solvent peak

[sonal]

I am developing a method for the analysis of an acidic drug. My mobile phase is methanol:buffer(phosphate pH 3.0)67:33. My stock solution is in acetonitrile.
When I injected a blank (ACN) I got a peak at Rt 2.45 and one at 2.72 which is like a small notch in the previous one. The one at 2.45 is due to methanol. When I injected methanol I obtained a single peak only. What could the peak at 2.72 be due to? Does it make any difference if I get a solvent peak like this one or is a single peak necessary? I am getting it in the analysis of the drug sample also.

Sonal

[Consumer Products Guy]

What's really important is the retention time, really the resolution, from your peak of interest. If your peak of interest is, for example, eluting at 6.37 minutes under your conditions, who gives a whiz? Just the sampler valve turning will cause a disruption, or "solvent peak" as well as any differences in your standard/sample solvent and the mobile phase itself.

[sonal]

Thankyou for your help. I changed my ACN and am using from a fresh bottle. What I am now getting is a separate very small peak just after my solvent peak but I guess that does not make any difference as mentioned by you in your reply. My retention time is around 6.00.

I have another problem. The back pressure varies from 1700-2100psi changing the retention time from 5.80-6.12 on the same day. Is this much variation acceptable? What is the range for variation in Rt.
Also as the pressure decreases the retention time should increase but the reverse is happening(5.80) and with an increase in pressure the retention time is increasing(6.12). Could you please explain. Can temperature variation cause this problem ? When the AC is on the temperature is around 20 degrees C and without the AC it is 35 degrees C. If not considering the temperature effect what could be the other possible reasons?

[tom jupille]

It's the temperature!! 15 degrees C is a huge difference. An increase in temperature will decrease pressure (lower viscosity) and usually also decrease retention (higher solubility).

[sonal]

Thankyou Tom.
1.Please could you explain how an increase in solubility decreases the retention time.
2.Also please let me know the limit that is permissible for variation in Rt. A RSD <2, does this apply for Rt's also?
3.Which buffer -acetate/phosphate is preferable for HPLC-UV and why?

[neelymw]

As mentioned above, your enivronmental temperature can have a big effect on the RT of your analyte. Just another thought. If you are running a gradient with a variable volume pulse damper, you can observe the tyoe of behavior as well. As the pressure goes up, the gradient delay volume goes up as well and vice versa.

You typically chose a buffer based on pKa of the analyte and UV-cutoff characteristics. Phosphate buffers have been used tradiionally since they have a UV-Cutoff <200nm and they have a wide buffering range. Acetate has a UV-cutoff around 210nm at 10mM and narrow buffering range 3.8-5.8. Another consideration is the solubility of the buffer in your organic mobile phase. High concentration of buffer can precipitate, clog, and cause increases in your system pressure.

[tom jupille]

Neelymw answered the buffer question quite well.

Re solubility: retention in reversed-phase HPLC is based on the equilibrium distribution between the liquid mobile phase and the stationary phase. All other things being equal, as solubility in the mobile phase increases, the equilibrium shifts in that direction and the analyte washed off the column more quickly. In my original answer, I was careful to qualify the statement ("usually"), because there are exceptions.

Re permissible limits in retention: depends on the complexity of the sample and the possibility of mis-identifying a peak. If you are doing a potency or content uniformity assay, for example, and there is effectively only one large peak in your chromatogram, then you can have a considerable day-to-day variation with no problem. Within a single day, the retention time variation should be such as to not affect quantitation. If you run check standards interspersed in the run and they all fall within the precision limits, then no problem. If the check standards drift out of range, then there is a big problem. Check the thread on System Suitability per USP <621> (link: http://www.sepsci.com/chromforum/viewtopic.php?t=725) for additional comments in this regard.

If you are looking at or for impurities or degradants, then you will require much tighter control of retention in order to correctly identify the peaks. There is no hard-and-fast criterion, but 1-2% variation is probably considered "typical".

[sonal]

Thankyou for your replies. I could maintain a constant pressure today(by controlling the temperature). Your advice really helped.

1.I have to develop a method for the analysis of a drug (pure sample provided) and then use this method for the analysis of marketed formulations(tablets). The problem I am facing is that during the standard curve preparation when I inject samples in triplicates I do not get the same area, rather they are varying a lot(eg in one run I get 76173 and in the next 86597 or 97073). When the loop is of a fixed volume(20microlitre) the injection volume cannot change and I inject the samples from the same solution. Where could the problem be? I wash my syringe a sufficient number of times(approx 25 times) with ACN in which my drug is soluble.

2.When I extend my method to tablet formulations the percentage recovery I am getting is between 62-75%. The procedure I followed is - crushed my tablets(film coated), took a known amount of powdered sample (together with the film), added ACN, stirred on a magnetic stirrer for 15 min and then centrifuged for 15min at 2500rpm. From the supernatant I prepared the dilutions and injected them. Could you please guide me.

[sonal]

For performing the tests of robustness we have to deliberately change certain parameters. To what extent do we change them? eg if we work on a flow rate of 1ml/min do we change it to 0.9/1.1 or more than that. Similarly for wavelength how far away from our working wavelength should we take?

[HW Mueller]

sonal,
Assuming that you inject by hand,
on the 20µL loop: How much do you inject? If you don´t inject much more than 20µL you may get varying amounts in your loop due to varying laminar flow.
On the cleaning of the syringe: If you don´t take the piston out, clean it, then draw cleaning solution (for instance, by dipping the needle in the solution and applying vacuum to the other end) through the syringe you will never get it clean.

[sonal]

We have a 25 microlitre syringe and I inject that amount in a 20 microlitre loop. So I don't think there should any problem of inadequate sample.

Any solutions for the problem encountered in tablet fromulations? can I use a different solvent for extraction that is not used in the preparation of stock solutions?

[WK]

Sonal,
The 25uL syringe is not adequate - I would use a 50uL syringe - then maybe you can see sample coming out of loop drain when you load sample into loop. I would think this would help.
WK

[HW Mueller]

Yes, WK, Dolan and Snyder, in Troubleshooting LC Systems (1989: page 240) recommend injecting 2-3 times the loop volume if you want to get the loop volume of sample on the column.

[sonal]

On my tablet formulations problem, is it possible that my drug is not being completely extracted by the process which I follow as a result of which my percentage recovery is so low? But, my drug is soluble in ACN which I am using to extract the drug.

I was told that TLC should be performed prior to HPLC as the Rf factor can provide useful information regarding the choice of the solvent for preparation of the stock solution- the one giving a higher Rf value being used as the solvent for stock solution. Is this true and if so, if we do not use the solvent giving the highest Rf value how can it affect our process?