3. EXPERIMENTAL WORK
3.1 Materials andMethods
3.1.1 Chemicalsand standards used
Table.No.15 List of chemicalsand standards used
S.No. Chemicals Manufacturer Name Grade
1 Water Merck HPLC grade
2 Acetonitrile Merck HPLC grade
3 0. 22µ Nylon filter Advanced lab HPLC grade
4 0.45µ filter paper Millipore HPLC grade
5 Theo asthalin Cipla syrup form
6 Salbutamol sulphate and theophylline In - House In- House
3.1.2 Instruments used
Table.No.16. List of instruments used
S.No Instrument name Model number Soft ware Manufacturers
Name
1 HPLC-auto sampler –UV detector Separation module2695, UV.detector2487 Empower-software version-2 Waters
2 U.V double beam spectrometer UV 3000+ U.V win soft ware Lab India
3 Digital weighing balance(sensitivity …show more content…
Selection of flow rate
6. Selection of column temperature
7. Selection of diluent
8. Selection of test concentration and injection volume
3.2.1.Selection of mobile phase
Acetonitrile:water (40: 60 % v/v)
3.2.2. Selection of wavelength
10mg ofsalbutamol sulphate and theophyllinewas dissolved in mobile phase. The solution was scanned from 200 -400 nm the spectrum was obtained.The overlay spectrumwas usedfor selection of wavelengthfor salbutamol sulphate and theophylline. The isobestic point was taken as detection wavelength.The overlay spectrums are shown in Fig.No.20-22
3.2.3. Selectionof column
Heart of HPLC made of 316 grade stainless steel packed with stationary phase.
Silica based columns with different cross linking’s in the increasing order of polarity are as follows: ß------- Non-polar----------moderately polar--------Polar----------à C18< C8< C6< Phenyl < Amino <Cyano< Silica
In reverse phase chromatography, hydrophobic interaction between drug molecule and the alkyl chains on the column packing material.
Column is selected based on solubility, polarity & chemical differences among analytes and Column selected: i.e. Phenomenex Luna C18 column (250 X4.6 mm ID) , 5 μm particle …show more content…
Linearity
Preparation of stock solution
The standard stock solution was prepared by dissolving 1mg of standard drug of Salbutamol Sulphate and 50mg Theophylline taken in to 100ml volumetric flask to which add 40ml of mobile phase [Acetonitrile-Water (40:60,v/v)] and sonicated for about 10 min then the final volume was made upto100 ml with the mobile phase and shaken then filtered through 0.45µ membrane filter. The filtered solution was further diluted in the diluent to make the final concentration.
Preparation of Level – I (0.25ppm Of SS & 12.5ppm of TP)
2.5ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – II (0.50ppm of SS & 25ppm of TP)
5ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – III (0.75ppm of SS & 37.5ppm of TP)
7.5 ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – IV (1.0ppm of SS & 50ppm ofTP)
10 ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – V (1.25ppm of SS & 62.5ppm of
3.1 Materials andMethods
3.1.1 Chemicalsand standards used
Table.No.15 List of chemicalsand standards used
S.No. Chemicals Manufacturer Name Grade
1 Water Merck HPLC grade
2 Acetonitrile Merck HPLC grade
3 0. 22µ Nylon filter Advanced lab HPLC grade
4 0.45µ filter paper Millipore HPLC grade
5 Theo asthalin Cipla syrup form
6 Salbutamol sulphate and theophylline In - House In- House
3.1.2 Instruments used
Table.No.16. List of instruments used
S.No Instrument name Model number Soft ware Manufacturers
Name
1 HPLC-auto sampler –UV detector Separation module2695, UV.detector2487 Empower-software version-2 Waters
2 U.V double beam spectrometer UV 3000+ U.V win soft ware Lab India
3 Digital weighing balance(sensitivity …show more content…
Selection of flow rate
6. Selection of column temperature
7. Selection of diluent
8. Selection of test concentration and injection volume
3.2.1.Selection of mobile phase
Acetonitrile:water (40: 60 % v/v)
3.2.2. Selection of wavelength
10mg ofsalbutamol sulphate and theophyllinewas dissolved in mobile phase. The solution was scanned from 200 -400 nm the spectrum was obtained.The overlay spectrumwas usedfor selection of wavelengthfor salbutamol sulphate and theophylline. The isobestic point was taken as detection wavelength.The overlay spectrums are shown in Fig.No.20-22
3.2.3. Selectionof column
Heart of HPLC made of 316 grade stainless steel packed with stationary phase.
Silica based columns with different cross linking’s in the increasing order of polarity are as follows: ß------- Non-polar----------moderately polar--------Polar----------à C18< C8< C6< Phenyl < Amino <Cyano< Silica
In reverse phase chromatography, hydrophobic interaction between drug molecule and the alkyl chains on the column packing material.
Column is selected based on solubility, polarity & chemical differences among analytes and Column selected: i.e. Phenomenex Luna C18 column (250 X4.6 mm ID) , 5 μm particle …show more content…
Linearity
Preparation of stock solution
The standard stock solution was prepared by dissolving 1mg of standard drug of Salbutamol Sulphate and 50mg Theophylline taken in to 100ml volumetric flask to which add 40ml of mobile phase [Acetonitrile-Water (40:60,v/v)] and sonicated for about 10 min then the final volume was made upto100 ml with the mobile phase and shaken then filtered through 0.45µ membrane filter. The filtered solution was further diluted in the diluent to make the final concentration.
Preparation of Level – I (0.25ppm Of SS & 12.5ppm of TP)
2.5ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – II (0.50ppm of SS & 25ppm of TP)
5ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – III (0.75ppm of SS & 37.5ppm of TP)
7.5 ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – IV (1.0ppm of SS & 50ppm ofTP)
10 ml of stock solution was taken in to 100ml of volumetric flask and dilute up to the mark with diluent.
Preparation of Level – V (1.25ppm of SS & 62.5ppm of