Lecture #1
Clinical chemistry is that branch of chemistry which deals with quantitative and qualitative analysis of body fluids like blood, urine, spinal fluid, exudates, transudates as well as feces and calculi.
BASIC LABORATORY PRINCIPLES AND TECHNIQUES
Volumetric equipment
Most clinical chemistry procedure requires accurate measurements of volume. In very precise work, it is never safe to assume that the volume contained or delivered by any piece of equipment is exactly that amount indicated. Ideally, all volumetric equipment for precise work should be designated Class A, which means that it confirms to the specifications by NBS (National Bureau of Standard).
I. Pipettes
These are glass tubes used for transferring or measuring solutions. In the clinical laboratory, rubber tubing or bulbs are used when pipeting to avoid mouth contact of a pathogen.
A. Pipettes design and calibration
The design of pipettes differs according to the viscosity of the fluid measured. Most pipettes are marked “TD” (to deliver) while some are marked “TC” (to contain).
1. “To contain” (TC) – these pipettes are rinsed out with the diluting fluid. It is calibrated by introducing the exact weight of mercury equivalent to the aqueous volume desired.
Mercury is a non–wetting liquid that drains completely, thereby simulating the washing out technique employed in aqueous solutions; and since mercury is 13.5 times as heavy as water, it makes possible the accurate weighing of small volumes.
2. "To deliver” (TD) – these pipettes are not rinsed out with the diluting fluid. It is calibrated by measuring the amount of water delivered by the pipette.
Water is commonly used as calibration liquid because it is readily available and it is similar in viscosity and speed of drainage to the dilute solutions ordinarily employed in clinical chemistry. Small amount left in the tip should not be blown out.
Formula for calibration of a “to deliver” pipette:
Apparent weight of H2O in grams x stated capacity of x correction at
At ToC (equilibrated temperature) pipette in ml ToC
= actual capacity in ml at 20oC
3. “To blow out” – this is usually indicated by a frosted band or double ring near the mouth piece. This means that the last few drops remaining in the tip are expelled by blowing after drainage is complete.
4. “Between two marks” – exact volume is calibrated to fill the volume between two calibrated points on the pipette.
B. Types of pipettes
1. Volumetric or Transfer pipette
a. Calibrated to deliver a fixed volume of liquid or the volume specified.
b. Has cylindrical bulb in the midway between the mouth piece and the delivery tip. A calibration mark is etched around the upper suction tube and the delivery tip tapers gradually.
c. Used for accurate measurements of aliquots for non–viscous samples, protein free filtrates and standard solutions.
d. This pipette is allowed to drain only on the last drop and is never blown out.
Types of Volumetric pipettes:
a. Ostwald–Folin pipette
(1) A modified volumetric pipette with their bulbs closer to the delivery tip.
(2) They are use for measuring from 0.5 to 10 ml of viscous fluids such as whole blood, serum or plasma.
(3) The pipette has an etched ring near the mouthpiece indicating that the last drop be blown out (blow–out pipette).
b. Van Slyke pipette
(1) Constructed of thick walled capillary tubing with a bulb in the center and one mark above and one below.
(2) The specified volume is delivered between these two marks.
Requirement of Volumetric pipette:
a. Calibration mark should not be close to the top of the suction tube.
b. The bulb should merge gradually into the narrower tube.
c. The orifice should be such that the outflow of liquid is not too rapid in order to reduce drainage errors to negligible proportions.
2. Graduated or Measuring pipettes
a. It is plain, narrow tube drawn out to the tip and graduated uniformly along its length.
b. Intended for the delivery of predetermined volumes.
c. Used for measuring reagents and are not generally considered accurate enough for measuring samples and standards.
Types of Graduated pipettes:
a. Serologic pipette – has graduation mark down to the tip.
b. Mohr pipette – calibrated between two marks on the stem.
3. Micropipettes – available for measuring very small volumes (0.5 ml or less). Volumes are often expressed in lambdas. One lambda equals 0.1 ml or 0.001 ul. It is calibrated either TC or TD. The TC or the wash out pipette are more accurate of the two.
