Chukwuemeka Nwabugwu

CHEM 4553
HPLC REPORT
*Dr. Murray
HPLC ANALYSIS OF VITAMINS A AND D IN SOFTGEL CAPSULES

General Information
The title of this experiment is the HPLC Analysis of Vitamins A and D in
Softgel Capsules.

The date the experiment started was February 1st, 2011. The date the
experiment ended was February 10th, 2011.

The date the write-up was completed was February 28th, 2011.

Purpose
The purpose of this experiment is to gain an understanding of the way in
which a polarity separation is optimized using gradient evolution.

In this experiment, we will be using a UV/Vis detector and monitoring the

absorbance of the analysis (Vitamins A, D2 and D3) as it migrates through the Liquid
Chromatogram system.

The use of Beer-Lambert law holds in this case as well, and a calibration plot
can be constructed from which the concentration of an unknown can be determined.

Introduction
From an initial perspective of the analysis of Vitamins A, D2 and D3, it would
appear that a Gas Chromatogram would be more precise in this analysis, but the
HPLC provides separation of mixtures of semi-volatile organics.

HPLC means High Performance Liquid Chromatography. There are several

examples of Liquid Chromatograms stationary phases, but the most common are the
normal and reverse phase. In normal phase liquid chromatography, the stationary
 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray
phase is polar solvent and the mobile phase is non-polar solvent, while the
stationary phase in the reverse phase liquid chromatography is non-polar solvent
and the mobile phase is polar solvent.

In reverse liquid chromatography, the stationary phase could be Silica (5 –

10 micrometers in diameter) that is coated with an alkane and the mobile phase
consists of a mixed aqueous/organic solvent such as methanol.

In the case of this experiment, the stationary phase is a solid (adsorbent)

known as Octadecylsilane-silica (C18) and the mobile phase is a liquid known as
water/acetonitrile; hence it is the reverse stationary phase.

The band broadening mechanisms in LC

Band broadening mechanism in Liquid Chromatography refers to the
broadening of the column band. In order to understand the process, we must first
understand what the multiple path of an analyte through the column packing means.

The rate as which the mobile phase passes through the column may vary
significantly across the column diameter, perhaps because of the particle shape,
porosity or bed structure. Hence, the components of the mixture might go through
multiple paths through the column. Band broadening is a result of the different flow
velocities through the column.

Another thing we need to realize is the rate of diffusion of the molecules. The
diffusion across the column is referred to as longitudinal diffusion. It is commonly
known that molecules disperse or mix due to diffusion. The longitudinal diffusion
can also lead to band broadening of the chromatographic zone.

The last thing we need to consider about band broadening is the kinetics of
mass transfer. This refers to the rate of diffusion (mass transfer) and adsorption
 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray
kinetics inside the particles. Adsorption kinetics is almost disregarded compared to
the rate of diffusion inside the particles because it’s very small. Therefore, the
slower the velocity of the mixture, the more uniformly analyte molecules may
penetrate within the particle. This would also lessen the effect of the different
penetration on the efficiency.

The equation used is from Beer-Lambert’s law, which is as follows:

Where A is Absorbance, ε is Molar Absorptivity, l is the Path Length and C is the

Concentration

Theory
Chromatography is a general technique that separates a mixture into its
individual components. High Performance Liquid Chromatography (HPLC) is used
to analyze liquid samples or the liquid extract of a sample.

The basis of HPLC consists of passing a sample (analyte mixture) in a high-

pressure solvent (called the mobile phase) through a steel tube (called a Column)
packed with sorbents (called the stationary phase). The analyte retention time
varies depending on the strength of its interactions with the stationary phase, the
ratio/composition of solvent used, and the flow rate of the mobile phase.

The Beer-Lambert Law governs the theory behind this analytical field of
chemistry. The law relates the absorption of light to the properties of the material
through which the light is traveling. The law states that there is a logarithmic
dependence between the transmission (or transmissivity), T, of light through a
substance and the product of the absorption coefficient of the substance, a, and the
distance the light travels through the material (i.e. path length), l.
 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray
HPLC could also be used to find the amount of different components in a
mixture and it works in the same way as a paper chromatograph. How fast each
component moves depends on their affinity for the mobile and the stationary
phases.

