I am running a reverse phase HPLC method (C8 column)using 100% aqueous mobile phase. My problem is that I can only run this method without purge my injecter in between samples. In other words, if I purge my injector in between samples I will lose the retention of my compounds.
The compounds I am analyzing are Creatine and Creatinine.
The method I am using is: C8 column with 10mM CH3COONH4 mobile phase. I run gredient to 50% acetonitrile in between to wet the column.
I believe what happened is that when the system is purging the injecter, it stops flow and therefore the system loses pressure. The flow interruption caused column calapse (dewetting). Dose anyone know how to solve this problem?

CD

Dera CD,

just try one of those "aqua-phases". These RP-Phases don not lose retention capability when run with 100% aqueous eluents, even after flow has stopped. Almost every company who sell HPLC-columns have those filling material in stock. Just call your local rep. I made very good experince with the Waters ATLANTIS column due to high stability both at low and high pH levels. But others might be suitable as well.
Hope this helps.

FP

Hi, CD
look at articles

"Phase Collapse in Reversed-Phase LC" by M.Przybyciel and R.E. Majors in LC-GC Europe Volume 15, Number 10, October 2002

"The Retention Behavior of Reversed-Phase HPLC Columns with 100% Aqueous Mobile Phase" by N. Nagae, T. Enami, S. Doshi in LC-GC North America
Volume 20, Number 10, October 2002

I hope this will be useful

Thanks for helping.
I tried a Waters Atlantis column and I did not have dewetting problem. However, I could not get separation for my compounds creatine and creatinine.
I tried to inject a blank before each sample and no purge injector before the sample. It is working good. My run time is long but at least it is working.

Again thanks for your ideas.

CD

I'm unfamiliar with 'purging' the injector. I have to 'flush' one of the systems we have, because it develops air in the lines when it is shut down, but only when the system is turned on, not before every injection.

What kind of crazy system architecture shuts off the column flow between analytical runs??? That makes no sense, and I've never heard of a system that did that.

FP is right. If you must run 100% water, you need a new 'Aqua' column, and it will be money well spent in the instrument time it saves. Ask the column manufacturers to run your samples on the column they suggest, just to be sure.

If you have 'carryover' or 'ghost' peak problems, that is a different issue.

Although I am not that familiar with the reversed-phase separation of creatine and creatinine, I have seen references.

The Atlantis column will not dewet and will give you the best chance of achieving a successful RP separation.

I agree with ScottF, please tell us what kind of HPLC purges the injector and causes flow stoppage/loss. That way we can all make sure that we don't buy it.

Seriously, that doesn't sound right. Most, if not all, injectors have separate flow paths for mobile phase solvent(s) and purge solutions. In this way, they never come in contact with each other but the valve(s) are cleaned/purged. An example is the trusty Rheodyne 7125/9125.

Please let us know what type of RP method you are trying and maybe we can help.

To DM and Scott F

There are several brands of HPLC system that do, in fact, stop flow to the column while they perform other tasks. Why do this? Primarly to speed up re-equilibration time while running gradients. So, divert flow from the column to waste, ramp the flow to a high rate with initial conditions untill system is equilibrated, reduce flow to usual rate, divert flow back to column. End result, time saved.

Further to that, in some systems, to purge the injector, a portion of the flow is diverted to the washing funtion, thereby reducing (but not stopping) column flow. Purge functions like this are most often performed before a set of analysis, not usually between runs.

ScottF,DM and Anonymous,
The software I am using has a place called "autosampler purge". If I put a volumn in it will do the purge. Because both the software (for MS) and HPLC are new to me so I do not know how everything works yet. I called the software tech support and they told me that this is the only way to wash the needle.
I just find out from the HPLC vendor that the HPLC washes needle automatically in between the samples. I do not need to purge. I only need to do the purge if I change the mobile phase. The purge process does stop the flow.

