UNIT: pH and Blood Gases 11pH.

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Purpose

To become acquainted with theory and methods of measuring pH and blood gases.

Objectives

Upon completion of this exercise, the student will be able to:

1. Review classroom notes on pH, Henderson-Hasselbach equation, normal values and

expected ratios, and pH electrodes.
Discuss the basic principles involved in pH, pO2 and pCO2 determinations on whole blood.
2.
Interpret the basic clinical significance of blood gas values.
3.
Discuss the basic theory behind operation of the pH electrode.
4.
Discuss the operation of the pH meter and the blood gas machine.
5.

Principle I

Determination of blood gases enables the evaluation of a patient's acid-base balance. Blood gas
instruments in the laboratory are designed to measure the partial pressures of carbon dioxide
(pCO2 ) and of oxygen (pO2 ) as well as blood pH. Specialized electrodes designed for each gas
determination are placed within the instrument so that one small blood sample suffices for all
measurements.

After measuring pH and pCO2 directly, it is possible to obtain bicarbonate and CO2 content values
by calculation or with the use of a nomograph. From a direct measurement of pH and pO2 , blood
oxygen % saturation can be determined.

Principle II

The carbonic acid-bicarbonate buffer system is the most important buffer system in the regulation
of hydrogen ion balance in the body. In plasma, the relationship between pH and the bicarbonate-
carbonic acid buffer system is expressed by the Henderson-Hasselbach equation:

With this mathematical expression of the relationship between the bicarbonate ion and carbonic
acid, it is possible to calculate pH.

Principle III

The ratio of HCO3 – (salt) to H2 CO3 (acid) is normally 20:1. With this ratio, the blood pH is 7.40.
The pH falls (acidosis) as bicarbonate decreases in relation to carbonic acid. The pH rises
(alkalosis) as bicarbonate increases in relation to carbonic acid.

Principle IV

Four categories of acid-base imbalance may be encountered: metabolic acidosis; metabolic

alkalosis; respiratory acidosis; and respiratory alkalosis. In this context, “metabolic” refers to the

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UNIT: pH and Blood Gases (continued)

bicarbonate concentration in the Henderson-Hasselbach equation and the “respiratory” to the

carbonic acid.

Principle V

Blood gases and pH are generally performed on arterial blood to provide acid-base and respiratory
information on a sample that is a mixture of blood from all parts of the body to tell how well the
lungs are oxygenating the body.

1. pH is the only way to determine if the body is too acid or too alkaline.

a. Acidemia and alkalemia refer to a condition of the blood.

b. Acidosis and alkalosis refer to the process occurring in the patient which caused the
condition.

2. The respiratory parameter is the pCO2 , or pressure/ tension exerted by dissolved CO2 gas
in the blood. pCO2 is influenced only by the function of the lungs.

a. CO2 gas should be considered an acid substance because in water carbonic acid
(H2 CO3 ) is formed.

b. Transport forms of CO2

1) dissolved CO2 gas

2) combined with H2 O 6 H2 CO3 6 HCO3 – + H+
3) as carboxyhemoglobin

3. Under normal conditions, dissolved CO2 gas has a pCO2 of approximately 40 mmHg. pCO2
of 40 mm = 1.2 mEq/L. Normal HCO3 – = 24 mEq/L. Total CO2 content = 25.2 mEq/L (or
mmol/L).

4. The H+ produced by the breakdown of H2 CO3 6 HCO3 – + H+

a. is loosely carried and thus buffered with plasma proteins

b. H+ is excreted by the kidney as NH4 +

5. CO2 is removed by the lungs

High PCO 2 – hypoventilation – respiratory acidosis by decreased elimination of CO2 by

lungs.

Low PCO2 – hyperventilation – respiratory alkalosis by increased elimination of CO2 by

lungs.

Non-Respiratory Parameters: HCO3 – and Base Excess (BE)

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Bicarbonate ion and BE are influenced by metabolic causes. Metabolic acid-base is under the
control of the kidneys.

