Biochem lab exam 1

gram per cent solutions: weight/weight

for an example, 10% NaCl solution (w/w) is prepared by mixing 10 grams of sodium chloride and 90 grams of water

gram per cent solutions: weight /volume

the 10% NaCl solution (w/v) is prepared by mixing 10 gram of sodium chloride with water to make the final volume of the solution to 100 mL.

molar solutions

do not suffer from such discrepancy as gram per cent solutions since molar solutions are prepared by dissolving 1 gram molecular weight (mole) of a substance in 1 liter of solution.

gram molecular weight

also known as mole, means the amount of substance in gram numerically equal to its formula (molecular) weight.

Molarity

comes from the term mole, means the number of moles dissolved per liter. since 1 mole of any substance contains 6 X 10²³ molecules, molar solutions always contain that many molecules of a particular substance per liter.

How to make a 1 molar solution

• weigh out molar mass in grams of substance

• add water to this measured amount in grams until the solution is volumized to one liter

how will you prepare 300ml of 100 mM Tris buffer having pH of 8.5 (molecular weight of tris is 121.4)

M = 0.1M

V = 0.3L

0.1M x 0.3L = 0.03mole

m = 0.03 mol X 121.4 g = 3.642g

Add HCL until pH is 8.5 add water until total volume is 300 ML

400 mL of 0.85% of sodium chloride contains how many grams of sodium chloride (molecular weight of NaCl is 58.44g)

0.85g x 400mL/ 100 mL = 3.4g NaCl

0.85g NaCl per 100 mL solution

How does the gram percent solution of weight/weight and weight/volume differ

w/w = grams of solute / grams of solvent 

• Calls for both solute and solvent having the same units this gives you a percent to show grams/mL of the solution. 

w/v = grams of solute / volume of solvent 

• Grams of solute used with respect to the desired percent of the solution volume of solution is determined by adding enough solvent that it reaches the desired volume.

what is a mole

6 × 10²³  number of particles in one mole of an element or compound. depending on the element or compound more or less grams of substance are required to reach one mole. 

how do you define molarity

M = number of moles of solute / liters of total solution

How many molecules of a particular substance are contained in one mole

6 × 10²³

Why do most biochemical reactions take place in water based environments

the molecule is amphoteric and serves as a proton donor acid or a proton accepter base. pure water ionizes as under

H2O = H+ + OH- 

How is the concentration of ions in water ionized

[H+] = [OH-] = 10^7 M

define pH

the negative log of hydrogen ion concentration

What is the pH of pure water

7.0

• remeber -log[H+] = pH

How does equilibrium shift resulting in pH change

the acidic or basic molecules dissolved in water react either with H+ or OH-

buffers

• Maintain the pH of the solutions or media to a particular value specific chemicals known as these are added to the solutions or media these chemicals have capacity to resist pH change resulting from the addition of acidic or basic molecules

Examples of organic and inorganic buffers used in lab

phosphate buffer, HEPES, Bis-Tris, tricine, bicine, Tris, MES, MOPS, etc…

Most common and more accurate method of measuring pH

electrical pH meter

Three important aspects of measurement of pH using electrical pH meter

electrodes, buffers, and measurement procedures

electrodes

are made up of either plastic or glass body. They can be either single cells pH dependent in pH independent reference electrodes or combined in one body for convenience

Reference electrode

 supplies a constant equilibrium voltage

pH dependent electrode

Supplies a voltage based on the pH of the sample solution

What does the potential difference between the reference electrode and pH dependent electrode mean

This is measured as a voltage which is related to the pH of the solution

How was the pH meter standardized

using buffers having known pH such as 4.0 7.0 and 10.0 before measuring the pH of the unknown sample solutions

How does temperature affect pH and electrode measurement of pH

higher temperatures lower the pH of a solution making it more acidic. A lower temperature makes the sample more basic because it raises the pH. the pH meters available now automatically displayed temperature corrected pH value while in the past pH meter used to have a temperature control knob for adjustment

Why must distilled water be used to wash the electrodes of an electrical pH meter

The DI water contains almost no ions of its own so using this to rinse the electrodes reduces the risk of contamination from ions in water or previous acidic or basic solutions before testing a new sample

what temperature does pH buffer need to be stored at

4 degrees celsius until it is used

function of buffer

maintain the pH of the solution

why is buffer important for the isolation of proteins

 proteins are sensitive to pH. If the pH deviates too much the proteins can become denatured have altered charges and solubility changes. Maintaining pH keeps the proteins in their functional state so they can be isolated

pH of tris buffer is to be adjusted to 7.5 after dissolving the tris in  water you find that the pH is 9.9 what will you need to add to bring the pH to 7.5

A strong acid such as HCL

What are three examples of biological buffers

 bicarbonate buffer system, phosphate buffer system, and tris buffer

Glass electrodes in electrical pH meter

 Measure the concentration of hydrogen ions making them pH sensitive

Reference electrodes in electrical pH meter

Provide a stable reference potential

Biochemical research often requires the quantitative estimation of protein concentrations in the solution there are mainly two types of techniques used for the estimation of proteins

color metric and ultraviolent spectrophotomeric methods.

