The document summarizes experiments conducted by students to identify unknown solutions labeled 150 mM NaCl, 0.4 mM NaN, 0.25 mM Tyrosine, and pH: 7.4 0.4 mM PBS. Through tests such as flame, Griess, pH, and ninhydrin tests, the students determined Unknown 1 was NaNO2 and Unknown 4 was tyrosine. However, they were unable to identify Unknowns 2 and 3 conclusively. Further tests on samples to identify 16k prolactin and sodium nitrite contamination levels were also summarized.
1. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Problem Statement: Identify the true identity of the labeled solutions.
Label of each Solution
150 mM NaCl (Unknown 1)
0.4 mM NaN (Unknown 2)
0.25 mM Tyrosine (Unknown 3)
pH: 7.4 0.4 mM PBS (Unknown 4)
Procedural Analysis and Experimentation
We made the following solutions for reference in our experiments: sodium nitrite, sodium chloride, PBS,
and tyrosine. The given samples were treated as unknown solutions and were tested parallel to the known
solutions and controls in most of the tests.
Solubility testing w/
Reasoning: Solubility can be used to differentiate between inorganic and organic molecules in solution. A
common example is the introduction of olive oil into a solution of water, where a clear separation of the
two can be observed. The reason for this is that organic molecules and inorganic molecules are insoluble
in an aqueous solution, and separate visibly. We however did not use this test because tyrosine in the
unknown was in aqueous form. The low concentration of tyrosine might have caused it be soluble in
water. However, if the concentration of tyrosine was extremely concentrated, it would not be soluble in
water. Therefore, we could not use the solubility testing with water.
Flame Test
Reasoning: The flame test identified the presence of sodium. Initially, we used our previous knowledge of
sodium combustion properties to presume that NaCl, NaN, and PBS would show an orange flame. We had
no presumptions to make about the reaction of tyrosine with the flame test and as a result we set up a
control that verified there was no color flame.
2. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
We did do this test. We used solid forms of the four known solutions for reference and our positive
controls of each known to compare with the unknown solutions.
Presumption: (NaCl, NaN, PBS) + Flame Yellow Flame
Pos.Controls Test Result (+/-)Neg.Control Test Result
(+/-)
Unknowns Test Result (+/-)
NaCl Solid + Water - #1 +
NaN Solid + #2 +
Tyrosine Solid + #3 +
NaCl Aqueous Solution + #4 -
NaN Aqueous Solution +
Tyrosine Aqueous Solution -
PBS Aqueous Solution +
Analysis: This test helped us to distinguish Tyrosine from the other unknown solutions. This was the first
test that insinuated the possibility of unknown #4 being mislabeled PBS.
Carboxylate Acid Identification Test
Reasoning: The carboxylic acid identification test detects the carboxylic acid that reacts with sodium
hydrogen carbonate to form carboxylic salt precipitate, water, and carbon dioxide gas.
We decided to follow through with this test but had no conclusive results.
Presumption: Organic Molecule + Sodium Bicarbonate Carboxylic Salt formation
± Controls (Tyrosine & O) Known solutions (PBS, NaCl,
NaNO3)
Unknowns #1, #2, #3 & #4
- - -
Analysis: The reaction occurred at such a small scale within the solutions due to the low concentrations
that we could not detect precipitation in the known and unknown solutions. We were unable to use this
test.
3. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Griess Test
Reasoning: The purpose of this test is to detect NO2–
ion in solution.
Presumption: Griess Reagent + N Violet Product
Neg.
Control
(H2O)
Pos.
Control
(NaNO2)
Known
NaCl
Known
PBS
Known
Tyrosine
Unknown
#1
Unknown
#2
Unknown
#3
Unknown
#4
- + - - - + - - -
Analysis: Unknown #1 showed violet color with the presence of Griess Reagent as well, so it has nitrite
ion in it and it is NaNO2.
