Stainless steel needle
Answers
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Mike:
Needle surfaces do help, I would try the 100%, but I would like to review your method and conditions. There could be several easier recomendations before swapping the needle. CAn you share the gradient and a little about the compound...neutral, hydrophobic etc. small or large molecule.
Thank you,
Liz
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Thanks Liz
The analytes are Levetiracetam, Lamotrigine and Topiramate. It is basically this method here:
http://www.waters.com/webassets/cms/library/docs/720002302en.pdf
The only thing different is I added 0.1 FA to improve resolution and sensitivity.
A: 10mM Ammonium Acetate with 0.1%FA
B: Methanol with 0.1%FA
The gradient starts off at 98:2 to 25:75.
I have another method that uses 5 mM ammonium acetate and ACN where I have the weak (A) and strong (B) wash lines in. So I tried moving the strong wash to MEOH with 0.1% FA. Long story short it makes no difference which one I used and increasing strong to 300 uL and weak to 900 uL made no difference either.
The only thing that reduces carryover is not using PLNO, just partial loop, and injecting as little as possible. The issue there of course is that limits my lower limit of detection.
Attached is a TIC followed by water. Concentration of 10 ug/mL Levetiracetam, and 20 ug/mL Lamotrigine and Topiramate. I'm injecting 3 uL.
thx
Mike
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Mike:
Couple of things, PLNO mode has better recovery than Partial Loop mode, but as you are loading more sample will exhibit more potential for carryover than Partial loop where the sample is diluted with weak needle wash and you only deliver the aliquot specified. So in this case where it seems to me to be a relatively high concentration on a your MS, although you do not say what type. Just check my math, but you are loading 60/30 ng on column. Am I correct?
Alternately in PLNO as the weak wash is never co-injected you can have two different selectivities in your washes. So I would make the strong very strong and give the waek a different selectivity. What is your sample dissolved in and what would desolve it best? That's what should go in the strong wash. I will ask some of our chemists for some guidance here. I would tend to put strong organic IPA/Methanol/THF, and even more formic acid (2%) in the Strong wash just cleans the loop and fluidics. The weak wash we can tune a little and again I will get some alternative selectivity here. However, I would like to see what your sample diluent is and the gradient table too and also the size of your loop?
Many TX
Liz
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Hi Liz
The concentrations I'm injecting is 0 to 50 ug/mL. The ion abundance is 10e5 to 10e6. The (neat) samples are in 98:2 ammonium acetate:methanol.
The gradient is as follows:
A: 10mM Ammonium Acetate with 0.1% FA
B: Methanol with 0.1% FA
Flow rate is 0.45 mL/min, column temperature 50°C, injection volume 2 uL (partial loop). The sample loop is 10 uL
Time %A %B
Initial 98 2
0.5 98 2
3.0 25 75
3.5 5 95
4.0 5 95
4.1 98 2
5.0 98 2
It was just suggested to me by Crystal Holt to try adding IPA to the strong wash. So I made up a 40/40/15/5 solution of MEOH/ACN/Water/IPA. Using this for strong wash in addition to injecting no more than 2 uL in partial loop mode (no overfill) greatly reduced carryover. In fact there is very little or none at all now even after the highest calibrator.
regards
Mike
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Mike:
So I think two things helped as you stated, using Partial loop and making the strong wash very organic, Acetone, THF, IPA all have been used. Methanol is a stronger solvent than ACN. Also acidifying/adding base is also really helps, (which helps by formation of ion pairs by introducing counter ions.
But a final note, you will note that the poster had a longer run time and a different flow rate, so more volume and a shallower and longer organic hold were experienced by the column. To get to the 25:75 conditions with a 2.1 x 50 mm column at 0.6 mL/min they used a volume ( 5.5 mins at 0.6 mL/min) across that was both bigger and shallower and would have been very good at prolonging the greater organic conditions. Your method, traded this for speed, so when you get carryover, sometimes you simply haven't eluted the compounds off all the sites. Your final hold conditions at 5 /95 lasts for 0.5 mins or 225 uL, same for your return to aqueous, a total volume of 1.4mins or 630 uL that is significantly less than 5 column volumes (2.1x50 = 174 uL) + x3 system volumes the rule of thumb for re-equilibration.
Often the tradeoff for reducing carryover is cycletime, right now you have the best of both worlds for these compounds that did solubilize successfully in the strong wash. Sometimes really sticky compounds you need a good organic hold and a good and strong wash. But this was too good an opportunity to pass up to illustrate the issues around carryover, so please forgive me.
Best reagrds,
Liz
Liz
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