PHARMACEUTICAL ANALYSIS 2 – Lecture

Requirements to be submitted after ECQ:

I. Individual requirement: Legibly write your answers on a separate sheet of letter-sized bond paper. List down
your references if applicable.
A. Journal critique
1. Search for an online journal (preferably the non-reputable ones). Critique the journal based on the
following questions:
a. What is the title of the study?
Interference-free Determination of Carbamazepine in Human Serum Using High Performance
Liquid Chromatography: A Comprehensive Research with Three-way Calibration Methods
Author/s: Shiva Ghafghazia, Taraneh Moini Zanjania, Maryam Vosoug, Masoumeh Sabetkasaeia
Published date: November 2015

b. What are the objectives of the study?

 A comprehensive study that aims to perform a quantitative measurement of
carbamazepine in human serum samples with emphasis Parallel factor analysis
(PARAFAC), alternating trilinear decomposition (ATLD), self-weighted alternating
trilinear decomposition (SWATLD), alternating penalty trilinear decomposition (APTLD),
and unfolded partial least squares combined with the residual bi-linearization procedure
(U-PLS/RBL) through the use of high-performance liquid chromatography with
photodiode-array detection (HPLC-DAD).

 The study compares the accuracy and precision of each algorithm for analyzing CBZ in
serum samples using root mean square error of prediction (RMSEP), the recovery values
and figures of merits and reproducibility of the analysis.

c. What are the methodologies used in the study? (Focus on the instrumentation part such as the
use of UV-Vis Spectrophotometer, FTIR, HPLC or GC.)

METHODOLOGY
Materials The materials used by the researchers for HPLC-DAD are the carbamazepine, HPLC-
grade methanol (MeOH), acetonitrile (ACN) and ethyl acetate (EA), and the serum
samples.

Equipment
Chromatographic conditions
For analytical grade, phosphoric acid, dipotassium hydrogen phosphate, and sodium
hydroxide for chromatographic conditions. Moreover, they used nylon membrane
filters with 0.22µM pore size for filtering solvent before HPLC analysis.
For instrumentation of HPLC, the researchers used an Agilent 1200 Series system
equipped, composed of a Rheodyne 7725 manual injector, a degasser, a quaternary
pump, a column oven compartment, and a Hewlett-Packard 1200 series photo
diode-array detector (DAD).
The column oven temperature was set to 25 ºC and chromatographic separation was
carried through a column (15 cm × 0.46 cm, 5 µM particle size, Agilent).
Data which was collected by Chemstation software, exported as Microsoft Excel® file
for further processing. They used MATLAB for all calculations.
The standard instrumental system for an isocratic mobile mode were consists of
acetonitrile (45%, v/v) and 0.005 mol L -1 K2HPO4 (pH = 7.5) buffer solution (55%, v/v).
The condition of mobile phase flow rate was 1.0 mL min-1 and the injection volume
was 20 μL. DAD detector was set to record between 210-400 nm with the spectral
resolution of 1.5 nm and integration period of 0.4 s per spectrum. Total run time was
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Sample handling and
extraction procedure
less than 5 min.
The residue extracted from serum samples, whereby the combined organic layers
were evaporated to dryness, was dissolved in 500 µL of mobile phase in ultrasonic
bath and filtered through a 0.22 µM PTFE syringe filter and an aliquot of 20 µL (usual
standard) was injected into the HPLC system.

d. Are the methodologies used appropriate for the study? Explain. (For example, is the set
wavelength appropriate for the analysis of the sample? Should the mobile phase isocratic or
gradient elution?)

e. Do the researchers fully explain the results acquired in the experiment?

f. What is/are the conclusion(s) of the study?
g. Are there any typographical errors, misspellings, or wrong punctuations?
h. Is the title of the study appropriate? If not, suggest a revised title.
i. What should have been improved in the study? Give your recommendations.
Summarize your findings and indicate your reference. No need to print out the journal.

B. Activity No. 3: Principles of different instrumentation methods

C. Activity No. 4: Problem solving related to different instrumentation methods

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5423239/pdf/ijpr-16-120.pdf

Activity No. 3: Principles of different instrumentation methods

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Instruction: Read and analyze the following questions. Write the letter of your choice on provided answer sheet.

