Instrumental

Instrumental
Analysis
Analysis

Tutorial 3
1
 Announcement
1. The lecture of instrumental analysis by Dr. Rasha Hanafi due
on Tuesday 2nd of October will take place on Wednesday 3rd of
October as usual in H5 at 8:00 AM, based on Prof. Afifis wish
who will take the official time of the instrumental analysis lecture
for her lecture of pharmacokinetics.
2. Quiz 1 will take place on Thursday 11th of October from 9:00 to
9:30 and will cover the first 3 lectures and first 3 tutorials.
Locations will be available on the intranet
\\FS2\Intranet\Faculties\Pharmacy &
Biotechnology\Pharmaceutical Chemistry\Instrumental
Analysis\PHCM561 WS2012-2013\Announcements.
3. No make up quiz will be offered under any circumstances. 2
quizzes will be counted out of 3.

2
 Locations of quiz 1
Group Location
1 H10
2 H10
3 H11
4 H11
5 C5-108
6 C5-106
7 C5-112
8 C5-204
9 C5-206
10 C5-209

3
Objectives

Determine the proper for determination of a component in a mixture

Biomedical applications of UV-Vis spectrophotometry
Exercises on spectrophotometric analysis.
Describe spectrophotometric titration curves.

5
Spectroscopic riddle Acid form spectra
445 nm [HA]

Absorbance
575 nm

Basic form
spectra [A-]

379 nm
isosbestic point
503 nm

440 nm

Wavelength
Choose the proper for detection of a weak acid (HA H+ + A-) in the
following cases:
1- Determination of the acidic form in presence of the basic form: 445 nm
2- Determination of the basic form in absence of the acid form: 379 nm or
575 nm, what is the difference?
3- Determination of the basic form in presence of the acid form: 575 nm
6
4- Determination of both species: 503 nm (isosbestic point)
 Biomedical applications of UV-Vis Spectrophotometry

1bilirubinanalyzer
(biophotonicdevice)
BiliCheknoninvasiveBilirubin
Analyzeriscapableofusinglightto
painlesslymeasurebilirubin,the
causeofinfantjaundice
(hyperbilirubinemia).

Thedevicesendsseveralflashesof
variousspecificwavelengthstowards
theinfant'sforehead,thenanalyzing
lightthatisreflectedback.Itcanbe
usedasareplacementfortheoften
painfulheelstickbloodtest.
7
2- Immunoassays employ antibodies to detect analytes.
An antibody is a protein produced by the immune system of an
animal in response to foreign molecule called antigen. The antibody
recognizes the antigen that match in synthesis the antibody.
Enzyme-Linked Immunosorbent Assays (ELISA) is used for many
biomedical tests.

ELISA steps

Antibody 1, which is specific for the

analyte of interest (the antigen
protein of the virus causing disease),
is bound to a polymeric support.
Analyte-antigen (in blood sample)
is then incubated with the polymer-
bound antibody to form a complex.
antibody 1
The fraction of antibody that bind
analyte is proportional to the
concentration of the analyte in the 8
unknown
 The antibody-antigen complex is treated
with antibody 2 which is already
covalently attached to an enzyme. Note
that antibody 2 will recognize a different
region of the analyte.
The enzyme attached to antibody 2 is
vital for analysis. The enzyme can
transform a colorless reactant into a
colored product.
Because one enzyme molecule catalyze
the same reaction many times, many 5. Enzyme reacts with an added
molecules of colored product are colorless reactant to form
created for each analyte molecule. The colored product
enzyme thereby amplifies the signal in
chemical analysis.
If the purpose is just qualitative analysis
(+ve or ve test), we just check the
intensity of the color obtained.
If the purpose is to quantify, a calibration
curve is prepared from which the
concentration of antigen can be 9
determined.
10
ELISA Enzyme-Linked Immunosorbent
Assay

Positive
ELISA

11
 Exercise 1
The drug tolbutamine (FM 270) has a molar absorptivity of 703 L mol -1 cm-1 at
262 nm. One tablet is dissolved in water and diluted to a volume of 2 liter. If
the solution absorbs 79.44% of the radiation in 1 cm, how many grams of
tolbutamine are contained in the tablet? (Answer 0.527 g)

