1. FOOD CHEMISTRY-Practical-DemoBY
DR BOOMINATHAN Ph.D.
M.Sc.,(Med. Bio, JIPMER), M.Sc.,(FGS, Israel), Ph.D (NUS, SINGAPORE), PDF (USA)
PONDICHERRY UNIVERSITY
I lecture
6/7August/2012
Source: Collected from different sources on the internet and presented by Dr L. Boominathan

2. PREPARING LABORATORY SOLUTIONS AND REAGENTS I THE BASICS

3. TOPICS Where do solution recipes come from?
Concentration of solute: calculations
Preparing solutions
Making diluted solutions from concentrated ones
Buffers
Bringing solutions to proper pH
Calculations for solutions with more than one solute, next lecture

4. WHERE DO SOLUTION "RECIPES" COME FROM?
Original Scientific Literature
Lab manuals (instructional)
Lab Manuals (professional)
Handbooks
Manufacturers and suppliers

5. INTERPRETING RECIPES DEFINITIONS:
SOLUTES -- substances that are dissolved
SOLVENTS -- substance in which solutes are dissolved (usually water)
AMOUNT -- how much

6. CONCENTRATION versus AMOUNT
CONCENTRATION -- amount / volume
Fraction where:
Numerator, the amount of solute
Denominator, usually volume of entire solution
solvent + solute(s)

7. 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)? _____

8. 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 mL
? = 0.8 mg, so the concentration is 0.8 mg/mL

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

10. 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 %)

11. 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 %)

12. 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

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

14. 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?

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

16. 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.

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

18. 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 = g
1 sulfur atom X g = g
4 oxygen atoms 4 X g = g
98.06 g

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

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

21. FORMULA
HOW MUCH SOLUTE IS NEEDED FOR A SOLUTION OF A PARTICULAR MOLARITY AND VOLUME?
(g solute ) X (mole) X (L) = g solute needed
1 mole L or
FW X molarity x volume = g solute needed

22. EXAMPLE
How much solute is required to make 300 mL of 0.8 M CaCl2?

23. ANSWER
(111.0 g) (0.8 mole) (0.3 L) = g
mole L

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

25. 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.

26. 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.

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

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

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

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

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

32. BY EQUATION 1. Total volume required is 500 mL. 2. 5% = 0.05
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

33. % 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.

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

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

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

37. 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.

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

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

40. 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.

41. 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

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

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

44. 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? ___________

45. Each star represents 1 mg of dioxin
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

47. 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

48. 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

49. 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.

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

51. “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.

52. 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)
? = L
Use 0.25 L of orange juice, BTV 1L.

53. 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

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

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

56. 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)

57. 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.

58. Add a small amount of acid or base.
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.

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

61. 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

62. It’s all about the concentration
Solution Chemistry
It’s all about the concentration

63. Common units of concentration
% by mass – g solute /100 g solution
% by volume – mL solute/100 mL solution
% by mass-volume – g solute/100 mL solution
Molarity – moles solute/L solution
Molality – moles solute/kg solvent
Normality – equivalent moles of solute/L solution
ppt – grams solute/thousand grams solution
ppm –g solute/million g solution
ppb – g solute/billion g solution
lb solute/million gallons solution

64. Some conversion problems:
Convert 136 μg NaCl/mL pond water to lb NaCl/million gallons pond water .

65. Some conversion problems:
136 μg NaCl …. ? lb NaCl
mL pond water million gallons pond water
What do we need to know?

66. Some conversion problems:
136 μg NaCl …. ? lb NaCl
mL pond water million gallons pond water
What do we need to know?
How many μg in a lb?
How many mL in a million gallons?