Fourier Transform Infrared Spectroscopy

Given the FTIR Spectra below, match the following bands to its corresponding interpretation.

1. Band A A A. Aliphatic O-H stretch

2. Band B E B. C=C stretch
3. Band C D C. C=O conjugated stretch
4. Band D C D. C=O unconjugated stretch
5. Band E B E. C-H stretch
F. N-H amide stretch

6. Band A A A. Aliphatic O-H stretch

7. Band B E B. C=C conjugated
8. Band C D C. C=O conjugated ketone stretch
9. Band D C D. C=O unconjugated ketone stretch
10. Band E B E. C-H stretch
F. N-H amide stretch

11. It is the instrument used to measure optical C. Retention time

activity of a sample. D. % Transmittance
A. Flame photometer 13. Which of the following wavelength is
B. Polarimeter commonly used for structural confirmation?
C. Refractometer A. 3 – 15 µm
D. Spectrometer B. 8 – 15 µm
12. In Gas chromatography, the result of the C. 15 – 300 µm
assay is expressed in: D. 780 – 3000 nm
A. Absorbance
B. Rf value

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14. Using the following data, calculate the D. Spectrophotometer
concentration of the sample: Asx = 0.58; Astd = 22. Radiant energy required in drug analysis
0.55; Concstd = 16.5 µg using fluorometry.
A. 15.50 µg A. Infrared region
B. 15.90 µg B. Ultraviolet region
C. 17.40 µg C. Visible region
D. 17.55 µg D. X-ray region
15. Using the following data, calculate the 23. Which of the following electrode is usually
concentration of the sample: Asx = 0.42; Astd = used for measuring pH of a solution?
0.39; Concstd = 15 µg A. Calomel electrode
A. 15.75 µg B. Glass electrode
B. 16.00 µg C. Platinum electrode
C. 16.15 µg D. Quinhydrone electrode
D. 17.50 µg 24. Gas chromatography is used in the
16. In the formula E = hv, “h” is the __. separation of:
A. Energy A. Amino acids
B. Frequency B. Carbohydrates
C. Planck’s constant C. Nucleic acid
D. Wavenumber D. Volatile oils
17. Beer’s plot in spectrometry is prepared to 25. It gives a generalization which governs the
determine: distribution of a solute between two non-
A. Absorbance of the sample miscible solvent.
B. Blank concentration A. Beer’s Law
C. Wavelength to be used B. Beer-Bouguer’s Law
D. Drug concentration C. Lambert’s Law
18. Which of the following instruments is D. Nernst’s Law
suitable for faster analysis of trace minerals 26. In column chromatography, the separation
present in multivitamins? of the sample mixture into a series of narrow
A. Atomic Absorption Spectrophotometer bands in a column is called __.
B. Fourier Transformed Infrared A. Chromatogram
Spectroscopy B. Eluant
C. High Performance Liquid C. Elution
Chromatography D. Spectra
D. UV-Vis Spectrophotometer 27. A plot of absorbance against concentration
19. Which of the following is the fingerprint of a standard drawn in a straight line.
region in infrared spectroscopy? A. Beer’s Law
A. Near IR B. Lambert’s Law
B. Medium IR C. Nernst’ Law
C. Far IR D. Newton’s Law
D. Both B and C 28. Which of the following is not a unit of
20. In this type of chromatography, cellulose of wavelength in spectroscopy?
the filter paper is used as the adsorbent. A. Angstrom
A. Column chromatography B. Hertz
B. Gas chromatography C. Micrometer
C. Paper chromatography D. Nanometer
D. Thin layer chromatography 29. Substances that have a power of rotating the
21. Which of the following is not an optical plane polarized light are said to be:
device or instrument? A. Dextrorotatory
A. Colorimeter B. Light sensitive
B. pH meter C. Optically active
C. Polarimeter D. Organic
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 30. It states that the power of transmitted light IV. Distance traveled by the standard
decreases exponentially as the thickness of solution
the solution increases arithmetically. A. I and II
A. Beer’s Law B. III and IV
