Transcript Slide 1

Determination of caffeine
using gravimetric and HPLC
analyses methods
Gravimetric Determination
of caffeine in tea bag
Principle
Caffeine in tea bag
isolation
2 gm
Identification by
Murexide test
Weighing Caffeine
Principle:
1. In addition to caffeine, tea contain catechins, phlobaphenes
(which gives tea its reddish brown color) and other water soluble
compounds.
2. Caffeine is the only component in tea whic is soluble in DCM
dichloromethane. Therefore, caffeine is separated from other
water soluble components by shaking tea aqueous extract with
DCM.
3. DCM is eveporated to dryness to leave out caffeine crystals,
which is weighted.
Shake with
DCM
Caffeine
migrates
to DCM
layer
Drawbacks and limitations:
• It is difficult to detect and /or quantify compounds, e.g., alkaloids, in complex
extracts or pharmaceutical preparations.
• Conventional color reactions are limited in that field.
HPLC
(High Performance Liquid Chromatography,
High Pressure Column Chromatography)
Alternative:
• Using chromatographic techniques, the mixture is separated in its individual components,
where each is detected and can be then quantified.
• Separation is done on chromatographic column as in the following video.
• To get a rapid and satisfactory results, columns of small particle size (3-5 µM) are used.
Because of the tiny particle size, a pressure is applied to force the mobile phase through the
column and get results in resonable time.
Conventional column chromatography
Stationary phase:
50 µm particle
Mobile Phase:
Driven by graphity
Sample size:
1-5 ml contain grams of analytes
Column material:
Glass
(afford atmospheric pressure)
Separated compounds are
monitored visibly or under UV
HPLC column
Stationary phase:
3-5 µm particle
Mobile Phase:
Driven by high pressure
from pump
Sample size:
10 – 50 µl contaim µg-mg
analytes.
Column material:
Stainless steel
(afford high pressure)
Separated compounds
are monitored by means
of detector
Play the video: https://www.youtube.com/watch?v=mPO7Tv2mIJU
Uploaded by Phenomenex
Play the video: https://www.youtube.com/watch?v=kz_egMtdnL4
Uploaded by: Royal Society of Chemistry
Computer
software
Principle:
• Sometimes it is difficult to separate the anayte of interest from the extract or other
sample components (matrix).
• HPLC allow separation of individual components, where detector signs each eluted
analyte as a peak.
• Analyte of interest could be detected by comparing the Rt of the peak to the Rt of the
standard analyzed in the same condition. (Qualitative detection)
(-)-epigallocatechin gallate (EGCG), (-)epigallocatechin (EGC),(-)-epicatechin
gallate (ECG), and (-)-epicatechin (EC),
as well as thermal isomers, including ()-gallocatechin (GC), (-)-Gallocatechin
gallate (GCG), and (-)-Catechin gallate
(CG).
• The peak area is directly proportional to the analyte
concentration.
• Setting a curve between concentration and peak area.
• Analyze the sample and determine the peak area of the
analyte of interest.
• By extrapolation of the area, one can determine the
concentration. (Quantitative determination)
Standard curve of caffeine standard
Peak Area
measured at 273 nm
700
y = 31.491x - 4.2308
R² = 0.9997
600
500
400
300
200
100
0
0
5
10
15
Concentration (mg/l)
20
25
Overlay of caffeine peaks
of different concentrations
Note:
Peak area α concentration
Standard curve of caffeine standard
Area
1.25
38.97
2.5
78.03
5
156.13
10
312.31
20
624.67
700
Peak Area
measured at 273 nm
Conc
mg/l
Area = slope x concentration + intercept
Y =ab+x
600
500
400
300
y = 31.491x - 4.2308
R² = 0.9997
200
100
0
0
5
10
15
Concentration (mg/l)
20
25
• How to set a calibration curve?
1) Prepare
stock soln.
2) Make
serial
dilutions
3) Inject each concentration and
determine the peak area
Caffeine
99.7% pure
High
concentration
Low
concentration
Standard curve of caffeine standard
Peak Area
measured at 273 nm
700
Area = slope * concentration + intercept
Y =ab+x
600
500
400
Y = 31.49 X – 4.230
300
y = 31.491x - 4.2308
R² = 0.9997
200
100
0
0
5
10
15
Concentration (mg/l)
• Area of unknown concentration: 227.97
• Y = 31.49 X – 4.230
• 227.97 = 31.49 X – 4.230
• 227.97 + 4.23 =31.49 X
• X = 7.37 mg/l
20
25
What students will do:
1. Some groups will prepare the serial dilutions of the
standard.
