CLINICAL CHEMISTRY I

Title:
Copper reduction method

Objectives:
 To learn and compare the specificity and sensitivity
of different methods in glucose measurement
 To learn how applied copper - reduction method in
determine the glucose concentration

Principle:
In hot alkaline solution, the aldehyde group of glucose
reduces cupric ions to cuprous ions, which form mainly
cuprous oxide (Cu2O). Phosphomolybdic acid that was added
in the second step is reduced by the cuprous ion to form
compounds with lower oxidation states of molybdenum.

These products are blue and suitable for photometric

measurement.
The overall reactions are listed below:

Cu2+ alkali
 → Cu2+. (heat)

Cu+ + OH → CuOH

2 CuOH heat
→
 Cu2O + H2O

Sample & lab apparatus:

Glucose with standard concentration at 0.125 mmol/L and
0.25 mmol/L, control, sample A and sample B, isotonic
sodium sulphate – Copper sulphate (Na2SO4 – CuSO4), sodium
tungstate, alkaline tartrate (Harding’s B solution),
phosphomolybdic acid, centrifuge and spectrophotometer

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Method/ Procedure/ Assay Protocol:

4 5
1 2 3 (Standa (standa 6
Tube (contr (Sampl (Sampl rd rd 0.25 (Blank
ol) e A) e B) 0.125 mmol/L )
mmol/L) )
Serum 0.05 0.05
- - - -
sample ml ml
Control 0.05
- - - - -
ml
Isotonic
0.5
Na2SO4 – 1.9 ml 1.9 ml 1.9 ml - -
ml
CuSO4
Sodium 0.05 0.05 0.05
- - -
tungstate ml ml ml
Mix and centrifuge
Centrifug
(2000 rpm, at 10 -
e
minutes)
Supernat
0.5 ml 0.5 ml 0.5 ml - - -
ant
Standard - - - 0.5 ml 0.5 ml -
Harding’s 0.5
0.5 ml 0.5 ml 0.5 ml 0.5 ml 0.5 ml
B solution ml
Mix well; plug all test tubes lightly with cotton wool and heat
in boiling water for 10 minutes. Cool to room temperature
before adding the following:
Phospho-
1.5
molybdic 1.5 ml 1.5 ml 1.5 ml 1.5 ml 1.5 ml
ml
acid
Mix well and read after 5 minutes at 680 nm

Results:
Tubes Absorbance
1 0.964
2 0.256 X 10 = 2.56
3 0.848
4 0.381

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 5 0.331 X 5 = 1.655
6 0

Control range for 0.125 mmol/L: 0.248 ± 0.019 mmol/L

(2 SD)
Control range for 0.25 mmol/L: 0.285 ± 0.022 mmol/L (2
SD)

Discussion and calculations:

Calculation of the glucose concentration:
ODtest
For standard 1 = X standard concentration
ODs tan dard
(0.125mmol/L)
ODtest
For standard 2 = X standard concentration
ODs tan dard
(0.25mmol/L)

Glu cos e(mmol / L)

Glucose (mg/dl) =
0.05551

[Glucose] [Glucose]
Standard Standard
standard standard
Sample 1 in 2 in
1 2
mg/dl mg/dl
(mmol/L) (mmol/L)
0.964 0.316 0.964 0.146
× 0.125 = × 0.25 =
0.381 0.05551 1.655 0.05551
Control
= 0.316 5.693 = 0.146 2.630
mmol/L mg/dl mmol/L mg/dl
2.56 0.840 2.56 0.387
× 0.125 = × 0.25 =
0.381 0.05551 1.655 0.05551
Sample A
= 0.840 15.132 = 0.387 6.972
mmol/L mg/dl mmol/L mg/dl
0.848 0.278 0.848 0.128
× 0.125 = × 0.25 =
0.381 0.05551 1.655 0.05551
Sample B
= 0.278 5.008 = 0.128 2.306
mmol/L mg/dl mmol/L mg/dl
Standard
0.125 Reference Reference Reference Reference
mmol/L
Standard Reference Reference Reference Reference

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 0.25
mmol/L
Blank 0 0 0 0

Copper sulphate contains cupric ions which are reduced

to cuprous ions in boiling conditions in presence of glucose
and in fact all reducing sugars (and also non sugar reducing
substances).

The cuprous ions are once again oxidized to cupric ions

at the expense of phosphomolybdic acid which is itself
oxidised to molybdenum blue. Molybdenum blue imparts a
blue color to the medium and is estimated colorimetrically at
580 nm which gives us an estimation of the concentration of
sugar.

The overall reactions are listed below:

Cu2+ alkali
 → Cu2+. (heat)

Cu+ + OH → CuOH

2 CuOH heat
→
 Cu2O + H2O

Advantages:
 Increase the accuracy

Disadvantages:
 Consume more time for preparation and result
 Much more expensive compared to glucose
oxidase method
 Required more than one standard

Conclusion:
From this experiment, the control value that obtained
from this experiment are slightly higher when using
standard 1 (0.125mmol/L) and slightly lower when
using standard 2 (0.25mmol/L) than the control range
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that has been provided. The reference control range for
standard 1 (0.125mmol/L) is 0.248 ± 0.019 mmol/L (2
SD) but the value of control that we get from this
experiment is 0.316mmol/L (7 SD). For standard 2
(0.25mmol/L) the reference controls range is 0.285 ±
0.022 mmol/L (2 SD) but the value of control that we
get from this experiment is 0.146mmol/L (12 SD). This
error shows some mistake that occur during method
preparation. These errors include:
 Interference that occurs in the samples
 The cuvette that was used are not cleaned properly
 The interference that occur in the
spectrophotometer
 The serum that was used are contaminate with other
substances
 Error in taking the absorbance value.

From the experiment that was performed, the value for

glucose concentration in sample A are 0.840mmol/L
(15.132mg/dl) when using standard 1 and 0.387mmol/L
(6.972mg/dl) when using standard 2.

From the experiment that was performed, the value for

glucose concentration in sample B are 0.278mmol/L
(5.008mg/dl) when using standard 1 and 0.128mmol/L
(2.306mg/dl) when using standard 2.

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