analysis of pharmaceutical ingredients, excipients and dosage...

University of Szeged

ANALYSIS OF PHARMACEUTICAL INGREDIENTS,

EXCIPIENTS AND DOSAGE FORMS

(PRACTICAL WORKBOOK)

Edited by:

Dr. Zoltán Aigner

Dr. Piroska Szabó-Révész

Authors:

Dr. Mária Budai-Szűcs

Dr. Erzsébet Csányi

Dr. Katalin Kristó

Dr. Péter Láng

Szeged, 2015

This work is supported by the European Union, co-financed by the European Social Fund, within the framework of "Coordinated, practice-oriented, student-friendly modernization of

biomedical education in three Hungarian universities (Pécs, Debrecen, Szeged), with focus on the strengthening of international competitiveness" TÁMOP-4.1.1.C-13/1/KONV-2014-0001

project.

The curriculum can not be sold in any form!

Analysis of pharmaceutical ingredients, excipients and dosage forms 2_______________________________________________________________________________

Contents

Introduction.................................................................................................................4

I. Preformulation ............................................................................................................6

1. Determination of the dissolution rate of the active substance ....................................7 2. Determination of the spectral transmission of glass containers................................13 3. Investigation of the gel formation of Carbomera......................................................19 4. Investigation of the consistency of suppository bases – Determination of the resistance to rupture and flow point of suppositories ...............................................22

II. Investigation of dosage forms...................................................................................28

5. Determination of the drop-weight of dosage forms used dropwise ..........................29 6. Determination of the viscosity of dextran infusion and the average molecular weight of dextran .....................................................................................38 7. Investigation of emulsions ........................................................................................43 8. Investigation of suspensions .....................................................................................49 9. Investigation of ointments I. – Investigation of drop point, solidifying point, oil

number and water absorbing capacity.......................................................................56 10. Investigation of ointments II. – Investigation of the washability and rheological

characteristics of ointments.......................................................................................61 11. Investigation of ointments III. – Characterisation of consistency by viscosity,

spreadability and adhesion investigations.................................................................66 12. Investigation of the water uptake of tablets ..............................................................74 13. Investigation of tablets ..............................................................................................80 14. Investigation of the disintegration of rectal and vaginal suppositories.....................85 III. Investigation of stability ...........................................................................................88

15. Determination of the decomposition (caramelization) of glucose solution ..............89

16. Investigation of the viscosity changes of hydrophilic sols I. ...................................94 17. Investigation of the viscosity changes of hydrophilic sols II....................................99 18. Influence of humidity on the geometric parameters of tablets ...............................103 19. Investigation of the stability of tablets containing acetylsalicylic acid I. – Investigation of decomposition kinetics, calculation of shelf life using (real time) long-term stability test...........................................................................108 20. Investigation of the stability of tablets containing acetylsalicylic acid II. – Investigation of decomposition kinetics, calculation of shelf life using stress test .......................................................................................................113 IV. Biopharmaceutical investigations ...........................................................................117

21. Investigation of the drug release of emulsions by static diffusion method.............118

22. Investigation of drug release by agar plate diffusion method.................................124 23. Investigation of drug release from suppositories by dynamic diffusion method....131 24. Investigation of the mucoadhesivity of hydrogels ..................................................136


Appendix.................................................................................................................142

Helios Alpha UV-Vis spectrophotometer ...............................................................143 Anton Paar RHEOLAB MC 1 rheometer ...............................................................145


Introduction

Medicine is a special commodity, which means that besides the active pharmaceutical

ingredients (API) (equivalent terms: active substance, active ingredient, drug substance,

medicinal substance) and the excipients, the product also involves the sum of technological

processes and additional information which leads to effective therapy.

For the formulation of a medicine with the desired effect and stability, comprehensive

physical, physical-chemical and biopharmaceutical knowledge is needed, which must be

applied already during the API research and development, and later through the process of

development, licensing and production as well. It is very important for the professionals

participating in development, production and control to adopt the approach which leads to the

solution of the above problem.

As the first step of development, preformulation studies are made. During the

preformulation studies the physical-chemical properties of the API and excipients are

investigated, they are qualified from the aspect of pharmaceutical technology and the

compatibility test of the ingredients is performed. The investigation of polymorphism, the

selection of API salts, early method developments and the analysis of the effects of

technological procedures are carried out in this stage of development. Examples for

preformulation, such as determination of solubility and dissolution rate as physical-chemical

tasks; studying of the effect of salts and pH on hydrophilic sols and gels as compatibility tests,

and investigation of the consistency of suppositories are all included in this book.

After – or possibly parallel to – the preformulation studies, stability tests are started in the

formulation development. With regard to stability, we can speak of chemical, physical-

chemical, microbiological, therapeutic and toxicological stability. The book discusses the

degradation kinetics of the API in the dosage form and its interpretation; and then in view of

kinetics, the calculation of shelf life is presented by using long term and stress tests.

Parameters indicating stability need to be determined in order to establish the physical-

chemical stability of dosage forms, because international guidelines (ICH, EMA) regulating

stability tests do not specify special parameters for the description of systems, they must be

ascertained during the development. The book provides some examples for the

characterization of dosage form stability, such as phase separation rate (emulsions), half-time

(suspensions), oil number (ointments), rheological parameters (yield point, viscosity,

thixotropy), disintegration time (tablets, suppositories).


The investigation of dosage forms can be conducted on the basis of the methods of the

Pharmacopoeia (disintegration, dissolution, viscosity measurements), but in many cases there

are no specific investigations for the dosage form, and then we can opt for investigations not

listed in the Pharmacopoeia. Such investigations are also presented in this book, for example,

redispersibility of suspensions, determination of the spreadability, adhesion and washability

of ointments.

In the book, great emphasis is laid on biopharmaceutical investigations. Dissolution and

mucoadhesivity tests belong to this type of investigations. In case of dissolution and diffusion

tests, pharmacopoeial and non-pharmacopoeial investigations of emulsions, ointments and

suppositories are presented. As regards mucosal drug delivery, the adhesion of the form on

the mucosa is of key importance, because in the absence of this drug absorption is

questionable. The book presents in vitro rheological methods for the investigation of

mucoadhesion, which can indicate the in vivo adhesivity of the system.

The investigation of containers is discussed in the Pharmacopeia in great detail. This type

of measurements is represented by the light permeability measurements of glass containers.

The book summarizes the main pharmacopoeial and non-pharmacopoeial investigations.

Because of the complexity and manual needs of the tasks, the students perform them in pairs.

The execution of the tasks, the preparation of the report and the (statistical) evaluation of the

results help the students to adopt the proper approach and to acquire manual skills.

Szeged, 1st June, 2015

Dr. Mária Budai-Szűcs Dr. Piroska Szabó-Révész Dr. Zoltán Aigner


I. Preformulation


1. Determination of the dissolution rate of the active substance

Introduction

The development of the effect of an active substance depends on several factors. One of

the most important of these is dissolution rate, which determines what quantity of the

pharmaceutical product is dissolved during its residence time in the gastrointestinal tract. The

bioavailability of the active substance may be impaired greatly by its possibly slow

dissolution rate. During the practice, we are going to examine the time course of the

dissolution of active substances, which can provide important data for formulation.

The rate of dissolution is the amount of the dissolved material (dw) that diffuses on surface

(A), perpendicularly to the surface, through layer thickness (d(x)), at constant temperature, as

a result of concentration change (d(c)) in time dt:

d(x)d(c)AD-=

dtdw

⋅⋅ (1)

where D is the diffusion constant (Fick’s law).

The dimension of the dissolution rate constant is: mg · cm-2 ·hour-1.

According to the Noyes-Whitney equation, the dissolution rate constant can be determined

with the following equation in the case of first order kinetics:

][][

][log303.2CC

Ct

ks

s

−= (2)

where t is the time, C is the current concentration of the solute, Cs is the saturation

concentration of the solute (e.g. at room temperature 0.50 g of theobromine dissolves in 1000

mL of distilled water).

The rate of dissolution depends on the following factors:

Test factors:

– mixing intensity, mixer type, geometric factors;

– flow conditions;

– concentration gradient;

– material quality of the solvent;

– temperature of the solvent, etc.

Physical-chemical factors:

– amorphous or crystalline state of the active substance;


– polymorphism;

– particle size;

– complex formation, eutectic formation, solid solution;

– chemical structure;

– presence of surfactants.

In practice, the rate of dissolution is usually tested by measuring the concentration increase

in the solution surrounding the solid material, or more rarely by determining the mass

decrease of the solid material not yet dissolved. There are several methods in literature for

determining the rate of dissolution, but it is important to distinguish between the dissolution

rate of the pure active substance and the rate of release of the active substance from the

dosage form. The specific properties of the active substance are determined in the first case,

while in the second case the quantity of the active substance released from the dosage form is

measured.

Task

1. During the practice, you are going to examine the dissolution rate of a specific quantity

of theobromine with the rotating paddle apparatus described in the Hungarian

Pharmacopoeia. Determine the active substance concentration of the samples taken at

the given times spectrophotometrically. Include the obtained results in a table.

Apparatus

Several methods and apparatuses have been developed for testing the rate of dissolution,

and the two most common ones, the rotating basket and the rotating paddle procedures are

official both in the American and the Hungarian Pharmacopoeias (Figure 1.1) (Ph. Hg. VIII.

2.9.3.).


Investigation method

Measure theobromine samples for three parallel examinations. 0.2500 g of theobromine

measured with analytical precision is used during the examination. A powder form is tested, it

is either spread on the top of the dissolution medium or filled in a capsule.

Perform three parallel examinations in the paddle dissolution apparatus with the

observation of the given parameters. Start your work by heating up the dissolution apparatus,

measuring and warming the dissolution medium. Measure 900 mL of dissolution liquid

(distilled water) into the vessel of the apparatus and set the temperature to 37 ± 0.5 °C with

the thermostat. The rotational speed of the paddle should be 100 rotations/minute, and

samples should be taken 5, 10, 20, 30, 60 and 90 minutes after the start of the examination.

Take 10 mL of sample with a pipette and filter it on a paper filter. Sampling should be made

at half height of the vessel, from the middle between the shaft of the paddle and the wall of

the dissolution vessel. Do not supplement the volume of the samples taken.

Figure 1.1: Basket and paddle dissolution apparatus

Prepare appropriate dilutions from the filtered samples in volumetric flasks in order to

obtain a measurable extinction (0.3 – 0.8 A) and measure their light absorption with a

spectrophotometer. Perform the measurements at a wavelength of λ = 273 nm, 0.1 A = 1.6529

µg/mL.

Analysis of pharmaceutical ingredients, excipients and dosage forms 10 _______________________________________________________________________________

2. Calculate the dissolution rate constant of theobromine (k).

3. Plot the average of c* against sampling time.

4. Write a short evaluation of the results of the test.


Report 1. Determination of the dissolution rate of the active substance

Name: ..................................................... Group: ....................... Date: ............................

1. During the practice, you are going to examine the dissolution rate of a specific quantity

of theobromine with the rotating paddle apparatus described in the Hungarian

Pharmacopoeia. Determine the active substance concentration of the samples taken at

the given times spectrophotometrically. Include the obtained results in a table. (c* is the

amount of dissolved theobromine expressed as the percentage of the measured amount.)

2. Calculate the dissolution rate constant of theobromine (k).

Measurement of theobromine Test 1 Test 2 Test 3

_________ with material (g)

_________ empty (g)

Measurement (g)

Results of the dissolution test of theobromine Measurement Time (minute) 5 10 20 30 60 90

A

dilution

c (µg/mL)

dissolved theobromine (mg)

1.

c*

A

dilution

c (µg/mL)


2.

c*

A

dilution

c (µg/mL)


3.

c*

c* average

k


3. Plot the average of c* against sampling time.

4. Write a short evaluation of the results of the test.


2. Determination of the spectral transmission of glass containers

Introduction

Glass containers are most commonly used for storing and dispensing liquid dosage forms.

A great number of APIs, excipients and dosage forms are light-sensitive, that is why they

must be dispensed and stored in glass containers protecting against light, which are tested

according to the Pharmacopeia.

According to Ph. Hg. VIII., glass containers for pharmaceutical use are glass articles

intended to come into direct contact with pharmaceutical preparations. Coloured and

colourless glass can be distinguished. Colourless glass is highly transparent in the visible

spectrum. Coloured glass is obtained by the addition of small amounts of metal oxide(s),

chosen according to the desired spectral absorbance.

The Hungarian Pharmacopeia (Ph. Hg. VIII., 3.2.1.) makes the following

recommendations about glass containers: Preparations for parenteral administration are

normally presented in colourless glass, but coloured glass may be used for substances known

to be light-sensitive. Colourless or coloured glass is used for the other pharmaceutical

preparations. It is recommended that all glass containers for liquid preparations and for

powders for parenteral administration permit the visual inspection of the contents.

Concerning glass containers, the Hungarian Pharmacopeia prescribes physical and

chemical investigations.

Physical investigations:

– determination of filling volume – spectral transmission for coloured glass containers

Chemical investigations:

– investigation of hydrolytic resistance A. hydrolytic resistance of the inner surface of glass containers (surface test) B. hydrolytic resistance of glass grains (glass grains test) C. to determine whether the containers have been surface-treated (etching test) D. surface hydrolytic resistance - determination by flame atomic absorption

spectrometry (faas) – investigation for arsenic compounds


Task

Break the glass container or cut it. Select sections representative of the wall thickness and

trim them as suitable for mounting in a spectrophotometer. Make sure the length of the

specimen is greater than that of the slit. Before placing in the holder, wash, dry and wipe the

specimen with lens tissue. Take care to avoid leaving fingerprints or other marks.

Apparatus: UV-VIS spectrophotometer, in transmittance mode

Place the specimen in the spectrophotometer in such a way that the light beam is

perpendicular to the surface of the section (Figure 2.1). Measure the transmission

(transmittance, T) of the specimen with reference to air in the spectral region of 290-450 nm,

at intervals of 20 nm.

Figure 2.1: Placing of the specimen into the cuvette holder of the spectrophotometer


Limits (Ph. Hg. VIII.; 3.2.1.)

