Introduction to biochemical practicals

Vladimíra Kvasnicová

• arrangement of practicals

• laboratory safety regulations

• laboratory equipment

• dealing with automatic pipette

• instructions : http://vyuka.lf3.cuni.cz/

plastic tip pipetting button

AUTOMATIC PIPETTE

filling emptying

Pipetting step by step

calibrated volume

standby

calibrated lock

bottom lock

1. SPECTROPHOTOMETRY

2. CHROMATOGRAPHY

3. VOLUMETRIC ANALYSIS

4. POTENTIOMETRY

Topics of laboratory tasks

HINTS to individual tasks

EXPLOSION?!?

PRINCIPLE

• interaction between a compound of interest

and a monochromatic radiation

• a part of the radiation is absorbed

by the compound and a rest

of the radiation is detected

by a detector

• quantity of the absorbed radiation is directly

proportional to the quantity of the compound

of spectrophotometry

spectrophotometer

Spectrophotometry in thepractical training

„Determination of urine creatinine“

analysed sample : own urine

1. colorless creatinine is transformed to a

colour compound by chemical reaction

2. absorbance of the compound is used to

establish creatinine concentration using a

calibration curve

Calculations in this practical

• preparation of calibration solutions from the stock solution of known concentration (dilution)

• dilution of urine sample

• conversion of the mass concentration to the molar concentration

Calculation of concentration:

1. Beer-Lambert´s law2. Calibration curve3. Calculation based on values

of standard solutions

Calculation of concentration:

Beer-Lambert´s lawA = εεεε x l x c

orT = 10- (εεεε x l x c)

A = absorbance (A = -log T)

T = transmittance (T = 10-A)

εεεε = molar absorption coefficient

l = thickness of cuvette (in cm), c = molar concentration

Calibration curve

3 or more standards

processed by the

same method

linear calibration curve

A = εεεε x l x c

y = kx + q

Calculation using standards

Ast = cst x l x εεεε Aus = cus x l x εεεε

Ast / cst = l x ε Aus / cus = l x ε

l x ε = l x ε

Ast / cst = Aus / cus

cus = Aus x (cst / Ast)

cus = Aus x f

f = average of all (c st / Ast) used in the experiment

Problems

1) standard: A = 0.600, c = 15.0 mMsample: A = 0.200, c = ?

2) standard: T = 0.30sample: T = 0.60

Is cs lower or higher than cst?

3) c1 = 0.1 mM → c2 = 0.01 mMHow many times was the sample diluted?

4) sample of c = 0.2 mM was diluted by 100 timesWhat is its final concentration?

Homework

1) As = 0.25 Cs = ?Ast = 0.40 Cst = 4mg / L

[2.5mg/L]

2) 1000 mg/L glucose standard (C st ) reads T = 0.49. T of asample is 0.55. What is glucose concentration in th esample? (in mg/L and mmol/L) MW = 180g

[839mg/L = 4.7mM ]

3) Protein standard: T = 0.33; patient’s sample: T = 0.44Compare the patient’s protein concentration with t hestandard

[4/3]

PRINCIPLE

Seperation of a mixture of solutes is based on a

differential distribution of the solutes between

two immiscible phases:

• stationary phase (solid or liquid)

• mobile phase (liquid or gase)

The mobile phase carries solutes through the stationary phase

with different velocities according to their mutual affinity.

of chromatography

Chromatography in the practical training

„ TLC of fat-soluble dyes“

• adsorption plane liquid chromatography

• mobile phase: toluene (nonpolar)

• stationary phase: plate of silica gel (polar)

• stadards of dyes → comparison of Rf

• unknown sample: composed of 2 unknown dyes

TLC chromatography = task of the practical training

„Show of HPLC and GC- a visit of the analytical laboratory“

HPLC = High Performance Liquid Chromatography

(or High Pressure LC)

• normal or reversed phase HPLC

GC

= Gas Chromatography

Scheme of HPLC

Mobile phase

Degasser

Pump

Sample injection

Column

Detector

Waste

Gas Chromatography (GC)

The figure was found at http://www.cofc.edu/~kinard/221LCHEM/ (November 2006)

Evaluation of chromatogram

Spots are compared with

standards:

Rf = a /b

Rf = retardation factor

or „rate of flow“

a = start to center of spot

b = start to solvent flow

1) Plane Chromatopgraphy (TLC)

The figure was found at http://sms.kaist.ac.kr/~jhkwak/gc/catofp/chromato/tlc/tlc.htm (November 2006)

2) Column Chromatography (HPLC, GC)

Peaks are compared with

standards:

tR = retention time

≈ identification of solutes

h = height of the peaks

≈≈≈≈ concentration of solutes

PRINCIPLE

A solution of known concentration is slowly added from the burette into the flask

containing a sample until a stoichiometric ratio of the reactans is reached

(= point of equivalence )

point of equivalence = reactants are present in a stoichiometric ratio given by the chemical equationdescribing the reaction used for the analyses

of volumetric analysis

The method is based on a chemical reactionbetween a solute of interest and

a titrimetric reagent

burette:titrimetric reagent

titrimetric flask: diluted sample of a solute of interest

titration=

determination of exact

concentration

Titration in the practical training

„Determination of acidity of gastric juice“

• analyte: HCl found in gastric juice

• titrimetric reagent: NaOH

→ neutralization titration (= alcalimetry)

• indicator: phenolphtaleine (colourless → violet)

