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Course: Analytical Chemistry

HPLC Coursework

1 HPLC in British Pharmacopoeia


1.1 Normal phase HPLC

Noscapine BP (C22H23NO7) is chosen to illustrate the application of normal phase


HPLC (British Pharmacopoeia Commission 2007, p. 1166). Under the monograph of
Noscapine BP liquid chromatography is used for the detection of related substances.
The stationary phase used in the test is nitrile silica gel for chromatography R. It is
a very finely divided silica gel with surface modified by the introduction of cyanopropylsilyl groups. It has
strong adsorption capacity and relatively polar due to the presence of -CN groups. The mobile phase is a
mixture of methanol R and phosphate buffer solution pH 6.0 R1. It is less polar than the adsorption phase.
The use of polar stationary phase and a less polar mobile phase indicates normal phase chromatography.

1.1.1 Retention mechanism


Normal phase chromatography is the oldest kind of HPLC. It separates noscapine
impurities based on compound polarity and the adsorption process. Retention of
noscapine and its impurities in the adsorption layer requires displacement of an
equivalent number of solvent molecules (Valko 2006, p. 46). Hence polar
compounds are better retained and elutes slower. This is how different substances
in Noscapine BP are separated. Methanol is included in the mobile phase as a polar
modifier. They are preferentially adsorbed onto the adsorption sites leaving a more
uniform population of weaker sites (Valko 2006, p. 51). This causes less betweenrun variation and higher column efficiency.
1.1.2 Suitability
The basic criteria for normal phase HPLC are that the testing material has molecular weight < 2000
and is moderately soluble in water or water miscible organic solvents. According to BP2007 the molecular

weight for Noscapine BP is 413.4; the sample is soluble in acetone but not water (British Pharmacopoeia
Commission 2007, p. 1166). Hence normal phase HPLC is suitable for separation of related substance in
Noscapine BP.
Normal phase HPLC allows easy separation of isomers or mixture of compounds with varying
functional groups (Valko 2006, p. 51). If the solute functional groups are in suitable conformation to
interact with the absorbent surface the retention time will be much greater. The majority of

Noscapine BP impurities is papaverine (impurity A; Figure 2). It is structurally similar


to noscapine as it shares the same chemical backbone (benzylisoquinoline). As
there is only one major impurity in the sample normal phase HPLC is sufficient
enough for the separation.

Figure 1. Impurity A (Papaverine) of Noscapine BP (British Pharmacopoeia Commission


2007, p. 1166).

1.1.3 Predicted retention order


The elution order should be: 1- papaverine, 2 noscapine; as noscapine is more
ploar than papaverine. Papaverine has four ether groups while noscapine has five
ether groups, one carboxylic group and one tertiary amine group.

1.2 Reversed phase HPLC

Nifedipine BP (C17H18N2O6) is chosen to illustrate the application of reversed phase


HPLC (RP-HPLC) (British Pharmacopoeia Commission 2007, p. 1142). Under the
monograph of Nifedipine BP liquid chromatography is used for the detection of
related substances. The stationary phase used in the test is octadecylsilyl silica
gel for chromatography R. It is a very finely divided silica gel (3um to 10um), chemically modified at
the surface by the bonding of octadecysilyl (C-18) groups. It is inert and non-polar. The mobile phase is a
mixture of acetonitrile R, methanol R and water R. These solvents are mixed in a pre-defined

proportion (9:36:55 V/V/V) to provide sufficient range of dipolar and hydrogen


boding interactions with solutes to separate a vast number of compounds. Methanol R
is the organic modifier to adjust the hydrophobicity. This allows a suitable balance between peak-to-peak

separation as required by BP and retention time. The mobile phase in general is more ploar than the
stationary phase. The use of non-polar stationary phase and a polar mobile phase indicates RP-HPLC.

