Separation of Urinary Compounds by High Performance Liquid Chromatography

Theory

Liquid chromatography involves the partitioning of an analyte between two phases, a mobile phase and a stationary phase. The chemical identities of these phases dictates the type of chromatography being performed:

Type of Chromatography	Stationary Phase	Mobile Phase	How Separation Occurs
Adsorption	solid polar material like silica or alumina	various depending on relative strengths of solvents and solutes	Competitive equilibria where stronger mobile phase drives solutes off stationary phase: too strong yields no separation; too weak yields no elution.
Partition	solid core has liquid layer coated or chemically bound; must be immiscible with mobile phase	‘normal’ phase has a nonpolar mobile phase (more polar column). ‘reversed’ phase has a polar mobile phase (less polar column)	Species dissolve between bound and mobile liquids. A higher affinity for mobile phase yields rapid elution. Higher affinity for bound yields slower elution.
Ion-Exchange	cross-linked polymer beads with charged groups (anionic or cationic forms available)	ionic buffer solutions	Competitive equilibria between analytes and buffer components for charged sites on stationary phase.
Size Exclusion	polymer beads with internal pores of tightly regulated size	any liquid that does not dissolve the beads nor react analytes	Species are separated purely on size as they traverse pores. Larger species elute first as they enter fewer pores.

As illustrated by this listing, several different types of chromatography are possible. All of them share some common attributes. A sample is injected at the head of a column and driven through by a mobile phase that is usually pumped at moderate to high pressures. It passes a stationary phase and partitions between the two phases according some degree of separation. The sample elutes from the tail of the column and solutes are passed by some form of detector. Final output is sent to integrator or computer for display.

As with GC, the area under a peak is proportional to concentration. For sharp peaks and carefully controlled flow rates, peak heights will also be proportional to concentration. In either case, a calibration curve is the method of quantitation.

In this experiment, the separation of urinary metabolites will be performed by reversed phase chromatography. The stationary phase packing will be small beads saturated with a chemically bound octadecyl hydrocarbon group (C₁₈). Nonpolar components, which have a high affinity for the C₁₈ environment, elute with relatively long retention times whereas more polar materials, including ionic species, will elute early. This experiment illustrates how five clinically important urinary compounds (uracil, uric acid, xanthine, allopurinol, and nicotinamide) can be separated and determined. The separation is achieved via isocratic elution (same mobile phase throughout) with ultraviolet detection. The order of elution is the same as the order of listing of the five species.

Experimental

Apparatus

• Liquid Chromatograph (Agilent 1260 Infinity II) with 25-μL sample injection system, UV (254 nm) detection, 30 cm × 4 mm C₁₈ column or whatever is currently available.

• Volumetric flasks, six 100 mL, one 10 mL

• Pipets, one 5 mL, four 4 mL, four 3 mL, four 2 mL, four 1 mL

• Mohr pipet, 2 mL

• Nalgene membrane filter, 0.2 μL

• Several syringe filters

• Centrifuge

Chemicals

• Stock solutions of

  • Uracil, 2.43 mg/100 mL

  • Uric Acid, 11.74 mg/100 mL

    Attention

    Must be fresh each day!

  • Xanthine, 4.67 mg/100 mL

  • Allopurinol, 5.43 mg/100 mL

  • Nicotinamide, 18.09 mg/100 mL

    Attention

    Made up with doubly distilled water and with several drops of 40% NaOH to enhance solubility; refrigerate until used.

• Urine samples — Unknown samples that contain the above components (plus uracil)

• Methanol, HPLC grade in LC solvent bottle

• Water, HPLC grade in LC solvent bottle

• 0.05 M acetic acid, buffered to pH 4.5; filtered (0.45 μm) into solvent bottle

• ZnSO₄ solution, 100 g/L

• 0.1 M NaOH

Procedures

Note

This below procedures have been written for an older model HPLC (Integral 4000). They will need to be adapted for the new Agilent 1260 Infinity II.

Instrument Parameters

Mobile phase will be the acetic acid buffer at approximately 2000 psi (or whatever yields 2.0 mL/min). This phase and all solvents must be degassed by helium purge for 15 minutes prior to starting the pump. The absorption maxima reported in the literature (Senftleber et al.) are uracil, 258 nm; uric acid, 292 nm; xanthine, 267 nm; allopurinol, 251 nm; and nicotinamide, 262 nm. The literature also reports that 254 nm is effective for detection. Since the Integral 4000 has a diode array, we can collect spectra for each peak. The manual has more information. A method should be prepared to run the 5 standards and 2 samples.

Calibration

• Uracil is used as an internal standard because of its stability and its short retention time.

• Pipet 5.00-mL aliquots of the uracil stock solution into each of six 100-mL volumetric flasks. Into one, pipet 1.00 mL of each of the other four stock solutions. Similarly pipet 2.00, 3.00, 4.00, and 5.00 mL of each stock solution into four other flasks. Ask the instructor to place an unknown into the sixth flask. Fill all flasks to the mark with doubly distilled water.

• Filter a few mL of each solution into an auto sampler vial and firmly attach the top. Place these into the tray being very careful not to touch the sampler arm (which easily knocks out of calibration costing needles and downtime). Run the method and collect the print outs.

Urine Sample

Pipet 5.0 mL of urine into a centrifuge tube that contains 15 mL of doubly distilled water. Precipitate the urinary proteins by addition of 1.5 mL ZnSO₄ solution (100 g/L) and 0.8 mL of 0.1 M NaOH. Centrifuge (see Dr. M) for 10 minutes at 5000 rpm. Filter the supernatant liquid through 0.2 μm Nalgene membrane filter to remove any remaining particulate matter. Dilute 1 mL of the filtrate to 10 mL and inject with no further pretreatment. Stop the run after 20 minutes of elution time.

After the run, purge the column with 1:1 methanol-water for 20 minutes followed by 20 minutes of pure methanol.

Treatment of Data

Determine the efficiency of the column by calculating the number of theoretical plates (\(N\)) using the retention time (\(t_r\)) and the base peak width (\(w_b\)):

(18)\[N = 16 \left( \frac{t_r}{w_b} \right)^2\]

Typical results for a reverse phase column vary from 1500 to 2000 plates for a flow rate of 2.0 mL/min.

Prepare standard additions working curves for allopurinol, nicotinamide, uric acid, and xanthine (plot all on the same axes). Determine the components in the unknown and the concentration of each from these curves. Similarly, identify the individual compounds and their concentrations in the urine sample.

Questions

1. Are any of the analytes (or other species in unknown) studied in this experiment amenable to separation on an ion-exchange column? Explain.

2. Calculate the analytical sensitivities for each analyte compound. For which species is the HPLC method used in this experiment most sensitive?

3. Calculate the efficiency of the column for each species as well as the height equivalent to a theoretical plate (see text, class notes, or GC lab for equations).

4. Explain the function of the mobile phase in GC vs. LC. Be specific with details — this is not a 1 or 2 point answer!

5. How could this experiment be performed to get better resolution between the peaks for uric acid and uracil?

References

1. Bastian, D.W., Miller, R.L., Halline, A.G., Senftleber, F.C., and Veening, H.,J. Chem. Ed., 54, 766(1977).

2. Senftleber, F.C., Halline, A.G., Veening, H., and Dayton, D.A., Clin. Chem., 22, 1522 (1976).