The observed spectral transmission for coloured glass containers for preparations that are

not for parenteral use does not exceed 10 per cent at any wavelength in the range of 290 nm to

450 nm, irrespective of the type and the capacity of the glass container. The observed spectral

transmission in coloured glass containers for parenteral preparations does not exceed the

limits given in the Table below.

Limits of spectral transmission for coloured glass containers for parenteral preparations

(Ph. Hg. VIII, Table 3.2.1.-5.)

Maximum percentage of spectral transmission at any wavelength between

290 nm and 450 nm Filling volume (mL) Flame-sealed

containers Containers with

closures Up to 1 50 25 Above 1 and up to 2 45 20 Above 2 and up to 5 40 15 Above 5 and up to 10 35 13 Above 10 and up to 20 30 12 Above 20 25 10

1. Measure each specimen in 4 different positions, in this way you will get 4 values for

each sample. Calculate the average of 4 measurements, and plot the data against the

wavelength. Evaluate the results according to the pharmacopoeial limits.

2. Correlate the different glass samples in such a way that their transmission refers to a

wall thickness of 1 mm, by using the following equation:

''%

1

''%

'%

% lglglglg Tll

TTT +⋅−= (1)

where T% is the light permeability of the minimum thickness of the glass container in %

transmittance, T’% is the maximum % light permeability measured in the investigated

wavelength range, T”% is the theoretical light permeability (92%) of the glass container

of a wall thickness of 0 mm (lg T’’% = 1.9638), l1 is the average thickness of the glass

sample in mm, l = 1 mm.

Measure the thickness of the glass samples at 10 points of the specimen with a

micrometer screw. Calculate the average values of wall thickness.


Report 2. Determination of the spectral transmission of glass containers

Name: ..................................................... Group: ....................... Date: ............................

1. Measure each specimen in 4 different positions, in this way you will get 4 values for

each sample. Calculate the average of 4 measurements, and plot the data against the

wavelength. Evaluate the results according to the pharmacopoeial limits.

Glass sample (code, characteristic):

Sample 1: .................................................................

Sample 2: .................................................................

Sample 3: .................................................................

Sample 4: .................................................................

Sample 1 Sample 2 λ

(nm) 1 2 3 4 average 1 2 3 4 average

290 310 330 350 370 390 410 430 450

Sample 3 Sample 4 λ(nm) 1 2 3 4 average 1 2 3 4 average

290 310 330 350 370 390 410 430 450


Evaluation:


2. Measure the thickness of the glass samples at 10 points of the specimen with a

micrometer screw. Calculate the average values of wall thickness.

Thickness (mm) Measurement

Sample 1 Sample 2 Sample 3 Sample 4

1

2

3

4

5

6

7

8

9

10

average

Transmittances referring to a thickness of 1 mm:

Sample 1, T% = ..................................................

Sample 2, T% = ..................................................

Sample 3, T% = ..................................................

Sample 4, T% = ..................................................

Evaluation:


3. Investigation of the gel formation of Carbomera

Introduction

Carbomers are high molecular mass polymers of acrylic acid cross-linked with polyalkenyl

ethers of sugars or polyalcohols (Ph. Hg. VIII. 04/2009:1299).

A wide variety of Carbomera product types are used in the field of pharmaceutical

industry. Carbomers are excellent viscosity increasing excipients, and they are used as

transparent aqueous or hydroalcoholic gel forming agents. Their favourable properties are

mostly due to the carboxylic groups of acrylic acid. Various degrees of neutralization are

needed for gelation in an aqueous phase. The aim of the present work is to investigate the

neutralization effect on gel formation.

The aim of the practice is the investigation of the gel formation of Carbomers depending

on pH and the determination of viscosity at different pH values.

Task

1. Prepare 50.0 g of 0.3 % Cabomera containing system. The polymer should be poured

onto the surface of the previously measured distilled water while stirring continuously.

Measure the pH of the aqueous suspension, then the viscosity at an increasing shear rate

(0.1 – 100 1/s) at 12 points.

2. Prepare again 50.0 g of 0.3 % Cabomera containing system. The polymer should be

poured onto the surface of the previously measured 40 g of distilled water while stirring

continuously. Start neutralization up to pH = 5 by adding 0.1 N NaOH in parts. After

reaching the desired pH, add the remaining distilled water up to 50.0 g and stir

vigorously to homogenize it. Measure the viscosity at an increasing shear rate (0.1 –

100 1/s) at 12 points.

3. Prepare again Carbomera containing systems according to point 2. Adjust the pH up to

pH = 6; 7; 8; 9 and 10 by adding 0.1 N of NaOH in parts. Measure the viscosity at an

increasing shear rate (0.1 – 100 1/s) at 12 points.


Report 3. Investigation of the gel formation of Carbomera

Name: ..................................................... Group: ....................... Date: ............................

1. Prepare 50.0 g of 0.3 % Cabomera containing system. The polymer should be poured

onto the surface of the previously measured distilled water while stirring continuously.

Measure the pH of the aqueous suspension, then the viscosity at an increasing shear rate

(0.1 – 100 1/s) at 12 points.

2. Prepare again 50.0 g of 0.3 % Cabomera containing system. The polymer should be

poured onto the surface of the previously measured 40 g of distilled water while stirring

continuously. Start neutralization up to pH = 5 by adding 0.1 N of NaOH in parts. After

reaching the desired pH, add the remaining distilled water up to 50.0 g and stir

vigorously to homogenize it. Measure the viscosity at an increasing shear rate (0.1 –

100 1/s) at 12 points.

3. Prepare again Carbomera containing systems according to point 2. Adjust the pH up to

pH = 6; 7; 8; 9 and 10 by adding 0.1 N of NaOH in parts. Measure the viscosity at an

increasing shear rate (0.1 – 100 1/s) at 12 points. Include the obtained results in a table. pH without

adjustment

5 6 7 8 9 10

measured

pH

D(1/s)

α η(Pa·s)

α η(Pa·s)

α η(Pa·s)

α η(Pa·s)

α η(Pa·s)

α η(Pa·s)

α η(Pa·s)

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13.


4. Plot the viscosity values measured at the highest shear rate against pH.

Give the minimum pH value needed for gel formation. Use the curve-fitting method.

pH = .......................................................................

Evaluation:


4. Investigation of the consistency of suppository bases –

Determination of the resistance to rupture and flow point of

suppositories

Introduction

The investigation of the consistency and hardness of suppositories is important with

respect to applicability. This investigation gives useful information on how the suppository

can retain its shape during packaging, storage and shipping. It is also important concerning

administration, as suppositories with not enough hardness cannot be administered easily,

while the ones that are too hard can cause pain.

Task

1. Determination of resistance to rupture

Apparatus: Apparatus for the determination of the resistance to rupture of suppositories

This test determines, under defined conditions, the resistance to rupture of suppositories

measured by the mass needed to rupture them by crushing (Ph. Hg. VIII. 2.9.24.) (Figure 4.1)


Check that the apparatus is vertical. Heat the thermostatted chamber to 25 °C. The dosage

form to be tested has been maintained for at least 24 h at the required measuring temperature.

Place the suppository vertically between the jaws in the sample holder with the point

upwards. The top pressure block of the suspension loading rod is carefully positioned and the

test chamber is closed with its glass window; for each determination, position the suppository

in the same manner with respect to the direction of the force applied. Wait for 1 min and add

the first 200 g disc. Again wait for 1 min and add another disc. Repeat the operation until the

suppository collapses. The mass required to crush the suppository is calculated by the sum of

the masses weighing on the suppository when it collapses [including the initial mass of the

device (600 g)] assessed as follows:


– if the suppository collapses within 20 s of placing the last disc, do not take this mass into

account,

– if the suppository collapses between 20 s and 40 s of placing the last disc, use only half of

this mass in the calculation, i.e. 100 g,

– if the suppository remains uncrushed for more than 40 s after the last disc is placed, use all

the mass in the calculation.

Figure 4.1: Apparatus for the determination of the resistance to rupture of suppositories

Carry out each measurement on five suppositories, making sure that no residue remains

before each determination (Ph. Eur.). Fill the attached table.

2. When the measurement is finished, prepare the different types of suppositories of the

composition given by your practice leader. Calculate 3.0 of base per suppository

together with excess. Use the moulding technique and place the finished suppositories

into boxes.


Compositions of series:

Series A:

1. Adeps solidus compositus 2. Adeps solidus compositus + 30 % Talcum 3. Adeps solidus compositus + 10 % Paraffinum liquidum 4. Adeps solidus compositus + 15 % Paraffinum liquidum + 30 % Talcum

Series B:

1. Adeps solidus 2. Adeps solidus + 30 % Talcum 3. Adeps solidus + 10 % Oleylum oleinicum 4. Adeps solidus + 15 % Oleylum oleinicum + 30 % Talcum

3. Determination of consistency by penetrometry

Apparatus: penetrometer (Ph. Hg. VIII. 2.9.9.) (Figure 4.2)


Determination of the consistency of test samples prepared from suppository bases with and

without excipients by measuring the flow point of suppositories. We measure the penetration

depth of the penetrating object into the test sample prepared from the series to be investigated.

The flow point ( pF (N/cm2)) is calculated from the weight of penetrating object (G (N)) and

the depth of penetration (cm):

π⋅

= 2

4s

GFp (1)

Place the test sample on the base of the penetrometer. Verify that its surface is

perpendicular to the vertical axis of the penetrating object. Bring the temperature of the

penetrating object to 25 ± 0.5 °C and then adjust its position such that its tip just touches the

object and hold it free for 5 s. Set the zero reading, release the penetrating object and measure

the depth of penetration in mm scale. Carry out 5 parallel measurements with each sample,

include the results in the table and evaluate them.


Figure 4.2: Penetrometer

4. When the measurements are finished, prepare the test samples of the composition given

by your practice leader in a small Petri dish. (Calculate with 30.0 g of base per test

sample.)

Composition of series:

Series A:

1. Adeps solidus compositus 2. Adeps solidus compositus + 30 % Talcum 3. Adeps solidus compositus + 10 % Paraffinum liquidum 4. Adeps solidus compositus + 15 % Paraffinum liquidum + 30 % Talcum

Series B:

1. Adeps solidus 2. Adeps solidus + 30 % Talcum 3. Adeps solidus + 10 % Oleylum oleinicum 4. Adeps solidus + 15 % Oleylum oleinicum + 30 % Talcum


Report 4. Investigation of the consistency of suppository bases – Determination of the resistance

to rupture and flow point of suppositories

Name: ..................................................... Group: ....................... Date: ............................

1. Determination of resistance to rupture

Letter symbol of series: ..........................................................

Measurement

1

Measurement

2

Measurement

3

Measurement

4

Measurement

5

Average Mark

of

supp. Mass

(g)

F

(N)

Mass

(g)

F

(N)

Mass

(g)

F

(N)

Mass

(g)

F

(N)

Mass

(g)

F

(N)

Mass

(g)

F

(N)

......./

1

......./

2

......./

3

......./

4

2. Determination of the flow point of suppository

Mark of

supp. ....... / 1 ....... / 2 ....... / 3 ....... / 4

Measurement Pit

(cm)

Fp

(N/cm2)

Pit

(cm)

Fp

(N/cm2)

Pit

(cm)

Fp

(N/cm2)

Pit

(cm)

Fp

(N/cm2)

1.

2.

3.

4.

5.

average


3. Evaluation:


II. Investigation of dosage forms


5. Determination of the drop weight of dosage forms used

dropwise

Introduction

Numerous dosage forms administered in drop form are known. Ph. Hg. VIII. lists the

following in this field:

– Liquid preparations for oral use: oral drops

– Ear preparations: ear drops

– Nasal preparations: nasal drops

– Oromucosal preparations: oromucosal drops

– Eye preparations: eye drops

Drops are solutions, emulsions or suspensions that are administered in small volumes such

as drops by the means of a suitable device.

Ph. Hg. VIII. prescribes dosage form investigations, such as dose and uniformity of dose,

only for oral drops in the monograph (Ph. Hg. VIII., p. 2985). In the case of other types of

drops, there is no prescribed investigation related to the drop form.

The weight of the drop can be modified by the excipients in the formulation. The weight of

drops (m) dropped out from the dropping tube depend on the external diameter of the

dropping tube (d) and the surface tension of the liquid (γ) (Tate 1864):

g

d=m xγπ ⋅⋅ (1)

where m is the drop weight in g, d is the diameter of the dropping tube in cm, π = 3.14, g =

981 cm·s-2, γx is the surface tension (mN/m).

Besides the above factors, the drop weight also depends on the property of the dropping

tube, the speed of dropping, the way the dropping tube is held and the force between the

surface of the dropping tube and the liquid dropping out (e.g. adhesion, wetting).

When checking the dose of drops, the drop number and the drop weight also have to be

taken into consideration. The drop number shows how many drops can be prepared from 1 g

of solution with a standard dropping tube (Figure 5.1).


Both the drop weight and the drop number change with changing surface tension.

Figure 5.1: Standard dropping tube


The aim of the investigation is to examine the changes of the drop weight of the dosage

form used dropwise as a result of the changing of the surface tension with different excipients.

There is proportionality between the drop weight and the surface tension of the purified

water and the solution containing different excipients:

γγ

x

v

x

v =mm (2)


where mv is the drop weight of purified water (g), mx is the drop weight of the examined

solution (g), γv is the surface tension of purified water (Aqua purificata) (mN/m), γx is the

surface tension of the examined solution (mN/m).

There is inverse proportionality between the drop number prepared from 1 g of solution

and the surface tension. The surface tension of purified water is known (73 mN/m at 20 °C),

mv and mx can be determined experimentally, so γx can be calculated:

73mm=

v

xx ⋅γ (3)

Tasks

1. Prepare two of the solution series of the following composition, chosen by your practice

leader.

Components A/1 A/2 A/3 A/4 A/5 B/1 B/2 B/3 B/4 B/5

Alcoholum 96% (g) 1.5 3.0 4.5 6.0 7.5 – – – – –

Mucilago methylcellulosi (g) - - - - - 0.75 1.5 2.25 3.0 3.75

Aqua purificata (ad g) 15 15 15 15 15 15 15 15 15 15

Components C/1 C/2 C/3 C/4 C/5 D/1 D/2 D/3 D/4 D/5

Polysorbatum 20 (g) 0.15 0.45 0.75 1.05 1.35 – – – – –

Tinctura aromatica (g) – – – – – 1 2 3 4 5

Aqua purificata (ad g) 15 15 15 15 15 15 15 15 15 15

2. Determine the drop number of the prepared solutions and the purified water with the

given dropping tube. Make 10 parallel measurements.

