Lab 1

 Bronested Lowry theory:

 Acid: Proton Donor (produces H+ in solutions).
 Base: Proton acceptor (accepts H+ in solutions).
 Arrhenius theory:
 Acids: substances (Hydrogen containing) which produce hydrogen ions (H+) in solutions
 Bases: substances (Hydroxyl containing) which produce hydroxide ions (OH-) in solutions.
 Weak Vs strong:
 Strong acid or base: they dissociate completely
 Weak acid or base: partial dissociation
With weak acid we use 𝐊𝐚 = [𝐇 +][𝐀 −]/ [𝐇𝐀] ,pKa= -logKa
 pKa ↑ ,(dissociation, ka ,strength of the acid, acidity) ⬇︎
 pH
 pH =7 for neutral solution.
 pH>7 for alkali (basic) solution.
 pH<7 for acidic solutions.
 a pH meter: measure pH of a liquid
 pH= -log[H+ ]
 Blood concentration of H+ is very low, about 40 nEq/L (4x10-8 Eq/L)
 Blood pH= -log 4x10-8 = 8-log 4 = 8-0.6 = 7.4
 Handerson-Hasselbach equation: pH= pKa + log ([𝑨−]/[𝑯𝑨])
 Buffer system:
 most important application for Henderson-Hasselbach equation
 buffer=weak acid+ C.base or weak base +C.acid
 resist pH change
 If an acid (H+ ) is added, it will be neutralized by the conjugate base, the reaction will be
shifted to reactant(substrate) side
 If a base (OH-) is added it will be neutralized by the acid, the reaction will be shifted to the
product side
 buffering zone: the best zone of pH during which the buffer resists pH change
- ranges from pKa-1 to pKa +1
- maximum buffering ability when pH=pKa.
 Note:
- If pH=pKa,maximum buffering and Acid=conjugate base (HA=A-).
- If pH> pKa, acid is lower than the conjugated base(HA<A-).
- If pH<pKa, acid is more than the conjugated base (HA>A-)
 Amino acids
 Human has about 20 aa types (but around 300 aa in nature).
 The basic structure of amino acids contains:
- α- carbon: a chiral carbon, Except for glycine
- α-Amine
- α-Carboxyl
- R- group: is the side chain or the functional group.
  two isomers (called enantiomers): L-isomer (Left or counterclockwise) and D-isomer (Dextro
which means right or clockwise)
 position of amine group determines whether its L(left) or D(right) conformation.
 in our bodies are in L conformation.
 Amino acid features:
 For most amino acids (biprotic: 2 titrable groups):
- α-Carboxyl (COOH): has a pKa of around 2-3
- α-Amine (NH3 + ): has a pKa of around 9-10
 Acidic amino acids (glutamate and aspartate are triprotic) have a third titrable group that is
carboxyl group in the side chain with a pKa of around 4.
 Basic amino acids (Histidine, Lysine and Arginine are triprotic): have a third titrable group
that is an amine group in the side chain with pKa of around 11. (Except for Histidine whose
side chain pKa is around 6).
 Amino Acids Titration
 At very low pH (less than 2 which is the starting point of titration): ALL titrable groups of the
aa are protonated.
 Adding NaOH (called the titrant) will remove “steal” H+ from the titrable groups starting
with the titrable group with the lowest pKa, then the higher and so on.
 Deprotonation converts the acid to its conjugated base.
 At very high pH (around 14): ALL the titrable groups are deprotonated
 the pH at which the net charge of the amino acid is zero called Isoelctric Point (PI) or the
Zwitterion point
 Neutral amino acids have only 2 titrable groups, also called simple AA:
 At very low pH, +1.
 More NaOH is added to reach the middle of a flat area, here the pH=pKa
 NaOH steals all the H+ from the first titrable, net charge zero
 PI= (pKa1+pKa2)/2
 More NaOH is added to reach the middle of a flat area, here the pH=pKa2
 At very high pH, the net charge is -1
 Titration of acidic amino acids as Asparatic acid and Glutamic acid:
 three titrable groups: α carboxyl with pKa1 around 2, R-carboxyl with pKa2 around 4 and an
α amine with pKa3 around 9.
 At very low pH, Net charge is +1
 Full deprotonation of the first group, The net charge is zero
 PI=(pka1+pkaR)/2
 Full deprotonation of the R COOH, The net charge here is -1.
 Full deprotonation of all groups, The net charge is -2.
 Titration of basic amino acids (lysine, arginine and histidine)
 third titrable group at the R which is an amine with a pKa around 11. (Except Histidine which
has pKaR of around 6.5)
 PI=(pKa2+pKa3)/2
Always the isoelectric point lies between the 2 closest pKa to each other, number of plateaus can determine the number of ionizable groups

