SPE columns

Hello, i'm a Milan university student in thesis in an analytical lab.

I'm currently trying to develop an analytical method to isolate and analyze Niaprazine in GC-FID, by using as internal standard Phenacetin. But as i know we, in this laboratory, have no experience with this type of extraction!

I need the SPE extraction because Niaprazine is in a complex matrix (Syrup). 

Now the problem is that by doing the extraction using some spare SPE columns of agilent we have in the lab (From what i read the solid phase is a mixture of C18+cation exchange), it seems i don't retain those compounds, since the last extraction with a mixture of CH2Cl2 doesn't seem neither of my target analytes.(I used some standards to make sure it was not a GC-FID method problem, and it does seem to be an extraction problem.

First of all i'm not an expert of SPE extraction since i always studied those on paper, and applied already existing extraction methods, so i might be doing something wrong for sure. My professor suggested to use the Methadone's extraction method certified to work with this type of columns.

This is the Methadone's SPE method applied:

  1. Sample preparation: 0.5 mL Niaprazine std in MeOH + 0.5 mL Phenacetine in methanol + 2 mL of phosphate buffer pH 6
  2. Columns conditioning: 2 mL of Methanol + 2 mL of water mq (flow 1 mL/min)
  3. After the conditioning i take the whole sample solution (after Vortex), and i start the SPE extraction with a flow of 1 mL/min
  4. Washing of the columns: 1 mL of water + 2 mL of HCl + 1 mL of MeOH, then i let the columns in high vacuum for 5 minutes.
  5. After the 5 minutes i use 2 mL of a mixture of IPA+CH2Cl2 to extract the content of the SPE solid phase.

As i said when i try to do this procedure it doesn't work. 

Since i have no experimental background on this type of experiments, i decided to do some considerations.

I used CHCl3 to repeat the extraction on a column, and i understood that the problem was not the extracting solvent since on the solid phase of the columns my target analytes are not retained.

So i decided to do a further test, decided to do a TLC on the 1st extract and i saw that there my target analytes were not retained at all since the concentration of those in the solution was the same as the initial concentration in the sample prepared during the sample preparation.

Now since i wanted more evidence of this, i decided to prepare a STD of phenacetin in methanol with a buffer and did only the 1st step of the extraction. Then i did a GC-FID analisys and saw that the major part of this standard was in the solution below the column, so it was not retained at all.

So i did started questioning the method used since i was told to apply that, and it had to be working. 

After a little chat with my professor we thought that we could use another method by applying the nicotine extraction method from urine.

The difference from the methadone's method is that the columns are conditioned with the buffer pH 6 and not with methanol. I still need to test this, and i'll probably do it next week, but i wanted to know what you think about this.

I've done a lot of tests and it seems to be really hard to extract those two molecules with this type of columns. 

I have a feeling that a big chunk of the problem is the methanol used in the IS preparation, and in the washing phase. And i'm pretty sure it's him that, even tho i let the flow of 1 mL/min, doesn't let my target compounds to interact with the solid phase.

I'm really sorry for the wall of text, but i thought here i could find something to start from. If you can help me with anything i would really appreciate!

Do you think i'm using the wrong solid phase columns, or am i doing something wrong with the extraction methods, like wrong solvents, or wrong pH?

Ty for the help, and for reading all of this!

(e.g. When i say that i don't see Niaprazine and Phenacetine, i mean that i don't see them at all, like 0 concentration at their RT(verified by using standard samples on my GC column)

  • Hi Nia,

    not a good step to just use a random method on something else. A first step in the development of a new SPE method should be looking at the log D charts of your analyte and the standard, in your case it looks like this:

    And you see, that the hydrophobicity of niaprazine changes drastically with pH. You need a stable plateau to work with SPE or LC. For niaprazine this is the pH range 10-12, quite basic, or zero, which is not an option at all.

    A rule of thumb is, that you can use reversed phase interactions if the log P > 1.5 - here it is 2.21 for niaprazine and 1.41 for phenacetin (a border case). Remember log P is the hydrophobicity, i. e. the log of the partitioning coefficient of an analyte between oil and water (oil meaning the reversed phase interaction), 2.21 means your distribution is 1:162, 1.41 is 1:26.

    Still on an end-capped C18 SPE cartridge you have enough retention to separate both from aqueous carbohydrate matrix.

    At pH 10-12 niaprazine is not ionized and therefore an ion-exchanger will not work (and you would need a stable pH range somewhere for the ionized analyte).

    Elution from the SPE phase could be done by pure strong organic solvent like acetonitrile of methanol.

    Maybe this handbook can help you.



  • i Thank you for the answer, for future tests i'll keep that in mind!

    I already did some tests by looking at the pka, and found out the problem was methanol! 

    Now i have a way to reason about those things! 

    You're SUPER

  • One last thing may i ask how to get LogD tables? Is there a site where i can access them?

  • Hi Nia,

    most logD tool you need to buy, but there is a Chemaxxon demo site you can use.

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