Hemoglobin is an iron-containing protein in red blood cells that carries oxygen from the lungs to tissues and returns carbon dioxide back to the lungs. Each hemoglobin molecule contains four heme groups that can each bind one oxygen molecule. Hemoglobin is made up of approximately 3.8% heme and 96.2% globin, which are polypeptide chains composed of amino acids. The document describes Sahli's method for determining hemoglobin content in blood, which involves converting a known volume of blood into acid-hemoglobin and comparing the color to a standard. Normal hemoglobin levels vary based on factors like age, sex, altitude, and medical conditions.

1. M.Pandian
Dept of Physiology
DYPMCKOP

2. What is Hemoglobin?
 Hemoglobin (abbreviated Hb) is a red
substance made of iron and protein
 Carries Oxygen to the cells
 Carries Carbon dioxide away from the cells

3.  Each molecule of hemoglobin contains four
groups of heme.
 Heme is the iron component of hemoglobin.
 Each group of heme can carry one molecule
of oxygen.

4.  A Hemoglobin molecule contains four
globin polypeptide chains composed of
amino acids.
 Amino acids are a group of chemical
substances that form proteins.
 Each hemoglobin molecule consists of
about 3.8% heme and 96.2% globin.

5. Practical
 Aim
 To determine the Hb content of blood (sahli’s method
its indirect method.)
 Principle
 The amount of Hb can be estimated by the
conversion of the known volume of blood into
acid-haematin by the addition of dilute N/10
Hcl and subsequent calorimetric comparison
with suitable standard.

6.  Requirements
 Sahli’s hemoglobinometer
 N/10 Hcl
 Distilled water
 Two Dropper
 Materials for sterile finger prick

9. How to prepare N/10 Hcl
 Normality (N)
 The normality of a solution is the number of
gram equivalents in 1 litre of water.
 A 1 N solution of Hcl contains 1 + 35 = 36 g of
Hcl in water made to 1 liter.
 Diluting this solution 10 times will give N/10
Hcl.

10. Procedure

11. Observation & Result
 Compare your colour matching and record the
observations in your work book.
 Take average of 3 readings as shown below, and report
your results,
 1st reading , when the colour is slightly darker than the
std……………..g/dl.
 2nd reading, when, after adding a few drops of distilled
water the colour exactly matches with std ………….g/dl.
 3rd reading, when, after adding some more drops, the
colour becomes a little lighter than the
std……………g/dl.
 Hb=………………g/dl.

12. Oxygen carrying capacity
 Knowing your Hb Conc. and that 1.0 g of Hb can
carry 1.34 ml 0f O2, calculate its O2 carrying
capacity as ……………..ml O2/dl.
 Normal values :- Males – 21mL/dl ; Female –
18mL/dl
Iron Content
 1g of Hb contains 3.35 mg iron, the amt of Hb can
be calculated.

13. Advantage
 Sahli’s method (indirect method)is easy to
perform and convenient.
 The cost is minimal.
 It is not very time consuming (maximum
fifteen minutes)

14. Disadvantage of Sahli’s method
 The colour of the std may not always be reliable,
especially with old apparatus.
 Sahli’s acid haematin method does not estimate all
the Hb. It estimate only oxyHb and reduced Hb,
but not the carboxyHb, methemoglobin and
sulfphhemoglobin.
 Finally, the acid haematin is not true solution.
Some degree of precipitation may be present at
times, which may interfere with colour matching.

15. Normal Levels
 12 to 16 (14 ± 2) grams per deciliter (g/dl)of blood in
women
 13.5 to 18 (16 ± 2) g/dl of blood in men
 New born, 16 – 22 g/dl. It decreases to 9 – 14 g/dl by
abt two months of age. By 10yrs of age, the normal Hb
Conc. Will be 12 - 14 g/dl.
 There may be a slight decrease in Hb level after 50 yrs
of age.

16. Other Methods
 Visual method
 Dare’s method
 Haden’s method
 Wintrobe’s method
 Haldane’s method
 Tallquist’s method
 Gastrometric method
 Spectrophotometric (these methods are rapid and give accurate result)
 Oxyhemoglobin method
 Cyanmethemoglobin method
 Automated hemoglobinometry
 Non-Automated hemoglobinometry
 Alkaline- hematin method
 Specific gravity method
 Comparator method

17. Precautions

18. Conditions that decrease Hb Conc.
I. Physiology
 Pregnancy (due to hemodilution)
 Children have lower values than adults
 Women have lower values than men because the total
RBC count is less.
 For male its due to testostreone stimulates
erythropoiesis in males.
II. Pathology
 Different types of anemia
 Relative decrease in Hb Conc. Occurs in different
pathological conditions that produce hemodilution, for
e.g excess ADH secretion as seen in pituitary tumors.

19. Conditions that increase Hb Conc.
I. Physiology
 High altitude (due to hypoxia)
 New borns and infants
 Excessive sweating (due to hemoconcentration)
II. Pathology
 Conditions that produce hemoconcentration (due to
loss of body fluid) for example, severe diarrhea,
vomiting.
 Conditions that produce hypoxia (e.g. congenital heart
disease, emphysema.
 Polycythemia vera.

20. Questions
1. What is N/10 Hcl and how will you prepare it?
2. Can strong acids ( such as Nitric, Sulphuric,& Hcl) or
alkalins be used in place of decinormal Hcl.
3. Why is the Hb level high in the new born?
4. What would happen if Hb present freely in the plasma
instead of in the red Cells?
5. Why should ten mins be allowed before diluting the
solution of blood & N/10 Hcl?

21. THANK YOU . . .