operating procedure for HPLC

Operation And Calibration Of HPLC SYSTEM

1.0.            OBJECTIVE:

The objective of this SOP is:

1.1               To ensure that the instrument performs satisfactorily and gives accurate and         reproducible data. 

2.0.            RESPONSIBILITY:

2.1               Officer or above of Quality control  - 

3.0.            ACCOUNTABILITY:

          

Head - Quality Control

4.0.            PROCEDURE:

4.1              PROCEDURE FOR GENERAL CLEANING:

Ø      Ensure that the power supply to the instrument is switched off and main cord is removed from supply.

Ø      Clean the instrument with a clean dry cloth everyday. A wet cloth dipped in dilute soap solution may be used occasionally.

Ø      Precaution must be taken to clean the instrument immediately with dry cloth.

                       

4.2              OPERATING INSTRUCTIONS:

Ø      Ensure that the instrument is properly connected to the power supply.

Ø      Fix the column and prepare the mobile phase. 

4.3              PREPARATION OF MOBILE PHASE

Ø      Use HPLC grade solvent / water & AR/ HPLC grade reagent only.

Ø    Filter the mobile phase through 0.45 membrane filter after mixing it in required proportion and de-gas on ultrasonic bath for about 5 minutes.

4.4              PROCEDURE TO GET STARTED

Ø      Switch on pump, auto injector, detector and then system controller.

Ø      Turn the drain valve knob to 180 in anti- clockwise to open the drain valve to run the purge system.

Ø      After the purge is over close the drain valve.

Ø      Set desired flow rate by pressing function key.

Ø      Press pump key. The pump will run and indicator will glow.

Ø      Purge the auto injector by pressing purge key on auto sampler display.

Ø      Enter the desired wavelength on detector using function key

4.5              PROCEDURE TO OPERATE CLOSE UP

Ø      Switch on the computer monitor and printer.

Ø      Double click on the main Menu of software which is you use for your hplc system .

Ø      It will show Confrigration,Real Time ,Sample Shedule & Post Run Analysis.

Ø      Open Real Time.

Ø      From the “file menu” create a new method or load the existing method.

Ø      Create a new sample shedule and feel sample name ,batch number,,sample volume , method name and file name ,after then save the shedule .

Ø      Run Sample Shedule .

Ø      For system suitability test, inject six continuous injections of same standard and RSD should not be more than 2.0 %, otherwise it is specified in standard test procedure.

Ø      After every ten injection of sample (5 samples in duplicate), the standard solution shall be injected 3 times and the RSD shall be calculated and ensure that it is within the limit. 

4.6              SHUT DOWN PROCEDURE

Ø      Close Sample Shedule ,Real Time and then CLASS LC 10 to Software.

Ø      From Start button select “ Shut down” and click yes.

Ø      Switch-off the computer.

Ø      Switch-off CBM .

Ø      Switch-off Detector then, auto sampler.

Ø      Stop pump by pressing, “Pump” key and switch off the pump module.

4.7              CALIBRATION PROCEDURE.

4.7.1          FOR PUMP:

Ø      Disconnect the column and connect the inlet and outlet tubing’s with a union.

Ø      Prime all the lines at 5 ml/min flow rate with water and ensure that flow line is free from air bubbles.

Ø      Set the flow rate at 1ml / min and collect the mobile phase (water) in a dry preweighed beaker and collect the mobile phase for 10 min. Weigh the beaker to get the weight of mobile phase.

Ø      Calculate the flow rate by dividing the weight obtained with weight per ml and 10 (run time).

Ø       Calculate the corresponding flow rate. Carry out the experiment in duplicate.

Ø      Repeat the same procedure for Pump- B

Ø      Record the observation in Annexure -1

Ø      Acceptance criteria : Flow rate should be in between 0.99 to 1.01 ml / min..

4.7.2          FOR GRADIENT VALVE:

Ø      Install union in place of column & flush solvent lines (A&B) at flow rate of 2ml/min with water.

Ø      Prepare the mobile phase.

Ø      Prepare 0.3% acetone with HPLC grade water.

Ø      Fill reservoir A with 100% HPLC grade water & reservoir B with 0.3% acetone in HPLC grade water as mobile phase.

4.7.3          INSTRUMENT SET UP:

Ø            Enter the following time program:

TIME	FUNCTION	VALUE
0.01	B CONC	10
10.00	B CONC	10
10.01	B CONC	50
20.00	B CONC	50
20.01	B CONC	90
30.00	B CONC	90
30.01	B CONC	100
40.00	B CONC	100
40.01	B CONC	0
50.00	B CONC	0

Ø            Use detector at wavelength of 254 nm.

Ø            Record the printout of gradient valve test as per Annexure - 2.

Ø            The gradient valve test shall be accepted if actual concentration with ±1% of set concentration.

