Chromatography Forum

Hi everyone

I am currently developping an HPLC method for the analysis of a high viscosity sample, which I cannot dilute because of the already too low level of the analyte.
The problem is, I cannot get through a single run, the pressure rises above the limit during injection (but drops back to almost normal afterwards). I work on Waters 717 plus or 2695 and cannot change mobile phase (85% aqueous phase/15% acetonitrile) or column since the analyte is already very hard to catch.

Does anyone know how I could reduce the injection flow rate on any of these injectors? It is by now my only lead...

Many thanks

Hi ,Who is high wiscosity ? High molecular substancies (proteins as blood globulin, high paraffines of petroleum, base of pharmaceutical ointment) ?

Looks like you "zasrali" (put a bunch of ) in starting zone of the sorbent in a column or filter. Need to to wash the reverse flow, but for this to know what to launder

If you have enough sample please do a concentration step via SPE cartridges. If that is not possible, would it be an option to increase the temperature on your injector, to reduce viscosity?

Thanks for the tips.

The viscosity is due to a pharmaceutical base.
Cartridges are not an option because of the analyte, which is adsorbed on virtually everything and has already been a real problem for prep development. I will try to increase the injector temperature, though I don't know if my samples will be stable enough to be stored at high temperatures. But it seems like a good idea.

I was also considering reducing the mobile phase flow rate during the first minutes of the injection. I just hope it will not have too much effects on the separation...

Try to dilute 1:10 with Water/ACN 95/5 and inject 10-fold. Maybe that works. In a similar challenge we injected onto a precolumn, washed and then switched the precolumn in the flowpath of the main column. you would need at least 2 pumps and a 6-port 2-position valve.

I agree with Alex. Please don't try any kind of flow rate gradient. That will end up with problems on the column equilibration, baseline drift and just ugly chromatogramm. You don't need to store your samples at elevated temperatures, just the injection system should be heated. Both injection system and column should have the same temperature. 30°C instead of room temperature will help.

pharmaceutical ointment (and suppositories) base?
Its may be
PEG 4000-8000- vary good solved in iso-propanole wich high temerature (70-80 C)
Paraffines and animal fat (Lanoline also ) - vary good solved in hexane & chloroformum, solved in iso- propanol with high temperature (50 and more C)
Fullens derivate such as Carbomer 940 - full insoluble, but good wash by isopropanol

in oral solution viscosity component also may be sample sugars, polisaccharides ( camidies such as persian and guar gum, dextrines, KMC -Na) - soluble only in water- dextrines and gum in hot, KMC only in cold (or alkaline solutions).
In solutions from infusion may be also dextrane and proteine ( such as depirogenisated gelatin or human albumine) - its also solved in cold water (with DMSO).

I'm recommendate read old book by regeneration of column , selected wash method (see properties of substances). Reverse column (stand without detector), heat (?) and wash its.

For degradation viscosity - may be use.
1) Full dissolve sample in simular solvent (need big number of dilution)
2) Exctraction active components
3) Cold sample solution by ice (paraffines) or - 30C (PEG and Carbomers), centrifugate or quicly filtrate liquid phase by cotton.

@DSP007: Soory for not being more precise about my matrix, but it is kind of confidential. Plus I don't consider it as relevant information since it is not the analyte.

@Gerhard: I don't think my autosampler (Waters 717 plus) makes any difference between sample storage temperature and injector temperature. But I will try this anyway.

All in all, I will try the higher injector temperature and the dilution (we have already tried this but only with a 2-fold dilution, which wasn't enough). Thanks all.

When I used to work with sticky plant extracts in prep. HPLC I injected additionaly some air. Compression of the air would stop the injetion pressure increase. Never tried that on analytical scale. The 2695 has the ability to do 'auto additions' from other (empty) vials.