BASIC HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
 
 
 EFFECTS OF pH ON THE RETENTION OF NEUTRAL, BASIC AND ACIDIC COMPOUNDS IN A REVERSED-PHASE CHROMATOGRAPHIC SYSTEM
 

 The main purpose of this experiment is to observer and understand the effects of different pH on the retention of neutral, basic and acidic compounds in a reversed-phase chromatographic system.

 The pH is generally defined as pH = - log [H⁺], where [H⁺] is the activity of H⁺. In most approximate applications, the pH is take as – log [H⁺]. A solution is acidic if [H⁺] [OH^-]. At 25^oC, an acidic solution has a pH below 7, and a basic solution has a pH above 7. Although pH generally falls in the range of 0 to 14, these are not limits of pH. A pH of –1.00, for example, means – log [H⁺] = 1.00; or [H⁺] = 10 M.
 The pH of solution can be controlled by a buffering solution. A buffering solution resists changes in pH when acids or bases are added or when dilution occurs. The buffer is a mixture of an acid and its conjugate base. There must be comparable amounts of the conjugate acid and base to exert significant buffering.
 In reversed-phase chromatographic system the component’s pH measured are acid, neutral and basic and the effects of pH in a chromatographic separation is noted when one changes the pH of a mobile phase and observe the effects of such change. At  low pH, the acidic components have a higher capacitor factor together with the neutral components. However, the basic components of a reversed-phase chromatographic system have a lower capacitor factor at low pH. In the pH 5.5 the acidic components and the basic components have the same capacitor factor and it is known as co-elution point. At pH over the co-elution point the basic components have a higher capacitor factor than the acidic ones. Consequently the acidic components eluted first that the basic at pH higher than 5.5.
 When one is using buffers in column packed at reverse-phase chromatography needs to be aware that it can not use buffers in mobile phase with a high concentration of methanol because the salts from the buffer solution will precipitated consequently causing damage to the column.
 

Materials and System Set-up:

Column: C18, 5 cm x 4.6 mm, 3 m m.
Detection: UV 254 nm
Chart Speed: 0.5cm/min.
Sample:250 m g/ml phenol
 250 m g/ml aminophenol
 12.5 m g/ml p-hydroxybenzoic acid
 50 m g/ml Uracil
 Mixture of the above.
 All samples prepared individually in 50% MeOH and 50% water.
Injection Volume : 20 m l
Flow rate: 1.5 ml/min (depending on pressure)
Mobile phase: 200 ml each:
  40% MeOH in 50mM buffers of pH 2.5; 4.0; 6.0 and 7.5 respectively. (80 ml MeOH + 120 ml buffers).

Procedure:

 The Seneca College technician already prepared the buffer solutions p 2.5; 4.0; 6.0 and 7.5. So, it was mixed 80 ml of MeOH with 120 ml of each respectively buffer solution. After that the mixture was filtered through filter in paper by vacuum to avoid formation of bubbles of air and to do the filtration faster and in addition to purify against a particles that could contaminate the solution.
 All solutions to the mobile phase were prepared in the high standard of purity to avoid the damage the column and consequently to lose the efficiency of the column by contaminating the column with solutions containing big particles.
 The standards 250 m g/ml phenol, 250 m g/ml aminophenol, 12.5 m g/ml p-hydroxybenzoic acid and 50 m g/ml Uracil were prepared individually in 50% MeOH plus 50% water.
 When in our case the solution, which contained a buffer pH 6.0 the base line equilibrated in 5 minutes after start to run in the HPLC. However, it was observed that other solutions with different pH it had a longer time to reach a satisfactory base line.
 Back Pressure for each run at each pH mobile phase:
 

	pH 2.5	pH 4.0	pH 6.0	pH 7.5
Pressure	246~260	260~270	273~280	287~300

The pressure increases with pH increases.

 The column was clean with a solution, which contained ACN (15%) because the high concentration of water dissolves the salt from the buffer and after 5 minutes of purge system is ready to be shut down.
 The pH of a mobile phase is an important tool in HPLC because silica-based packing are stable, in general, to all common organic solvents and to aqueous buffers in a pH range of 2.0 to 7.5. At pH values below 2.0, the bonded phases can be stripped off the packing, in extreme cases leaving the bare silica substrate. At pH values above 7.5, the base silica can dissolve, even to the extent that voids or channels are created in the packed bed. However, some newer silica-based packing have extended pH tolerance.
 In the pH range of 2.0 to 7.5 a pH control through buffer can be an useful tool for improving resolution in developing a chromatographic separation, since substances if acid or base change the capacity factor when the pH of the mobile phase changes. A change of as little as 0.1 pH units in a buffer can result in a retention time shift around 10% from one substance. So, it is very important to measure and keep track of the pH of a mobile phase in reversed-phase chromatography.

