Questions:
- for the modified NaOH digestion: add only sulfuric first, or also
h2o2?
- For the measuring mL by weight, did she just add 30 g or did she do
the proper calculation?
Modified Phosphorus Fractionation
Prep Work
To begin with, I made 5 L of 0.5M NaHCO3, 0.1 M NaOH, and 1M HCl, and 6 L
of 0.9M H2SO4. When I had finished all four fractionations, I had extra of all the
reagents, especially the 0.9 M H2SO4, but some of my modifications to the
method may account for the difference. Calculating the amount of reagent
needed is straightforward: you will need 30 mL of NaHCO3 for each sample,
blank, and check, as well as enough to make the NaHCO3 calibrants (15 in total);
30 mL of NaOH per sample, blank, and check, and enough to make the 15 NaOH
calibrants; 30 mL of HCl per sample, blank, and check, as well as enough
reagent to make the 5 HCl calibrants. Depending on how you approach the
determination of organic Phosphorus for the bicarb and NaOH fractions, you will
need either ~20 mL or ~30 mL per sample, blank, check, and calibrant. Be sure
to err on the side of caution when deciding the volume of reagent to make,
especially if you intend to do multiple fractionations from the same batch of
chemicals -- you will need extra to rinse soil off filters and should assume that
you will lose some along the way.
**0.5M NaHCO3: dissolve 42 g NaHCO3 + 0.5 g NaOH to 1 L with nanopure DI
0.1M NaOH: dissolve 4 g NaOH to 1 L with nanopure DI
1 M HCl: bring 88.5 mL concentrated HCl to 1 L with nanopure DI.
0.9M H2SO4: bring 50 mL concentrated H2SO4 to 1 L with nanopure DI.
Once you’ve decided how many liters you need, make the appropriate
multiplication (all the formulas above are based on 1 L), and prepare your
extracts. Add roughly half the volume of water you’ll need to the container you’ve
chosen, then add your chemical or acid, and finally bring to volume. Be sure to
prepare the acids in the hood.
**The 0.5M NaHCO3 should have a pH of 8.5, although anything between 8.45
and 8.55 is acceptable. To adjust the pH of your solution, prepare 1M NaOH, and
add dropwise. It’s easiest if your flask is being stirred on a stir plate with a pH
meter electrode suspended in the solution. Be sure to leave enough time
between additions; it may take between 30 seconds and a minute for the latest
addition to be dispersed throughout the solution and for the pH meter to regeister
the change.
The number of calibrants seems overwhelming at the start, but the reason for all
of them will become clearer as the fractionation progresses. A basic set of
calibrants, made with inorganic phosphorus - 0.1 ppm, 0.5 ppm, 1.0 ppm, 1.5
ppm, and 2.0 ppm - is needed for each reagent (NaHCO3, NaOH, and HCl). The
different calibrants are made in the different reagents (e.g. inorganic P +
NaOH, organic P + HCl, etc.) not ultrapure DI water. An additional 10
calibrants are required for the bicarb and NaOH fractions because both inorganic
and total P are determined at these steps. The initial range of calibrants made
with inorganic P is all that is needed to find inorganic P for each fraction. The
digestion of the bicarb and NaOH extracts that determines total P dilutes our
samples by a factor of 3 (10 mL of H2SO4 + 5 mL of sample) and requires
another set of calibrants. For both NaHCO3 and NaOH, we also make 0.3 ppm,
1.5 ppm, 3.0 ppm, 4.5 ppm, and 6.0 ppm calibrants for both inorganic and
organic P. The organic calibrants function as a check to see how well our
digestions work. (When it is time to analyze your data, you will compare your
organic calibrants to the inorganic calibrants to find a percent organic recovery,
which you will use to correct your data, thereby taking into account organic
matter that was not completely digested).
When making calibrants, I began with two stock solutions: one of 100 mg
inorganic P/ L and one of 100 mg organic P/ L. Both my stock solutions were
made with nanopure DI. The directions for making the inorganic P stock solution
are in the total P section of the Alpkem manual. Making the organic P stock
solution requires calculating what percent (by weight) of the organic-P-containing
compound is actually phosphorus. The chemcial formula of the disodium 6Fructose salt that I used was C6H11O9PNa2.
