pKa Estimator Elite - MicroSolv Technology Corp.
Contents
1. Figure 5 Calculate Apparent pK form prior to pK determination 12 buffers pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc pKa Estimator Elite les File About z o Edit Default Yalues Calculate Apparent pKa I M Ta s Tm s Use Y C cePRO Manager pKa pKa Data by Compound Quinine 24 Pt Aqueous1 2004 10 07 R 0 0000 Ld cm 33 00 0 050 9 0 050 414 0 050 419 0 050 406 0 050 406 05 88 391 85 80 370 353 38 30 24 25 32 334 341 335 Le cm 55 00 y 3500 Molecular Weight J 324 00 Dutput 1 pKa p 3 pKa i Acid Base Zwitterion 50 ppm in 1 mM HCI 0 1 DMSO Buffer lot 040902 A Compound Name Initial Guess Best Fit Apparent pKa Original data Quinine 3 4 00 pKa iL 4 35 pKa Best Fit Start Calculation 7 99 00 pka2 8 54 pka2 Message Report Generation dane Figure 6 Calculate Apparent pK form prior to pK determination 24 buffers 4 1 Entering the Compound Molecular Weight The pKa Estimator Elite software selects from nine different model equations for determining a best fit line to the data The software applies the best fitting equation to the migration data based on the maximum positive and or negative effective mobility of the sample compound along with its molecular weight Therefore for the calculation to determine2. 11 that contains 24 buffers Calculate Apparent pKa form prior to pK determination 13 12 buffers Calculate Apparent pKa form prior to pK determination 14 24 buffers Prompt to remind user to enter a molecular weight value eee 15 pK control with 1 pKa highlighted eee ceeeeeceseeeceeeeeenteeeeneeeenee 15 Initial Guess and Best Fit Apparent pK controls when 16 3 pK is selected Pop up when the software is unable to find a best fit curve eee 16 Prompt to select new model equation Yes or accept calculated 17 model equation No Equation Selection form for user to select a different 18 model equation Calculate Apparent pKa form after pK calculation 12 buffers 18 Calculate Apparent pKa form after pK calculation 24 buffers 19 Indicator showing how compound was fitted acid base or zwitterion 20 Calculate Apparent pKa screen showing output fields 21 Microsoft Excel report generated from the pKa results c ccscceescesseeeeeees 23 pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc List of Tables Table 1 Instrument Configuration for pK Measurement by CE oooconcocccccnccccnonncnnnnos 25 Table 2 Approximate CE Run Parameters for Generic CE pK Method 27 Table 3 Experimental Results for pK Determination by CE oooonoocccnccccccna
3. a table format click on the Output option in the Input Output control to the left of the graph see Figure 16 pKa Estimator Elite Eile About 1 Edit Default Yalues Calculate Apparent pKa m fit TA 4 C Program Files PKa Estimator Elite data 2004 10 12 Benzoic Acid Run 1 R 2 0 9977 Ld cm 9 39 00 Le cm 9 49 00 RAR ofeis 18 4 48 1 191 9 w ip y 15000 274 2 Molecular 276 7 Weight Erri 9 122 00 277 1 277 1 277 1 277 1 S bo ES te go D B FEEEEREREEEE 5 Mobility 10 6 cm 2 Y s 5 e p 2 pKa 3 pKa Base 1 Comment 2witterion 2 0 3 0 4 0 5 0 6 0 7 0 Benzoic Acid Opt 12 pt run 1 10 12 2004 P pH Compound Name Initial Guess Best Fit Apparent pKa Originaldata Benzoic Acid p 4 00 pKal 4 08 Beste et n pKa morros E 2 Message File Save 0 96 Report Generation w Figure 16 Calculate Apparent pKa screen showing output fields pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 22 4 11 Saving a File To save all of the experimental information migration times pH values La Lo V molecular weight value comments compound name user name and pK results to a file click the File Save button A standard file save dialog box will appear for specifying the name to save the file to As with the Default Values files the default directory is C Program Files pKa E
4. tables pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc pKa Estimator Elite File About Edit Default Yalues Calculate Apparent pKa Experimental Parameters bos T pH Count 12 do Ld cm Le cm Jooo Yoo 24 Buffers Figure 1 Edit Default Values form with pH values and first I M value entered pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc pKa Estimator Elite File About Edit Default Yalues Calculate Apparent pKa Experimental Parameters pH Count Y 12 Jo aten AE 0 00 0 00 24 Buffers gt Figure 2 Edit Default Values form after Fill button has been pressed 2 Enter the experimental values for Lg cm effective capillary length to the detector in centimeters Le cm total capillary length in centimeters and v voltage in volts 3 Enter a descriptive phrase in the Experimental Parameters field for future reference e g lot of the pH buffers used pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 3 2 Saving Default Values To save the values entered into the Edit Default Values tab click on the Save to File button A standard file save dialog box will appear for specifying a name to save the file to see Figure 3 The default directory that files are saved to is C Pr
