HPLC

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amit_chem
amit_chem's picture
HPLC

Hi sir,
my question is ,I am using mobile phase (0.02 M kH2PO4 in water ,and adjust the pH to 6.8 with tri ethyl amine) I got the all 8 stages separated of our scheem.the peak shape & resolution is also good.
But is it ok combination of KH2PO4 & TEA for seting pH 6.8.
Or I should change the combination for getting pH 6.8.

Dr. Analytical
Dr. Analytical's picture
The purpose of using a buffer

The purpose of using a buffer is to control the pH and the ionization state (neutral or ionized) for an acid or base.  How you make the buffer is usually not important.

However, TEA is also used to cover the surface of the stationary phase, which often gives better results for basic compounds.  But new stationary phases are much better now and TEA is not necessary with many columns.  You can continue to use it or you could try makeing the adjustment with NaOH.

The only problem with using NaOH is that the phosphate salts are not soluble in acetonitrile, so if you have too much acetonitrile in the mobile phase you might precipitate the buffer.

amit_chem
amit_chem's picture
Thank you sir, for u r reply.

Thank you sir, for u r reply.
I also try,0.02 M K2HPO4 in water and adjst the pH 6.8 with ortho phosphoric acid.
I got the similar peak pattern for all the satges and rt also near about same.
In these two cases which mobile phase is better for analysis. and also for column life.

Dr. Analytical
Dr. Analytical's picture
I do not think that there is

I do not think that there is a difference in stability for the two buffers.  However, the TEA buffer is more complex (and it smells bad ), so I would prefer the simple buffer.

To get the maximum lifetime for a column, observed the following procedures:

1. Filter your buffer after you have adjusted the pH.
2. Never let the flow stop for more than a short time (a few hours) when there is buffer present in the system.  You should always have a slow flow or flush out the buffer with water if you do not need to use the column.
3. Regularly flush out the system with water.  This step will help extend the lifetime of your pump also.

NNGG
NNGG's picture
Hello,

Hello,

I am using shimadzu and using a colum of 4.6mm C18 .
But  the pressure at the specified flow exceeds the maximum pressure and
even the peak area fluctuates with each chromatogram.

Please suggest.

Thank you

Dr. Analytical
Dr. Analytical's picture
Please start a new discussion

Please start a new discussion topic.

Also, include more information about your column dimensions, the mobile composition, and operating conditions (flow, injection volume, detection).  What compounds are you trying to measure?