1. pKa PRO™ System Overview

2. Disruptive Technologies Product Range and Manufacturers Represented
Biolgical Sample Preparation
Nucleic acides, protein and small molecules extraction from hard-to-lyse tissue samples (Pressure Biosciences)
Dissolution/Formulation
Dissolution baths, friability and disintegration instruments (Distek)
Physico Chemistry
HT Log P and pKa analyzer (AATI)
Kinetic solubility instruments (Analiza)
Rapid microbiology with MicroPRO (AATI)
Service
Agilent Channel Partner to service their range of HPLC, UV spectrophotometers and CE systems
Preparative
OPLC chromatography solutions for semi preparative applications (OPLC systems, pumps, sample applicator, video imaging and densitometry instruments, reagent sprayer (OPLC-NIT)
Flash chromatography (Gyan)
Automated SPE system (HTA)
Analytical
HT oligonucleotides purity analyzer (AATI)
HT proteins analyzer (AATI)
HT DNA analyzer (AATI)
HT Chiral analyzer (AATI)
Spotter for MALDI and tissue MALDI imaging (SunChrom)
Type-C silica hydride HPLC columns Flat sorbent beds for OPLC (MicroSolv)
Accessories and consumables for CE and HPLC (MicroSolv)
Validation kits for HPLC systems (MicroSolv)

3. Outline Background and Importance of Measuring pKa Values
Overview of pKa PRO™ Technology
Measurement of Aqueous pKa Values
Cosolvent pKa Extrapolation of Aqueous Insoluble Compounds
Log P Measurements
Chiral Separations
Summary
Literature References 3

4. pKa Values (Acid Dissociation Constants)‏
Many drugs are either weak acids or weak bases
The pKa value is a measure of the ionization ability of a weak acid or base:
HA  H+ + A- Ka = [H+][A-] / [HA]
pH = -log [H+] pKa = -log Ka
pKa = pH – log ([A-] / [HA])‏
From the above relationship, it is observed that the pKa value = the pH at which 50% ionization has occurred ([A-]/[HA] = 1; log 1 = 0)‏
Ka is an equilibrium constant; the time scale of this equilibrium is much faster than the separation process so the compound appears as a single peak
The pKa is the pH where an acid or base is 50% ionized. It is an equilibrium constant, but the equilibrium is so fast you cannot separate both the neutral and ionized forms. This is why you always see a single compound peak in the analysis. 4

5. Why is pKa Important?
From 80% - 95% of commercial drugs are ionizable by some estimates
The pKa value of a compound strongly influences its solubility, ability to permeate cell membranes, complexation to drug targets, and bioactivity
The pKa value is of fundamental importance in early discovery and development processes for:
Prediction of ADMET (absorption, distribution, metabolism, excretion, toxicity)‏
Assessment of potential challenges in formulation/process development
Prediction of chromatographic/electrophoretic separation behavior
pKa is a core compound property, and strongly influences chemical and biological behavior as a function of pH.
Earlier assessment of drug physicochemical properties (pKa, log P, solubility, permeability) helps to reduce compound attrition rates and shorten development times 5

6. Biologically Relevant pH Range for Pharmaceutical Products12 6 2 4 8 10
stomach
blood
small intestines
vinegar
orange
juice
milk
bleach
cola
pharmaceutical products
colon
urine
When a drug is taken orally, it experiences a variety of pH environments. The biologically relevant pH range is usually considered pH 1 – pH 9. 6

7. How pKa Affects Membrane Permeability
B + H+ B
BH+
The neutral form of a drug permeates cell membranes better and is usually lower in aqueous solubility than the charged/ionized form.
The neutral form (B) of an ionizable drug generally has a higher lipophilicity and membrane permeability as compared to the ionized form (BH+)‏
The neutral form (B) of an ionizable drug is most always of lower aqueous solubility 7

8. Common Issues Encountered During pKa Measurements
Limited amounts of sample available
Traditional potentiometric methods require mg amounts of pure compound
Purity and/or stability of sample has not been precisely evaluated
Traditional methods provide “batch” analysis of entire sample and cannot resolve individual components
Relatively low aqueous solubility
Traditional methods require relatively high sample concentrations, leading to compound precipitation
Number of ionizable groups in pH range of interest unknown
UV spectrophotometric methods are structurally sensitive and may miss pKa values for ionizable groups 2-3 bonds or more from chromophore
This slide highlights some common challenges to measure pKa. With traditional methods, a lot of sample is consumed and compound precipitation is very common resulting the common use of organic solvents for measurement. 8

