1. What is the pKa Determinations Software used for?



2. How does the system work?



3. Can I use this kit with my current CE Instrument? Even if it only holds one capillary?



4. Who is the manufacturer of the software and the Buffers? Who does the technical support when I am installing the software and using the kit?



5. What is the pH range of the pre-made buffers?



6. Does the software come with installation instructions?



7. While running according to the instructions, I noted the currents were a little high at low pH. Would this be remedied by using a 50 um capillary?



8. What do you enter into the software program if the solute molecular weight is unknown?



9. Do you have any guidelines for selecting the appropriate equation for an unknown compound?



10. You recommend variable pressure for different pHs, what is the reason for this?



11. Please clarify the ionic strength correction. Do you just plug the numbers into the equations on page 29 to generate the data in Table 4? Is the pKa data produced by the software already corrected for ionic strength effects?



12. What are the buffers in the kits? What are their concentrations?



13. Can I get a demo version of the pKa Determination Software to evaluate?



14. What is the pH value and components of each of the buffers for pka?



15. Do you control the ion strength in different pHs? I got different currents when running pHs and I am not sure if it affects the result?



16. 2 mL of H20 in the vial was used to rinse the capillary after the capillary was conditioned by 0.1N NaOH. Does the H20 become very basic after a lot of injections and the capillary would carry over the basic H20 to the running buffer and affect the running buffer pH, especially at low pH? Is 0.1N NaOH required between each injection?

1. What is the pKa Determinations Software used for? [top]
The software and buffers for the determination and estimation of pKa by CE is a computer program that can be run under Windows® 98, ME, XP or 2000 environments. This software is adapted to manually accept migration time information from CE instrumentation that incorporates single capillaries such the Beckman P/ACE 5000™, Beckman MDQ™, Beckman PA800™, Agilent 3DCE™ and others (multi capillaries not required). Pre-made CE buffers are available that make the analysis and determination easy and reliable.

2. How does the system work? [top]
Using the pre-made buffers, run your analytes in a variety of different buffers (different pH) on your current CE instrument using a new bare fused silica capillary. Take the migration times of your analytes and the EOF as measured by your instrument for each pH. Enter the data into the software program which runs on your PC. Calculate the pKa. The software uses sophisticated algorithms and estimates the pKa automatically.

3. Can I use this kit with my current CE Instrument? Even if it only has one capillary? [top]
Yes, the software and buffers can be used with any CE instrument. Simply record EOF migration time and your analytes migration times in different buffers. The software does the rest.

4. Who is the manufacturer of the software and the Buffers? Who does the technical support when I am installing the software and using the kit? [top]
CombiSep, Inc. is the manufacturer of the software and the buffers. They are packaged and sold exclusively by MicroSolv and supported by MicroSolv and CombiSep. Contact Technical Service for assistance.

5. What is the pH range of the pre-made buffers? [top]
The pre-made buffers range from pH 2.1 to 10.8 for the 12 point kit or pH 1.77 to 11.31 for the 24 point kit.

6. Does the software come with installation instructions? [top]
Yes, the software has complete installation instructions, troubleshooting tips and operating instructions.

7. While running according to the instructions, I noted the currents were a little high at low pH. Would this be remedied by using a 50 um capillary? [top]
Yes, for the low pH phosphate buffers the currents are quite high under the operating conditions (15 kV, 300 V/cm). However, we prefer the 75 um i.d. capillaries for the pKa application for a couple of reasons. One, they are easier to work with and are less prone to plugging. Most importantly, we need to maximize the detection sensitivity as much as we can in order to analyze low solubility compounds. The lower concentration we can detect, the more dilute we can prepare the low solubility compounds and analyze them without precipitation in the capillary. This is a very important issue for many labs working with low solubility, high log P compounds.

8. What do you enter into the software program if the solute molecular weight is unknown? [top]
Usually, the operator would have knowledge of the compound molecular weight so it could be entered. However, if you do a ballpark guess within 50 daltons or so this should suffice and allow the software to estimate the compound charge.

9. Do you have any guidelines for selecting the appropriate equation for an unknown compound? [top]
If you prepare the compound at a low concentration (100 ppm or less) and the DMSO concentration is 0.1% will help in this process. If you correctly assign the peaks and plot the data, the software will make a best guess what the limiting charge on the compound is from the highest mobility and MW. This is why it is important to enter an accurate MW for the compound. The basis for this calculation is fully described in the paper by Miller et al from Pfizer. They did measuree the electrophoretic mobility for 80 or so drug compounds of widely varying molecular weights, and empirically derived a relationship between MW, mobility, and charge for acids and bases. This relationship is written into the software and works quite well. Usually, for a monoacid or monobase assuming the pKa falls in the pH range between 2 and 10.5, the software will predict a model equation number or "charge" between 0.9 - 1.2. For diacids and dibases, it is usually 1.5 - 2.1. So, most of the time, the software chooses the correct equation. Sometimes, when a second pKa value is close to the extreme ends of the pH range (for example, a dibase with pKa1 of 2.0 and pKa2 of 8.5), the model equation number will guess below 1.5, but it is usually obvious there is a second pKa present.

