REGENERATION
During given HPLC analysis following steps should be followed:
Ø Flush the column, first with filtered and de-gassed water for 30 minutes at the rate of 1.0ml per Minute.
Ø Then with either Methanol or Acetonitrile for further 30 minutes at the flow rate of 1.0ml per minute.
Ø Condition the column with a given mobile phase for at least 30 minutes.
Ø Ensure that base line signal is stable, carry out the analysis.
Ø Check the responses of the Chromatogram of standard component with previous analytical report. If both satisfactory with respect to peak response, resolution, capacity factor, theoretical plates continue the analysis.
Ø If any of the above conditions are not satisfactory, regenerate the column as per the procedure given in 6.0 .
Ø After analysis is complete flush with the mobile phase for 15 minutes (flow rate 1.0ml/min) ,followed by water for at least 45 minutes and finally with organic solvents Methanol or Acetonitrile.
Ø Extra precaution should be taken when organic buffers are being used in a mobile phase; flush the column with filtered de-gassed water for at least 1 Hour.
Note: When inorganic buffers are used in mobile phase do not flush directly with organic solvents like Methanol, Acetonitrile as these reduces the performance & life of the column.
PROCEDURE FOR REGENERATION (REVERSED PHASE) : Whenever Column fails in System
Suitability Test as per pharmacopoeia Regeneration should be done.
Ø For columns C18, C8, C6, C4, Phenyl, Amino, Polymeric follow the sequence given below with a 1.0ml flow per minute flow rate.
Ø Flush with filtered and de-gassed water for 30 minutes. Inject 20 m liter of 1.0% of Acetic acid in Water.
Ø Methanol for 30 minutes, inject 20m liter of 1.0% DMSO (spectroscopic grade) in Methanol.
Ø Acetonitrile for 15 minutes.
Ø Isopropanol for 15 minutes.
Ø Chloroform for 10 minutes.
Ø Acetonitrile for 10 minutes.
Ø Methanol for 30 minutes.
Ø Water for 15 minutes. Check pH of water at inlet and outlet, which should be identical.
Ø Mobile phase for the compound to be analysed.
PRECAUTIONS:
Ø Column should be kept with proper end fitting during storage.
Ø Do not over tighten the column end fittings.
Ø Frits of the column should be checked for cleanliness/disposition of the excipients in consultation with the Department Head.
Ø Use dedicated columns only for a given product.
Ø Protect the column from rapid changes in solvent’s composition.
Ø Do not change the flow rate at increment greater than 0.2ml/min.
Ø Prevent the column from mechanical shocks.
Ø Always use the column in the specified direction.
Ø pH of the mobile phase should not be less than 2.0 and not more than 7.0.
Ø Backpressure of the column should be monitored for variation.
Ø Clean the solvent reservoir filter in the mobile phase after every analysis by sonication in the Methanol for 30 minutes.
Ø Test sample should be clean homogeneous and free from particulate matter.
I.D. No. | Name of column & Dimension (Mm) | Mobile Phase | Flow (ml/min) | Test sample & Concentration | Wave length | Min. no. of theoretical plates |
1. | Kromasil 100–5C1– 5 µ 250 ´ 4.6 mm | 75: 25 Methanol: Water | 1.0 | Dimethyl phthalate, Toluene & Biphenyl | 254 nm | NLT 30% of the suppliers specification |
2. | Kromasil 100 – C18, 5 µ 250 ´ 4.6 mm | Acetonitrile: Water (80 : 20) | 1.0 | Toluene, Naphthalene | 254 nm | NLT 30% of the suppliers specification |
3. | Kromasil 100 – C18, 5 µ 250 ´ 4.6 mm | Acetonitrile: Water (80 : 20) | 1.0 | Toluene, Naphthalene | 254 nm | NLT 30% of the suppliers specification |
4. | Waters Spherisorb S5C6 12.5 X 4.6 mm | Methanol: Water 70: 30 | 1.0 | Dimethyl phthalate, Toluene & Biphenyl | 254 nm | NLT 30% of the suppliers specification |
5. | Zorbax TMS, 5 µ, 4.6 x 250 mm Sr.No. | Methanol : Water 48 : 52 | 1.0 | Uracil, Acetone, Bezyl Alcohol | 254 nm | NLT 30% of the suppliers specification |
6. | Waters Spherisorb S5C6 150X 4.6 mm | Methanol : Water 70 : 30 | 1.0 | Dimethyl phthalate, Toluene & Biphenyl | 254 nm | NLT 30% of the suppliers specification |
7. | Waters Symmetry C18, 5µ (3.9 x 150 mm) | Acetonitrile : Water 60: 40 | 1.0 | Acenaphthene, Acetone | 254 | NLT 30% of the suppliers specification |
8. | Kromasil C18, 250 ´ 4.6 mm | Acetonitrile : Water (80 : 20) | 1.0 | Toluene, Naphthalene | 254 nm | NLT 30% of the suppliers specification |
9. | Kromasil C18, 250 ´ 4.6 mm | Acetonitrile : Water (80 : 20) | 1.0 | Toluene, Naphthalene | 254 nm | NLT 30% of the suppliers specification |
10. | Kromasil C18, 250 ´ 4.6 mm | Acetonitrile : Water (80 : 20) | 1.0 | Toluene, Naphthalene | 254 nm | Min. no. of theoretical plates |
11. | Kromasil C18, 250 ´ 4.6 mm | Acetonitrile : Water (80 : 20) | 1.0 | Toluene, Naphthalene | 254 nm | NLT 30% of the suppliers specification |
12. | Kromasil KP – 100 C18 - 25406 mm | Acetonitrile: Water (80 : 20) | 1.0 | Toluene, Naphthalene | 254 nm | NLT 30% of the suppliers specification |
13. | Waters Novapck C18 4 µ (3.9 x 300 mm) | Acetonitrile : Water 60: 40 | 1.0 | Acenaphthene, Acetone | 254 nm | NLT 30% of the suppliers specification |
14. | Waters Novapck C18 4 µ (3.9 x 300 mm) | Acetonitrile: Water 60: 40 | 1.0 | Acenaphthene, Acetone | 254 nm | NLT 30% of the suppliers specification |
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