No-column injection for peak area determination

[holmerz]

Hi

Sorry if the title of the post is a bit cryptic!
I'm in the process of developing a method that aims at separating 2 compounds by HPLC with UV-VIS detection. However neither of the 2 compounds displays profound UV activity, and sometimes I'm not really sure whether the compounds have eluted or not.
Hence, I inject the reference sample with no column installed, and use that as a reference point with respect to the peak area, just to have a bearing with regards to the expected peak area.
But is there any pitfalls to this approach, and does any body else use this?

In advance thanks
Erik

[lmh]

Yes... depending on your injection solvent and wavelength, there is a risk that you'll get a peak that is caused by the injection solvent having a different extinction coefficient to the solvent flow from the pump (in a very few autosamplers there may be a loop that may be partially filled with some other solvent too; this used to be a feature of Thermo's old Surveyor autosampler).

You should definitely compare the peak area of injections of standard and injections of the same volume of the solvent in which you are dissolving your standard.

Be aware, even then, that the extinction coefficient and wavelength maximum of an analyte in water may be different to its extinction coefficient and wavelength maximum in acetonitrile (and of course pH matters too), so you won't necessarily get the same result from the peak with a column and the peak without.

[holmerz]

Thank you for the swift reply. I'm using an aqueous buffer (10mM KH2PO4 pH 3) and acetonitrile as eluents, and the reference is dissolved in water. I'll make a reference sample using the eluent and see if it makes a difference.

Thanks again
Erik

[HPLC chemist]

You have no evidence that these molecules elute under those chromatographic conditions OR even absorb in the UV which means you have to select a different detector! I would suggest you make up a 100 ppm standard and scan it using a spectrophotometry. Select the peak maximum and inject it into the HPLC. Use 95% organic and add water (Buffer) until these molecules elute with a 'capacity factor' >2 (usually between 5-10 minutes using a 5 cm column flowing at 1 mL/minute) where the 'real' chromatography occurs..

[holmerz]

Thanks for the reply. I know that compound #1 absorb at 215nm as it has a carboxylic acid functionality, whether it's the compounds max wl I'm not sure. It's compound #2 that's the problem, as it only have cyano functionality. I'll try to determine the max abs wl (I actually think the UV detector of the HPLC system has that capacity, as it can do 3D UV-plots! does that sound right?).

I have to rule out LC-MS, as we only 1 MS system, and we have to have a backup in our QC methods. Choosing GC, the compounds needs to be derivatized, and thats a no-go as well.

Any suggestions for alternative detection methods are appreciated.

\Erik

[HPLC chemist]

Assuming the 2nd compound has a UV spectrum, a PDA plot will detect it. As I recall this is common for the Waters PDA but less so for the Agilent DAD.

This also assumes that the pair of electrons of the cyano group interact with the column and prevent its elution. You cannot change this chemistry but rather use it to your advantage.

Try using a phenyl column which has a pi/electron cloud and prevent this interaction. Thus the compound would elute in the solvent front, in which case you would have to add buffered water to your mobile phase to allow a later elution and the chromatographic 'magic' to happen.

[Multidimensional]

Your method if injecting the sample WITHOUT THE COLUMN is completely unscientific and not valid. The fact that the sample has such poor chromophore activity is trying to tell you something. You are using the wrong detector. Can you run the sample isocratically? If so, use RID and you should be able to create a proper HPLC method. Is the sample non-volatile? If so, MS, ELSD and CAD detection are options. If derivatization is possible, then you could also go back to UV/VIS, but don't develop a UV method for a sample that you can not detect.

[James_Ball]

Your method if injecting the sample WITHOUT THE COLUMN is completely unscientific and not valid. The fact that the sample has such poor chromophore activity is trying to tell you something. You are using the wrong detector. Can you run the sample isocratically? If so, use RID and you should be able to create a proper HPLC method. Is the sample non-volatile? If so, MS, ELSD and CAD detection are options. If derivatization is possible, then you could also go back to UV/VIS, but don't develop a UV method for a sample that you can not detect.

Sometimes we as chromatographers have to work with less than ideal conditions such as limited resources or only one type of detector so we have to try our best to make an analysis work with what we have even when it isn't optimal.

Injecting without a column will give a rough idea of what to expect, but once the column is introduced, you will have things that cause differences in peak area. Even peak width will cause changes in the integrated area of the peak. After you get an idea of how sensitive the instrument will be as a certain concentration, try injecting 10x that concentration with the column installed, it might be an overloaded peak but you will then be certain you have the peak eluting from the column. When starting from scratch on and unknown analyte I normally inject the highest concentration I can then work backwards. Even a totally unusable overloaded peak gives you a starting point you can work from, starting at a very low concentration and hoping you have identified your target in the baseline noise just causes a lot of guessing and uncertainty until you move up in concentration enough to prove you are seeing the correct peaks.

[lmh]

I have to disagree completely with the criticism of doing an injection with no column. Of course it's not a good way to quantify something, but there is was no suggestion that the final assay should be done without a column. It's merely a diagnostic experiment to check for a drastic disaster in the chromatography.