10. You recommend variable pressure for different pHs, what is the reason for this? [top]
The variable pressure for different pH is mostly a convenience factor to try and push down the total time to analyze a compound over 12 or more pH values. Setting up the different pressures in a single capillary system is pretty straightforward. Also, having the DMSO come out at approximately the same time sometimes helps to discriminate between compound and DMSO. However, in the end it does not affect the results and the different methods (with and without variable pressure) generally yield the same pKa results.

11. Please clarify the ionic strength correction. Do you just plug the numbers into the equations on page 29 to generate the data in Table 4? Is the pKa data produced by the software already corrected for ionic strength effects? [top]
The pKa data produced by the software is NOT corrected for ionic strength, and is labeled as an apparent pKa value (I = 50 mM). We used to have a toggle function to show the apparent or corrected pKa values, but some users simply wanted the apparent pKa values. To make things simpler, we set up the software to just give the apparent pKa value and let the user make the corrections themselves if they desire.

This is a confusing issue - a lot of times papers in literature do not fully elucidate whether the pKa values are apparent or thermodynamic, and many authors simply state the apparent pKa values. It also makes it a little more challenging to compare data between studies, although the differences in the correction are pretty minor for mono- and diprotic compounds (e.g., the correction is only 0.04 or 0.11 between 50 mM and 150 mM for monoprotic and diprotic compounds, respectively). This difference is easily on the same order as the scatter in the literature between methods already.

The data in Table 4 is generated from the equations on page 29, but for diprotic and triprotic compounds, you have to subtract the first correction from the second. This is because the ionic strength terms for the two different species are split between the numerator and denominator when you write out the equilibrium equations. If you write out the equilibria and break out the ionic strength from the concentration terms, you can see how it works.

12. What are the buffers in the kits? What are their concentrations? [top]
The buffers are designed specifically for pKa determinations. For a listing of the buffers, please click here. All buffer concentrations are adjusted to have a consistent and standard ionic strength of 50mM.

13. Can I get a demo version of the pKa Determination Software to evaluate? [top]
Yes, we offer a 15 day free trial version of the software. If you would like a copy, please click here to complete a request form.

14. What is the pH value and components of each of the buffers for pka? [top]
All the buffers are made to a common Ionic strength of 50mM and the pH and components are listed below.

Buffer	pH Value	Components
1	1.75	Phosphoric Acid, NaCl
2	2.10	Phosphoric Acid, NaCl
3	2.50	Phosphoric Acid, NaCl
4	2.90	Formic Acid, NaCl
5	3.40	Formic Acid, NaCl
6	3.90	Formic Acid, NaCl
7	4.40	Acetic Acid, NaCl
8	4.80	Acetic Acid
9	5.20	Acetic Acid
10	5.60	Acetic Acid
11	6.00	Sodium Phosphate, NaCl
12	6.40	Sodium Phosphate, NaCl
13	6.80	Sodium Phosphate, NaCl
14	7.20	Sodium Phosphate, NaCl
15	7.60	Sodium Phosphate, NaCl
16	8.00	Boric Acid, NaCl
17	8.40	Boric Acid, NaCl
18	8.80	Boric Acid, NaCl
19	9.20	Boric Acid, NaCl
20	9.60	Boric Acid, NaCl
21	10.00	Boric Acid, NaCl
22	10.40	Boric Acid
23	10.80	Boric Acid
24	11.20	Boric Acid

Note: the ionic strength of all buffers is leveled at I = 50 mM

15. Do you control the ion strength in different pHs? I got different currents when running pHs and I am not sure if it affects the result? [top]
Yes, the ionic strength is leveled to I = 50 mM in each buffer across the pH range. This is important to eliminate any effects of ion strength on effective mobility of your sample.

The reason why you may observe different currents is that we use different buffer species at varying concentrations depending upon the pH of the buffer (phosphate, acetate, borate, etc.). To level the ionic strength sodium chloride is added at varying amounts in some of the buffers. So, although the ionic strength is equal you may see different currents because the chloride ion has a lot higher conductivity than say a phosphate ion.

16. 2 mL of H20 in the vial was used to rinse the capillary after the capillary was conditioned by 0.1N NaOH. Does the H20 become very basic after a lot of injections and the capillary would carry over the basic H20 to the running buffer and affect the running buffer pH, especially at low pH? Is 0.1N NaOH required between each injection? [top]
I would suspect that the carryover of NaOH to the water vial and then on to the buffer would have a negligible effect because of the dilution between each step (the capillary has a total volume of only a few microliters; this is being diluted into 2 mL of water and then microliters of this is being diluted into buffer).

We have found by experience that it is not really necessary to perform a base and water flush in between each different sample injection. In our protocol on the Beckman MDQ, we flush the capillary for 1 min @ 80 psi with the next buffer to be analyzed into a waste vial prior to performing the sample injection. In the user manual for the pKa Estimator Elite software, there is an example protocol with suggested CE and pressure settings for each pH. It is recommended to start by analyzing the highest pH value first and moving successively down in pH, to minimize any pH drift associated with CO2 exposure of the buffer vials during the time course of the experiment.