method development

[ajey]

hi pravin,
thanks for ur reply
if mixture contains analyte Salbutamol sulphate, Guaiphenesin and Ambroxol HCl
Which buffer should be used & Why?
Regards,
Ajay

[SIELC_Tech]

I don't have this exact application, but this is typical combination of cough and cold compositions (acetaminophen, guaifenesin and two basic drugs). I am sure that you can adopt one of the following methods. Just look at the general structure of your compounds" neutral and lightly hydrophobic and basic and hydrophobic (terbutalin is similar to salbutamol for example)

http://www.sielc.com/compound_119.html


Regards,

Vlad

[mohan_2008]

I checked the link and surprised, how they get a pH 3.0 with TEA phosphate. Is the pH re-adjusted with a phosphate buffer to pH 3.0.

My suggestion is to try out a [u]neutral pH situation [/u](pH = 6.0) using a phosphate buffer with Methanol as organic modifier. A 20 mM buffer strength is ok.

Apparently, selectivity is pretty high at neutral pH conditions and resolution may be better. However, Try using an ACE Phenyl or PFP column at this application.

[praveenpaliwal]

Hi, Ajay sorry for late

I think u should use 0.01M KH2PO4 buffer in water and take ACN as modifier with C-18 column. U can get proper separation. Remember In all three molecule there is comon hydrophylic group -OH
The Guaiphenesin has only two -OH group other wise it has only lypophylic moity. so excess of water it can eluted easily.
Salbutamol sulphate : It has also -OH group but has one ionizable group -NH whic is very easily ionize when u use pH near to 4.5 and rest of mioty help it to retain inside the column.
Ambroxol HCl: It has also lypophylic moity but very strong ionizable -NH2 group.

so if u can run a gradient from 70% aq. to 30 % aq. i hope u will get sharper and seperate peaks.
Probably the Ambroxol HCl should eluted Ist then Salbutamol sulphate and finally Guaiphenesin sholuld eluted.

I hope this solvent system sholud work

Thanks and regards

Praveen
Executive analytical

[mohan_2008]

praveen's analysis regarding chemical structures is good.

However, to my knowledge, the use of a buffer is irrelevant to the column chemistry, or separation chemistry.

Buffer only maintains the appropriate pH and nothing more.

Phosphate buffer may be the number one choice, as it has a low UV cut-off and has a wide buffer range.

Selectivity of the drug moieties on the column, and hence the separation is totally a different issue.

[Vlad Orlovsky]

Could not disagree more with both previous posts (Mohan and Praveen)
Here are my 2 cents:
1. Regarding structure: hydroxyl groups are not influencing polarity in this case because protonation of amino groups changes polarity of compounds to the huge magnitude. Ionization changes polarity and ionic properties of the molecule (just find calculator for LogP and LogD and you will see how polarity changes when you protonate amine).
Here are examples proving the point:
benzylamine which is not protonated has log p of 1.12
4-hydroxybenzylamine not protonated has Log P of 0.64 (3 times "more polar" than benzylamine)
benzylamine protonated has logP of minus 1.42 (almost 350 times difference from non-protonated benzylamine)
4-hydroxybenzylamine protonated has Log P of minus 1.9 (same 350 times difference from non-protonated hydroxybenzylamine)
so based on this you can neglect presence of hydroxy group in these basic drug.

you can clearly see that in case of bases hydroxy group is not making as much difference as protonation. If you want to retain polar bases you either need to go above pKa (or close) for reverse phase column or below pKa for mixed-mode ion-exchange. With pH below pKa of your base peak shape might suffer due to residual silanols and retention will suffer due to polarity associated with protonated base.

The whole approach of analyzing basic molecules at higher pH is based on making molecule less polar by going to pH above pKa (for bases).

2. Regarding effect of buffer on retention: this is probably true for reverse phase chromatography, but in ion-exchange and mixed-mode ion-exchange chromatography buffer pH and buffer concentration is the most powerful tool which can help you moving compounds in any direction (increase or decrease retention time, change peak shape, etc.). You can increase retention time several folds by changing buffer concentration in mixed-mode reverse phase chromatography. In certain cases you can change order of elution for compounds by playing with buffer concentration alone. Check our web site you will find numerous examples how to adjust retention/selectivity by using such powerful tool as buffer concentration.

[mohan_2008]

Thanks Vlad.

I was referring to the "Buffer affect" w.r.t Reversed phase only.

I agree with you in other modes of retention mechanism,
such as ion pair, buffer concentration can infact affect your separation chemistry. Infact, buffer control (counter ion strength) is very critical in such mixed mode mechanisms.

[praveenpaliwal]

Thanks mohan , I agree that buffer is not affect the column chemistry in RP- mode. But in the presence of buffer the pH if mobile phase will change and it will effect the retention of molecule.
The pH play's important role in RP-chromatography and specially the compound which has -NH2 group show's easly ionization[In case of acidic pH]
In case of simple 4-Hydroxy Biphenyl the retention is also little bit affect by the pH of mobile phase. As we know that phenols are slight acidic so if we select the pH near to 2.5 this slight ionization will also supressed because in acidic phase the slight involvation of lone pair of oxygen elcetron's in pheyl ring is suppressed. and that's why we found more retention in high acidic mobile phase. Here retention will also affect the presence of phenyl ring also but in acidic phase hydrophylic charector of phenyl is also suppressed and we got proper retention.

Thanks and regards

Praveen

[HW Mueller]

As many have stated before, pH is probably the most powerful variant in retention, etc. in RP. It also influences stationary phase chemistry via the silanols. SiOH = SiO- + H+ is controlled by pH.