5. 2 Running pKa Estimator Elite Using the Start button on the computer navigate to Programs pKa Estimator ElitespKa Estimator or the directory the user installed it to if the default directory was not used and select 2 3 Exiting pKa Estimator Elite An Exit button is available on each tab of pKa Estimator Elite for exiting the program To stop running the software click the Exit button and the software will close automatically pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc Chapter 3 Using the Edit Default Values Tab 3 1 Entering Default Values If this is the first time the user has used pKa Estimator Elite or the user has just completed a series of CE runs and experimental parameters such as the pH ionic strength I M capillary length or voltage are different than previously used a Default Values file can be created in the Edit Default Values tab 1 Enter in the pH and ionic strength I M values for the experiment in the table form If the same ionic strength was used for all pH values recommended simply enter the value in the first row and then click the Fill button see Figures 1 and 2 The buffer pH values should be entered in increasing pH order from top to bottom in the form If 24 pH values were used in the experiment then click on the 24 Buffers button This will increase the number displayed in the pH Count field to 24 and will display 24 rows in the pH and I M
6. Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 20 Acid Zwitterion Figure 15 Indicator showing how compound was fitted as an acid base or zwitterion 4 5 Comment Field Information about the pK experimental results e g sample diluent pH points used buffer lot can be entered and edited using the Comment field This information will be saved with the pka file and will be displayed whenever the file is reopened 4 6 Compound Name Field The compound name or identifier can be entered into the Compound Name field This information will be saved with the pka file and will be displayed whenever the file is reopened 4 7 User Name Field If desired the name of the analyst performing the pKa experiment can be entered into the User Name field This information will be saved with the pka file and will be displayed whenever the file is reopened 4 8 Reset The Reset button will clear the table and graph on the Calculate Apparent pKa tab Likewise if the user loads a new pka file see Section 4 12 the values will be cleared and replaced with the new values pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 21 4 9 Refresh The Refresh button on the graph clears the best fit line from the graph leaving only the scatter plot of pH vs effective mobility 4 10 Output To view the calculated effective mobilities and the best fit effective mobilities in
7. J Compo Guinine Repalcreaed 111020064357 PM Analyst Hename C ogram Ale cPha Estmator Bite Yatn 2004 10 07 Guinine 24pt040002pka Comment 50 ppm mbt HCI 0 1 DASO Buf bol 20402 as 2 ES Calculated Pocttve Chage 1 S Cakcuakd Megatve Charge 000 1 bbae Figure 17 Microsoft Excel report generated from the pK results pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 24 Chapter 5 Summary This manual walks the user through how to use CombiSep s pKa Estimator Elite software This software enables the user to easily consolidate experimental conditions with CE migration time data The consolidated data is then used to find a non linear best fit line to the pH vs effective mobility plot from which a compound s apparent pK value can be obtained Microsoft Excel reports can be generated and saved to better archive experimental results Appendix provides a generic CE protocol to help the user get started generating data and become familiar with pK determination by CE For further questions regarding the use of pKa Estimator Elite software please contact CombiSep technical support at 1 888 822 7949 toll free or by email at tech support combisep com pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 25 Appendix 1 Example Procedure for pK Measurement by CE The following is a generic procedure for pK meas
