Amino Acid Analyzer - Manual for CHROMuLAN
Contents
1. INGOS s r o Page 15 400 Section 3 ASSESSMENT In the case of the Amino Acid Analyzer it is necessary to use always a computation with the standard This computation is carried out according to the following formula Area Mutiply Factor A t x Usr Peak Fact Response Divide Factor where Area is the peak area UsrPeakCoef is the coefficient from the peak table Th factor is set in the method heading and is the same for all peaks and for all samples assessed by using the method in question MutiplyFactor and DivideFactor are set for each sample separately in a sequence or in the heading of the sample they are all for all peaks Response is computed from the standard according to the formula Area Mutiply Factor UsrPeakCoef a Factor sta Response Amount Divide Factor std std The meaning of individual members is the same they are only taken from the standard 3 5 Comparison of analyses The program makes it possible to enter several analyses into a single graph This can be made by pressing the button serving for comparison of analyses see fig 7 and then open a subsequent analysis of functions File Open Individual analyses can be moved and enlarged by using the functions from the submenu Math in the chro matogram menu Another possibility of displaying several analyses into a single graph is by using the function Open selected in one window in t2. If you did not have in the sequence 2 4 a correctly set method use the function Method gt Load From for reading the right method for the standard see B 3 3 and activate the function Peak Autodetect 4 Check whether all peaks have been assessed If not correct the method see B 3 2 and activate the function Peak Autodetect 5 Save the standard File gt Save 6 Press the button Graph in the technological window and select the analysis which you want to assess 7 If you did not have in the sequence 2 4 a correctly set method use the function Method Load From for reading the right method for the analysis see B 3 4 and activate the function Peak Autodetect 8 Check whether all peaks have been assessed If not correct the method see 8 5 2 and activate the function Peak gt Autodetect 9 Read the standard of functions Method gt Load Calibration File and activate the function Peak Calculate Amounts 10 Call out the peak table fig 9 Peak Browse where it is possible to check the results possibly to print them by using the function Print Report 3 2 Chromatogram window It is possible to open the chromatogram window in two ways By pressing the button Graph in the technological window or from the menu by using the command File gt Open You can carry out basic operations with the graph with the help of buttons in the lower part of the window see fig 7 By pressing the button at the same time with the Shift
3. key you may use the function in question on a repeated basis By pressing the right mouse button in this window call out the chromatogram menu which would have the following items Setup setting the analysis parameters Method method submenu Baseline baseline submenu Peaks peak submenu INGOS s r o Page 11 AAA400 Section 3 ASSESSMENT MMD ULAN DATA Sequences 010117 1 Sample1 ULF E lol x kcys 6 36 asp 21 31 mets 24 13 thr 26 89 ser 29 12 ali 24 AA pro 36 58 aly 41 07 ala 43 23 1f2cys 46 55 val 50 54 met 54 78 ila 57 53 leu 59 32 tyr 65 01 phe 67 18 his 69 77 lys 72 99 NH3 75 79 1 Button for manual creation of a 5 Button for the cursor position dis baseline 2 1 play mode 2 Button for manual creation of 6 Button for the comparison of anal peaks B 2 1 yses see B J 3 Button for the setting of a cut out 7 4 Button for the returning of the previous cut out 9 Activation of the baseline display Activation of the axes display Activation of the peak description display go 10 Activation of the report display Fig 7 Okno chromatogramu Math conversion submenu to be used in the case of overlapping of analyses see B 5 View setting of visible items axis peak description baseline and oth ers Scale basic scale Copy to clipboard graph copying to other applications Print printing 3 2 1 Editing peaks You can edit peak p
4. Program Sample State Running 7147 h Samples Waiting 121 00 Reactor Displa Program Method Graph Status 118 00 126 00 i Current puffer number display Column temperature display Pump 1 pressure display Reactor temperature display State line see Chapter Control button panel Pump 2 pressure display onou Current sample test tube number display Fig 2 Technologick okno The left part of the technological window contains a chart of the analyzer in which the status of individual apparatuses is displayed The lower right part contains buttons serving for controlling individual functions a 5 2 2 on of the Analyzer Check the apparatus and operation chemicals see the User s Manual for AAA 400 and its chapter on the PUTTING INTO OPERATION Check the setting of parameters 2 2 Turn on the apparatus by using the button On Off situated in the technological window Insert samples into the dispensing disk and prepare the sequence 2 4 Wait until the apparatus is ready 2 5 Turn on the sequence by using the Start button Setting INGOS s r o Page 5 AAA400 Section 2 CONTROL By pressing the button Setting you will call out the dialog serving for the setting of basic parameters of the program and starting parameters of the apparatus Seguences directory Directory which contains all seguences Programs directory Dir