1. HCO3 – is an alkaline (base) substance excreted or conserved by the kidneys.

2. A high HCO3 – or positive BE indicates metabolic alkalosis caused by loss of non-volatile acid
or gain of HCO3 –

a. excess vomiting
b. excess diuretic therapy
c. excess bicarbonate intake

3. A low HCO3 – or negative BE indicates metabolic acidosis caused by loss of HCO3 – or its
neutralization by a non-volatile acid.

a. seen in conditions causing the accumulation of organic acids as in diabetic

ketoacidosis, lactic acidosis, renal failure, etc.

b. deficit of bicarbonate due to diarrhea, renal tubular acidosis, ammonium chloride

treatment.

Oxygen

The relationship between pO2 and hemoglobin O2 (HbO2 ) is an s-shaped curve, the hemoglobin-
oxygen dissociation curve.

To fulfill its function as respiratory pigment, hemoglobin must specifically bind with high affinity to
large quantities of oxygen, transport them, and release them in appropriate tissues. It is the
tetrameric structure of hemoglobin that provides its unique oxygen-binding capacity and makes
it superior.

Each molecule of hemoglobin contains four (ferrous) iron-containing heme groups, each capable
of binding one molecule of O2 (ˆ Hb + 4 O2 º Hb(O2 )4 ), each O2 added depends on the (pO2 )).

The position of the oxygen-

dissociation curve is
determined by a number of
factors in addition to the
previously me ntioned
partial pressure of oxygen;
body temperature, red cell
2, 3, DPG concentration,
and pH (Bohr effect).

Top left, oxygen

dissociation curves for
human blood with different
pH(S) but constant pCO2
(40 mm Hg). DPG
concentration in

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UNIT: pH and Blood Gases (continued)

erythrocytes (4.8 mmol/L), and temperature (37°C).

Principle VI

Review of Potentiometric Methods

1. Potentiometric methods are based on the measurement of a potential (voltage) difference

between two electrodes immersed in a solution under the condition of zero current. The
electrodes and the solution constitute an electrochemical cell. Each electrode is
characterized by a half-cell reaction with a corresponding half-cell potential. The potential
difference between the two electrodes is usually measured using a pH/milli-volt meter.

2. Indicator/measuring electrodes' half-cell potential responds to changes in the activity or

concentration of the substance/species in the solution being measured.

3. Reference electrodes serve as an internal standard as their half-cell potential does not
change. They consist of a metal and its salt in contact with a solution of the same ion (being
measured) of known concentration. Reference electrodes are usually a calomel or silver-
silver chloride type.

4. The salt bridge or liquid junction is a device that allows ionic movement between
compartments of an electrochemical cell to maintain electrical contact and at the same time,
prevents mixing of the separate solutions of the half-cells.

Principle VII

pH Electrodes

– The first ion-selective electrode to be widely used was the pH electrode designed to measure
hydrogen ion activity. This was made possible by the development of a special pH-sensitive glass.
When a thin membrane of this glass separates two solutions of differing hydrogen ion
concentrations, a hydrogen ion exchange takes place in the outer hydrated layers of the glass,
causing a potential to develop across the glass membrane. If a calomel half-cell (reference
electrode) is also immersed in the solution and the two are connected through a pH meter, the
meter can measure the potential difference (in millivolts) between the electrodes and convert this
to pH units.

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1. Calomel or silver-silver chloride electrode

2. Buffer-chloride solution
3. Saturated (3.5M) chloride solution
4. pH sensitive glass membrane
5. Liquid junction

Principle VIII

The pCO2 Electrode – The pCO2 electrode consists of a pH electrode with a CO2 permeable
membrane covering the glass membrane surface. Between the two is a thin layer of dilute
bicarbonate buffer. The aspirated blood sample is in contact with the CO2 -permeable membrane,
and as CO2 diffuses from the blood into the buffer, the pH of the buffer is lowered. The change
of pH is proportional to the concentration of dissolved CO2 in the blood. The glass electrode
responds to the buffer pH change, and the meter is calibrated to read the pCO2 in mm of mercury.
This type of pCO2 electrode is known as the Severinghaus electrode.

In a patient's blood the three values, pH, pCO2 and bicarbonate, are all inter-related according to
the Henderson-Hasselbach equation. If any two of the values are known, the third can be
calculated. Since blood gas instruments designed today measure the pH and pCO 2 , the
bicarbonate value may be calculated. Often a total CO2 , (which includes dissolved CO2 , carbonic
acid, and bicarbonate) is ordered.