Colormetric method

relies upon the formation of colored products of protein formed due to its reaction with a particular reagent. the intensity of color is proportionate to the concentration of protein present.

Calibration curve

 first planted using standard protein of known concentrations. The concentration of unknown protein solution is then calculated from the standard curve

What are different types of colormetric methods that are commonly used

biuret / lowery method, the Bradford assay method, and bicinchoninic acid method

Mechanism of BCA method

the protein reduces CU2+ to CU+. CU+ then reacts with Apple green color reagent to produce purple color complex. Formation of purple color is proportionate to the concentration of protein present. The color is measured at 562 nm wavelength

UV spectroscopic method

This method relies upon the fact that protein absorb UV light with absorbance maximum of 280 nm.

UV spectroscopic method calibration curve

 Standard UV absorbance calibration curve is plotted taking absorbance of standard protein solution of known concentration at 280 nm wavelength. Then the absorbance of unknown solution is measured at the same wavelength in the concentration of unknown is calculated by comparison to the standard curve.

How do you prepare the solution for a BCA test

Solution of standard protein having concentration of 1 mg / 1 mL.

What volume do all of the tubes in the BCA test have to be

100 µL add distilled water to the tubes containing less than the volume so they all have this volume

How much and what do you do after you add the BCA reagent

add 2 mL of BCA reagent and then incubate all tubes at 37°C for 30 minutes

What is the general principle of Colorimetric estimations

Color intensity formed as proportional to the concentration of protein or DNA. The absorbance of the color is measured using spectrum meter at a certain 260-280 nm wavelenght using beer Lambert law

What is the mechanism of BCA method of protein estimation

Proteins reduced copper ions under alkaline conditions. When the reagent BCA is added to the copper ion and protein solution the purple colored complex is formed.

What is standard curve and why is it necessary to plot

this represents the known concentration of proteins against its absorbed values. This provides linear calibration to compare test samples.

What is the mechanism of estimation of DNA in protein by UV spectroscopy

 DNA and RNA can absorb 260 nm because of their aromatic bases. Proteins absorb 280 nm due to aromatic amino acids. This provides the values needed to use the beer Lambert law. A = epsilon(d) this checks for purity.

Give one example of color metric method used for protein estimation besides BCA method

Bradford assay, blue dye vines to proteins and absorbance happens at 595 nm

First step for the separation and identification of amino acids in protein/peptide

Hydrolyze the sample to obtain free amino acid which can be further identified using a variety of techniques

What techniques are used to further identify free amino acids

high performance liquid chromatography (HPLC), capillary electrophoresis (CE) , gas chromatography, paper chromatography or thin layer chromatography (TLC).

what can hydrolyzed the amide bonds in peptides and proteins

strong acid or base

Acid catalyzed hydrolysis

 destroys tryptophan and cystine, causes some loss of serine and threonine 

What amino acids does acid catalyzed hydrolysis convert

aspargine and glutamine to aspartic acid and glutamic acid respectively

Base catalyzed hydrolysis

 leads to destruction of serine, threonine, cysteine, and cystine and also results and racemization of the free amino acids

racemization

is the process of converting an amino acid from one enantiomer to its mirror image, resulting in a mixture of both forms.

What is the preferred method of hydrolysis

Acid hydrolysis because it is less destructive

Common procedure of acid hydrolysis

Dissolving the sample in 6M of HCL, and heating the solution either in autoclave or in a sealed evacuated vial

At what temperature and for how long is the solution heated in an autoclave

At 121 degrees Celsius for 6 hours

At what temperature and for how long is the solution heated when in a sealed evacuated vial

100 degrees Celsius for 12 to 24 hours

After the free amino acids are hydrolyzed what are the different paper chromatography’s that they can be separated and identified on

TLC, CE, GC, or HPLC

Which methods of chromatography are more sensitive

CE, GC and HPLC and quantitative methods, paper chromatography and TLC are still used due to the demand for rapid and qualitative routine analytical methods.