B. Bouguer’s Law C. I, II, III
C. Nernst’ Law D. I, II, IV
D. Both A and B 40. Which of the following mobile phases is/are
used in Gas chromatography?
For numbers 29 – 33: Prednisolone is to be eluted from I. Acetonitrile
an ODS column. Arrange the following solvent systems II. Helium
in order of decreasing rate at which they will elute III. Methanol
prednisolone. IV. Nitrogen
A. Acetonitrole/H2O (50:50) A. III only
B. Methanol/H2O (20:80) 3 B. I and III
C. Methanol/H2O (50:50) 2 C. II and IV
D. Methanol/H2O (80:20) 1 D. None of the above
E. Acetonitrole/H2O/THF (50:40:10) 41. Which of the following statement is/are true
31. 1st D for Atomic Emission Spectrophotometry?
32. 2nd C I. Determination of alkali and alkaline
33. 3rd B earth metals
34. 4th E II. More sensitive than Atomic Absorption
35. 5th A Spectrophotometry -AAS
III. Solid samples are incorporated in KBr
36. The index of refraction of a volatile oil can be pellet. -FTIR
determined by: IV. Requires different hollow cathode for
A. Colorimeter each element. –AAS
B. Polarimeter
C. Refractometer A. I only
D. Spectrophotometer B. II and IV
37. Turbidimetric and nephelometric methods of C. I, II, IV
assay are applicable to: D. All of the above
A. Colored samples 42. Calcium and sodium content of the blood
B. Solutions can be determined by:
C. Suspensions A. Colorimetry
D. Volatile oils B. Flame spectrometry
38. In thin layer chromatography, finding the C. Nephelometry
spot of the colorless compound in the D. Turbidimetry
chromatogram can be done by: 43. It is the number of complete cycles that pass
I. Plate exposure to iodine vapor. a given point per second.
II. Plate exposure to UV light. A. Frequency
III. Use of ninhydrin solution. B. Wavelength
IV. Use of H2SO4 spray. C. Radiant energy
A. II only D. Spectrum
B. I and IV 44. Which of the following is the heart of HPLC
C. I, II, III and GS?
D. All of the above A. Injection valve
39. What are the data needed for calculating the B. Column
Rf value in paper chromatography? C. Detector
I. Distance traveled by the solute D. Pumps
II. Distance traveled by the solvent 45. It is the most widely used chromatographic
III. Distance traveled by the blank solution method in drug analysis.
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 A. Column chromatography
B. Gas chromatography
C. High performance liquid
chromatography
D. Thin layer chromatography
46. A fundamental vibration of atoms in which
the distance increases or decreases along the ANSWER SHEET: Write the
bond axis: letter of your choice for each
A. Deformation number (50 points).
B. Stretching
C. Twisting 1. A 26 A
D. Wagging .
47. It refers to the ratio of the radiant power 2. E 27 A
transmitted by the solution to the radiant .
power transmitted by the blank.
A. Absorbance 3. D 28 B
B. Concentration .
C. Molal absorptivity
4. C 29 C
D. Transmittance
.
48. A spectrophotometric method of analysis
which is used in the standardization of 5. B 30 B
bacterial inoculums in microbial assays. .
A. Fluorometry
B. Nephelometry 6. A 31 D
C. Refractometry .
D. Turbidimetry
7. E 32 C
49. It is the term given to the functional group of
.
an organic molecule that absorbs maximum
radiation in UV or IR region. 8. D 33 B
A. Carbonyl .
B. Chromophore
C. Moiety 9. C 34 E
D. Nitrile .
50. What is the relationship between 10 B 35 A
absorbance and transmittance of light . .
through a sample in a spectrophotometer?
A. An increase in transmittance usually 11 B 36 C
results in an increase in absorbance. . .
B. A decrease in transmittance usually
results in a decrease in absorbance. 12 C 37 C
C. A decrease in transmittance usually . .
results in an increase in absorbance. 13 A 38 D
D. Transmittance and absorbance should . .
remain equal.
14 C 39 A
. .