3. Fill in the tables. Calculate the values of γx. Evaluate the results statistically. (Calculate

standard deviation, relative standard deviation according to the equations in Table

2.9.40-2 of Ph. Hg. VIII.)


4. Plot the measured drop weight values against the concentration of the examined

excipients on a graph paper. How does the surface tension of the solutions change with

the concentration of the excipients?

5. If 15 g of excipient-free aqueous solution contains 0.30 g of codeine hydrochloride

dihydrate, how is the codeine hydrochloride dihydrate content in one drop of solution

changed by adding the excipients in different concentrations? Fill in the table. Evaluate

the data.


Report 5. Determination of the drop weight of dosage forms used dropwise

Name: ..................................................... Group: ....................... Date: ............................

1. Fill in the tables. Calculate the values of γx. Evaluate the results statistically. (Calculate

standard deviation, relative standard deviation.)

Name of the excipient used: .....................................................

Measurements

Drop

number of

water

Drop

number of

solution

..........

Drop

number of

solution

..........

Drop

number of

solution

..........

Drop

number of

solution

..........

Drop

number of

solution

..........

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. average

stand. dev. rel. stand. dev.

drop weight (cg) γ (mN/m)



Measurements

Drop

number of

solution

..........

Drop

number of

solution

..........

Drop

number of

solution

..........

Drop

number of

solution

..........

Drop

number of

solution

..........

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. average

stand. dev. rel. stand. dev.

drop weight (cg) γ (mN/m)


2. Plot the measured drop weight values against the concentration of the examined

excipients on a graph paper. How does the surface tension of the solutions change with

the concentration of the excipients?




3. Fill in the table corresponding to the composition series chosen by your practice leader.

Evaluate the results.

Alcoholum 96%

(m/m %)

Amount of codeine

hydrochloride dihydrate in 1

drop (mg)

Mucilago methylcellulosi

(m/m %)

Amount of codeine


drop (mg)

10 5

20 10

30 15

40 20

50 25


Polysorbate 20

(m/m %)

Amount of codeine


drop (mg)

Tinctura aromatica

(m/m %)

Amount of codeine


drop (mg)

1 6.6

3 13.3

5 20.0

7 26.7

9 33.3

Evaluation:


6. Determination of the viscosity of dextran infusion and the

average molecular weight of dextran

Introduction

On the basis of their origin or synthesis, macromolecular compounds can be natural (e.g.

starch, gelatine, etc.), semisynthetic (e.g. dextran, cellulose derivatives, etc.) or synthetic (e.g.

polyvinyl-alcohol, polyethylene glycol, etc.). Spherical and linear macromolecules can be

distinguished according to the shape of the macromolecules. The original structure and

concentration of the dissolved macromolecular material have a profound effect on the

properties of the formed colloidal solution, among which flow behavior is very important.

Threadlike macromolecules are inordinately located in all directions of the space or they may

adhere to each other, which is determinative in terms of viscosity.

Dextran is produced by the fermentation of sucrose using the bacterial strain and substrains

of Leuconostoc mesenteroides. In the 8th Edition of the Hungarian Pharmacopoeia 4 dextrans

with different average molecular weight are official. For medical purposes, the fraction

having a molecular weight of 30,000 – 80,000 is the most advantageous, because it has the

least harmful side effect. One of the requirements for dextran used as a plasma substitute is

that its viscosity should be constant and approximately identical to the viscosity of the blood

plasma, the other is that its molecular weight should not change during storage and

sterilization.

The average molecular weight of dextran can be determined with simple physical

investigations by using the dextran solution.

Task

Determination of the density and dynamic viscosity of dextran solution. Calculation of the

average molecular weight of dextran from the results by using a formula.


1. Determination of density

Apparatus: pycnometer

For the determination of the density of the liquid, use a pycnometer with a volume of 10.00

mL. Measure the weight of the empty and dry pycnometer on an analytical balance, then fill it

up with the test liquid of ~20 °C until the meniscus of the liquid is under the circular mark.

Then put the pycnometer into a water bath of 20 ± 0.1 °C. After 10 minutes, fill up the

pycnometer in the water bath with a pipette so that the lowest point of the meniscus touches

the circular mark. If necessary, dry the neck of the pycnometer inside with a filter paper and

close it with its plug.

After taking out the pycnometer, dry it carefully, place into the balance box for 10 minutes,

then measure its weight. The difference between the weight of the empty pycnometer and that

of the liquid-filled one gives the weight of the liquid enough to fill the flask (mf).

After that, empty the pycnometer and wash it carefully with water of ~20 °C; then fill it up

with water in the way described above and calculate the weight of water (mv).

From the quotient (mf/mv) of the weight of liquid (mf) and water (mv), measured in the same

way, the density at 20 °C (ρ20 °C) can be calculated with the following formula:

0012.0997003.020 +⋅=°v

fC m

mρ (1)

Give the calculated value of density rounded to 3 decimal places.

2. Determination of viscosity

Apparatus: Ostwald-Fenske capillary viscometer No 5 (Figure 6.1)

The Hungarian Pharmacopoeia (Ph. Hg. VIII., 2.2.9.) prescribes the following for the

determination of viscosity using a capillary viscometer:

– the determination of viscosity using a capillary viscometer is carried out at a

temperature of 20 ± 0.1 °C, unless otherwise prescribed;

– the time required for the level of the liquid to drop from one mark to the other is

measured with a stop-watch to the nearest one-fifth of a second;

– the result is valid only if two consecutive readings do not differ by more than 1 %;

– the average of not fewer than three readings gives the flow time of the liquid to be

examined.


Figure 6.1: Capillary viscometer

The dextran infusion is diluted with isotonic sodium chloride solution up to 0.6 %. Then,

by using the Ostwald-Fenske capillary viscometer No. 5, the viscosity of the diluted infusion

is measured at 20 ± 0.1 °C.

For the determination of viscosity, the flow time of the defined volume of the liquid to be

examined in the capillary of a given length and diameter is measured. A certified capillary

viscometer should be used for the measurements. The capillary viscometer selected according

to the viscosity of the sample to be examined or its solution of the required concentration

should be placed into a water bath at constant (± 0.1 °C) temperature. The size of the water

bath should be sufficient for the examined sample in the capillary viscometer to be at least 20

mm under the surface of the water bath and also at least 20 mm from the bottom during the

entire examination.

Water should be used for filling the thermostat. During the measurement, the middle of the

thermometer's mercury reservoir should be at the same height as the middle of the capillary.

The flow time is measured with a stop-watch.

The bubble-free liquid to be examined is filled into the viscometer that has been degreased,

washed and dried carefully. About three-quarters of the spherical reservoir on the thick pipe


should be filled with the liquid to be examined. The viscometer is placed into the bath in such

a way that the stems ending in the gap are upright. The measurement can be started only if the

sample to be examined has taken over the temperature of the bath (approximately 10

minutes). After this time, the liquid to be examined has to be absorbed through the rubber

tube over the circular mark between the two balls above the capillary, then the absorption has

to be finished. The stop-watch should be started if the surface of the liquid sinking reaches the

circular mark between the two balls and it should be stopped if it reaches the circular mark

above the capillary. The time measured in this way is the flow time (t). The flow time should

be measured at least 5 times, and for the calculation of the result only the flow times which do

not show any one-way differences and differ not more than ± 0.5 % from each other should be

considered. The dynamic viscosity of the examined sample can be calculated by using the

following formula:

tk CC ⋅⋅= °° 2020 ρη (2)

where η20°C is the dynamic viscosity in mPa·s at 20.0 °C, k is the constant of the instrument at

20.0 °C (according to the certification, it is: 8.65 °C·10-4 mm2/s2), t is the mean value of the

flow time in seconds (s), ρ20°C is the density of the sample to be examined at 20.0 °C in g/mL

(determined in an earlier examination).

Based on the results obtained, the average molecular weight can be calculated by using the

following formula:

awMk ⋅=η (3)

where η is the dynamic viscosity of the solution, k is a constant (9.78·10-4), a is the index

(0.5), Mw is the average molecular weight.


Report 6. Determination of the viscosity of dextran infusion and the average molecular weight

of dextran

Name: ..................................................... Group: ....................... Date: ............................

1. Prepare 50 mL of 0.9 % sodium chloride solution. How much NaCl should be used for

the preparation? .................................... g

2. Determine the density of the dextran infusion by using a pycnometer according to the

Hungarian Pharmacopoeia, with three parallel measurements, and fill in the table.

Number of pycnometer Weight of empty pycnometer (g) Weight of pycnometer filled with water (g) Weight of pycnometer filled with dextran infusion (g) Weight of water (g) Weight of dextran infusion (g) Density (g/cm3)Average of density (rounded to three decimal places)

3. Determine the dynamic viscosity of the diluted dextran infusion with the help of a

modified Ostwald-Fenske capillary viscometer according to the Hungarian

Pharmacopoeia, with 5 parallel measurements. Include the results in the table below:

Number of measurement Flow time (s) Average of flow time (s) Viscosity (mPa·s)

1. 2. 3. 4. 5.

4. Calculate the average molecular weight of the dextran infusion according to the formula

given. The average molecular weight of dextran: ............................................

Evaluation:


7. Investigation of emulsions

Introduction

Emulsions are thermodynamically unstable systems. The reason for this is the large

specific surface area of the particles. The emulsified state lasts until the layers separating the

liquid drops retain their structure. Thus the constancy of the degree of dispersion is one of the

most important aspects concerning the stability of emulsions, because depending on the drop

size distribution – depending on time – they may float. The drop size is increasing with the

confluence of the floated or flocculated drops (coalescence); this leads to the breaking of the

emulsion, then the dispersed part begins to form a continuous phase, therefore a

thermodynamically favourable condition results. From the point of view of pharmaceutical

production, an emulsion is regarded stable if its degree of dispersion remains unchanged or

changes only within the prescribed limits until use.

During the investigation of the stability of emulsions, the rheological properties, which are

determined by the following factors, are also of great significance: the dispersed phase, the

viscosity of the dispersion medium, the amount and the material quality of the emulsifier

used. If the volumetric concentration of the dispersed phase is small enough, the system

behaves as a Newtonian or ideal viscous fluid. The yield curve of ideal viscous fluids is

characterized by a baseline starting from the origin, and they give a straight line. The flow

becomes pseudoplastic if the volumetric concentration is increased, as a result of which the

resistance of the system to flow increases. The yield curves of such materials also start from

the origin, but they are not linear. Viscosity changes depending on shear stress. The reason for

this is the instantaneous and reversible disaggregation or deformity of the emulsified drops

caused by shear force. The aim of this practice is the investigation of the characteristic

properties of emulsions in case of changing the factors influencing stability.

Task

1. The determination of the yield properties and viscosity of emulsions

The Hungarian Pharmacopoeia (Ph. Hg. VIII., 2.2.10.) lists the following types of

instruments for the determination of viscosity by using a rotating viscometer: spindle


viscometers, cone-plate viscometers and concentric cylinder viscometers. During the practice,

an Anton Paar Rheolab MC 1 Rheometer is used, which is a concentric cylinder type

viscometer. The fluid to be examined has to be located in the gap between the inner and outer

cylinders. Viscosity can be measured by rotating the inner cylinder ("Searle" type viscometer)

or by rotating the outer cylinder ("Couette" type viscometer).

Apparatus: Anton Paar Rheolab MC 1 Rheometer


Sample holder "Z3" (Figure 7.1) is filled hermetically with the emulsion to be examined up

to the inner mark. The cylindrical probe is affixed with the help of the retaining ring. The

device is installed according to the instructions of the manual and the required program is

started.

Figure 7.1: Sample holder "Z3" of the Anton Paar Rheolab MC 1 Rheometer

2. Investigation of spontaneous separation (not an official investigation method in Ph. Hg.

VIII.)

Apparatus: 25.0-mL measuring cylinder with a ground stopper



25.0 mL of emulsion is filled into a measuring cylinder with a ground stopper. The sample

is shaken a few times. The volume of the water separated and collected in the bottom of the

measuring cylinder is determined after a certain time (one and a half hours).

Calculation of the speed of separation:

V-VV+V

t1=v

t

t

0

0log (1)

where v is the speed of separation (mL/hour), t is the time of observation (hour), Vt is the

volume of water separated during t time (mL), V0 is the original volume of the emulsion (25

mL).

Compositions to be examined:

Materials I. II. III. IV. V.

Paraffinum liquidum 10.0 g 30.0 g 60.0 g 30.0 g 30.0 g

Aqua purificata 68.0 g 48.0 g 8.0 g 47.0 g 49.5 g

Polysorbatum 20 2.0 g 2.0 g 2.0 g 3.0 g 0.5 g

Mucilago methylcellulosi 20.0 g 20.0 g 20.0 g 20.0 g 20.0 g

Apparatus: Mixer (stage 1, 1 min of mixing time)


Report 7. Investigation of emulsions

Name: ..................................................... Group: ....................... Date: ............................

