed in the sample compartment clean them thoroughly 6 Close the sample compartment cover 7 Switch off the spectrometer See also Wakeup page 5 42 2 3 20 2 0 Single Cell Holder 2 3 Single Cell Holder 2 3 1 Description Locking screw for horizontal alignment Vertical alignment screw Milled post Lifter horizontal align ment Figure 2 1 Single Holder B050 5071 Note Depending on the spec
124. values if ENTER gt required and press START BACK CORR Insert cell s containing a blank solution PRESS START and press START WAVPROG SMPL1 Insert a cell containing the sample PRESS START solution and press START 5 Continue to insert samples when asked until they have all been measured Example of the Display Shown During the Measurement WAVPROG SMPLn 200 nm Wavelength xxx nm ABS ABS Measured value ordinate as selected gt Repeat measurement cycles still to be performed This appears on the top right when cycles 7 1 Printout The result is printed out at the end of the analysis 2161 20 Relesse 2 0 EE 5 11 Concentration Methods 5 7 Concentration Determination You use CONCENTRATION 1 and CONCENTRATION 2 methods to determine the sample concentration Using CONCENTRATION methods you first establish a calibration curve and then measure the sample concentration The instrument calculates the calibration curve from the corrected or uncorrected values at defined wavelengths via the peak heights CONCENTRATION 1 or the peak areas CONCENTRATION 2 or the 2nd derivative CONCENTRATION 2 of the spectrum 5 7 1 CONCENTRATION 1 Method Peak heights Summary of the procedure for creating a CONCENTRATION 1 method Determine the measurement wavelength s see page 5 13 Create a CONCENTRATION 1 method see page 5 14 Esta
125. version of a substrate into a product whereby the enzyme acts as a catalyst The reaction can be followed photometrically an added coen zyme e g NADH is oxidized or reduced in the course of the reaction and the resulting change in absorbance measured Or the substrate or product may be photoactive and the absorbance will change with the concentration In enzyme kinetics the substrate is added in excess and the reac tion rate as d4 dt measured With excess substrate it is constant and directly proportional to enzyme activity substrate kinetics the substrate reacts completely The substrate concentration can then be calculated from the change in absorbance AA A4 1 Enzyme Kinetics A4 2 Enzyme Kinetics In enzyme kinetics the enzyme activity of a sample solution is determined the sample solution containing enzyme e g serum is reacted with a high excess of substrate The substrate is converted to product by the enzyme the rate of the reaction can be followed photometrically and is a direct measure of the enzyme activity Enzyme activity is given as International Units U 1U the enzyme activity required to convert 1 jumole of substrate per minute under optimal conditions The following reaction can be assumed for the conversion of substrate S into product P k 5 5 2 Where E enzyme and reaction rate constant The rate of reaction S E

Perkin Elmer Lambda 12-14-14P Operation manual

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