8. corrections for acids and bases for up to three ionizable groups Note that the correction factor for the second ionization of a diprotic compound takes into account the monoprotic species it is in equilibrium with 1 e for a diacid the correction is 0 33 0 08 0 025 The same applies to triprotic compounds The correction factor for zwitterionic compounds is more complex and in general apparent pK values are reported in the literature Ideally literature pK values should be clearly stated as apparent or thermodynamic values and the ionic strength of the buffer medium provided although this is not always the case pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 30 Table 4 Ionic Strength Corrections Apparent pK to Thermodynamic pK pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 31 Literature References Listed below are several selected references describing the use of CE for pKa analysis This list is not comprehensive rather it is intended to give the user a general idea of how CE has been applied for the determination of compound pK values Review Paper on pK Determination by CE 1 Poole SK Patel S Dehring K Workman H Poole CF Determination of acid dissociation constants by capillary electrophoresis J Chromatogr A 2004 1037 445 454 Additional References 2 Miller J Blackburn AC Shi Y Melzak AJ Ando HY Semi em
9. has been chosen Do you wish to choose a different Equation Figure 11 Prompt to select new model equation Yes or accept calculated model equation No pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 18 Monobase Monoacid Dibase Diacid Monoacidic Monobasic Ampholyte Tribase Triacid Diacidic Monobasic Ampholyte Monoacidic Dibasic Ampholyte Edit Default Yalues Calculate Apparent pKa IM Ta s Tm s use Cs Program Files PKa Estimator Elite data 2004 10 121 Benzoic Acid Runt Ld cm 39 00 0 050 129 129 0 050 139 136 0 050 135 127 0 050 134 112 0 050 150 116 0 050 153 115 0 050 162 120 i 0 050 156 116 8 47 0 050 C178 128 9 27 0 050 177 128 10 06 0 050 192 135 10 86 0 050 194 136 Lc cm y 49 00 y 15000 Molecular Weight 9 122 00 EE ENE ENE NE NENE NES Comment Benzoic Acid Opt 12 pt run 1 10 12 2004 meal Cocnnound Necie Initial Guess Best Fit Apparent pKa Benzoic Acid 4 00 pKal 4 08 pKa TIE Anion Model E Smith Equation Message 0 96 pace Figure 13 Calculate Apparent pKa form after pK calculation 12 buffers pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc pKa Estimator Elite D TEJ File About oo Edit Default Yalues Calculate Apparent pKa pH I M
10. select a different model equation see Figure 12 The pop up form will close automatically shortly after a selection is made The software will then proceed to find a best fit line to the data points using non linear regression A progress bar will appear in the upper center of the scatter plot to inform the user of the status of the best fit calculation When the calculation is complete the progress bar will disappear After the best fit line has been determined the R value for the fit will be displayed in the R field that is located in the upper right corner of the Calculate Apparent pK form This value gives the user a statistical measurement of how closely the best fit line matches the measured data points The best fit line is plotted on the existing graph and the apparent pK value s appear in the Best Fit Apparent pKa field s see Figures 13 and 14 Note The apparent pK value often written as pK is the value displayed by the software Under typical experimental conditions the ionic strength is set to level ionic strength of I 50 mM In order to convert the apparent pK value to a thermodynamic value at I 0 mM or to a different ionic strength a correction factor should be employed Correction factors for acids and bases are listed in Appendix 1 Correction factors for zwitterionic compounds are less straightforward and as aresult apparent pK values are most commonly listed in the literature Model equation Monoacid
11. ti ustic ase aes aan da oes tao god sate eats E eae weet 20 AO ROSES AN 21 pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc A TO SOUU eae NEA RS e do En e du o eee 21 AN O Qusiaaaucs E wed oa aa ES E 22 ND AD RNA AR AAE E E E E E E 22 4 13 Report Generat lomos lata IA RE labs 22 Chapter Dc SUI 24 Appendix 1 Example Procedure for pK Measurement by CE ooooonnccnocccocccincnnnnnnnonncnnns 25 1 1 CE Instrument Conti giiratiomssccis s scsastecscvavecatassssvaesadeasacassedsedoasncondeetebsnctensnceciens 25 1 2 Sample Preparation sne nne e a E ees se E E EEO 26 1 3 PH B tf r Preparation e E 26 1 4 CE Run Sequence Par dea AS RS 26 1 5 Example Res init tdi EEE iia 28 Appendix 2 Ionic Strength Correction Factors oooooccconcnnocnnonncoonnnonnnonnnonrncnoncnnnc crac crnn anno 29 Literat re Reterenco sionistas 31 pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 iv List of Figures Edit Default Values form with pH values and first I M 7 value entered Edit Default Values form after Fill has been pressed 8 Standard Save Dialog BOX uti idad 9 Edit Default Values form after loading a file ooooon nnnnnnn n n