1. The effect of the weight ratio of the dispersed phase on the properties of the emulsion:

Composition Speed of separation Viscosity (D = 656 1/s)

II. (10 %)

I. (30 %)

III. (60 %)

Evaluation:

2. The effect of the concentration of emulsifiers on the properties of the emulsion:

Composition Speed of separation Viscosity (D = 656 1/s)

V. (0.5 %)

I. (2 %)

IV. (3 %)

Evaluation:


3. Rheological characterization of the compositions tested (determination of flow type):

I. II. III. IV. V.

D

(1/s)

α τ

(N/m2)

D

(1/s)

α τ

(N/m2)

D

(1/s)

α τ

(N/m2)

D

(1/s)

α τ

(N/m2)

D

(1/s)

α τ

(N/m2)


4. Plot and evaluate the yield curves of the compositions.

Evaluation:


8. Investigation of suspensions

Introduction

The two most important requirements to be met by suspensions are: the homogeneous

distribution of the active ingredient at least for the period between the mixing of the

suspension and the pouring of given dose, and the easy redispersibility of the sediment

formed during storage. Sedimentation cannot be prevented, but it can be delayed with various

technological methods. One possibility for this is to reduce the size of the non-dissolving

particles, or to enhance the viscosity of the dispersion medium with various macromolecular

materials (e.g. mucilages). With respect to the sediment formed, suspensions can be

flocculated or deflocculated. Flocculation occurs if the macromolecules adsorbed on the solid

particles are cross-linked, or if the surface parts of the particles not covered with

macromolecules stick together due to the high adhesion. The sediment of flocculated

suspensions is loose and bulky. Its advantage is that the floccules (grain aggregates) form a

sediment which is easily redispersible, but sedimentation is fast in this case. In contrast, the

sediment of deflocculated suspensions consists of particles in a close packed arrangement (so-

called "cemented sediment"), therefore it is difficult to shake, but its sedimentation is slower.

The sedimentation of suspensions can be free or hindered (see Figures 8.1 and 8.2).

Sedimentation is free if the suspension is sufficiently washy, in which the particles do not

hinder each other during sedimentation and a sediment with a gradually increasing volume is

formed, where the supernatant is cloudy because of the floating particles. Free sedimentation

is characterized by a high half-life and a low sedimentation rate. However, hindered

sedimentation is more typical of pharmaceutical suspensions. In this case, the particles hinder

each other during sedimentation. The reason for this is the higher concentration of the

particles in the given volume and the interactions between them, and also the presence of the

viscosity increasing excipient. Hindered sedimentation is characterized by a clear supernatant

and a gradually decreasing phase boundary between the sedimenting particles. The aim of this

practice is to determine the half-life (t1/2) of flocculated and deflocculated suspensions, and to

investigate the resuspensibility of the sediment.


Figure 8.1: Hindered settling suspension

Figure 8.2: Free settling suspension

Task

1. Investigation of the sedimentation of suspensions (not an official investigation method

in Ph. Hg. VIII.)

Apparatus: 25.0-mL measuring cylinder with a ground stopper



Pour the homogenous suspension into a measuring cylinder with a ground stopper and

shake it well. Observe the separation of the dispersed part and the dispersion medium. Note

the height of the sediment column in every 5 minutes during 30 minutes, then in every 15

minutes during 75 minutes. If the suspension becomes foamy after being poured into the

cylinder, the total distance should be measured from the bottom of the foam layer.

Determination of half-life: plot the height of the sediment column against time. In case of

hindered sedimentation:

2

l-l-l=l E1/2

00 (1)

In case of free sedimentation:

2l=l E

1/2 (2)

where l1/2 is the height of „half-distance”, l0 is the initial height, lE is the equilibrium height.

Plot the sediment volume against time. Mark the l1/2 values on the curve, and project them

onto the time axis. The value thus obtained is the half-life. During the determination of half-

life it is also acceptable if the volume of the sediment column is measured instead of its

height. The last measured value is regarded as the equilibrium height (lE) and the equilibrium

volume (VE) .

2. Redispersibility of the sediment (not an official investigation method in Ph. Hg. VIII.)

Apparatus: resuspension device


Carefully fix the cylinder containing the sedimented suspension into the stand of the

sample holder. Rotate the measuring cylinder until the sediment is uniformly distributed in the

medium. The number of rotations is the value of resuspension. The sediment in the bottom of

the measuring cylinder can be observed better if the device is stopped during the investigation

when the measuring cylinder is with its mouth downwards (make sure that the glass stopper

closes the measuring cylinder).


Compositions to be examined

Instrument: Mixer (stage 1, 1 min of mixing time)

Series „A” A/1 A/2 A/3 A/4

Bismuthi subgallas 5.0 g 5.0 g 5.0 g 5.0 g Natrii dihydrogenophosphas dihydricus

0.0 g 0.5 g 10.0 g 20.0 g

Aqua purificata ad 50.0 g ad 50.0 g ad 50.0 g ad 50.0 g Series „B” B/1 B/2 B/3 B/4

Bismuthi subgallas 5.0 g 5.0 g 5.0 g 5.0 g Mucilago hydroxyaethylcellulosi 0.0 g 0.5 g 10.0 g 20.0 g Aqua purificata ad 50.0 g ad 50.0 g ad 50.0 g ad 50.0 g

Attention! Start the preparation of the suspensions in series „A” with the dissolution of

NaH2PO4.


Report 8. Investigation of suspensions

Name: ..................................................... Group: ....................... Date: ............................

1. Investigation of sediment volume (mL) depending on the time:

Time of sedimentation (min) Sample

5 10 15 20 25 30 45 60 75 A/1 A/2 A/3 A/4 B/1 B/2 B/3 B/4

2. Plot the sediment volume against time (see on the following pages). Calculate and mark

the half-lives on the graph. Complete the table below with the results.

3. Effect of the composition changes of the dispersion medium on the sedimentation of

suspensions and the structure of the sediment:

Note: over 250 rpm, the suspension should be considered unshakeable.

Sample Half-life

(min)

Value of resuspension

(rpm) Type of sedimentation

A/1 A/2 A/3 A/4 B/1 B/2 B/3 B/4


Series „A”


Series „B”

Write an evaluation about the results of the investigations. What cause and effect relationship

explains the observed phenomena?


9. Investigation of ointments I. – Investigation of drop point,

solidifying point, oil number and water absorbing capacity

Introduction

The determination of the physical properties of water-free ointments is very important for

cream development. It is essential to know the solidifying and drop points during the

manufacturing process. The oil number gives information about the binding force between the

solid and liquid lipophilic phases. By ascertaining the water absorbing capacity, the maximum

amount of water which can be emulsified into the water-free ointment can be determined.

Tasks

1. Determination of the drop point of ointments

The drop point is the temperature at which the first drop of the melting substance to be

examined falls from a cup under defined conditions.

Apparatus: Ubbelohde-thermometer (Ph. Hg. VIII. 2.2.17., Figure 9.1)

Figure 9.1: Ubbelohde-thermometer



Fill the cup to the brim with the substance to be examined without melting and bubbles.

Remove the excess substance at the 2 ends of the cup with a spatula. When sheaths A and B

have been assembled, press the cup into its housing in sheath B until it touches the supports.

Remove with a spatula the substance pushed out by the thermometer. Place the apparatus in

the water-bath. Heat the water-bath and, when the temperature is at about 10 °C below the

presumed drop point, adjust the heating rate to about 1 C/min. Note the temperature at the fall

of the first drop. Carry out at least 3 determinations, each time with a fresh sample of the

substance. The difference between the readings must not exceed 3 °C. The average of 3

readings is the drop point of the substance.

2. Determination of the solidifying point of ointments

Apparatus: rotating thermometer (Figure 9.2)

Figure 9.2: Determination of the solidification point of ointments with a rotating thermometer


Insert the thermometer through a one-bore cork into a 100-mL, wide-necked conical flask,

which serves as an air-bath. Heat the sample on the water-bath, while stirring, to a

temperature 8-10 ºC higher than the expected solidification point. Heat the air-bath with the


thermometer on the same water-bath and at the same temperature. When the ointment has

reached the desired temperature, remove the thermometer with the cork from the flask, and

immerse its mercury reservoir into the melted ointment so that one drop hangs on its tip.

Move the thermometer into the flask immediately and rotate it carefully together with the

flask, horizontally around its longitudinal axis at a speed of about 2 seconds per rotation. The

solidification point of the sample is temperature at which the drop on the mercury reservoir

makes the first rotation together with the thermometer (the ointment is solidified). Calculate

the average of 3 parallel measurements.

3. Determination of the oil number of ointments (Figure 9.3)

Figure 9.3: Determination of the oil number of ointments


Melt the water-free ointment base, fill it into a glass vessel and let it solidify. Put the vessel

on a 10 x 10 cm filter paper, with its broader mouth downwards. Place the sample pretreated

in this way on a watch-glass and let it stand at 36–37 ºC for 2 hours. After this period,

measure the smallest and largest diameters (mm) of the elliptical fatty stain on the paper. The

arithmetical average of these two values is considered to be characteristic of the oil number.

Calculate the average of 3 parallel measurements.

Attention: start the work with this determination.


4. Investigation of water absorbing capacity


Measure 10.00 g of ointment base with cg accuracy into a tarred metal pot with pestle and

heat it to 35–40 ºC. Add 1.0 g of water of the same temperature. Add more water in small

portions (0.5–1.0 g) emulsifying each portion before a further amount of water is added until

no more water is ready to emulsify. Decant the excess of water and blot the water droplets

cautiously from the surface of the ointment and the pot with filter paper. Determine the

weight of the ointment with 0.01 g accuracy and calculate the water absorbing capacity by

using the following equation. The w/w % water content is the water absorbing capacity of the

ointment. Calculate the average of 3 parallel measurements.

(%)100⋅−=b

abVf (1)

where Vf is the absorbed amount of water, a is the weight of the ointment, b is the weight of

the ointment and the absorbed water together.


Report 9. Investigation of ointments I. – Investigation of drop point, solidifying point, oil

number and water absorbing capacity

Name: ..................................................... Group: ....................... Date: ............................

1. Fill in the table. Calculate the average of the measurements. Evaluate the results.

Name of ointment 1: ................................................................

Name of ointment 2: ................................................................

Name of ointment 3: ................................................................

Investigation Ointment 1. Ointment 2 Ointment 3

1.

2.

3. Drop point (°C)

average

1.

2.

3. Solidifying point (°C)

average

1.

2.

3. Oil number (mm)

average

1.

2.

3.

Water absorbing

capacity (%)

average

Evaluation:


10. Investigation of ointments II. – Investigation of the washability

and rheological characteristics of ointments

Introduction

In the case of ointments and creams applied on the skin surface, it is very important to

know to what extent the ointment layer administered onto the skin surface is able to stay on

the skin surface when coming into contact with water. This information is particularly

important in the case of dermoprotective ointments. The aim of this investigation, besides

determining the rheological parameters, is to model to what extent ointments and creams

serve their application purposes concerning washability.

Tasks

1. Determine the washability coefficient of the creams received.


Apply a thin layer from the cream to be examined onto a plastic plate with a given depth of

notch (100 x 50 x 1 mm). Continuously pour 50 mL of distilled water dropwise from a burette

onto the surface of the prepared ointment. After dropping is finished, dry the plastic plate and

place it on a graph paper. Specify how many mm2 of ointment area is washed off by 50 mL of

distilled water. Knowing this, calculate the value of the washability coefficient (K) from the

results of 3 parallel measurements by using the following formula:

VFK = (1)

where F is the area of the ointment film washed off in mm2, V is the used water volume in mL

(Figure10.1).


Figure 10.1: Determination of the washability coefficient

2. Determine with a rheometer the flow curves (0.1-100 1/s) of Unguentum emulsificans

anionicum with three different water content values (10-80 %) and plot the flow curves.

Include the results of the measurements in separate tables.

Fill the measuring device with the ointment to be examined. Measure the shear stress with

increasing and decreasing shear rate (0.1-100 1/s) at 12 points. Plot the flow curves based on

the measurements.


Report 10. Investigation of ointments II. – Investigation of the washability and rheological

characteristics of ointments

Name: ..................................................... Group: ....................... Date: ............................

1. Determine the washability coefficients of the creams received and fill in the table.

Measurement Cream 1: ..................................................... Cream 2: .....................................................

1.

2.

3.

average

2. Rheological measurements of Unguentum emulsificans anionicum ointments with

different water content. Indicate the water content of the ointment.

Water content: ...........................................

Measuring

degree

D

(1/s) α „up”

τ „up”

(N/m2)α „down”

τ „down”

(N/m2)


Water content: ...........................................

Measuring

degree

D

(1/s) α „up”

τ „up”

(N/m2)α „down”

τ „down”

(N/m2)

Water content: ...........................................

Measuring

degree

D

(1/s) α „up”

τ „up”

(N/m2)α „down”

τ „down”

(N/m2)


3. Plot the flow curves of Unguentum emulsificans anionicum with different water

content (τ–D) and evaluate the rheograms.

Evaluation:

1. Connection between the washability coefficient and the application purpose of the

creams.

2. Evaluation of the rheograms.


11. Investigation of ointments III. – Characterisation of

consistency by viscosity, spreadability and adhesion investigations

Introduction

It is very important to know the consistency of ointments both from the point of view of

the manufacturing process and the field of application. Besides viscosity measurements, the

spreadability and the adhesion of ointments give information about their applicability on the

skin.

Tasks

1. Viscosity measurements

Apparatus: rotational viscometer


Fill the measuring device with the ointment to be examined. Measure the shear stress with

increasing and decreasing shear rate (0.1-100 1/s) at 12 points. Plot the flow and viscosity

curves based on the measurements.

2. Characterization of the spreadability of ointments

Apparatus: extensometer


Put 1.0 g of ointment on a glass plate in a circle of 1 cm radius. Place the glass plate on a

graph paper and cover it with a glass plate of the same size. After 1 minute, read the two

perpendicular diameters of the ointment spot with the help of the graph paper. Place weights

of 10, 20, 50, 100, 200 and 500 g in the centre of ointment spot, and after 1 minute of loading

determine the diameters of the ointment spot in the same way as mentioned above. Investigate


two different ointments with three parallel measurements (Figure 11.1). Calculate the area of

the ointment spots with the average of diameters.

Figure 11.1: Investigation of the spreadability of ointments

Characterisation of spreadability:

4

2 π⋅= dA (1)

where A is the area of the ointment spot, d is the average of the perpendicular diameters of the

ointment spot.

3. Investigation of the adhesion of ointments

Apparatus: instrument for adhesion investigation with stand


Spread 0.1 g of ointment in the middle of a glass plate and cover it with another one.

Remove the excess of ointment obtained by pressing the covered plate with 1 kg of weight for

1 minute to form a uniform film from the ointment. Place the sample on the stand and fix the

lower plate. Put the given weights (1-30 g) in the pan and measure the time required for the

upper plate to stop (Figure 11.2). Carry out 5 parallel measurements.


