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EXTRACTION AND DETERMINATION OF CAFFEINE CONTENT USING POLAR-

NONPOLAR SOLVENT TECHNIQUE.

Method · September 2017

DOI: 10.13140/RG.2.2.12335.64165

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 EXTRACTION AND DETERMINATION OF
CAFFEINE CONTENT USING POLAR-
NONPOLAR SOLVENT TECHNIQUE

AUTHORS: D.K. NKANSAH, O.A.K. AMANO, E. APPIAH-DANQUAH, Department of Chemical Engineering, KNUST.
E. ADOM +233549052820
DISCLAIMER
These method(s) involve the use of hazardous materials, operations and equipment. These test
method(s) may not address all of the health and safety problems associated with its usage. The
user is responsible for establishing appropriate safety and health practices and to determine the
applicability of regulatory limitations prior to performing this test method(s).

Access to and use of these test method(s) shall impose the following obligations on the user. The
user is granted the right, without any fee or cost, to use, copy, modify, alter, enhance and share
these methods for any purpose whatsoever, except commercial sales, provided that this entire
notice appears in all copies of the methods. Furthermore, the user agrees to credit the authors in
any publications that result from the use of these Methods. The names Biotechnology Laboratory
and/or The authors, however, may not be used in any advertising or publicity to endorse or
promote any products or commercial entity unless specific written permission is obtained from the
authors. Mention of trade names or commercial products does not constitute endorsement or
recommendation of use.

IN NO EVENT SHALL THE AUTHORS BE LIABLE FOR ANY SPECIAL, INDIRECT OR

CONSEQUENTIAL DAMAGES OR ANY DAMAGES WHATSOEVER, INCLUDING BUT NOT
LIMITED TO CLAIMS ASSOCIATED WITH THE LOSS OF DATA OR PROFITS, WHICH MAY
RESULT FROM AN ACTION IN CONTRACT, NEGLIGENCE OR OTHER TORTIOUS CLAIM
THAT ARISES OUT OF OR IN ASSOCIATION WITH THE ACCESS, USE OR PERFORMANCE
OF THESE METHODS.

1
Contents
1.0 Introduction ............................................................................................................................................ 3
1.1 Structural, Physical and Chemical Properties ..................................................................................... 4
2.0 Scope ....................................................................................................................................................... 5
2.3 Terminology ........................................................................................................................................ 5
3.0 Significance and use ................................................................................................................................ 5
4.0 Interferences ........................................................................................................................................... 6
5.0 Apparatus ................................................................................................................................................ 6
6.0 Reagents and materials........................................................................................................................... 7
7.0 Environmental Safety and Health (ES&H) Considerations and Hazards ................................................. 7
8.0 Test sampling .......................................................................................................................................... 7
9.0 Procedure ................................................................................................................................................ 7
9.1 Dissolution of Caffeine in Water ......................................................................................................... 7
10.2 Extraction of Caffeine Using Chloroform .......................................................................................... 8
10.3 Crystallization of Caffeine ................................................................................................................. 8
11Calculations ............................................................................................................................................... 9
12 Report Format .......................................................................................................................................... 9
13 Quality Control ......................................................................................................................................... 9
14 Appendices ............................................................................................................................................. 10
15 References ............................................................................................................................................. 11

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Laboratory Analytical Procedure
1.0 Introduction
Naturally, caffeine is available in coffee and cocoa beans, kola nuts, tea leaves and a variety of

exotic berries in a substantial amount. Caffeine is mostly used in pharmaceutical industry as

caffeine sodium benzoate, caffeine citrate and in combination with painkillers such as aspirin and

acetaminophen (Sama et al., 1994). Apparently, caffeine is widely consumed in the form of a

beverage all over the world, it is a cardinal stimulant of the central nervous system (CNS) with

specific action on blood vessels (Rogers and Dernoncourt, 1998). Extraction involves the

separation of a component from a matrix. The initial solvent used in the extraction of caffeine

from a given sample is water. Caffeine is sparingly soluble in water at ambient temperatures (2g/

100 mL) but highly soluble in water at 100 oC with a yield of 66g/ 100 mL (Subila and Shirley,

2016). Meanwhile, the solubility of caffeine in chloroform is relatively high at ambient

temperature. Therefore, caffeine can be easily extracted from an aqueous solution sample by the

use of chloroform. The chloroform - caffeine mixture can then be separated by utilizing their

variation in density. Chloroform is insoluble in water and has a higher density compared to water,

this makes it form a layer underneath water and can be separated using a separatory funnel.