For example, in our experiment, the mobile phase was more polar than the
stationary phase so the polar components of the mixture moved with the mobile
phase from one end of the chromatogram to the other. We would expect that the
more polar compounds, Vitamin A, would come out of the column first. The last to
leave the column should be column D3 since it is the least non-polar.

Procedure

Figure 1: The schematic of an HPLC instrument

Ten softgel capsules of Vitamin A and D were measured. The mass of ten
capsules was 2.705g. The mass of one capsule was 0.2705g. The sample was
transferred into 25mL volumetric flask and added to it was 15mL of methanol. Then
filtered before injecting in the HPLC.

The method file is C:\Star1\Chem 4553\Chem 4553 method\Spring2011-1.mth

 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray

The mobile phase reservoir, as shown in Figure 1, contains the solvents to

give a suitable polarity for the mixture being analyzed. The pumps produce a
pressure of 15000kPa (hence the name High Pressure LC). The mixture is injected
using a syringe. The pumps force the mixture through the column. In the column, the
components of the mixture are separated due to their affinity for the polar solvent.
The components of the mixture with a high affinity (or attraction) for the polar
solvent would come out almost as soon as the polar solvent leaves the column.
While the component of the mixture with a lower affinity for the polar solvent lag
behind and hence, come out of the detector at a much later time.

The separated components of the mixture enter the detector. In the detector,
ultraviolet light passes through it. The light is at a set wavelength that is absorbed
by all the components. The time that each component takes to come out of the
column is called a retention time, which helps identify the components. The
component with the greater polarity (or affinity) for the mobile phase would come
out first followed by the less polar component. We are expecting the Vitamin A
compound to come out first because it is much more polar than Vitamin D.

The standard solutions of the Vitamins were prepared and ran through the
column. The results were used as a test bed to determine the components of the
Unknown. The retention time, peak area, width and concentration of the standards
were recorded. The information from the above was then used to find the
concentration of the components in the sample from the Unknown. A graph of Peak
Area (Counts) VS Concentration would serve us well for this purpose.

Results
The experiment took a week and a half to complete. We set out to analyze
samples of Vitamin A, D2 and D3. The type of liquid chromatography we did for the
experiment was the reverse phase liquid chromatography. The solvent for the
 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray
mobile phase was water/acetonitrile. This means that the components that came
out of the column first are more polar than the others, indicating its affinity for the
polar solvent, while the slowest is the most non-polar.
The summary of the data collected is seen in Table 1 and Table 2. Please view
the discussion and conclusion for explanation of the results.

Table 1: Data Collected From the HPLC for Vitamin A, D2 and D3.

TRIAL SAMPLE CONCENTRATION RETENTION PEAK WIDTH

TIME AREA
1 Vitamin A 25 mg/mL 4.045 mins 2205964 7.4nm
counts
2 Vitamin A 10 mg/mL 4.105 mins 18299 7.1nm
counts
3 Vitamin D2 10 mg/mL 12.470 mins 3986 13.9nm
counts
3 Vitamin D3 10 mg/mL 13.611 mins 14730 18.0nm
counts
4 Vitamin D2 25 mg/mL 12.345 mins 6908 16.2nm
counts
4 Vitamin D3 25 mg/mL 13.485 mins 29039 18.5nm
counts

Table 2: Data Collected From the HPLC for the Unknown Mixture
COMPONENT CONCENTRATION RETENTION PEAK AREA WIDTH
TIME
Component 1 6.9898 mg/mL 4.708 mins 19426 counts 7.7 nm
Component 2 22.9774 mg/mL 11.887 mins 6514 counts 15.1 nm
Component 3 12.0735 mg/mL 13.576 mins 16708 counts 20.6 nm

Discussion and Conclusion

We set out to measure the elution order of the Vitamins in the unknown
sample. I gained more experience working with the HPLC instrument, even though
 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray
we had a lot of trouble with getting the concentration right. The concentration was
high at the beginning so we diluted the standards by more than half. We did about
10 trials for this experiment. We could not get the HPLC to run properly, so we had
to settle for two points from the spectrum of each Vitamin to graph our standard
calibration curves.