Figure 11.2: Investigation of the adhesion of ointments

Two of the following ointments are investigated:

I. Vaselinum album

II. Pasta zinci oxydata

III. Unguentum emulsificans nonionicum

IV. Unguentum hydrophylicum nonionicum


Report 11. Investigation of ointments III. – Characterisation of consistency by viscosity,

spreadability and adhesion investigations

Name: ..................................................... Group: ....................... Date: ............................

Name of the investigated preparations

Ointment 1: .........................................................

Ointment 2: .........................................................

1. Results of rheological measurements

Ointment 1

Measuring

degree

D

(1/s)

α „up” τ „up”

(N/m2)

η „up”

(Pa·s)

α „down” τ „down”

(N/m2)

η „down”

(Pa·s)


Ointment 2

Measuring

degree

D

(1/s)

α „up” τ „up”

(N/m2)

η „up”

(Pa·s)

α „down” τ „down”

(N/m2)

η „down”

(Pa·s)

2. Fill the spreadability results in the table.

Weight (g) Composition Spreadability 0 10 20 50 100 200 500

1.

2.

3.

d average (cm)

Ointment 1

surface (cm2)

1.

2.

3.

d average (cm)

Ointment 2

surface (cm2)


3. Fill the results of the adhesion investigations in the table. Calculate the standard

deviation and the relative standard deviation according to the parallel measurements. If

the measured time is longer than 180 s, consider it infinite.

Weight (g) Composition Time

(s) 1 5 10 15 20 25 30

1.

2.

3.

4.

5.

average

deviation

Ointment 1

relative

deviation

1.

2.

3.

4.

5.

average

deviation

Ointment 2

relative

deviation


4. Plot the flow curves (τ–D) and the viscosity curves (η–D) of the two investigated

ointments.

Flow curves


Viscosity curves

5. What are the flow types of ointments?

Flow type of ointment 1: ...........................................................................

Flow type of ointment 2: ...........................................................................


12. Investigation of the water uptake of tablets

Introduction

Affinity to the solvent – which can be described quantitatively by the amount of water

taken up by the polymer and by the rate of water uptake – is an important preliminary

examination with respect to the technological usability of macromolecules during granulation

and pelleting. This property is also important in the disintegration processes of tablets and

granules. From the technological point of view, the ideal polymer swells rapidly and is able to

take up great quantities of water.

The so-called Enslin number is determined for the measurement and the quantitative

description of the affinity of tablets to fluids. The Enslin number gives the amount of fluid

absorbed by 1 g of solid material, powder or tablet, in a given time. This procedure is not

official either in the Hungarian pharmacopoeia or in foreign pharmacopoeias, yet it is an

important part of preformulation studies.

The following empirical relationship was established between the volume of the fluid

absorbed by the tablet (V) and the time of the examination (t):

tm+V=V 0 (1)

where V0 is the volume of the fluid absorbed at time 0. If the quantity of the absorbed water is

plotted against the square root of time, a straight line is obtained. The slope of the line (m)

gives the rate constant of water uptake.

Task

1. Examine the water uptake of the two tablets with different compositions chosen by your

practice leader. Include the data in a table.

Apparatus

The apparatus presented in the figure is used for determining water uptake (Figure 12.1).

The apparatus consists of the following parts: a glass filter with appropriate pore size, in

which the material to be examined is placed; a pipette connected to the filter, which makes it


possible to quantitatively measure the amount of water absorbed by the tablet through the

filter. The amount of water absorbed depends mainly on the affinity of the examined material

to water, but naturally, the result is also influenced by the construction of the apparatus used

in the experiment. For example, the pore size of the filter, the position of the pipette as well as

the mass and diameter of the tablet placed on the filter all influence the results obtained.

Under the same test conditions, the data are well comparable and can be used for the

quantitative description of the water uptake of tablets with different compositions.

Figure 12.1: Apparatus for determining the Enslin number

(1 – tablet; 2 – filter paper; 3 – G filter; 4 – pipette)


Assemble the Enslin apparatus. Fill the pipette – through the filter – with the test fluid

(unless otherwise prescribed, with distilled water) and place it so that the glass filter and the

pipette will be in the same plane. Get a filter paper slightly larger in size than the area of the

glass filter and place it on its surface without leaving gaps. Set the fluid level to zero. Then

place the tablet to be examined on the filter paper concentrically and immediately start

measuring the time.

During the task, take care of the following:

– fill the pipette in a bubble-free way;

– make sure that the filter paper is placed tightly on the glass filter.

Measure the amount of the absorbed water after 10, 20, 30 seconds and 1, 2, 3, 4, 5, 6, 8

and 10 minutes. After finishing the examination, remove the solid material together with the

filter paper. Perform three parallel measurements.


2. Calculate the Enslin number of the tablets from the average.

3. Plot the )(tfV = and )( tfV = functions. From the latter one, calculate the rate constant

of the process (m), the slope of the function.

4. Compare the affinity of the tablets with different compositions to water. Explain the

difference between the Enslin numbers of the tablets.


Report 12. Investigation of the water uptake of tablets

Name: ..................................................... Group: ....................... Date: ............................

1. Examine the water uptake of the two tablets with different compositions chosen by your

practice leader. Include the data in a table.

2. Calculate the Enslin number of the tablets from the average.

Name of tablet 1: .................................................. Average mass: ............................(g)

Name of tablet 2: .................................................. Average mass: ............................(g)

Sample Time (s) 10 20 30 60 120 180 240 300 360 480 600

1. (mL)

2. (mL)

3. (mL)

average (mL) 1.

Enslin

number

1. (mL)

2. (mL)

3. (mL)

average (mL) 2.

Enslin

number


3. Plot the )(tfV = function.


3. Plot the )( tfV = function. Calculate the rate constant of the process (m), the slope of

the function.

Sample m

Tablet 1

Tablet 2

4. Compare the affinity of the tablets with different compositions to water. Explain the

difference between the Enslin numbers of the tablets.


13. Investigation of tablets

Introduction

In the course of tablet formulation, the dosage form must satisfy the requirements specified

in the technological documentation. These are partly the examinations prescribed in Ph. Hg.

VIII, but there may also be other examinations in the documentation. Such is, e.g., the

checking of the geometric parameters of the tablets, which is important for the setting of

automatic packaging machines. So the purpose of these tests is to evaluate the tablets on the

basis of some important examinations.

The Hungarian Pharmacopoeia contains the following requirements about the uniformity

of the mass of coated and uncoated tablets (Ph. Hg. VIII., 2.9.5.):

Tablet mass Accepted tolerances

< 80 mg ± 10.0 %

80 – 250 mg ± 7.5 %

> 250 mg ± 5.0 %

Only 2 individual masses can differ to a greater extent, and the deviation from the average

value cannot be more than two times the given value for any of the individual masses.

According to the Hungarian Pharmacopoeia, tablets must also comply with the

requirements of the uniformity of dosage units described in the examination (Ph. Hg. VIII.,

2.9.40.). The term “uniformity of dosage units” is defined as the degree of uniformity in the

amount of the active substance among dosage units, which can be demonstrated by either of

two methods: content uniformity or mass variation. In order to determine content

uniformity, the contents of ten units are assayed individually using an appropriate analytical

method. To examine mass variation, ten individual tablets are measured precisely. From the

mass of the individual tablets and from the result of the content assay, the active substance

content of the tablets is calculated individually, expressed as the percentage of the label claim.

The test for mass variation is applicable for tablets containing 25 mg or more of an active

substance comprising 25 % or more, by mass, of the dosage unit. The content uniformity


method may be applied in all cases. The requirements of the uniformity of dosage units are

satisfied if the acceptance value calculated from the first ten dosage units is L1 or lower. If it

is higher, another twenty dosage units are tested and the acceptance value is calculated again.

The requirements are satisfied if the final acceptance value calculated for thirty dosage units

is L1 or lower, and if during the calculation of the acceptance values of content uniformity

and mass variation no individual content lower than (1-L2·0.01) M or higher than

(1+L2·0.01) M is found among the dosage units. Unless otherwise prescribed, L1 = 15.0 and

L2 = 25.0.

Tasks

1. Determine the geometric parameters of 20 tablets (diameter, height) with a micrometer

screw. Calculate the average and the relative standard deviation.

2. Perform the mass uniformity test according to the Hungarian Pharmacopoeia (Ph. Hg.

VIII., 2.9.5.). Calculate the average, the standard deviation, the relative standard

deviation and evaluate the results according to the specifications of the Hungarian

Pharmacopoeia.


Measure the mass of each of twenty tablets selected at random. Use an analytical balance

for the measurement.

3. Perform the test of the uniformity of dosage units (Ph. Hg. VIII., 2.9.40.) by using the

method of mass variation, as prescribed by the Hungarian Pharmacopoeia. Evaluate the

tablets according to the specifications of the Hungarian Pharmacopoeia.


In the first step, determine the active substance content of the tablets. Powder 10 tablets in

a mortar and homogenize the powder. Measure an amount of the powdered sample equalling

the mass of 1 tablet with analytical precision and spread it into the flask which contains the


required quantity of dissolution medium. Mix the contents of the flask from time to time, then

30 minutes later take a sample, filter it on a filter paper and measure the active substance

content of the sample spectrophotometrically. Dilute the sampled aliquot quantity with a

filtered dissolution medium. Calculate the active substance content in relation to a tablet with

average mass. Perform three parallel measurements.

Calculate the active substance content of the individual tablets from the individual mass

and from the result of the average active substance content of the first 10 of the tablets

measured in point 2, expressed as the percentage of the label claim, then calculate the

acceptance value. The calculation of the acceptance value (AV) is the following:

skXMAV ⋅+−= (1)

where M is the reference value, X is the average value of individual contents as the percentage

of the label claim, k is the acceptance constant (in case of n = 10, its value is 2.4), s is the

standard deviation of the sample.

if 98.5 % ≤ X ≤ 101.5 %, then M = X, AV = k · s

if X < 98.5%, then M = 98.5 %, AV = 98.5 -X + k · s

if X > 101.5%, then M = 101.5 %, AV = X – 101.5 + k · s

Tablets to be examined

Name of tablet Dissolution medium Spectrophotometric data

Tabletta coffeini artificial gastric juice λ=277 nm; 0.100 A = 2.0641 µg/mL

Tabletta sulfadimidini artificial gastric juice λ=244 nm; 0.100 A = 1.9001 µg/mL

Tabletta paracetamoli artificial intestinal juice λ=243 nm; 0.100 A = 1.6260 µg/mL


Report 13. Investigation of tablets

Name: ..................................................... Group: ....................... Date: ............................

1. Determine the geometric parameters of 20 tablets (diameter, height) with a micrometer

screw. Calculate the average and the relative standard deviation.

2. Perform the mass uniformity test according to the Hungarian Pharmacopoeia (Ph. Hg.

VIII., 2.9.5.). Calculate the average, the standard deviation, the relative standard

deviation and evaluate the results according to the specifications of the Hungarian

Pharmacopoeia.

Name of tablet: ....................................................... Tablet Diameter (mm) Height(mm) Mass (g)

1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

average

minimum

maximum

standard deviation

relative standard deviation


Evaluation of mass uniformity:

3. Perform the test of the uniformity of dosage units (Ph. Hg. VIII., 2.9.40.) by using the

method of mass variation, as prescribed by the Hungarian Pharmacopoeia. Evaluate the

tablets according to the specifications of the Hungarian Pharmacopoeia.

Measurement A Average active substance content (mg)

1.

2.

3.

average

standard deviation

Tablet Mass (g) Active substance content (mg) Active substance content (%)

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

average

standard deviation

Evaluation of the uniformity of dosage units:


14. Investigation of the disintegration of rectal and vaginal

suppositories

Introduction

The suppository bases and excipients used in manufacturing can significantly modify drug

release, consequently the effect caused by the suppository. The suitable choice of the

suppository base is an important factor in achieving the desired effect produced by the

suppository and in determining the disintegration time of the dosage form. The aim of this

investigation is to determine whether the suppositories disintegrate within the prescribed time

when placed in a liquid medium in the experimental conditions (Ph. Hg VIII. 2.9.2).

Task

In vitro investigation of the disintegration time of suppositories and whether they satisfy

the requirements of the Pharmacopoeia.

Apparatus: Apparatus for the disintegration of suppositories (Figure 14.1)

Figure 14.1: Sample holder of the apparatus for the disintegration of suppositories


Apparatus description

The apparatus consists of a sleeve of glass or suitable transparent plastic, of appropriate

thickness, to the interior of which is attached by means of three hooks a metal device

consisting of two perforated stainless metal discs, each containing 39 holes 4 mm in diameter;

the diameter of the discs is similar to that of the interior of the sleeve; the discs are about

30 mm apart. The test is carried out using three such apparatuses, each containing a single

sample. Each apparatus is placed in a beaker filled with water maintained at 36-37 C. The

apparatuses are placed together in a water bath. The beaker is fitted with a slow stirrer and a

device that will hold the cylinders vertically not less than 90 mm below the surface of the

water and allow them to be inverted without emerging from the water.


1. Switch on the thermostat 20-30 minutes before starting the investigation. Set the knob

on the right side to VAR position, and the one on the left side to the desired

temperature.

2. Measure 3.00 kg of distilled water into 3 glass beakers (3,000 mL) and place them into

the apparatus. Wait until the desired temperature is reached in the acceptor phase (in the

beaker 37 ± 0.5 °C)

3. Switch on the Timer knob. The duration of the examination is 60 min.

4. Switch the knob to ALAP position, the rotation speed will be 1 turn/min.

5. Place the suppositories into the metal baskets and fix them to the rotating device.

6. Press the START knob.

The measurement is started at the moment the suppositories are immersed in the water and

lasts until complete disintegration.

Carry out two parallel measurements and calculate the average of the suppositories of the

same composition. Record the results in the table and evaluate them according to the

requirements of the Pharmacopoeia.


Report 14. Investigation of the disintegration of rectal and vaginal suppositories

Name: ..................................................... Group: ....................... Date: ............................

1. Investigation of the disintegration of rectal and vaginal suppositories

Sample name,

mark ........................................ ........................................ ........................................

Disintegration time min min min

1.