I think I am ok now. I do not have to do a blank injection in between every sample. It does sound creazy if I need to do that.
Thanks again for all your help.
CD

Yes the waters Alliance systems do a needle wash automatically between injections, I only run a purge "wet prime" when I am switching the lines to a new mobile phases and it has always worked fine.

Daren and others:

For your information and to clarify some terms;

On Waters Alliance

1-The needle is washed prior to every injection using the needle wash solvent. This needlewash solution uses its own pump and does not impact the mobile phase flow in any way.

2- Purge - This function has several purposes, it will remove traces of solvent in the sample loop from previous runs and is also used for removing air bubbles from the syringe and fluidic path. This function can also be used to test the needle seals via the compression check choice. It needs to be done after changing solvents, wet priming your system, and should really be done daily (at the start of the day). Flow to the column is stopped during this function.

3-Wet Prime - Wet prime pumps solvent through the fluidics at high flow rate to waste, use this when changing solvents.

CD---glad everything is working for you. These systems are not simple, and we all have stories to tell about new systems, software, methods, compounds, etc. Thanks for letting us know what happened.

Anon 8:07

It would seem to me that with a relatively modern, low-volume system running a gradient, switching to 'purge', ramping flow, etc., bypassing the column, wouldn't help much. The bulk of the system dead volume, and therefore mobile phase dilution, is in the column. My HP 1050 has about 150 uL between the pump and the column head, and that volume is replaced very quickly at typical 0.4-1.0 mL flow rates, without much dilution effect because it is all in very small tubing. However, to get the column back to 'equilibrium', where it started the gradient, takes several minutes, as judged by the UV change between 90/10 and 10/90 water/ACN.

If the column were not equilibrated, just the 150 uL volume replaced, the chromatography in the subsequent injection would be done at some combination of the old mobile phase/new mobile phase. In my case, almost always pretty poor. Many times when I've been in a big hurry I have aborted a run at the top of the gradient and immediately re-injected. It has never worked for my applications. But that doesn't mean it wouldn't work for some other application, on some other system. We've never had a system, or application, that we thought might benefit from the switching routine. I think I've read about multiple column switching systems that use a 2nd, cheap pump to do the equilibration, though.

I'm sure there are uses for this type of operation, but the problem presented didn't sound like one of them. And that seems to be how it turned out.

Scott F

Re:switching to 'purge', ramping flow, etc., bypassing the column, wouldn't help much

It is all a matter of sample throughput, lets say I had 5000 runs to do, a savings of 15 seconds per sample cuts the total analysis time by almost 20 hours. For some workers (combinatorial chemistry types) this savings is huge (time=money). I will freely admit that for the majority of HPLC users there is little need for this kind of strategy. I was just offering the information to those who had never heard of such a thing. Many of those who need very high throughput do employ column switching and off-line column re-equilibration as you suggested in combination with rapid system re-equilibration.

And just as a final note, the rule of thumb for re-equilibration after a gratient is 3X the system volume + 5X the column volume.

8:07

8:07, I figured it was something like that.

I don't even want to think about 5000 runs. Around here, the odds of that working without a hitch are zero. I think our record for continuous operation is probably between 2-3 weeks on one of our LC/MS systems.

Thanks for making me think about dual column systems. For quick re-equilibration purposes, a system wouldn't be expensive to put together. If there is a suitable LC available, the valves aren't expensive compared to the cost of the total system, and prudent chemists already have a spare column on hand. I think we just might put a system together to see what we can do with it!

On the edge....

Don't you agree that the use of C18 and a totally aqueous mobile phase is the "bitter end".

Why don't you test the completely ortogonal technique HILIC i.e. Hydrophilic Interaction Chromatography.

When using the ZIC™-HILIC column you will start with about 80-90% acetonitrile (if gradient run) and elute by an increasing content of water (buffer).

In this case it is certainly an interesting option!