5. Tuer ra comrae 1 a a E s s pH2 60 Status Program Sample State acNone pH3 00 aan Ready 532 h Samples Waiting Prg Lir 12 00 Zero Lir 31 gare 31 00 acNone 425 Start Settings Seguence On Off Pra Lir 46 00 acNone 1 5NAOF Prg Lir 69 00 acStartEq Display Method Graph Status Prg Lir 71 00 acNone 0 2 NAOH Prg Lir 76 00 acNone acH20 gt 29 a T start 53 A al chromuan 16 41 Fig 1 ChromuLan The program is structured in such a manner that the entire system can operate in a fully automatic mode Set a seguence in the program within the framework of which each sample is assigned an analytic program and assessment method The apparatus will process samples in an automatic manner ensures the eguilibration of the column during the passage between analytical programs and will automatically assess the results according to the method pre set INGOS s r o Page 4 2 CONTROL The control module for AAA 400 is activated through the function Aplication gt Amino Acid Analyzer At the same time when the control module is activated also the technological window of the Analyzer will open fg 2 Coho AAA 400 lol x BUFFERS Amino Acid Analyser o INGOS s r o MPa 200 00 9 00 0 10 4 00 www ingos cz AAA Status Time
6. INGOS s r o AMINO ACID ANALYSER AAA400 Manual for ChromuLan PRAGUE May 4 2001 Producer Development Chemical system Supplier and service INGOS PiKRON V Havl ek ZMBD chemik INGOS s r o K Nouzovu 2090 14316 PRAHA 4 c INGOS 2001 Tel 02 4097683 02 4097692 Fax 02 4097685 e mail ingos tnet cz www ingos cz INTRODUCTION CONTROL ASSESSMENT TABLE OF CONTENTS 1 INTRODUCTION This is a freely disseminated software serving for controlling the sets of apparatuses and subseguent assessment of results The project is initialized and subsidized by PiKRON whose instruments support communication and control through the uLAN communication protocol At present the system is developed in the DELPHI environment for WINDOWS NT or WINDOWS 2000 and an extension for LINUX is expected This manual deals with the use of the ChromuLan program for the Amino Acid Analyzer AAA 400 In this case the program uses standard assessment and a special program module for the control of the Amino Acid Analyzer ChromuLan v0 26 i File Applicaton Edit Setup Tools Windo eja xaje Cal D ULAN DATA Sequences 010117 1 x le x AAA 400 mets 24 13 BUFFERS Sequence 010117 1 Pi pk P2 pk Column Reactor Autosampler Detector thr 26 89 29 12 00199 0158 Pine rou
7. alibration File Use Internal Standard Factor 1 Calibration File Age No Unknown Peaks Cancel Parameters Fig 10 Hlavi ka metody Base max diff Specifies maximum noise which could appear on the section which is considered to be the baseline Min peak height Minimum peak height The peaks which are lower are ignored during the self detection INGOS s r o Page 14 AAA400 Section 3 ASSESSMENT Min peak width Minimum peak width The peaks which are narrower are ig nored during the self detection Use negative peaks Mark the field if you want to assess negative peaks It is not used in the case of the Amino Acid Analyzer Calc Amounts Mark the field if you want to compute the quantity automat ically In the case of the Amino Acid Analyzer it is always marked Use Calibration File Mark the field if you want to use the standard In the case of the Amino Acid Analyzer it is always marked Use Internal StandardMark the field if you want to use the internal standard Factor Conversion factor see B 4 Calibration File Age Date and time for the reading of the calibration file During computation it is necessary to check whether the calibration file was not modified after this date In the case that it was it is necessary to carry out its new reading No Unknown Peak If you mark this field only those peaks are assessed which are defined in the method in question 3 3 2 Method peaks By using the f
