Name: Jayden Thies

Partner: Sydney Gibson

Chem 254-TA: Logan Walsh
Experiment 11: HPLC and the detection of Vitamins
Date: 3/8/18

Purpose
The purpose of this experiment is to make a series of dilutions for the vitamins thiamine,
pyridoxine, and niacinamide, determine the concentration of one of the vitamins in a
commercial vitamin tablet.

Theory
High performance liquid chromatography is a powerful technique which allows
researchers to separate mixtures of complex molecules, such as vitamins, with relative ease. It
contains some elements of both column and gas chromatography. The principles of HPLC are
similar except that HPLC gives better separation and detection of compounds in a shorter
period of time. Similar to gas chromatography, the sample is injected into the column and
detected as it exists. Its presence is recorded as a series of peaks on a chromatogram. HPLC
resembles column chromatography in that the mobile phase, or eluant, is a solvent or a solvent
mixture which carries the sample through the column. The column is packed with fine particles
which interact with the components in the sample to different extents which cause them to
partition themselves into separate fractions. Sample components are partitioned between the
immiscible liquid mobile phase and the liquid stationary phase that depends on the solubility of
each compound. The ratio of partitioning in this heterogeneous equilibrium is known as the
partition coefficient.
The stationary phase may be a solid as in column chromatography or a liquid organic
compound which is bonded to small beads of silica gel within the column. The small particle size
used as the stationary phase provides enhanced separation and sensitivity to the method.
However, the very small particle size also has a downside in that it severely restricts flow of the
solvent through the column. The composition of the mobile phase can remain constant during
the run or it can vary from beginning to end.
HPLC, unlike gas chromatography, can be applied to the separation of nonvolatile liquids
and solids as well as heat sensitive substances which decompose at elevated temperatures. It is
an extremely powerful technique used in industry and research in the analysis of
pharmaceuticals, nucleic acids, pesticides, vitamins, amino acids, etc. Identification with HPLC
can be at the ppm or ppb level. Consequently, HPLC is one of the most sensitive identification
techniques available to a chemist.

Reaction
No reaction.
Compounds Used:
Calculations:
Thiamine

Y=1.8209x+27.924
Y=25.47799
X=-7.49mg/L = -7.49ppm

(-7.49mg/L) x 0.1L = (-0.749mg/60mg) = 0.0125 x 100 = 0.12% Thiamine

Pyridoxine

Y=4.341x+51.686
Y=15.68
X=15.52mg/L = 15.52ppm

(15.52mg/L) x 0.1L = (0.155mg/60mg) = 0.0026 x 100 = 2.6% Pyridoxine

Niacinamdie

Y=3.5861x+3.1444
Y=25.48
X=-1.34mg/L = -1.34ppm

(-1.34mg/L) x 0.1L = (-0.003mg/60mg) = -0.000024 x 100 = 0.02% Niacinamide

Methods/Procedures
The experiment was followed verbatim to the lab manual found on pages 89-90 with no
exceptions.
As for safety precautions, acetonitrile is a flammable liquid. Keep away from flames and
hot objects. Both riboflavin and niacinamide are irritants to the eyes and can cause skin rash.
Wear gloves and safety glasses when handling.
Observations/Results
Everything in this experiment went as planned during this experiment but we had come
up with negative “x” values for thiamine and niacinamide due to the most likely scenario of the
standards being made wrong. We ended up with 0.12% composition for thiamine, 2.6% for
pyridoxine, and 0.02% for niacinamide. We were able to calculate these percentages using the
areas under the curves. The vitamin solution that was used was a pale orange color and smelled
of oranges.

Discussion/Conclusion
We were able to make the dilution of all three of the vitamins and calculate the percent
concentration of each of the vitamins in the 60mg vitamin tablets using the area under the
peaks and linear equation from the calibration curve.

Exercises

2. Which type of high performance liquid chromatography is most widely used? Explain its
characteristics.

Reverse phase chromatography has the widest range of applicants in terms of HPLC types.
Using a nonpolar organic chain that is bound to an inert silica surface as the stationary phase
and a mobile phase composed of aqueous-organic or aqueous polar solvents with different
levels of polarity throughout. The elution sequence progresses from most polar first to least
polar eluting through last.

4. Why is it necessary to degas the mobile phase?

The mobile phase of HPLC has the potential to trap air from the atmosphere and this trapped
air is released as small bubbles under high pressure. The release of these bubbles can lead to
signal noise or blockage of flow through the column. Degassing of the mobile phase is essential
to avoid complications.

5. Which is the most commonly used detector in HPLC and why?

UV-VIS is the most commonly used detector type in HPLC. This detector gives specific responses
to particular compounds which makes it ideal. Specifically, it absorbs wavelengths of
compounds in the same range as many organic compounds are found, making it useful for
analysis of organic compounds.

7. What is the difference between Isocratic and Gradient Elution.

These terms refer to the operation type. Isocratic refers to not changing the mobile phase
composition during the chromatographic run with the solvent consisting of a single compound
or a mixture of compounds in a fixed proportion. A gradient elution refers to a changing
solvent composition over the time of the run meaning that the solvent from the start of the run
will change at a consistent rate throughout the run time.

Calibration Curves

Niacinamide
2000
1800
f(x) = 3.59 x + 3.14
1600 R² = 0.99
1400
1200
Area

1000
800
600
400
200
0
0 100 200 300 400 500 600
Concentration (ppm)

Pyridoxine
2500

2000 f(x) = 4.34 x − 51.69

R² = 0.99
1500
Area

1000

500

0
0 100 200 300 400 500 600
Concentration (ppm)
 Thiamine
1000
900 f(x) = 1.82 x + 27.92
800 R² = 1
700
600
Area

500
400
300
200
100
0
0 100 200 300 400 500 600
Concdentration (ppm)