12. Ta s Tm s Use Y C cePRO Manager pKa pKa Data by Compound Quinine 24 Pt Aqueous 2004 10 071 R 2 0 9984 1 77 0 050 9 551 Ld cm 2 08 0 050 414 558 233 00 q 2 51 0 050 419 566 2 93 0 050 406 546 3 40 0 050 406 534 05 518 Lc cm 55 00 88 475 A 391 463 3500 85 450 80 438 370 423 353 401 38 380 30 368 24 357 25 352 32 352 334 347 341 349 335 340 342 344 10 86 344 11 31 9 050 336 Molecular Weight J 324 00 Output 1 pKa p 3 pKa Acid Conner Zwitterion 50 ppm in 1 mM HCI 0 1 DMSO Buffer lot 040902 A Compound Name Initial Guess Best Fit Apparent pKa Original data Quinine E 4 00 pKal 4 35 pKa Ep User Name z Start Calculation 9 00 pka2 8 54 pka2 Cation Model Equation Message 1 94 Report Generation done Figure 14 Calculate Apparent pKa form after pK calculation 24 buffers 4 4 Model Equation Number The value reflecting the maximum fractional charge of the compound used to determine which model equation to use will be displayed in the Model Equation field This value is rounded to the nearest whole number to guess the charge valency of the compound 1 Mono 2 Di 3 Tri Acid Base or Zwitterion will be highlighted in the indicator to the left of the graph see Figure 15 to indicate if the compound was treated as an acid base or a zwitterion pKa
13. ccconnccnnnnos 28 Table 4 Ionic Strength Corrections Apparent pK to Thermodynamic pK 30 pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc vi Document Notation Within this document the following conventions are used Courier New Used to denote file path names and form names in CombiSep s pKa Estimator Elite software Italics Software names File names and extensions are denoted with italics bold Courier New Used to denote Windows commands and names of fields and buttons in pKa Estimator Elite General Software Information Throughout the software fields with a blue background are fields that require user input Fields with a yellow background are fields that the software will use to display computed values For example in Figure 1 the Date Created Time Created and Path fields will be filled in by the software The other fields shown require user input pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 1 Introduction 1 1 Evaluation of pK Values by Capillary Electrophoresis The acid dissociation constant pK value of an ionizable compound is defined as the pH value at which the dissociated and undissociated species are of equal equilibrium concentration and is a fundamental physicochemical property that strongly influences many properties For example the neutral form of the compound is less water soluble more lipophilic a
14. nd possesses higher membrane permeability than the ionized form Experimental knowledge of compound pK values provides a measure of the extent of compound ionization across the pH range of pharmaceutical relevance and is highly beneficial for predicting compound absorption distribution metabolism and excretion ADME properties The pK value also plays an important role in the development of drug delivery formulations Over the past decade capillary electrophoresis CE has emerged as a valuable tool for the evaluation of compound pK values as it possesses many favorable qualities e Potential impurities and degradants can be separated from the target compound e Intimate knowledge of sample concentration is not required for analysis e Sparingly soluble compounds with a suitable UV chromophore can be analyzed e Nochanges in spectral properties are required for detection of a pK value Minimal sample amounts are required for analysis ug amounts Numerous publications have appeared describing the use of CE for pK analysis including a recent review 1 Several of these references are listed at the end of this pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 2 manual Users are encouraged to study these articles to improve their knowledge of CE based pK analysis and best understand the strengths and limitations of the technique To determine the pK value s of a compound by CE it is necessary t
