1

Food Chemistry Practical Notes

Analysis of colorants in beverage products

Introduction
The objectives of this experiment are to:

Determine the concentration of the colorant additives in a beverage;

Establish whether the product conforms the current regulatory standards;

Provide experience in using a spectrophotometer. This is one of the instruments used most
widely in the analysis of food molecules;

Illustrate the Beer-Lambert Law; and

Apply this law to the determination of the concentration of colorants in a food.

Background
When light is shone through a coloured solution some light is absorbed by the solution and some
passes straight through. We see the light that passes through. A red solution is red because red light
passes through and most other wavelengths are absorbed.
Absorbance and transmittance
The spectrophotometer actually measures the transmittance and then calculates what we are really
interested in, which is the absorbance (the internationally accepted abbreviation is A). Older
instruments will provide us with the option to record either transmittance or absorbance. The latter is
all that we need because it is directly related to how much of a component is present. Note also that
the term optical density is an older name and has now been replaced by absorbance.
Beer-Lambert Law
The amount of light absorbed by a solution is proportional to the concentration of the solution.

A = c l
where is the extinction coefficient
c the concentration; and

the length of the light path (cm)

Note that the units of c will depend upon those of the value used.

Experimental procedures
Prior to carrying out any of the further steps check that the spectrophotometer and weighing balance
are turned on to allow these to warm up for at least thirty minutes.
Note that there are spaces on the attached form for recording of laboratory data. This form will
become the basis of your report for the practical.
1.

2.

The products are provided as powder and so you need to dissolve these. So firstly look
at the level, decide how much you will weigh and the volume to be prepared. Some of the
aspects you may consider are:
The procedure recommended on the product label

3.

If the label does not indicate how much to weigh then check whether there is
information on serving size, number of servings in the package and the total weight in the
pack. These can often tell us the concentration.

4.

For the measurements to be made in the spectrophotometer we need only 3.0 mL.
Therefore it makes sense to prepare either 50 or 100 mL.

5.

The analysis you are doing will be used to establish regulatory compliance. Therefore
also ensure that your plan gives you good reliability in your measurements. So for example,
you need to be able to trust the accuracy of the weighing and the volumes being prepared.
Consider what is the most reliable glassware to use for this purpose.

Measurements using the spectrophotometer

Your demonstrator will explain the Varian Cary IE Spectrophotometer and the associated Varian
software. Briefly the procedure involve:
1.

Turn on the instrument to allow a period of at least 15 minutes warm-up.

2.

Using the Set-up option of the Scan software, select the wavelengths you want to scan.
The instrument is able to cover the whole visible spectrum (350 to 800 nm wavelength) as well
as the ultraviolet (UV) in the range of 200-350nm. As we are interested in visible colorants,
scan the wavelengths for the visible region after entering the highest first as this is where the
instrument commences. Select a scan rate of 200nm/min.

3.

Once the software set-up options have been selected we need to balance the light beams
that are passing through the two positions where we place our blank or reference and also our
sample. To zero the instrument, place two cuvettes both containing distilled water into the
positions.

4.

To record the spectrum of the beverage sample, place a cuvette of the solution into the
position closest to the front of the Spectrophotometer. After closing the lid start the scan

5.

Save the scan as this can also be plotted using Excelsoftware as long as we save it into a
CSV file format.

6.

Inspect the spectrum and then record the maximum(peak) wavelength for each major
absorption band.

7.

Using the Simple Reads software, record absorbance measurements of the sample at each
wavelength after adjusting Zero in between readings.

Table 1

Selected literature data for synthetic colorants

Additive Name

Tartrazine
Quinoline yellow
Sunset yellow FCF
Azorubine (Carmoisine)
Amaranth
Ponceau 4R
Erythrosine
Red 2G
Allura red AC
Patent Blue V
Indigotine
Brilliant blue FCF
Green S
Fast green FCF
Brilliant black BN
Brown FK
Brown HT
Notes

1
2

INS
Number

CI
designation
(Marmion3)

FD and C designation
(Marmion3)

max
(nm)
(Socaciu2)

Extinction
(1%, 1cm)
(Socaciu2)

max
(nm)
(Marmion3)

Extinction
(g per L)
(Marmion3)

102
104
110
122
123
124
127
128
129
131
132
133
142
143
151
154
155

19140
47005
15985
14720
16185
16255
45430
18050
16035
42051
73015
42090
44090
42053
28440
NA
20285

Yellow No 5
D & C Yellow No 10
FD & C Yellow No 6

426
413
481
516
520
506
526
520
520
638
610
630
608

530
865
555
510
440
430
1100

428
413
484

53
92
55

527

110

2050
480
1630

502

55.6

610
630

47.8
164

625

156

FD & C Red No 2
FD & C Red No 3
FD & C Red No 40
FD &C Blue No2
FD &C Blue No1
FD & C Green No 3

570

530

Not all of these colorants are currently approved for use in Australia, NA not available
Socaciu, C. 2008 Food colorants: Chemical and functional properties, CRC Press, Boca Raton, Florida, USA p 540.
Marmion, DM. 1979. Handbook of U.S colorants for foods, drugs, and cosmetics. New York. Wiley.

Food Chemistry - Analysis of colorants in beverage products

Form for recording of laboratory data
This form should be attached to your final report as an appendix. Remember to include units
when you record your results and in the calculations wherever appropriate.
Your name:
Group:

Student number:
Partner(s) for this exercise:

Name of your food product:

Record here any label information from your product which may be useful when you write your
report. You may want to include relevant nutrition panel data and the ingredient list:

From the spectra determine the wavelength value ( max) for each peak and record these.
Based upon the literature values given (Table 1) determine which colorants you appear to have
found in each of the products.

Sample

max values

Colorants found

Then for each peak you have identified calculate the concentration of the colorant present.
These calculations use the equation

A = c l
which you need to rearrange because we want to calculate the concentration.
Remember that the amount you calculate will be expressed in units corresponding to those of
the extinction value you select from Table1 .Attach your calculation and complete the
following table. Note that in expressing results you need to include units. You have flexibility in
choosing units consider using the same units as the reference values that you will compare your
data with.
Product

Colorant

Concentration

Conclusion: (Include another page if required.comment on your results in relation to any

relevant aims on page one of the notes).