By Derek Menefee and Nathan Tompkins
MEASURING THE CHANGES OF
ANION CONCENTRATIONS OF
SALIVA
What is Saliva?
 Saliva is produced in response to stimulation
(usually taste) as well as movements of the jaw
muscles, joints, and pressure from chewing.
 It is produced mainly by three glands: the parotid,
submandibular, and the sublingual.
 Saliva controls the environment that the teeth are
in, as well as protects against bacteria, and helps
break down certain foods by using enzymes.
 Saliva is made up mostly of water, electrolytes,
mucus and enzymes.
What is Saliva Cont.
 Contains 2 main enzymes: Amylase (starch
breakdown) and maltase (maltose
breakdown)
 Saliva also contains bicarbonate, which acts
as a pH buffer by neutralizing acid production
and controlling plaque pH (Hogg, 2011).
What is being tested and
Why?

1.) Human Salivary Anionic Analysis Using Ion Chromatography Z.F. Chen,
B.W. Darvell and V.W. Leung
- 10 subjects were tested and analyzed for pH of saliva after they have
been fasting overnight.
- Saliva samples looked for concentrations of chloride, phosphate,
nitrate, sulfate, thiocyanate, and acetate were analyzed. Acetate ion
was the most abundant ion in saliva samples with nitrate as the second
most abundant.
 2.) Effectiveness In Learning Complex Problem Solving and Salivary Ion
Indices of Psychological Stress and Activation P.Richter, J.W. Hinton, S.
Reinhold
-33 students were tested for samples of saliva fasting and a rest, and
after being stressed after playing a challenging computer game for 2-3
hours.
-Studies showed that a change in ion concentration (increase in
potassium ion and decrease in sodium ion) in saliva occurred after
psychologically stressful activity (Hinton, 1998).
Our Experiment/Hypothesis
 In this experiment, involving the use of Ion
Chromatography, is testing the concentrations of
ions in saliva during different stimuli and analyzing
the changes (if any) that take place.
 The hypothesis is that the different samples that
may induce enzyme reactions, stimulation, and
changes in pH change may also result in changes in
ion concentrations in saliva after the sample is
consumed.
 Reference Instrument:
Varian Inductively Coupled Plasma
 Used to identify element intensities between saliva
after fasting and after ingestion of sample.
Materials and Methods
 Use of the Dionex ICS-90 instrument to run the samples
of saliva
 Parameters:
-Pressure: at 890 Psi
-Flow Rate: 1 mL/min
-Pressure Gauge: 9
 4 different experiments are run on the IC to determine
ion concentration of saliva after different stimuli
 Samples Used:
1.) Monster Energy Drink
2.) Protein/starch meal
3.) Vegetable/fruit meal
4.) Coffee (no sugar or milk added)
Materials and Methods Cont.
 A sample of saliva is taken from the experimenter, who




has been fasting for 10-12 hours prior to consuming
food or drink. This is done so the saliva may be used as
a control to compare with the sample analysis.
Experimenter must rinse with DI water before each
sample is taken as to not induce any false readings
from the sample itself, but only to obtain the salivary
reaction to the sample.
2 different samples are taken for each experiment (one
from each experimenter).
A pH sample is also be taken before and after
consuming to check for any differences.
The same procedure is also taken for the analysis of
the ICP instrument.