• c(HCl) → calculation of pH of gastric juice

• pH before and after a stimulation of the stomach

is determinated

Calculation of sample concentration

• based on knowledge of a stoichiometry of chemical reaction

a A + b B → c C + d D

a, b, c, d = stoichiometric coefficients = substance amounts (n)A = „titrimetric reagent“, B = analysed sample

a / b = n(A) / n(B)

a / b = n(A) / n(B)

c = n / V → n = c x Vc = molar concentration (mol/l)

n = substance amount (mol)V = volume of a solutiona, b = stoichiometric coefficients

a x n(B) = b x n(A)

a x cB x VB = b x cA x VA

a x cB x VB = b x cA x VA

• stoichiometry of the reaction is known • concentration and consumed volume of the

titrimetric reagent at a point of equivalenceis known

• sample volume used for the analyse is known

the only unknown value is

cB

Calculations in this practical

• calculation of the substance amount of NaOH consumed during titration

• calculation of the substance amount of HCl in the whole sample volume

• calculation of BAO and MAO

• calculation of gastric juice pH from the substance amount of HCl and the whole volume of the gastric juice (before and after stimulation)

PRINCIPLE

it is an electrochemical method based on the

measurement of voltage of an

electrochemical cell when no current flows .

two electrodes :

• working (indicating) electrode

• reference electrode

of potentiometry

Potentiometry in the practical training

„ Measuring pH of phosphate buffer“

• various solutions of phosphate buffer

• pH determination by pH -meter

• calibration of the instrument by standards

• glass combination electrode („twin “)

Glass combination

electrode

The figure was found at http://www.ph-meter.info/img/combination-electrode.png (October 2007)

potentiometer

BUFFERSBUFFERSBUFFERSBUFFERS

= solutions which have the ability to absorb small solutions which have the ability to absorb small solutions which have the ability to absorb small solutions which have the ability to absorb small additions of either a strong acid or strong base additions of either a strong acid or strong base additions of either a strong acid or strong base additions of either a strong acid or strong base with a with a with a with a very little change of pHvery little change of pHvery little change of pHvery little change of pH....

• buffers are used to maintain stable pHbuffers are used to maintain stable pHbuffers are used to maintain stable pHbuffers are used to maintain stable pH• composition of buffers:composition of buffers:composition of buffers:composition of buffers:

„„„„conjugated pair: acid /baseconjugated pair: acid /baseconjugated pair: acid /baseconjugated pair: acid /base““““* weak acid + it`s salt* weak acid + it`s salt* weak acid + it`s salt* weak acid + it`s salt* weak base + it`s salt* weak base + it`s salt* weak base + it`s salt* weak base + it`s salt* 2 salts of a polyprotic acid* 2 salts of a polyprotic acid* 2 salts of a polyprotic acid* 2 salts of a polyprotic acid* amphoteric compound (e.g. protein)* amphoteric compound (e.g. protein)* amphoteric compound (e.g. protein)* amphoteric compound (e.g. protein)

„bicarbonate buffer“ HCO3- NaHCO3 ↔ Na+ + HCO3

-

H2CO3 H2CO3 ↔ H+ + HCO3-

NaHCO3

mixed→ Na+ + HCO3-

H2CO3 H+ + HCO3-

+ H2CO3

+ HCl + NaOH(H+ + Cl-) (Na+ + OH- )

Na+ + HCO3- Na+ + HCO3

-

H+ + H2CO3 H2O + HCO3-

Cl- + H2CO3 Na+ + H2CO3

HCO3- + H+↔ H2CO3 H+ + OH- ↔ H2O

HendersonHendersonHendersonHenderson----Hasselbalch equationHasselbalch equationHasselbalch equationHasselbalch equationpH = pKpH = pKpH = pKpH = pKaaaa + log (+ log (+ log (+ log (ccccssss / / / / ccccaaaa)))) (for acidic buffer )

pOH = pKpOH = pKpOH = pKpOH = pKbbbb + log (+ log (+ log (+ log (ccccssss / / / / ccccbbbb )))) (for basic buffer)

pH = 14 - pOH

pK = dissociation constant of the weak acid (pKa) or base (pKb)cs = actual concentration of the salt in the bufferca = actual concentration of the weak acid in the buffercb = actual concentration of the weak base in the bufferc = c´ x V´ c´ = concentration before mixing the components

V´ = volume of a component (acid or base or salt)

Calculations in this practical training

• calculation of pH of all buffer solutions

pH = pK(H2PO4-) + log c(HPO4

2-) / c(H2PO4-)

pH = pKa + log n(HPO42-) / n(H2PO4

-)

pH = pK a + log (c s´x Vs´) / (ca´x Va´)

c = n/V n = n´ = c´ x V´ s = HPO42- a = H2PO4

-

(V = volume of the buffer in which ns and na are present)

• calculation of pH-changes of these buffer solutions after addion of HCl

1. SPECTROPHOTOMETRY - B1

2. CHROMATOGRAPHY - B2

3. TITRATION - B3

4. POTENTIOMETRY - B4

4 tasks / 4 weeks / 8 working places

- division of the study group to 8 working groups -

Homework

1) titrimetric reagent: 23.8 ml NaOH, (factor = 0.9685; C = 0.1M), sample = 10ml H 2SO4; C = ?

[0.12M]

2) titrimetric reagent: 10ml KMnO 4 (0.1M), sample: 20ml FeSO4 ; C = ? (mol/ L, % ), MW = 152g

[0.25M = 3.8% ]

3) H3PO4 → Na2HPO4sample: 20ml H 3PO4 (C = 0.3M ), titrimetric solution: 0.2M NaOH V = ?

[60ml]