From the Tables represented in the data section, it shows that Vitamin A was
Component 1, Vitamin D2 was Component 2 and Vitamin D3 was Component 3 from
the unknown mixture. The elution order of the vitamins were Vitamin A @
4.708mins, Vitamin D2 @ 11.887mins and Vitamin D3 @ 13.576mins.

This is because Vitamin A is the most polar of the three vitamins. Thus
Vitamin A had the greatest affinity for the mobile phase in the liquid chromatogram.
The mobile phase in our reverse phase liquid chromatogram was a polar molecule
of Water and Acetonitrile.

The concentrations of the Vitamins in the unknown were calculated using

their peak counts and interpolating the value of concentration from the graphs. In
Graph 1: Peak Area VS Concentration for Vitamin A; the equation of the line was y =
145844x – 1E+06. The peak area for Component 1 (Vitamin A) was 19426 counts.
Extrapolating from the graph gave us a concentration of 6.9898 mg/mL. This
concentration is very low, but the intensity of the peak as shown in the spectrum is
lower than the intensities of the Vitamin A runs. In Graph 2: Peak Area VS
Concentration for Vitamin D2; the equation of the line was y = 194.8x + 2038. The
peak area for Component 2 (Vitamin D2) was 6514 counts. Through interpolation,
the value of concentration was 22.9774 mg/mL. The equation of the line for Graph
3: Peak Area VS Concentration for Vitamin D3; was y = 953.93x + 5190.70. The Peak
Area for Component 3 (Vitamin D3) was 16708 counts. Through interpolation, the
concentration of Vitamin D3 in the Unknown was 12.0735 mg/mL.
 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray
I would conclude that only the concentration of Vitamin A was determined
correctly. The spectrum shows that the concentration and intensity match the data
collected, unlike the spectrum of concentration and intensity for Vitamin D2 and D3.

Bibliography
1. Rapid Analysis of Water-Soluble Vitamins Using Smartline HPLC by KNAUER

2. HPLC Assay of Water-Soluble Vitamins, Fat-Soluble Vitamins, and a

Preservative in Dry Syrup Multivitamin Formulation

3. www.wikipedia.org

Additional Questions
1) In the analysis of Vitamin A2, after we varied the chromatogram
conditions, we observed that the peak at 2mins was at a greater intensity
than the intensity of the initial settings.

Also, the peak at 4mins was at a lower intensity than the intensity of the
initial settings. I think the affinity of the components of the mixture
changed as a result of the change in chromatogram settings.

2) The paper I read is cited as [1] in the bibliography. In the article, Vitamins
C and four B – complex vitamins in micronutrient tablets were achieved
on a C18 phase by use of Smartline HPLC and SmartMix static mixer.

The analysis of water-soluble vitamins using traditional reversed-phase

HPLC was done because the highly polar compounds are not retained on
conventional silica C18 columns. I think this is where they went wrong.
 Chukwuemeka Nwabugwu
CHEM 4553
HPLC REPORT
*Dr. Murray
First of all, they used modified silica C18 hydrocarbon as opposed to the
Octadecylsilane-silica we used in this experiment. Then they carried out
the experiment in reverse phase Liquid Chromatography as opposed to
normal phase Liquid Chromatography.

The other paper I read is cited as [2] in the bibliography. In the article,
fat-soluble vitamins were extracted with DMSO and ethyl acetate. They
should have used Methanol and Acetonitrile.

3) Yes. This is because the wavelength for absorption of Ultraviolet light is

between 300 – 400nm. Putting the wavelength at 270nm is below the
range of detection.

4) The elution order if it were normal phase would be Vitamin D3 first, then
Vitamin D2 second and Vitamin A last. This is because the stationary
phase is the polar and the mobile phase is non-polar. Vitamin D3 has the
greatest affinity for the non-polar solvent.

5) Five standards are best to produce an accurate result in HPLC. The reason
is because you have a better chance of having a smaller Relative Standard
Deviation of the five results. Also, you would be able to fit the components
of the Unknown in your calibration curve.