By Anonymous on Thursday, June 19, 2003 - 08:40 pm:

I have a problem with high pressure in HPLC. High pressure is not coming from the column. And it's not a problem with pressure transducer. When disconnect the column, the pressure is not high. But when connecting the column, the pressure is high (twice as it should be). When connect this column with another HPLC unit, the pressure is just normal. I'm using Shimadzu LC-10AD pump.

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By HW Mueller on Thursday, June 19, 2003 - 11:44 pm:

Could be a bad fitting that closes partially when connected to the column?

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By ab on Friday, June 20, 2003 - 12:54 am:

Is this problem occuring just with this particular column? Maybe the source of the high pressure is not the column, but a tubing after column, or detector... Try to disconnect the column's outlet and if pressure doesn't drop change the tubing before the column (as sugested by HW Mueller).

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By Anonymous on Tuesday, July 15, 2003 - 01:47 am:

I have the same problem. Also, I'm using Shimadzu pump. This is not a problem of bad fitting.
Cutting the end of tubing did not affect the system pressure.
I'm washing the pump unit for three days. But the pressure is high.
I'm very confused.

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By Chris Pohl on Tuesday, July 15, 2003 - 12:29 pm:

If, as mentioned above in the original post you are observing higher pressures than expected but only when the column is connected, it would seem that the only logical explanation is a calibration problem on your pump transducer. I've never used a Shimadzu pump, so I can't comment on whether or not the transducer can readily be recalibrated, but I would check the manual to see if there is an SOP for recalibration of the pressure transducer.

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By Anonymous on Tuesday, July 15, 2003 - 06:03 pm:

If you use dual pump, check the pre-mixer.

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By Recep on Thursday, July 24, 2003 - 01:17 am:

Did you checked the flow rate from the output of the column via a measuring cylinder?
May be a flow rate problem.

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By tom jupille on Sunday, July 27, 2003 - 03:18 pm:

Could the orginal poster clarify a point:

does the problem occur with *all* columns on this system or only with *one particular* column?

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By Anonymous on Sunday, February 8, 2004 - 09:18 am:

Retention time change with reverse phase HPLC using buffer m.p.

Hi all,

I am seeing the following issue and need some insight.

In a number of tests I am seeing retention time shifts all peaks(of 0.7 mins, 7 hours into an 11 hour run) with both the standard and sample and during other tests (maybe not related to first issue) a pressure increase.

The mobile phase is: a buffer soln,(10g of citric acid in 1L, pH 7.4)/ACN/Tetrahydrofuran (53/27/20).
Filter the MP with 0.45um nylon filter.
The test is run at ambient temp.

Note:
The system is cleaned (after buffer, 30mins water then 30mins 50/50 ACN/Water).
Column is (C18 5u 4.6 X 250mm), System is a HP Agilent LC system.

Questions:
- Can the temperature of the room affect the retention time?
- Should a buffer m.p be continuously stirred while running the batch of tests (11 hours long)? I think that the retention time change may have to do with the composition of the m.p changing.
- With the pressure increase; do you think the salt could be precipitating? Any good pointers on this?


Thanks in advance for any help. Chris.

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By Uwe Neue on Sunday, February 8, 2004 - 04:08 pm:

Your buffer is not very good. Retention can be affected by room temperature. Is the retention going up or down, and does the direction agree with the room temperature change? The typical change with temperature is around 2% per degree C.

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By Anonymous on Sunday, February 8, 2004 - 04:58 pm:

Hi Uwe,

many thanks for your response.

Retention time is increasing. Very interesting rule of thumb I assume you mean inverse(ie -2%/C).

One line of thinking I am following is that the salt is coming out of solution. If this were happening over time the column would gradually become blocked, causing an increase in pressure. Would this cause the retention time to increase? Some tests have been aborted by the system because of a leak (perhaps because of high pressure eg blockage). We have already adjusted the flow rate from 1.5 to 1.4 ml/min.

Question: would keeping the mobile phase stirred constantly prevent the m.p. precipitating? Any other ideas?

Thanks Chris.

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By Uwe Neue on Monday, February 9, 2004 - 03:53 pm:

Hi Chris,

Retention is increasing, pressure is increasing... Both would agree with a change in temperature, i.e. the room is getting colder. Evaporation of the organic solvents with an increase in pressure and retention is possble as well, but with a 3-component mixture like yours there are no data that predict the viscosity. I have seen citrate buffers clog frits, but this results in either unchanged retention or even a decrease in retention due to a slight heating of the mobile phase.

Since you got a continuing problem, one may exclude the temperature. If it were getting colder, you would have realized it by now yourself and put a coat on, I guess...

Pressure increases can also affect the flow rate. This can compensate for the temperature change mentioned above.

If the frit is the problem, can you replace the frit?

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By anu gupta on Thursday, June 10, 2004 - 11:19 pm:

hi
can any one tell me how to prepare citrate and phosphate buffers with molarity 10-20mM having ph of 3-5.

any reply will be highly appreciated.

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By Anonymous on Friday, June 11, 2004 - 09:28 am:

There are a number of free buffer calculators available on the web. For example:

http://www.zirchrom.com/pass.asp

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By Uwe Neue on Friday, June 11, 2004 - 12:16 pm:

Phosphate is not a buffer at pH 4.