15. nt autosampler such that the highest pH value buffer is analyzed first and subsequent pH buffers are analyzed in order to the lowest pH value Analysis of the highest pH value first will serve to minimize changes in pH due to absorbed CO from the environment throughout the time interval of the experiment 1 4 CE Run Sequence Parameters It has been demonstrated that the use of pressure assisted CE can significantly reduce migration times while preserving data quality for pK analysis 1 5 Table 2 lists example pressure settings as function of pH under the above instrument configuration along with approximate migration times for a neutral marker EOF and current levels for a buffer series of I 50 mM pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 27 Table 2 Approximate CE Run Parameters for Generic CE pK Method pH Value Pressure Level psi Migration Time for EOF min Total Current uA Using the above parameters the analysis of basic compounds can be completed in approximately 2 5 min per CE run pH value and acidic compounds can be analyzed in approximately 3 min per CE run pH value The resulting total time to complete an unattended analysis at 12 different pH points using a 3 min CE run time was 80 min The above method was found to provide a reasonable compromise between speed and separation resolution An increase in the pressures used in conjunction with CE can slightly dec
16. o measure the migration times of the compound in relation to a neutral marker usually DMSO over a range of pH values From the migration times of the compound ta and neutral marker tm in seconds the effective mobility ueff can be calculated via Equation 1 Eqn 1 4 LiL V GA 3 A Where La is the length of the capillary to the detector L is the capillary total length in cm and V is the applied voltage V A plot of Hers vs pH yields a sigmoidally shaped titration curve from which the inflection point s define the pK value s Using non linear regression analysis a best fit line can be applied to the data using a set of standard fitting equations depending on the number of ionizable groups and the pK value determined Equations used for the evaluation of up to three ionizable groups have been described in the literature references available at the end of this manual 1 2 1 2 pKa Estimator Elite Software Overview The introduction of pKa Estimator Elite software from CombiSep provides a simple straightforward method for calculation of pK values from CE data Equations for performing non linear regression analysis of up to three ionizable groups along with an empirical estimation of compound valency have been incorporated into the software to pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 3 simplify data analysis All that is required of the user is to enter the migration time
17. ogram Files pKa Estimator Elite data If desired the user can navigate to a different directory using the standard file save dialog box The file that is created is saved as a comma delimited file with a csv extension The file can be opened and edited in MicroSoft Excel or text editing software if desired Please note that changing the format or layout of the file within Excel or a text editor may make the file unreadable by the pKa Estimator Elite program The date and time when the file is created and the path the file is saved to will be stored in the file and these values read in and displayed in the appropriate fields the next time the file is loaded Save As 2 x Save in a data e Ed History Desktop e My Documents My Network Pe Save as type Custom Pattern cs y Cancel Mee Figure 3 Standard Save Dialog Box pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 10 3 3 Loading Default Values To load experimental parameters from an existing csv file click on the Load Values button and use the standard file open dialog box to navigate to the folder the file is in and select it The default directory that files are read from is C Program Files pKa Estimator Elite data The software will automatically determine from the csv file that is loaded whether 12 or 24 pH buffers were used thus the user does not need to click the 24 Buffers button prior
18. on 2 0 Copyright 2005 CombiSep Inc 16 Initial Guess Best Fit Apparent pKa 972 00 pka1 0 00 pka 75 00 pKa2 0 00 pKa2 8 00 pKa3 0 00 pka3 Figure 9 Initial Guess and Best Fit Apparent pK when 3 pK is selected Occasionally the pKa Estimator Elite software is unable to find a best fit line for the data If this occurs a pop up window will be displayed to inform the user of the situation and will prompt the user for new initial guess values Figure 10 Look closely at the scatter plot of pH vs effective mobility to determine which initial guess values need to be modified to more closely match the location of the inflection points Often one of the initial guess values needs to be modified by as little as 0 5 pH units to enable the software to find a best fit curve After modifying the values in the Initial Guess fields click Start Calculation to find the best fit line Figure 10 Pop up when the software is unable to find a best fit curve 4 3 Determining the Apparent pK Value When ready click on the Start Calculation button to calculate the apparent pKa value s A dialog box will appear to inform the user which model equation has been chosen and to ask if the user would like to choose a different model equation see Figure 11 To choose a different model equation click Yes Clicking yes will open a pop up pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 17 form where the user can