15 C 40 C
. .

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16 C 41 A
. .
17 A 42 B
. .
18 C 43 A
. .
19 B 44 B
. .
20 D 45 C
. .
21 B 46 B
. .
22 B/C 47 D
. .
23 B 48 D
. .
24 D 49 B
. .
25 D 50 C
. .

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 Activity No. 4: Problem solving related to different instrumentation methods
Instruction: Read, analyze and compute for the following word problems. Write your solutions on separate sheets of letter-
sized bondpaper. Round-off your final answers to three decimal places. Box your final answer for each number.
(Nb. No need to copy the given questions).

Formulas for Spectrophotometry

Absorptivity (a) = Absorbance (A) Molar absorptivity = Absorbance (A) = Absorbance (A)
(g/L)(cm) (mol/L)(cm) (g/MW*L)(cm)

Spectrophotometry
1. A solution of c = 2.0 x 10 -5 moles/L of chlordiazepoxide dissolves in 0.1N NaOH was placed in a fused silica cell
having an optical path of 1 cm. The absorbance (A) was found to be 0.648 at a wavelength of 260 nm.
a. What is the molar absorptivity?

Given:
A
c=2 x 10−5 moles/ L ε=
bc
A=0.648
b=1 cm
0.648
ε=
2 x 10−5 moles
(
(1 cm )
L )
ε =32400

b. If the solution has A of 0.298 in a 1cm cell at 260n, what is the concentration (c)?
A
A=0.298 c=
εb
b=1 cm
0.298
c=
( 32400)(1 cm)

c=9.198 x 10−6 moles /L

2. A single tablet of colchicine is dissolved in 100mL of ethanol and its absorbance maximum of 243 nm is
measured as 0.438 in a 1cm pathlength cell. The molar absorptivity is 29200, what is the amount in mg of
colchicine in the tablet? (MW of colchicine = 399.4 g/mole)

Given:
A=0.438
b=1 cm
ε =29200
Solution:
A
c=
εb

0.438
c=
( 29200)(1 cm)
c=1.5 x 10−5 moles/ L
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 moles 399.4 g 5.991 x 10−3 g 5.991mg x
1.5 x 10
−5
x = ≈ = x=0.5991 mgColchicine
L mole L 1000 ml 100 ml

UV-Vis Spectrometry
3. Calculate the percentage of stated content of promazine HCl in promazine tablets using the data below:
a. Tablet powder containing 80 mg of promazine HCl is ground to a paste with 10 mL of 2M HCl.
b. The paste is then diluted with 200 mL of water, shaken for 15 minutes and finally made up to 500mL.
c. A portion of the extract is filtered.
d. Five mL of the filtrate is taken and diluted to 100 mL with 0.1M HCl.
e. The absorbance is read at a wavelength of 251 nm.
o A (1%, 1 cm) value of promazine HCl at 251 nm = 935
o Stated content of promazine HCl per tablet = 50 mg
o Weight of 20 tablets = 1.667 g
o Weight of tablet powder taken for assay = 0.1356 g
o Absorbance reading = 0.755
Solution:

wt . of tablet powder taken

 Expected content in tablet powder taken: x Stated content x no .of tablets
wt . of tablets
0.1356 g
x 50 x 20=81.344 mg
1.667 g

 Dilution factor: 5−100 ml=20

absorbance reading
 Concentration in diluted tablet extract: conc=
A
0.755 8.075 x 10−4 g
= =0.808 mg/100 ml
935 100 ml
 Concentration in original tablet extract: conc .∈diluted x dilution factor
0.808 x 20=16.16 mg/100 ml
 Volume of original extract: 500 ml
 Amount of promazine HCl in original extract:
16.16 mg
x 500 ml=80.8
100 ml
Amount∈original extract
 Percentage of stated content: x 100
expected content ∈tablet powder taken
80.8 mg
x 100=99.331 %
81.344 mg