By summing the atomic weights of each element, I found the weight for the whole
molecule.
6 C + 11 H + 9 O + 1 P + 2 Na = 6*12 + 11*1 + 9*16 + 1*31 + 2*31 = 304
The fraction of P by weight = 31/304 = 0.10197. Using the formula
0.10197x=100mg, I calculated the amount of disodium 6-fructose salt I would
need to make a solution that contained 100 mg of P. However, 100/0.10197 =
980.68 mg, a quantity much larger than the amount that I had purchased. I could
maintain the same concentration of P, but make a much smaller volume of stock
solution if I had 25mg of organic P in 250 mL of nanopure DI.
100 mg/1000 mL = 25 mg/250 mL
This required 245.17mg of 6-fructose. If 0.10197x = 25 mg, then x = 25/0.10197
= 245.17.
Because the bottles that I made my calibrants in could only hold 60 mL, the
weights of stock solution that I used for each calibrant were based on a 60 mL
total volume.
For instance, using the equation V2 = (C1 * V1) / V2 a 1.0 ppm calibrant requires
.6 mL of stock solution because (1 mg/L * 60 mL)/ 100 mg/L = 1*0.6 = 0.6 mL. If
the bottles you’re using hold a different volume, calculating new weights is easy
using the above equation and substituting a new value in the place of V1.
*
As is standard, I added the appropriate amount of stock solution and then filled to
volume with the appropriate reagent. I did not record the actual weights of stock
solution that I used; although this didn’t end up causing any problems when I was
creating calibration curves in Winflow, I nonetheless recommend recording the
weights.
Procedure
Day 1: I weighed out 0.5 g soil (+/ - 0.0025g) into 50-mL centrifuge tubes. I
recorded the weights in a blank digestion table. I labelled this first set of tubes,
which is used throughout the fractionation, on both the side and lid with my
initials, the sample number, and a number that indicated which fractionation it
was (either 10, 20, 30, or 40). 10 is the first, 20 the second, and so on. The
remaining tubes were labelled only on the sides and according to the following
numbering system. In each case, the first digit represents the fractionation and
the second digit a particular fraction.
*
Using a styrofoam centrifuge tube holder on top of one of the lab scales to hold
individual tubes, I placed the tube without a lid on the scale, tared it, and then
added 30 mL of NaHCO3 (measured by weight). When NaHCO3 had been
added to all the samples, I designated two tubes as blanks and added NaHCO3
to them. I was not consistent with the amount of reagent I used to make up
blanks, and I generally added closer to 50 mL than 30 mL because it seemed like
it might be advantageous later to have more blank. Then because I had only 60
mL total of each of my calibrants I put 20 mL of calibrant in the centrifuge tubes
that had been marked for calibrants.
During the first fractionation I ran all 15 NaHCO3 calibrants. For later
fractionations I used only the high and low. It was a mistake not to have enough
of the calibrants to run the high and low calibrant in all four fractionations. For my
fourth fractionation, I used the second highest and lowest calibrants instead, but
this inconsistency made it difficult to calculate percent inorganic and organic
recovery later. When all the tubes were ready I put them on the shaker and left
them to shake overnight (for a total of 16 hours).
Katherine Tully 9/20/2007 1:40 PM
Comment [1]: By the weight of NaHC03?
Or to 30g?
Day 2: I came in about 45 minutes before the tubes would finish shaking to rinse
the filters with nanopure DI water. I placed funnels over a set of tubes that I
always used for rinsing, then wearing gloves, I wet the enter surface of the filter
with nanopure DI water using a squirt bottle, then folded the filter into quarters,
and set in the funnels to drain. By the time the tubes were off the shaker and had
been centrifuged the filters and funnels were ready to use. Once the samples
were off the shaker, I put them in the centrifuge for 10 minutes with the speed set
above 4000 rpm to ensure that it would go its maximum speed (somewhere
around 3700 rpm). While the tubes were in the centrifuge, I would label the next
set of tubes (11, 21, 31, or 41) with my initials, the sample number, and the
fractionation ID number. When one batch was finished in the shaker, I would
transfer the funnels and filters onto the newly labelled tubes and empty the tubes
from the centrifuge into the appropriate centrifuge tubes. The old tubes, which
still contained the sample, I would set aside. I always had enough samples that I
needed to do a second batch in the centrifuge (it onlys holds 16 tubes at a time).
