Paper Order or Assignment Requirements please find all the instructions in the

Paper Order or Assignment Requirements

please find all the instructions in the attached document
SPECTROPHOTOMETRICESTIMATION OF HAEMOGLOBIN

Youare provided with a standard blood Z containing 12.2 g/dl of Heamoglobin (based on
1:200dilution). Follow the method below to construct a calibration curve using this sample.

Method

Step1: Calibration Curve Makea 1:100 dilution of blood Z by taking 1mL of well mixed blood and making up to100mLwith ammonium chlorideMixwell 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
101:100
dilution
0.5mL
1.0mL
1.5mL
2.0mL
2.5mL
3.0mL
3.5mL
4.0mL
4.5mL
5.0mLAmmonium
chloride
4.5mL
4.0mL
3.5mL
3.0mL
2.5mL
2.0mL
1.5mL
1.0mL
0.5mL
0.0mLPreparea reagent blank consisting of ammonium chloride only
Readthe blank and calibrate the spectrophotometer at wavelength 540nm.
Readall the remaining tubes at 540nm and record the absorbance; remembering youhaveset the tubes up in duplicate.

Step2: Determine Hb concentration in patient samples Youare provided with samples for 6 patients samples A-F
Induplicate aliquot 20mL into 4mL of ammonium chloride.
Performthe same step with sample Z as a quality control measure
Mixwell and leave for 10mins
Readthe absorbance for each sample at 540nm and record in a suitable table
Results

CalibrationCurve

Completethe table below and calculate the relative dilution factors and concentrations for
eachtube.
Tubes
1
2
3
4
5
6
7
8
9
101:100
dilution
0.5mL
1.0mL
1.5mL
2.0mL
2.5mL
3.0mL
3.5mL
4.0mL
4.5mL
5.0mLAmmonium
chloride
4.5mL
4.0mL
3.5mL
3.0mL
2.5mL
2.0mL
1.5mL
1.0mL
0.5mL
0.0mLDilution Factor1:200HbConc (g/dL)12.2Abs1

0.095

0.195

0.300

0.375

0.477

0.578

0.666

0.761

0.850

0.947
Abs2

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

Determinationof Hb Concentration in patient samples

Completethe table below and provide concentrations for each of the samples as determined by
thecalibration curve.
Sample
A
B
C
D
E
F
ZAbs1

0.187

0.208

0.296

0.192

0.312

0.258

0.643
Abs2

0.189

0.218

0.297

0.193

0.315

0.259

0.645
Mean
HbConc (g/dL)

8

Questions:

Answerthe following questions in the boxes provided:
Whydo you dilute sample Z 1:100 initially? Whydo you include testing sample Z with the unknown patient samples? Whydo you need toread absorbance of thedilutionsat 540nm?

Areyour results accurate and how can you tell? Whatare the Hb reference ranges for men women and neonates? Whatis the definitionof anaemia? SampleD is female is she anaemic?

What effectwould a high white cell count have onyour haemoglobin measurement? How is haemoglobin measured in thelaboratory? Describe the structure of the haemoglobin molecule and describe its function.

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