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Thursday, March 1, 2012

Spectrophotometer and Electrolytes Handouts: Clinical Chemistry Laboratory

DIASYS STARDUST FC

Basic components of a spectrophotometer:

Light source, entrance slit, monochromator, exit slit, cuvette, detector and read –out device.

Some of the basic components may also be found in your operating features. Operating features are the parts seen outside and the basic components may be seen outside but some are seen inside the machine.
Beer-Lambert’s Law

This is the law applicable to the spectrophotometer, in which the concentration of a solution is directly proportional to the absorbance (optical density) and depth of a solution, and inversely proportional to the transmitted light or transmittance.

There are three methods in Unknown concentration determination:

1. Ratio of standard to unknown – use formula to compute for Cu (unknown concentration)

Cu= Au X Cs / As

2. Standard curve = where you extrapolate the Au in the standard curve chart to find the equivalent concentration.
The concentration is plotted in the X axis and the absorbance readings in the Y axis.
Plot the As and Cs in your chart (use a graphing paper with small lines) before you look for Cu.

YOU DON’T COMPUTE FOR ANYTHING IN YOUR STANDARD CURVE. YOU JUST NEED A RULER TO BE ACCURATE IN DETERMINING YOUR Cu.

3. Molar absorptivity value – constant absorptivity values given to each substance. No computations and no charts.

N.B. CONTROL VALUES ARE USED IN DETERMINING THE RELIABILITY OF METHODS , WHILE STANDARD VALUES ARE USED IN DETERMINING THE UNKNOWN CONCENTRATION.

If there is only one standard concentration then use the “ratio of standard to unknown” formula to determine your Cu.

If there are more than 2 standard values , then make use of the standard curve to determine your Cu.

WHY DO YOU HAVE TO WARM UP YOUR SPECTRO?

This is to stabilize the machine and avoid fluctuation in readings to come up with reliable results.

(READ YOUR LAB MANUAL FOR ADDITIONAL NOTES)

POTASSIUM

Potassium is an element (and an electrolyte) that's essential for the body's growth and maintenance.
It's necessary to keep a normal water balance between the cells and body fluids.
Potassium also plays an essential role in the response of nerves to stimulation and in the contraction of muscles.
Cellular enzymes need potassium to work properly.

SOURCES
bananas, cantaloupe, grapefruit, oranges, tomato or prune juice, honeydew melons, prunes, molasses and potatoes. Some foods high in potassium are also high in calories. When weight control is important, eat more low-calorie foods.

A. Specimen

Serum or plasma
Unhemolyzed, nonturbid, nonlipemic ,.non-icteric, clear

HEPARIN: anticoagulant of choice

PRECAUTIONS for potassium determination:

• Use unhemolyzed, nonturbid, nonlipemic , non-icteric, clear serum
• Use heparin if plasma is used
• Don’t test if the patient has undergone strenuous exercises. This could increase the concentration of potassium because of the tendency of the intracellular potassium to be forced outside the cell and increase its serum concentration.
• Avoid prolonged application of tourniquet because of the danger of extracellular and intracellular shift of electrolytes.
• Avoid coffee, alcohol, cigarette

(read your lab manual and the kits’ literature for additional notes)

C. Procedures

Methods of Determination:

Colorimetric/ Chemical Method (Lockhead and Purcell)

Lockhead and Purcell Method (Lochhead Purcell)

a. Principle:
Potassium in serum is reacted with sodium tetraphenyl boron (2.1 mM) to produce a colloidal suspension which is measured spectrophotmetrically. The amount of colloidal suspension is directly proportional to the concentration of the solution.

b.Reagents and Results
Sodium tetraphenyl boron (2.1 mM

c.Clinical significance:

1. Decreased potassium levels (hypokalemia/hypotassemia)
a. decreased in take as seen in chronic starvation
b. increased loss of potassium-rich body fluid in prolonged diarrhea and vomiting
c. redistribution of extra cellular potassium into the intracellular fluid
d. alklosis
e. hyperaldosteronism
f. primary and secondary hypertension

2. Increased potassium levels (Hyperkalemia/ Hyperpotassemia)
a. Dehydration
b. shock with tissue hypoxia
c. severe burns
d. diabetic ketoacidosis
e. massive hemolysis
f. status epilepticus
g. Addison’s disease

Normal Values
3.6-5.4 mmol/L (serum)


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