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Operating Procedure

A. Turn on the spectrophotometer (left-hand knob on the front of the instrument (A)) and allow it
to warm up for at least 15 min.

B. Adjust the wavelength to the appropriate value. The knob on the right top (C)of the instrument
controls the wavelength, which is indicated at the left of the digital display.

C. With the sample holder empty and the lid closed, adjust the Zero Adjust Knob (A) until the
instrument reads 0% on the transmittance scale. Be sure that the display function is set to
transmittance, if not push the "Mode" button until the display is set to transmittance.

D. Carefully insert the appropriate blank tube (cuvette) into the sample holder (E) and close the
cover. Be sure you are using a cuvette with white markings. The cuvette's outside surface must
be dry and clean, including free of fingerprints!! Use a Kimwipe to clean the cuvette before
inserting. The white markings should line up with the notch on the sample holder. It is important
to line up the markings. The cuvettes will be scratched otherwise.

E. Adjust the 100% Adjust Knob (B), on the right front of the instrument, until the display reads
100% on the transmittance scale.

F. Remove the blank cuvette and immediately insert the sample cuvette as described in step d
above. Do not change any instrument setting! Switch the display to read absorbance by pushing
the "Mode"(D) button.
G. Record the value indicated on the absorbance scale.

H. Repeat this procedure for additional cuvettes or wavelengths as required. Always adjust the
blank transmittance to 100% before inserting and reading a new set of cuvettes.

Now Complete the Pre-Lab Quiz

THE SPECTROPHOTOMETER
Background
.

A spectrophotometer is an instrument designed to detect the amount of radiant


light energy absorbed by molecules. To do this, the instrument must have five
basic components:

1. a light source;

2. a prism or diffraction grating;

3.an aperture or slit;/ monochromator

4. a detector (a photoelectric tube);

5.a digital meter to display the output of the

phototube.

The arrangement of these parts is shown below.

When light is reflected from a diffraction grating,


it is split into its component colors or
wavelengths, which then diverge. Sections of the
projected spectrum can be either blocked or allowed to pass through the slit so that
only one wavelength will pass to the other sections of the spectrophotometer (The
position of the grating is adjustable so that the region of the spectrum projected on
the slit can be changed.). Light that passes through the exit slit travels to the
phototube/sample, where it creates an electric current proportional to the number
of photons striking the phototube.

THE SAMPLE IN A CUVET , ABSORB A PORTION OF LIGHT , AND


THE REST IS TRASMITED THROUGH THE SAMPLE. THE TRANSMITTED
BEAM STRIKES A DETECTOR, WHICH CONVERT RADIANT ENERGY
INTO ELECTRICAL ENERGY PROPORTIONAL TO THE AMOUNT OF
LIGHT. A READ OUT DEVICE PRESENTS THE ELECTRICAL SIGNAL IN
USEFUL UNITS.

Note that the relationship between absorbance and concentration is linear. As


concentration increase the absorbance also increases. This relationship allows
one to convert an absorbance value into a concentration.

EXAMINATION SERUM CHOLESTEROL TOTAL & TRIGLISERIDE


1. Preparing the patient

Patient must be fasting 12 – 14 hours before taking sample.

2. Prepare : 1.syringe or vacutainer tube


2.torniquet kit
3.Seventy percent isopropyl alcohol on cotton balls
4.gauze pads sterile or sterile cotton
5.band aid.

3. Take the sample according the picture (below)