Unknown #2, #3, and #4 did not change their color. They are probably NaCl, PBS and Tyrosine.
pH Meter Test
Reasoning: We reasoned that this test would distinguish NaCl, and PBS from NaN. We went off of the
presumption that NaCl and PBS are both very close to neutral pH and NaN is a base. From the molar
calculations we expected NaN to have a relative pH of 11, making NaN distinguished among the rest of
the solutions.
We decided to follow through with this test because it was a quick and reliable. We did an qualitative test
with pH paper and did a quantitative test with the pH meter.
4. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Presumption: NaCl neutral, NaN basic, Tyrosine acidic, PBS neutral
+ Controls pH Unknowns pH
NaCl 7.53 #1(Supposedly NaCl) 6.13 ± 0.1
PBS 9.37 #2 (Supposedly NaN) 5.69 ± 0.1
NaN 4.99 #3 (Supposedly Tyrosine) 7.24 ± 0.1
Tyrosine 7.88 #4 (Supposedly PBS) 6.96 ± 0.1
Analysis: The pH test only measures the pH of each solution and does not demonstrate its chemical
properties such as PBS’s ability as a buffer. The pH of each solution was not representative of trustworthy
data so the test was inconclusive.
Ninhydrin Test
Reasoning: The purpose of the Ninhydrin test is to isolate and identify the presence of an organic
molecule in solution. This works when the Ninhydren reacts with the amino acid in the organic molecule
which then changes the pigmentation of the solution to a dark blue color.
Presumption: Ninhydren solution + Carbon Chain -- OH Blue Product
- Control (O) + Control
(Tyrosine)
Unknown #1 Unknown #2 Unknown #3 Unknown #4
- + - - False + (yellow) -
5. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis: We used our Tyrosine solution as a positive control for the test because tyrosine changed the
color of the solution to blue. From there we did the experiment with each of our unknowns and observed
results. There was slight change in pigmentation present, yellow and eventually copper after cooling,
when we introduced our unknown labeled Tyrosine. We assumed that was a confirmation for the presence
of tyrosine but this was an incorrect assumption. After further and research, we found an article talking
about the effects of PBS on ninhydrin produce a yellow product, courtesy of the phosphate ion being
released during the heating period of the test. This confirmed that PBS was in the bottle labeled Tyrosine.
Chlorine Ion Test
Reasoning: Chlorine and phosphate in the presence of silver will from a white precipitate. The amount of
precipitation depends on the amount of chlorine present.
Presumption: NaCl + AgN ACl precipitate & PBS + AgN AgP precipitate
- Control
(O)
+ Control
(NaCl)
Known
PBS
Known
NaNO2
Known
Tyrosine
Unknown
#1
Unknown
#2
Unknown
#3
Unknown
#4
- + + + - + + + -
6. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis: Since unknown 1, 2, and 3 formed a precipitate, we could infer that unknown 4 is tyrosine.
Nitrite Test
Reasoning: The nitrite test is intended to isolate the nitrite ion in solution and initiate a reaction with
copper sulfate that would result in a blue color of varying intensities. The chemical reaction for this
system is shown below:
200 µL NaN (aq) + 800 µL HCl (aq) NS(aq) + HN(aq)
NS (aq) + HN(aq) + 500 µL CS (aq) Dark Blue Product
We decided to follow through with this test.
Presumption: NaN + HCl NS + HN + CS Dark Blue Solution
+ Control
NaN
(.4mM)
+Control
NaNO2 (.628
M)
Known
PBS
Known
NaCl
Known
Tyrosine
Unknown
#1
Unknown
#2
Unknown
#3
Unknown
#4
+ + - - - + - - -
7. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis: The initial concentration of the control solution was not high enough to continue the reaction to
completion which resulted in a lighter blue than the unknown. A separate control with high concentration
had the same hue of blue as the unknown 1. With this test, unknown 1 is observed to be NaNO2.
