A comprehensive presentation on Blood Gas Analyzer and Blood Gas Analysis for self- learning undergraduate medical ,dental, ,pharmacology and biotechnology students . Laboratory determination of blood gas analysis –Micro method & technical errors involved are described. Blood sample collection for blood gas analysis is illustrated. Principle & Important components of Blood gas Analyzer are presented in lucid language. Polari graphic method for PO₂ Measurement using pO₂ electrode is explained. Integral parts of pO₂ electrode ie platinum electrode, silver /silver chloride reference electrode & their working (reaction at electrode) is presented here. Design of pCO2, & pH electrodes along with their working principles are elucidated for perusal of technologist. Typical reference ranges in blood gas analysis are mentioned and are useful to classify acid base imbalance. Nomogram of acid base disorder is illustrated for clinical applications. Laboratory determination of blood gas analysis along with its standardization is presented step wise. The Henderson’s Hassel Balch equation pursuing interrelation of TCO₂, Bicarbonate, Carbonic acid, PCO ₂, & p H is presented for manual calculation of certain parameters. Google images are used for impact of subject on self-learners.

2. ASTRUP BLOOD GAS ANALYZER
Procedure
• Heparinized blood ( capillary /arterial )
• Wax sealing
• Remix before use
• Poor peripheral circulation –caution
• eg ABL /Roche blood gas analyzer

3. Laboratory determination of blood gas analysis –Micro method 1.
•Procedure :By using heparinized capillaries ,blood can be
collected from fingertips , a lobe of ear or a heel. After filing
with the blood capillaries are sealed by using modelling
wax. Immediately before use blood is remixed & by cutting
the sealed ends the blood is introduced into each
equilibration chamber of apparatus. After determining
blood pH & p CO₂ ,the bicarbonate & base deficit ( or
excess ) are determined by using a Nomograms.

4. Laboratory determination of blood gas analysis –Micro method 2.
•Although under ordinary conditions capillary blood is
similar to arterial , it can be assumed to so in patients with
poor peripheral circulations . For reliable results a good
flow of capillary blood is essential. If this cannot be
obtained arterial blood must be used.
•In hypothermia following corrections has to be made. The
measurements are made at 38 ⁰ C.
pH at T ⁰ C = p H at 38 ⁰ C+ 0.0146 ( 38 –T )
PCO₂ at T ⁰ C = Antilog ( log PCO₂ at 38 ⁰ C – 0.021 (38-T )
• Blood must not be exposed to air in any circumstances before its
analysis .

6. Blood sample collection for blood gas analysis
•

7. PRINCIPLE OF
BLOOD GAS ANALYSER
BLOOD GAS ANALYSER works with three in-built
electrodes
1. pco₂ electrode
2. pO ₂ electrode
3. pH sensitive glass electrode

8. pco₂ electrode
1. A. Glass electrode-sensitize to p H
B.silver /silver chloride reference
electrode
2.Bicarbonate Buffer
3. Plastic membrane selectivity permeable
to gaseous CO₂ but not dissolved ions
4.pH α p CO₂, inverse relationship between
p H & log p CO₂
pO₂ Measurement
1.Polari graphic method
A. platinum electrode
B.silver /silver chloride
reference electrode
2.NaCl / phosphate buffer
3. Membrane permeable to
gaseous O₂
4.A .O₂ + H₂O +4 e⁻  4 OH⁻
B. 4 Ag + 4 Cl⁻  4AgCl +4 e⁻

9. Important components of Blood gas Analyzer -1.
pH sensitive glass electrode
Reference electrode ( silver –silver chloride ) immersed in bicarbonate buffer
• Principle :The pCO₂ Electrode : Test solution –Arterial blood separated by plastic membrane
permeable to gaseous CO₂ but not permeable to dissolved ions .
• CO ₂ of blood diffuses through plastic membrane & react with the buffer system to change
pH.
• The pCO ₂ electrode takes advantage of linear correlation between p H & log PCO₂ over the
range 11-90 mm Hg .
• The hydrogen ion concentration change due to the dissolution of CO₂ is detected by the pH
sensitive glass electrode .
• A potential difference exist between glass electrode & Reference electrode This is measured
on the meter. The meter’s scale is usually calibrated for in semi logarithmic fashion, since pH
is inversely proportional to the log of pCO ₂.