Although there was nothing to separate in the blanks and calibrants, I centrifuged
them as well, so they would undergo the same process as the samples.
While the samples, calibrants, and blanks were filtering I would weigh out 0.5g of
ammonium persulfate on a piece of weighing paper for each sample, calibrant,
and blank. The weighing out does not have to be particularly accurate -- Tiessen
said that they used a constant volume scope. If your samples are not being
completely digested, Tiessen suggests that you add more ammonium persulfate.
Once that was done, I would calibrate a large pipette to 5 mL and transfer 5 mL
of sample into glass test tubes that had been labelled (with the number 12, 22,
32, or 42, my initials, and the sample number) and had a mark showing 15 mL.
Blanks and calibrants were also included in this process. The next step was done
in the hood. There I added 10 mL of 0.9M H2SO4 in four parts: 1 mL + 1 mL with
a pipette, then 4 mL + 4 mL with a repipettor. This is a fairly reactive process,
and the danger of spilling is high because the glass tubes are so small. Be
careful. Once the additions were complete, I covered each tube with a square of
tinfoil, and autoclaved them for 60 minutes.
Next, I rinsed the dirt from the filters back into the original test tubes. To do this, I
placed the old test tube on the scale, returned the funnel and filter, and tared the
weight of the whole. Then I removed the tube, funnel, and filter from the scale,
placed a drop of 0.1M NaOH on the filter, and used a straight, blunt, metal tool to
poke hole at the base of the filter. Then using a transfer pipette with 0.1 M NaOH
I proceeded to rinse the dirt from the side of the filter back into the test tube.
When all the dirt had been washed down, I threw away the old filter, and set the
funnel aside. Then I filled the tube to 30 mL by weight with 0.1M NaOH. I made
sure the tubes were tightly capped, so they would not spill on the shaker, and
also shook them by hand until the dirt at the bottom had loosened. After I had
poured the NaOH calibrants into a labelled centrifuge tubes and made up two
blanks with NaOH, everything was ready to shake overnight. It is important to
label the blanks and calibrants at this stage with the reagent, so you can keep
them apart later on.
Finally, the samples that have been autoclaved need to be filled to 15 mL with
nanopure DI.
Day 3: The process today is very similar to that on Day 2. I rinsed the filters
before the samples had finished shaking. I labelled a new set of tubes, while the
samples were in the centrifuge. Once the centrifuged tubes had been emptied
into the next set of tubes (13, 23, 33, or 43) and the extract was filtering, I
weighed out 0.6 g of ammonium persulfate. Following the same procedure, I
added 5 mL of sample (as well as calibrants and blanks) and the ammonium
persulfate to the labelled glass test tubes. Then in the hood I added 10 mL of
0.9M H2SO4 in the same order as before: 1mL + 1 mL with the pipette, and 4 mL
and 4 mL with the repipettor. I always found that this reaction was much less
reactive than it had been with the NaHCO3. These samples were then covered
with tin foil and autoclaved for 90 minutes. Both times I tried this method, not all
my samples were competely digested. The second time, I added more
ammonium persulfate, as Tiessen had suggested, but it didn’t fix the problem.
His other suggestion was to do a normal digestion with these samples instead of
doing the persulfate digestion in the autoclave. I found this much quicker and
more effective than the persulfate digestion and would recommend it. When
doing a regular digestion, I added 5 mL of sample, blank, or calibrant to a
digestion tube, a boiling chip, and then I added 5 mL of concentrated sulfuric
acid. I placed these on a warm block and heated to 360 degrees. i removed them
from the block, let them cool for 15 minutes, and then added 0.5 mL of H2O2.