%Absorption=79.44% %Transmission= 20.56 % A= - log (0.2056) = 0.699

A= b c C=A/ b C=0.699/(703)(1) C=9.94 10-4 M

Wt in grams = M M.wt Vol. in liters Wt. of tolbutamine = 0.527 gms

12
 Exercise 2
Amines (weak base) form salts with picric acid, and all amine
picrates exhibit max= 359 nm with a molar absorptivity of 1.25x104
L mol-1 cm-1.
A 0.2 g sample of aniline, C6H5NH2, (FM 93) is dissolved in 500 ml
water. A 25 ml aliquot is reacted with picric acid in a 250-ml
volumetric flask and diluted to the volume. A 10 ml aliquot of this
is diluted to 100 ml and the absorbance is read at 359 nm in a 1-
cm cell. If A=0.425, what is the percent purity aniline?(Answer 79.05%)
A= b c C diluted =A/ b Cdiluted = 0.425 / (1) (1.25 104) Cdiluted= 3.4 10-5 M
To calculate dilution factor: 0.2 g Dissolved in 500 mL
Total dilution factor
25 10 250 mL 10 10 =100
10 10 100 mL
C (original) = Cdil 100 Absorbance is measured for this soln.
Mass of aniline = 3.4 10-3 93 500 10-3 = 0.1518 g
= 3.4 10 M -3
% Purity = 0.1518 / 0.2 100 = 79 %
13
Exercise 3
Standard solutions of compound Y were used to construct a
calibration curve: ( provided b = 1 cm )

Concentration 1 2 3 4
(mg/L)
Abs. 0.2 0.37 0.54 0.71

If 0.2 ml of an unknown solution of Y was diluted to 10 ml and

the absorbance of this solution was found to be 0.5 in a 1-cm
cell, what is the concentration of Y? (Answer: 147 mg/L)
A = b C (if conc. is in mole/liter i.e. M ) A = a b C ( if conc. is in g/L ,
( molar absorptivity) (absorptivity)
mg/mL , mg/L , etc.)

To get a : (Y2-Y1) / (X2-X1) a= 0.17 L mg-1 cm-1

50
Cdil = 0.5 / 0.17 = 2.94 mg/L 0.2 ml 10 ml
Coriginal = Cdil 50 = 147 mg / L
14
Exercise 4 (self study)
Several standard solutions of a compound were prepared and the
absorbance of each was measured in a 5-cm cell. The absorbance
of each at 534 nm was plotted as a function of its concentration.
The plot was linear with a slope of 973 L mol-1 and went through
the origin. The absorbance of the analyte solution was 0.493 in a
1-cm cell at 534 nm. Calculate the concentration of the absorbing
compound in the analyte. (Answer 2.5x10-3 M)

Slope = b = 973 L mol -1 b= 5 cm = 973 / 5 = 194.6 M-1 cm-1

A sample = 0.493 A= bC C = 0.493 / (194.6)(1)

= 2.53 10 -3 M

15
Exercise 5 (self study)

The molar absorptivities of compounds X and Y were measured with pure

samples of each:

X Y A Eqn.
(nm)

272 16 440 3 870 0.957 0.957 = 16440 X + 3870 Y

327 3 990 6 420 0.559 0.559 = 3990 X + 6420 Y

A mixture of compounds X and Y in a 1.0 cm had an absorbance of 0.957 at

272 nm and 0.559 at 237 nm. Find the concentrations of X and Y in the
mixture. (Answer: [Y] = 5.96x10-5 M, [X] = 4.42x10-5 M)

X= 0.957- 3870Y / 16440 Substitute in 2 to get Y

16
Spectrophotometric titration

If Analyte and
product absorb

analyte > product

If Analyte and
product absorb

product > analyte

If Analyte and
product absorb

product = analyte

17
Exercise 6
In the following reaction:
A+T P
T = p and A = 0 (at certain ). Explain why cant the reaction be used
for the spectrophotometric titration of A with T, what will be the shape
of the titration curve in case of T > A and p = 0?
End point End point

A AA
A
T

p
Blank Blank p= 0

Vol. of titrant Vol. of titrant

No change in absorbance before and after

equivalence point
18
 Exercise 7 (self study)

KMnO4 was titrated with H2C2O4 in H2SO4 as acid medium yielding

colorless MnSO4. Sketch the spectrophotometric titration curve, at max
of KMnO4, considering it once as a titrant and other as being titrated.

