Question6 A patient is tested for a deficiency of water - soluble vitamins (B1,

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Question6 A patient is tested for a deficiency of water - soluble vitamins (B1, B2, B3 and Vitamin C). A urine sample is collected and quantification is performed using a HPLC method. In addition to the four vitamins an internal standard, 5 - methyl cytosine, is also added. The structure of internal standard and vitamins are shown below; After a filtration step, samples were spiked with internal standard and run on a HPLC using normal phase Hydrophilic Interaction Liquid Chromatography (HILC) column. The mobile phase was 80:20 acteonitrile:aqeous buffer pH 6.2. a) Is the mobile phase used in this application weak or strong? Explain your logic. b) Two of the compounds above are quite acidic, and hence have two values for Log P (vitamins B3 and C). Does the second value (given In parentheses) correspond to the acidic or basic form of the compound? Explain. In the mobile phase suggested here which Log P value is most appropriate to consider for vitamins B3 and C? c) Use the log P values above to predict the elution order of the compounds on this HILC column with this mobile phase. d) Of the detectors listed below which would be most suitable for this analysis? Why?

Explanation / Answer

SOLUTION:

a)

A typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. To obtain reproducible results, at least 3% water should be maintained in the mobile phase. This amount of water is necessary in order to ensure sufficient hydration of the stationary phase particles. In addition, remember that, a higher concentration of organic solvent in the mobile phase will increase the retention.

Several polar, water-miscible organic solvents are possible to use in HILIC, although acetonitrile is by far the most popular. The solvent strength is roughly inverted to what is observed for RPLC separations, and the relative solvent strength can be outlined as:

• Acetone < acetonitrile < iso-propanol <ethanol < methanol < water

Acetonitrile, one of the weaker solvents in HILIC, provides a much higher increase in retention compared to methanol. The latter is yet a usable alternative, and may possibly provide a higher solubility for certain compounds, although it will give less retention. From an efficiency point of view, acetonitrile is typically the best choice.

b) The mobile phases had different buffer pHs and different percentage. when using a mobile phase having 30% acetonitrile with a buffer pH of 3.0. phosphate, a pro-drug with greater aqueous solubility and a marked relates pH with the pKa of a weak acid and pH 6.2-8.2 and pH 11.3-13.3. Mobile phases was dependent upon the type and amount of organic eluent. This had counteranion of the acidic modifier on the retention of ionized basic analytes. Effect of Salt, Buffer Concentration and Ionic Strength. Acetonitrile- Aqueous adjusted with phosphoric acid, pH=2.4 (80:20); flow rate, 1.0 ml/min.

c) Under HILIC conditions, a water-enriched liquid layer will be established within the stationary phase. The separation is achieved by partitioning of solutes from the eluent into this hydrophilic environment, a process that typically is exothermic. Hence, both hydrogen bonding, the extent of which depends on the acidity or basicity of the solutes, and dipole-dipole interactions, which depend the dipole moments and polarizability of the solutes, are factors governing retention. The primary function of HILIC stationary phases is thus to bind water.

d) Although of limited importance for the primary HILIC retention, charged stationary phases adds a second, very significant, dimension of selectivity due to the opportunities of electrostatic interactions with the analytes. The downside of electrostatic interactions is the need of salts or buffers in the mobile phase to disrupt these interactions for analyte elution. Higher buffer concentrations may be negative to MS detection sensitivity. With zwitterionic stationary phases, however, the electrostatic forces of each charge are partly counterbalanced by the proximity of an ion with opposite charge, i.e., the combined overall effect is weaker electrostatic interactions.

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