9. Capillary Electrophoresis (CE) Technology Overview+ -
Bulk Flow: EOF + Vacuum UV N + N -
Time
Charge-based separation by application of high voltage across capillary filled with aqueous-based buffer
Narrow bore, bare fused silica capillaries (75 m i.d., 200 m o.d.)‏
Electroosmotic flow (EOF) provides bulk flow towards cathode (detector) at pH > 4
Application of vacuum provides bulk flow to detector at all pH values
Migration time depends on analyte charge-to-mass ratio; neutral compounds migrate with bulk flow
Many publications dating back >15 years describe single capillary CE for the measurement of compound pKa values
CE is a widely explored and accepted technique for measuring pKa. The charge-based separation is ideal for measuring pKa. 9

10. Measuring pKa by Capillary Electrophoresis (CE)‏
Time + N
Bases
Acids
Time N -
Acid/Base
Time + N -
Low pH
Intermediate pH
The separation profile for the three different types of common compounds are shown here. Bases go from positive to neutral, acids go from neutral to negative, and acid/base ampholytes go from positive to negative as pH increases.
High pH
Neutral marker (DMSO) is added to sample
Plot of migration time difference vs. pH yields titration curve
pKa value corresponds to inflection point of titration curve

11. Key Advantages of CE for Measuring pKa
Often only small quantities (mg) of relatively impure compounds are available in early discovery
CE Approach:
Requires only small amounts of material (g range – only ng consumed)‏
Sample purity not as critical (CE is separation technique)‏
Measurement of migration time (ionic mobility) vs. pH (intuitive)‏
No spectral differences between ionic and neutral species required; only UV absorbance at low UV wavelength
Sparingly soluble compounds can be investigated with aqueous buffers
Knowledge of sample concentration not required
This highlights significant advantages of CE… however, conventional single capillary CE does not have the throughput or user friendliness needed to compete. The pKa PRO system does!
However, conventional single capillary instruments can only analyze a few samples/day and do not have software for pKa data analysis 11

12. pKa PRO™ System
A dedicated 24 or 96-channel CE system developed for performing rapid pKa measurements
Simple user interface and predefined CE methods for ease-of-use and streamlined operation
Advanced, fully integrated data analysis software for pKa calculation and report generation
Designed with feedback from scientists directly involved in pharmaceutical research and pKa analysis
The pKa PRO provides a rapid, dedicated means to measure pKa values using CE technology. 12

13. Principles of pKa PRO™ Operation
Advanced Analytical’s patented, parallel CE technology. 24 or 96 capillaries are imaged with UV light onto a linear photodiode array detector. This technology provides CE separation with 24 – 96 times the sample throughput of conventional single capillary systems!
24 or 96 capillaries are arranged in a linear array at detection window
UV light is passed through capillary array and imaged onto photodiode array detector
Capillary inlets are arranged 8 x 12 for direct sample injection from 96-well micro plates
Capillary outlets are bundled and connected to a syringe pump for buffer filling
Different pH buffers are injected into capillary array prior to CE separation
24 or 96 individual CE-UV separations are performed in parallel
Four samples can be analyzed over 24 pH values in a single experiment 13

14. 96-Capillary Array Viewed from Detector Position
This is a 96-capillary array with the covers removed. The brown coloring on the capillaries is from polyimide, applied to the capillary to provide increased strength and flexibility. The polyimide must be removed at the detection window to pass UV light.
Capillary Outlets
(12 Bundles of 8 Capillaries)‏
Capillary Inlets
(Arranged in 8 x 12 Format)‏
Detection Window
(Polyimide coating removed)‏

15. Inside View of the pKa PRO™ Instrument
Capillary Array Cartridge
Lamp
Housing
HV Power Supply
Syringe
Pump
Optical Platform Housing
Capillary Array Detection Window
This is inside view of the cePRO system, showing the major components. They include a high voltage power supply; syringe pump and distribution valve fluid handing system; UV light source; optical detection platform; and capillary array assembly.