1.2.1 Retention mechanism


RP-HPLC is the most popular separation method in HPLC. Comparing to the
normal phase chromatography it is more versatile, reproducible and with a much
shorter equilibration time. The retention order of RP-HPLC is the reverse of that in
normal phase chromatography: the higher the lipophilicity of the compound the
later it elutes from the column. This order is governed by the partition of the
molecules between the mobile and the stationary phase. The impurities of
Nifedipine BP have different lipophilicity and hence varied retention order. The
retention order k can be expressed as:
'

'

log k =slope+log k 0

where slope is the slope of

log k

'

against organic phase concentration,

volume fraction of organic modifier in the mobile phase and


of the

log k '

'

log k 0

is the

is the intercept

against organic phase concentration plot.

1.2.2 Suitability
The basic criteria for RP-HPLC are that the testing material has molecular weight < 2000 and is
soluble in non-polar to weakly polar solvents (Harris 2000, p. 723). According to BP2007 the molecular
weight for Nifedipine BP is 346.3; the sample is insoluble in water but soluble in acetone (British
Pharmacopoeia Commission 2007, p. 1142). Hence RP-HPLC is suitable for separation of related
substance in Nifedipine BP.
RP-HPLC allows separation of isomers or mixture of compounds with varying functional groups at a
much quicker speed than its normal phase counterpart. The majority of the impurities (specified impurities
A and B) share the same chemical structure as nifedipine with only two function group variations (Figure
2). Minor impurities C and D are sub-fragments of nifedipine (Figure 3; Figure 4). Due to a high number of
impurities the use of normal phase HPLC is ineffective; it is likely that impurity signals overlap with each
other. RP-HPLC is therefore an better method of differentiating these structurally-related compounds.

Figure 2. Common structure of specified impurities A and B of Nifedipine BP. A R = NO 2, B


R = NO (British Pharmacopoeia Commission 2007, p. 1142).

Figure 3. Structure of impurity C of Nifedipine BP (British Pharmacopoeia Commission


2007, p. 1142).

Figure 4. Structure of impurity D of Nifedipine BP (British Pharmacopoeia Commission


2007, p. 1142).

1.2.3 Predicted retention order


The elution order should be: 1- impurity A ( nitrophenylpyridine analogue), 2 Impurity
B (nitrosophenylpyridine analogue), 3 Nifedipine. Pyridine analogues (A and B) are less
lipophilic than nifedipine as the nitrogen atom on pyridine segments has an extra
lone pair. A is more polar than B due to the presence of an extra electronegative
oxygen atom.

1.3 Ion-pair HPLC

Tioconazole BP (C16H13Cl3N2OS) is chosen to illustrate the application of ion-pair


HPLC (British Pharmacopoeia Commission 2007, p. 1670). Under the monograph of
Tioconazole BP liquid chromatography is used for the detection of related
substances. The stationary phase used in the test is end-capped octadecylsilyl
silica gel for chromatography R. It is a very finely divided silica gel (3um to 10um), chemically
modified at the surface by the bonding of octadecysilyl (C-18) groups. The free acidic silanol groups are
present in the stationary phase which may react with basic compounds. To minimise such reactions the
silica is end-capped to cover the remaining silanol groups. As a result the stationary phase is inert and
non-polar. The mobile phase is a mixture of tetrabutylammonium dihydrogen phosphate R and methanol
R. Tetrabutylammonium dihydrogen phosphate R is used as ion pair reagent for retaining

acidic substances in Tioconazole BP. It is buffered with dilute ammonia R to pH 7.4.


Methanol R is the organic modifier as described in the previous section. The mobile phase is more ploar
than the stationary phase. The use of non-polar stationary phase and a polar mobile phase with ion pair
reagent indicates ion-pair reversed phase chromatography.