10. Important components of Blood gas Analyzer 2
Principle : pO ₂ electrode –Polarographic method
Reference platinum & ( silver –silver chloride ) electrode is immersed in a buffer containing ( phosphate &
sodium chloride ) .These electrodes are separated from test solution.
• Test solution –Arterial blood separated by plastic membrane permeable to gaseous O₂ but not permeable to
dissolved ions .
• O₂ of blood diffuses through plastic membrane & react with the buffer Following reactions take place –
• At platinum electrode ( cathode ) electro reaction occurs .
O₂ + 2H ₂ O+ 4 e⁻  4 OH ⁻
• The electrons necessary for electro reduction are produced at reference electrode (anode ) as follows-
4 Ag⁺ + 4 Cl ⁻  4 AgCl + 4 e⁻
• A potential difference exist between glass electrode & Reference electrode This is measured on the meter.
The meter’s scale is usually calibrated for in semi logarithmic fashion. The current through the system is
directly proportional to PO ₂ & can be recorded directly after amplification into PO ₂.

20. pH electrode
Principle of p H electrode :
The measurement of pH is called potentiometric analysis. It has been
reported that a difference in electrical potential could be measured between
two solutions of different pH separated by a thin glass membrane. The
potential thus produced varies with the hydrogen ion concentration of two
solutions .The glass membrane is sensitive for H ⁺ions. It is on this principle
that the glass electrode is constructed for pH measurement.

21. Combined pH electrode
Integral Parts of combined p H electrode :
1. Simple glass electrode
2. Calomel reference electrode

22. Combined pH electrode
Integral Parts of combined p H electrode :
1. A simple glass electrode:
Modern glass electrodes are constructed from glass containing Lithium oxide
which is soft ,hygroscopic imparting low resistance. The inner surface of glass
membrane is in contact with a buffer pH. Into this buffer dips a silver /silver
chloride electrode ,the internal glass electrode .The glass electrode functions
like semi permeable membrane selectively permeable only to H⁺ ions.
2.Calomel reference electrode : consist of Mercury (Hg ⁺²) in contact with a
solution of potassium chloride saturated with Calomel (HgCl ).It is
surrounded by a outer vessel holding saturated Potassium chloride which
acts as a salt bridge between reference and test solution. This electrode is
not sensitive to pH .

23. Combined pH electrode
Only when glass electrode is coupled with a Calomel reference electrode the
potentiometric measurements are possible. The potential difference ( or
electrical voltage ) between two electrodes depends upon the hydrogen ion
concentration of test or standard solution .
It is the logarithmic response measured in millivolts on which pH meter is
calibrated in both millivolts and pH.
pH measurements vary with temperature and all measurements should be
made at a temperature of 25° C . With increasing temperature there will be fall
in of pH. It is therefore important to record the temperature of liquid before
measuring pH by adjusting dial on p H meter and then record p H .
Some pH meters have facilities whereby a temperature thermometer can be
incorporated into circuit and variation above and below 25 °C are automatically
corrected by pH meter itself.

27. Combined pH electrode
Scehatic representation of combined electrode
Ag /AgCl /HCl/glass membrane Test solution KCl/HgCl/Hg
Glass electrode Reference electrode
is the boundary between the two immiscible solutions.