16. pKa PRO™ System Specifications
Sample Throughput: pKa PRO™ 96XT
12 compounds/h for aqueous 24-point pKa measurement
pKa PRO™ 24HT
3 compounds/h for aqueous 24-point pKa measurement
Detection: UV absorbance at 214 nm; other wavelengths available
Detection Sensitivity: 5 g/ml (ppm) depending on chromophore; working concentration 50 g/ml
Sample Required: Working volume 50 l/well; 24 wells per 24 pH analysis (< 100 g)‏
Sample Format: DMSO concentration < 0.2% (v/v); higher DMSO concentrations tolerated at higher wavelength
pKa Measurement Range: 1.8 – 11.2
Software: Proprietary pKa PRO™ software for system control/data analysis
Data Export Format: Microsoft® Excel spreadsheet
Environmental Conditions: Indoor use, normal laboratory environment; lab temperature 15–25º C
Relative Humidity Range: < 80% (non-condensing)
Electrical: 100–200 VAC; Hz (200–230 VAC; 50–60 Hz available); 15 A
Instrument Dimensions: Fully configured requires 96” W x 30” D x 39” H
Instrument Weight: 195 lbs. (88.6 kg)‏
General specifications of the pKa PRO. Note typical sample amount required is less than 100 ug, and that DMSO should be removed and re-added to a low % to maximize sensitivity. 16

17. pKa PRO™: Some Equations for pKa Measurement
Effective Mobility (Meff)‏ Z
MWX
Meff = m
Ltot  Leff V
(1/ta – 1/tm)‏
Meff =
MW = Molecular weight
Z = Compound charge
m, x = Determined empirically by experiment
Charge is Directly Related to Meff!
Charge (# pKa) predicted from Meff and MW
Ltot = Total length of capillary
Leff = Length to detector
V = Applied voltage
ta = Migration time of analyte
tm = Migration time of neutral marker (DMSO)‏
The important points are that mobility is directly related to the difference in migration time between the compound and neutral marker. Compound charge is directly related to mobility. Also, you can predict the number of pKa values from MW and mobility.
Relationships between Meff, pH, and Apparent pKa
Meff =
Mb10-pH
10-pKa + 10-pH
Monobase:
Ma10-pKa
Monoacid:
Ma, Mb = Meff of completely ionized species
Non-linear regression of Meff vs pH plot is performed with appropriate equation to yield pKa
Equations in: J. M. Miller et al. Electrophoresis, 2002, 23, 17

18. Sample and Buffer Tray Configuration for pKa Analysis
12 pH Point pKa Analysis (8 Samples)
Analyte
Compound 1
Compound 2
Compound 3
Compound 4
Sample Tray
Compound 5
Compound 6
Compound 7
Compound 8
This shows how the sample and pH buffer plates should be configured for a 12 pH point pKa experiment.
The marked well (E7) corresponds to compound 5 analyzed at pH 6.80
Inlet Buffer Tray
2.10
2.91
3.40
4.40
5.20
6.00
6.84
7.60
8.40
9.20
10.00
10.83 pH 18

19. Experimental User Interface Screen
This is one example of how the pKa PRO is designed with user in mind. This interface allows quick loading of sample information needed to calculate the final pKa value and generate a complete results report.
User selects experimental mode (12 or 24 point aqueous, 12 or 24 point co-solvent)
Compound names, molecular weights and predicted pKa values (if available) are entered
Buffer pH information file is loaded
Information is saved for pKa calculation and report generation 19

20. Results for 4-Aminopyridine (monobase)
As pH increases going from the top left to bottom right, the 4-aminopyridine becomes neutral and co-migrates with the neutral DMSO marker.
4-Aminopyridine (red cursor) is a basic compound; therefore it migrates before the DMSO neutral marker (black cursor)‏
Software automatically selects two highest peaks above threshold 20

21. Results for 4-Aminopyridine (monobase)
pKa
The software predicts the proper equation to use (monobase, dibase, etc) based on the predicted charge (in this case = monobase). pKa value is halfway point equal to 50% ionization along titration curve (inflection point).
Mobility vs. pH plot yields titration curve; inflection point = pKa value (9.23)‏
Software automatically predicts compound charge from MW and Meff
Charge(4-AP) = = monobase. 21

22. Results for Benzoic Acid (monoacid)
As pH increases going from the top left to bottom right, the benzoic acid becomes negative and migrates after the neutral DMSO marker.
Benzoic Acid (red cursor) is an acidic compound; therefore it migrates after the DMSO neutral marker (black cursor)‏ 22

23. Results for Benzoic Acid (monoacid)
pKa
Predicted charge in this case is -1.07, corresponding to a monoacid.
pKa value: 4.06
Charge (benzoic acid) = = monoacid 23

