determination of ph, ca 2+ and mg 2+ in saliva
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Determination of pH, Ca 2+ and Mg 2+ in Saliva. Cody Chalker David Julia Real World Project Fall 2009. Electrolytes in Saliva. Na + K + ,Ca 2+ Mg 2+ (mM): Cu 2+ Pb 2+ (µM), Electrolyte Conc. affected by flow rate and disease Only bound and free Ca 2+ can be measured simultaneously - PowerPoint PPT PresentationTRANSCRIPT
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Determination of pH, Ca2+ and Mg2+ in Saliva
Cody ChalkerDavid Julia
Real World Project Fall 2009
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Electrolytes in Saliva
• Na+ K+,Ca2+ Mg2+(mM): Cu2+ Pb2+ (µM),• Electrolyte Conc. affected by flow rate and
disease• Only bound and free Ca2+ can be measured
simultaneously– 10% bound to proteins, 40% to phosphates,
citrates and lactates, 5% to bicarbonate; Thus approx. 45% is in ionic form
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Bicarbonate and pH• Bicarbonate a product of cell metabolism • Bicarbonate does not buffer it mainly
neutralizes• Bicarbonate concentration in saliva is linked to
flow rate
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Reasons for testing pH of saliva• The ability to monitor health status, disease
onset and progression, and treatment outcome through noninvasive means is a highly desirable goal in health care promotion and delivery.
• Oral fluid is a perfect medium to be explored for health and disease surveillance.
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Importance• A healthy individual should have a saliva pH of 6.5-
7.4• Most ailments such as cancer, kidney stones, heart
disease, etc. are associated with an acidic pH in the body
• As you age the pH of your saliva changes• Over half of adults are acidic at 6.5, symbolizes signs
of aging i.e. calcium deficiency.• Most children are around a pH of 7.5• Most cancer patients have a pH of 4.5 while in
terminal condition.
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Why Hasn’t This Been Studied Before?
• One of the main reasons this is just becoming a research topic is that the amount of ions in saliva are so small that not until recently were techniques used to be able to make affirmative quantitative assumptions
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Techniques Used Today• Microfluidics and Microelectromechanical systems
are MEMS used for salivary diagnostics
• MEMS are integrated systems composed of mechanical elements, sensors, actuators and electronics on a common silicon substrate developed through micro fabrication technology
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Other Techniques Used Today
• Electrochemical methods have been devised• Thiocynate is an example of an ion being
studied• Process includes high performance liquid
chromatography, silica gels, and ion specific electrodes
• Pros of procedure: that it is quick, precise, and requires few samples
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Techniques Used• pH of saliva: – Saliva was taken from volunteer who were
asked to build up approx. 1-2mL of saliva which was then extracted with a disposable pipette. The saliva was then immediately immersed in paraffin oil
CO2 + H2O H2CO3 HCO3- + H+
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pH cont’d• To finish testing the pH of saliva 8mL of water was
added to each sample. The water added was boiled for an hour and found to have a pH of 6.75
• pH glass-electrode probe was calibrated with different pH buffers to obtain pH values 4,7, and 10
• Following the calibration the pH probe was immersed into each sample
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Determination of Magnesium and Calcium
• Determination of the two ions cannot be found exclusively through an EDTA titration since both Magnesium and Calcium complex to EDTA
• Volunteers were asked to collect as much spit in their mouths(approx. 5mL), sans mucus, and spit only once into the beaker
• 8mL of buffer-indicator was added. Standard was made of 8mL of water and 1mL of indicator.
• Standard was titrated with EDTA solution to determine approximate end point. Each sample was titrated to an end point
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Determination of Magnesium
• Thiazole yellow was used as an indicator• Ammonium Oxalate was used to precipitate
out calcium • UV Vis instrument
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Procedure for Use in UV Vis• Saliva samples were mixed with ammonium
oxalate and centrifuged (suspected problem area)
• 0.75mL of each sample was added to cuvettes. 0.75mL additions were added of water, polyvinyl alcohol, thiazole yellow and of NaOH
• The UV Vis was calibrated with magnesium concentrations of .1mM, .2mM, and .3mM. Each sample was run against these concentrations
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Results• Using a glass-electrode the pH of 5 samples
were found to be 7.25, 7.4, 7.45, 7.3, 7.3. • EDTA titration of saliva involves small amounts
of either ion concentration. To determine the concentration of the two ions a comparison is made against a standard
Sample mM Mg/L .05% EDTA mL added1 0.11 0.242 0.13 0.293 0.23 0.354 0.34 0.415 0.54 0.616 0.83 0.95
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Standard Calibration Graph
0 0.2 0.4 0.6 0.8 1 1.20
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
f(x) = 0.950484848484849 x − 0.00106666666666677R² = 0.996431237628571
Mg Standard
.05% EDTA Mg-Standard mL
Linear (.05% EDTA Mg-Standard mL)
mM Mg/L
.05%
EDT
A M
g -S
tand
ard
mL
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Results
1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.91
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
f(x) = 0.949386768008292 x + 0.180669372948696R² = 0.98500725413631
Concentration of Mg2+ and Ca2+
.05% EDTA mL addedLinear (.05% EDTA mL added)
Concentration of Mg mM
0.05
% E
DTA
titra
tion
mL ti
tran
t add
ed
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Results Fitted with 4th degree Polynomial
1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 21
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2
f(x) = − 15.3283630912421 x⁴ + 88.575235561734 x³ − 189.24918570414 x² + 178.124465354143 x − 61.1637863510535R² = 0.999144906936939
Concentration of Mg + Ca
.05% EDTA mL addedPolynomial (.05% EDTA mL added)
Concentration of Mg mM
.05%
EDT
A tit
ratio
n m
L add
ed
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Magnesium Results
• The results for magnesium concentration were obtained via UV Vis.
• The experimental results proved to be elusive. Quantitative data was unable to be obtained.
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Conclusion
• The results show:– An equation that describes the Mg concentration
in saliva at low concentrations– Can effectively determine pH of Saliva with a glass
electrode pH meter• What techniques are used today to determine
pH, [Mg2+], and [Ca2+].• Real world application and importance.
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References
• Starr, Henry. "Studies of Human Mixed Saliva". The Journal of Biological Chemistry. Philadelphia: Robert Hare Laboratory school of medicine, 1922.
• Wong, David. "Salivary diagnostics powered by". American Dental Association 2006: 313-321.
• Ericsson, Yngve. "Simplified Methods for Determination of Calcium and Magnesium in the Saliva". Journal of Dental Research 1955: 104-112