Precipitation test involving predetermined amounts of concentrated HCl and Co
Reasoning: The purpose of this test is to differentiate between the only two remaining unknowns left:
NaCl and PBS. The test includes two steps. The first step is to express the presence of phosphate ion in
PBS solution. The second step is to form precipitate of Co3(PO4)2 in the PBS solution while NaCl solution
is still aqueous.
Presumption:
Chemical reactions for the first step (with HCl 0.1mM)
- NaCl does not react with HCl
NaCl (aq) + HCl (aq) Aqueous solution containing Na+
, H+
, and Cl–
ions. (1)
- PBS (Na2HPO4 / NaH2PO4) reacts with HCl to form PO4
3–
ions.
HP (aq) + HCl (aq) Na+
Cl–
(aq) + 3H+
PO4
3–
(aq) (2)
8. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
NaH2PO4 (aq) + HCl (aq) --> Na+
Cl–
(aq) + 3H+
PO4
3–
(aq)
Chemical reactions for the second step (with CoCl2 0.7mM)
- (1) does not react with CoCl2:
NaCl (aq) + HCl (aq) + CoC(aq) Aqueous solution containing Na+
, H+
, Cl–
, and Co2+
ions
- (2) reacts with CoCl2 to form precipitate:
2PO4
3–
(aq) + 3Co2+
Co3(PO4)2 precipitate
The presence of Co3 precipitate will be used to conclusively identify which solution is PBS.
Known solutions Precipitation Unknown solutions Precipitation
NaCl - 1 -
PBS - positive control - 2 -
NaNO2 - 3 -
Tyrosine - 4 -
Water -
Analysis: Theoretically, the precipitate Co3(PO4)2 should have formed in the PBS and the positive control,
but there was no form of precipitation in any solution . To explain this result, we think about the
concentration of the unknowns, HCl and CoCl2 were so small so that the precipitation was form but not
clear enough to observation under naked eyes. Without any notable results, the test was inconclusive and
could not be used in identifying the unknown solutions.
Buffer Test
Reasoning: A buffer is able to neutralize the amount of acid or base added to the solution whereas a
neutral or acidic or basic solution cannot.
Analysis: We were unable to obtain complete data for this test and could not make any conclusions.
Conclusion:
9. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
For our observations we could conclude that unknown 1 is NaNO2and unknown 4 is tyrosine.
Unfortunately, our observations were not sufficient enough to identify unknown 2 and 3.
Problem Statement:
Griess Test
Procedure: 20 µL of the sample was pipetted into 20 µL of Griess reagent.
Reasoning: Griess Reagent reacts with nitrite ions in solution to produce a purple product. This test will
show if samples that probable amounts of NaN contamination.
Griess Reagent + N Violet Product
Results:
11. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis:
BCA Test
Procedure: 20 µL of the sample was pipetted into 20 µL of 50:1, BCA Reagent A: Reagent B solution.
Reasoning: BCA reagent solution reacts with Amino acids in solution to produce a purple pigmentation.
This test will identify the presence of both amino acid and polypeptides thus can be positive for Tyrosine
and/or 16k Prolactin.
BCA Working Reagent + Amino Acid Violet Product
Results:
Solution Test Result (+/-) Concentration Level
(Scale 0 – 5)
Tyrosine (0.25mM)
(Positive Control)
+ 5
13. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Bradford Test
Procedure: 20 µL of the sample was pipetted into 20 µL of Bradford reagent.
Reasoning: The test alters from red coloration to blue to determine the amount of protein present in the
solution by ionizing the proteins to expose its hydrophobic pockets within the tertiary structures. This test
helps separate the samples with only protein from samples that include both proteins and amino acids.
