IQC Lesson 5-2

PIPETTING AND LABORATORY

MATHEMATICS

ANTONIO F. LAUDE JR, RMT MPH

SCHOOL OF MEDICAL TECHNOLOGY

Pipetting Technique using Manual

Pipettes

Develop good technique

Through practice

Improper pipetting – laboratory accidents

Mouth pipetting – potential hazard

Never acceptable

Caustic reagents

Contaminated specimens

Poisonous solutions

Pipetting with Manual Pipettes

1. Check the pipette to ascertain its correct size.

2. Wearing protective gloves.

3. Place the tip of the pipette well below the surface

of the liquid to be pipetted.

4. Use aspirator bulb – pipette until the level of

liquid is well above the calibration mark

5. Quickly cover the suction opening at the top of the

pipette with the index finger


6. Wipe the outside of the pipette dry with a piece of

gauze or tissue to remove excess fluid.

7. Hold the pipette in a vertical position with the

delivery tip against the inside of the original vessel.

Carefully allow the liquid in the pipette to drain by

gravity.

Miniscus it the concave or convex surface of a column

of liquid as seen in a laboratory pipette, buret, or

other measuring device.


8. While still holding the pipette in a vertical position,

touch the tip of the pipette to the inside wall of the

receiving vessel.

Remove index finger, permit free drainage.

TD pipettes – small amount will remain in the delivery

tip.


9. To be certain that the drainage is as complete as

possible, touch the delivery tip of the tip to another

area on the inside wall of the receiving vessel.

10. Remove the pipette from the receiving vessel, and

place it in appropriate place for washing.

Automatic Micropipettors

Micropipettor – most common type of micropipette

used in many laboratories

Allow repeated, accurate, reproducible delivery of

specimens, reagents, and other liquids requiring

measurement in small amounts.

Delivery volume is selected by adjusting the settings

on the device.

0.5 to 500 L – allow volume

Automatic Micropipettors

Contain or deliver from 1 to 500 L

Follow manufacturers instructions

Steps apply for use of a micropipettor

1. Attach the proper tip to the pipettor, and set the

delivery volume.

2. Depress the piston to a stop position on the

pipettor.

3. Place the tip into the solution, and allow the piston

to rise slowly back to its original position.

4. Some tips are wiped with a dry gauze at this step,

and some are not wiped. Follow the

manufacturer’s direction.

Steps apply for use of a micropipettor

5. Place the tip on the wall of the receiving vessel,

and depress the piston, first to stop position where the

liquid is allowed to drain, then to a second stop

position where the full dispensing of the liquid takes

place.

6. Dispose of the tip in the waste disposal receptacle.

Some pipettors automatically eject the used tips, thus

minimizing biohazard exposure.

Reading Meniscus

1. With a clear colorless solution, read the bottom of

the meniscus

2. With colored solutions, read the top fluid column.

3. Reading must be made with eye level to avoid

parallax error.

LABORATORY MATHEMATICS

Minimum numbers of digits needed to express a

particular value in scientific notation without loss

of accuracy.

728.4 contains how many significant figures?

7.284 x 102

0.000532 contains how many significant figures?

5.32 x 10-4

Significant Figures

Is equal to parts per 100 or the amount of solute per 100 total units of solution

It is determined in the same manner regardless of whether it is w/w, v/v or w/v units are used

Percent solution

1. Weight/Volume (w/v) % solutions

The most common type of solution prepared in clinical

laboratory

Refers to the number of grams of solute per 100 mL

of solution

Grams of solute = % solution desired x total volume desired

100

2. Volume/Volume (v/v)% solutions

Used when both solute and solvent are liquid

It refers to the amount of solute in mL in 100mL of

solvent

mL of solute = % solution desired x total volume desired

100

3. Weight/Weight (w/w) % solutions

Refers to the number of grams of solute per 100 gms

of solution

Grams of solute = % solution desired x grams of the total solution

100

When preparing concentrated acid solutions,

always add acid to water.

MOLARITY

The number of moles of solute per liter of solution.

