Lars de Bruin
5V1, Dominicus College Nijmegen
April 2nd 2009
Summary It’s known for quite a while, that the body
Rumor has it that, after you cook spinach converts nitrate into nitrite by itself. But we
twice, it would become toxic. But how can also discovered that nitrate is converted to
spinach become toxic? nitrite when vegetables are cooked! So,
The substance in spinach that would be when we cook vegetables more often,
toxic, is nitrite. The molecule formula for even more nitrate is being converted into
nitrite is NO2-. The molecule formula for nitrite!
nitrate is NO3- and as you see, these two Nitrite is a toxic substance. The
molecules are very much alike. The reason why nitrite is toxic: is that it causes
hypothesis is the following: nitrate is being oxygen to less easy to assimilate from the
converted to nitrite when cooked twice. blood. Nitrite can, with some specific
To test this hypothesis, we cooked proteins, form the really toxic nitrosamine.
spinach in water after let it weak in water. These nitrosamine are carcinogenic. And
One beaker is filled with uncooked that’s the main problem when cooking
spinach. This beaker serves as a spinach twice: these carcinogenic
reference. nitrosamines. We’ll explain these
The second beaker contains spinach that nitrosamines further now.
is cooked once and the third beaker
contains spinach cooked twice.
The idea is that we can indicate nitrite in
this third beaker because there is twice
cooked spinach in it.
A long time ago people thought that you
could warm up vegetables as long as you
wanted. And even as often as you wanted.
But after a period, people discovered that Nitrite
there often was a huge amount of nitrate (http://www.chemistry.wustl.edu/~courses/genchem/
in vegetables. And then people also
discovered that this gave some risks.
Nitrosamines can be formed under certain
circumstances. They are formed out of
some nitrites and secondary amines,
which are often in proteins.
The circumstances in which proteins are
being transformed to nitrosamines, are
often there when spinach is cooked. But Let’s find out…
how is this working exactly?
Quoted from http://en.wikipedia.org/wiki/Nitrosamine
“Under acidic conditions the nitrite forms
nitrous acid (HNO2), which is protonated
and splits into the nitrosonium cation N=O+ Experimental procedure and approach
and water: H2NO2+ = H2O + NO+. The
nitrosonium cation then reacts with an Well, what have we done exactly?
amine to produce nitrosamine.” Right here, our work method, in items:
These nitrosamines are often in nutrition. Buy some spinach at the
But the government made some laws for supermarket
limits of nitrosamines that may be in the
nutrition. Store the spinach in the fridge
There are though many cases of cancer
due to this nitrosamines in trivial food. Pulverize the spinach
How are nitrosamines toxic? Weigh the spinach
These nitrosamines are being converted
into formaldehyde and later to a
carbenium ion. These are metabolites that
can disturb the replication in cells. These
can namely connect to our DNA. Due to
changes they make in the DNA, there can
occur tumours by reading mistakes.
Besides, nitrosamines can easily pass a
cell membrane and can than cause
problems anywhere in the body.
Don’t cook 50,17 grams spinach
Cook 49,94 grams of spinach once
Cook 50,00 grams of spinach
Drop all these in 400 mL of water
Well, will spinach be toxic after cooking
once and even more after cooked twice?
We do think so, yes.
Cook the spinach for two minutes
Curvet NO2- H2O Buffer
Sift it after two days Nr. (mL) (mL) Sulfanil-
Filtrate it after sifting 1 0,0 5,0 2,0 1,0 2,0
2 1,0 4,0 2,0 1,0 2,0
3 2,0 3,0 2,0 1,0 2,0
4 3,0 2,0 2,0 1,0 2,0
5 4,0 1,0 2,0 1,0 2,0
6 5,0 0,0 2,0 1,0 2,0
7 5,0 0,0 2,0 1,0 2,0
8 5,0 0,0 2,0 1,0 2,0
9 5,0 0,0 2,0 1,0 2,0
10 5,0 0,0 2,0 1,0 2,0
11 5,0 0,0 2,0 1,0 2,0
12 5,0 0,0 2,0 1,0 2,0
Table 1 Measuredata
Take an aquarium Table 1 shows how much we need
test for the samples for Nitrite testing.