24. Sample and Buffer Tray Configuration for pKa Analysis
24 pH Point pKa Analysis (4 Samples)
Acyclovir
4-Aminopyridine
Cefadroxil
Quinine
Analyte
Sample Tray
This shows how the sample and pH buffer trays are arranged to perform 24 point pKa analysis on four samples.
Inlet Buffer Tray 24

25. 24 Point Results for Cefadroxil (diacid/monobase zwitterion)
This shows CE traces for 24 pH point analysis of cefadroxil. Note at low pH, cefadroxil is positive, at pH 4 – 6 it is neutral, and at pH above 6.4 it is negative.
At low pH, cefadroxil migrates before DMSO neutral marker
At high pH, cefadroxil migrates after DMS neutral marker

26. 24 Point Results for Cefadroxil (diacid/monobase zwitterion)
24 Point buffer series increases resolution and expands measurable pKa range
Note the
pKa values: 2.56,7.24,9.67
pI value: 4.90
Charge (cefadroxil) = +0.85; = monobase/diacid 26

27. Exported Excel Report for Cefadroxil
The Excel report contains:
Compound Name
Date of measurement
Analyst information
Assay type
User comments
Measured pKa value(s)‏
Measured pI value (if applicable)‏
Titration curve
R2 value (goodness-of-fit)‏
M.W.
Predicted charge
Buffer information
Structural image file can be inserted if available
Electropherogram traces (separate tab)‏
Software generates a very comprehensive report for notebook or electronic storage. 27

28. pKa Results Data Table
Data table provides quick access to previously determined results.
Each saved pKa result is entered into sortable indexed data table 28

29. Results Obtained with the pKa PRO™
Compound MW
Type n pK a
PRO ™
' (I = 50 mM)‏ SD
Literature Values
Acebutolol
336 B 15
9.51
0.09
Acyclovir
225
A/B 13
2.19
0.03
9.20
0.01
4-Aminopyridine 94 8
9.22
Benzoic Acid
122 A
4.07
Betahistine
136 2B 11
3.88
0.02
9.97
0.04
Cefadroxil
363
2A/B
2.57
7.21
9.70
0.05
9.89
Cefuroxime
423 2A 9
2.12
2.04
11.19
0.15 NR
Clomipramine
315
9.56
Furosemide
331 24
3.61
10.39
Imipramine
280 5
9.60
Indomethacin
358
4.02
0.08
Piroxicam
1.87
5.35
0.06
Procaine
300 16
2.13
9.06
Quinine
324 18
4.33
8.50
Tyrosine
181 10
2.23
8.85
10.05
0.07
Results obtained with pKa PRO correlate very well with literature data obtained using traditional methods.
Literature pKa values were reported at ionic strengths from 0 – 150 mM
To date, the pKa values for >100 compounds have been measured
Average SD ± 0.06 units; typical agreement to literature ± 0.2 units or better 29

30. pKa Analysis of Tyrosine (monobase/diacid zwitterion)‏*
NOTE: the pKa PRO correctly determined that there were 3 pKa values present from compound charge and can measure all three. The lower pKa (2.21, -COOH) cannot be measured using UV spectroscopy because this functional group is too far away from the benzene ring which absorbs UV light.
pKa Values: 2.21, 8.79, 10.08
pI Value: 5.54
Charge: +0.71, = monobase/diacid
–COOH pKa value not observed by UV spectrophotometry 30

31. pKa Analysis of Procaine + Impurity
A 4-aminobenzoic acid hydrolysis impurity (20%) of procaine was present
The pKa values for both species were determined in the same experiment
4-ABA pKa’ Values:
2.37, 4.38
Procaine pKa’ Values:
2.20, 9.04 +
pH Value
Effective Mobility (x 106 cm2/V•s)‏
Procaine
4-ABA
pH 1.78 (Top Left) – pH 6.46 (Bottom Right)‏
pH 6.82 (Top Left) – pH (Bottom Right)‏ *
The pKa PRO could measure BOTH the target compound procaine AND its degradation impurity! CE separates the impurity out so it does not interfere in the measurement!!! * * 31

32. pKa Analysis of a Peptide: Asp-Phe
H-Asp-Phe-OH
pKa Values: 2.13; 3.71; 7.95
pI Value: 2.96
The pKa PRO is uniquely suited for pKa AND pI determination, because the CE separation can identify where the compound is overall neutral (pI value). Traditional methods can only identify a pKa value but cannot tell what the overall charge state of the molecule is.
Charge-based measurement provides indication of isoelectric point as well as pKa values 32