8. ands may appear in the AA Command column Inject Dispense the sample from the loop This command should always be in the time 0 Zero Zero the detector This command should be addressed to the place for which it is sure that there is a baseline StartEquil From this command the equilibration analysis starts 2 4 2 H20 Switch the pump 2 entry to water NHD Switch the pump 2 entry to ninhydrin StopAcq terminates the data record Load Prepares another sample to the loop None No action is being made After modification it is necessary to save the program Saving will be carried out by using the Save command from the menu which is called out with the help of the right button If you are correcting a program while an analysis is running the changes will only be applied during the next sample 2 4 Sequence INGOS s r o Page 7 AAA400 Section 2 CONTROL The seguence specifies an arranged set of samples during processing Each line of the seguence corresponds to one sample The seguence is automatically named according to the date of creation At the same time when the seguence is created a directory of the same name is created on the disk and individual analyses are stored into it By pressing the Seguence button in the technological window call out the menu which has three items New Makes it possible to create a new seguence Before actual editing it is necessary to fill the heading of the sequence firs
9. arameters directly in the graph or in the peak table In the graph you can edit also the baseline and the integration marks of the peaks see 8 If you want to edit peak parameters in the graph mark the peak by clicking on the description and by further clicking on the description call out the dialog for editing peak parameters You can change the position of the end points of the baseline and integration marks directly by using the mouse INGOS s r o Page 12 400 Section 3 ASSESSMENT Peak description Data measured Baseline Baseline end point Peak integration marks mets 24 13 oF WN F Fig 8 Editace piku Addition of further peaks and sections of the baseline is carried out with the help of buttons in the lower part of the chromatogram window 8 2 Call out the peak table by using the function Peak gt Browse see fig 9 3 3 Method The method contains information for the assessment of the analysis The method forms a part of any analysis But it can also be saved in a separate file and to be read again from this file This can also be made by using the function Method Save To and Method Load From In the case of the Amino Acid Analyzer the method set in the sequence is automatically inserted into the analysis see 2 4 The data in the method can be divided into three parts method heading peak description ad baseline description 3 3 1 Method heading Call out the meth
10. ding serves for the entering of parameters common for the entire se quence The dialog for its editing will be called out automatically during the creation of a new sequence alternatively it can be opened by using the command Header in the menu which is called out by using the right button in the sequence window In the case of the Amino Acid Analyzer only the Program and Method items are used from the sequence heading 2 4 2 Equilibration analysis During the sequence processing and on any change in the analytical program an equili bration analysis is automatically put on the beginning This analysis serves for the cleaning and stabilization of the column The equilibration analysis runs according to the program of the following analysis It does not begin at zero time but at the place where the program contains the StartEquil 2 3 command 2 4 3 mass assessment In the sequence window it is possible to carry out mass assessment of several samples The samples which we want to assess are marked and by using the function Show INGOS s r o Page 9 AAA400 Section 2 CONTROL result for sel from the menu under the right mouse button the result window is called out Samples for mass assessment must have the correct method assigned to them B 3 and a standard The standard can be assigned to selected samples by means of a function Assign calib file 2 5 State information The right part of the technological
11. ectory which contains analytical programs Methods directory Directory which contains assessment methods Current Seguence Current sequence name 2 4 Default Prog Name Name of the program to be set in the heading during the creation of new sequence 2 4 1 Default method for new seqName of the method to be set in the heading during the creation of a new sequence 2 4 1 P1 Flow ml min Pump 1 flow rate 0 3 P1 Press Min MPa Lower limit of the pump 1 pressure 2 0 P1 Press Max MPa Upper limit of the pump 1 pressure 9 0 P1 Deviation Permitted oscillation of the pump 1 pressure 50 P2 Flow ml min Pump 2 flow rate 0 2 P2 Press Min MPa Lower limit of the pump 2 pressure 0 1 P2 Press Max MPa Upper limit of the pump 2 pressure 3 0 P2 Deviation Permitted oscillation of the pump 2 pressure 80 Column Temp Column temperature 60 Col Temp Min Dif C Maximum permitted column temperature drop 5 Col Temp Max Dif C Maximum permitted column temperature increase 5 Colum Temp Dev Permitted oscillation of the column temperature 10 React Temp C Reactor temperature 121 React Temp Min Dif C Maximum permitted reactor temperature drop 5 React Temp Max Dif C Maximum permitted reactor temperature increase 5 React Temp Dev Permitted oscillation of the reactor temperature 10 2 3 Analytical program The analytical program serves for controlling the ap