1.3.1 Retention mechanism


The retention mechanism can be explained by i) the ion-exchange process
between the solute and ii) the adsorbed ion-pair molecules in the stationary phase
(Valko 2006, p. 46). The buffer in the mobile phase allows certain compounds in
Tioconazole BP predominantly exist in their deprotonated form. These deprotonated
compounds will then form natural ion-pair with the tetrabutylammonium cation.
These amphiphilic ion-pairs then goes through reverse phase retention by adsorbing
into the non-polar stationary phase. The result of ion-pair interaction is increased
retention time and improved peak shape. In conclusion ion-pair chromatography
improves separation of impurities by differentiating their lipophilicity as well as their
charge under certain pH.
1.3.2 Suitability
This type of chromatography follows the same criteria as RP-HPLC: testing material of molecular
weight < 2000 and is soluble in non-polar to weakly polar solvents (Harris 2000, p. 723). According to
BP2007 the molecular weight for Tioconazole BP is 387.7; it is soluble in methylene chloride and alcohol
and less soluble in water (British Pharmacopoeia Commission 2007, p. 1670). Hence RP-HPLC is
suitable for separation of related substance in Tioconazole BP.
Ion-pair chromatography allows separation of widely different components in a sample, including
neutral and ionic compounds. The majority of the impurities (specified impurities A, B, C) share the same
chemical structure as tioconazole with only two function group variations (Figure 2). The remaining
impurity D is a sub-fragment of tioconazole (Figure 3). These impurities are structurally-related with
similar lipophilicity and hence separation by RP-HPLC is ineffective. Ion-pair chromatography allows
improved separation by exploiting the different charge states of the impurities in Tioconazole BP under pH
7.4.

Figure 5. Common structure of specified impurities A, B and C of Tioconazole BP. A R1 =


R2 = H, B R1 = R2 = Cl, C R1 = Cl and R2 = Br. (British Pharmacopoeia Commission 2007,
p. 1670).

1.3.3 Predicted retention order


The elution order should be: 1- impurity B, 2 impurity C, 3 tioconazole, 4
impurity A. The order is similar to RP-HPLC with better separation. A is the least polar due to
the absence of bromo- / chloro- groups. C is less polar than B as the bromo- group is
less electron withdrawing than the chloro-group. C is more polar than tioconazole as
it has two electron withdrawing groups instead of one in tioconazole.

Figure 6. Structure of impurity D of Tioconazole BP (British Pharmacopoeia Commission


2007, p. 1670).

2 Calculation
2.1 Concentration of unknown paracetamol solution
Let the concentration of unknown paracetamol solution be x.
Due to the same retention time the peak areas (10500mAu and 8750mAu) are
compared.
As same amount of solution is injected, by simple ratio,
10500mAu = x : 8750mAu

500uM

x = 416.67uM

Answer: the concentration of unknown paracetamol solution is 416.67uM.

2.2 Amount of paracetamol injected


2.2.1 From standard paracetamol solution
By conversion of paracetamol concentration,
litre

500uM

= 500 umol in 1

= (500umol x 10u) in 10ul


= 5nmol in 10ul
Molecular weight of paracetamol according to BP = 151.2g
Weight of paracetamol = 5nmol x 151.2g = 0.756mg
Answer: the amount of paracetamol injected from the standard paracetamol
solution is 0.756mg (5nmol).
2.2.2 From unknown paracetamol solution
By simple ratio, mass of paracetamol injected from the unknown paracetamol
solution
= 0.765mg x 8750 / 10500
= 0.630mg
Similarly the number of mole of paracetamol = 5nmol x 8750/10500 =
4.167nmol
Answer: the amount of paracetamol injected from the standard paracetamol
solution is 0.630mg (4.167nmol).

3 Bibliography
British
Pharmacopoeia
Commission,
2007.
Monographs.
Pharmacopoeia 2007. 2007th ed.: Stationery Office Books. p. 1670.

In

British

British
Pharmacopoeia
Commission,
2007.
Monographs.
Pharmacopoeia 2007. 2007th ed.: Stationery Office Books. p. 1142.

In

British

British
Pharmacopoeia
Commission,
2007.
Monographs.
Pharmacopoeia 2007. 2007th ed.: Stationery Office Books. p. 1166.

In

British

Harris, D. C., 2000. HPLC. In Quantitative Chemical Analysis.: W. H. Freeman and


Company. p. 723.
Valko, K., 2006. High Performanace Lquid Chromatography.: School of Pharmacy,
University of London. p. 46.
Valko, K., 2006. High Performanace Lquid Chromatography.: School of Pharmacy,
University of London. p. 51.