but in most instruments the intensity is below the

danger level. Methods in which photochemical reactions

are likely to occur usually mention the precautions to be

taken against light exposure. For example, the reconstituted glucose reagent kit is recommended to be stored in a

dark bottle, because on exposure to light a photochemical

reaction takes place and the reagent gets oxidised and

develops a pink color.

c. Color Instability

In some colorimetric reactions, the color may be stable for

only a short period of time. It is then necessary to time the

reaction carefully so that the readings of all samples and

standards are made during the time that the color remains

constant. Instability of color may be due to temperature

or absorption by the walls of the container where these

factors have an influence, they must be kept constant for

test samples and standards.

d. Foreign Matter and Air Bubbles

Solutions in the cuvettes must be free of lint or other

foreign matter, and air bubbles. A scrupulous cleaning

of the cuvettes and other glassware used in the analysis

should help to eliminate foreign matter from the solution.

For example, in a flow through cuvette of most semiautomated analyzers an air bubble trapped in it, will lead

to a decrease in optical density of the solution.

e. Errors of Weighing and Dilution

Simple errors of weighing and dilution in preparing

reagents, sample and standards can affect photometric

results appreciably. A good analytical balance and reliable

volumetric glassware should be used. For example, while

reconstituting a control serum care should be taken while

dispensing the volume of distilled water to the control

sera bulb. An error in dilution will result in change in the

concentration of the constituents.

Instrument

Instruments are capable of considerable precision of

measurement. The instrumental precision is of a higher

order than that normally resulting from the development

of color in the test solutions. Inherent errors of the solution

as mentioned above actually cause greater deviation than

do instrumental errors.

a. Light Source

Unless a double cell photometer is being used, the

consistency and reproducibility of the light source is

important. Fluctuations in voltage should be overcome by

the use of a voltage stabilizer in line-operated instruments.

Lamps should be allowed to warm up for at least 5 minutes

before steady output can be expected.

b. Stray Light

Stray light from windows or overhead lighting striking

the instrument can cause error since invisible particles

suspended in solutions can reflect these rays. The covering

of the cuvette compartment with a light-tight cover (as

usually provided with the instrument) before taking

readings will reduce this error.

c. Slit Width

In the prism spectrophotometer, the purity of the

monochromatic band depends on the width of the

entrance and exit slits. The use of a narrow slit width will

produce more accurate results.

d. Moisture

Moisture can be the cause of fluctuating readings in

spectrophotometric operation. In instruments employing

desicant, it is advisable to change the freshly dried silica

gel at regular intervals. This is particularly important in an

environment of high humidity.

e. Linearity of Photocell Response

Reliable results depend upon the current output of

the photocell being proportional to the light striking

the photocell. This relationship can be disturbed if the

photocell is not adequately protected from moisture and

from overheating. Some instruments are fitted with heat

absorbing filters in the optical system and provided with

thermal insulation of the light source.

Clinical Chemistry 465

f. Cuvettes

An important source of error and one which requires

constant checking is the cuvette. It is necessary that cuvettes

be optically matched, so that readings will not be influenced

by their individual variation when a series is used for making

measurements. When it is necessary to reuse cuvettes that

have not had adequate time to dry after cleaning, a rinse

with alcohol and ether or acetone may be used to speed up

the drying process. If cuvettes are dirty, etched, scratched

or marked with fingerprints, erroneous readings will result.

g. Wavelength Calibration

A wavelength scale which is off calibration can be a source

of error in spectrophotometers.