Introduction to biochemical practicals. Vladimíra Kvasnicová

Transcription

1 Introduction to biochemical practicals Vladimíra Kvasnicová

2 arrangement of practicals laboratory safety regulations laboratory equipment dealing with automatic pipette instructions:

3 plastic tip pipetting button AUTOMATIC PIPETTE

4 Pipetting step by step calibrated volume standby calibrated lock bottom lock filling emptying

5 Topics of laboratory tasks 1. SPECTROPHOTOMETRY 2. CHROMATOGRAPHY 3. VOLUMETRIC ANALYSIS 4. POTENTIOMETRY

6 HINTS to individual tasks EXPLOSION?!?

7 PRINCIPLE of spectrophotometry 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

8 spectrophotometer

9 Spectrophotometry in the practical 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

10 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

11 Calculation of concentration: 1. Beer-Lambert s law 2. Calibration curve 3. Calculation based on values of standard solutions

12 Calculation of concentration: Beer-Lambert s law A = ε l c x x or T = 10 - (ε x l x c) A = absorbance (A = -log T) T = transmittance (T = 10 -A ) ε = molar absorption coefficient l = thickness of cuvette (in cm), c = molar concentration

13 Calibration curve 3 or more standards processed by the same method linear calibration curve A = ε x l x c y = kx + q

14 Calculation using standards A st = c st x l x ε A st / c st = l x ε A us = c us x l x ε A us / c us = l x ε l x ε = l x ε A st / c st = A us / c us c us = A us x (c st / A st ) c us = A us x f f = average of all (c st / A st ) used in the experiment

15 Problems 1) standard: A = 0.600, c = 15.0 mm sample: A = 0.200, c =? 2) standard: T = 0.30 sample: T = 0.60 Is c s lower or higher than c st? 3) c 1 = 0.1 mm c 2 = 0.01 mm How many times was the sample diluted? 4) sample of c = 0.2 mm was diluted by 100 times What is its final concentration?

16 Homework 1) A s = 0.25 C s =? A st = 0.40 C st = 4mg / L [2.5mg/L] 2) 1000 mg/l glucose standard (C st ) reads T = T of a sample is What is glucose concentration in the sample? (in mg/l and mmol/l) MW = 180g [839mg/L = 4.7mM ] 3) Protein standard: T = 0.33; patient s sample: T = 0.44 Compare the patient s protein concentration with the standard [4/3]

17 PRINCIPLE of chromatography 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.

18 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 R f unknown sample: composed of 2 unknown dyes

19 TLC chromatography = task of the practical training

20 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

21 Scheme of HPLC Mobile phase Degasser Pump Sample injection Column Detector Waste

22 Gas Chromatography (GC) The figure was found at (November 2006)

23 Evaluation of chromatogram 1) Plane Chromatopgraphy (TLC) Spots are compared with standards: R f = a /b R f = retardation factor or rate of flow a = start to center of spot b = start to solvent flow The figure was found at (November 2006)

24 2) Column Chromatography (HPLC, GC) Peaks are compared with standards: t R = retention time identification of solutes h = height of the peaks concentration of solutes

25 PRINCIPLE of volumetric analysis 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 equation describing the reaction used for the analyses

26 The method is based on a chemical reaction between a solute of interest and a titrimetric reagent titration = determination of exact concentration burette: titrimetric reagent titrimetric flask: diluted sample of a solute of interest

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29 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

30 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)

31 a / b = n(a) / n(b) c = n / V n = c x V c = molar concentration (mol/l) n = substance amount (mol) V = volume of a solution a, b = stoichiometric coefficients a x n(b) = b x n(a) a x c B x V B = b x c A x V A

32 a x c B x V B = b x c A x V A stoichiometry of the reaction is known concentration and consumed volume of the titrimetric reagent at a point of equivalence is known sample volume used for the analyse is known the only unknown value is c B

33 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)

34 PRINCIPLE of potentiometry 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

35 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 )

36 Glass combination electrode The figure was found at (October 2007)

37 potentiometer

38 BUFFERS = solutions which have the ability to absorb small additions of either a strong acid or strong base with a very little change of ph. buffers are used to maintain stable ph composition of buffers: conjugated pair: acid /base * weak acid + it`s salt * weak base + it`s salt * 2 salts of a polyprotic acid * amphoteric compound (e.g. protein)

39 bicarbonate buffer HCO 3 - NaHCO 3 Na + + HCO 3 - H 2 CO 3 H 2 CO 3 H + + HCO 3 - NaHCO 3 mixed Na + + HCO 3 - H 2 CO 3 H + + HCO H 2 CO 3 + HCl + NaOH (H + + Cl - ) (Na + + OH - ) Na + + HCO 3 - Na + + HCO 3 - H + + H 2 CO 3 H 2 O + HCO 3 - Cl - + H 2 CO 3 Na + + H 2 CO 3 HCO H + H 2 CO 3 H + + OH - H 2 O

40 Henderson-Hasselbalch Hasselbalch equation ph = pk a + log (c s / c a ) (for acidic buffer ) poh = pk b + log (c s / c b ) (for basic buffer) ph = 14 - poh pk = dissociation constant of the weak acid (pk a ) or base (pk b ) c s = actual concentration of the salt in the buffer c a = actual concentration of the weak acid in the buffer c b = actual concentration of the weak base in the buffer c = c x V c = concentration before mixing the components V = volume of a component (acid or base or salt)

41 Calculations in this practical training calculation of ph of all buffer solutions ph = pk(h 2 PO 4- ) + log c(hpo 2-4 ) / c(h 2 PO 4- ) ph = pk a + log n(hpo 2-4 ) / n(h 2 PO 4- ) ph = pk a + log (c s x V s ) / (c a x V a ) c = n/v n = n = c x V s = HPO 4 2- a = H 2 PO 4 - (V = volume of the buffer in which n s and n a are present) calculation of ph-changes of these buffer solutions after addion of HCl

42 4 tasks / 4 weeks / 8 working places - division of the study group to 8 working groups - 1. SPECTROPHOTOMETRY - B1 2. CHROMATOGRAPHY - B2 3. TITRATION - B3 4. POTENTIOMETRY - B4

43 Homework 1) titrimetric reagent: 23.8 ml NaOH, (factor = ; C = 0.1M), sample = 10ml H 2 SO 4 ; C =? [0.12M] 2) titrimetric reagent: 10ml KMnO 4 (0.1M), sample: 20ml FeSO 4 ; C =? (mol/ L, % ), MW = 152g [0.25M = 3.8% ] 3) H 3 PO 4 Na 2 HPO 4 sample: 20ml H 3 PO 4 (C = 0.3M ), titrimetric solution: 0.2M NaOH V =? [60ml]