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 Post subject: Retention Time Shift.
PostPosted: Wed Jun 06, 2007 6:12 pm 
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Joined: Thu Nov 02, 2006 11:57 pm
Posts: 16
Location: Loma Linda, CA
My mobile Phase is 50% methanol, 40% water, and 10% buffer. Flow is 1.0 ml/min. It is premixed. My Instrument is Agilent 1100 with quaternary pump. If I let the quaternary pump mix the solution with same percentage, I see a change in retention time from 2.95 to to 4.05. I checked each pump's flow rate at 1.0 ml/min and It is accurate to within 2%. However If I checked the flow rate using 2 pumps; A and B,(50/50 methanol water) my flow rate drops 20%. Is there a problem with my mixing valves? How do you fix this?

Jamie


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 Post subject:
PostPosted: Wed Jun 06, 2007 9:52 pm 
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Joined: Tue Jan 11, 2005 11:37 pm
Posts: 974
Location: Sunnyvale, CA
When you wrote "pumps" I assume you meant "channels." First, is your degasser working? bubbles could be forming in at the proportioning valve which would cause low, irregular flow. Your gradient proportioning valve could be bad. Try moving the water to a different channel and try the flow rate at 50:50 again.

There is a routine test for proportioning valves where you put 0.1% acetone in B & D, and put clean water in A & C. Monitor UV at 270nm. Then run a stair-step program at 0, 1, 5, 50, 95, 99, 100 and compare the levels of the steps to a theoretical gradient; do A-B and C-D.

Your buffer might be precipitating in the proportioning valve and the crystals get stuck in the check valve. If your mobile phase is right on the edge of maximum solubility, this can happen.

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Mark Tracy
Senior Chemist
Dionex Corp.


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 Post subject:
PostPosted: Thu Jun 07, 2007 9:56 am 
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Joined: Thu Jul 07, 2005 12:38 pm
Posts: 130
Location: Denmark
Also make sure that all your channels are purged and filled with solvent of some sort... even if your only using 2 channels all 4 have to be purged. Water or MeOH will do fine.

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Leadazide


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 Post subject:
PostPosted: Thu Jun 07, 2007 10:49 am 
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Joined: Sat Sep 24, 2005 10:54 am
Posts: 31
Location: INDIA
Dear Jamie

I fully agree with Marc Tracy . If u cannot check that you may

Please call service engineer of your HPLC & ask him to check Board of Pump whether any malfunctioning is taking place electronically . ( Basically solvenoid valve functioning is proper or not is to be checked )

Also ask him to check Composition of Pump which is most of the time probable reason for Retention time shifting .

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SUNIL PANDYA


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 Post subject:
PostPosted: Thu Jun 07, 2007 9:45 pm 
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Joined: Thu Nov 02, 2006 11:57 pm
Posts: 16
Location: Loma Linda, CA
Mark Tracy:

Thank you for your input. I tried other channel combinations and measured the flow rate. Here are the results with the first letter as methanol and second letter as water:

% flow rate

AB-82%
AC-82%
AD-82%
BC-83%
BD-82%
CD-100%

It appears that the use of channels A and B results in lower flow rate.
I will perform the step gradient next.

Jamie


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 Post subject:
PostPosted: Fri Jun 08, 2007 3:47 pm 
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Joined: Thu Jul 07, 2005 12:38 pm
Posts: 130
Location: Denmark
If you have an online degasser try purging and cleaning channel A and B.. It could very well be the reason!

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Leadazide


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 Post subject:
PostPosted: Fri Jun 08, 2007 4:11 pm 
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Joined: Fri Jun 08, 2007 2:24 pm
Posts: 30
<B>Jamie,

This shows channels A & B are having trouble. You can try following things.
1. Purge channels A & B first with 100% water each for few minutes followed by 100% IPA.
2. Repeat the same with 50-50% each channel.
3. Rinse the channels with water & try using your mobile phase.
4. If the problem persists try passivation as suggested by manufacture.

If the above steps doesn't work. The gradient valves needs replacement. </B>

Regards,




Jamie Wrote:
% flow rate

AB-82%
AC-82%
AD-82%
BC-83%
BD-82%
CD-100%

It appears that the use of channels A and B results in lower flow rate.
I will perform the step gradient next.


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 Post subject:
PostPosted: Fri Jun 08, 2007 4:46 pm 
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Joined: Tue Aug 17, 2004 7:59 pm
Posts: 1549
Location: 38.6075°N 90.3493°W
Just a note - 1100s are available in 4 channel single pump and 4 channel binay pump flavors. So, if your phases are arriving via different pumps, you could have a problem w/ just one of them.

Looking at the data just above this, I'm betting that you have a dual pumper with a blown seal or stuck check valve in the A-B side.

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Thanks,
DR
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 Post subject: Step Gradient Test
PostPosted: Fri Jun 08, 2007 8:15 pm 
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Joined: Thu Nov 02, 2006 11:57 pm
Posts: 16
Location: Loma Linda, CA
Hi All:

Before I implement your suggestions let me report my results for the step gradient test I did. Channels A and C are pure water and channels B and D are filled with 0.1% acetone in methanol. The steps I performed are 0,25,50,75,100% of the acetone solution. The graph was printed on a 3395B Agilent integrator and the height of the percentages was measured in mm measured manually.

A=Water and B= .1% acetone

25%= 27 mm
50%= 55 mm
75%= 78.5 mm
100%= 98.5 mm

C=water and D= .1% acetone
wavelenght= 270nm; Flow= 1.0 ml/min

25%= 27.5 mm
50%= 53 mm
75%= 79.5 mm
100%= 98 mm

I do not know if these numbers are good enough but I can see that they are slightly higher than the predicted height based on the height at 100%.

I have done enough HPLC to predict that if the aqueous part of my mobile phase increased from 50% to 55% the retention time of my hydroquinone peak should not increase from 2.95 to 4.05 minutes. And I certainly will do a premix solution of 55% aqueous to see just how much will change.

I did not expect the C-D channels test to be so close to the results of A-B test since the flow rate C-D channels was accurate.

I will implement your cleaning suggestions. I do not know if the tests that I have done have somehow started the cleaning of the proportioning valves.
If you have a different insight on the data presented above thank you for sharing them.

Jamie


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 Post subject:
PostPosted: Fri Jun 08, 2007 8:21 pm 
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Joined: Thu Nov 02, 2006 11:57 pm
Posts: 16
Location: Loma Linda, CA
DR:

I have the model G1311A. It is 4 channel with a single pump that has 2 pistons. At least that is what I see from the outside and the diagram in the manual.

Jamie


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