67. Some conversion problems:
136 μg NaCl …. ? lb NaCl
ml pond water million gallons pond water
453.6 g = 1 pound
1 μg = 10-6 g
1 mL = 10-3 L
1.057 L = 1 quart
4 quarts = 1 gallon

68. Some conversion problems:
136 μg NaCl * g * 1 lb = 2.998x10-7 lb
mL pond water 1 μg g mL pond water
2.998x10-7 lb * 1 mL * L = 3.17 x10-4 lb
mL pond water L qt qt
3.17 x10-4 lb * 4qt * 106 gal = x103 lb
qt gal million gal million gal

69. Some conversion problems:
Convert 36% by mass of HCl to Molarity.
How do we start?

70. Some conversion problems:
Convert 36% by mass of HCl to Molarity.
How do we start?
Units! Units! Units!

71. Some conversion problems:
Convert 36% by mass of HCl solution to Molarity.
36 g HCl …… Moles HCl
100 g solution L solution
What do we need to know?

72. Some conversion problems:
Convert 36% by mass of HCl solution to Molarity.
36 g HCl …… Moles HCl
100 g solution L solution
What do we need to know?
Molar mass of HCl
Density of HCl solution

73. Some conversion problems:
Convert 36% by mass of HCl solution to Molarity.
36 g HCl …… Moles HCl
100 g solution L solution
What do we need to know?
Molar mass of HCl (36.46 g/mol – from Periodic table)
Density of HCl solution (from where???)

74. Density – your critical judgment
For a solution, sometimes you know the density, sometimes you don’t.
There are tables, but they are not all inclusive.
You might, for example, find in a table that:
Density (30% HCl) = 1.12 g/mL
Density (40% HCl) = 1.23 g/mL
Density (36% HCl) = ???

75. Interpolate or Assume Density (30% HCl) = 1.12 g/mL
You could assume that 36% is closest to 40% and use 1.23 g/mL. This is legitimate, although not 100% accurate. Results may vary, depending on how good the assumption is.

76. Interpolate or Assume
Density (30% HCl) = 1.12 g/mL Density (40% HCl) = 1.23 g/mL
Density (36% HCl) = ???
You could assume that density changes linearly with concentration (it doesn’t, but it is pseudo-linear for small changes). In that case, you would “linearly interpolate” the density.
1.23 g/mL – 1.12 g/mL = g/mL = g
40% HCl-30%HCl % mL%
1.12 g/mL g/mL% * 6% = g/mL = 1.19 g/mL
This is legitimate, although still not 100% accurate, but probably better than the previous assumption.

77. If I don’t have Density tables…
For dilute solutions, you can get pretty close by assuming the density of the solution is the same as the density of pure water.
For concentrated solutions (like 36%), this is probably not a good assumption, but it is better than nothing!

78. Solving the problem (finally)
Convert 36% by mass of HCl solution to Molarity.
36 g HCl …… Moles HCl
100 g solution L solution
What do we need to know?
Molar mass of HCl (36.46 g/mol – from Periodic table)
Density of HCl solution (1.19 g/mL – by assuming linear change)

79. Solving the problem (finally)
36 g HCl * 1 mol * g * 1000 mL
100 g sol g 1 mL 1 L solution
= 11.7 mol HCl = 11.7 M HCl
L solution
(if you don’t specify solvent, usually assumed to be water)

81. Common units of concentration
% by mass – g solute /100 g solution
% by volume – mL solute/100 mL solution
% by mass-volume – g solute/100 mL solution
Molarity – moles solute/L solution
Molality – moles solute/kg solvent
Normality – equivalent moles of solute/L solution
ppt – grams solute/thousand grams solution
ppm –g solute/million g solution
ppb – g solute/billion g solution
lb solute/million gallons solution

82. All are important, but… Moles! Moles! Moles!
Molarity – moles solute/L solution (most common)
Molality – moles solute/kg solvent (not very common)
Normality – equivalent moles of solute/L solution (specialized usage)
What’s “equivalent moles”?

83. Normality vs. Molarity Molarity = moles solute/L solution
- generic, just the moles folks
Normality = equivalent moles of solute/L solution
- specific, it takes into account the actual chemistry of the solute.

84. Acids
What’s an acid?

85. Acids
What’s an acid?
Within the Bronsted-Lowry theory of acids/bases, an acid is a proton (H+) donor and a base is a proton acceptor.
Can you think of examples of acids or bases?