What are the plates used in chromatography coded with or made of

silica gel or cellulose in common use

Purpose of ninhydrin

Free amino acids can be detected on the developed chromatographic plates via a reaction with this reagent

What color forms due to the detection of amino acids when Ninhydrin is used

a pink purple color. the exception is proline which develops a yellow color

What was the standard solution of amino acids for this experiment

amino acid standard solution and one percent water

What was the chromatography solvent for this experiment

 methanol and 0.1 molar ammonium acetate and a 60:40 ratio at pH between 4.0 to 5.0

How are the silica gel plates prepared

• The plates are heated at 100°C for 15 minutes

• At one end of the plate about 1 to 2 cm away from the margin a thin line with pencil is drawn and this will be used for spotting the sample

Describe the preparation of the chromatographic chamber

 the chamber should be filled with at least 1 cm high layer of solvent and allowed to become saturated with solvent vapor the chamber also needs to be closed air tightly

When should the chromatography plate be removed from the chamber

the solvent must run through the plate until it reaches about 2 to 3 cm from the top mark the level that the solvent has traveled.

What has to be done after the chromatography plate has been removed from the chamber for the spots to develop

the plate must be air dried and then sprayed with Ninhydrin reagent. Then the plate should be placed in an oven for the development of color spots

How do you calculate RF value

Measure the distance traveled by the solvent. Measure the distance traveled by the amino acid by starting at the origin of the spotting point and then measure upwards the distance traveled by the amino acid. Value is calculated by dividing the distance traveled by the amino acid by the distance traveled by the solvent. 

Name the three components of any chromatographic system

Stationary phase, mobile phase, and, analyte 

For the amino acid analysis proteins are hydrolyzed by dissolving in 6M HCl and heating the solution at what temperature

121°C for six hours

What is RF value and what is the significance of RF value

 used to determine characteristics of a compound and analytes under a given solvent system. RF value can also be used to identify compounds and provide a measure of mobility of the analytes in the compound

Spraying what reagent is used to develop the TLC plate for the detection of amino acids

Ninhydrin reagent

TLC separates amino acids on the basis of what

molecular weight and solubility in the solvent

Why are carbohydrates important

 essential part of human diet since they are the primary source of energy and are also used for the synthesis of the cell wall components

What agency test for the types of carbohydrates in food

the FDA and other food industry agencies

Reactive aldehyde and keto groups

because of the presence of these sugars participate in varieties of chemical  reactions which can be used for their detection and estimation

What tests are used to detect the presence of sugars in a sample

Molish, benedict’s, seliwanoff’s, and bials 

What were the sugar solutions used in this experiment

 one percent sugar solutions of glucose, fructose, ribose and starch or glycogen

What was the acid used in these experiments

Concentrated sulfuric acid

Molisch test

general test for the presence of carbohydrate in a sample. The positive test indicates the presence of carbohydrate in the sample

Mechanism of molish test

carbohydrates are dehydrated to furfural in the presence of concentrated acid, which then condenses with alpha-naphthol to form red to violet color complex

step 1 in procedure for Molish test

play five ML of sample in a test tube and add two drops of molish reagent 5% alpha-naphthol in alcohol and mix.

step 2 in procedure for Molish test

carefully pour 3 MLs of concentrated sulfuric acid along the side of the tube so as to form a layer under the water

step 3 in procedure for Molish test

Development of reddish violet color at the junction of the two liquids indicates positive results

Benedict’s test 

This test is positive for the reducing sugars, which have either aldehyde or keto group available for the reduction reaction

aldehyde or ketone groups

in number of sugars especially diasaccharides or polysaccharides these reactive groups although present are involved in the formation of glycosidic linkage and thus not available for the reduction reaction. Such sugars behave as nonreducing sugars.

mechanism of benedict’s test

When the solutions of reducing sugars are heated under alkaline conditions in the presence of certain metallic ions, the carbonyl group of the sugar is oxidized while the metal ion is reduced forming a colored precipitates.

benedict’s reagent 

a mixture of Copper sulfate (CuSO4), Sodium carbonate (Na2CO3) and Sodium Citrate.

step 1 in procedure for benedict’s test

Place 1 ml of sample in a test tube.

step 2 in procedure for benedict’s test

Add 5 ml of Benedict’s reagent to the 1 ml sample.

step 3 in procedure for benedict’s test

Place the tube in a boiling water-bath for 5-15 minutes.

step 4 in procedure for benedict’s test

Formation of yellow, green or red precipitate indicate the presence of

reducing sugar