Lab 2
 Radiation
 energy propagating into a space
 Wavelength (λ)= the distance between 2 crests ‫ قمتين‬or troughs ‫قاعين‬
 The spectrum (Electromagnetic spectrum):
- The total range of wavelengths of the light with all its components
- including gamma rays, X-rays, UV rays, visible light, infra-red and radio rays.(from
shortest to longest)
- Ultraviolet (UV): λ<400nm
- Visible: λ between 400-700 nm, each visible color has specific wavelength.
- Infrared: λ >700 nm
 Spectroscopy
 the study of electromagnetic radiation emitted or absorbed by a given substance
 for virtually every type of electromagnetic radiation
 Spectrophotometry
 Is a type of spectroscopy that deals with visible light, nearby UV and nearby infra-red.
 Helps in diagnosis of many diseases (Ex, bilirubin, hemoglobin and glucose in the serum of
the blood)
 The pathway:
- Apply light to a sample (chemical)
- Measure how much a sample absorbs light (based on I)
- The more light absorbed, the more sample concentration
 analytical technique that uses light absorbance to measure concentration
 quantitative study
 also known as colorimetry (it measures the concentration based on the amount of a colored
product)
 easily and quickly& most cost-effective option
 The spectrophotometer:
- instrument that measures the amount of light that passes the sample
- It has a light source that emits light and a lens to concentrate light into a prism. The
prism (monochromator) then breaks light to its different wavelengths
- single wavelength is allowed to pass the sample by a wavelength selector
- UV spectrophotometers: uses UV light source (Deuterium lamp)
- Visible light spectrophotometers: uses visible light source (Tungston Halogen lamp
that emits light at 400-700 nm).
- Light Intensity (I): the number of photons passing a point during a unit of time
 Cuvettes:
 mall containers hold the samples in spectrophotometry.
 different shapes: squared (rectangular) or tubular (rounded or circular).
 Are made of different:
- Glass: for visible light spectrophotometry. Used when the accuracy is more important
than the speed of experiment.
- Plastic: for visible light spectrophotometry. Used when the speed (fast experiments) is
more important than the accuracy of experiment. They are disposable.
- Quartz: for UV light spectrophotometry (expensive but accurate).
 - The length of the cuvette is generally 1 cm
 Transmittance
 Is the fraction of incident light (Io)that passes the sample
 Calculated as 𝐓𝐫𝐚𝐧𝐬𝐦𝐢𝐭𝐭𝐚𝐧𝐜𝐞 = 𝐈 (𝐭𝐫𝐚𝐧𝐬𝐢𝐦𝐭𝐭𝐞𝐝)/ 𝐈𝐨 (𝐢𝐧𝐜𝐢𝐝𝐞𝐧𝐭)
 Percent Transmittance =( 𝐈 (𝐭𝐫𝐚𝐧𝐬𝐢𝐦𝐭𝐭𝐞𝐝)/ 𝐈𝐨 (𝐢𝐧𝐜𝐢𝐝𝐞𝐧𝐭)) 𝐱𝟏𝟎𝟎%
 Absorbance:
 A=-log T. A=log (1/ T). A=log (Io/ I). A=2-logT%.
 ranges from 0 when the transmittance is 100% to 2 when the transmittance is 1%
 As the wavelength increases, the absorbance increases until it reaches the maximum point
called Amax, the wavelength that corresponds with the maximum absorbance is called λmax
 Each substance has a special λmax used in spectrophotometry
 Beer-Lambart Law
 A= ε X L X C as:
- A: Absorbance (has no unit).
- ε: is the molar absorptivity (in M-1 cm-1 or 𝐿 /𝑚𝑜𝑙𝑒.𝑐𝑚 ), the amount of light that is
absorbed by a unit concentration (molar) of a sample.
- C: sample Concentration (in mole/L). and L: Length of the cuvette (in cm).
- higher the ε, the better light absorbance
 straight line equation
 Slope of the curve = m = εL
 Absorbance-concentration curve is linear and is preferred to be used in labs.
 transmittance-concentration curve is logarithmic and not preferred in labs
 Application of Beer-Lambart law: determining a sample concentration:
- 𝑪𝟐 = ( 𝑨𝟐/ 𝑨𝟏) 𝒙𝑪1
 Dilution
 reduces the concentration of a substance in a solution.
 Stock solution:
- concentrated solution in lab
- Dilution equation: Cstock (initial)x Vstock (initial) = C desired (final) X V desired (final)
- DF=V final/V initial
 Determination of Glucose concentration:
 to diagnose diabetes.
 Indirect Enzymatic Colorimetric test.
 Glucose itself is colorless and cannot absorb light
 we have to add a special glucose to reagent:
- giving a color “Red-Pink”
- Glucose oxidase: it oxidizes glucose in the presence of O2 to produce Gluconic acid
and H2O2.
- Peroxidase: it converts H2O2 + reduced 4-AP (colorless 4 AminoantiPyrine)+ phenol 
H2O and Quinonimine (it is an Oxidized 4-AP and its color is pink-red).
- Glucose level can then be estimated indirectly based on the fact: The more glucose
monosaccharide, the more Quinonimine and the more the color.
- the absorbance of Quinonimine is measured at λmax of 500nm
Lab 3
 Biuret protein assay
 cupric ion “Cu+2”. So Cu+2 is the main player in this assay.
 Biuret compound is obtained by heating 2 moles of urea at 180 C
 doesn’t actually use biuret
 peptide bond is an amide type of covalent bonds .so, cupric ion has the ability to chelate
with peptide bond
 Biuret reagent: blue solution composed of:
 CuSO4: provides the cupric ions and gives the reagent its blue color
 NaOH : provides the alkaline medium needed for the reaction between cupric ions and
peptide bonds
 Sodium-Potassium tartrate: stabilizes the Cu +2 ions and prevents it’s precipitation
 KI : an antioxidant
 The cupric ion chelates with 4 peptide bonds forming cupric -peptide complex which has a
violet color. (at least 2 peptide bonds)
 It has maximum absorbance at about 540 nm
 Cx = Ax. Cs/As
[x stands for sample and s stands for standard ]