When using micropipettes, the sample is drawn just above the mark, the outer surface of the pipette is wiped clean with tissue paper and the sample is adjusted to the mark by touching the tip repeatedly to the paper. The contents are then delivered rinsing several times into a diluent. The diluent should not be drawn above the calibration mark
because this will add a film of sample in excess of normal value.
a. Sahli hemoglobin pipette – calibrated to contain .02 ml (20ul).
b. Kirk pipette – tip drawn out to the finer point (TC).
c. Lung–Levy pipette – self–adjusting and may either be calibrated TC or TD.
d. Self–filling or Overflow pipette – smaller in size and automatically fills by capillary action (TC).
4. Automatic pipettes – dispenses a pre–rated sample when plunger is moved through its complete stroke.
a. Peristaltic type
b. Piston type
c. Seligson type
II. Burettes
Burettes are long tubes of uniform bore with graduation along the length for measuring titration volumes. The volume, however are larger than the pipettes.
The outflow of liquid from the burette is controlled by:
A. Glass stopcocks which is used for acids.
B. Rubber stopcocks which is used for alkalines.
III. Volumetric flasks
These are glassware used primarily in preparing solutions of known concentration (standard solutions).
IV. Graduated cylinders
Commonly called graduates, are used when less accurate measurements are required.
V. Separatory funnels
Employed for simple extraction procedures. This involves the bringing of a given volume of solution into contact with a given volume of solvent (immiscible in the solution) by vigorous shaking until equilibrium has been attained, followed by the separation of liquid layers.
General laboratory supplies
I. Glassware
There are five types of glasses in general:
1. High thermal resistant glass (borosilicate glass with low alkali content – kimax, pyrex).
a. Corex brand – glass which has been strengthended chemically.
b. Vycor brand – recommended for use involving high temperature; drastic heat shock. It is primarily used in ashing and ignition techniques.
2. High silica glass
3. Glass with higher resistance to alkali
4. Low Acitinic Glassware
This contain materials that imparts an amber or red color to the glass and reduce the amount of light passing to the substance within the glasswares.
It was developed to provide a highly protective laboratory glassware for handling materials sensitive to light (bilirubin, carotene and Vitamin A).
5. Standard Flint Glass
This is a soda–lime glass composed of mixtures of the silione, calcium and sodium. This type of glass is lowest in cost and can readily be fabricated in a variety of shapes. Glass of choice for weighing bottles.
II. Special glasses
1. Colored and Opal glasses – made by adding small amounts of coloring materials to glass bottles. These glasses are used in light filters, lamp bulbs and lighting lenses.
2. Coated glass – used in electronics.
3. Optical glass – used in making prisms, lenses and optical mirrors.
4. Glass ceramics (Pyroceram) – useful in hot plates, table tops and heat exchange.
5. Radiation absorbing glass – useful in preventing transmission of high energy radiation.
III. Plastic wares
1. Polyclefins (polyethylene, polypropylene)
2. Polycarbonate resins
3. Tygon
4. Teflon Fluorocarbon resins
IV. Cleansing solutions for glassware:
1. Diluted detergent
2. 10% KOH
3. potassium dichromate
FILTERS AND FILTRATION
Filter papers are used to separate precipitates from fluid solutions. The purpose of filtration is to separate a solid from a liquid component. Filtration is usually accomplished by passing the liquid through a porous barrier possessing openings smaller than the solid particles being filtered out.
Based on speed of filtering, they can be classified as:
a. Slow–fine retention
b. Medium–medium retention
c. Rapid–coarse retention
Filter papers with diameter 9.0 ml and 12.5 cm are commonly used because they fit funnels generally used for preparation of blood and urine filtrates. Precipitates are poured into the paper with the aid of a glirring rod to avoid spillage and to keep the precipitate near the apex of the filter paper cone.
Phase separating paper – a water repellant cellulose acetate that acts as phase separator in place of conventional seperator in place of conventional separatory funnels.
Glass fiber filters – produced from borosilicate fibers when used in Buchner funnel, it gives a combination of fine retention with rapid filtering speed.
CHARACTERISTICS FEATURE OF FILTER WHATMAN NO.
I. Unwashed
Medium medium weight, speed and retentiveness 1
Dense more retentive, less rapid 2
Thick heavy, strong, quite retentive 3
Soft very rapid, less retentive 4
Very dense very retentive, filter slowly 5
II. Single acid washed (HCl)
Medium fairly rapid and retentive 30
Soft more rapid, less retentive 31
Retentive 32
III. Double acid washed (HCl and HF)
Medium medium speed and retentiveness 40
Soft more rapid, less retentive 41
Hard rapid, strong 42
Dense more retentive, less rapid 43
Thin very retentive 44
IV. Hardened
Retentive 50
Medium medium speed and retentiveness 52
More rapid, less retentive 54
CHEMICALS
Degree of purity of chemicals:
1. Primary standards – highly purified standard chemicals which maybe weighed out directly for the preparation of solution selected concentration or for the standardization of solution of unknown strength.