DATA
Nate Fasting Day 1 Sample
Anion
Injection Peak
Chloride
Nitrate
N/A
N/A
Phosphate
N/A
Ret. time
0.46
4.86
6.65
7.34
8.42
8.91
12.06
Peak Height
0.315
0.527
0.059
11.265
1.34
228.652
0.475
ppm
N/A
N/A
0.002
N/A
N/A
N/A
N/A
Nate Monster Sample
Anion
Injection Peak
Fluoride
Chloride
N/A
N/A
Phosphate
N/A
Ret. time
0.33
3.17
3.93
N/A
N/A
8.85
N/A
Peak Height
0.915
0.046
0.035
N/A
N/A
67.398
N/A
ppm
N/A
0.009
N/A
N/A
N/A
N/A
N/A
Anion
Ret. time
Peak Height
ppm
Injection Peak
N/A
N/A
Nitrite
Phosphate
N/A
0.49
N/A
N/A
7.5
9.08
N/A
.413
N/A
N/A
12.859
242.354
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Ret. time
0.49
N/A
7.33
9.02
N/A
N/A
Peak Height
0.268
N/A
20.031
465.608
N/A
N/A
ppm
N/A
N/A
N/A
N/A
N/A
N/A
Derek Fasting Day 1 Sample
Derek Monster Sample
Anion
Injection Peak
N/A
Nitrite
Phosphate
N/A
N/A
Derek Fasting Day 2
Anion
Injection Peak
N/A
N/A
Nitrate
Phosphate
N/A
N/A
Ret. time
0.37
N/A
N/A
N/A
8.97
N/A
N/A
Peak Height
0.365
N/A
N/A
N/A
350.996
N/A
N/A
ppm
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Derek Protein Saliva Sample
Anion
Injection Peak
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Retention Time (min)
4.83
6.53
N/A
7.30
N/A
N/A
8.57
10.28
Peak Hight (µS)
0
.40
N/A
18.442
N/A
N/A
454.834
324.391
Amount ppm
N/A
N/A
N/A
N/A
N/A
N/A
41.268
N/A
Nate Fasting Day 3 Sample
Anion
Injection Peak
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Retention Time (min)
4.84
N/A
N/A
N/A
N/A
N/A
8.65
9.78
Peak Hight (µS)
0
N/A
N/A
18.442
N/A
N/A
355.265
4.137
Amount ppm
N/A
N/A
N/A
30.238
N/A
N/A
30.238
N/A
Retention Time (min)
0.41
N/A
N/A
4.88
N/A
7.21
8.57
10.28
Peak Hight (µS)
0
N/A
N/A
18.442
N/A
N/A
N/A
324.391
Amount ppm
N/A
N/A
N/A
N/A
N/A
N/A
26.429
N/A
Peak Hight (µS)
0
N/A
.4
N/A
N/A
18.821
470.635
6.513
Amount ppm
N/A
N/A
N/A
N/A
N/A
N/A
42.092
N/A
Nate Vegetable Sample
Anion
Injection Peak
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Derek Day 3 Fasting Sample
Anion
Injection Peak
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Retention Time (min)
1.83
N/A
4.84
N/A
N/A
7.32
8.73
9.83
Derek Fruit Sample
Anion
Injection Peak
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Retention Time (min)
0.41
N/A
N/A
7.39
N/A
N/A
8.953
N/A
Peak Hight (µS)
0
N/A
N/A
22.285
N/A
N/A
343.433
N/A
Amount ppm
N/A
N/A
N/A
30.238
N/A
N/A
N/A
N/A
Retention Time (min)
2.83
N/A
4.39
N/A
N/A
N/A
5.20
N/A
Peak Hight (µS)
0
.40
6.674
18.442
N/A
N/A
244.846
N/A
Amount ppm
N/A
N/A
.383
N/A
N/A
N/A
N/A
N/A
Retention Time (min)
0.56
N/A
N/A
N/A
N/A
N/A
6.867
N/A
Peak Hight (µS)
0
N/A
N/A
N/A
N/A
N/A
323.026
N/A
Amount ppm
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
Derek Day 4 Fasting Sample
Anion
Injection Peak
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
Derek Coffee Sample
Anion
Injection Peak
Fluoride
Chloride
Nitrite
Bromide
Nitrate
Phosphate
Sulfate
ICP RESULTS
Nate Monster
Nate Fasting Sample
Element
Intensity
Al
278.88
As
Element
Intensity
Al
383.52
1.4510
As
2.0548
B
142.37
B
141.39
Br
1073.4
Br
960.38
C
138903
C
252743
Ca
19933
Ca
56832
Cl
2165.3
Cl
1948.5
Co
41.341
Co
40.517
Cu
152.12
Cu
159.73
Fe
155.54
Fe
161.35
H
454.17
H
194.54
I
119.43
I
270.18
K
2293814
K
1395923
N
742.58
Na
810814
Ni
30.703
O
4445252
P
13783