86. Some acids and bases NaOH – base Mg(OH)2 – base
HCl – acid (hydrochloric acid)
HF – acid (hydrofluoric acid)
H2SO4 – acid (sulfuric acid)

87. Acid – what’s it good for?
????

88. Acid – what’s it good for?
Protons
If we define an acid as a proton donor, the proton is what makes it what it is.

89. Consider two solutions:
1 M HCl
1 M H2SO4
How are they the same? How are they different?

90. Consider two solutions:
1 M HCl 1 M H2SO4
1 mole molecules/L mole molecules/L

91. Consider two solutions:
1 M HCl 1 M H2SO4
1 mole molecules/L mole molecules/L
H+ Cl- in solution H+ and SO42- in solution
HCl(aq) → H+(aq) + Cl-(aq) H2SO4 (aq) → 2 H+ (aq) + SO42-(aq)
HCl(aq) + H 2O(l) → H3O+(aq) + Cl-(aq) H2SO4 (aq) + 2 H2O(l)→ 2 H3O+ (aq) + SO42-(aq)

92. Consider two solutions:
1 M HCl 1 M H2SO4
1 mole molecules/L mole molecules/L
H+ Cl- in solution H+ and SO42- in solution
HCl(aq) → H+(aq) + Cl-(aq) H2SO4 (aq) → 2 H+ (aq) + SO42-(aq)
HCl(aq) + H 2O(l) → H3O+(aq) + Cl-(aq) H2SO4 (aq) + 2 H2O(l)→ 2 H3O+ (aq) + SO42-(aq)
1 mol H+/L solution 2 mol H+/L solution

93. Consider two solutions:
1 M HCl 1 M H2SO4
1 mole molecules/L mole molecules/L
1 mole H+/L solution 2 mol H+/ L solution
They are both acids, they are defined by their ability to donate protons. The protons are the “equivalents” for an acid.
1 N HCl 2 N H2SO4

94. Carbohydrate Analysis: Estimation of Sugars Lab
Lab Exercise 0ne
Carbohydrate Analysis: Estimation of Sugars
Lab

95. Biochemical Assay
Biochemistry deals with the identification and quantification of bio-molecules from a variety of living systems
Rely on the chemical reactivity and physical properties of bio-molecules to make identification and quantification.
Primary tool is the spectrophotometer
Uses absorption of mono chromatic light

96. Spectrophotometer

98. Measure quantity
Some bio-molecules have properties which allow direct measurement.
proteins have aromatic amino acids (280nm)
Nucleic acids have unsaturated ring structures (260nm)
Other molecules have chemical properties which can be used in indirect measurement.

99. Introducing concept of standard curve
Uses dilutions of a solution of known concentration to determine concentration of unknown

100. Standard Curve
Assumes that unknown will respond in assay the same as the known
Valid in todays assay as they (the reactive groups. glucose) are the same
Problem in other assay as they may not contain same amount of reactive groups
Protein assays (have to choose)
But usually close

101. Our model carbohydrate is the sugar glucose
We will exploit its ability to reduce other compounds to produce a product which can be measured optically

102. Reducing Sugars Have aldehyde group Can be oxidized to acid
Reduces another compound

103. Requirement placed on sugar
Must be an aldehyde
Ketones and hemiacetal configurations are not reducing
Conditions of reactions favor conversion to aldehyde by lowering aldehyde concentration

104. Sugars as Reducing Agents
Equilibrium between
hemiacetal and open chain
is driven to open chain as
oxidation to acid form takes
place. This ensures a
quantitative conversion with
time and a stoicheometric
production of reduced copper.

105. Nelson Assay (a two step Rx)
In the Nelson assay Cu+2 is reduced to Cu+1 by the reducing activity of the sugar (step 1)
Cu+1 is oxidized to Cu+2 by addition of arsenomolybdic acid (colorless) (step 2)
Results in blue (reduced) arsenomolybdous acid
Amount is directly related to [CU+1]
Will detect any reducing sugar (concentration of sugar must be limiting factor)

106. We will do the DNS assay Is a direct assay
Measures the reducing capability of glucose
Uses a color conversion reaction from yellow to red brown @ A540
Conversion of moles of DNS equals moles of glucose.