12. he menu which will be called out by using the right button in the seguence window It is possible to switch active analyses by using the function Overlay from the chro matogram menu INGOS s r o Page 16 4 TABLE OF CONTENTS LI INTRODUCTION da nie netaha Koa der te a BA CONTROL assassin L 21 turning on of the Analyzer sess Setting PE PASS P re imam V e e eo eda o i 2 Analytical Prana o dk A o o aut L E SEQUENCE O R e aks 1 E 2 4 1 Sequence heading zzz ___ 24 Equilibration analysis nan g mass assessment seas RKA A oes g State information ae ove niece rested seuss TO BJ ASSESSMENT wesselam oo al Heid eae L 3J ASSENU proc d te sees covet eerie sese es E L 3A Chromatogram weiss e a se KL B2N Editing peaks ee e a soon 12 Method persse aan a eee 13 rectal Method heading sss 3 E 334 Method peaks Agden bie e ba e 5 Method for the standard eat nd datace p 3 34 Method for the sample 5 E Comp AONE dok av n s o a de bg EE 5 L_ 3 5 Comparison of analyses 16 EJ TABLE OF CONTENTS zast A ad T L_ 43 1 List of pictu
13. is added the num INGOS s r o Page 8 AAA400 Section 2 CONTROL ber which follows by the highest number used will be filled in automatically greater by 1 Sample Desc Sample description Not obligatory User name Name of the user who has been analyzing the sample File name Name of the file on the disk It is obligatory if a sample is added it will automatically be filled in as Sample together with the ordinal number Program name Analytical program name It is obligatory If a sample is added the program name from the sequence heading is automatically added 2 4 1 State Sample processing state Individual states are marked in red for the purpose of fast orientation Error Error state There was an error during the sample analysis Done The sample was processed in order OK Running The sample is currently being processed InLoop The sample is ready in the loop Waiting The sample is waiting for processing Method name The name of the method which will be used for the sample assessment B 3 It is obligatory but during assessment it is possible to change the method on an additional basis B 3 When the sample is added the method name is filled in auto matically from the sequence heading 2 4 1 Inserting new lines and deleting lines can be carried out by using the Insert and Deletekeys or from the menu called out by means of the right mouse button 2 4 1 Sequence heading The sequence hea
14. od heading by using the function Method gt Edit see fig 10 The meaning of individual items is as follows Method Template of the file from which the method was created Calibration File Name of the standard Calibration File Method file name Duration This parameter is not used for the time being Base min interval be used during automatic detection of the baseline it says how long the straight section must be so that it can be consid ered as a baseline INGOS s r o Page 13 AAA400 Section 3 ASSESSMENT 0 9553 25 28 mets 23 7 1 2 00 1 066 25 42 thr 23 74 1 2 00 1 071 25 7 ser 23 9 1 2 00 1 075 2858 glu 2403 1 1 00 1 151 6 803 pro 2391 1 1 00 0 02645 28 22 gly 23 52 1 1 00 1 2 1 Retention time 7 Response see 2 Peak area 8 Name of the line from which the 3 peak name peak is assessed If the field is 4 amino acid quantity empty it is assessed according to 5 Coefficient for the computation of a green color while the B letter quantity see and means that it is assessed from a 6 Window for the assignment of the blue color method peaks see 3 3 2 Fig 9 Tabulka p k OLT EJ Method template Samplet Calibration file D ULAN DATA S _ Method File Name h ULM Dilution Base min interval min 0 30 Base Max Diff 0 006 Min peak height 0 01 Min peak width min 0 20 Use Negative Peaks Calc Amounts Use C
15. paratus during the sample analysis Each line of the program defines activities of the apparatus at a given time By pressing the Program button in the technological window call out the menu which has three items New Calls out the window for editing a new program Open Calls out the menu for opening a program Edit Calls out the window for editing the program which was edited dur ing the last session The program editing window is in fig 3 The program lines are automatically arranged according to the time Inserting new lines and deleting old lines can be carried out by using the Insert and Deletekeys or from the menu which can be called out by using the right mouse button With regard to the automatic arrangement of the lines it is possible that at a change of time the line will move to another position During modifications being made in INGOS s r o Page 6 400 Section 2 CONTROL 1 Time 2 Column tem perature 3 Puffer num ber 50 acNone pH3 00 4 Command 50 acZero 5 Note 50 60 60 74 74 acZero acNone pH4 25 acNone 1 5 acStanE quil acNone 0 2 NAOH C wo wo NH S amp S A A acNone acH20 acLoad 50 1 acAcgStop 50 1 acNHD acNone Fig 3 Okno editace programu a program it is necessary to work with this function carefully and always after a change in time to check whether the right line is being edited The following comm