2. Secondary standards – are solutions whose concentration cannot be determined directly from the weight of solute and volume of solutions. The concentration of secondary standard is usually determined by analysis of an aliquot of the solution by the acceptable reference method using a primary standard.
3. Reagent Grade or Analytical Reagent Grade (A. R.) – these chemicals meet specifications to permit use of qualitative analysis.
4. Chemical Pure Grade (C. P.) – the degree of purity of materials carrying this label which is shared by term, “highest purity” is used by manufacturers for organic chemicals that they have purified to as great a degree as they find practical. The purity is usually determined by measurement of melting point or boiling point.
5. USP or NF grade – these chemicals are produced to meet specifications set out in the United States Pharmacopoeia (USP) or the National Formulary (NF).
6. Purified, Practical or pure grade – these chemicals can be used as starting material for laboratory synthesis of other chemicals of greater purity, but probably require purification and analysis before they can be used as analytical reagents. For certain analysis when reagents are not available, practical chemicals can be used if a blank is also run. In general, this chemical reagents should not be used in clinical chemical.
7. Technical or Commercial Grade – these chemicals are generally used only in manufacturing.
8. Reference Standards – these consist of spectroanalysed solvents, chromatographically pure reagents and calibrating reagents such as bilirubin and cholesterol assay.
9. Certified Standards – there a few standards that are certified by the College of American Pathologist. Examples are bilirubin and cyanmethemoglobin.
DISTILLED AND DEIONIZED WATER
Deionized water is water from which mineral salts have been removed by a process of ion exchange. Commercial deionizers are often used for this process.
To obtain water of the highest purity, distilled water is redistilled from an alkaline permanganate solution which oxidizes the nitrogenous matter present. When redistilled water is prepared in this manner, it is termed, conductivity water.
Property Requirements
Residue after evaporation Not more than 1 mg/L after drying for 1 hr at 105oC
Chloride content Not more than 0.1 mg/L
Ammonia content Not more than 0.1 mg/L
Heavy metals Not more than 0.01 mg/L
Consumption of permanganate When 0.03 ml of 0.1 molar potassium permanganate is added to 500 ml of water containing 1 ml of concentrated sulfuric acid, the color should not completely disappear on standing for 1 hr at room temperature.
Silicate Not more than 0.01 mg/L
Sodium Not more than 0.1 mg/L
Carbon dioxide Not more than 3 mg/L
PREPARATION AND CONCENTRATION OF SOLUTIONS
Components of a solution:
1. Solute – dispersed phase on the dissolved substance.
2. Solvent – dispersion medium or the substance in which the solute is dissolved.
Types of solution:
1. Dilute solution – contains a relatively small proportion of solute.
2. Concentrated solution – contains a relatively large proportions of solute.
3. Saturated solution – solution in which the dissolved and the undissolved portions of the solute are in equilibrium with each other.
4. Supersaturated solution – solution in which there is more solute in solution than is present in a saturated solution of the same substance at the same temperature and pressure.
5. Standard solution – a solution whose precise concentration is known. The process of determining of adjusting the concentration of the standard solution is known as standardization.
Three methods of standardization:
1. Direct preparation of the standard solution by dissolving a weighed amount of a pure, dry chemical and diluting the solution to an exactly known volume.
2. Titration of a solution of a weighed portion of pure, dry chemical by the solution to be standardized.
3. Titration against a primary standard such as hydrochloric acid that has been made up from a constant boiling hydrochloric acid. The solution standardized is known as secondary standard.
DILUTIONS
Dilution involves the preparation of a weaker solution from a stronger one. Usually, this is done by adding a diluent such as water which contains none of the solute, to a solution.
Reasons for dilution:
1. Concentration of the material in solution is too high to be accurately measure.
2. Removal of undesirable substances like protein in PFF preparation.
3. Preparation of working standards from the stock solutions.
Dilutions are usually expressed as a ratio, e.g., 1:10. This means that 1 unit of the original solution was diluted to a final volume of 10 units and will give a concentration of 1/10 of the original solution.
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