19. pK Estimator Elite User Manual pK Analysis Software for Capillary Electrophoresis CombiSe High Throughput Separations for Today s Laboratory Version 2 0 11 Table of Contents Chapter TMC ON 246 S55 co ccae eas vesev aus cedeectilnc on seupaan qua easy AE A A E LA Eae 1 1 1 Evaluation of pK Values by Capillary ElectrophoresiS oocoonnccccnoncccnonccconancnnnnoss 1 1 2 pK Estimator Elite Software Overview sii AAA badenis 2 Chapter 2 Instalados 4 2 L System Required EEPE SA SEERE ESETE ASET 4 2 2 Running pKa Estimator Elite A A NA AA a A A A AAA 5 23 EXAMS pKa Estimator ELC usas iia setts 5 Chapter 3 Using the Edit Default Values Taba rada 6 3 1 Entering Default V aluesijicccisccecaicssdsasvesesaasstegcaetasaesaatnarsd nnan i 6 32 Saving Default Values 0 ha Me aera tae ee del alee 9 3 3 Lords Default Values dt la latin 10 Chapter 4 Determining the Apparent pKa Value oooooonnococnnccccnoncnononcnononcnononcnnnnnaconnnaconn 12 4 1 Entering the Compound Molecular Welght ooonncccnnncconocicononcccnoncnononcnonnnnccnnnncnnns 14 42 Tnitiar Guess Vas as 15 4 3 Determining the Apparent pK Value cee eeececeenceceeeeeceeeeeceeeeeeeeeeecsteeeenteeeees 16 4 4 Model Equation Numbers ooe AS E aaeyees 19 4 5 Comment Field z postean O 20 4 6 Compound Name EFleld o oooo occccccnnonononanononocnononnnnnnonononnonnnnnno no nnncnnononnananannnos 20 4 User Name ld as 20 Bh RES EEE A AE ET
20. pirical relationships between effective mobility charge and molecular weight of pharmaceuticals by pressure assisted capillary electrophoresis Applications in drug discovery Electrophoresis 2002 23 2833 2841 Jia Z Ramstad T Zhong M Medium throughput pKa screening of pharmaceuticals by pressure assisted capillary electrophoresis Electrophoresis 2001 22 1112 1118 Ishihama Y Nakamura M Miwa T Kajima T Asakawa N A Rapid Method for pKa Determination of Drugs Using Pressure Assisted Capillary Electrophoresis with Photodiode Array Detection in Drug Discovery J Pharm Sci 2002 91 933 942 Wan H Holmen A Nagard M Lindberg W Rapid Screening of pKa values of pharmaceuticals by pressure assisted capillary electrophoresis combined with short end injection J Chromatogr A 2002 979 369 377 Avdeef A In Absorption and Drug Development Edited by John Wiley amp Sons Inc 2003 22 41 pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc
21. rease the total analysis time at the expense of reduced resolution between the analyte and EOF marker pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 28 1 5 Example Results Table 3 lists experimental results for several test compounds achieved using the above method with a comparison to some literature results 2 6 These results can serve as a good measure of CE system and buffer performance when users are attempting to validate their method Table 3 Experimental Results for pK Determination by CE EEE pK Value I 50 mM pK Value I 0 mM Literature Value I 0 mM 4 29 0 12 3 93 4 13 4 14 3 88 2 3 5 6 Quinine 324 5 8 55 0 05 8 27 8 39 8 39 8 43 2 3 5 6 Note All values were converted to I 0 mM for comparative purposes References 2 5 are by CE reference 6 is by potentiometry pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 29 Appendix 2 Ionic Strength Correction Factors To correct for the effects of ionic strength on compound pK values equations 2 and 3 can be applied for acids or bases respectively 1 0 508527 VIT Eqn 2 Acids Ka pKa anes E BIR la dE 0 508522 VT Eqn 3 Bases pKa pKa 1 0 328la 1 In each equation z is the valency of the ion I is the ionic strength of the buffer M and a is the ion size parameter usually assumed to be 5 angstroms Table 4 displays the ionic strength
