Basic Concepts in Clinical Biochemistry-A Practical Guide.7z

16.5 Procedure

Take nine test tubes in duplicate, and label them as blank, S 1
S 7 (standard) and T
(test). Add standard glucose solution of 25, 50, 75, 100, 200, 300, and 500 mg % in
test tubes S 1
S 7 , respectively. Then add reagents according to protocol given in the
table. Read absorbance at 430 nm.

Reagents Blank S 1 S 2 S 3 S 4 S 5 S 6 S 7 T

N/12 H 2 SO 4 (ml) 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6 1.6

Glucose standard (ml) – 0.2 0.2 0.2 0.2 0.2 0.2 0.2 –

Glucose concentration

(mg/dl)

• 25 50 75 100 200 300 500 –

Glucose amount (mg) – 0.025 0.05 0.075 0.1 0.2 0.3 0.5

Distilled water (ml) 0.2 ––––––––

Serum/plasma (ml) ––––––––0.2

Sodium tungstate (ml) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2

Mix well and wait for 10 min, and then centrifuge at 3000 rpm for 10 min. Collect supernatant

Supernatant (ml) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

Alkaline CuSO 4 (ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Mix well and keep in boiling water bath for 10 min and cool

Phosphomolybdate (ml) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0

Shake well to remove CO 2 completely

Distilled water (ml) 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5

14.4 Calculations

Plot a graph between amount of glucose at x-axis and absorbance at y-axis. Extrap-
olate glucose amount in the given sample from the graph or calculate using equation
as explained in previous experiment.

62 14 To Determine the Blood Glucose Levels by Folin and Wu Method