4.7.4          CALIBRATION OF INJECTOR:

4.7.4.1    CHECK FOR PRECISION:

Ø      Purge the injector system with 100% water to ensure the complete washing of injector.

4.7.5          STANDARD PREPARATION:

Ø      Transfer about 50 mg of Uracil to a 250ml volumetric flask. Add 100ml of Methanol, sonicate to dissolve and make up the volume with Methanol to obtain a solution containing about 0.02 mg/ml of Uracil.

Ø      Filter the solution through 0.45m membrance filter. 

Ø      Use HPLC grade Methanol as the mobile phase.

Ø      Instrumental Set Up

                        Column                         :  Hypersil ODS or equivalent 150 x 4.6 mm, 5.0m

Flow rate                      :  1.0 ml/min

Injector Volume            :  20m litre

Detector                       :  254 nm

Ø      Inject the standard preparation six times in the system. The peak areas observed shall be consistent.

Ø      The relative standard deviation for area counts calculated shall not be more than 1.0%.

Ø      Record the observation in the format as mentioned in Annexure – 3.

4.7.6          CHECK FOR LINEARITY:

Ø      Inject 10, 20, 30, 40 & 50m litre of standard preparation in duplicate. Calculate the average area counts corresponding to each set of injection.

Ø      Tabulate the average area against each injection. Plot a graph for area counts vs mlitre the resulting graph shall be linear.

Ø      The correlation co- efficient calculated shall be not less than 0.99.  

Ø      Record the observation as per the Annexure - 3.

4.7.7          CALIBRATION OF DETECTOR:

Ø      Standard preparation. Mobile phase, Instrument set- up same as mentioned in calibration of Injector.

Ø      Run the chromatograph at different wavelength (252 nm – 262 nm with 2 nm increment)

Ø      The largest peak response shall be at 258 nm ± 2nm.

Ø      Record the results in the detection calibration record as per the Annexure - 4.

Ø      Affix a calibration status label on the instrument containing “Calibrated On”, “Due On” and “Signature”.

Report to Head – QC, if any discrepancy observed during calibration or operating the instrument and affix ‘Under Maintenance’ label on the instrument

    FREQUENCY OF CALIBRATION:

Ø      Detector :      Once in three months and after each maintenance job.

Ø      Pump     :       Once in three months and after each maintenance job.

Ø      Injector  :       Once in six months and after each maintenance job.

Ø      Gradient :       Once in six months and after each maintenance job in the pump.

about noise and drift of HPLC calibration

Noise and drift

In HPLC we deal with the time-dependent process. The appearance of the component from the column in the detector represented by the deflection of the recorder pen from the baseline. It is a problem to distinguish between the actual component and artifact caused by the pressure fluctuation, bubble, compositional fluctuation, etc. If the peaks are fairly large, one has no problem in distinguishing them. However, the smaller the peaks, the more important that the baseline be smooth, free of noise, and drift.
Baseline noise is the short time variation of the baseline from a straight line caused by electric signal fluctuations, lamp instability, temperature fluctuations and other factors. Noise usually has much higher frequency than actual chromatographic peak. Noise is normally measured "peak-to-peak": i.e., the distance from the top of one such small peak to the bottom of the next. Sometimes, noise is averaged over a specified period of time. Noise is the factor which limits detector sensitivity. In trace analysis, the operator must be able to distinguish between noise spikes and component peaks. A practical limit for this is a 3 x signal-to-noise ratio, but only for qualitative purposes. Practical quantitative detection limit better be chosen as 10x signal-to-noise ratio. This ensures correct quantification of the trace amounts with less than 2% variance. Figure below illustrates this, indicating the noise level of a baseline(measured at highest detector sensitivity) and the smallest peak which can be unequivocally detected.

Definition of noise, drift, and smallest detectable peak.

Another parameter related to the detector signal fluctuation is drift. Noise is a short-time characteristic of a detector, an additional requirement is that the baseline should deviate as little as possible from a horizontal line. It is usually measured for a specified time, e.g., 1/2 hour or one hour. Drift usually associated to the detector heat-up in the first hour after power-on. Figure also illustrates the meaning of drift. 

Dissolution test

Acceptance criteria for dissolution test for tablets according to Indian pharmacopoeia

Level                      Number of                                         Acceptance criteria
                               tested
S1                              6                                          Each unit is not less than D* + 5 per
                                                                              cent**.
S2                              6                                          Average of 12 units (S1 +S2) is equal to
                                                                              or greater than D, and no unit is less
                                                                              than D –15 per cent**.
S3                             12                                         Average of 24 units (S1+S2+S3)is equal
                                                                              to or greater than D, not, More than 2
                                                                              units are less than D – 15 per cent** and
                                                                              no unit is less than D – 25 per cent**.

where
*D is the amount of dissolved active ingredient specified in the individual
  monograph, expressed as a percentage of the labelled content.
**Percentages of the labelled content.