2.

average

conclusion

Evaluation:


III. Investigation of stability


15. Determination of the decomposition (caramelization) of

glucose solution

Introduction

Sugars (glucose, fructose, sucrose, etc.) are known to decompose due to heat (preparation

of syrups, sterilization of infusions containing sugar). During chemical

decomposition/oxidation, yellowish-brownish substances with bitter a taste (so-called

humins) are formed. During the preparation of syrups, local overheating can be prevented by

using a duplicator, thereby preventing the degradation of sugar. In case of infusions, the

solution can be sterilized without degradation if the pH of the glucose infusion is adjusted to

3-4.

Spectrophotometry is a well suitable analytical method for the detection of decomposition

products characterized by light absorption. The purpose of this practice is to investigate

spectrophotometrically the degradation of glucose solutions with different pH values during

sterilization (121 °C, autoclaved for 20 minutes).

Task

Adjust the pH of glucose solutions to different pH values by using a buffer. Determine the

changes occurring during sterilization after appropriate dilution with spectrophotometry.

1. Preparation of buffer stock solutions

Preparation of buffer stock solutions according to Mc Ilvaine. The buffers are prepared by

mixing two buffer stock solutions.

Stock solution Volume Mt

0.2 M Na2HPO4 100 mL (Na2HPO4 12 H20) = 358.14 (Na2HPO4 2 H20) = 177.99

0.1 M citric acid 100 mL (C6H8O 7 H2O) = 210.14


2. Preparation of glucose solutions using different buffers

Dissolve 5 g of glucose in distilled water of a volume of ~70 mL each time, then pipette

the following volumes from the buffer stock solutions into volumetric flasks of 100 mL to

reach the appropriate pH:

pH 0.2 M Na2HPO4

(mL)

0.1 M citric acid

(mL)

3.0 4.11 15.89

4.0 7.71 12.29

5.0 9.00 11.00

6.0 12.63 7.37

7.0 16.47 3.53

8.0 19.45 0.55

Complete the flasks with distilled water, then homogenize them. Pour the contents into

infusion bottles, close and sign them properly. The infusions should be sterilized in autoclave

at 121 °C for 20 minutes according to the Hungarian Pharmacopoeia (it is done by assistants)

(Figure 15.1).

Figure 15.1: Colour changes of glucose solutions after sterilization between pH 3-8


3. After appropriate dilution, determine the absorbance of the solutions at the given

wavelength.

The absorbance of the solutions prepared and sterilized the previous week should be

investigated at the absorption maximum of the formed decomposition products, at a

wavelength of 281 nm, against distilled water. If it is necessary, make a dilution from the

solution to be measured, using distilled water.

4. Plotting and evaluation of graph

Plot the results against the pH values.


Report 15. Determination of the decomposition (caramelization) of glucose solution

Name: ..................................................... Group: ....................... Date: ............................

1. Prepare the following stock solutions of buffer solution in a volume of 100 mL each,

then indicate the amount of the substances to be measured in the appropriate place.

The amount of substance needed for the preparation of 100 mL of 0.2 M Na2HPO4 solution:

.......................... g

Indicate which crystal water-containing substance was used. Mt = .....................................

The amount of substance needed for the preparation of 100 mL of 0.1 M citric acid solution:

.......................... g

2. Prepare the 100-100 mL of glucose solution concentration of 5 % using buffer solutions

having different pH-values, and pour them into infusion bottles of 100 mL.

3. Measure the absorbance of the sterilized glucose solutions with a spectrophotometer, at

a wavelength of 281 nm against distilled water. If it is necessary, prepare dilutions to

volume percent with the absorbance value measured between 0.3 and 0.8. Calculate the

product of absorbance and dilution. Include the results obtained in the table below.

pH A dilution A · dilution

3

4

5

6

7

8


4. Plot the results in the last column of the table against the pH (A · dilution).

Evaluate the results obtained. Which pH range can ensure the proper stability of glucose?

Evaluation according to the colour intensity:

Evaluation according to the data:


16. Investigation of the viscosity changes of hydrophilic sols I.

Introduction

Methylcellulose (MC) is a commonly used gel- and sol-forming additive, which dissolves

in cold water colloidally. Its aqueous solution is opalescent because of the not sufficiently

hydrated polymer fibres. Exposure to heat results in a reversible coagulation, which can be

converted back by cooling. Similarly, the reversible coagulation of MC occurs due to larger

amounts of electrolytes (e.g. iodide, chloride and nitrate salts).

Hydroxyethyl cellulose (HEC) is the monoglycol ether of cellulose in its main mass. It is

specifically hydrophilic, therefore it dissolves in water colloidally at a low degree of

substitution. It solvates quickly and completely, the colloid solution obtained is clear and does

not precipitate even when exposed to heat. HEC is used for bonded granulation in the

pharmaceutical industry. The basic compound and especially its derivatives are also used for

film coating. Its colloid solution is one of the most commonly used viscosity enhancing

excipients during the production of suspensions and emulsions. It is incompatible with the

concentrated solutions of inorganic salts and with tannates.

Carmellose sodium (also known as carboxymethyl cellulose sodium, CMCNa) is the

sodium salt of a partially O-carboxymethylated cellulose. In the 8th Edition of the Hungarian

Pharmacopoeia Carmellosum natricum substitutum humile (low-substituted carmellose

sodium) is also official, which may contain short fibrils. The calcium salt of partially O-

carboxymethylated cellulose is also official. It swells in water and becomes opalescent, it

forms a viscous colloidal solution. It is primarily used as a viscosity enhancing excipient

(stabilizer of emulsions and suspensions), as a hydrogel in different concentrations and as a

disintegrant in tablets. It is incompatible with different salts, acidic medium and cationic

surfactants.

These polymers are usually used for preparing colloidal solutions. The resulting gel

structure is very sensitive even to slight changes in the environmental parameters (e.g. pH,

temperature, ion concentration), which is also reflected in the changes of the physical-

chemical properties (e.g. shape, viscosity).

The purpose of the task is to study the viscosity changes of one of the mucilages

(methylcellulose/hydroxyethyl cellulose/carboxymethyl cellulose) due to the effect of sodium


chloride (investigation of a NaCl-free composition and two NaCl compositions containing

different ingredients).

Task

Apparatus: Höppler-type viscometer (Figure 16.1)

Figure 16.1: Höppler-type viscometer


Dissolve an appropriate amount of sodium chloride in the given type of mucilage so that

the total weight will be 100 g and the product will have the given sodium chloride

concentration. Add the salt to the mucilage in portions and apply intensive stirring.

After dissolution, determine the apparent viscosity of the samples with the Höppler-type

viscometer. The salt-free mucilage serves as the basis for comparison. Plot the calculated


viscosity values against the load. Make 3 parallel measurements and always use a new sample

for each measurement.

Process of viscosity measurement

Fill the measuring cup with the colloidal solution to be tested in a bubble-free way. Choose

the ball rod and mount it onto the measuring arm. Make the device ready for measurement.

(levelling, offsetting the buoyancy with sliding weight). Put the smallest weight on the weight

holder plate, release the locking screw, measure the time needed to cover the distance

corresponding to the 0-30 scale. After that, remove the weight, pull back the ball rod carefully

and fix the measuring arm with the eccentric. Wait for a short time and repeat the

measurement with greater load. You can increase the load as long as the descent time is

higher than 4 seconds.

The equation of the calculation of viscosity:

tpk= ⋅⋅η (1)

where η is the apparent viscosity (mPa·s), (Pa = 0.01 g/cm2), k is the instrument constant

(cuvette labelled 01, k = 0.1053), p is the load, shear stress (p = 10, 20, 30, 40 and 50 g/cm2), t

is the descent time (s).


Report 16. Investigation of the viscosity changes of hydrophilic sols I.

Name: ..................................................... Group: ....................... Date: ............................

1. Name of mucilage: ........................................................

Load (g/cm2) 10 20 30 40 50

NaCl conc. Measurement Descent time (s)

1.

2.

3. 0 %

Average

1.

2.

3. ....... %

Average

1.

2.

3. ....... %

Average

NaCl conc.

(%) 0 % ......... % ......... %

Load

(g/cm2)

t

(s)

η

(mPa·s)

t

(s)

η

(mPa·s)

t

(s)

η

(mPa·s)

10

20

30

40

50

2. Plot the viscosity against the load.


3. Evaluation:

Write down what you experienced and find a correlation between the concentration of

sodium chloride and the viscosity of the mucilages based on the results. What kind of

colloidal physical phenomenon is it? Give reasons for your answer.


17. Investigation of the viscosity changes of hydrophilic sols II.

Introduction

Carmellose sodium (also known as carboxymethyl cellulose sodium, CMCNa) is the

sodium salt of a partially O-carboxymethylated cellulose. In the 8th Edition of the Hungarian

Pharmacopoeia Carmellosum natricum substitutum humile (low-substituted carmellose

sodium) is also official, which may contain short fibrils. The calcium salt of partially O-

carboxymethylated cellulose is also official. It swells in water and becomes opalescent, it

forms a viscous colloidal solution. Its application is diverse: for internal use it can be used

only in the form of a colloidal aqueous solution. It is beneficial as a coating agent in the case

of gastro-intestinal disorders. As it can adsorb several substances, it can also be used as an

antidiarrheal drug. It also has an antacid effect, and it is used as a food additive as well (E

466).

In pharmaceutical technology, it is used as a binder agent during the production of

granules. In tablets, Carmellosum natricum conexum (croscarmellose sodium) containing

cross-links can be applied as a disintegrant and it has good swelling properties. In different

concentrations it can also be used as a hydrogel, as the stabilizer of emulsions and

suspensions, furthermore, as a protective colloid, too. It is incompatible with acidic medium,

heavy metal salts, cationic surfactants, alkaloid salts and phenols.

The purpose of the task is to present an incompatibility: to study the viscosity change of 3

% of carmellose sodium mucilage due to the effect of hydrochloric acid.

Task

Apparatus: Höppler-type viscometer


Determine the apparent viscosity of the different available – stored for 24 hours –

carmellose sodium mucilages containing hydrochloric acid of 2 M (1, 2, 3 %) with the

Höppler-type viscometer. The hydrochloric acid-free mucilage serves as the basis for


comparison. Plot the calculated viscosity values against the load. Make 3 parallel

measurements and always use a new sample for each measurement.

Process of viscosity measurement

Fill the measuring cup with the colloidal solution to be tested carefully, in a bubble-free

way. Choose the ball rod and mount it onto the measuring arm. Bring the device state ready to

measure. (levelling, offsetting the buoyancy with sliding weight). Put the smallest weight on

the weight holder plate, release the locking screw, measure the time needed to cover the

distance corresponding to the 0-30 scale. After that, remove the weight, pull back the ball rod

carefully and fix the measuring arm with the eccentric. Wait for a short time and repeat the

measurement with greater load. You can increase the load as long as the descent time is

higher than 4 seconds.

The equation of the calculation of viscosity:

tpk= ⋅⋅η (1)

where η is the apparent viscosity (mPa·s), (Pa = 0.01 g/cm2), k is the instrument constant

(cuvette labelled 01, k = 0.1053), p is the load, shear stress (p = 10, 20, 30, 40 and 50 g/cm2), t

is the descent time (s).


Report 17. Investigation of the viscosity changes of hydrophilic sols II.

Name: ..................................................... Group: ....................... Date: ............................

1. Results of the rheological measurements:

Load (g/cm2) 10 20 30 40

HCl conc. Measurement Descent time (s) 1.

2.

3. 0 %

Average

1.

2.

3. 1 %

Average

1.

2.

3. 2 %

Average

1.

2.

3. 3 %

Average

HCl conc.

(%) 0 1 2 3

Load t

(s)

η

(mPa·s)

t

(s)

η

(mPa·s)

t

(s)

η

(mPa·s)

t

(s)

η

(mPa·s)

10

20

30

40


2. Plot the viscosity against the load.

3. Evaluation:

Write down what you experienced and find a correlation between the concentration of

hydrochloric acid and the viscosity of the mucilages based on the results. What kind of

colloidal physical phenomenon is it? Give reasons for your answer.


18. Influence of humidity on the geometric parameters of tablets

Introduction

Humidity in the air may affect the stability, the physical parameters of tablets, such as their

mass, geometric parameters, mechanical strength, disintegration and also active substance

release. The geometric parameters of tablets (diameter, height) must be within the threshold

limits prescribed in the detailed specifications of the given preparation. The uniformity of size

and shape has great significance when packaging with automatic packaging machines.

The reason for the effect of humidity lies in the hygroscopicity and water uptake capacity

(Enslin number) of the excipients and active substances of the tablets. For example, excipients

used as disintegrants or superdisintegrants, such as starch and sodium starch glycolate, have a

high Enslin number. Because of this, these tablets must be stored carefully away from air, or

with the use of a moisture absorber.

The aim of this investigation is to examine how 100% relative humidity influences the

mass and the geometric parameters of tablets containing various excipients.

Task

1. Measure the mass, diameter and height of the disintegrant- and superdisintegrant-

containing tablets given by your practice leader. Measure the mass with an analytical

balance and the geometric parameters with a micrometer screw.

2. Measure the above parameters also for tablets stored at room temperature, under normal

circumstances and for tablets stored for different periods of time at room temperature at

100% relative humidity (storage time: 24, 48, 72, 96 hours). Perform 10 parallel

measurements for each. Include your results in a table. Calculate the averages, the mass

and volume changes.

3. Plot the volume change against time.


Report 18. Influence of humidity on the geometric parameters of tablets

Name: ..................................................... Group: ....................... Date: ............................

1. Measure the mass, diameter and height of the disintegrant- and superdisintegrant-

containing tablets given by your practice leader. Measure the mass with an analytical

balance and the geometric parameters with a micrometer screw.

2. Measure the above parameters also for tablets stored at room temperature, under normal

circumstances and for tablets stored for different periods of time at room temperature at

100% relative humidity (storage time: 24, 48, 72, 96 hours). Perform 10 parallel

measurements for each. Include your results in a table. Calculate the averages, the mass

and volume changes.

Tablet 1: .................................................................. Mass (mg) Tablet 1

0 hours 24 hours 48 hours 72 hours 96 hours 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

Diameter (mm) Height (mm) Tablet 1 0

hours 24

hours 48

hours 72

hours 96

hours 0

hours 24

hours 48

hours 72

hours 96

hours

1. 2. 3. 4. 5. 6. 7. 8. 9.

