By Bharat on Thursday, July 29, 2004 - 02:40 am:

Why PDA detector is less sensitive than UV detector.

Why Fluoresecnece detector is more sensitive than UV ?

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By DR on Thursday, July 29, 2004 - 06:37 am:

PDA detectors tend to be less sensitive than UV because of the way that they are designed to work. They are built to 'simultaneously' monitor a broad range of wavelengths. What they actually do is scan a series of narrow wave bands very quickly. This limits the dynamic range and sensitivity. (I'm not an expert, but this crude probably flawed explanation may be helpful)

That said, I have noticed that current PDA detectors (we just bought 2 Agilent systems) really do not suffer much in terms of sensitivity compared to their UV counterparts.
Older ones were a lot less sensitive than their contemporary UV detectors.

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By Bharat on Thursday, July 29, 2004 - 12:04 am:

What is difference between ODS-3 And ODS 3V column

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By Bharat on Thursday, July 29, 2004 - 12:02 am:

Dear Chromatographer,

What is the role of buffer concentration in HPLC mobile phase and what is the effect on Retention time of an analyte, if we increase or decrease the buffer concentration in mobile phase.Does it effect the pH of mobile phase. Is it there any significane of that parameter ?

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By readski on Thursday, July 29, 2004 - 11:26 am:

ODS-3V
GL-Sciences has developed a program to manufacture, test and guarantee validated HPLC columns that will provide high performance results column after column, lot after lot. In addition to the test certificate included with each analytical column, all validated columns are supplied with a packing material certificate including all the manufacturing and test data done for that material lot. Every step in the manufacture and testing of Inertsil ODS-3Vcolumns is critically controlled and monitored.

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By Uwe Neue on Friday, July 30, 2004 - 10:33 am:

The buffer concentration usually has only a very small effect on retention, even under critical conditions. Both an increase and a decrease in retention are possible. Usually, a higher buffer concetration means an increase in retention. As an example, a change in buffer concentration by 5% changed retention times by 0.15% for well behaved compounds.

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By SIELC_Tech on Friday, July 30, 2004 - 01:10 pm:

Our next newsletter which is coming next week is titled "Effect of Buffer Type and Concentration on Retention of Charged Analytes". Please contact us at mail@sielc.com if you would like to receive it.

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By MarkosP on Monday, August 2, 2004 - 02:34 pm:

Uwe,

Is your statement true for any compound and any HPLC column or not, I am observing decreased retention at higher concentrations of buffer (very small peptide, phosphate buffer, RP column with polar embedded groups)

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By Uwe Neue on Monday, August 2, 2004 - 03:19 pm:

Markos,

The study was done with small neutral molecules and bases. For the bases the retention shift was roughly 2-3 times larger. I do not have a study for peptides at hand. It is also possible that in your case the retention shift is caused by ion-exchange (decreased retention with increased buffer concentration) instead of RP.

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By HW Mueller on Monday, August 2, 2004 - 11:06 pm:

Markos,
are you sure that your higher concentration buffer has the same pH as the lower....? What are the retention time and concentration diffs?

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By MarkosP on Tuesday, August 3, 2004 - 10:12 am:

DEar HW

My concentrations are 20, 30 and 50 mmol with the same pH, I am seeing the difference in retention of up to 3 minutes on 150 mm column (20 vs 50 mmol)

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By HW Mueller on Wednesday, August 4, 2004 - 01:05 am:

A "salting out" effect would cause an increase of retention, as Uwe mentioned. A "salting in" effect would cause a decrease, now your concentrations are all in a range where one would expect a salting in effect, albeit I strongly doubt that this is causing your retention change. Your buffering capacity certainly should be quite low.... maybe there is a pH change involved? It would be interesting to see what happens if you keep your ionic strength constant by adding a neutral salt (I suspect you still would get the shift).

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By Bharat on Saturday, August 7, 2004 - 11:22 pm:

What is Type A and Type B silica column ?