Lab 12 Vitamin C Quantification Using Fluorescence Spectroscopy and HPLC

Question 1

What can column chromatography be used for? [3]

Answer 1

To (1) separate, (2) identify and (3) quantify compounds in a sample.

Question 2

What is the principle of column chromatography?

Answer 2

• Sample is pumped in a solvent (mobile phase) at high pressure through a column with chromatographic packing material (stationary phase).
• Compound retention times vary depending on their interactions with the stationary and mobile phase.
• As compounds elute from the column, they are detected by a detector.

Question 3

What is reversed phase chromatography?

Answer 3

• Uses nonpolar column and polar mobile phase
• C18 molecules attached to silica column
• Nonpolar molecules elute later than polar molecules

Question 4

What are the main components in an HPLC system? [5]

Answer 4

• Solvent reservoir(s)
• High-pressure pump
• Column
• Injector system
• Detector

Question 5

If the stationary phase is C18 and the mobile phase is aqueous KH2PO4 and methanol, will vitamin C elute early or late?

Answer 5

• Early around 2.2 min since it is a fairly polar molecule with lots of hydroxyl groups

Question 6

How is [vitamin C] determined by HPLC?

Answer 6

• Use the peak area produced by HPLC software to construct a standard curve

Question 7

What is the principle of fluorescence spectroscopy?

Answer 7

1. Photons of light are absorbed by molecules
2. The molecules are elevated to a higher energy level
3. Once the excited molecules return to a ground level, energy is emitted

Question 8

The emission spectrum has lower energy than the absorption (excitation) spectrum.
True or False?

Answer 8

True.
Due to some loss of vibrational energy as a result of molecular collisions.

Question 9

The absorption (excitation) spectrum has lower energy than the emission spectrum.
True or False?

Answer 9

False.
The emission spectrum has lower energy than the absorption (excitation) spectrum, due to some losses of vibrational energy as a result of molecular collisions.

Question 10

What is a fluorophore? [3]

Answer 10

• Molecules which exhibit fluorescence
• Absorb light energy of a specific wavelength and re-emit at a longer wavelength
• Can form covalent bonds with some molecules, allowing them to be detected using fluorescence.

Question 11

Why do fluorophores have a high degree of resonance stability?

Answer 11

• They typically contain several aromatic groups and multiple conjugated double bonds, which have pi bonds.
  
  • Pi bonds are covalent chemical bonds where two lobes of an orbital on one atom overlap two lobes of an orbital on another atom.
    
    • This results in a high degree of resonance stability.
    • Such molecules are said to have low energy to begin with and can therefore absorb energy, resulting in a higher energy state.

Question 12

What are the components of a spectrophotometer? [4]

Answer 12

1. Light source
2. Monochromator - splits light into each colour
3. Adjustable aperture - selects wavelength
4. Detector - detects how much light is transmitted

Question 13

What are the components of a fluorescence spectrophotometer? [4]

Answer 13

1. Excitation source (xenon lamp) and a fluorophore
2. Monochromator + adjustable aperture
3. FIlter to isolate emission photons from excitation
4. Detector - registers emission photons and produces output

Question 14

Why are the transparent plates used in absorption spectroscopy not used in fluorescence spectroscopy?

Answer 14

• Reflections cause unspecific signals that significantly impair sensitivity.

Question 15

Fluorescence is read from the top down while absorbance is read from the bottom up.
True or False?

Answer 15

True.

Question 16

Fluorescence is read from the bottom up while absorbance is read from the top down.
True or False?

Answer 16

False.
Fluorescence is read from the top down while absorbance is read from the bottom up.

In a fluorescence measurement, the excitation light is directed onto the sample from above and the emitted light is detected from above. In an absorbance measurement, the light is directed onto the sample from below and the amount of light that passes through the sample and reaches the detector above is measured. So, in this context, “top down” means that the measurement is taken from above the sample, while “bottom up” means that the measurement is taken from below the sample.

Question 17

How can ascorbic acid be detected by fluorescence spectrophotometry?

Answer 17

• OPD and DHA bind to form a highly fluorescent tricyclic compound that becomes excited at 340 nm, and emits at 400 nm.

Question 18

Describe in detail the fluorescence spectroscopy method for vitamin C quantification.

Answer 18

In the fluorescence spectroscopy method, ascorbic acid is converted to cis-dehydroascorbic acid (DHA) in the presence of oxygen with carbon as the catalyst.

DHA then reacts with ortho-phenylenediamine (OPD) to give a fluorescent quinoxaline compound, measured at an excitation wavelength of 340 nm, and an emissions wavelength of 400 nm.

Borate is used in the blank buffer to bind to DHA so that it cannot react with OPD, thus allowing only contaminants in the samples/standards to be measured.

Question 19

Describe in detail the HPLC method for vitamin C quantification.

Answer 19

In the HPLC method, vitamin C is extracted from food samples and then filtered into HPLC vials that are placed into an autosampler that will deliver specific volumes to the HPLC column to be analyzed.

The HPLC column used for this experiment consists of silica coated with C18 molecules which makes the column hydrophobic or non-polar.

A polar mobile phase is then used to carry samples onto the column. This set up of having a non-polar column and polar mobile phase is known as reversed phase HPLC.

The polar mobile phase is usually water or a buffer solution, and then a less polar organic solvent such as methanol is gradually added to elute more hydrophobic compounds from the column.

A diode array detector (DAD) set at 260 nm is then used to detect the absorbance of compounds as they elute from the column, producing peaks.

These peaks can then be used to create standard curves to calculate the unknown concentration of compounds in samples.

Question 20

What will the ascorbic acid be converted to upon reaction with oxygen and acid washed carbon?

Answer 20

Dehydroascorbic acid (DHA)

Question 21

In spectrofluorometry, what wavelength does the compound in the sample absorb energy from?

Answer 21

Excitation wavelength = 340nm

Question 22

In spectrofluorometry, what wavelength is measured?

Answer 22

Emission wavelength = 400 nm

Emission wavelengths are longer than absorption wavelengths (because some energy was lost through heat or vibration)

Question 23

Why do you have to shake vigorously when you mix the charcoal with your sample?

Answer 23

To allow oxygen and carbon to catalyze the conversion of ascorbic acid

Question 24

What chemical is added to the blank buffer to inhibit the vitamin C derivative from binding to OPD?

Answer 24

Borate

Question 25

What is the purpose of the 10 min waiting period in spectrofluorimetric quantification of vitamin C?

Answer 25

Allows time for boric acid to bind to DHA

Question 26

Excitation wavelengths are […] than emission wavelengths and […] in energy.

Answer 26

Excitation wavelengths are shorter than emission wavelengths and higher in energy.

Question 27

What are the mobile phases used in the HPLC analysis of vitamin C?

Answer 27

methanol + potassium dihydrogen phosphate

Question 28

What HPLC data will you use to construct your vitamin C standard curve?

Answer 28

Peak area

Question 29

What is the purpose of metaphosphoric acid and acetic acid in HPLC quantification of vitamin C?

Answer 29

Extraction of vitamin C from sample