10.


Volume (mm3)Tablet 1

0 hours 24 hours 48 hours 72 hours 96 hours 1. 2. 3. 4.5. 6. 7. 8. 9.

10.

Average values of the tested parameters of Tablet 1:

Storage time (hours)

Average mass (mg)

Average diameter

(mm)

Average height (mm)

Average volume (mm3)

Mass change (mg)

Volume change (mm3)

024 48 72 96

Tablet 2: .................................................................. Mass (mg) Tablet 2

0 hours 24 hours 48 hours 72 hours 96 hours 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.


Diameter (mm) Height (mm) Tablet 2 0

hours 24

hours 48

hours 72

hours 96

hours 0

hours 24

hours 48

hours 72

hours 96

hours 1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

Volume (mm3)Tablet 2 0 hours 24 hours 48 hours 72 hours 96 hours

1. 2. 3. 4. 5. 6. 7. 8. 9.

10.

Average values of the tested parameters of Tablet 2:

Storage time (hours)

Average mass (mg)

Average diameter

(mm)

Average height (mm)

Average volume (mm3)

Mass change (mg)

Volume change (mm3)

024 48 72 96


3. Plot the volume change against time.

Evaluation:


19. Investigation of the stability of tablets containing

acetylsalicylic acid I.

– Investigation of decomposition kinetics, calculation of shelf life

using (real time) long-term stability test

Introduction

The suitable stability of the dosage form provides the efficacy of the medicine up to the

expiration date. A basic requirement is that neither the effect, nor the side effect of the

medicine is allowed to change. The drug content may change between the values prescribed

by law, the maximum decrease permitted is usually 10%.

The kinetic investigation of drug stability gives information about the time course of drug

decomposition. The main decomposition ways of drugs follow first or zero order kinetics.

In case of zero order kinetics, the reaction rate is independent of the concentration of the

reaction partners (Figure 19.1):

[ ] [ ] tkCCt ⋅−= 0 (1)

where Ct is the concentration of the drug in time t, C0 is the initial concentration of the drug, k

is the rate constant.

Figure 19.1: Decomposition of drug following zero order kinetics


According to the above, the reaction rate is constant during the reaction.

[ ]0k

dtCdv =−= (2)

On the basis of equation (1), in case of zero order kinetics, the values of t1/2 (half-time) and

t10% (shelf life) can be calculated as follows:

[ ]0

02/1 2k

Ct = (3)

5

2/1%10

tt = (4)

In case of first order kinetics, the reaction rate depends on the concentration of the

reaction partners (Figure 19.2):

[ ] [ ] tkt eCC 1

0−= (5)

[ ] [ ]Ckdt

Cdv 1=−= (6)

On the basis of equation (5), in case of first order kinetics, the values of t1/2 and t10% can be

calculated as follows:

11

2/1693.02lnkk

t == (7)

90

100lg303.2

1%10 k

t = (8)

Figure 19.2: Decomposition of drug following first order kinetics


Task

The aim of the investigation is to determine the half-time and the shelf life (t1/2, t10 %) of

tablets containing acetylsalicylic acid (ASA).

Measure the ASA content of tablets stored in well-sealed containers for different periods of

time by means of spectrophotometry. Make reaction kinetics calculations on the basis of 3

parallel measurements. Determine the order of the reaction (zero or first order). Knowing the

rate constant, calculate the values of t1/2 and t10 %.


Pulverize 5 tablets from each batch of tablets stored for a given time. Weigh the amount

corresponding to 1 tablet from the pulverized sample by using an analytical balance and wash

it into a 100-mL volumetric flask with 15 mL of 96% alcohol. Dilute it with purified water,

shake it for 5 minutes and complete it to 100.00 mL with purified water. Filter the solution

immediately and prepare a dilution in the given way.

Make 3 parallel measurements, preparing 3 dilutions from each filtered solution. Measure

the absorbance of the solutions (A) at λ = 276 nm. The reference solution is purified water.

On the basis of the calibration curve, 0.100 A corresponds 25.66 µg/mL of ASA.


Report 19. Investigation of the stability of tablets containing acetylsalicylic acid I.

– Investigation of decomposition kinetics, calculation of shelf life using (real time) long-

term stability test

Name: ..................................................... Group: ....................... Date: ............................

Nominal weight of 1 tablet: ................................... g

ASA content of fresh tablet: ................................... g

Tablet number

Storage time (month)

2

10

30

60

Pulverize 5 tablets from each batch of tablets stored for a given time. Weigh the amount

corresponding to 1 tablet from the pulverized sample by using an analytical balance and wash

it into a 100-mL volumetric flask with 15 mL of 96% alcohol. Dilute it with purified water,

shake it for 5 minutes and complete it to 100.00 mL with purified water. Filter the solution

immediately and dilute 5 mL of the filtered solution to 100 mL with purified water. Prepare 3

dilutions from each filtered solution.

1. Results of the spectrophotometric measurements:

Measurement 1 Measurement 2 Measurement 3

Sample A

conc. (µg/mL) A

conc. (µg/mL) A

conc. (µg/mL)

Average conc.

(µg/mL)

ASA content

in 1 tablet (mg)


2. Calculation of reaction kinetics parameters:

sample C0 (%) Ct (%) k1 k2 t1/2 t10%

AVERAGE:

Evaluation:


20. Investigation of the stability of tablets containing

acetylsalicylic acid II.

– Investigation of decomposition kinetics, calculation of shelf life

using stress test

Introduction

Tablets are usually stored at room temperature. Investigations lasting for months or years

are often needed to determine how long they remain stable and contain the effective drug

concentration (>90%) when stored at room temperature. Stress tests can be performed in order

to shorten these investigations.

There is proportionality between the decomposition rate of the drug and the storage

temperature, which is known as the Arrhenius equation:

RT

EAk a

303.2lglg −= (1)

where k is the rate constant of decomposition, A is the pre-exponential factor/frequency factor,

Ea is the activation energy (kJ/mol), R is the universal gas constant (8.314 J/mol·K), T is the

temperature (K).

If log k is plotted against 1/T, it will result in a linear correlation, which is called Arrhenius

plot (Figure 20.1). The slope of this line is -Ea/2.303·R, from which the activation energy can

be calculated.

Figure 20.1: Arrhenius plot


Knowing the value of the activation energy (Ea) and the rate constant (k1) at a given

temperature (T1), the rate constant (k2) at a desired temperature (T2) can be calculated and the

shelf life (t10%) can also be determined at this temperature.

−−=

121

2 11303.2

logTTR

Ekk a (2)

2

%,10105.0

2 kt T = (3)

In case of first order kinetics, if the drug concentrations are known, the rate constant can be

calculated as follows:

tC

Ct

k 0log303.2= (4)

where C0 is the initial drug concentration, Ct is the drug concentration in time t, t is the time

(days).

Task

The aim of the investigation is to determine the half-time and the shelf life (t1/2,) of tablets

containing ASA by using stress test. The tablets were stored at different temperatures (40, 50,

60, 70, 80 °C) and the drug content was measured after 1, 2 and 3 days.

1. Calculate the average drug content of the tablets from the data at the given time and

temperature. On the basis of equation (4), calculate the rate constant of the reaction as a

first order reaction at each measuring point, by using the substitution method. Calculate

the rate constant for each temperature.

2. Draw the Arrhenius plot from the mean value of the rate constants belonging to the

given temperature. Calculate the activation energy from the slope of the plot.

3. Calculate the rate constant for 25 °C on the basis of equation (2). Knowing the rate

constant, calculate the shelf life of the tablet referring to 25 °C.


Report 20. Investigation of the stability of tablets containing acetylsalicylic acid II.

– Investigation of decomposition kinetics, calculation of shelf life using stress test

Name: ..................................................... Group: ....................... Date: ............................

1. The ASA concentrations in tablets with different storage times and temperatures are

listed below. Calculate the rate constant for each measuring point and for each

temperature.

ASA concentration (%) Temp.

(T)(°C)

1/T

(K-1)

Storage time (t)(days)

1 2 3 mean k kmean log kmean

1 99.4 99.9 99.8

2 99.1 98.4 98.9

40

3 97.8 98.1 98.7

1 98.9 99.2 98.9

2 97.0 96.9 96.5

50

3 95.4 95.4 95.7

1 96.4 96.3 95.9

2 92.8 92.9 92.7

60

3 89.9 89.9 92.0

1 88.7 89.1 89.2

2 82.0 82.2 81.8

70

3 78.9 78.7 78.2

1 76.2 75.9 75.9

2 60.5 59.9 59.6

80

3 47.2 46.8 47.0


2. Arrhenius plot, determination of activation energy:

Ea = ...................................................................

3. Rate constant and shelf life of tablet referring to 25 °C:

k25°C = ...............................................................

t10%, 25°C = .........................................................

Evaluation:


IV. Biopharmaceutical investigations


21. Investigation of the drug release of emulsions by static

diffusion method

Introduction

Although the excipients used in formulation do not have an effect on their own, they can

influence the release of the API, and thereby the efficacy of the drug significantly. The type of

the emulsion is chosen on the basis of the physical-chemical properties of the API. Based on

these factors, the proper choice of the type of the emulsions and the emulsifiers applied is

very important. In the case of emulsions for external use, both the o/w and the w/o types are

used, depending on the extent of penetration into the deeper layers of the skin and on the

duration of drug release.

Polysorbate 20 is a nonionic, water-soluble surfactant, which can be used in o/w emulsions

and emulsion ointments. It increases the solubility of poorly water-soluble drugs. In water it

forms micelles belonging to the colloidal size range and it encloses the non-soluble solid and

liquid components (e.g. essential oils). It is used as a wetting agent in suspensions and

suppositories. It is applied in a concentration of 1-10 % as an emulsifier and 0.1-3 % as a

wetting agent.

Span 80 is a nonionic, practically water-insoluble surfactant, which can be used to produce

w/o type emulsions by itself. It can increase the water absorption of creams. It is also applied

as a solubilizer and a wetting agent in lipophilic medium.

The static diffusion method is a model by which the release of the drug can be determined.

It consists of three main parts: a donor and an acceptor phase, and a semipermeable dialysis

membrane separating the two phases. The donor phase contains the emulsion to be tested,

from which only the API can get through the dialysis membrane into the acceptor phase.

Concerning the latter one, the physical-chemical properties of the API should be considered

and the appropriate temperature of the phase is also an important aspect (it is not an official

method in the Hungarian Pharmacopoeia).

Because the HLB-value significantly influences drug release, during the task the rate of the

release of the API incorporated in different types of emulsions is determined by the static

diffusion method under in vitro conditions, using an emulsifier with a lower (Span 80) and a

higher (Polysorate 20) HLB-value.


Figure 21.1: Investigation of the drug release of emulsions by static diffusion

Task

The release and the membrane diffusion of lidocaine hydrochloride, as a model API, is

determined using different types of emulsions (o/w and w/o). The API is dissolved in the

aqueous phase in every case. During the investigations, the drug release of the emulsion is

measured, which means the diffusion of the API through the dialysis membrane into the

acceptor phase in such a way that the amount of lidocaine hydrochloride is determined

spectrophotometrically in the acceptor phase (Figure 21.1).

For the investigations, the students have to make the emulsions and arrange the membranes

themselves.

1. Preparation of the diffusion tests

Cut a 10-cm piece from the membrane used, soak it in water for 15 minutes and affix the

plastic closures to the membrane. The two emulsions to be prepared are investigated


simultaneously, so altogether 6 membranes should be arranged for the 3 parallel

measurements each. Put the membranes into 6 beakers of 250 mL, previously filled with 100

mL of distilled water of room temperature (~ 25 °C).

2. Preparation of the emulsions to be examined

Prepare the emulsions with the given ingredients properly by using 2 medicine bottles of

100 g.

Sample "A" weight (g)

Lidocaini hydrochloridum 5.00

Polysorbatum 20 1.00

Mucilago hydroxyaethylcellulosi 6.00

Paraffinum liquidum 20.00

Aqua purificata 18.00

Preparation: dissolve the lidocaine hydrochloride in the aqueous phase, mix the emulsifier,

then emulsify the oily phase into this in portions. Shake vigorously.

Sample "B" weight (g)

Lidocaini hydrochloridum 5.00

Span 80 1.50

Mucilago hydroxyaethylcellulosi 5.00

Paraffinum liquidum 20.00

Aqua purificata 18.50

Preparation: dissolve the lidocaine hydrochloride in the aqueous phase. Mix the emulsifier

into the oily phase, then emulsify the previously prepared aqueous phase in portions. Shake

vigorously.


3. The carrying out of dialysis

Carefully measure out 5 mL of the emulsions prepared into each dialysis membrane with a

digital pipette. Make sure that the dialysis membranes are closed bubble-free.

When you have finished the insertion of the sample, the investigation is started. Start the

first measurements after 15 minutes by taking out 5 mL of each sample. Filter it on a filter

paper, then determine spectrophotometrically the amount of lidocaine hydrochloride dissolved

in the acceptor phase and replace the amount of the sample taken out (5 mL) with distilled

water.

The spectrophotometric evaluation is made possible by the light absorption maximum of

the model API at 263 nm. At this wavelength, the values of the calibration curve were

recorded previously, and by using them the measured extinction values can be recalculated

into the concentration of the API (0.100 A = 154.262 µg/mL). During the measurements,

make dilutions if necessary and consider them when making the final calculations. Repeat the

sampling at the times given (15, 30 and 60 minutes).


Report 21. Investigation of the drug release of emulsions by static diffusion method

Name: ..................................................... Group: ....................... Date: ............................

1. Fill in the table below with the results of the investigation and calculate the appropriate

values. Give the total amount (in mg!) of the API dissolved after 60 minutes.

Sample Time

(min) A dilution

c

(mg/mL)

API in the sample

(mg/5 mL)

API in the dissolution

medium (mg/95

mL)

Total API dissolved

(mg)

A

B


2. Show your results graphically by plotting the total amount of the API diffused against

diffusion time.

