Chemistry

Chromatography is the separation of mixture by distributing its components between mobile and stationary phase over time .It is use to determine characteristic composition of sample. Whereas Analgesic is drugs or medicines given to reduce pain without resulting in loss of consciousness. It is sometimes referred to as pain killer medications. Examples of analgesic drugs include Aspirin, ibuprofen. Analgesics act on body by acting on the central nervous system. In this chromatography is utilized to find the components of analgesics.

In this experiment modern liquid Chromatographic techniques are applied to separate the components of analgesic tablets like Aspirin, Caffeine and Paracetamol and Salicylic acid.

HPLC (High Performance Liquid Chromatography) is a form of Column Chromatography, which is used to separate, identify and quantify compounds on the basis of their idiosyncratic polarities and interaction with columns stationary phase, HPLC utilizes various stationary phase like hydrophobic saturated carbon chains, a pump that moves the mobile phase and analyte through the column, and a detector that provides a characteristic retention time for the analyte. The detector may also provide characteristic information. Analyte retention time varies depending on the strength of its interactions with the stationary phase, the ratiocomposition of solvent used, and the flow rate of the mobile phase.

Experiment Setup and Procedure
1. In this experiment HPLC separation takes place using ion suppression in the reverse phase mode ODS( Octadecylsilance, C18) column.C18 is used in this experiment because C18 will help in increasing the  retention time because it gives non-polarity and is more hydrophobic than other alkyl group all these properties of C18 helps in the separation.

2. To make the Internal Standard Solution dissolve 0.3gms Salicylic acid in 25ml of HPLC methanol in a conical flask and add 25ml distilled water and mix well. (Internal standards standards are used because they are the substance used as reference in quantitative analysis, the internal standard is first mixed with standard solution, later it is added to the unknown and the ratio of peak heights of internal standard and analyte is used for quantitative analysis).

3. Label three 100ml volumetric flasks no s 1, 2, 3. To each flask add the volumes of the stocks prepared and internal standard as indicated in the table below.


          Flask(1)         Flask(2)           Flask(3)Volume of Uracil                  1                   1                   1Volume of Aspirin                  6                   3                    9Volume of Caffeine                 3                   6                    9Volume of Paracetamol                 9                   6                    3Volume of Internal Std.                 3                   3                    3


Uracil
4. To make mobile phase mixture take 400ml of MeOH H2O by mixing 160ml HPLC methanol with 240 ml of 1 acetic acid.
5. To make sample solution weigh out 60 mg of powdered sample, transfer it quantitatively to a     100 ml volumetric flask using a maximum of 20 ml of HPLC methanol. Then add to it 3 ml of internal standard solution and make up to the mark with distilled water.

Materials Required
                                               Pump                           0.1mlmin flow rate
                                               Spectrophotometer      UV detector set at 280 nm
                                               Column                        15cm, 5um, reverse phase C18 column
                                               Mobile Phase               initially as above

Result and Discussion
Resolution of Peak  tr  Wavelength (average)

                       Sample 1 Peak 23 Resolution0.740.164.625
                       Sample 1 Peak 23Resolution 0.740.154.93
                        Sample 2 Peak 23Resolution0.650.154.33
                        Sample 2 Peak 23Resolution0.620.163.875
                        Sample 3 Peak 23Resolution0.650.173.823
                        Sample 3 Peak 23Resolution0.640.173.764
The most important way of determining the operational parameters in chromatography is through resolution. Hence it is very necessary to determine the resolution between two consecutive peaks. The resolution between peaks 2 and 3 is nearly same which means their compositions are nearly same. The brand broadening is a major factor that influences the efficiency and resolution of chromatographic separations.
Plate height HLN
 Plate number
The number of theoretical plates in a given column is known as plate number. It is the measure of capacity of column for restraining peak depression.
Sample 1N5.54x9.42.04411183.37
Sample 1N5.54x9.48.04   1312.98
Sample 2N5.54x8.88.04   1229.88
Sample 2N5.54x8.52.04841180.02
Sample 3N5.54x8.82.0576977.2
Sample 3N5.54x8.82.0576977.2
Plate height H0.251143.44.0002186

Optimal value flow rate is very important for HPLC column of different diameters. Flow rate directly impacts HPLC system pressure. Factors that effect optimal value flow rate are column dimensions, stationary phase particle, size, temperature.
To obtain optimal separations, sharp, symmetrical chromatographic peaks must be obatained.this means that Band broadening must be limited. It is also beneficial to measure the efficiency of the column.
                      Internal diameter                       Standard flow rate                                   4.6                                   1000                                   2.1                                     200                                   1.0                                       50                                   0.30                                       4                                   0.15                                       1
                                                                   Conclusion
200mg of Aspirin, 50 mg of caffeine and 75mg of Paracetamol was observed.
The experiment as successfully performed and the peaks were successfully observed. Hence by this method highest performance was obtained. Thus this method shows very good reproducibility.