Einer,
I actually found this method from literature. So I bought the column and try the method from a paper. I do not have many columns since I am just starting to do a lot of method development. I would like to have a whole set of HPLC columns for my method development. I just bought a HILIC column for underivatized amino acids analysis. I can try my compounds on this column.

CD

Einar,

Seeing as how you are from a new, small company that is trying to break into the column market with HILIC columns, I can understand your attempts to suggest HILIC at every turn. However, most separations can be done on RP columns. Columns such as the Atlantis column mentioned above work wonderfully at high aqueous mobile phases and are very retentive of polar compounds. That is just one of these columns. Others have been mentioned in other threads.

HILIC does have its place for very, very polar compounds (underivitized amino acids mentioned above). But there are problems with HILIC such as sample solubility (e.g., attempting to dissolve very polar analytes in ACN), column equilibration, column lifetime, and injection solvent strength effects on peak shape(s), etc.

HILIC is not a panacea but a method to use as a last resort.

HILIC is my preferred methodology for polar analytes by LC-MS. It is usually much more sensitive than RP, has lower pressure and can be run at much faster flow and under gradient conditions. It also has the advantage of being compatible with elution solvents from RP SPE products when making online plasma injection. Actually I have heard that a lot of LC-MS people are moving into HILIC from RP. For very polar analytes, we typically need 20% aq. as mobile phase. So we can use 20%aq./80% MeCn as diluent. I haven't seen solubility problem yet. However, I do see peak shape distortion when injecting extracts from Oasis MCX cartridges. However, there are tricks available to this problem.

Returning to the creatine and creatinine separation problem. Was the resolution problem solved by the “aqua phase” Atlantis? I am quite sure that these will be retained on the ZIC™-HILIC column.

I agree,
with JL Shen that HILIC is a useful tool from time to time, with few associated problems. With respect solid phase extraction it may also be valuable to clean the sample by using a HILIC SPE cartridge. The polar compounds are then finally eluted by water/buffer and peak compression will be obtained when injected on a RP analytical column, although using a low content of organic modifier (that is likely to be required for wetting?).

I disagree,
with “dm” that my comment just is a cheap attempt to break into the “column market” promoting something associated with numerous of problems. My experience is hands on work with chromatography for 20 years in pharmaceutical analytical chemistry, academic research and product development. According to our estimates 10-20% of all RP separations may be performed in HILIC mode. I just don't think it is a good idea for scientists to only use one tool, especially when there are several available in the toolbox.

Einar,
How does one do HILIC SPE from a plasma sample?
Best regards
Uwe

Einar,
I have not get separation of creatine-creatinine from the Atlantis column. I have many projects with only one LC/MS system. Need to move onto a different project. I will let you guys know if I do get a chance to try it.

Thanks

CD

I wouldn’t dare to say that one procedure fits all, but here are some thoughts and ideas.

An approach for plasma sample pre-treatment is to more or less "rapidly inject" the sample into acetonitrile. Most, but not all, proteins precipitate and may then be removed by means of centrifugation. This procedure results in a sample diluted in acetonitrile that then can be concentrated by evaporation (into dryness). Else, a very small injection volume or another dilution with water is required to attain peak compression in a RP system.

If the analyte is a polar hydrophilic compound it may instead be trapped, from the acetonitrile solution, on a HILIC SPE. The analyte is eluted by an aqueous solution and then injected into the RP system.

One of the standard procedures for the sample pretreatment of a plasma sample is protein precipitation, which is carried out by adding a large amount of acetonitrile to the sample. The proteins precipitate, and are removed either by centrifugation or by filtration. Normally, the remainder is injected onto a column, with few problems, except for interferences (no problem, if one has a MS detector). With reversed-phase columns, the difficulty is now that the large amount of acetonitrile limits how much one can inject onto the column. Therefore the standard approach is to evaporate the sample to dryness and redissolve it in water, or whatever is compatible with the RP chromatography. With HILIC as the final chromatography method, one does not need to do this, and can inject the acetonitrile sample directly.
This is beautifully simple, if the HILIC column provides retention. Weng Naidong has written many publications on this technique. As an advocate of HILIC, I would have thought that you are familar with this method.
I can not see how a HILIC SPE method could help in this approach. If one needs to do additional sample clean-up after the protein precipitation, one of the better approaches would be mixed-mode SPE. But if I need to do SPE, I would use SPE to start with and not do the unnecessary complication of protein precipitation.