16. rtes and tables 2 2 22 0005 seeeenessseasdeeeeeraeeseeeenes 1 4 1 List of picturtes and tables Fig I ChromuLan sa43 a ose Kk SAGA eee ess ddd da dan Fig 2 Technologick okno Fig 3 Okno editace programu ict eludes 1 Fig 4 Window for editing the sequence sussssusseessssesssssnnns 8 Fig 5 Stavov dek z lt lt lt kenenin and on PEE LEE EEE EE ES Fig State E M S S L E mahal 10 Fig 7 Okno chromatogramu an 8x88 epee asd ta 2 Fig 8 Editace Diku R R VO O O O ES O O 3 Pig 9 Tabulka Pik ase shod aji deo ad Ss aa Pigs 10 Hlavi ka metody yssazasisidedildidd dsn d usa s ckak sdida pktod daje INGOS s r o Page 17
17. t 2 4 1 Open Calls out the menu for the opening and editing of a sequence which has already been saved At the same time it will set this sequence as the current one Edit Calls out the window for editing the current sequence If the sequence is being executed you will call out directly the editing of the current sequence In the sequence being executed it is only possible to change the samples which are in the Waiting state The window for editing the sequence is in fig 4 Individual columns have the following meaning a Sample Name Sample Desc 1 VH Testl Sample 2 Test2 VH Sample s AAP ULM 3 Test3 VH Sample3 5 ULM 4 Test4 VH Sampled s AAP ULM 5 Tests VH Samples ULM 21 WH Sample6 ULM 22 Test VH Sample ULM 8 Test WH Samples ULM 11 Test Sampled hAAP ULM 12 VH Sample10 hAAP 13 11 Mn Sample11 hAAP h ULM 15 Testl2 VH Samole12 h AAP gt 1 Name of sample 5 File name 2 Number of test tube 6 Analytical program name 3 Sample description 7 Sample processing state 4 Name of the user who has been 8 Method name analyzing the sample Fig 4 Window for editing the sequence Sample name Sample name If it remains undefined the file name will be filled in automatically Vial Nr Number of test tubes from which the sample in question will be taken It must be filled in if a sample
18. unction Method gt Peaks gt Browse you will call out the method peak table This table is the same as the peak table You can add peaks into this table by using the Insertkey alternatively you can copy the peaks marked from the analysis by using the command Peaks Copy Selected To Method The peaks of the method are assigned to the peaks measured with the help of the retention time and window If the peak is not assigned correctly change the retention time in the method peak table or enlarge the window If the window is changed be careful that the windows should not overlap 3 3 3 Method for the standard The method for the standard must have in the heading B 3 1 the following setting Faktor 1 Moreover the method peak table must have in the column Amount the settings of quantities of individual amino acids in the standard The column UsrPeak Coef must be 1 Also Multiply Factor and DivideF actor in the sequence must be set to 1 3 3 4 Method for the sample If you want to know the results in grams you must set molar weights of individual amino acids in the method peak table in the column UsrPeakCoef If you are using a constant base and dilution include it into Faktoru in the method heading if you do not use Multiply Factor and DivideFactor in a sequence The name of the peak in the method for the sample and in the method for the standard must be the same otherwise the peak in question will not be assessed 3 4 Computation
19. window contains a state line see fig 5 AAA Status Time Program Sample State Ready 27 42 h Sample Waiting 2 4 5 1 Apparatus state 4 Current sample name 2 Analysis time 5 Current sample state 3 Current program name Fig 5 Stavov dek Other information concerning the state of the apparatus can be called out by pressing the button State see Hg 6 Detector Dispenser state Detector state Absorbance values G 0 0003 B 0 0010 Sequence mon 441 1 Current sequence P1 o 2 Pump 1 state S 6 5 3 Pump 2 state Column Dk e Hore 4 Column state 5 Reactor state 6 7 8 NZ 8 Fig 6 State information INGOS s r o Page 10 3 ASSESSMENT For AAA400 the assessment with the help of a standard is used With regard to the fact that chemistry used in the Analyzer changes in the time NHD is getting old it is necessary to arrange a standard after each 5 to 10 samples For the purpose of elimination it is possible to use also an internal standard It is mainly used for the elimination of an error arising during the preparation of the sample 3 1 Assessment procedure 1 Open the analysis of the standard Press the button Graph in the technological standard and select a standard 2 If you did not mark the standard in the sequence mark it additionally in the headings function Setup mark the field Cal Standard 3
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