2. Others will preapre the tea extract.
3. Others will prepare the instant coffee extract
4. Last group will prepare samples of beverage.
5. Each group will take the table of (concentration vs
area) and set a graph using EXCEL software.
• Print the graph showing the trendline and its linear
regression equation and bring it before coming to
the lab.
6. Each group will be supplied with peak area of the
unknowns (tea, coffee, beverage) and asked to
calculate the amount of caffeine in the samples.
The method is adopted from the following sources:
1. Srdjenovic B, Djordjevic-Milic V, Grujic N, Injac R, Lepojevic Z (2008)
Simultaneous HPLC determination of caffeine, theobromine, and theophylline
in food, drinks, and herbal products. Journal of Chromatographic Science, (46),
144-149.
2. Naegele E. Determination of caffeine in coffee products according to DIN 20481.
by Agilent Technologies.
http://www.chem.agilent.com/Library/applications/5991-2851EN.pdf
3. Determination of caffeine in beverages by high performance liquid
chromatography.
http://www.oswego.edu/~kadima/CHE425/CHE425L/HPLC_08.pdf
A) Prepartion of the stock solution and the serial dilutions
1. Acuurately weight 10 mg caffeine .
2. Dissolve in about 50ml water for HPLC.
3. Transfer to a volumetric flask (100 ml) and complete to the mark
with water (HPLC grade). This represents (…?….
100 mg/l).
• (In lab, you have this stock solution prepared for you)
4. Make double fold dilutions.
• In series of test tubes add 5 ml of the stock soln + 5ml water
to give dilution A.
• Dilution B (5 ml dilution A + 5ml water (HPLC grade))
• Dilution C (5 ml dilution B + 5ml water (HPLC grade))
• Dilution D (5 ml dilution C + 5ml water (HPLC grade))
• Dilution E (5 ml dilution D + 5ml water (HPLC grade))
TIP:
. Begin with adding 5 ml of water in all the test tubes.
A
B
C
D
E
B) Prepartion of the tea and coffee extracts
1. Weigh 0.5 g tea or coffee powder. (In lab, This is preweighted for you)
2. Transfer to around 70 ml hot water, stirr and leave to cool down (at least 5 min).
3. Transfer to volumetric flask (100 ml) through filteration, wash the funnel and
cotton, and complete to the mark with water (HPLC grade).
4. Dilute to 20-fold (5 ml of the extract + 95 ml water) in another 100 ml
volumetric flask.
5. Filter around 2 ml of the extract through membrane filter.
C) Prepartion of the beverage for analysis
1. Get rid of gas bubbles by vacuum aspiration or ultrasonic.
• In our case the bottle is left open overnight.
2. Dilute to 20-fold. (5 ml of the beverage + 95 ml water).
3. Filter around 2 ml of the extract through membrane filter.
How to use Excel to make the standard curve?
1.
2.
3.
4.
Open new Excel sheet.
make a table of two columns corresponding to conc. and area.
Select the table
Insert ........ Scatter........
5. Select the cahrt then go to ..... chart tools.... Layout .......
Trendline ........ More trendline options
6. In the Format Trendline dialog box,
Select linear, display equation and the
R-square value
This will be your result
Students Data for the Standard Curve
Conc mg/l
3.125
6.25
12.5
25
50
100
Area
10994.67
21720.60
43892.48
88536.23
170023.73
337998.73
A. In excel sheet:
1. Set up a calibration curve of the above given data.
2. Determine the equation of the line and the R square value.
B. Print the graph and bring it next lab.
(Each group is reponsible to submit it next lab, to use it in calcultion)
Conventional column chromatography
Stationary phase:
50 µm particle
Mobile Phase:
Driven by graphity
Sample size:
1-5 ml contain grams of analytes
Column material:
Glass
(afford atmospheric pressure)
Separated compounds are monitored
visibly, under UV, or by TLC
HPLC column
Stationary phase:
3-5 µm particle
Mobile Phase:
Driven by high pressure
from pump
Sample size:
10 – 50 µl contaim µg-mg
analytes.
Column material:
Stainless steel
(afford high pressure)
Separated compounds
are monitored using
detector