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• arrangement of practicals
• laboratory safety regulations
• laboratory equipment
• dealing with automatic pipette
• instructions : http://vyuka.lf3.cuni.cz/
1. SPECTROPHOTOMETRY
2. CHROMATOGRAPHY
3. VOLUMETRIC ANALYSIS
4. POTENTIOMETRY
Topics of laboratory tasks
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
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
„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
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)
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]
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