Equivalence point
Equivalence

A
point A

blank
blank

Vol. of titrant Vol. of titrant

Using KMnO4 as titrant Using KMnO4 as analyte

19
Exercise 8 (self study)

Ammonia can be determined spectrophotometrically by reaction with phenol in

the presence of hypochlorite (OCl-). A 4.37 mg sample of protein was chemically
digested to convert its nitrogen to ammonia and then diluted to 100 ml. Then 10
ml of the solution were placed in a 50-ml volumetric flask and treated with 5 ml
of phenol solution plus 2 ml of sodium hypochlorite solution. The sample was
diluted to 50 ml, and the absorbance at 625 nm was measured in a 1 cm cuvet
after 30 min. FOR REFERENCE, a standards solution was prepared from 0.01 g
of NH4Cl (FM 53.49) dissolved in 1 L of water. Then 10 ml of this standard were
placed in a 50-ml volumetric flask and analyzed in the same manner as the
unknown. A reagent blank was prepared using distilled water in place of the
unknown.
(a) Calculate the molar absorptivity of the blue product.
(b) Calculate the weight percent of nitrogen in the protein.

Sample Absorbance at 625 nm

Blank 0.140
Reference 0.308
Unknown 0.592 20
 Solution of Exercise 8

(a) Concentration of NH4Cl std. = 0.01(g/L) X 1/ 53.49 (mol/g)

= 1.869x10-4 M
Concentration of std. (after dilution) in the colored solution measured
=(1.869x10-4) x 10/50 (this is the inverted dilution factor, because we need to
calculate the diluted conc. not the original conc.)
=3.739 x 10-5 M
Since:
A =b c

(0.308 0.140) = x 1 x (3.739x10-5)

= 4.49x103 L mol-1 cm-1 (M-1 cm-1)

A std c std
(b)
A unk c unk

0.308 0.140 1.869 10 4

0.592 0.140 Cunk

Cunk= 5.029 x 10-4 M 21

 Since [N] = [NH3] = 5.029 x 10-4 M
Mass of nitrogen in 100 mL protein sample
= 5.029 x 10-4 mol/L x 14 g/mol x 0.1 L
= 7.043 x 10-4 g

7.043 10 4
%N 100 16.1%
4.37 10 3

22
 SAMPLE COLLECITVE PROBLEMS AND QUESTIONS ON UV-VIS (NON- GRADED)

Exercise 1: Cu+ reacts with neocuproine to form a colored complex (neocuproine) 2Cu+, with an
absorption maximum at 454 nm. Neocuproine is particularly useful because it reacts with few
other metals. The copper complex is soluble in isoamyl alcohol, an organic solvent that is
immiscible with water. If (neocuproine)2Cu+ is present, virtually all of it goes into the organic
phase.

For analysis of Cu in a rock sample, the following procedure is carried out:

Step1. A rock containing copper is pulverized, and all metals are extracted from it with
strong acid. The acidic solution is neutralize with base and made up to 250 mL in flask A.

Step 2. Next, 10.00 mL of the solution are transferred to flask B and treated with
10.00 mL of a reducing agent to reduce all Cu2+ into Cu+. Then 10.00 mL of buffer are
added to bring the pH to a value suitable for complex formation with neocuproine.
Step 3. After that, 15.00 mL of this solution are withdrawn and placed in flask C.
To the flask are added 10.00 mL of an aqueous solution containing
neocuproine and 20.00 mL of isoamyl alcohol. After shaking well and allowing the phases to
separate, all (neocuproine)2Cu+ is in the organic phase.
Step 4. A few milliliters of the upper layer are withdrawn, and the absorbance at 454 nm is
measured in a 1.00-cm cell. A blank carried through the same procedure gives an absorbance of
0.056.

23
a) Suppose that the rock contained 1.00 mg of Cu, what will be the
concentration of Cu (moles per liter) in the isoamyl alcohol phase?
b) If the molar absorptivity of (neocuproine)2Cu+ is 7.90x103 M-1cm-1, what
will be the observed absorbance? Remember that a blank carried the
same procedure gave an absorbance of 0.065.
c) A rock is analyzed and found to give a final answer of 0.874
(uncorrected for the blank). How many milligrams of Cu are in the rock?
24
Exercise 2: A portable photometer with a linear response to radiation registered
73.6 A with a blank solution in the light path. Replacement of the blank with an
absorbing solution yielded a response of 24.9 A. Calculate
(a) The percent transmittance of the sample solution.
(b) The absorbance of the sample solution.
(c) The transmittance expected for a solution in which the concentration of the
absorber is one-third that of the original sample.

Exercise 3: Describe the difference between the following and list any
particular advantages possessed by one over the other:
(a) Deuterium and tungsten lamp as sources of radiation.
(b) Filters and monochromators as wavelength selectors.
(c) Phototube and photomultiplier tubes.
(d) Single-beam and double-beam instruments for absorption measurements.
(e) Conventional and diode-array spectrophotometers.

Now, try to solve the following problems:

Harris Text Book, Chapter 18, pp 429:
18-12, 18-15, 18-16, 18-17, and 18-18 25