1 mole of substance equals its gram molecular weight

(gmw)

Gram Molecular Weight (GMW) – is obtained by

adding the atomic weights of the component elements.

Molarity of solution (M) = Grams of solute .

GMW x volume of solution (L)

Sample Problem

Solution

Solution

MOLARITY

To prepare a molar solution:

Grams of solute = Molarity x GMW of the solute x

Volume (liter) desired

To convert % w/v to Molarity (M):

M = %w/v x 10

GMW

MOLARITY

The amount of solute per 1 kilogram of solvent

Expressed in terms of weight/weight or moles per

kilogram (mol/kg)

Molecular Weight (MW) is obtained by adding the

atomic weights of the given compound.

Molality (m) = Grams of solute

MW x kg of solvent

NORMALITY

Is the number of equivalent weight of solute per liter of solution

It has been used in acid-base calculations

Normality (N) = Grams of solute

EW x volume (L)

Equivalent weight (EW) = MW

valence

NORMALITY

To prepare a Normal solution of Solids:

Grams of solute = EW x Normality x Volume (L)

To convert % w/v to Normality (N):

N = % w/v x 10

EW

Relationship between Molarity and Normality:

Normality = Molarity x valence

Molarity = Normality

valence

Sample Problem

Solution

Milliequivalents

The most common way of expressing electrolytes

mEq/L= mg/dL x 10 x valence

MW

Millimoles

Molecular weight in millimoles (mmol/L)

mmol/L= mg/dL x 10

MW

RATIO and DILUTION

Ratio = Volume of solute

Volume of solvent

Dilution = Volume of solute

Volume of solution

Represents the ratio of concentrated or stock

material to the total/ final volume of a solution

DILUTIONS

(volume increases, concentration decreases,

amount of solute remains the same)

DILUTIONS

3 Reasons for Doing Dilution

1. The concentration of material is HIGH to be

accurately measured.

2. Removal of undesirable substances like in PFF

(Protein-free filtrate) preparation

3. Preparation of working standard from stock

solution

Dilution factor

The ration of a concentrated solution to the total

solution volume equals the dilution factor.

Is made by adding the concentrated stock to a

diluent

Example 1

What is the dilution factor needed to make a 100 mEq/L sodium solution from a 3000 mEq/L stock solution? The dilution factor becomes

100 = 1

3000 30

This means that the ratio of stock is 1 part stock made to a total volume of 30.

To make the solution: 1mL of stock is added to 29 mL of diluent.

The sum of the amount of the stock material plus the

amount of diluent must equal the total volume or

dilution fraction denaminator.

The dilution factor may be written as a fraction or

can be expressed as 1:30

1/30 or 1:30 either may be used

Example 2

If in the preceding example, 150 mL of the 100

mEq/L sodium solution was required,

The dilution ratio stock to total volume must be

maintained.

Set up a ratio between the desired total volume

and the dilution factor to determine the amount of

stock needed.

Equation

1 = x

30 150

5/150 = 1/30

To make this solution:

5mL of stock is added to 145 mL of the

appropriate diluent

Making the stock volume to diluent equal to 5/145

Simple Dilutions

The laboratorian must decide on the total volume desired and the amount of stock to be used

Examples:

A 1:10 (1/10) dilution of serum can be achieved by using any of the following approaches:

a. 100 uL of serum and 900 uL of saline

b. 20 uL of serum and 180 uL of saline

c. 1 mL of serum and 9 mL of saline

d. 2 mL of serum and 18 mL of saline

Dilution factor

Is used to determine the concentration of a dilution

or stock material by multiplying the original

concentration by the dilution factor.

When determining the original stock or undiluted

concentration, multiply the concentration of the

dilution by the dilution factor denominator.

Example

A 1:2 dilution of serum with saline had a creatinine

result of 8.6 mg/dL. Calculate the actual serum

creatinine concentration.

Dilution factor ½

Dilution result = 8.6 mg/dL

Because this result represents ½ of the

concentration, the actual serum creatinine value is

2 x 8.6 = 17.2 mg/dL

IQC Lesson 5-2

Education