Experiment 6 HPLC Determination of Ascorbic Acid in Wine

Introduction

High Performance Liquid Chromatography is an analytical technique being used to

differentiate between different molecules and their concentrations based on their
composition and different affinities with the stationary and mobile phases. As the solution
enters the column, the molecules are being separated based on their affinities with the
stationary phase and mobile phase. Molecules that have a higher affinity with the mobile
phase will pass through the column more faster, whereas molecules that have a high
affinity with the stationary phase will remain in the column for a longer time, which enable
the separations to occur. In this experiment, reverse-phase HPLC was used and which
means that the stationary phase was non-polar and the mobile phase was polar.（Figure
1 shows a diagram of HPLC）

Figure 1. Systematic diagram of HPLC

Ascorbic acid is usually added into wine as it can act as an antioxidant. Which helps to
prevent against oxidative spoilage. It is often added to white wine or other wine with
lighter colour to prevent discolouration. Ascorbic acid is a crucial element in wine making,
which needs to be monitored to ensure the most desirable effect is achieved. HPLC was
a suitable technique for this experiment as ascorbic acid is a non-volatile and thermally
unstable molecule. A UV detector was selected particularly as ascorbic acid is a
conjugated system and it can absorb light.

Aims

In the experiment of ‘HPLC Determination of Ascorbic Acid in Wine.’ The concentration

of ascorbic acid in wine using reverse phase HPLC was firstly being determined. Then,
the concentrations were being determined using the calibration curve method.

Experimental
Equipment
Table 1. Conditions used for the HPLC analysis for ascorbic acid in wine.

Pump Marker：Shimadzu
Model no：LC-10AT

Column Marker：Ascentis

Dimension：7.5cm x 4.6mm x 2.7μm

Mesh：Silica gel

Stationary Phase：C18

Detector Marker：Shimazdu

Model：SPD-10A

Set wavelength：UV at 245nm

Mobile phase identity：0.3% w/v metaphosphoric

acid in deionised water

Flow rate：0.600 ml/min

Preparations of solutions
Solutions were prepared by making a stock solution of 1.00g/L of ascorbic acid with
0.3% metaphosphoric acid. Mass of 0.010g of ascorbic acid was weighed and made up
to 100ml with metaphosphoric acid. The stock solutions were diluted into standard
ascorbic acid concentrations of 10.0, 20.0 and 30.0 mg/L subsequently. The wine sample
was prepared by diluting it with 0.3% of metaphosphoric acid.

Experimental Procedure

The procedure detailed on pages 3 to 4 in the Chem30012 Laboratory Manual was

followed in determining the concentration of ascorbic acid in wine using reverse phase
HPLC. As well as in determining the concentrations using the calibration curve method.

Results and Discussion

Solutions were prepared according to their prescribed method and their peak areas were
measured by HPLC. （see below for table 2.）

Table 2. Calibration data from HPLC

Concentration [mg/L] Retention Time [s] Peak Area (10^3)

0 1. 1.715
1. 0.00489

2. 1.716
2. 0.00316

Mean：1.716 Mean：0.00403

10.0 1. 1.714
1. 0.64033

2. 1.714
2. 0.59473

Mean：1.714 Mean：0.61753

20.0 1. 1.712
1. 1.061

2. 1.711
2. 1.241

Mean：1.712 Mean：1.150

30.0 1. 1.709
1. 1.764

2. 1.709
2. 1.712

Mean：1.709 Mean：1.741
These results were then used to plot a calibration curve （Figure 1）, whose best fitting
line was used to determine the concentrations of ascorbic acid in wine.

Figure 2. Calibration curve of peak area vs. concentration for ascorbic acid.

Table 3. Table data for diluted wine

Runs of diluted wine Retention time Peak area (10^3)

1 1.674 0.412

2 1.674 0.386

3 1.674 0.346

The equation from the calibration curve from Figure 1 was then used to calculate the
concentration of ascorbic acid in the diluted wine.

Calculations
The peak area from run 1 was substituted into the equation to find the concentration of
ascorbic acid in the diluted wine.

y=17.192x

y=17.192 x 0.412

=7.08mg/L

Taking into account the dilution factor of 5, the concentration of ascorbic acid in the
undiluted wine was found.

7.08mg/L x 5 =35.42 mg/L

The same calculation method was used to calculated runs 2 and 3 of the diluted wine.
The results are shown in table 4.

Table 4. Wine analysis results for ascorbic acid

Run 1 Run 2 Run 3 Mean Stand. Dev. 95%
conf.interval

Conc. [mg/L] 35.42 33.18 29.74 32.78 2.86 High: 20.58

Low: 17.27
Standard deviation and the 95% confidence interval was calculated using excel.

Wine Ethanol Retention Time Peak Area (10^3)

1 1.517 0.731

The retention time for the diluted wine had a mean of 1.674 seconds, whereas the
retention time for the diluted wine sample containing ethanol was 1.517 seconds.

The experiment could be improved by rinsing the equipments more thoroughly in order to
obtain less impurities in the solutions were testing the results. It can also be repeated
several times to get a more accurate result. The injection time could occur more rapidly
after the solutions were prepared in order to obtain a more steady result. Air bubbles in
the syringe could have affected the measurements too. The weighing of the ascorbic acid
was 0.010g instead of 0.1000g might have also affected the result.

Questions
1. The standard method has separated the ascorbic acid well. Since in the HPLC the
higher the concentration of the ascorbic acid added, the higher the retention time and
the peak area. The concentrations in the three run times foe the wine analysis for
ascorbic acid were also similar. The method could be optimised further by adding
higher concentrations of the ascorbic acid into the wine sample and testing it in the
HPLC, the injection of each sample could be injected as soon as the solutions were
prepared, as well as to repeat the procedure a several times.

2. The difference in step 6 comparing to step 4 in the retention time was not significant,
in step 4 the average retention time was 1.674s and the retention time in step 6 was
1.517s. However, the difference in the peak areas for the wine ethanol sample was
doubled when comparing to the pure wine sample. Therefore it had the ability o
detect that the difference between metaphosphoric acid and the ethanol as the peak
area for the ethanol wine sample was double the size of the metaphosphoric acid
sample. It could be an issue if detecting a complex sample as it might damage the
machine itself by overlapping the retention time and the peak areas. The column also
has a chance to be blocked if a too complicated sample was being analysed. The
implications for a laboratory analysing multiple types of wine can be diluting them in a
lower concentration and extra and purify them before they get injected into the
machine.

Conclusion

The concentration of ascorbic acid in the wine sample was determined to be 32.78 mg/L
through the use of HPLC and constructing a calibration curve. The experiment outlined
that there was a difference in retention time between the pure wine sample （1.674s）
and the wine ethanol sample （1.517s）, which demonstrated that the effect of
concentration on the retention time. The results also identified that solutions with a higher
concentration resulted in a faster retention time due to the fact that the column being
loaded and an access of the analyte molecules flooding into the column.

References

Skoog, West, Holler, and Crouch, Fundamentals of Analytical Chemistry, 9th Edition,
2014, Chapter 33.