High Spike Recovery problem

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Lysander's picture
High Spike Recovery problem

Hello there guys
I have a problem with High spike recovery- It's been consistent- yet on high side- (104.5%)
My calibration curves with two component matrix that I'm running couldn't be looking better-I'm getting 99.99%
My Mobile phase is  Isocratic and it is : 90% Methanol, 8% Water  and 2% of  Phosphoric Acid( 0.5%)  
So the Mobile Phase pH is about 3.75 to 3.95 and my  0.5 % Phosphoric Acid is about 1.95%- It is too low in my opinion-
My column is C18 15cmx 4.6 mm x 5 Phenomenex
My flow rate is about 1 ml/min and my dtecetion in my ten minute run changes at 5.65 minutes from 310 nm to 357 nm
My other compound of interest is Avobenzone-
The pH of the finished product should be between 6.8 and 7.2 and the two analytes are coming on right time according to the method. However; I'm getting lower numbers on my Octinoxate compound compare to a different lab. There is a 0.4 % difference in our results. My low recovery numbers are ok but the high spike recovery numbers are around 104.5%. So, I'm getting almost 0.4% less in my assay compare to a differnt lab , but my hish spike recovery is about 104.5%.Aside from this my System Suitability test is failing ecause of low number of Theoretical plates.
Some Ideas that could  alter to make my theoretical plates higher numbers and my hish spike recovery within 98-102 ?
Thanks in advance guys!

Dr. Analytical
Dr. Analytical's picture
Please give us some

Please give us some information on your system - manufacturer,  modules, injection volume, column temperature and exact column description.
There are several things to try now. 
First, let's start with the column efficiency.  When you say you have "low" efficiency, what does that mean?  What is your plate count?  Are the peaks tailing or not?  Can you analyze a test mix or other simple solution to see if your column is bad?  Can you compare the peak shape with the other lab? If you can find some other test mix, or even something simple like toluene or benzene, inject that solution and observe the peak shape.  Also calculate the plate count, and compare it with your analytes of interest.  Have you "cleaned" the column lately?  Flush it with 100% acetonitrile for about 30 minutes.
Your analytes (avobenzone and octyl methoxycinnamate, right?) are not acidic or basic, so the pH adjustment will have no impact on their retention.  But if you want to simplify your mobile phase, I will usually prepare a 0.1% solution of phosphoric acid in water, and mix this with my organic solvent.  Or you could even try not using the acid at all.  The pH of your sample should also not be important, but I hope you are dissolving/diluting the sample in mobile phase.  If not, try that next.
These steps should get your system working as well as possible.  If you system suitability still does not pass, send us a chromatogram.
For your spike recovery, give us some details on your spiking procedure - concentration, volume, etc.  Have you tried a spike into just a mobile phase blank?  This will verify that your concentration, calculations, and procedures are correct. 
Try some of these things and then write back.
Thanks for posting your question on scientistsolutions.com.

Lysander's picture





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Dr. Analytical
Thank you so much for replying back sir
My system is Dionex  with following 4 components
 Low Pressure P680 Pump  
ASI 100 Auto sampler- PDA 100 Detector and STS 585 Column Compartment
Dionex  names  it a tower system- with Chromeleon 6.4
The injection volume is 10 µl and the column is LC- ABZ  Supelcosil  15 cm x 4.6 mm x 5 µ
I  can't recall what was the number on the plates but the SST in summary page shows that it's failed. With that being said; my resolution, peak symmetry, capacity factor and signal noise are all ok.
(I will give the plate number when I get back to my lab)
Now then,
The peaks are not tailing and the column is relatively new with less than 500 injections on it. I received the column in September of 2007. I haven't checked the other labs chromatograms and I have cleaned my column every single time by running 100% MeOH for 15-20 minutes or so.
I was thinking maybe the mobile phase  pH is too low and therefore; that's why my system suitability is giving a low count number- ( I don't know why this is happening). The samples are being dissolved in the mobile phase.
For Low Spike recovery - I'm diluting my sample with water
and for high spike recovery-
 I'm making a sample with high concentration dissolving both standards in MeOH  ( weight/weight) and  afterwards make a more concentrated sample.
Then  I'm running the samples and calculate the 
< Actual in my sequence run / Theoretical  x  100> and that is how I get the recovery
The Low Spike recovery for the AVB has been 99.5 and for OMC it's been 100%
The high spike recovery for the  AVB has been 100% and for OMC it is about 104.5%.
The high spike recovery is been consistent. But like I said-
The weight percent calculation shows my OMC at 5.65% where as the other lab reports 5.9% and  the spec sheet asks between 5.4 and 6.6 - So we both are in range - But intermediate precision is about 0.35% off.
Is this something I should worry about?
And lastly
No Dr. A
I have not tried spiking into mobile phase by itself- Maybe I'll do that this coming Monday.
The RSD values  for both calibration curves are less than 2.0%- in fact,  way below 2.0% and the R2 values are 0.9999.
Have a good weekend guys- I'm going to drink some wine and sleep!

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

I do not think that pH is the problem here.  As I mentioned, I regularly run my systems with 0.1% phosphoric acid.  You could try to remove the acid completely, just to check, but I do not expect it to help.  In rare cases, some columns are less stable in acidic solution, but I do not think your column would behave in this way.
I notice that your low and high spikes are in different solutions (low in water and high in methanol).  Try spiking into mobile phase at both levels, and compare the results.

Lilly Ashton
Lilly Ashton's picture
Spiked sample hplc

I spiked a bark sample with 5ml of alkaloids the extraction was over 100% why?