33. pKa Analysis of an Insoluble Compound (Clomipramine)
Analyzed at 100 ppm (100 g/ml)‏
Precipitation from solution at pH
Analyzed at 10 ppm (10 g/ml)‏
No ppt. observed, pKa’ = 9.54  0.05 (n = 8)‏
ppt
MW: 314.9
Calculated log P: 5.53 ± 0.51
Measured log P: 5.19
Calculated solubility at pH 10.0: 16 g/ml
Values obtained from ACD I-Lab V. 7
This slide shows what precipitation of an insoluble compound looks like. The peak broadens due to compound crashing from solution. In this case, the sample concentration can be diluted further by 10X and successfully measured with aqueous buffers!
Low solubility compounds can often be analyzed at lower concentration without the use of cosolvent 33

34. Cosolvent pKa Extrapolation of Insoluble Compounds
Method:
pH values of methanol containing buffers were measured using aqueous standards ( pH) and converted to pH values as previously described*
The pKa’ values are determined for compounds using 30%, 40%, 50% and 60% (v/v) methanol-containing buffers
pKa’ values are plotted as a function of solution dielectric constant ( ) and extrapolated to 0% cosolvent to yield the pKa’ value (Yasuda-Shedlovsky Method)‏
Four compounds can be run in parallel over 24 pH values or eight compounds can be analyzed over 12 pH values (2 - 4 compounds/h) w s
For very low solubility compounds, the pKa PRO can measure pKa with methanol co-solvent buffers. The pKa is measured at several decreasing concentrations of methanol, and extrapolated to 0% methanol to yield the aqueous pKa value (Yasuda-Shedlovsky method).
* Roses, M.; Bosch, E. J. Chromatogr., A 2002, 982, 1-30. 34

35. pKa Analysis of an Aqueous Insoluble Compound
ppt
pH 7.20
pH 6.80
Tamoxifen
MW: 371.5
Calculated log P: 7.88 ± 0.75
Calculated solubility: 0.05 g/ml
Measured solubility: 0.01 g/ml
Calculated values from ACD I-Lab V. 7
Measured value from Avdeef (2003)‏
Tamoxifen has very low solubility. When analyzed at the detection limit, it still precipitates. Cosolvent is required in this case.
24-Pt aqueous pKa Analysis at 30 ppm (30 g/ml)‏
Precipitation from solution at pH 6.8 – 7.2
Sample dilution to detection limit = ppt 35

36. pKa Analysis of Tamoxifen in 30% (v/v) Methanol
When analyzed in 30% (v/v) methanol buffers, tamoxifen stays in solution and its pKa can be measured!
Tamoxifen stays in solution when analyzed at ~20 g/ml in 30% (v/v) cosolvent buffers 36

37. Yasuda-Shedlovsky Extrapolated pKa’ Value for Tamoxifen
This shows final results for tamoxifen after analysis with 30%-60% methanol cosolvent buffers and extrapolation. The pKa PRO software enables easy determination of the final pKa value.
Extrapolated pKa’ value (I = 50 mM):8.53 ± 0.07 (n = 9)‏
Literature pKa’ value (I = 150 mM): 8.58 (Avdeef, 2003)‏
Software performs entire analysis with minimal input 37

38. Cosolvent pKa Results for Test Compounds
Results for insoluble compounds agree very well with literature data using traditional methods. But, pKa PRO can measure using a fraction of compound and much less time!
Compounds marked (*) required cosolvent; other compounds could be analyzed with aqueous buffers (^ tamoxifen, terfenadine analyzed from 40%-60% CS; # amiodarone analyzed at 50%-60% CS)
Overall, extrapolated pKa’ values agree well with available literature values
pKa PRO™ requires much less sample (<100 g) than potentiometry (mg)‏
pKa PRO™ analysis time much faster than potentiometry 38

39. pKa Paper in Collaboration with Pfizer
An exhaustive study was performed over several years with two different generations of technology to validate the multiplexed CE method for pKa analysis
Excellent correlation found between pKa values measured with multiplexed CE-UV and available literature values, using both aqueous and cosolvent methods
Shalaeva M, Kenseth J, Lombardo F, Bastin A Journal of Pharmaceutical Sciences, Accepted for Publication.

