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PREPARING LABORATORY SOLUTIONS AND REAGENTS I
Dr Ramakrishna

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TOPICS Atomic structure
Basic concept of solution
Preparing solutions
Molarity, Normality, Molecular weight etc.
Examples

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Atomic structure Atoms are made up of 3 types of particles electrons , protons and neutrons.
Electrons are tiny, very light particles that have a negative electrical charge (-).
Protons are much larger and heavier than electrons and have the opposite charge, protons have a positive charge.
Neutrons are large and heavy like protons, however neutrons have no electrical charge. Each atom is made up of a combination of these particles.
E P N

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Atomic structure p + e _
Structure of Hydrogen
Structure of Helium

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Atomic structure We can measure an atom's size in two ways: using the atomic number (Z) or using the atomic mass (A, also known as the mass number).
The atomic number describes the number of protons in an atom. For hydrogen Z= 1, For helium Z = 2. Since the number of protons equals the number of electrons in the neutral atom, Z also tells you the number of electrons in the atom.
The atomic mass tells you the number of protons plus neutrons in an atom. Therefore, the atomic mass, A, of hydrogen is 1. For helium A = 4.

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Atomic structure

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Atomic structure

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Atomic structure

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BASIC CONCEPTS OF SOLUTIONS SOLUTE – The part of a solution that is being dissolved (usually the lesser amount)
SOLVENT – The part of a solution that dissolves the solute (usually the greater amount)
Solute + Solvent = Solution

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BASIC CONCEPTS OF SOLUTIONS Solutions can be classified as saturated or unsaturated.
A saturated solution contains the maximum quantity of solute that dissolves at that temperature.
An unsaturated solution contains less than the maximum amount of solute that can dissolve at a particular temperature

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BASIC CONCEPTS OF SOLUTIONS SUPERSATURATED SOLUTIONS contain more solute than is possible to be dissolved
Supersaturated solutions are unstable. The super saturation is only temporary, and usually accomplished in one of two ways:
Warm the solvent so that it will dissolve more, then cool the solution
Evaporate some of the solvent carefully so that the solute does not solidify and come out of solution.

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BASIC CONCEPTS OF SOLUTIONS One application of a supersaturated solution is the sodium acetate “heat pack.”

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The amount of solute in a solution is given by its concentration.
Fraction where:
Numerator, the amount of solute
Denominator, usually volume of entire solution
solvent + solute(s)
CONCENTRATION versus AMOUNT

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Each star represents 1 mg of NaCl.
What is the total amount of NaCl in the tube? _____
What is the concentration of NaCl in the tube (in mg/mL)? _____

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Each star represents 1 mg of NaCl.
What is the total amount of NaCl in the tube?
4 mg
What is the concentration of NaCl in the tube (in mg/mL)?
4 mg = ?_
5 mL 1 mL
? = 0.8 mg, so the concentration is 0.8 mg/mL

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WAYS TO EXPRESS CONCENTRATION OF SOLUTE Source of confusion: more than one way to express concentration of solute in a solution

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CONCENTRATION EXPRESSIONS 1. WEIGHT PER VOLUME
2. MOLARITY
PERCENTS
a. Weight per Volume %
(w/v %)
b. Volume per Volume %
(v/v %)
c. Weight per Weight %
(w/w %)

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MORE CONCENTATION EXPRESSIONS 4. PARTS
Amounts of solutes as "parts"
a. Parts per Million (ppm)
b. Parts per Billion (ppb)
c. Might see ppt
d. Percents are same category (pph %)

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STILL MORE CONCENTRATION EXPRESSIONS TYPES NOT COMMON IN BIOLOGY MANUALS:
MOLALITY
6. NORMALITY
for NaOH and HCl, molarity = normality, however, this is not always true for all solutes

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1.WEIGHT / VOLUME Means a fraction with:
weight of solute in numerator
total volume in denominator

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EXAMPLE:
2 mg/mL proteinase K
2 mg of proteinase K in each mL of solution.
How much proteinase K is required to make 50 mL of solution at a concentration of 2 mg/mL?

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PROPORTION PROBLEM
2 mg proteinase K = X
1 mL solution 50 mL solution
X = 100 mg
= amount proteinase K needed.

