Clinical Chemistry II DATE : 15.01.

2008

TITLE
INSTRUMENTATION : SPECTROPHOTOMETRY

OBJECTIVES
To learn the method for simple spectrophotomete usage

PRINCIPLE

Serum albumin in the presence of bromcresol green at a slightly acid pH produces

a color change of the indicator from yellow-green to green-blue.

PROCEDURE

1. Albumin standard of 50 mg/ml are provided. A series of dilution is set up and

albumin is assayed using the Bromcresol green method.
2. The assay protocol is followed. The absorbance for each dilution is taken.
Absorbance versus concentration graph is plotted. The concentration of albumin
in sample is determined.

ASSAY PROTOCOL
Wavelength : 546nm
Measurement : against reagent blank
Only one reagent blank per series is required

PIPETTING SCHEME

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 SAMPLE / STANDARD /
REAGENT BLANK
CONTROL
Distilled water 10μl -
Sample / standard / control - 10μl
Color reagent 1000μL 1000μl
Mix, incubate for 10 minutes at 20-25°C. The absorbance of the sample and the
standard against the reagent blank is measured within 60 minutes.

CALCULATION

C1V1 = C2V2

C1 = Albumin standard (50mg/ml)

C2 = Standard concentration
V1 = Volume of standard
V2 = Total volume (20μl)

• For standard concentration of 0 mg/ml

(50 X V1) = (0 X 20)
V1 = 0 μl of standard volume + 20 μl of distilled water

• For standard concentration of 10 mg/ml

(50 X V1) = (10 X 20)
V1 = 4 μl of standard volume + 16 μl of distilled water

• For standard concentration of 20 mg/ml

(50 X V1) = (20 X 20)
V1 = 8μl of standard volume + 12μl of distilled water

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 • For standard concentration of 40 mg/ml
(50 X V1) = (40 X 20)
V1 = 16 μl of standard volume + 4 μl of distilled water

• For standard concentration of 50 mg/ml

(50 X V1) = (50 X 20)
V1 = 20 μl of standard volume + 0 μl of distilled water

RESULTS

Standard concentration (mg/ml) Absorbance

0 0.007
10 0.080
20 0.153
40 0.297
50 0.401
Control 0.275
Sample A 0.234
Sample B 0.164

DISCUSSIONS

Spectrophotometer is a types devices that measuring the light of intensity with

based on monochromatic light over a continuos range of wavelength. There are many

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types of spectrophotometer; where the simplest instrumentation of spectrophotometer are
based on single-beam. This apparatus are divided into seven basic components, which
includes a stable source of radiant energy; an entrance slit to focus the light; a wavelenght
selector; an exit slit to focus the light; a device to hold the transparent container (cuvette);
a radiant-energy detector and a device to read out the electrical signal generated by the
detector. Nevertheless, the more features of all types spectrophotometer is the spectral
bandwidth and the linear range.

From the graph, we obtain that the graph line of concentration for albumin against
the absorbance seem to be increasing. This showed that the concentration of albumin is
proportional to the absorbance. From the graph plotted, the control value is 37 mg/dl and
this is not valid because slightly highof range of the true value in between of 36.27 ±
0.2343 mg/ml. Thus, the both sample A and B concentration also are not valid, since the
result of sample is lesser from the true value. Some possibilities could be occur during
this experiment which are; pippeting technique; inaccurate of timing process; dirty
cuvette; error in mixing of solution and temperature of incubation.

CONCLUSIONS

Spectrophotometry has becomes a widely used as intrumentations in laboratory

where the function as for measuring the concentration of analyte within a solution. From
this experiment, the concentration of albumin for sample A is 31 mg/ml, while sample B
is 21.5 mg /ml. Eventhough, both this sample is not valid because of certain errors that
occurs during this experiment as the control on this experiment is slightly high that could
not be expected as the range given (36.27 ± 0.2343 mg/ml).

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