[snowbunny]

I think this is the right place for this...Alright....I'm a master's student just starting my thesis project which is looking at nitrogen and phosphorus allocation in flower parts.  I know...biologists doing chemistry.... Anywhooooo... I'm using a protocol from a 1994 paper doing a similar analysis.  So I am digesting my flower parts (which have been dried and ashed at 500C and weigh from .2 to 1 microgram after drying) by adding 25 microliters concentrated (99.8%) sulfuric acid and heating at 80C for one hour, letting cool for 20 minutes, and then 50 microliters 30% hydrogen peroxide and heating at 80C for another hour.  The samples are then brought to 140 microliters total volume.  This is divided in half and 70 microliters is used for N analysis and 70 for P...Right now neither is working.  Soooooo.....problems with the N...

My protocol for this is 750 microliters DI water, 250 microliters 10% NaOH, 50 microliters Nessler's reagent (from Sigma) and then 70 microliters from my sample, mix and read at 425 nm on a spec.  So when I mix my sample and all this I get bubbles, which is not so hot for trying to read on a spec... 0.080 ...0.089...0.086....0.087...0.090...and so on...yeah bubbles kind mess up absorbency... who would have thought? Interestingly when I mix it in the clean pyrex test tube the bubbles stop after a few seconds.  As soon as I put it in the quartz cuvette, they start and will not quit.  So I thought maybe I can't get the cuvette clean enough and tried using methylcrylate disposable cuvettes (brand spankin new), nope same problem.  I have no idea what is reacting, but it's something in my digestion solution because when I try without the digestion solution things work perfect.  Next problem with this part, I'm getting turbidity and I don't know why. This has been steadily getting worse so I'm assuming it's my reagents, but I don't have a clue which one. 

Now for my issues with the phosphorus analysis...using the ascorbic acid method, 500 microliters water 500 microliters reagent C (2 part DI water, 1 part 6N sulfuric acid, 1 part 2.5% ammonium molybdate, 1 part 10% ascorbic acid) and 70 microliters sample.  Add the sample it turns yellow, and I mean not the reagent C yellow but bright a** yellow, so we think pH, start to tritrate.  HCl doesn't do anything so we go the other way with NaOH, get it to turn colorless but can't get the purdy blue color change we are looking for.  This protocol for this usually uses HCl for the digestion, so I'm assuming it's the H2SO4 causing the issue, but I'm not sure how to resolve this issue.

I so wish that I could use some different methods for this but I'm having a hard time finding any other method that will give the resolution I need without having to split my sample (samples are waaaaay to small to try to aliquot before digestion and too small for some of the other methods we have looked into) Anyway I'm about ready to rip my hair out and my chemistry knowledge is fairly limited so any help, ideas, suggestions, wild stabs in the dark would be greatly appreciated.

[Borek]

So when I mix my sample and all this I get bubbles

Hydrogen peroxide decomposition?

[snowbunny]

I was thinking that, but I don't get why it stops in the pyrex tube, but as soon as I put it in the cuvette it just goes and goes and goes forever

[MOTOBALL]

Reading between the lines, you are trying to perform trace analysis (x2) with procedures that you are not familiar with.

I would suggest that you first get very comfortable (and good) using a reference compound e.g. an aminoacid (ca. 1 mg) for N and H3PO4 (ca. 5 uL) for the P test.  When you can get accurate (say +/- 5-10%) and reproducible results (RSD<10%) for the knowns, then scale down in steps (say 100 ug, 10 ug, 1 ug) to the level of your flower parts.

When the results at the 1 ug level for the knowns are acceptable, then you should have resolved the issues with the method (reagent quality/ contamination/ handling techniques etc.).  Then go to the flowers.  Please let us know if you do get the assays working (or not).

[PS.  I would think that 80 C/1 hr would destroy the H2O2]