SPECTROPHOTOMETRIC ESTIMATION OF HAEMOGLOBIN
Paper, Order, or Assignment Requirements
please find all the instructions in the attached document
SPECTROPHOTOMETRIC ESTIMATION OF HAEMOGLOBIN
You are provided with a standard blood ‘Z’ containing 12.2 g/dl of Heamoglobin (based on
1:200 dilution). Follow the method below to construct a calibration curve using this sample.
Method
Step 1: Calibration Curve
- Make a 1:100 dilution of blood ‘Z’ by taking 1mL of well mixed blood and making up to
100mL with ammonium chloride
- Mix well and leave for 10mins
- Set up, in duplicate, 2 rows of 10 tubes and follow the dilution table below:
| Tubes | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 1:100
dilution |
0.5mL | 1.0mL | 1.5mL | 2.0mL | 2.5mL | 3.0mL | 3.5mL | 4.0mL | 4.5mL | 5.0mL |
| Ammonium
chloride |
4.5mL | 4.0mL | 3.5mL | 3.0mL | 2.5mL | 2.0mL | 1.5mL | 1.0mL | 0.5mL | 0.0mL |
- Prepare a reagent blank consisting of ammonium chloride only
- Read the blank and calibrate the spectrophotometer at wavelength 540nm.
- Read all the remaining tubes at 540nm and record the absorbance; remembering you
have set the tubes up in duplicate.
Step 2: Determine Hb concentration in patient samples
- You are provided with samples for 6 patients, samples A-‐F
- In duplicate, aliquot 20mL into 4mL of ammonium chloride.
- Perform the same step with sample Z as a quality control measure
- Mix well and leave for 10mins
- Read the absorbance for each sample at 540nm and record in a suitable table
Results
Calibration Curve
Complete the table below and calculate the relative dilution factors and concentrations for
each tube.
| Tubes | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 1:100
dilution |
0.5mL | 1.0mL | 1.5mL | 2.0mL | 2.5mL | 3.0mL | 3.5mL | 4.0mL | 4.5mL | 5.0mL |
| Ammonium
chloride |
4.5mL | 4.0mL | 3.5mL | 3.0mL | 2.5mL | 2.0mL | 1.5mL | 1.0mL | 0.5mL | 0.0mL |
| Dilution Factor | 1:200 | |||||||||
| Hb Conc (g/dL) | 12.2 | |||||||||
| Abs 1
|
0.095
|
0.195
|
0.300
|
0.375
|
0.477
|
0.578
|
0.666
|
0.761
|
0.850
|
0.947
|
| Abs 2
|
0.094
|
0.196
|
0.301
|
0.375
|
0.478
|
0.582
|
0.674
|
0.762
|
0.858
|
0.949
|
| Mean
|
Present your data as a calibration curve using the graph paper provided over leaf
Determination of Hb Concentration in patient samples
Complete the table below and provide concentrations for each of the samples as determined by
the calibration curve.
| Sample | A | B | C | D | E | F | Z |
| Abs 1
|
0.187
|
0.208
|
0.296
|
0.192
|
0.312
|
0.258
|
0.643
|
| Abs 2
|
0.189
|
0.218
|
0.297
|
0.193
|
0.315
|
0.259
|
0.645
|
| Mean
|
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| Hb Conc (g/dL) |
8
Questions:
Answer the following questions in the boxes provided:
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