107. 3,5-dinitrosalicylic acid (DNS)
Sugar reduces the organic DNS which absorbs maximally at yellow wave length
Results in change (shift) in absorption spectrum from red/orange to red/brown at 540nm
Different from Nelson reaction
Measured at 540nm
Unreacted DNS not seen at this wavelength
Amount of absorbance directly related to amount of reducing sugar

108. The DNS reagent From the MSDS:
LABEL PRECAUTIONARY STATEMENTS TOXIC (USA) HARMFUL (EU) HARMFUL BY INHALATION, IN CONTACT WITH SKIN AND IF SWALLOWED. IRRITATING TO EYES, RESPIRATORY SYSTEM AND SKIN. IN CASE OF CONTACT WITH EYES, RINSE IMMEDIATELY WITH PLENTY OF WATER AND SEEK MEDICAL ADVICE.
3,5-dinitrosalicylic acid is reduced to 3-amino,5-nitrosalicylic acid

109. The DNS assay Experimental design and flow charts
Be sure to read “Hazards”
Data analysis

110. Today's Experiment
Measure the concentration of glucose by detecting the reducing end of the monosaccharide.
This group converts the oxidized form of 3,5-dinitrosalicylic acid, DNS, to reduced form which absorbs at 540nm.
Amount of reduced DNS proportional to amount of glucose.

111. What are we doing today?

112. Important
Pipetting technique is critical to accuracy and to preventing cross contamination of samples
Pipetters have two stops
First to take up selected volumes
Second to deliver
Choose pipetter “in the range” that you need.

113. You will create a standard curve
You are provided a stock solution which contains 1.2 mg/ml
You will dilute this stock solution in a specified manner always producing a 4 ml solution
You will read the absorbance of each solution at 540 and plot vs concentration
You will compare the A540 of unknown to standard curve

114. Glucose “Standard” Volume
Table A.1-2. DNS Assay Components
Tube Number
Water Volume
(ml)
Glucose “Standard” Volume
Unknown Volume
DNS
A540
Amount
(mg)
[Glucose]
(mg/ml) 1
3.000
0.000
1.00 - 2
2.750
0.250 3
2.500
0.500
1,00 4
2.250
0.750 5
2.000
1.000 6 7 8

115. Standard curve
Uses dilutions of a solution of known concentration to determine concentration of unknown

116. Important
Careful handling of Cuvettes is essential for accuracy and prevent contamination
Handle only with gloves
Touch only the areas not in the light path
Rinse carefully with DH2O after each use
Always go from lowest concentration to highest concentration.
Wipe clear surface if necessary with “Kimwipe”

117. Extremely Important
Put cuvette into Spec slot that is in the beam path
Be certain that clean panes face the beam path
Measure only with the lid closed
Always set the spec with a blank (line 1 table A.1-2, page 38)
Contains all components of reaction except that which is to be measured
Always use same cuvette

118. PLEASE DO NOT SLAM THE SPEC LIDS

119. Important 1. Wear Gloves and Safety Glasses
2. Record the code number of your unknown
3. Be certain that test tubes are clean
4. Water/H2O always means distilled water
5.Have TA initial your data before you leave. See lab exit requirements page

120. Lab reports for this class
Abstract. Statements regarding:
WHAT you are doing (-> procedure)
WHY you are doing it (-> your hypothesis)
WHAT you hope to accomplish (-> also hypothesis)
Cf. ‘purpose/goal’ in a good lab notebook! Might think of it as a very short introduction
Background information and theory
Results/Data/Data Analysis
Discussion MUST relate data analysis to hypothesis!

121. Application quiz Address in your report
What does the portable glucometers used by diabetics measure?
How do they measure it?

122. Reminder
Lab Reports are PERSONAL

123. Grading for This Experiment
Number of lab periods = 1
Lab Report = points
Pre lab= points
Total = points

124. Clean up (Please) before you go
See page 44. Waste Disposal & Clean up
Return pipetts to rack

125. Good luck with your lab !!!