Bradford Reagent + Polypeptide Blue Pigment
Results:
Solution Test Result (+/-) Concentration Level
(Scale 0 – 3)
Bovine Serum Albumin
(Positive Control)
+ 3
Water
(Negative Control)
- 0
Sample #1 - 0
Sample #2 + 1
Sample #3 + 1
Sample #4 + 1
Sample #5 + 3
Sample #6 + 3
Sample #7 + 3
Sample #8 + 3
Sample #9 + 2
Sample #10 + 2
Sample #11 - 0
14. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Sample #12 - 0
Analysis:
Analysis: Based on the results of our testing we came to the conclusion that 16k prolactin spans from
sample 2 to sample 10, where the peaks from the BCA and Bradford Tests overlap. Based on the range of
the Sodium Nitrite contamination curve we can infer that there is low and moderate Sodium Nitrite
contamination in the heavy 16k prolactin saturated samples (namely samples 5 through 8). Our plan is to
treat the salvaging process in four segments, samples 2 through 4, samples 5 and 6, samples 7 and 8 and
lastly samples 9 & 10.
15. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Recovery Period: BCA Test
Procedure: Refer to previous page for procedures. The only alterations were the testing of the filter and
solution separately.
Reasoning: Refer to previous page
Results:
Round 1 Round 2 Round 3 Round 4 Round 5
Group A
Solution
1 1 2 2 2
Group B
Solution
1 1 2 3 3
Group C
Solution
1 1 2 3 3
Group D
Solution
1 1 1 2 2
16. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis: The BCA test increased as the cleaning of the solution progressed and indicates that the tyrosine
in the protein was being washed off into the solution.
Recovery Period: Bradford Test
Procedure: Refer to previous page for procedures. The only alterations were the testing of the filter and
solution separately.
Reasoning: Refer to previous page.
Results:
Round 1 Round 2 Round 3 Round 4 Round 5
Group A
Filter
1 2 2 2 3
Group B Solution N/A 0 0 0 0
Group B Filter 3 3 3 3 3
Group B Solution N/A 0 0 0 0
Group C Filter 2 3 3 3 3
Group C Solution N/A 0 0 0 0
Group D Filter 2 3 3 3 3
Group D Solution N/A 0 0 0 0
19. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis: The Bradford test demonstrates that the protein became more concentrated as we continued to
wash out the contamination. Also, the zero concentration of the solutions concludes that there was no
protein that was washed out into the solution.
Recovery Addition Period: Griess Test
Procedure: Refer to previous page for procedures. The only alterations were the testing of the filter and
solution separately.
Reasoning: Refer to previous page.
Results:
Round 1 Round 2 Round 3 Round 4 Round 5
Group A
Filter
0 0 0 0 0
Group B Solution 1 1 1 1 1
Group B Filter 1 1 1 1 0
Group B Solution 1 1 1 1 1
20. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Group C Filter 2 1 1 1 1
Group C Solution 2 2 2 2 2
Group D Filter 2 2 1 1 1
Group D Solution 2 2 2 2 2
23. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis: The nitrate continued to be washed off the protein, which was shown by the low numbers for
the filter of each group, and was added into the solution, which was shown by the high or increasing
numbers for the solution of each group.
Cleaning Period: Griess Test
Note: The nitrate continued to be washed off the protein, which was shown by the low numbers for the
filter of each group, and was added into the solution, which was shown by the high or increasing numbers
for the solution of each group.
25. Shrishti Bhattarai, Monica Nguyen, Doan Pham, Ryan Taylor
Analysis: After calculating the probable starting concentrations using a system of equations based on the
known concentration of the BSA sample we prepared and the number of microliters we where able to
retain, we concluded that we were able to salvage about 1725 µg from group B (samples 5 and 6), 1112
µg from group C (samples 7 and 8), 866 µg from group A (samples 2 through 4) and 833 µg from group
D (samples 9 and 10)
*should data be included?*
In conclusion we learned to identify potential chemical entities, purify chemically contaminated protein
and quantitate an approximation for the amount of protein that we recovered. All in all we feel that this
experiment was success and we were successfully able to salvage the “16k prolactin”.