Residual water is separated by filtering the chloroform through reverse-phase filter paper with the

aid of a vacuum pump. The reverse-phase filter paper allows the nonpolar solvent, thus chloroform,

to be filtered while water which is the polar solvent is retained. The caffeine is then crystallized

on a watch glass. The crystallized caffeine can be isolated and quantified using UV/ Vis

Spectrometry and chromatographic methods.

3
1.1 Structural, Physical and Chemical Properties
Molar mass: 194.19 g/mol
IUPAC ID: 1,3,7-Trimethylpurine-2,6-dione
Melting point: 238 °C
Boiling point: 178 °C
Density: 1.23 g/cm³
Formula: C8H10N4O2

1.2 Portions of this procedure is similar to ASTM F2988 - 12

1.3 This LAP provides an easy and efficient method for extraction of caffeine.

4
2.0 Scope
Extraction of caffeine using polar-nonpolar solvent technique.

2.1 All analyses should be conducted in accordance with stipulated guidelines established in an

appropriate laboratory specific quality assurance plan (QAP).

2.2 Results of the weight extraction and percent yield can be calculated using this method.

2.3 Terminology
2.4 Extraction: This involves the withdrawal of an active component or waste substance from a

solid or liquid mixture with a liquid solvent.

2.5 Filtration: This is the process of removing suspended solids from liquid by passing the liquid

through a permeable fabric or porous bed of materials.

2.6 Evaporation: The process in which all or a portion of a liquid is turned into vapour in order to

increase concentration of a solid component in a mixture.

2.7 Mixture: A combination of two or more substances that are mixed together but not chemically

bonded.

2.8 Boiling point: This is known to be the temperature at which the vapour pressure of the liquid

is equal to the atmospheric (or applied) pressure.

2.9 Concentrated solution: A solution in which the stable solubility limit has been reached.

3.0 Significance and use

3.1 This method is applied, in conjunction with other procedures for extraction, isolation and

quantification of caffeine.

5
4.0 Interferences
4.1 Tannins can interfere with extraction of caffeine because of its solubility in both water and

organic solvents. A weak base such as calcium carbonate or sodium sulphate can be added to

breakdown tannins ester bonds into glucose and calcium or sodium salt of gallic acid, both of

which will not be extracted into the organic solvent.

5.0 Apparatus
5.1 1000 mL Beaker 5.10 Stirring rod

5.2 250 mL Graduated Cylinder 5.11 125 mL Erlenmeyer flask

5.3 Funnel 5.12 Ice water bath

5.4 Hot pads 5.13 Whatman filter paper

5.5 Distilled Water 5.14 Watch glass

5.6 Separatory funnel stand 5.15 Vacuum flask

5.9 Hot Plate 5.16 500 mL separatory funnel

5.7 Analytical balance 5.17 Vacuum pump

5.8 Reverse-phase filter paper 5.18 Buchner funnel

5.9 Pipette

6
6.0 Reagents and materials
6.1 Chloroform, (CHCl3)

6.2 Anhydrous Sodium Sulphate, (Na2SO4)

7.0 Environmental Safety and Health (ES&H) Considerations and Hazards

7.1 Adhere to all applicable Material Safety Data Sheet (MSDS) chemical handling techniques.

7.2 These techniques may not address all of the safety concerns associated with the use of this

procedure. It is the duty of the user of this technique to establish suitable health and safety

measures and determine the appropriateness of regulatory limits before use.

8.0 Test sampling

Samples must be obtained using a reliable sampling procedure and stored at room temperature

throughout the analysis.

9.0 Procedure
9.1 Dissolution of Caffeine in Water
9.1.1 Obtain a 1000 mL beaker.

9.1.2 Weigh 25 g of sample, W0 into the beaker using an analytical balance.

9.1.3 Add 250 mL of distilled water to the beaker.

9.1.4 Add substantial amount of Anhydrous Sodium Sulphate to the solution.

9.1.5 Boil the beaker containing the sample on a temperature controlled water bath for 30 minutes

at 100 oC while stirring occasionally intermittently.

7
10.1.6 Remove the beaker from the heat source after the boiling period and allow to cool to ambient

temperature.

10.1.7 Using vacuum filtration, filter the solution through Whatman filter paper to remove any

solid particles.

