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Introduction: Haemoglobin is a globular protein tetramer consisting of two alpha (α) and two beta (β) chain subunits which gives its speciality function in binding and transporting oxygen (O2) from the lungs to the respiring tissues and in a reverse way of transporting CO2 from the tissues. Haemoglobin is also involved in transporting other gases such as nitric oxide, hydrogen sulphide and sulphide out of the respiring tissues. As haemoglobin absorbs radiation, spectrophotometer is therefore ideal to determine its erythrocytes (red blood cells) concentration level in the solution. Anaemia is defined as a low Hb level than expected value (i.e. the normal range). Anaemia is broadly cause by the impaired red blood cells (RBC) production, increased RBC destruction (haemolytic anaemia), blood loss and fluid overload (hypervolemia). The most commonly cause of anaemia is blood loss but this does not have any lasting symptoms unless a relatively impaired RBC production develops. It is therefore necessary to determine the Hb level in patient blood to enable anaemia investigation. Aims: To determine the Haemoglobin (Hb) concentration of an unknown female patient blood sample by comparing its light absorbance with the light absorbance of a standard stock solution of known Hb concentration.
Method: Determination of peak wavelength: plotting absorbance spectrum.
Two spectrophotometer cuvettes were used. One filled with deionised water and the other with stock solution of Hb (0.125mg/ml). Both cuvetters were filled approximately 3ml with the deionised water as a reference. Spectrophotometer set at 380nm then zero with reference (deionised water) cuvette, and then measurement taken for absorbance of the haemoglobin stock solution. Measurement was taken at the intervals of 5nm from 380 nm to 420 nm and zeroing the instrument with reference (water) at each wavelength.
The absorbance of a substance in solution is directly dependent on its concentration (Beer-Lambert law). However, this approach is not always accurate because of the limitations with Beer-Lambert law, particularly at higher concentration. A series of reading was used to produce a calibration graph by comparing the absorbance of the unknown with the known sample under same condition. Concentration of the unknown haemoglobin can be determine by measuring its absorbance at the wavelength and read its concentration from the calibrated graph.
PREPARING THE CONCENTRATION:
Series of duplicates dilutions were prepared of the stock Hb solution in clean dry test tubes as follows and mixed well.
Table of 7 duplicate samples ranging from 0 (tube 12) to 0.125 mg/ml haemoglobin (tube 1) were shown at regular intervals. This ensures that points are evenly spaced on the calibration graph.
0.1ml of the unknown stock solution was mix with 0.9ml deionised water covering with parafilm and inverted gently three times. Sample was incubated for 5mins at room temperature.
0.1ml stock solution was taken from thee incubated sample and mix with 0.9ml deionised water again as before and incubated for 5mins at room temperature.
0.3ml stock solution was taken from the second incubated sample (100 fold dilution) and mix with 2.7ml deionised water and incubated as before.
From the results obtained, the absorbance is proportional to wavelength as the (Beer-Lambert law). The more concentrated the stock solution, the higher the absorbance.
A table below shows concentration of stock solutions with respective absorbance.
ml of standard haemoglobin solution (0.125 mg/ml)
Final concentration (mg/ml)
Work e.g. Tube 2 and 3.
2,5ml stock sol/3ml total vol - 0.125 mg/ml stock sol
= 0.104 mg/ml
At optimum wavelength 405nm the concentration of unknown female patient haemoglobin was determine on the graph as 8.3 g/dl. It was workout as follows: 0.083mg/ml multiply by 1000 fold which equal to 83mg/ml, is equal to 83g/L. As there is 10 decilitre (dL) in 1 litre (L), therefore by dividing 83 by 10 equal to 8.3g/dL of Hb concentration.
Discussion: The unknown female patient blood Hb level was determined to be 8.3g/dl which indicate anaemic condition. From the results it shows that the higher the concentration of the solution, the better the value of the absorbance. The graph shows optimum wavelength increases from 380 to 405nm and decreases from 410 to 420nm. The experiment was successful in achieving the aim of the experiment, which was to determine the unknown Hb concentration by comparing it to the series of the known solutions absorbance on the graph. Though the result obtained could be limited to the following sources of errors. There may be slight error in the calibrated spectrophotometer during the experiment which could have an impact on the result even though this could be negligible. As the volume measured was very small, there could be pipetting error which could also have an impact of the overall result. A future modification by using automatic pipettes and plate readers could minimize the level of error in the experiment. The aim of the experiment was achieved as result proved patient anaemic condition.
A low Hb range achieved from the conducted experiment prove patient being anaemic but offers no information regard to the aetiology. Supplementary research is required in order to determine the cause of the anaemia and to establish its sequent treatment.