Results:

Part A:

Table #1. Raw Data: Retention Time

	pH 2.5	pH 4.0	pH 6.0	pH 7.5
Uracil	0.554	0.549	0.550	0.549
Phenol (cpd P)	1.392	1.362	1.395	1.370
Aminophenol (cpd A)	0.575	0.582	0.570	0.594
p-hydroxybenzoic (cpd H)	0.906	0.806	0.515	0.492
Peak 1 in mixture	0.559--A	0.567--A	0.513--H	0.525--H
Peak 2 in mixture	0.893--H	0.820--H	0.581--A	0.590--A
Peak 3 in mixture	1.400--P	1.358--P	1.381--P	1.372--P

A = AMINOPHENOL; P = PHENOL H = p-HYDROXYBENZOIC
 

Calculated Results:

CAPACITY FACTOR OF COMPOUNDS

The mathematical expression for K’ is:

K’ = T/T_o -1 or (V_s – V_o) / V_o

Part B:
 

Table #2. Capacity Factor (k’) for Each Compound at Each pH.

	pH 2.5	pH 4.0	pH 6.0	pH 7.5
Formula	(tR/0.554)-1	(tR/0549)-1	(tR/0.550)-1	(tR/0.549)-1
cpd A in mixture	0.009	0.033	0.056	0.075
cpd H in mixture	0.612	0.381	0	0
cpd P in mixture	1.527	1.474	1.510	1.499

A = AMINOPHENOL; P = PHENOL; H = p-HYDROXYBENZOIC
 

A = AMINOPHENOL; P = PHENOL; H = p-HYDROXYBENZOIC

Discussion:

 The graph where was plot K’ versus pH the neutral compound (phenol) is identified by the curve with showed that the capacitor factor (K’) did not show a strong variation when the pH of the mobile phase was changed. However, the acidic substance (p-Hydroxybenzoic acid) was identified because in an acidic mobile phase the substance showed a high capacity factor (K’) but when the mobile phase change to more neutral pH the capacity factor (K’) from the substance became drastically low. Opposite behavior was observed to aminophenol that is an basic substance, at low pH the aminophenol had a low capacity factor (K’) but when the pH of the mobile phase became more neutral the capacity factor (K’) from the aminophenol became higher.
 The equilibrium constant for the ionization of a weak acid in water is called an acid ionization constant (Ka). An example, acetic acid ionizes in water.

 CH3COOH (aq)       «            H+ (aq)   + CH3COO- (aq)

And its ionization at 25^oC is

Ka = [H+] [CH3COO-] / [CH3COOH] = 1.76 x 10-5
 The pKa of acetic acid at 25^oC is
 pKa = - log 1.76 x 10-5 = 4.754

 Acid is the substance that ionizes in water to form H+ (aq) ions. Base is the substance that ionizes in water to form OH- (aq) ions. Neutral substance in which hydrogen ion and hydroxide ion are in equilibrium or absence of both ions.
 The pKa from phenol is 9.89, from p-Hydroxybenzoic acid is 4.48 and from aminophenol is 10.46.
 A buffer contains the ion and the acid. If they are present in equal amounts, the resulting pH is the pK of the buffer. A buffer has always has its highest buffering capacity at its pK.
 At low pH 4.48 the p-Hydroxybenzoic the substance is protonated. Consequently the substance retains longer in the column. However when the pH of the mobile phase increases the p-Hydroxybenzoic has a low retention because the substance is less protonated. Opposite behavior is observed with aminophenol that has a pK of 10.46 at low pH the aminophenol is non-polar the consequently retains shorter. However, at a higher pH the retention changes to a higher retention because the substance is more protonated. However the phenol does not show a great changes in retention because it is a neutral substance. So, in reversed phase, the retention of acid decreases and the retention of bases increase with increasing pH. In another words when the stationary phase does not have polarity as in reversed phase chromatography the capacity factor (k’) of acids increases when the mobile phase has a low pH and the basis shows a low capacity factor (k’) when the mobile phase has a low pH.
 

Index