Placed them back on block for 30 minutes. Removed and cooled for 15 minutes,
added 0.5 mL of H2O2, returned to block etc. It should only take 1 or 2 additions
of hydrogen peroxide to have the digestate turn clear. On several occasions I did
unneccessary additions because I wasn’t sure if my samples had been
completely digested. When the digestion was over I filled the tubes to 75 mL with
DI water, covered, inverted several times, and left over night to settle. I found that
filling to 75 mL made many of the samples too dilute. In the future I would
recommend only filling to 50 mL, and making calibrants for this digestion the
same way you would for a normal digetions (i.e. based on 50 mL). The calibrants
that you made initially will be very dilute, although you’ll still need to incorporate
some inorganic/organic pairs to calculate recovery.
Once your extracts have been digested you need to finish by rinsing the filters.
This is done the same way as before. The funnels and filters are returned to the
original tubes, and the dirt is rinsed off with 1M HCL. Once the dirt has been
washed into the orginial tube, the filter is discarded and the tube is filled to 30 mL
by weight with HCl. Again you must add new tubes for the calibrants and blanks.
These are all, then, set to shake overnight.
Day 5:
Katherine Tully 9/20/2007 1:43 PM
Comment [2]: No H2O2 in this step?
As before, the tubes are removed from the shaker, centrifuged, and filtered
through rinsed filters into a new set of tubes (labeled 15, 25, 35, or 45, with my
initials, and the sample number). While the extract is filtering, you must wash the
soil from the original test tubes into digestion tubes using nanopure DI. It is
difficult to get the soil out of the centrifuge tubes, especially with very little water.
You want to use as little water as possible (the method says 10 mL) because all
of the water you add needs to evaporate before the digestion can proceed. When
forced to choose between using only 10 mL or leaving soil behind, however, I
always decided to use extra water and get as much soil as possible. For this
digestion use calibrants and blanks just as you would for a normal total P
digestion. When all the soils has been washed into tubes, add 1 boiling chip, and
place on cold block. Although it will be very slow, it’s important to put your tubes
on a cold block because they will boil over easily and you need to give them as
much time as possible to have the water evaporate. You will need to watch these
carefully because they will try to boil over often. I found that I had to turn the
block off several times as it was heating to 360. When all the water has
evaporated, remove from block, cool for 15 minutes and 0.5 mL hydrogen
peroxide. When I did these digestions, I would 0.5 mL of hydrogen peroxide
several times and then begin adding 1.0 mL of hydrogen peroxide because that
was what I had seen in Deborah’s notes. Although this eventually seemed like a
bad idea, I felt like I couldn’t stop once I had started for consistency’s sake. In the
future I would recommend, adding 1.0 mL two times at the beginning and the
switching over to 0.5 mL for the remaining additions. You could always only ever
add 0.5 mL of hydrogen peroxide but that lengthen the process by several
additions. when this digestion was complete, I would fill to 75 mL, cover, invert,
and leave over night to settle. Again, I would recommend, only filling these to 50
mL, so as not to dilute your samples too much.
Degassing:
I degassed my bicarb and NaOH PI fractions after I had completed the rest of the
fractionation, but you could always do it as you go, if you feel you have enough
time. To degas, pipette 10 mL of solution (from the tubes marked 11, 21, 31, 41,
or 13, 23, 33, or 43) into a 50 mL centrifuge tube. For the bicarb solution, add 6
mL of 0.9M H2SO4. Again, the NaHCO3 tends to be reactive so you want to add
it slowly. I think I added 1 mL and then 5 mL slowly. Do what works best for you possibly 1 mL + 1 mL + 4 mL. The NaOH is much less reactive. To it you add 1.6
mL 0.9M H2SO4. You can add it all at once, although it’s always a good idea to
do it slowly. After the 0.9M H2SO4 addition, cap the centrifuge tubes loosely and
place in the fridge for 30 minutes. Then centrifuge for 10 minutes. The
centrifuging will separate the precipitate from the extract. Once the centrifuging is
completed it’s important to pour off the supernatant into an acid washed
scintillation vial. The scintillation vials we use at the end of the digestion do not
have to be acid washed because they get rinsed twice; these do have to be
washed because they are not rinsed.