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Preparation of solution 1.0 L of water was used to make 1.0 L of solution. Notice the water left over.

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2. MOLARITY
Molarity is: number of moles of a solute that are dissolved per liter of total solution.
A 1 M solution contains 1 mole of solute per liter total volume.
1 mole (GMW) in 1000 ml = 1M

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MOLE How much is a mole?
From Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, Seidman and Moore, 2000

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EXAMPLE: SULFURIC ACID
For a particular compound, add the atomic weights of the atoms that compose the compound.
H2SO4:
2 hydrogen atoms 2 X 1.00 g = 2.00 g
1 sulfur atom 1 X 32.06 g = 32.06 g
4 oxygen atoms 4 X 16.00 g = 64.00 g
98.06 g

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EXAMPLE CONTINUED
A 1M solution of sulfuric acid contains 98.06 g of sulfuric acid in 1 liter of total solution.
"mole" is an expression of amount
"molarity" is an expression of concentration.

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DEFINITIONS
"Millimolar", mM, millimole/L.
A millimole is 1/1000 of a mole.
"Micromolar", µM, µmole/L.
A µmole is 1/1,000,000 of a mole.

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EXAMPLE 1. Prepare 75 ml of 0.1 M CaCl2
Molecular Weight of CaCl2 = 111
Gram Molecular Weight of HCl = 111 gms
Weight required to make 1L of 1 M HCl
= 111 gms
Weight required to make 1 L of 0.1 M HCl is = 0.1 x 36.5 ? 1 = 3.65 gms
Weight required to make 75 ml of 0.1 M solution =
1000 ml ----------- 3.65
75 ml ---------------?
75 x 3.65 ? 1000 = 0. 273 gms

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ANSWER

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From Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, Seidman and Moore, 2000

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TO MAKE SOLUTION OF GIVEN MOLARITY AND VOLUME 1. Find the FW of the solute, usually from label.
2. Determine the molarity desired.
3. Determine the volume desired.
4. Determine how much solute is necessary by using the formula.

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PROCEDURE CONT. 5. Weigh out the amount of solute.
6. Dissolve the solute in less than the desired final volume of solvent.
7. Place the solution in a volumetric flask or graduated cylinder. Add solvent until exactly the required volume is reached, Bring To Volume, BTV.

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PERCENTS X % is a fraction
numerator is X
denominator is 100
Three variations on this theme.

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WEIGHT/VOLUME %
TYPE I:
Grams of solute
100 mL total solution
Most common in biology.

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EXAMPLE
20 g of NaCl in
100 mL of total solution
= 20% (w/v) solution.

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EXAMPLE: BY PROPORTIONS How would you prepare 500 mL of a 5 % (w/v) solution of NaCl?

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ANSWER By definition: 5 % = 5 g
100 mL
5 g = ?
100 mL 500 mL
? = 25 g = amount of solute
BTV 500 mL

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BY EQUATION How would you prepare 500 mL of a 5 % (w/v) solution of NaCl?
1. Total volume required is 500 mL.
2. 5% = 0.05
3. (0.05) (500 mL) = 25

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% EXAMPLE CONTINUED 4. 25 is the amount of solute required in grams.
5. Weigh out 25 g of NaCl. Dissolve it in less than 500 mL of water.
6. In a graduated cylinder or volumetric flask, bring the solution to 500 mL.

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From Basic Laboratory Methods for Biotechnology: Textbook and Laboratory Reference, Seidman and Moore, 2000

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TWO OTHER FORMS OF % v/v mL solute
100 mL solution
w/w g solute
100 g solution

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WEIGHT/WEIGHT How would you make 500 g of a 5% solution of NaCl by weight (w/w)?

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ANSWER Percent strength is 5% w/w, total weight desired is 500g.
5% = 5g/100g
5g X 500 g = 25 g = NaCl needed
100 g
500 g – 25 g = 475 g = amount of solvent needed
Dissolve 25 g of NaCl in 475 g of water.

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PARTS Parts may have any units but must be the same for all components of the mixture.

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EXAMPLE:
A solution is 3:2:1 ethylene:chloroform:isoamyl alcohol
Might combine:
3 liters ethylene
2 liters chloroform
1 liter isoamyl alcohol

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PPM AND PPB ppm: The number of parts of solute per 1 million parts of total solution.
ppb: The number of parts of solute per billion parts of solution.