Lab 4
 Enzymes
 are biological molecules (mainly) proteins
 speed up the rate of a biochemical reaction by decreasing the activation energy
 Activation energy
 energy barrier separating the reactants and the products.
 energy difference between the reactants and the high energy intermediate occurring during
the formation of the products.
 Transition state: where the high-energy intermediate is formed during the conversion of
reactants to products.
 There must be sufficient energy to overcome the energy barrier (transition state) for the
reaction to occur.
 How enzyme work?
 enzymes DON’T change the energy of the reactant or the products
 ONLY lower the activation energy.
 doesn’t affect ∆G or whether the reaction is spontaneous or not.

 Factors affecting enzyme activity

 Substrate concentration: positive regulator until reach maximum velocity then increasing
the con. Will not affect rate of reaction since all active sites of enzymes are occupied
  Temperature: increasing temp. will increase the rate until reach the optimal temp. after it
the enzyme start to denature
- Note: Increasing the temperature will increase the number of molecules having
sufficient energy to overcome the energy barrier.
 pH level: pH at which maximal enzyme activity is achieved is different for different enzymes.
- pepsin is maximally active at pH 2, trypsin 6, and alkaline phosphatase 8
 Co-Factors:
 small non-protein helper or accessory molecules that are necessary to activate the enzyme.
 can be
- inorganic (Mg+2, Cu+2...)
- organic coenzymes such as vitamins (B-complex).
 Enzyme inhibitors:
 can be
- reversible which bind non-covalently
- irreversible which bind covalently to the enzyme.
 competitive or non-competitive:
- competitive: when the inhibitor binds to the same site that the substrate bind (active
site).
Can be over-come by increasing substrate Con. (can reach Vmax)
- Non-competitive: when the inhibitor and the substrate bind at different sites.
Can’t be over-come by increase substrate con concentration and Vmax can never be
reached
- Competitive shift the curve to the right
- Non-competitive shift the curve rightward and downward (non-competitive inhibitors
decrease Vmax.)
 Measuring enzyme activity:
 spectrophotometric assay change in the intensity of the light absorbed by the reaction
solution is measured.
 measured by international unit.
 U or IU is the amount of enzyme that catalyzes the conversion of 1 micromole of substrate
into product per minute under specified conditions of the assay method:
specific condition will usually be the optimum conditions including the
- substrate concentration - temperature - and pH.
 Alkaline phosphatase enzyme activity:
 enzymes that split off a terminal phosphate group from an organic ester
 most effective in alkaline environment.
 Found in hepatic, renal and skeletal tissues (increased in bone and liver diseases)

Ahmad Alsawaie