Pb
130.05
Pb
100.92
S
888.14
S
848.26
Zn
97.298
Zn
90.453
N
794.63
Na
306923
Ni
33.288
O
4357401
P
27941
Derek Fasting
Derek Monster
Element
Intensity
Al
278.88
As
1.4510
B
142.37
Br
1073.4
C
138903
Ca
19933
Cl
2165.3
Co
41.341
Cu
152.12
Fe
155.54
H
454.17
I
119.43
K
1395923
N
742.58
Na
810814
Ni
30.703
O
4445252
P
13783
Pb
100.92
S
848.26
Zn
90.453
Element
Al
As
B
Br
C
Ca
Cl
Co
Cu
Intensity
377.27
2.0997
149.15
1578.4
441089
45925
2046.5
41.636
156.63
Fe
H
I
K
N
Na
Ni
O
P
Pb
S
Zn
153.91
877.46
238.73
1927251
817.16
1839628
32.872
3829018
24472
97.376
2001.4
181.28
Discussion of Results
•In regards to the changes in ion concentration the only changes
witnessed were due to the pH and protein samples. However the
concentrations varied between the two different experimenters.
•In the first Nathan fasting sample a nitrate peak of .002 ppm appeared
at retention time 6.65 minutes. When the Nathan Monster Sample was
run the nitrate peak vanished and a fluoride peak of .009 ppm
appeared at retention time 3.17 minutes.
•Derek’s phosphate group nearly doubled in height after drinking the
Monster sample.
•Derek’s protein also showed an increase in peak height an a
concentration of 41.268 pmm
• Average number peaks obtained by a sample was 8.3
(lowest number being 6, highest number being 11)
•ICP was used to look at the changes in element intensity.
The ICP data for “fasting” to “Monster” matched up with the same
pattern of changes in the “fasting” to “Monster” data for the IC.
Changes in pH while testing
•Standard “Fasting” pH for Derek : 6.5
•Standard “Fasting” pH for Nathan: 7.2
•pH of Monster being used: 3.5
•Monster Sample pH for Derek : 6.0
•Monster Sample pH for Nathan: 6.8
•All other sampling’s pHs only moved by .1 or by nothing at all.
Things that could be changed and Improved upon
•Use a good 7 anion standard.
• Have all the test subjects drink the same water while fasting. Most likely
DI water.
•Consume the same exact meals
•Stress / sickness
•Time restraints, waiting on the IC to warm up. Having time to run more
samples on the ICP. Only running 4 samples a day.
•Better Storage.
Future Research
•More test subjects (to look at more samples, to see if there is more
sample similarities.
•do more test with varying pH’s , and use more sensitive pH tests.
•Calculate the actual amount of time needed to produce a “fasting
sample”. We could have run more than two samples a day, which would
have saved on time.
•Further speculation of different ions inducting thiocyanate and acetate.
Works Cited
 Chen, Z.F., Darvell, B.W., Leung, V.W.H. Human saliva
anionic analysis using ion chromatography. Archives of Oral
Biology. (2004) Vol 49 (11). Pgs. 863-869.
 Richter, P., Hinton, J.W., Reinhold, S. Effectiveness in
learning complex problem solving and salivary ion indices in
psychological stress and activation. International Journal of
Psycology (1998). Vol 30 (3). Pgs. 329-337.
 Hogg,Steve. Salivary Buffering, bicarbonate, and pH. The
Oral Environment. (2011)
http://www.ncl.ac.uk/dental/oralbiol/oralenv/tutorials/bicar
bonate.htm
 Kaufman, Eliaz., Lamster, I.B. Diagnostic Applications of
Saliva-A Review. International and American Associations for
Dental Research. (2002) Vol 13 (2). Pgs. 197-212.