High Performance Liquid Chromatography

4. Calculate the percentage content using the data obtained below upon HPLC analysis of Paracetamol 500 mg
tablet:
Weight of 20 tablets = 12.2243 g
Weight of tablet powder taken = 152.5 mg
Stated content per tablet = 500 mg
Initial extraction volume = 200 mL
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 Dilution steps: 20 to 100mL; 10 to 100mL
Area of chromatographic peak for paracetamol extracted from the tablets = 44519
Calibration curve of paracetamol: y = 35656x + 80; r = 0.999 (where x is in mg / 100 mL)

Solution:

 Substituting the area obtained for the paracetamol peak obtained from the analysis of the tablet powder extract
into the equation for the line: 44519=35656 x+ 80
 Concentration of paracetamol in diluted tablet extract:
44519−80
x= =1.246 mg/100 ml
35656
 Dilution steps:
20−100=5
10−100=10
Total = 50
 Concentration of paracetamol in undiluted tablet extract:
1.246 mg/100 ml x 50=62.3 mg /100 ml
 Amount of paracetamol in undiluted tablet extract:
200 mL (volume of undiluted tablet extract)
62.3 mg in 100 mL extract

Amount of paracetamol in 250 mL extract:

62.3 mg/100 mL=x /200 mL
(200 ml)(62.3mg)
x= =124.6 mg(amount of paracetamol found in tablet powder assayed)
100 ml

 Amount of paracetamol expected in the tablet powder taken for assay

Weight of 20 tablets: 12.2243 g
Weight of one tablet: 12.2243 g/20 = 611.215mg
Stated content per tablet: 500mg
Amount of paracetamol expected in the weight of tablet powder taken for assay
152.5mg( wt of powder taken)
x 500 mg=124.752mg
611.215 mg(wt /tab)

 Percentage of stated content

( 124.6 mg/124.752mg ) x 100=99.878 %

5. Data below were obtained upon HPLC analysis of tablets containing Naproxen 100 mg and Aspirin 250 mg per
tablet:
Weight of 20 tablets = 10.3621 g
Weight of tablet powder assayed = 257.1 mg
Volume of initial extract: 250 mL
Dilution steps: 10 to 100mL; 20 to 100 mL
Calibration curve of naproxen: y = 174040x + 579; r = 0.999 (where x is in mg / 100 mL)
Calibration curve of aspirin: y = 54285x + 1426; r = 0.999 (where x is in mg / 100 mL)
Area of peak obtained for naproxen in diluted sample extract = 72242
Area of peak obtained for aspirin in diluted sample extract = 54819
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 Calculate the percentage of stated content for both naproxen and aspirin.

5. SOLUTION

Substituting the area obtained for the naproxen and aspirin peak obtained from the analysis of the tablet powder
extract into the equation for the line:

naproxen: 72242= 174040x + 579

aspirin: 54819 = 54285x + 1426
Concentration in diluted tablet extract

Naproxen:( 72242−579 ) /174040=¿ 0.4112 mg/100 mL ¿

Aspirin: (54819−1426) /54285=0.984 mg/100 mL

Dilution steps

10mL – 100mL: 10
20mL – 100mL: 5
Total = 50

Concentration in undiluted tablet extract: (conc. diluted extract x dilution)

Naproxen: ( 0.4112 mg
100 mL )
x 50=20.6 mg/100 mL

0. 984 mg
Aspirin: (
100 mL )
x 50=49.2 mg/100 mL

Amount of naproxen and aspirin in 250 ml undiluted tablet extract

(25 0 ml)(20.6 mg)

Naproxen: x= =51.5 mg
100 ml
(250 ml)( 49.2 mg)
Aspirin: x= =123 mg
100 ml
Amount of expected in the tablet powder taken for assay

Weight of 20 tablets: 10.3621 g

Weight of one table: 10.3621 g/20 = 0.518105 g = 518.105 mg
Stated content per tablet: Naproxen 100 mg, Aspirin 250 mg
Weight of tablet powder assayed = 257.1 mg

257.1mg
Naproxen: x 100 mg=49.623mg
518.105mg
257.1mg
Aspirin: x 250 mg=124.058 mg
518.105mg
11 | P a g e
Percentage of stated content

Naproxen: ¿ ¿

Aspirin: ¿ ¿

12 | P a g e