I fully agree with UWE that using both protein precipitation and then SPE on plasma samples are too much. However, some analytes are so basic (pKa 12.6 or so) that you will need to use 0.2N NaOH, 0.4 N methylammonium hydroxide or 400 mM salt added to eluant to elute them from Waters MCX. Those eluates would NOT be good for the steps that follow. In addition, highly aq. eluant is very difficult to dry. Protein precipitation will result in dilution; evaporation enriches samples but don't remove junk. LC-MS still requires clean (and sometimes very clean) samples if we need very rugged methods or very low LLOQ (we have a method with LLOQ 0.5pg/ml in plasma), especially with ESI. An alternative would be to use weak ion exchange with RP (mixed mode), which would not be very rugged for very polar analytes. First, we usually add phosphate or so to remove protein binding. That increases salt concentration in plasma samples, which will reduce ion exchange effects. Those analytes have no retention at all on HLB. I also read an article from IST that ion exchange alone is not rugged enough for SPE, additional forces like hydrophobic would be needed to get really rugged methods. So doing proein precipitation and then SPE is indeed a choice ocassionally. This is seen from some conference posters from some famous big Pharmas.
Good news is that as I have heard, Varian Focus SPE works with almost any molecules. I haven't tried yet but definately will.

Jinlin,
In our standard approach for mixed-mode cation SPE, you wash the adsorbed sample with acidified water, then with acidified organic solvent (e.g. acetonitrile). Afterwards, you elute the sample in an organic solvent with base added. As an optional step, you can also wash the adsorbed sample in water at alkaline conditions, but we have not found a need for this, since the eluate is very clean, even from plasma samples. With this approach, we get about 10 to 20 times less ion suppression than with protein precipitation, and at least 5 times less than with one-dimensional SPE.
In the context of the discussion above, the sample is eluted in acetonitrile, and can be injected directly into the HPLC instrument running in HILIC mode. No evaporation to dryness - no additional steps - nothing! Fast and simple!
If you are interested, I'll send you an article on this, in combination with a microelution 96 well plate (currently in print).

Uwe:

One analyte (we have worked on) is very polar and has a pKa of 12.7. They are not eluted with 5% ammonia in various percentage of aq.methanol. It is obvious that it is still bound to MCX and 5% of ammonia is not strong enough to keep the analyte neutral. We needed stronger base to keep the analyte in neutral form and to get it out from the MCX cartridges. The analyte is not a quaternary amine, but behaves very similar to it. So to disrupt the ion exchange, we either need stronger bases or high conc salt added to the eluting solvent, either of which are not good for us. Different percentage of organic can be usd to further clean up samples. If the analyte is very polar and we elute with acetonitrile (basified), we will get a lot of junks that otherwise would have been removed. It would be advisable to elute with basified 10%methanol (for example) instead of basified acetonitrile. 10% methanol would be difficult to evaporate.

Basically, my point is that it is very difficult to clean up samples on MCX for analytes like quaternary amines and compounds that behave like quaternary amines.

We have enjoyed a lot of great work from your group. Please send the article to me.

HILIC SPE -> RP

Sorry for being "off-line" from this interesting discussion.

My point was the -ortogonal approach- where a HILIC SPE step as sample clean up may preced the use of another selectivity, e.g., a RP aqua phase for a polar compound, or vice versa. In addition, serial purification steps using the same selectivity does not attract me (or you).