22. s of the compound being evaluated and the neutral marker compound molecular weight and experimental information regarding capillary lengths and voltage The pKa Estimator Elite program then generates a plot of uef vs pH and using a previously described empirical relationship between pef and molecular weight 2 determines which ionization equation best fits the data A best fit line is then plotted and the apparent pKa value pK is displayed along with a goodness of fit R value Version 2 0 of pKa Estimator Elite provides additional reporting capabilities Comments describing the experiment can be entered and saved with the pK result along with entries for the compound name and analyst name Report generation capabilities have been added allowing the user to generate a printable Excel based report The single page report contains all pertinent experimental information and results and also contains a field for the user to paste in a compound structure image file if desired The Excel format provides a means to export the numerical results to a user defined database and better archive experimental results By utilizing the automated capabilities of pKa Estimator Elite users can save valuable time and resources otherwise required for data analysis of pK values by CE The remainder of the manual provides the user a complete description of the various functions and operations of the pKa Estimator Elite software At the end of the man
23. stimator Elite data The file that is created is a comma delimited file however it is given a pka extension by the software 4 12 Opening a File To open an existing pka file click on the File Open button A standard file open dialog box will appear for selecting the file to open The default directory is C Program Files pKa Estimator Elite data All of the experimental information will be loaded along with the pK results To work with an existing pka file the user does not need to go through the step of entering the default values 4 13 Report Generation Version 2 0 of the pKa Estimator Elite software introduces the capability to create a report of the pK results in a Microsoft Excel format To generate a report press the Report Generation button A standard file save dialog box will appear for specifying the name to save the file to As with the Default Values files the default pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 23 directory is C Program Files pKa Estimator Elite data The file that is created is given an x s extension by the software The Excel report contains all of the experimental information a plot of effective mobility vs pH value and a space at the bottom to paste in an image file of the compound structure if available Figure 17 EJ Microsoft Excel Quinine Report xls Net Previous Zoom Prnt setup Marons Page Break Previn cose Cer
24. to loading a file that contains 24 buffers The 24 Buffers button will be set to True automatically when a file containing 24 buffers is loaded see Figure 4 Note The user must use the Load Values button to open a saved default values file for the information on the Edit Default Values tab to be transferred to the Calculate Apparent pKa tab Alternatively the user can enter this information directly in the Calculate Apparent pK tab without creating a default values file pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc pKa Estimator Elite File About Edit Default Yalues Calculate Apparent pKa 10 25 2004 8 56 AM 4 C Program Files PKa Estimator Elite data 040902 24 pt Aq meas 10 07 Experimental Parameters lot 040902 meas 10 7 04 pH Count Vv 24 3500 Ld cm ie cm 933 00 955 00 24 Buffers Figure 4 Edit Default Values form after loading a file that contains 24 buffers pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 12 Chapter 4 Determining the Apparent pK Value Click on the Calculate Apparent pKa tab to move to that screen The La L and V values from the first tab will automatically be displayed in the appropriate fields on this form see Note in Section 3 3 above Alternatively experimental information can be entered directly into this form without using the Edit Defa
25. ual a generic experimental CE protocol with example results is described for collecting migration time data necessary for pK determination pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc Chapter 2 Installation 2 1 System Requirements This software must be installed on a computer with a Windows operating system to function properly If using an Intel Pentium 4 computer with Hyper Threading Technology the Hyper Threading feature must be disabled for the pKa Estimator Elite software to operate properly The Hyper Threading feature can be disabled by entering the BIOS settings of the computer on start up and selecting Disable for the Hyper Threading option To properly generate Microsoft Excel reports the macro security settings must be set to trust Visual Basic Project To change this setting open Microsoft Excel and navigate to Tools Macro Security Select the Trusted Publishers tab and ensure that the Trust access to Visual Basic Project setting is enabled 1 To install pKa Estimator Elite place the CD in the CD drive Navigate to pKa Es se timator Elite Installer gt setup ex and double click on tup exe 2 Follow the setup instructions provided by the installation wizard The default installation directory is C Program Files pKa Estimator Elite pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 2