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PPM EXAMPLE: 5 ppm chlorine = 5 g of chlorine in 1 million g of solution,
or 5 mg chlorine in 1 million mg of solution,
or 5 pounds of chlorine in
1 million pounds of solution

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CONVERSIONS To convert ppm or ppb to simple weight per volume expressions:
5 ppm chlorine = 5 g chlorine = 5 g chlorine 106 g water 106 mL water
= 5 mg/1 L water
= 5 X 10-6 g chlorine/ 1 mL water
= 5 micrograms/mL

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PPM TO MICROGRAMS/mL For any solute:
1 ppm in water = 1 microgram
mL

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Each star represents 1 mg of dioxin.
What is the concentration of dioxin in tube expressed as ppm (parts per million)? ____________
What is the total amount of dioxin in beaker? ___________

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Each star represents 1 mg of dioxin. What is the total amount of dioxin in tube? 25 mg What is the concentration of dioxin in tube expressed as ppm? ____________ 1 ppm in water = 1 µg mL 25 mg/500 mL = 0.05 mg/mL = 50 µg/mL so the concentration is 50 ppm

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PREPARATION OF SOLUTIONS Preparing Dilute Solutions from Concentrated Ones (C1V1=C2V2)
Biological Buffers
Preparing Solutions with More Than One Solute
Assuring the Quality of a Solution

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PREPARING DILUTE SOLUTIONS FROM CONCENTRATED ONES Concentrated solution = stock solution
Use this equation to decide how much stock solution you will need: C1V1=C2V2
C1 = concentration of stock solution
C2 = concentration you want your dilute solution to be
V1 = how much stock solution you will need
V2 = how much of the dilute solution you want to make

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EXAMPLE How would you prepare 1000 mL of a 1 M solution of Tris buffer from a 3 M stock of Tris buffer?
The concentrated solution is 3 M, and is C1.
The volume of stock needed is unknown, ?, and is V1.
The final concentration required is
1 M, and is C2.
The final volume required is 1000 mL and is V2.

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SUBSTITUTING INTO THE EQUATION:
C1 V1 = C2 V2
3 M (?) 1 M (1000 mL)
? = 333.33 mL
So, take 333.33 mL of the concentrated stock solution and BTV 1 L.

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“X” SOLUTIONS The concentration of a stock solution is sometimes written with an “X”.
The “X” is how many more times the stock is than normal.
You generally want to dilute such a stock to 1X, unless told otherwise.

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EXAMPLE A can of frozen orange juice is labeled 4X. How would you dilute it to make 1L of drinkable drinkable juice?
Using the C1V1=C2V2 equation:
C1 V1 = C2 V2
4X (?) = 1X (1L)
? = 0.25 L
Use 0.25 L of orange juice, BTV 1L.

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BIOLOGICAL BUFFERS Laboratory buffers
solutions to help maintain a biological system at proper pH
pKa of a buffer
the pH at which the buffer experiences little change in pH with addition of acids or bases = the pH at which the buffer is most useful

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TEMPERATURE Some buffers change pH as their temperature and/or concentration changes
Tris buffer, widely used in molecular biology, is very sensitive to temperature

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DILUTION Some buffers are sensitive to dilution
Phosphate buffer is sensitive to dilution

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ADJUSTING THE pH of a BUFFER This is done to set the buffer to a pH value which is...
somewhat close to its pKa
useful for the biological system the buffer is to be used with
Often adjust pH using NaOH or HCl
Not method used for phosphate buffer (see textbook)

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BRINGING A SOLUTION TO THE PROPER pH Adjust the pH when the solution is at the temperature at which you plan to use it.
Mix the solute(s) with most, but not all, the solvent. Do not bring the solution to volume.
Stir solution.

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Check the pH.
Add a small amount of acid or base.
The recipe may specify which to use.
If not, HCl and NaOH are commonly used.
Stir again and then check the pH.

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Repeat until the pH is correct, but don’t overshoot.
Bring the solution to volume and recheck the pH.

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ASSURING THE QUALITY OF A SOLUTION Documentation, labeling, recording what was done
Traceability
SOPs
Maintenance and calibration of instruments
Stability and expiration date recorded
Proper storage