Friday, May 10, 2013

Quantitative analysis by using high performance liquid chromatography


1. To determine the concentration of acetophenone in a mixture by reversed phase performance liquid chromatography using standard calibration



High performance liquid chromatography (HPLC) is a form of liquid chromatography that used to separate non volatile compounds in a mixture in which they are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a high pressure pump, an injector, a separation column, and a detector. HPLC is one of the most widely used analytical techniques in industry. It is used to separate and analyse compounds through the mass-transfer of analyte between stationary phase and mobile phase. The technique of HPLC utilizes a liquid mobile phase to separate the components in a mixture.

First of all, the components themselves are dissolved in a solvent. Then, they (in mobile phase) are forced to flow through a column (stationary phase) under high pressure. The mixture is resolved into its components within the column. Due to the different partitioning behavior of the different components in the mixture, they will pass through the column at different rates and hence separation of compounds can be done based on the individual retention time. The interaction of the solute with mobile phase and stationary phase can be controlled by choosing different mixture of solvent and column.

HPLC mainly categorized into two types: normal phase and reversed phase. For normal phase, polar stationary phase (commonly silica) is used to retain the polar components which will stick at the column for a longer time. Hence, non polar component will pass through the column faster than polar component. The primary difference between reversed-phase chromatography and normal phase chromatography is that its stationary phase utilizes a non-polar or hydrophobic surface (typically long chain hydrocarbon) as opposed to a polar (Si-OH) surface used in normal phase chromatography. In this column, polar component will pass through it faster compared to non polar component.

The principles of high performance liquid chromatography are generally similar with gas chromatography. This analyse is based on a comparison of either peak height or peak area observed. The area under a peak is proportional to the mass or concentration of the compound giving rise to that peak, the formula is shown as below:

A m

A = f m

where f is called the detector response factor for the compound. A plot of peak area versus concentration should be linear in which it can be used as a calibration curve to determine the concentration of compound that present in an unknown solution.



Stock solution of 1000ppm acetophenone in methanol, mixture M52 containing an unknown amount of acetophenone



Pipette, volumetric flask, beaker



High performance liquid chromatography-mass spectrophotometer




Result and calculation:

Calculating the volume of 1000ppm acetophenone needed in each standard solution

By applying the forlmula of M1V1 = M2V2

M1 = Molarity of stock solution, 1000ppm

V1 = Volume of stock solution needed

M2 = Molarity of standard solution

V2 = Volume of standard solution

(1000ppm)(V1) = 50ppm (10ml)

V1 = 0.5ml

Applying the same formula to obtain the volume of stock solution needed to prepare standard solutions with 100, 150, 200, 250 and 300ppm.

Table 1: Concentrations of acetophenone and the peak areas in chromatogram

Volume of 1000ppm acetophenone needed (ml)

Concentration of acetophenone (ppm)

Peak areas in chromatogram

Retention time (min)

























Table 2: The peak area of unknown M32 in the chromatogram


Peak areas in chromatogram

Retention time (min)

Unknown M32



A calibration curve (as shown in Graph 1) has been plotted. The concentration of acetophenone in unknown M32 has been determined by using the calibration curve, which is 205ppm.


The concentration of acetophenone in unknown M32 is determined by using the calibration curve obtained. The peak areas of unknown M32 obtained is 6658.05 with the concentration of acetophenone of 205ppm. Based on the chromatogram obtained, we found that there is only one compound presents in the unknown M32 since the chromatogram showed only one peak. The identity of this compound can be further confirmed by using liquid chromatography mass spectroscopy.

Although we can get the calibration curve, however the calibration is not so accurate. This can be seen at the deviation of the retention time of last two standard solutions which are 250ppm and 300ppm. In order to obtain a better calibration curve, the calibration should be repeated until we obtain the retention time of 1.58min for all the standard solutions. For the sample unknown M32 should be repeated again in order to get the retention time of 1.58min instead of 1.57min. However, we cannot repeat the experiment because we are lack of time.

Precaution steps:

1. Make sure there is no bubble present in the syringe during injection.

2. The syringe used should be washed for few times before use to ensure there is no contamination.

3. Parallax error should be avoided to ensure the concentration of acetophenone is accurate during preparation.