Evaluation:


22. Investigation of drug release by agar plate diffusion method

Introduction

The ointment bases and excipients used in formulation can modify drug release, and

thereby the efficacy of the drug significantly. Their proper choice is very important with

regard to effect.

During the experiment the rate of the release of the incorporated API depending on the

properties of the ointment bases and drug concentration is determined under in vitro

conditions.

Task

Investigate the drug release from ointments containing salicylic acid. The iron (III)

chloride dissolved previously in the agar medium will form a coloured complex with salicylic

acid after its diffusion from the ointment samples. The size and the intensity of this violet-

coloured ring around the samples are proportional to the amount of salicylic acid released

(Figure 22.1).

Figure 22.1: Salicylic acid diffusion in iron (III) chloride containing agar gel


1. Preparation for the diffusion investigation

For the investigation, the students get the ready agar gels in Petri-dishes and the different

ointment samples in marked tubes. In each Petri dish six cylindrical holes should be punched

with a special puncher, spaced at equal distances from each other and not too close to the wall

of the Petri dish. Carry out 2 parallel measurements with the same ointment with different

salicylic acid contents. Mark the holes on the bottom of the Petri dish to avoid mixing up the

samples. Use the short markings on the tubes.

2. Measurement of drug (salicylic acid) release from different ointments

Carry out the experiments with 4 different ointments containing 3 different salicylic acid

concentrations (1 w/w %, 5 w/w %, 10 w/w %). Fill each hole completely with the samples.

Use a small spatula to help the filling. Take care to avoid the ointment getting onto the surface

of the agar gel. The samples must be in contact with the agar gel on the side of the holes.

Start measuring the release time as one Petri dish is filled. The first reading time is after 30

minutes. Measure 2 perpendicular diameters of the coloured ring by means of a graph paper.

Be careful to read the Petri dishes in the same order as they were filled. Measure the size of

the ring by putting a graph paper under the Petri dish and reading the diameter of the coloured

ring through the transparent agar gel.

The reading times of the coloured rings are: 30, 60, 90 and 120 minutes.


Report 22. Investigation of drug release by agar plate diffusion method

Name: ..................................................... Group: ....................... Date: ............................

1. Fill in the table below and calculate the average of the two parallel measurements.

30 min.

average diameter

(mm)

60 min.

average diameter

(mm)

90 min.

average diameter

(mm)

120 min.

average diameter

(mm) Ointment API

%1. 2. 3. average 1. 2. 3. average 1. 2. 3. average 1. 2. 3. average

Evaluation:


2. Plot the results.

Ointment: ...............................................................


Ointment: ...............................................................


23. Investigation of drug release from suppositories by dynamic

diffusion method

Introduction

Suppository masses do not have an effect on their own, but they can modify drug release –

and thereby therapeutic effect – significantly. In this practice the rate of the release of the API

incorporated in different suppository masses and in different concentrations is determined by

dynamic diffusion.

Task

The drug release of readily water-soluble chloroquine phosphate and its diffusion through a

membrane from different suppository masses are determined. During the investigation the

drug release from the suppositories through a membrane is measured by determining the

chloroquine phosphate content in the acceptor phase spectrophotometrically.

Investigation method:

1. Measure the 3 suppositories received from your practice leader with mg accuracy and

mark them as Sample A, B, and C. You have 3 Petri dishes for the storage and the

identification of the suppositories. The base and the API content are the same in all 3

samples.

2. Cut off a piece from the membrane tube equalling the suppository length + 2 cm. Place

the suppositories into the tubes and close them with the closing device.

3. Place the samples prepared in this way into the apparatus.

4. Start the apparatus. The rotational speed is 1 turn / 30 s.

5. Take samples after 15, 30 and 60 minutes in a volume of 20 mL from the acceptor

phase with the rubber tube connected to the equipment. You will need 3 x 3 pieces of

Erlenmeyer flasks for the sampling.

6. Use an aliquot of the 20-mL samples and make a dilution to measure the value of

quenching. The spectrophotometric evaluation is made possible by the light absorption

maximum of chloroquine phosphate at 220 nm. At this wavelength, the values of the


calibration curve were recorded previously, and by using them the measured quenching

values can be recalculated into the concentration of the API. Consider the dilutions

when making the calculations.

7. The volume of the acceptor phase taken during sampling was supplied; do not forget

about it when you make the final concentration calculation.

Apparatus: Apparatus for the disintegration of suppositories

Use of apparatus

1. Switch on the thermostat, set the knob on the right side to VAR position, and the one on

left side to the desired temperature.

2. Measure 3.00 kg of distilled water into 3 glass beakers (3,000 mL) and place them into

the apparatus. Wait until the desired temperature is reached in the acceptor phase.

3. Place the wrapped suppositories into the metal baskets, immerse them into the acceptor

phase and fix the rotating device.

4. Switch on the timer.

5. Switch the knob to ALAP position.

6. Press the START knob (to set the rotating device to the right position).

7. Press the ‘STOP’ knob.

8. Switch the knob to ‘STOP’ position.

9. Set the timer: 00, 30. (In ALAP position the two numbers mean hours and minutes, in

TIMER position minutes and seconds.)

10. Press the LOAD knob.

11. Switch the knob to ‘TIMER’ position.

12. As the switch is turned to ‘TIMER’ position, the investigation is started. Half a

rotation is made, repeated every 30 seconds.

The apparatus should work continuously during the 1 hour of investigation time. Take the

samples after 15, 30 and 60 minutes. When the experiment is finished, switch off the

apparatus and the thermostat.


Start the work by switching on the thermostat and measuring the acceptor phase. You have

time to prepare the suppositories for investigation till the acceptor phase reaches the desired

temperature.


Report 23. Investigation of drug release from suppositories by dynamic diffusion method

Name: ..................................................... Group: ....................... Date: ............................

1. Give the following data.

Number of composition: .............................................

API concentration: ...................................................... %

Weight of suppository A: ............................................ mg

Weight of suppository B: ............................................ mg

Weight of suppository C: ............................................ mg

2. Fill in the table with the results of the three parallel measurements (aliquot, dilution,

quenching values).

Sampling time (min)

15 30 60 Supp.

Aliquot Dilution Quenching Aliquot Dilution Quenching Aliquot Dilution Quenching

A

B

C

3. Calculate the released amount of API in µg/mL. Use the values of quenching, and the

slope and the intersection of the calibration curve. Give the released amount of API in

mg and w/w%, too. During the calculation, take care to consider the dilution, the

starting amount of the acceptor phase and the supplied amount of the sampling volume.

Equation of the calibration curve:

bxay +⋅= (1)

where y is the quenching, a is the slope (0.0618), x is the concentration (µg / mL), b is the

intersection (- 0.0776).

The absorption maximum of chloroquine phosphate is at 220 nm.


Released API (mg) Sampling time

(min) Supp. A Supp. B Supp. C Average

15

30

60

Released API (%) Sampling time

(min) Supp. A Supp. B Supp. C Average

15

30

60

3. Plot the released API % against sampling time.

Evaluation:


24. Investigation of the mucoadhesivity of hydrogels (Calculation task)

Introduction

The mucoadhesivity of a pharmaceutical preparation means the adhesion between the

preparation and the mucosa, in the course of which physical and chemical interactions can

form. Physical interactions mean the diffusion of the polymer chains into the mucin chains

and their interpenetration. Chemical interactions mean the formation of primary (e.g.

covalent) and secondary (e.g. hydrogen, van der Waals) bonds between the entangled chains

(Figure 24.1).

Figure 24.1: Mechanism of mucoadhesion

A: contact stage; B: consolidation stage;

C: formation of primary and secondary chemical bonds between the mucin and polymer chains

As mucoadhesion is a complex mechanism, numerous theories exist to explain it, such as:

– electric;

– adsorption;

– wetting;

– diffusion and

– fracture theory.


Because of these complexities, there is no standardized method of measurement; numerous

methods are known and developed to investigate mucoadhesive behaviour. One of the most

accepted methods is rheology. The rheological synergism between the polymer and the mucin

chains can be regarded as an in vitro parameter of mucoadhesion. The base of the

methodology is that the rheological parameters change after mixing the polymer gels with

mucin. This change is greater than it could be expected from the sum of the rheological

parameters.

Hassan and Gallo were the first to describe the relationship which uses a viscosity

parameter (ηb) to characterize the strength of mucoadhesivity:

pmtb ηηηη −−= (1)

where, ηt is the viscosity of gels containing polymers and mucin, ηp is the viscosity of the

polymer gels without mucin, and ηm is the viscosity of mucin dispersion.

Besides the equation above, the relative synergism parameter can be also used, where the

changes in viscosity refer to the original viscosity:

pm

brelb ηη

ηη+

=, (2)

Task

The mucoadhesivity of polymer gels containing different amounts of hydroxyethyl

cellulose polymer (HEC) is investigated by means of rotational viscometry. From the

measured data listed below, the synergism and relative synergism parameters can be

calculated, and the changes of mucoadhesivity can be characterized by increasing the polymer

concentration.

The flow and viscosity curves of the mucin (porcine stomach type II) dispersion, and gels

containing HEC alone and HEC and mucin together were recorded in the range of 0.1-100 1/s

shear rate.


The composition of the samples is listed in the table below:

Sample Mucin

(%) HEC(%)

1 4 02.1 0 1 2.2 0 2 2.3 0 3 2.4 0 5 3.1 4 1 3.2 4 2 3.3 4 3 3.4 4 5


Report 24. Investigation of the mucoadhesivity of hydrogels

(Calculation task)

Name: ..................................................... Group: ....................... Date: ............................

1. Shear stress (τ) and viscosity data (η) (at 100 1/s) derived from the flow and viscosity

curves can be seen in the table below. Three parallel measurements were made.

Calculate the mean values.

Measurement 1 Measurement 2 Measurement 3 Mean Sample τ

(Pa) η

(mPa·s) τ

(Pa) η

(mPa·s) τ

(Pa) η

(mPa·s) τ

(Pa) η

(mPa·s) 1 1.5 15.1 1.6 15.5 1.3 13.2

2.1 2.1 21.1 2.1 21.3 2.1 20.8

2.2 14.3 143 14.4 144 14.3 143

2.3 43.5 435 45.5 455 50.7 507

2.4 163 1630 182 1820 148 1480

3.1 7.32 73.2 7.6 75.7 8.9 88.8

3.2 35.6 356 35.1 351 33 330

3.3 89.6 896 88.1 881 90.8 908

3.4 244 2440 306 3060 296 2970


2. Plot the values of the shear stress against the HEC concentration both for the sample

containing HEC alone and for the sample containing HEC and mucin together.

3. Evaluation:


4. Calculate the value of ηb and ηb rel.

HEC conc. (%) ηb(mPa*s)

ηb rel (-)

1

2

3

5

5. Plot ηb and ηb rel against the HEC concentration. Use a different y-axis for the ηb (left

side y-axis) and the ηb rel (right side y-axis) values.

Evaluation:


Appendix


A.1. Helios Alpha UV-Vis spectrophotometer

The spectrophotometer is an optical instrument, which measures the intensity of

monochromatic light and its changes.

Spectrophotometers can be classified on the basis of:

– applied wavelength:

ultraviolet

visible

infrared

– operation way:

single beam

double beam

The Helios Alpha UV-Vis spectrophotometer (Figure A.1.1) is a double beam

spectrophotometer, which means the optical system divides the light from the light source into

two paths, one of them goes through the reference, and the other one goes through the sample.

This operation way allows the simultaneous measurements of light intensity passing through

the sample and the reference.

Figure A.1.1: Unicam Helios Alpha UV-Vis spectrophotometer


Parts list:

– light source (wolfram, deuterium lamp, λ = 190-1100 nm, stepwise: 2 nm)

– sample cell with holder (quartz cuvettes)

– monochromator

– detector

– display

Operations:

– scanning mode (SCAN)

absorbance (and its 1st-4th derivative)

transmittance

intensity

– at fixed wavelength

absorbance

transmittance

concentration (knowing the correlation between the concentration and the absorbance).

– plotting of the calibration curve


A.2. Anton Paar RHEOLAB MC 1 rheometer

The Anton Paar Physica RHEOLAB MC 1 rheometer is a rotational rheometer, which can be

used to measure the flow curve, yield point, structure breakdown and recovery (CREEP test),

viscosity of Newtonian and non-Newtonian systems. The instrument can be used in CSR

(controlled shear rate) and CSS (controlled shear stress) mode, too (Figure A.2.1).

Figure A.2.1: Anton Paar Physica RHEOLAB MC 1 rheometer

The measuring device is a concentric cylinder. The sample is placed in a gap between a fixed

cup and a rotating cylinder (Searle system). The measuring controller developed for the

device is based on a dynamic system which consists of a drive unit and an optical encoder

without gear wheel and mechanical power converter, therefore it measures the rotary torque

without deformation, so without loss. The measuring controller is such that the revolving

body rotates at the desired speed and it measures the rotary torque – which comes from the

flow resistance of the sample – acting on the measuring body. The investigation of shear

stress or the CREEP test is also possible by setting the desired shear stress, and the shear

deformation of the sample is measured through the angle rotation.

Controlled shear investigations (CSS) for the determination of the flow properties of

plastic materials can also be performed by the Anton Paar Physica RHEOLAB MC 1 device,


which allows the accurate measurement of the flow point without deformation, so without the

shearing of the internal structure of the sample (rotation).

Handling of the device, measurement:

1. In case of the investigation of emulsions, sample holder "Z3" is filled hermetically with

the sample up to the inner mark (Figure A.2.2).

1. In case of the investigation of the gel-forming of polymers and the investigation of

ointments, sample holder "Z4" is filled hermetically with the sample up to the inner

mark (Figure A.2.3).

2. The cylinder measuring head is fitted and fixed with the help of the retaining ring.

3. The device is switched on at the back.

4. The "PROG" function is chosen and the "OK" button is pressed.

5. In case of the investigation of emulsions, the "PROG 02" function is chosen and the

"OK" button is pressed.

5. In case of the investigation of the gel-forming of polymers, the "PROG 01" function is

chosen and the "OK" button is pressed.

6. The measurement is started by pressing the "ST" (START) button.

7. A piping sound will indicate the end of the measurement.

Figures A.2.2 and A.2.3: Measuring heads "Z3" and "Z4"

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