Make the samples for the nitrite
Make a calibration curve with a
Thin the cooking water
Measure the amount of NO2- with
this calibration curve.
Results Curvet Concentration Transm. Extinction
Nr. NO2- (mg/L) I (%) Log(I0/I)
Figure 1 shows the concentration of 1 0 100 0
Nitrite. The diamond shows the standard 2 0,100 86 0,066
Nitrite test. The 2 squares show the test 3 0,200 69 0,161
we did. In table 2 you see the transmission 4 0,300 53 0,276
we measured with the spectrophotometer. 5 0,400 41 0,387
The results are that the spinach we 6 0,500 36 0,444
cooked once the same concentration has uncooked 1 7 0,367 46,5 0,333
as the spinach we cooked twice. Spinach
Cooked 1 8 0,072 86 0,066
Concentration Nitriet 2x Test
Standard Nitriet Test y = 0,9053x
Cooked 1 9 0,072 86 0,066
Test 1 R2 = 0,9888
0,50 not Test
0,45 Cooked 2 10 0,119 78 0,108
Cooked 2 11 1,877 2 1,699
0,25 2x Test
0,20 Cooked 2 12 1,350 6 1,222
0,10 Table 2 Measureresults
0,05 We converted this to the extinction en then
0,00 to the concentration NO2- (mg/L). The
0,00 0,10 0,20 0,30
spinach we haven’t cooked has a bigger
concentration, but this sample is thinned
Figure 1 Concentration Nitrite 50 times. The other samples, spinach
cooked once and twice, are thinned 200
times. The final results, the concentration
times the thin factor, are showed in table
Data analysis 3.
The table shows the test results. We did 2 Concen
tration Thin Concentration
tests, but we only used test 1. The first test NO2-
makes a difference to the second test. We (mg/L) factor NO2- (mg/L)
did the first test after 2 days when we Spinach Test
uncooked 1 0,367 50,0 18,367
cooked the spinach. We did the second Spinach 1x Test
test after a week because the first test was cooked 1 0,072 200,0 14,471
Spinach 2x Test
not reliable. There was a big difference cooked 1 0,072 200,0 14,471
between the results of test 1 and test 2.
We think that test 1 is the most believable, Table 3
because we think that there is more nitrate
converted to nitrite in the week we did the We think that the concentration NO2-
second test. (mg/L) are not reliable, because the
concentration is very high. We can’t
compare it with the standard nitrite test.
When we pulverized the spinach, there
was lost some spinach next to the beaker.
Also after weighing the spinach, there was
spinach falling next to the beaker.
The results are, due to that, not reliable on
Cooking is off course a subjective
occurrence. Sometimes you find the water
cooking earlier on than the other time. So
maybe the spinach is not cooked exactly
The samples we made for the calibration Bibliography
curve were hard to make. We needed a lot
of substances and it was not easy to http://www.steljevraag.nl/viewquestion?
measure the right amount of it. That can questionID=970
also be gone wrong.
The colour of the samples of our test were http://www.food-info.net/nl/qa/qa-
not blank. The water was green and light fp7.htm
green of the samples. We think that that’s
the reason that the aquarium test didn’t http://nl.wikipedia.org/wiki/Nitriet
give the right results.
The problem we discovered, was that time http://en.wikipedia.org/wiki/Nitrosamine
was really important in our experiment.
When the spinach was laid down for a http://www.translate.google.com
long time in water, especially when it was
cooked, there was being formed more and
more nitrite. That’s also the main reason
why we did not get reliable results out of
Yes, there is formed nitrite after cooking
spinach once and after cooking twice.
There was formed the same concentration
Nitrite by once cooking as by twice
cooking. This means that cooking spinach
twice is not worse as cooking spinach
once. The best, if looking to the quite toxic
nitrite, is to eat spinach uncooked.
For further research, we have important
Keep eye on the time: you need to do the
tests fast after each other because time is
a really important factor in this research.
You need to filtrate the spinach better than
we did because the green colour of the
spinach itself gave not reliable results
when doing things as an aquarium test.