10.2 Extraction of Caffeine Using Chloroform

10.2.1 Transfer the solution obtained from step 10.1.7 above to a 500 mL separatory funnel. Add

100 mL of chloroform and swirl gently for 5 minutes.

10.2.2 Allow the chloroform extract to settle to the bottom. Carefully drain the chloroform layer

into a beaker.

10.2.3 Filter the chloroform-caffeine solution through reverse-phase filter paper under vacuum.

This will allow the chloroform to filter through but will trap any water and residual matter.

10.2.4 Transfer the solution into a 125 mL Erlenmeyer flask.

10.3 Crystallization of Caffeine

10.3.1 Place the chloroform solution over a water bath at a temperature of 62 oC in a fume hood.

The boiling point of chloroform is known to be 62 oC.

10.3.2 Evaporate the solution down to approximately 20 mL. Remove from the heat source

afterwards.

10.3.3 Weigh a clean watch glass and record its weight (W1).

10.3.4 Fill the watch glass with a portion of concentrated caffeine solution. Place the watch glass

over the water bath to evaporate. Repeat this process until all the concentrated solution is finished.

10.3.5 Remove the watch glass from the water bath and let it cool. Wipe the moisture from the

bottom of the watch glass.

8
10.3.6 Reweigh the watch glass (W2) to give the weight of the caffeine.

10.3.7 The percentage of caffeine can now be calculated.

11Calculations

11.1 Weight of caffeine, W3= W2-W1

11.2 Percent yield of caffeine, Y can be calculated as:

(𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑐𝑎𝑓𝑓𝑒𝑖𝑛𝑒, 𝑊3 )
𝑃𝑒𝑟𝑐𝑒𝑛𝑡 𝑌𝑖𝑒𝑙𝑑, 𝑌 = 𝑥 100%
(𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒, 𝑊𝑂 )

Where:
Wo = Weight of sample
W1 = Weight of watch glass
W2 = Weight of watch glass with caffeine
W3 = Weight of caffeine

12 Report Format
12.1 Report the results of weight of caffeine in grams (g) and yield of caffeine in percentage.

12.2 Express percent to the nearest 0.01.

12.3 For replicate analyses of the same sample, report the average value obtained.

13 Precision and Bias

13.1 The precision and bias of polar-nonpolar solvent extraction technique has not been

determined.

13 Quality Control
13.1 Replicates: Run all samples at a minimum of duplicate.

9
13.2 Method verification standard (MVS): Run reference sample with every batch sample. Check

published data to know the expected results for the analytical sample(s).

13.3 Sample size: Determined by user of this method.

13.4 Sample storage: Not applicable.

13.5 Relative percent difference criteria: Not applicable.

13.6 Sample storage: Not applicable.

13.7 Standard storage: Not applicable.

13.8 Definition of a batch: A number of samples analyzed and recorded together. The maximum

size of a batch will be restricted by equipment limitations.

13.9 Control charts: MVS material should be controlled and charted for reproducibility

verification.

13.10 Others: Caffeine samples have high potential of gaining or losing moisture when in contact

with air. During the weighing steps, minimize the amount of time the sample is opened to air.

14 Appendices
None

10
 15 References
1. Sama Venkatesh, Swamy M. M., Reddy Y.S.R, Sursh B. and Suresh B. and Sethuraman M.,

July 1994. “A Simple Method for Determination of Caffeine Content in Tea Samples.”, Ancient

Science of Life, Volume XIV, No. 1 & 2, page 35- 38.

2. Rogers P. J. and Dernoncourt C., April 1998. “Regular caffeine consumption: a balance of

adverse and beneficial effects for mood and psychomotor performance.” Pharmacol Biochem

Behav; 59(4):1039-45.

3. Subila S. and Sirley Navis, December 2016. “Determination of Caffeine in Different Tea

Samples.”, IOSR, Journal of Applied Chemistry (IOSR - JAC), Volume 9, Issue Version 1, page

75-78. www.iosrjournals.com

4. ASTM F2988-12, Standard Specification for Commercial Coffee Brewers, ASTM International,

West Conshohocken, PA, 2012, www.astm.org

5. Jenway, February 2014. “Determining the Concentration of Caffeine Using UV/Vis

Spectrometry.”, Jenway, Staffordshire, United Kingdom.

https://www.azom.com/article.aspx?ArticleID=10654

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