26. ult Values tab If data has been previously entered on the Edit Default Values tab the user can modify the La Le and V values on this form Enter in the migration times for the analyte ta and EOF marker tm in seconds for each pH value and then press the Refresh button If the user wants to exclude the migration time data from a particular pH value in the table from the pK calculation click on its corresponding Use light to turn off the light and then press the Refresh button At this point the graph will display a scatter plot of pH vs calculated effective mobility Herr for the data see Figures 5 and 6 pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc pKa Estimator Elite File About Edit Default Yalues Calculate Apparent pKa pH IM Ta s Tm s use 0 050 0 050 0 050 4 43 0 050 5 23 0 050 6 04 0 050 6 84 0 050 7 64 0 050 8 47 0 050 9 27 0 050 10 06 0 050 10 86 0 050 Comment Benzoic Acid Opt 12 pt run 1 10 12 2004 Start Calculation Report Generation 4 C Program Files PKa Estimator Elite data 2004 10 12 Benzoic Acid Run 1 Ld cm 9 39 00 Le cm 3 49 00 Y p 15000 Molecular Weight 9 122 00 Mobility 106 cm 2 s R 2 0 0000 Base 1 1 T Zwitterion 2 0 3 0 4 0 5 0 Compound Name Initial Guess Best Fit Apparent pKa Benzoic Acid 3 4 00 pKal 0 00 User Name pKa J Smith Message done
27. urement by CE It was adapted from several previously published literature protocols 1 3 This method is intended to serve as a Starting point for users gaining experience in using CE for pK determination and can be readily adapted to different instruments capillary lengths and pH buffer systems It is assumed the user has a working knowledge of CE 1 1 CE Instrument Configuration This method was developed using a Beckman MDQ single capillary CE system but is readily translatable to any single capillary CE instrument The instrument configuration is summarized in Table 1 Table 1 Instrument Configuration for pK Measurement by CE pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 26 1 2 Sample Preparation Weigh out 0 1 mg ml of compound into a tared vial Dissolve basic compounds in 1 mM HCI and acidic compounds in 1 mM NaOH and add 0 1 v v DMSO neutral marker Sonicate and filter if necessary for compounds that are not fully dissolved If the sample is presented as a 10 mM stock solution in DMSO dilute to 0 2 mM 20x 2 DMSO sample concentration with 1 mM HCI bases or 1 mM NaOH acids and monitor at 229 nm and or 240 nm 1 3 pH Buffer Preparation Add 1 25 ml each of 12 different pH buffers of equal ionic strength kits available from Microsolv or CombiSep to a series of inlet and outlet vials 2 vials per pH 24 vials total Load the different pH buffer vials into the instrume
28. which model equation to use monobase dibase etc the molecular weight of the compound should be entered into the Molecular Weight field Contributions from counter ions should be subtracted from the total molecular weight if a salt form is being pKa Estimator Elite Software Manual Version 2 0 Copyright 2005 CombiSep Inc 15 analyzed If the user attempts to calculate the pK value without first entering a molecular weight a prompt will appear reminding the user to input a value see Figure 7 Figure 7 Prompt to remind user to enter a molecular weight value 4 2 Initial Guess Values Below the scatter plot is a column of one or more fields labeled Initial Guess These values are used as initial seed values for the non linear regression analysis The use of initial guess values may aid the software in situations where a best fit solution is not easily calculated see below To change the values in the Initial Guess field s before calculation select the number of fields pK values to display by selecting 1 pKa 2 pK or 3 pK in the pK control to the left of the plot see Figure 8 and enter new values in the Initial Guess fields see Figure 9 If a pK calculation has already occurred simply press the Start Calculation button again after changing the initial guess values to re determine the best fit line ETT 2 pKa 3 pKa Figure 8 pK control with 1 pK highlighted pKa Estimator Elite Software Manual Versi
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