If you carry out chromatography on a pure standard and fail to detect anything, one quite reasonable possibility is that the analyte got lost in the column and failed to elute. Trying an injection without the column is the ideal way to test if this has happened. If you still don't manage to detect anything without a column, then it's more likely that the analyte can't be detected at the concentration you're using, with the chosen detector. Doing an injection without a column is part of the diagnostic procedure of working out why there was no peak in the original run.

Since injecting pure solvent or water can also make a peak on a non-specific detector, it makes perfect sense to compare the size of the peak in identical injections with and without the analyte, to make sure that it's the analyte that's responsible for the peak.

It's always a good idea to solve one problem at a time. If you have no idea how your analyte will behave in chromatography and no idea how to detect it, then it's hard to choose a chromatographic system that will give reasonable retention time and look for the compound simultaneously.

In terms of finding the wavelength maximum, a PDA is merely an online spectrophotometer, so it's a reasonable option for checking spectra. The only reservation is that the spectrum will depend on solvent/pH, so a spectrum determined in an injection won't necessarily be an exact match to the spectrum in a real peak from half way through a chromatogram. But it's a first good guess.

Yes, Agilent PDA will also give you a spectrum; the limitation of Agilent's PDA, at least in Chemstation, is that you can't subsequently build from the collected spectra a chromatogram at any chosen wavelength - you only get chromatograms at the fixed wavelengths you asked for in advance - frustrating, but that's how it is.

In terms of other detectors - well, yes, if you have the luxury of a range of detectors, by all means try a different one if the analyte isn't likely to have good absorbance at a nice long wavelength, but not everyone has CAD and MS available, and if PDA's all you've got, and the analyte has at least some absorbance, it might be adequate to use this detector even though in an ideal world you'd choose something else.

[lmh]

... post-script: I agree with James Ball about injecting a reasonably concentrated standard when using PDA because for a first assessment of the chromatography, an overloaded peak isn't a catastrophe, but no peak at all leaves you none the wiser about what happened.

My only exception to this is MS, where a badly contaminated instrument is a pain to clean, so with MS I usually start dilute, but prepare myself to run a few times at higher concentrations if nothing appears.

[holmerz]

Thank you for your replies.

As mentioned, injecting without a column was intended to be a guide. I don't have access to a CAD detector, although it sounds like a nice tool.

Just to make it easier to understand where I'm going with this, I'll try to explain what I intend to do.

I'll radio label a compound with 11-carbon, which will yield compound #2, which will be hydrolysed to the final product #1.
So eventually I'll be using a radio detector, but I need to be sure that I can separate compound #1 and #2, prior to performing any "hot" experiments.

I guess I'll have to use MS instead of UV to verify the ability, of a given HPLC method, to separate the two compounds. Later on when performing QC on the hot productions, I only have to quantify compound #1, and this compound seems sufficiently UV-active, and the literature reports sensitivities of UV-methods at 0.50 ng.

Thanks again
Erik

[lmh]

If you're working with radioactive samples, it's a whole different ball-game. I'd definitely stick to PDA if you possibly can. It's much nicer for containment and contamination. With PDA, what goes in one end, comes out the other end, and the only place that can get contaminated (without a leak) is the flow-cell. ELSD and CAD may be universal detectors, but both involve evaporating the sample. Who knows where the radioactive material will end up? MS is even worse; you'll end up with radioactive vacuum pump oil, and a complete instrument any part of which may be contaminated. Of course you may know that the levels involved are trivial, but if you need an engineer to carry out maintenance/repairs on your system, you'll have to convince them that it's safe to do so... not easy. Also, of course, there will be all the local regulations about containment of radioactive materials, and issues about where the vapours from the spray-chamber are going. A little bit of radioactivity converts a simple analysis into an organisational nightmare.

[dap]

Yes, Agilent PDA will also give you a spectrum; the limitation of Agilent's PDA, at least in Chemstation, is that you can't subsequently build from the collected spectra a chromatogram at any chosen wavelength - you only get chromatograms at the fixed wavelengths you asked for in advance - frustrating, but that's how it is.

Yes, we can: http://www.chromforum.org/viewtopic.php?t=23202 !

[lmh]

Thanks for that! But why, oh why, can't Agilent put it in as a standard intuitive feature like every other manufacturer??? Frustration!

[holmerz]

If you're working with radioactive samples, it's a whole different ball-game. I'd definitely stick to PDA if you possibly can. It's much nicer for containment and contamination. With PDA, what goes in one end, comes out the other end, and the only place that can get contaminated (without a leak) is the flow-cell. ELSD and CAD may be universal detectors, but both involve evaporating the sample. Who knows where the radioactive material will end up? MS is even worse; you'll end up with radioactive vacuum pump oil, and a complete instrument any part of which may be contaminated. Of course you may know that the levels involved are trivial, but if you need an engineer to carry out maintenance/repairs on your system, you'll have to convince them that it's safe to do so... not easy. Also, of course, there will be all the local regulations about containment of radioactive materials, and issues about where the vapours from the spray-chamber are going. A little bit of radioactivity converts a simple analysis into an organisational nightmare.

Luckily it's a rather short lived nuclide (~20mins), so this is not a concern as such.

I'm thinking of utilizing LC-MS to develop a HPLC method to separate the intermediate and the product. My concern is whether it's possible to detect the intermediate with MS as it's molecular weight is 85.01. Any thoughts on this?

In advance thanks
Erik