28. Combined electrode of p H meter

29. Combined electrode of pH meter

33. Modern Combined glass electrode
for p H determination.

34. Single blood sample is in contact
With pCO2, pO₂ & pH electrodes
for their Simultaneous
measurement .

35. Typical reference ranges in blood gas analysis-1
Parameter Normal range
p H 7.35 -7.45
H⁺  ions ( 35-45mmol/ lt )
Arterial pO₂ 95- 100 mm of Hg
Arterial pCO₂ 4.7 -6.0 Kpa ( 1.02 -1.35 mmol/ lt or 35-45 mm Hg )
Total CO₂ 25-39 mmol /lt
Carbon dioxide combining power 53-75 ml per 100ml of plasma
HCO₃⁻ 21- 28 mmol /lt ( mequ/lt )
Base excess of blood 2.3 to + 2.3 mequ /lt
T O₂ Sum of O₂ dissolved in plasma & chemically bound
to Hb
H₂ O + CO ₂ ↔ H₂CO₃ ↔ H⁺ + HCO₃ ⁻ uncompensated phase – CONC CO₂ ↑ pCO₂ ↑, Total CO₂ ↑(RESPIRATORY ACIDOSIS )

36. Typical reference ranges in blood gas analysis-2
Parameter Normal range Acidosis Alkalosis
p H 7.35 -7.45 < 7.35 > 7.45
H⁺  ions ( 35-45mmol/ lt ) > 45 mmol/ lt < 35 mmol/ lt
Arterial pO₂* 95- 100 mm of Hg
9.3-13.3 kPa
Arterial pCO₂ ◊ 4.7 -6.0 Kpa ( 1.02 -
1.35 mmol/lt at 35-45
mm Hg )
High ( respiratory
acidosis –under
ventilation )
uncompensated phase
low ( respiratory
alkalosis –Hyper/over
ventilation )-
uncompensated phase
T O₂ Sum of O₂ dissolved in
plasma & chemically
bound to Hb
H₂ O + CO ₂ ↔ H₂CO₃ ↔ H⁺ + HCO₃⁻
* PO ₂ Low =Hypoxemic,< 60 mm Hg- supplemental O ₂ needed ,< 26 mm Hg-Risk of death requires immediate O ₂
supplementation

37. Typical reference ranges in blood gas analysis-3
Parameter Normal range Acidosis Alkalosis
Total CO₂ ( HCO₃⁻ +
PCO₂)
Conc HCO₃⁻ : m M
PCO₂ :Kpa
25-30 mmol /lt (
Total CO₂= (HCO₃⁻ )
+α PCO₂) α =
0.0226mmol /Kpa
 mM /Kpa
Carbon dioxide
combining power
53-75 ml per 100ml
of plasma
HCO₃⁻ 21- 28 mmol /lt
( mequ/lt )
Low( metabolic
acidosis )
High ( metabolic
alkalosis )-kidney
function normal
compensating for
respiratory issues
Base excess of blood 2.3 to + 2.3 mEq /lt

38. Typical reference ranges in blood gas analysis-3
• ◊ Arterial pCO₂ ◊- can become abnormal when respiratory system is working to
compensate for metabolic issue so as to normalize the blood p H & elevated
pCO₂ level is desired in some disorders associated with respiratory failure ,this
is known as ‘Permissive hyper apnea ’. (↑ CO₂ )
• HCO₃⁻ : levels can become abnormal when kidneys are working to compensate
for respiratory issues so as to normalize blood pH.

39. Laboratory determination of blood gas analysis
(1) The pH of blood : heparinized whole arterial blood ( or heparinized capillary
blood ) is used. The pH determination is performed immediately after collection of
blood .The blood can be stored at 0-4 ⁰ C up to 2-3 hrs,without significant change in
pH .
(2) PCO₂ : ( The respiratory parameter ) plasma carbonic acid can not to be found
directly .But it is determined by measuring PCO₂ . The PCO₂ of arterial blood is
usually directly proportional to the amount of carbon dioxide which is being
produced in the body & inversely proportional to the rate of alveolar ventilation in
the lungs .
(3) TCO₂ : Total CO₂ is mainly bicarbonate & also includes dissolved carbon dioxide.
Measurement of CO₂ ,carbonic acid & bicarbonate of plasma derived from blood
plasma ,collected under liquid paraffin gives measure of CO₂ content . It is reported
as CO₂ per 100ml at standard conditions of temperature & pressure . If the plasma is
equilibrated with normal alveolar air ( 40 mm Hg ) before it is measured ,the CO₂
combining power is obtained.Ordinarily the CO₂ content & CO₂ combining power
are practically identical.

