By column on Saturday, September 27, 2003 - 12:19 pm:

What is a good time for column use?

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By Anonymous on Saturday, September 27, 2003 - 06:45 pm:

Most columns can be used any time of the day!

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By Anonymous on Monday, September 29, 2003 - 07:41 am:

Maybe you mean column lifetime: use until the resolution, peak, shape, pressure, etc., works for the application it is used for.

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By Anonymous on Tuesday, September 30, 2003 - 06:41 am:

IMHO, if the number of theoretical plates is decr. ~30% than it time to replace.
If You need more visit me at sim-gmbh.de under training, write to the "theory person" in contacts.

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By Anonymous on Tuesday, September 30, 2003 - 10:30 am:

see:
http://www.lcgcmag.com/lcgc/data/articl ... rticle.pdf

There is the explanation of John Dolan.

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By lucia on Tuesday, November 18, 2003 - 09:58 am:

How is it possible the a new column C18 gives values of area for ascorbic acid nearly the half of the previous column ( the mobile phase hasn't change), the method is validated and we have a problem with that, what can be the reason?

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By Chris Pohl on Wednesday, November 19, 2003 - 02:03 pm:

What's your method and how do you condition the column before putting it in use?

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By Uwe Neue on Wednesday, November 19, 2003 - 07:24 pm:

DO you get consistently half the value, or is there a trend from injection to injection?

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By Anonymous on Thursday, November 20, 2003 - 01:24 pm:

How about your pressure - any leaks? How is your lamp energy?

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By readski1 on Monday, November 24, 2003 - 08:40 am:

How clean is your flow cell? Check lamp energy with and without flow cell. If ratio of with/without is <0.5 try cleaning your cell.

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By yader Hernandez on Wednesday, April 7, 2004 - 02:09 pm:

Yo estoy trabajando con una columna LC-ABZ con fase movil 0.1M acido fosforico y acetonitrilo para determinacion de cocaina, benzoilecgonina y cocaetileno, de un dia a otro los tiempos de retencion disminuyeron significativamente. Ejemplo: cocaina que era determinada a 3.98 minutos ahora se presenta a los 2.95 minutos. Que esta sucediendo con mi columna.

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By Anonymous on Sunday, April 11, 2004 - 01:05 am:

Hey Spiko no hablo in espanolo

pendehoe!!!!

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By Anonymous on Sunday, April 11, 2004 - 03:47 pm:

Moi, je prefere parler Francais...

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By Anonymous on Thursday, April 15, 2004 - 11:46 am:

I use a LC-ABZ column and now a days the pressure is very high ! I don't nkow what to do. What can I do to clean it? thank you.

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By Anonymous on Friday, April 16, 2004 - 11:54 am:

please, can any one help me? I'm using a Supelco Lc-ABZ column, what can I do to clean it? Please!

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By tom jupille on Sunday, April 18, 2004 - 09:59 pm:

If it's an old column (500-1000 injections or so), it may be more cost-effective to simply replace it. Certainly if there are other problems (e.g., peak shape or selectivity changes), then replacement is the best choice.

If your column is relatively new and the high pressure is the only problem, the first thing is to backflush the column (disconnect the column and reconnect the outlet where the inlet was). Run your regular mobile phase at your usual flow rate for 10-15 minutes, then turn the column back to the original direction.

If that doesn't help, then try changing the inlet frit (but be aware that changing the frit has the potential to kill your column if you're not careful.

Good luck!

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By Anonymous on Thursday, May 20, 2004 - 11:32 am:

thank you Tom Jupille, I'll try this.

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By Anonymous on Thursday, May 20, 2004 - 12:26 pm:

Tom Jullip,
May I ask you one more question?
How can I try changing the inlet frit? It seams to be impossible. I try but it seams to be fixed into the column.
Can you expalin me?
Thank you again.
Mara

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By tom jupille on Wednesday, May 26, 2004 - 07:29 pm:

Depends on the column end-fitting design. On some columns, the frit is, indeed, pressed into the column tube and is not replacable.

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By gerd on Monday, June 7, 2004 - 02:19 am:

HAllo!
Can anyone recommend a column for analysing fermentation broth by HPLC. Especially organic acids and carbohydrats are of interest, detection by RI-detector. At the moment I am using an ORH801 column (now with Transgenomics label I think) but I do not know if this is really the best for this purpose (and of course not the cheapest.) My dealer offered me as alternatives a benson polymeric organic acid column, ICSept ICE-ION-300, TG-Coregel 87H Ionexclusion H+form, or CARBOSep H plus.
Comments please!
Many thanks in advance.
Gerd

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By Chris Pohl on Monday, June 7, 2004 - 11:01 am:

In general, the idea of doing carbohydrates in organic acids simultaneously on a gel based column is a bit of a stretch. The organic acid separation requires a sulfonic acid form resin. Carbohydrate separations on such media work better when the resin is in a salt form, loaded with potassium or calcium, for example. It's a significant compromise in the resolving power of the resin to try using the acid form for carbohydrate separations. Having said that, however, the Aminex HPX-87P from Bio-Rad looks like a reasonable alternative. They have examples of fermentation broth assays on this column in their catalog.