40. Correlation of Multiplexed CE-UV pKa Values to Literature
Over 150 pKa values for 100+ compounds were measured in this study over a wide pKa range from < 2 to > 11. Excellent correlation to average literature values was obtained.
98 compounds (>150 pKa values) measured by aqueous buffers compared to average literature values
23 compounds (26 pKa values) measured by co-solvent buffers compared to average literature values

41. pKa Measurement Pre-made Buffer Plates
Pre-made plates offer reduced labor, ease of use and reduce potential for errors.
Pre-made buffer trays provide savings in customer labor and time, reduce error
Aqueous and cosolvent buffer plates now commercially available 41

42. pKa Summary
The pKa PRO™ system provides a rapid approach for pKa measurements of drug compounds
Reproducible pKa results in good agreement to literature values can be obtained over a wide range of pH values (1.8 – 11.2)‏
Impurities, degradants or UV absorbing counterions can be successfully resolved from the target compound
pKa values undetectable by UV spectrophotometry can be successfully measured
Charge-based separation provides clear, intuitive indication of overall charge state and compound isoelectric point
Compound charge can be predicted, allowing for assessment of number of ionizable groups and detection of closely spaced pKa values
Insoluble compounds can be analyzed for pKa using methanol cosolvent buffers and linear extrapolation to 0% cosolvent 42

43. Log P Measurements on the pKa PRO™ System

44. Octanol-Water Partition Coefficients (log P Values)‏
log P is a measure of how well the neutral, unionized form of a drug partitions between a lipid phase (e.g., n-octanol) and water
P is defined as the partition coefficient:
P = Co / Cw
If log P = 5, Co / Cw = 100,000:1 at equilibrium!
where Co and Cw are the equilibrium drug concentrations measured in the n-octanol and water phases, respectively
Traditional method for determining log P is the shake flask method; HPLC also widely used
Log P is a commonly measured property for estimating how well a compound can cross a cell membrane.
n-octanol
water 44

45. Log P Analysis of Neutral/Basic Compounds
Multiplexed, microemulsion electrokinetic chromatography (MEEKC) was employed for indirect log Pow evaluation.
Microemulsion Buffer: 8.0% (w/v) 1-butanol, 1.2% (w/v) n-heptane, 2.0% (w/v) sodium dodecyl sulfate, with phosphate/borate buffer (pH 10.0). Validated to correspond to octanol-water shake flask
MEEKC is similar to MEKC. There is a moving “oily” phase which migrates against the EOF. Hydrophobic compounds spend more time in the oily phase, so they reach the detector later than more hydrophilic compounds.
MEEKC is based on the partitioning of analyte between an aqueous phase and an immiscible microemulsion (ME) phase comprised of oil droplets + surfactant
More lipophilic compounds favor the ME phase and migrate slower
Order of migration: DMSO (EOF marker), Analyte, Dodecylbenzene (ME marker)‏
Poole, S. K.; Durham, D.; Kibbey C. J. Chromatogr. B 2000, 745,
Figure adapted from (Author Kevin Altria)‏ 45

46. Experimental Design for Log Pow Measurement
A standard mixture of compounds with known log Pow values is used to calibrate the system
The standard mixture and other test solutes are dissolved in microemulsion buffer containing DMSO (EOF marker) and dodecylbenzene (microemulsion marker).
Capacity factors (log k’ values) are calculated for standards and sample using Equation 1:
(1)‏
where ts, teof, and tme are the migration times of the solute, EOF marker (DMSO), and microemulsion marker (dodecylbenzene), respectively.
log k’ values for the standard compounds are plotted vs. literature log P values to calibrate the system via Equation 2:
log POW = A  log k’ + B (2)‏
where A is the slope and B is the y-intercept.
Sample log P is calculated by entering experimental log k’ value into Equation 2.
The user measures the migration times of the standard mixture to calibrate the system. Then, using the migration time of sample, calculates the log P from the calibration equation 2. 46

47. 96-Capillary MEEKC Measurement of Log P
All results are obtained in parallel simultaneously!!
Migration Order: DMSO, Solute, Dodecylbenzene
96 samples analyzed simultaneously 47

48. Separation of Log P Standard Mixture
DMSO
(EOF Marker)‏
Dodecylbenzene
(ME Marker)‏ 1 2 3 4 5 6
The standards have a range of log P values, with the higher log P value samples migrating closer to the ME marker.
Standards: 1. Pyrazine, 2. Benzamide, 3. Nicotine, 4. Quinoline, 5. Naphthalene, 6. Imipramine
MMEEKC has also been employed as a generic purity screening approach 48

49. Typical Standard Log P Calibration Plot
Calibration curve is used to determine sample log P from measured log k’ value.
Averaged (n = 4) log k’ values for the six standards were used to construct the calibration plot 49

