Electrolytes

Sodium (Na or Natrium)

- Major cation of the extracellular fluid (ECF)

Methods:
1. Flame emission spectrophotometry (FES)
2. Atomic absorption spectrophotometry (AAS)
3. Ion selective electrode (ISE) – glass aluminium silicate
4. Colorimetric / chemical method of Albenese and Lein
Principle:
Sodium is made to react with zinc uranyl acetate to produce a sodium uranyl
acetate precipitate after the addition of polyvinyl alcohol. With the addition of water, a
yellow solution is formed which is then measured spectrophotometry.

Reagents and results:

TCA – protein precipitant
Zinc uranyl acetate
Polyvynil alcohol
Distilled water
Sodium uranyl acetate precipitate (yellow end product)

Precautions in sodium determination:

1. Sodium excretion increases during exercise.
2. Anticoagulants lower sodium levels like oxalates which tends to increase plasma
volume
3. Unpurified distilled water contains traces of sodium ions which will erroneously
elevate sodium levels
4. Mixing tubes using thumb as cover causes an elevation of sodium levels since NaCl
is present in the skin.
5. Glasswares may be contaminated with oxides of sodium.

Potassium (K or Kalium)
- Major cation of the intracellular fluid (ICF)

Methods:
1. Flame emission spectrophotometry (FES)
2. Atomic absorption spectrophotometry (AAS)
3. Ion selective electrode (ISE) – valinomycin gel
4. Colorimetric / chemical method of Lockhead and Purcell
Principle:
Potassium is made to react with sodium cobaltinitrite to produce sodium
potassium cobaltinitrite. With the addition of phenol, a blue color is produced and
determined spectrophotometrically.
 Reagents and results:
Sodium cobaltinitrite
Sodium acetate
Glycine
Sodium carbonate
Phenol – color developer
Blue – end color

Specimen considerations:
1. Hemolysis of 0.5% of RBC can falsely increase serum potassium levels by 0.5
mmol/L.
2. Levels of potassium in plasma have been shown to be 0.1-0.7 mmol/L lower
than in serum due to release of potassium from ruptured platelets during
coagulation process.
3. Plasma potassium increases by 10-20% as a result of tourniquet application
and muscle activity if the patients opens and closes his fist repeatedly prior to
venipuncture.
4. Heparinized plasma is preferred over serum due to potassium release during
blood clotting

Chloride (Cl)
- The major anion of the ECF

Methods:

1. Mercuric titration (Schales and Schales)

Principle:
The chloride in a tungstic acid PFF is titrated with standard solution of mercuric
ions to form a soluble compound of mercuric chloride which does not dissociate to
mercuric ions. The excess mercuric ions combine with diphenylcarbazone to form a
blue violet colored complex.

Reagents and results:

Indicator - s-diphenylcarbazone
Titrating agent - mercuric nitrate
End color - blue violet
End product - mercuric chloride

2. Spectrophotometric methods
a. Mercuric thiocyanate (Whitehorn titration mtd.)
Principle:
The specimen is mixed with a solution of mercuric thiocyanate. As a
result of high affinity of mercuric ions for chloride, non-dissociating
mercuric chloride is formed. In the process, free thiocyanate ions are
 released and react with the ferric ions present in the ferric nitrate reagent
to form reddish brown complex of ferric thyocyanate.

b. Ferric perchlorate
Principle:
Based on the reaction between ferric perchlorate and chloride
forming a colored chloro-complex of ferric ions.

3. Coulometric-amperometric titration (Cotlove Itratos method)

Principle:
The sample is diluted with an acid solution and placed in a small titrating
vessel containing gelatin, into which two pairs of electrodes are dipped. The first
electrode is a generating electrode which produces silver ions at a constant rate
and the other electrode is a sensing electrode which detects electrical
conductivity changes in the solution. When turned on, the silver ions combine
with the chloride ions to form an insoluble silver chloride. The time necessary to
reach the titration end point is measured as chloride concentration.

Reagents:
Nitric acid acetic acid solution – serves as a diluent and prevents reduction
of precipitated silver chloride at the sensing electrode.
Gelatin – equalizes the reaction rate over the entire electrode surface.

4. Ion-Selective Electrode (ISE) – ion exchange membrane (tri-n-octylpropylammonium

chloride decanol)

Precautions in chloride determination:

1. All chloride methods measure bromide to some extent.
2. In the Schales and Schales method, the color produced is stable, but difficult to
determine with urine, pleural fluids and serum with elevated bilirubin levels.

Calcium (Ca)
- Three physicochemical states:
o Protein-bound calcium - 45%
o Ionized calcium - 45% physiologically active form
o Complexed with small diffusible ligands - 10%

Methods:
1. Atomic absorption spectrophotometry (AAS)
o In terms of accuracy, precision and speed, this determination of calcium is the
method of choice for routine analysis and as a reference method.

