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please : how can i test the solubility in hplc please ?

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

23 posts Page 1 of 2
Hi my dear ,

i have made nine compostion of methanol water mixture .to make it very clear , i have 9 ( 10 ml flasks) , i put in each flask 0.5 g from pesticide (solid crystal ), then i prepared mixture of methanol water as the following

Mass%of methanol
0
10
20
30
40
50
60
80
100

please note that the pesticide weight in all flasks was 0.5 gram.It is clear that the solubility in 100 %methanol is better and has the highest solubility and 0% methanol has the lowest solubilty becuase all compostion is water .The aim is to measure solubility by RP-HPLC ( microgram/ml) please.

do i need calibration curve ??

can you give me any papers or book to understand this because all my work depends on this please

30 people have seen my topic with no answer :(

I think you'd need a calibration curve to get decent accuracy, and you'd need to filter your samples, because you don't want precipitated solids to gunk up your system and column. I think you should dilute your samples after filtration in mobile phase to minimize matrix effects.

EM

There's a chapter in "Drug discovery and evaluation: safety and pharmacokinetic assays" called physicochemical properties and if I remember there was something about it but I no longer have the book

I think you'd need a calibration curve to get decent accuracy, and you'd need to filter your samples, because you don't want precipitated solids to gunk up your system and column. I think you should dilute your samples after filtration in mobile phase to minimize matrix effects.

EM
Thanks dear Eric , what i did i dilute my samples with the same solvent ( methanol-water%) because when i inject my sample ( 0.5g/10 ml ) the peak of pesticide was very huge and wide. when i diluted 1000 times with same solvent compostion i saw a good peak but i have proplem of reproducibility i think that because i did not do filtration , i will do it and see.

my dear i heared a lot about matrix effect but i don't know what is it , and is the dilution that i did would be enough or not ??

is the calibration curve must be pesticide in pure methanol ? or mixture of methanol water because i want to measure the solubility of my pesticide in methanol water ?

i am sorry for all these question dear

There's a chapter in "Drug discovery and evaluation: safety and pharmacokinetic assays" called physicochemical properties and if I remember there was something about it but I no longer have the book
thanks for you dear for telling me the book but can make sure because i want to buy the book and i spend four days looking how to measure the solubility by hplc but i didnt find!!

thanks for you

"thanks for you dear for telling me the book but can make sure because i want to buy the book and i spend four days looking how to measure the solubility by hplc but i didnt find!! " - please use googlebooks, maybe there's no need to buy the book :)
Hi!
I will tell you how I measure the solubility by HPLC for formulation/ dissolution tests development purpuses – and I hope I do it right.
I shake excess quantity of substance with the solvent in order to form saturated solution – the concentration of the saturated is the solubility.

- To make the saturated solution you shake the solvent with excess amount for quite long time – if it is for formulation/other practical purposes few hours, if for theoretical purposes – even longer. Elevated temperatures better be used (30, 40 C) to overcome kinetic barrier – if the substance is stable. After shaking – let the saturated solution to stay at room temperature for some time (2 hours) – in case of super saturation. At the end of this process – it should be undissolved substance in the solution (proving saturation). If there is not, add more substance and repeat the whole process.
- Now that you have your saturated solutions, you can determine their concentration. To do it you should first remove all undissolved particles from solution (because they will further dissolve on dilution leading to lack of reproducibility). I centrifuge the samples and then filter the supernatant.
- The clarified saturated solution should be now diluted to bring it to concentration practical for HPLC (0.5-1mg/ml) is a good guess. So diluting 1:500, 1:1000 as you do is good. I think it is better first to inject the samples with just 1 standard (0.5mg/ml or 1mg/ml). After you do it and know approximately your range – calibration curve may be prepared to cover the range.
- After you know the concentration of the diluted samples – the solubility is of course obtained by multiplying it by the dilution factor.
Hope the answer is not too long and sorry for my English.

"is the calibration curve must be pesticide in pure methanol ? or mixture of methanol water because i want to measure the solubility of my pesticide in methanol water ? "

it depends on your method, calibration curve is done in the diluent specified in the method (which you developed), and this diluent is used for calibration solution as well as for the solution from solubility samples

so you could prepare your samples as suggested by marina1111 and you dilute them with diluent which is the same for all samples
Hi!
I will tell you how I measure the solubility by HPLC for formulation/ dissolution tests development purpuses – and I hope I do it right.
I shake excess quantity of substance with the solvent in order to form saturated solution – the concentration of the saturated is the solubility.