40. Laboratory determination of blood gas analysis
(4)The plasma bicarbonate : ( non respiratory parameter )can be determined
by finding out the actual bicarbonate concentration of plasma separated from
blood taken anaerobically & expressed as mill equivalents per liter .This can be
calculated from the p H & PCO₂ by using the Henderson's –Hassel Balch
equation or from total carbon dioxide & the PCO₂ .
(5) Standard bicarbonate : is expressed as milliequivalents per liter, is the
concentration of bicarbonate in plasma separated from whole blood taken
anaerobically which has ben equilibrated at 37⁰ C a PCO₂ of 40 mm Hg with
oxygen to give full saturation to the hemoglobin.The plasma concentration
bicarbonate is influenced by change in PCO₂ & degree of oxygen saturation .
Alkali reserve is plasma bicarbonate . It is this fraction of plasma which is
used to neutralize all the acidic compounds entering the blood & tissue.
Plasma bicarbonate can also be calculated ( in terms of mequ /lt ) by
dividing CO₂ combing power by 2.24.

41. Laboratory determination of blood gas analysis
(5)PO₂ : determination is carried out to assess the oxygen carrying capacity of
blood hemoglobin . The increased oxygen affinity of hemoglobin is indicated
by elevated PO₂ values. The measurement of arterial PO₂ is also used in
conjunction with that of PCO₂ in assessment of respiratory disorders .A low
PO₂ is a measure of anoxia. It may also occur with a high PCO₂ when there is
alveolar hypo ventilation due depression or obstruction of respiration . A low
PO₂ with low PCO₂ may also be observed in pulmonary edema .
( 6 ) Base excess : is the amount of acid required to titrate blood to p H 7.4 at
37⁰ C & PCO₂ at 40 mm Hg . Base deficit is the reverse concept.

42. Henderson’s Hassel Balch equation :

43. Henderson’s Hassel Balch equation for pH determination

47. BLOOD GAS ANALYSIS
The interrelation of TCO₂ , Bicarbonate , Carbonic acid , PCO ₂, & p H in the
Henderson’s Hassel Balch equation :
p H = p ka + log (congugate base= HCO₃⁻)
(non ionized acid = H₂CO₃)
At 38⁰ C p ka = 6.1 therefore pH=6.1 + log (HCO ₃⁻ )/ H₂CO₃
H₂CO₃ ( mequ /lt ) =(0.0301 X pCO ₂)
pCO ₂= partial pressure of CO ₂ in mmHg equilibrated with plasma at 38⁰ C.
For normal plasma at 38⁰ C at pCO ₂ of 40mm Hg
H₂CO₃ = 0.0301 X 40 (pCO ₂)
H₂CO₃ = 1.204 mequ /lt
p H of plasma at 38⁰ C=
6.1 + log ( total CO ₂ )- (0.0301 X PCO ₂)
( 0.0301 )X 40 (pCO ₂)

48. Laboratory determination of blood gas analysis
• Standardization : by measuring the p H or the blood at its actual PCO₂ & at
accurately known PCO₂ values ,one higher & one lower than the normal
PCO₂. This can be achieved by equilibrating two portions of the blood with
carbon dioxide & oxygen mixture with PCO₂ values between 30 -60 mmHg
respectively .This reduces experimental error since the actual PCO₂ values
are between 30-60 mmHg.
• 2. A Nomogram is constructed by plotting log PCO₂ against p H .It is possible
to calculate standard bicarbonate & base excess ( or deficit ) .

51. Laboratory findings in compensated acidosis & alkalosis
Clinical conditions pH PCO₂ Bicarbonate PO₂
Respiratory acidosis Fall Rise Rise Rise
Respiratory alkalosis Rise Fall Fall Fall
Metabolic acidosis Fall Fall Fall Fall
Metabolic alkalosis Rise Rise Rise Rise

52. pH determination for urine , saliva
and low volume body fluids

53. Pulse oximetry