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By Ameroid on Friday, June 25, 2004 - 07:42 am:

Hello, I am new here.
I am using a Varian C18 column to perform a calibration curve of some phenolic compound. The matrix containing the phenolic cmpd is a carbonate buffer at pH 9.9
The isocratic eluting solvent is a 60:40 (v/v) methanol:water mixture

I notice my column registers abnormally high pressure of close to 5000 psi after a few runs. Is my column stuck due to the buffer salt? what precautions should i take to minimize this blockage?

Please advice. Thank tou.

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By Chris Pohl on Saturday, June 26, 2004 - 02:26 pm:

You don't say anything about the pH of your mobile phase. Are you working with an unbuffered system? If so, your problem might be due to precipitation of the sample at the head of the column (assuming the phenolic compound was dissolved in the carbonate buffer for solubility reasons). If this is the cause of the problem, you should be able to remove the precipitated components by adjusting the mobile phase to one that is suitable for dissolving your matrix (you should be able to sort this out with a few test tube experiments).

Alternatively, if the column isn't particularly tolerant of high pH, the development of high pressure might be due to damage to the silica in your column. What is the recommended operating pH limit for the column you are using? Can you work with a less alkaline matrix? This might avoid the problem in the future. Unfortunately, if this is the cause of the problem you probably won't be able to restore your column to its original backpressure.

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By Ameroid on Tuesday, July 6, 2004 - 10:44 pm:

Dear Chris,

Thanks for your help. My phenolics are in a carbonate buffer matrix (pH10). The reason why they were made in the carbonate buffer was to construct a calibration curve for the phenolic compound in its deprotonated (anionic) state. As you mentioned, a particularly high pH will damage the silica. However, the retention time of my phenolic compound has not changed after many sample injections while keeping a high pH 10 value. So am I right to say that there is no damage done to the silica packing?

Thank you very much indeed.

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By Ameroid on Tuesday, July 6, 2004 - 10:52 pm:

One more question please:

The pressure reading of my Varian C18 column was hovering around 4700 psi when I usually run my samples. The elution conditions are as follows:

Mobile composition: 60 HPLC Grade MeOH :40 H2O
(v/v)

Flow Rate: 1.0 mL/min

Column Temperature: Not Set

Sample Temperature: 25 deg C plus/minus 1 deg C

Sample matrix: Water

Analyte: phenolic compound.

Run time: 5 minute/sample

Ever since I have stopped using my HPLC about 1 week ago, I have observed a unusually hugh drop in column pressure hovering around 2000 psi with exactly the same elution conditions. This figure also fluctuated widely (for example, between 1800 to 2200 within 5 seconds). Eventually, the pressure edged up to about 4700 psi which is more "normal".

What is happening? Can anyone advice?

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By tom jupille on Wednesday, July 7, 2004 - 08:38 am:

What column dimensions? 4700 psi @ 1 mL sounds rather high unless you are using a long, small-particle column.

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By Ameroid on Saturday, July 10, 2004 - 06:34 am:

Hi Tom, you are right on. 4700 psi is abnormally because when I began using it, the pressure registers only around 2500psi. The column has maintained this pressure until I took it out of the HPLC module for more than a month. When I hooked it back to the HPLC after that,the presurre shot up.

By the way, the column dimensions are:
Varian Nucleosil?C-18 non-polar column
100 ?average pore size, 3 micron average particle size,150 X 4.60 i.d. mm

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By Chris Pohl on Saturday, July 10, 2004 - 01:13 pm:

Ameroid,

I'm still a bit unclear as to why you are operating at high pH in order to analyze phenolic compounds. They can easily be done at neutral or acidic pH with good peak shape and thus avoid operating under conditions corrosive to the column material. I'm not sure you can conclude that your operating conditions are not responsible for your high-pressure just because you don't see a change in retention time. Did you store the column in your mobile phase when you took it out of service? If so, your high-pressure is probably connected to damage of the support particles. I think you would be much better off working under acidic conditions. At the very least, you should displace your working eluent and replace it with an unbuffered eluent in your column prior to storage. Otherwise, this will only exacerbate the silica corrosion problem.

The other problem you describe (low pressure, gradually moving back to the expected pressure) is symptomatic of a priming problem. Sometimes if the problem is due to small bubbles of gas it can be self-correcting. Do you have an online eluent degasser? If not, you may need to more thoroughly degas your eluent prior to operation in order to avoid such problems. If you pull a vacuum on your eluent container while simultaneously placing it in an ultrasonic bath, this is quite efficient at removing dissolved gas.

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By Ameroid on Monday, July 19, 2004 - 04:47 am:

Good day. Just a very simple question. Does it make a difference if i allow the mobile solvent to flow from either one end of the column? I observed a flow arrow on my column. If I do not follow the direction of the arrow, will I be damaging the silica packing?

Thank you

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By judd on Monday, July 19, 2004 - 09:23 am:

Switching directions should be fine in most cases. You should always be careful in your mobile phase and sample preparation to eliminate contaminants and particulates that can build up on the head of the column, however, as any significant build up can cause irreproducible results if you swap ends down the line. Always wash the column with a buffer-free mobile phase, then a "strong" solvent after use too, just to be sure it's clean. Your column longevity will astound you. I have a C8 column with well over 8,000 injections from 1997 that will STILL perform well. A chromatogram using that column is nearly indistinguishable from that of a new one and the quantitative results are similarly indistinguishable...and I swap directions all the time. No joke. YMMV, however.

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By Ameroid on Tuesday, July 20, 2004 - 07:05 am:

Judd, Thanks for sharing your experiences. I appreciate it :)