As mentioned by JL Shen "evaporation enriches samples but don't remove junk". Evaporation for enrichment may, as you are aware, be substituted by on-column peak compression, that in turn requires a very weak solvent strength by the injected sample solvent. Sometimes centrifugation can be omitted if the sample is applied on a SPE before injection on the analytical column.

So, the schemes I had in mind were these general procedures for combining SPE and an analytical column separations:

HILIC SPE -> RP analytical column
A. The sample is diluted in acetonitrile and applied on SPE.
B. Elution by water or a buffer.
C. Peak compression when injected onto RP column.

RP SPE -> HILIC analytical column
A. The sample is diluted in water and applied on SPE.
B. Elution by organic solvent
C. Peak compression when injected onto HILIC column.

Seems to be fast and simple to me. By the way, the zwitterionic phase is in some sense “mixed-mode” and the sample work-up of quaternary amines on a ZIC™-HILIC SPE is an interesting application.

Einar,

The problem is that HILIC is not orthogonal to RP, but retrograde. Ion exchange is orthogonal…

Let us take the second case first, because I think we agree on this (remember we were working from a plasma sample).

Case 2: The sample is acidified, applied to a reversed-phase SPE column. The polar interferences, including proteins and sugars, are washed off. The analyte is reextracted in an organic solvent like acetonitrile. The extract is injected onto a HILIC column. If the analyte is sufficiently polar, or has other interactions with the HILIC column, it will be retained and it can be analyzed. The rest of the world will wash through the HILIC column unretained.

Case 1: You suggest to add acetonitrile to the plasma sample. You need to add at least 4 volumes of acetonitrile to get any retention in the following SPE, maybe more. Now you have protein goo in your sample, and you need to filter the stuff or better centrifuge the stuff. You apply the supernatant to the HILIC SPE. Unless the HILIC SPE has some magic side properties (like ion exchange), most of the compounds will break through unretained, and only very polar compounds are retained on the HILIC SPE. You reextract from the HILIC SPE using water (or buffer), and you inject the aqueous sample now onto the C18 column. If you are lucky, the compound will be retained on the C18 column, and you can do your analysis. The problem is that HILIC works best for stuff that has little to no retention on C18. If you were successful to retain it on your HILIC column, you have much less of a chance to get it retained on a C18.

The method of Case 1 is not only more complicated than the method in Case 2, it is also limited to a smaller polarity range. In real life, most people will do the acetonitrile addition to accomplish the protein precipitation, and then inject the stuff onto an LC column – done.

The best approach is to use true orthogonal methods in SPE and HPLC. You do this by using ion-exchange for SPE, while using either RP or HILIC for the final separation. There are several functional methods around, but the simplest is to use a two-step mixed-mode SPE for the clean-up of the sample from the plasma, extraction from the SPE in acetonitrile, and injection of the extract onto a HILIC column, if the analyte is suitable for this, i.e. sufficiently polar. If the analyte is too non-polar to be injected onto a HILIC column, you can still inject it onto a reversed-phase column, unless you need to inject a large volume. There are SPE solutions to this as well. I guess, I have written enough for today. I should not start to write novels.

I was analysis creatinine in urine by HPLC, but my problem are the back ground is not so good. In my Question is, Can I run creatinine in urine using column C18 and using mobile pharse 20mM acetate acid with 20% methanol and PH 6.25?

You probably need to clean the sample using another technique. Solid-phase extraction can be very specific, and you can get very good results. I think for your compound, solid phase extraction with a cation exchanger or mixed-mode cation exchanger will do well.

There is another thing that I do not like about your HPLC method. How do you get to pH 6.25 with acetic acid? Please explain, and maybe we can fix this!

And, creatinine is very polar, so it might not be retained on a C18 column.

Here is a method for analysis of creatinine in mixture with neurotransmitters (5-HTP, DOPA, serotonine and creatinine)

http://www.allsep.com/makeCmp.php?cmp=Cmp_112