Principle IX

The pO2 Electrode – The Clark electrode for measuring the partial pressure of oxygen in the blood
is based on a different principle from that of pH measurement. While the pH electrode measures
a voltage difference when no current is flowing; the pO2 electrode measures the current that flows
when a constant voltage is applied to the system. The current is the stream of electrons that flow
as the oxygen molecules are reduced at the cathode:

½ O2 + H2 O = 2e- + 2OH-

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The source of the electrons is the silver-silver chloride anode where the silver molecules are
oxidized:

Ag 6 Ag+ + e-

The amount of current that flows through the system is a direct measure of the number of
electrons released to the oxygen and is consequently a measure of the number of oxygen
molecules available for reduction. The current is directly linear with O2 concentration as long as
the constant voltage is maintained.

A platinum wire forms the cathode; the anode is a silver wire in AgCl. The contact between the
poles is an electrolyte solution that is separated from the test sample (blood) by a membrane
permeable to O2 molecules. Dissolved O2 diffuses from the blood through the membrane and is
reduced at the cathode. The rate-limiting factor in the system is the diffusion of oxygen molecules
through the membrane. The diffusion rate depends directly upon the pO2 of the sample, so the
change in current flow offers a direct measurement of the pO2 .

Principle X

Blood Gas Instruments Calibration and Maintenance – Blood gas instruments must be monitored
constantly and calibrated frequently. All three electrodes (pH, pO2 , and pCO2 ) are calibrated by
setting with two standard concentrations. Two buffers in the physiologic range are used for pH
calibration, and two gases (high and low concentrations of O2 and CO2 ) are used for the gas
electrodes. The gases are bubbled through water in the instrument to saturate them with water
vapor. Gases dissolved in the blood would be comparably saturated. Corrections must be made
for water vapor pressure and for the barometric pressure, which must be checked regularly
throughout the day.

Since the pressure of gases and pH is dependent upon temperature, the temperature of the bath
surrounding the electrodes must be carefully monitored and closely controlled. Usually the bath
is maintained at 37°C ± 0.1°C.

Both the pO2 and pCO2 electrodes require regular maintenance to keep the membranes intact,
taut, and clean. Obstruction to diffusion, such as protein build-up on the membrane, slows down
the response and may give low results.

Principle XI

Blood Gas Measurement

Normally arterial blood is drawn for blood gas studies since it will provide the best information
concerning overall acid-base balance as well as lung efficiency. Arterialized capillary blood is
acceptable and preferred when working with infants and small children.

The heparinized sample must be drawn, handled, and measured anaerobically, as any exposure
to atmospheric gases will change the patient's sample.

The sample is transported in crushed ice to slow cell metabolism. Testing should begin ASAP.

The sample should be evaluated for the presence of clots or air bubbles, then well-mixed before
introducing it into the blood gas instrument. The blood gas instrument is maintained at 37°C to

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determine the pH of the blood as it existed in the patient's body. Temperature control is very
important because pH is temperature dependent and decreases about 0.015 units for each degree
rise in temperature.

Principle XII

Siggard-Andersen Nomograph

In routine blood gas measurement, three parameters (pH, pCO2 , and pO2 ) are measured. The
total CO2 or bicarbonate ion (HCO3 – ) and another parameter known as base excess (BE) can be
obtained by calculation or through the use of the Siggard-Andersen nomograph.

To obtain total CO2 (or bicarbonate ion), and base excess values from the Siggard-Anderson
nomograph, use a straight edge to mark a line that crosses pCO2 , pH, BE grid, and CO2 scales.

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UNIT: pH and Blood Gases (continued)

Siggaard-Andersen nomograph.

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Table 1
Primary Blood Gas Classifications

Base
Primary Ventilatory pCO2 pH HCO3 Excess
1. Acute ventilatory failure I D N N
2. Chronic ventilatory failure I N I I
3. Acute ventilatory insufficiency D I N N
4. Chronic ventilatory insufficiency D N D D

Primary Acid-Base
1. Uncompensated acidosis N D D D
2. Uncompensated alkalosis N I I I
3. Partly compensated acidosis D D D D
4. Partly compensated alkalosis I I I I
5. Compensated acidosis/alkalosis I or D N I or D I or D
I = increased; D = decreased; N = normal

Normal Blood Gas Values

Arterial Blood Mixed Venous Blood
pH 7.35 - 7.45 7.31 - 7.41

pO2 80 - 100 mmHg 35 - 40 mmHg

O2 saturation $ 95% 70 - 75%

pCO2 35 - 45 mmHg 41 - 51 mmHg

HCO3 – 22 - 26 mEq/L 22 - 26 mEq/L

Total CO2 23 - 27 mmol/L 23-27 mmol/L

Base Excess -2 to +2 -2 to +2

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Name

Date

Study Questions

Instructions: Unless otherwise indicated, each question is worth one point. Indicate your
answers in an appropriate manner or in the space provided.