- To make the saturated solution you shake the solvent with excess amount for quite long time – if it is for formulation/other practical purposes few hours, if for theoretical purposes – even longer. Elevated temperatures better be used (30, 40 C) to overcome kinetic barrier – if the substance is stable. After shaking – let the saturated solution to stay at room temperature for some time (2 hours) – in case of super saturation. At the end of this process – it should be undissolved substance in the solution (proving saturation). If there is not, add more substance and repeat the whole process.
- Now that you have your saturated solutions, you can determine their concentration. To do it you should first remove all undissolved particles from solution (because they will further dissolve on dilution leading to lack of reproducibility). I centrifuge the samples and then filter the supernatant.
- The clarified saturated solution should be now diluted to bring it to concentration practical for HPLC (0.5-1mg/ml) is a good guess. So diluting 1:500, 1:1000 as you do is good. I think it is better first to inject the samples with just 1 standard (0.5mg/ml or 1mg/ml). After you do it and know approximately your range – calibration curve may be prepared to cover the range.
- After you know the concentration of the diluted samples – the solubility is of course obtained by multiplying it by the dilution factor.
Hope the answer is not too long and sorry for my English.
your English is perfect as same as your answer .I really have rproducibility problem after i did many filtration and dilution

thanks indeed for u

"is the calibration curve must be pesticide in pure methanol ? or mixture of methanol water because i want to measure the solubility of my pesticide in methanol water ? "

it depends on your method, calibration curve is done in the diluent specified in the method (which you developed), and this diluent is used for calibration solution as well as for the solution from solubility samples

so you could prepare your samples as suggested by marina1111 and you dilute them with diluent which is the same for all samples
thanks dear grzesiek for continuous help

best regards

Hi Newchromatographer,

If I were you I wouldn’t employ HPLC or any other form of chromatography in this type of investigations.
You would be better off if you just determine the concentration by means of spectrophotometry.

Best Regards
Learn Innovate and Share

Dancho Dikov
Hi my dear ,

i have made nine compostion of methanol water mixture .to make it very clear , i have 9 ( 10 ml flasks) , i put in each flask 0.5 g from pesticide (solid crystal ), then i prepared mixture of methanol water as the following
.....
The aim is to measure solubility by RP-HPLC ( microgram/ml) please.

do i need calibration curve ??

can you give me any papers or book to understand this because all my work depends on this please
The first requirment is you need to obtain a totally solved solution of your analyte and then you could compare the peak area of this solution with the peak area of your unknown sample.
Of course, depending on the precision you need, you could also prepare a complete calibration curve but pay attention to the detector saturation.
The problems you had working with the dilutions, during GLP method development, is part of the method validation. You have to prepare a very concentrated sample and then dilute it with your matrix (i.e. plasma or in your case methanol) in order to bring its concentration inside your linear range.
Samuele Pedraglio
Developability Dept.
NiKem Research S.r.l.
Italy

marina gave a good summary of solubility studies, one thing I didn´t see, though: a lot of substances form relatively stable supersaturated solutions. That´s why I never attempted to do such things other than semiquantitatively, for own rough use only.

I've found that solubility studies can be very dependent on the analyte crystal size, and good, consistent mixing. If you are not certain, choose a cheap compound with similar properties, and practice with that analyte first to ensure that you get the same values as published literature.

I prefer to use ultrasonication and vortex mixing, with some warmth, then allowing the solutions to cool. I also reanalyse aliquots of the solutions several times, to ensure each result is consistent. If I see variation, I know that the dissolution or clarification methods are not working properly.

To clarify the solution, I prefer to use strong/quick centrifugation in a sealed tube in a controlled-temperature centrifuge, without any filtration.
Longe centrifuagtion times in some centrifuges may change the temperature of the solution.

If you filter, ensure that only pressure filtration is used. If you use vacuum filtration, the solvent can evaporate, cool the solution, and more analyte precipitates.

Provided you have crystals present at all stages, as recommended by Marina1111, you are extremely unlikely to form supersaturated solutions.
If you want to be certain, you can sprinkle a few crystals onto the solution, and gently swirl as they descend. Then centrifuge, sample the supernatant, and dilute.

Like Danko, I would not use chromatography for solubility testing of a pure compound, but some other property - eg spectroscopy, optical rotation etc. If you do want to use chromatography, you should dilute samples and standards with a solvent that will completely dissolve the highest concentration of the analyte.

Please keep having fun,

Bruce Hamilton
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