50. Log P Calculator Software
The log P calculator software calibrates the separation results and determines log P for each sample.
Advanced data analysis software calculates log P and tabulates results

51. Long Term (> 8 months) Reproducibility of Log P Values
2-aminopyridine 34
-0.41 ± 0.01
2.44
0.41 ± 0.01
0.49
-0.08
aniline 36
-0.12 ± 0.02
16.67
0.90 ± 0.02
2.22
0.9
benzamide 50
-0.17 ± 0.02
11.76
0.81 ± 0.02
2.47
0.64
0.17
4-chloroaniline
0.62 ± 0.03
4.84
2.16 ± 0.04
1.85
1.88
0.28
chlorpromazine 7
2.21 ± 0.04
1.81
4.74 ± 0.06
1.27
5.19
-0.61
coumarin 26
0.22 ± 0.02
9.09
1.48 ± 0.05
3.38
1.39
0.09
3,5-dimethylaniline 15
0.57 ± 0.03
5.26
2.04 ± 0.05
2.45
2.17
-0.13
ethylbenzoate 38
0.97 ± 0.04
4.12
2.75 ± 0.04
1.45
2.64
0.11
hydroquinine 42
1.26 ± 0.06
4.76
3.23 ± 0.10
3.10
3.43
-0.2
imipramine 52
1.86 ± 0.08
4.30
4.23 ± 0.08
1.89
4.42
-0.19
indazole 46
0.38 ± 0.03
7.89
1.75 ± 0.08
4.57
1.77
-0.02
lidocaine
0.89 ± 0.04
4.49
2.62 ± 0.03
1.15
2.26
0.36
3,5-lutidine 14
0.42 ± 0.02
1.77 ± 0.03
1.69
1.78
-0.01
naphthalene 53
1.36 ± 0.07
5.15
3.40 ± 0.09
2.65
3.3
0.1
nefopam 32
1.14 ± 0.05
4.39
3.04 ± 0.04
1.32
3.05
nicotine
0.18 ± 0.02
11.11
1.40 ± 0.02
1.43
1.17
0.23
nitrobenzene 35
0.40 ± 0.02
5.00
1.79 ± 0.04
2.23
-0.06
phenanthrene 13
1.92 ± 0.06
3.13
4.29 ± 0.11
2.56
4.46
-0.17
MMEEKC log k'
MMEEKC log P ow
Solute n
avg. ± SD
%RSD
Lit. log P OW 
log
acebutolol
0.41 ± 0.03
7.32
1.80 ± 0.04
1.71
pyrazine
-0.96 ± 0.01
1.04
-0.51 ± 0.03
5.88
-0.26
-0.25
pyrene 8
2.21 ± 0.23
10.41
4.75 ± 0.38
8.00
4.88
pyrilamine
1.18 ± 0.06
5.08
3.11 ± 0.05
1.61
3.27
-0.16
pyrimidine
-1.05 ± 0.02
1.90
-0.67 ± 0.03
4.48
-0.4
-0.3
quinoline
0.54 ± 0.03
5.56
2.00 ± 0.04
2.00
2.03
-0.03
tetracaine
1.42 ± 0.07
4.93
3.52 ± 0.10
2.84
3.73
-0.21
Results are reproducible and in good agreement to other methods, which consume more sample and take longer.
Good reproducibility and agreement to better than 0.5 log units to literature data 51

52. Sample Throughput for Indirect Log P Methods
7 – pKa PRO™
46*
1.25
MMEEKC 4 5 6
2-3
100 (per week)‏ 2
18-23 - 30
MEEKC 3 15
MEKC
1,2 20
RP-HPLC
Reference
Approximate Throughput (samples/h)‏
Average Analysis Time per Sample (min)‏
Method
The pKa PRO can provide greatly improved throughput for log P analysis over traditional methods!
Lombardo F.; Shalaeva M.Y.; Tupper K.A.; Gao F.; Abraham M.H. J Med Chem 2000, 43,
Lombardo F.; Shalaeva M.Y.; Tupper K.A.; Gao F. J Med Chem 2001, 44,
Smith J.T.; Vinjamoori D.V. J Chromatogr B 1995, 669,
Mrestani Y.; Neubert R.H.H.; Krause A. Pharm Res 1998, 15,
Kibbey C.E.; Poole S.K.; Robinson B.; Jackson J.D.; Durham D. J Pharm Sci 2001, 90,
Jia Z.; Mei L.; Lin F.; Huang S.; Killion R.B. J Chromatogr A 2003, 1007,
Wong, K-S; Kenseth J.R.; Strasburg, R.S. J Pharm Sci 2004, 93,
* 4 of 96 capillaries are used for the standard mixture 52