2. Precipitation and Redox titration methods

a. Clark Collip precipitation method
Principle:
 Ammonium oxalate is added to the diluted serum sample wherein calcium
is precipitated as calcium oxalate. The precipitate is then washed with diluted
ammonium hydroxide to remove excess oxalate. This prevents interference of
magnesium which precipitates with the excess oxalates to form magnesium
oxalate. The excess oxalates are dissolved in sulphuric acid forming oxalic acid
which is titrated with a standardized potassium permanganate (K2MnO4). The
appearance of a purple color is the end point.

Reagents and results:

Ammonium oxalate - precipitant
Ammonium hydroxide - washes the precipitate and provides
alkalinity
Sulfuric acid - dissolves excess oxalate
K permanganate - titrating agent
Oxalic acid - end product
Purple - end color

b. Ferro Ham chloranilic acid precicpitation mtd

Principle:
Calcium is precipitated as calcium chloranilate by adding sodium
chloranilate. The precipitate is washed with isopropyl alcohol to remove excess
chloranilic acid and then titrated with EDTA which chelates calcium and release
chloranilic acid giving a purple color.

3. Colorimetric method
a. Using Ortho-Cresolphthalein Complexone dyes
Principle:
Serum is mixed 0.3M HCl to dissociate calcium from proteins then dialyzed
into a reagent stream containing Ortho-Cresolphthalein Complexone and
hydroxyquinoline in diluted HCl. Hydroxyquinoline is added to bind magnesium
which would otherwise cause interference. A colored complex between calcium
and the dye is formed and maintained after the addition of diethylamine buffer.
Dyes: alizarin, calcein, murexide and nuclear fast red

4. EDTA Titration method (Bachra, Dawer and Sobel)

Principle:
A diluted serum sample is titrated with EDTA in the presence of an indicator
(calcein red) at an alkaline pH (this prevents magnesium interference). The initial yellow
green fluorescence caused by the calcium-calcein complex changes to a non-
flourescent salmon pink color (free calcium) when all calcium present has been
chelated with EDTA.

Chelation or sequestration – is the reaction involved when metallic ions combine with
an organic chelating agent

Reagents and results

Calcein red - indicator
 Versene EDTA - chelating titrant
KOH - provides alkalinity
Ca-EDTA complex- end product
Salmon pink - end color

5. Emission Flame Photometry

6. Calcium Ion-Selective Electrode

Inorganic phosphorus

Method:
1. Fiske and Subbarow method
Principle:
A TCA filtrate of serum or urine is treated with molybdate reagent which reacts
with phosphates to form ammonium phosphomolybdate. A reducing agent is added to
form a blue color of hetero-polymolybdenum blue.

Reagents and results:

TCA – protein precipitant and for acidity of medium
Pictol (amino naphthol sulfonic acid) – reducing agent
Other reducing agents are:
a. Elon (p-methyl amino phenol)
b. Sonidine (n-phenyl-p-phenylene diamine)
c. Ascorbic acid
d. Stannous chloride
e. Ferrous sulphate
Ammonium molybdate – color reagent
Heteropolymolybdenum blue – end product/color

Magnesium (Mg)
- 2nd major intacellular cation

Methods:
1. Atomic Absorption Spectrophotometry (AAS)
Principle:
This is the method of choice for the determination of magnesium. After
deproteinization with either TCA or HCl, and removal of phosphate ions with lanthanum
salt, the diluted filtrate is analysed using 285 nm lime of magnesium hollow cathode
lamp.

2. Fluorometric and complexometric methods

Principle:
Magnesium ions and 8-hydroxyquinoline sulfonic acid react to form fluorescence.
 3. Dye-Lake method (Titan Yellow method)
Principle:
A TCA filtrate is treated with a titan yellow dye in an alkaline solution. The red
lake formed is absorbed at the surface of the magnesium particles which are kept in
solution with the addition of polyvinyl alcohol.

Titan yellow method dye (Clayton yellow or Thiazole yellow)

- Methylbenzothiazidediazoaminobensoldisulfonic acid dye

4. Colorimetric methods
a. Calmagite method – (+) reddish violet complex
b. Formazen dye method – (+) colored complex
c. Magnesium thymol blue method – (+) colored complex

Iron
- Important for the synthesis of haemoglobin

Method:
1. Serum iron
a. Colorimetric method
Principle:
Sample is reacted with hot TCA to dissociate iron from its binding proteins.
The reduced iron is then reacted with chromogen such as sulfonated
bathophenanthroline 2,4,6-tripyridyl-s-triazine (TPTZ), ferrozine, terosite, and
thiocyanate to produce an iron chromogen complex. A color reagent is added to
the complex with an absorbance maximum in the visible region.
b. AAS
c. FES
d. X-ray Fluorescence Spectrometry