UV/VIS Application Note

Calibration with caffeine

Summary Sample preparation

Caffeine is most commonly used primary standard. Preparation of stock solution
In this application it is used to verify: Caffeine standard 1000 ppm (mg/L):
1. Wavelength accuracy in UV region at 275 nm Weigh 100 mg of caffeine standard in 100 mL
2. Micro volume techniques using two different volumetric flask. Dissolve the standard by taking
sample volumes (1.0 L, and 3.0 L) sufficient amount of deionized water. Fill up to the
accuracy and reproducibility volume of 100 mL with deionized water.
3. Path length accuracy
This is achieved by using 3 different path lengths Preparation of standard solution
(1 cm, long and short path length) for 3 different Prepare 3 different concentration ranges of standards
concentration range (low, mid and high) standard as mentioned in table1, 2 and 3 using the 1000 ppm
solution. Here a series of standard concentration standard stock solution.
ranging from 2 – 1000 mg/L are prepared. They Standard Volume stock Water to final
are intended to be used to create a calibration curve concentration
solution (mL) volume (mL)
of absorbance versus concentration of standard (mg/L)
solution. It is found to be linear and having a 0 0.0
coefficient of determination greater than or equal to 2 0.1
0.999. 4 0.2
50
6 0.3
8 0.4
Sample
10 0.5
Caffeine standard, Sigma Aldrich, No. C 6035 Table1: Low concentration range caffeine standard

Standard Volume stock Water to final

concentration
solution (mL) volume (mL)
(mg/L)
0 0.0
10 0.5
20 1.0
40 2.0 50
60 3.0
80 4.0
100 5.0
Table2: Mid concentration range caffeine standard

Standard Volume stock

Instruments and Accessories Water to final
concentration
solution (mL) volume (mL)
 UV5Nano Spectrophotometer (ME-30254729) (mg/L)
 XP 205 Analytical balance (ME-11106027) 0 0.0
 Rainin pipettes 100 1.0
- 1000 L (ME-17011782) 200 2.0
- 10 mL (ME-17011790)
400 4.0 10
 12 x 50 mL volumetric flask
600 6.0
 6 x 10 mL volumetric flask
800 8.0
 2 x 100 mL volumetric flask
1000 10.0
Table3: High concentration range caffeine standard

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 Procedure Results
Fill a cuvette of 1 cm path length with low
UV/VIS Application Note

concentration of caffeine standards. Blank

measurement is performed using deionized water.
Sample volume used for mid concentration solution
is 3.0 L (long pathlength) and for high
concentration 1.0 L sample volume (short
pathlength) is used. Blank measurement is
performed using deionized water.
Note: To prevent sample carryover, clean the micro
volume platform after every measurement using a lint
free tissue wipe. A final cleaning is recommended Standard solution Coeffient of determination of
2
with distilled water. Do not use organic solvents or of caffeine R
abrasive cleaning agents.
Low Concentration 0.9997

Measurement Mid Concentration 0.9990

Method parameters High

0.9996
Concentration
Methods: Quant
Wavelength: 273 nm Conclusion
Background correction: 1-point Wavelength accuracy is possible with a Quant
Reference wavelength: 400 nm measurement on UV/VIS spectroscopy. Caffeine
No of standards: 6 standard solutions were found to be linear in the
Unit: mg/L concentration range of 2 - 1000 mg/L. Specified
Fit type 1: Linear acceptance criteria are met: the coefficient of
Measurement cell and path length (depending on the determination meets the requirements and
working solution concentrations) measurements are repeatable. Comparable results
were obtained at different sample volumes as well as
Low concentration: None
different path lengths. Calibration curve shows that it
1.0 cm
obeys Lambert-Beer law within the concentration
Mid Concentration: Micro volume platform
range.
Long path length
High Concentration: Micro volume platform Reference
Short path length S.Sethuraman et al., Analytical Method Development
and Validation Of Caffeine In Tablet Dosage Form By
Using UV/VIS Spectroscopy.
Acceptance Criteria
Coefficient of determination of Further information
2 0.999
(R ) www.mt.com/uv-vis

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