53. Chiral Separations on the pKa PRO™ System

54. Chiral Separations with the pKa PRO™ System
The different enantiomeric forms of chiral drugs can often possess dramatically different potency or toxicity
CE is an attractive technique for separating the different +/- enantiomers of chiral molecules:
Minimal sample and reagent consumption
Different chiral resolving agents can simply be added to the run buffer to optimize the separation; no separate columns required as in HPLC
The pKa PRO™, equipped with the thermoelectric cooling option, can perform chiral CE separations in parallel with dramatically improved throughput
Useful for:
Identifying best chiral selector/condition for achieving best resolution
Screening chiral reactions for enantiomer excess (EE)

55. Chiral Selector Screening Results for p-Chloroamphetamine
S--CD
HS--CD
HS--CD
HS--CD
PTS Internal Standard
In this experiment, several different chiral additives were screened for the same compound. The HS-gamma-CD provided the best resolution of the two enantiomer peaks.
All samples contained pyrenetetrasulfonate (PTS) internal standard (peak #1)‏
Migration time could be reduced by use of vacuum assisted CE 55

56. 96-Capillary Chiral CE: Mixture of (+/-) Isoproterenol
PTS Normalized Migration Time
(+) Isoproterenol: 0.52% (n = 96)‏
(-) Isoproterenol: 0.72% (n = 96)‏
(+)/(-) Normalized Peak Area
0.952 ± (RSD = 2.68%)‏
96 samples analyzed in
< 25 min
This demonstrates that the pKa PRO can analyze up to 96 chiral samples in a single run with good reproducibility. 56

57. Measurement of Enantiomeric Excess
( + )‏
PTS
( - )‏
The system can be used to monitor amounts of excess enantiomer for different chiral reaction products.
Sample: 1000 ppm (+) isoproterenol
BGE: 5% sulfated--CD (Aldrich) in 25 mM H3PO4/TEA pH 2.5
Contains a minor (-) isoproterenol enantiomer impurity
Normalized corrected peak area of (-) impurity: ± (RSD = 6.30%; n = 24)‏ 57

58. Other Applications on the pKa PRO™ System

59. Other Potential Applications
In addition to :
pKa
Log P
Chiral separation
Protein purity and size (Protein PRO)
The pKa PRO can be used for the determination of :
Log D
Impurity profile
Drug binding to plasma proteins
Etc ...
Any CE separation can be transferred to the pKa PRO platform to accelerate throughput

60. Literature References
Reviews Describing pKa and log P Measurement by CE
Weinberger R: Determination of the pKa of Small Molecules by Capillary Electrophoresis. American Laboratory 2005, August:36-38.
Jia Z: Physicochemical Profiling by Capillary Electrophoresis. Curr. Pharm. Anal. 2005, 1:41-56.
Poole SK, Patel S, Dehring K, Workman H, Poole CF: Determination of acid dissociation constants by capillary electrophoresis. J. Chromatogr. A 2004, 1037:
Poole SK, Poole CF: Separation methods for estimating octanol-water partition coefficients. J. Chromatogr. B 2003, 797:3-19.
Papers Describing pKa PRO™ Core Technology
Zhou C, Jin Y, Kenseth JR, Stella M, Wehmeyer KR, Heineman WR: Rapid pKa Estimation Using Vacuum-Assisted Multiplexed Capillary Electrophoresis (VAMCE) with Ultraviolet Detection. J. Pharm. Sci. 2005, 94:
Pang H, Kenseth J, Coldiron S: High-throughput multiplexed capillary electrophoresis in drug discovery. Drug Discovery Today 2004, 9: 60

61. Key Benefits and Summary
Parallel CE-UV technology can be applied to a broad range of high throughput applications spanning pharmaceutical and biotechnology markets
Parallel CE provides many benefits:
Significantly increased sample throughput
Improved laboratory efficiency
Lower turnaround times
Decreased reagent and sample consumption
Reduction in labor and operational/maintenance costs
Many methods previously developed for single capillary CE instruments can be successfully transferred to a parallel format
The parallel CE configuration format provides an open, flexible format to vary capillary length, i.d., # capillaries and/or separation conditions to adjust resolution or accommodate different applications as needed

62. THANK YOU