1. pH is defined as the
A. H+ concentration
B. log of the H+ concentration
C. negative log of the H+ concentration
D. log of the reciprocal of the OH concentration
E. sum of the OH– and H+

2. The pH meter actually measures

A. current
B. pH
C. voltage
D. ion numbers

3. Most of the CO2 present in blood is in the form of

A. carbonic acid
B. dissolved CO2
C. calcium carbonate
D. a protein complex
E. bicarbonate ion

4. Which of the following are compensatory mechanisms used by the body in acid-base
disturbances?
A. blood buffer systems
B. respiratory mechanisms
C. renal mechanisms
D. all of these

5. In uncompensated metabolic acidosis, which of the following would be observed?

A. pH decreased, HCO3 – increased
B. pH decreased, HCO3 – decreased
C. pH increased, HCO3 – decreased
D. pH increased, HCO3 – increased

6. Making an electrode selective for an ion other than hydrogen is made possible by
A. increasing the buffer pH
B. changing the reference electrode
C. using stronger buffer solutions
D. decreasing the buffer temperature
E. changing the chemical composition of the glass membrane

7. What is the difference between an indicator electrode and a reference electrode? (2 points)

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UNIT: pH and Blood Gases (continued)

8. Using classroom and laboratory notes, define/describe a “salt bridge.” Be sure to include
its purpose.

9. State the Henderson-Hasselbach equation. What is it? (2 points)

10. What is the normal bicarbonate ion to carbonic acid ratio in the body?

11. What is the most important single factor or system responsible for keeping the pH of the
blood within normal range?

12. Which compensatory mechanism will respond the quickest to a sudden change in the acid-
base status of a patient?

13. Use the Siggard-Anderson nomograph to determine the total CO 2 ; bicarbonate level and
base excess (BE) of a patient with 7.47 pH and a PCO2 of 37 mmHg.

Total CO2 bicarbonate base excess

14.
The Severinghaus electrode measures

while the Clark electrode measures .

15. State the normal body pH range.

16. Normally, the body is slightly (acidic / alkaline). Circle the correct response.

Case Study Questions

17. (5 points) A medical laboratory technician obtained the following blood-gas results from a
well-iced arterial specimen: pH = 7.14, pCO2 = 51 mmHg, and HCO3 – = 10 mmol/L.

1. Are each of the individual results consistent with acidosis? alkalosis?

pH ___________________
pCO2 _________________
HCO3 _________________

2. Which of the results indicate respiratory acid / base?

3. Which of the results indicate metabolic acid / base?,

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4. Are these results consistent with a metabolic or respiratory problem?

The specimen is re-examined, and small clots are noted. A repeat blood-gas specimen is
obtained and analysis yields the following data: pH - 7.23, PCO2 = 22 mmHg, PO2 = 55
mmHg, and HCO3 = 10 mmol/L.

1. What type of acid-base disorder do these results indicate, if any?

2. What other laboratory results do you think may be abnormal in a patient with these
blood-gas results?

A half hour later a request for a stat lactic acid is received. The result is 21 mmol/L (21
mEq/L). (NV = 0.5 - 1.9 mmol/L)

1. Is this result consistent with the above results?

CASE STUDY: Acid-base Balance

18. (5 points) A 27 year old man was comatose with depressed respiration upon arrival at the
ER. A friend stated that the man had overdosed on “sleeping pills.” The following blood gas
results were obtained:
pH 7.29
pCO2 58 mmHg
HCO3 25 mmol/L
pO2 72 mmHg

1. Evaluate these ABGs and determine the patient’s acid-base balance. Is there any
compensation occurring?

2. What has caused the acid-base state of this man?

3. Using the Siggard - Anderson nomograph, calculate the total CO2 and BE.

4. What parameter would change if complete compensation occurs?

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