Chromatography Forum

Hi Chromatographers,

I am analysing impurities by gradient of Salbutamol + Bromhexine expectorent. Sodium Benzoate is present as Preservative.

My mobile phase is A. 0.2 molar KH2PO4 + ACN 95:5 (PH 4.7)
B. 0.2 molar KH2PO4 + ACN 60:40 (PH 5.4)
Gradient run is
0minnute 100%A , 10minutes 100%A, 15minutes 85%A, 28minutes 45%A, 30minutes 50%A, 33minutes 100%A, 40minutes 100%A

Temp: Ambient

As expected I am getting peaks of 11 impurities with active ingredients and preservatives. Problem is posed by excepients whose peaks are merging with some of the impurities thus giving problems. Excipients used in expectorant are Aspartame (major culprit), Cherry Sweet, Propylene Glycol, HCL.

I have used waters symmetry C18 (Brand: Symmetry®, Particle Size: 3.5 µm , Inner Diameter: 4.6 mm , Length: 75 mm, Pore Size: 100Å , pH Range: 2 - 8
Surface Area (m2/g): 335 , Endcapped: Yes , % Carbon Load: 19.1)

but results are not satisfactory. With C8 column (Princeton 250x4.6mm, carbon load 11%, particle size 5u, pore size 60A) also results are even worse than symmetry.

Can you help me out so that I get results with pure peaks without merging with each other?

Hi Harry,

It’s not that easy to identify the problem without seeing the chromatogram you aren’t satisfied with.
Besides, you don’t mention whether the method has been running satisfactory up to a point and then degraded, or you just perform some test runs – kind of developing/transferring a new method. The further discussion will be very much depending on the answer to the question above.
But something bothers me regardless the task: Your 2 eluents (A and B) have 2 different pH values i.e. 4.7 and 5.4, which is very unusual in Reverse Phase chrom. This is a major source of instability and should be avoided almost at any cost. Another thing I see as a problem is that none of these 2 pH values are within phosphate’s buffering ability.
So, unless you are willing or able to share some more information, my advice will be: Work/experiment with the pH value of the eluents. Try pH between 2.3 and 2.6 and preferably the same value for both of them

Good luck
Danko

I agree with the previous comment that you should not work at this pH with a phosphate buffer. Actually, I do recommend to first explore the changes in the separation that you get with a pH change. I recommend to work at pH 2.5 with phosphate buffer, at pH 7 with phosphate buffer and at pH 4.75 with an acetate buffer. This might change the selectivity sufficiently to get you into a better spot. Afterwards, you can then finetune the gradient to get a complete separation of everything. This is my standard way of exploring and optimizing a separation.

There's probably a zillion things that you could do that would make small but signicant changes in the selectivity - and then it would be a matter of trial and error.

I agree with the above postings that your pH is not ideal (not much buffer capacity) for phosphate. And you have about 12g/ litre of buffer (0.2M). That is a huge amount - are you sure that it doesnt precipitate in mobile phase B?

Before I started using high ph columns (XTerra Gemini etc), for amines like salbutamol (albuterol for us in the US). I would generally use pH 2.5 to 3 (phosphoric acid with little or no buffer) - If separation didnt work well, I would often add a sulphonic acid at different chain lengths and conc until my separation worked out OK.

To amplify a bit on some of the earlier comments: if you want to move peaks around with respect to one another (change selectivity) in reversed-phase, you essentially have six possibilities:
- change the steepness of the gradient
- change the temperature
- change the organic solvent
- change the pH
- change the column
- use additives (e.g., ion-pair reagents) in the mobile phase.

With a moderately complex sample like yours, there is no a priori way to tell which will be "best", because a change that improves the separation for one pair of peaks may move a different pair together. My general approach is a "matrix" of 4 runs (steep/shallow gradients at high/low temperature) repeated with different organic solvents, different pH values, and different columns; the details are a matter of personal preference and experience. You can accumulate a lot of useful data by automating an overnight series of runs (assuming you have an instrument with a column switching valve and temperature control!). For example with 6-minute and 18-minute gradients, you could do the 4-run matrix in an hour (allowing time for re-equilibration), and screen two solvents (ACN & MeOH) at high/low pH on two columns, with all runs in duplicate in an overnight run (16 hours).

Specific comments: as has already been pointed out, phosphate is not a good buffer choice for pH 4.7, and 200 mM is almost 10X higher concentration than most people would use. I'll add that your gradient seems very shallow for that short a column (but that depends on your flow rate).

only add one comment after these excellent comments from others:
look into the structure of the API, excipients, impuritys you should be able to know how pH will altere the retention for many of them before run any
experimnets

Hi,

Thanks for all your suggestions. I will definitely try this and get back to again in case any help is required.