Question and answer for pharma interview

11. Define process flow of API manufacturing?

12. Define process flow of Tablet manufacturing?

13. Define process flow of Sterile Lyophilized manufacturing?

14. Define process flow of Biotech product manufacturing?

15. Define Troubleshooting approach for problems related to particulate matter:

16. Preformulation research involves pharmaceutical and analytical investigations that both precede and support formulation development efforts for all dosage forms, please provide some example:

• Interview question mostly ask during HR round:

1. Tell me about yourself.
2. What are your two best points?
3. What are your two weakest points?
4. What are three things you want to change about yourself?
5. How do you handle conflict?
6. Explain your leadership/research/volunteer experiences?
7. What extracurricular activities are you engaged in?
8. Where do you see yourself in five/ten years?
9. What do you do in your spare time?
10. What do you do to relieve stress?
11. What qualities do you have that you think are important for an effective leader to have?

Interview question mostly ask for fresher in B.pharm or M.pharm:

1. Describe your project work during M.Pharm.

2. Equipment knowledge and handling during project B.pharm or M.pharm

Question and answer for pharma interview

 Q. Which type of tablets are exempted from Disintegration testing?

A. Chewable Tablets

02. Q.What are the common variables in the manufacturing of tablets?
A.
·       Particle size of the drug substance
·       Bulk density of drug substance/excipients
·       Powder load in granulator
·       Amount & concentration of binder
·       Mixer speed & mixing timings
·       Granulation moisture content
·       Milling conditions
·       Lubricant blending times
·       Tablet hardness
·       Coating solution spray rate

03. Q. Whether bracketing & validation concept can be applied in process validation?
A.Both Matrixing & Bracketing’s can be applied in validation studies.
Matrixing
Different strength of same product
Different size of same equipment
Bracketting - Evaluating extremes
Largest and smallest fill volumes
Fastest and slowest operating speeds

04. Q. What is the difference between calibration and Validation?
A. Calibration is a demonstration that, a particular
Instrument or device produces results with in specified limits by comparisons with those produced by a reference or traceable standard over an appropriate range of measurements.

Where as Validation is a documented program that provides high degree of assurance that a specific process, method or system consistently produces a result meeting pre-determined acceptance criteria.

05. Q. WHAT ARE GOOD MANUFACTURING PRACTICES (GMP)?

A.  Good Manufacturing Practices are a set of regulations, codes, and guidelines for the manufacture of: Drug substances and drug products, Medical devices, In vivo and in vitro diagnostic products, Foods

The term "cGMP" is used by the federal government as current good manufacturing practices. By definition, "cGMP" indicates that the current GMP - which is "state of the art" - can change. "GMP" and "cGMP" are often used interchangeably and essentially they have the same meaning.

06. Q. WHO ENFORCES GOOD MANUFACTURING PRACTICES (GMP)?

A.  Good Manufacturing Practices are enforced in the United States by the FDA (Food   and Drug Administration)

Good Manufacturing Practices are enforced in the United Kingdom by the Medicines and Healthcare Products Regulatory Agency (MHRA)

Good Manufacturing Practices are enforced in Australia by the Therapeutical Goods Administration (TGA)

Good Manufacturing Practices are enforced in India by the Ministry of Health, multinational and/or foreign enterprises and those individuals in the following positions:

Each of the inspectorates carry out routine GMP inspections to ensure that drug products are produced safely and correctly.

 

 07.Q.LIST OUT THE APPEARANCE DEFECTS OF TABLES DURING COMPRESSION ACTIVITY ?

Capping:- ‘Capping’ is the term used, when the upper or lower segment of the tablet separates horizontally, either partially or completely from the main body of a tablet and comes off as a cap, during ejection from the tablet press, or during subsequent handling.

Laminating:- ‘Lamination’ is the separation of a tablet into two or more distinct horizontal layers.
   
Sticking/filming: ‘ Sticking’ refers to the tablet material adhering to the die wall. Filming is a slow form of sticking and is largely due to excess moisture in the granulation.

Cracking:-  Small fine cracks observed on the upper and lower center surface of the tablets, or very rarely on the side wall are referred to as cracks.
   
Chipping:- ‘ Chipping’ is defined as the breaking of tablet edges, while the tablet leaves the press or during subsequent handling and coating operation.
   
Mottling:‘ Mottling’ is the term used to describe an unequal distribution of colour on a tablet.
   
Double Impression: ‘ Double impression’ involves only those punches,which have a monogram or other engraving on them.

8. Q What is the standard number of rotations used for friability test?
A. 100 rotations

9. Q What is the fall height of the tablets in the friabilator during friability testing? 
A. 6 inches.Tablets falls from 6 inches height in each turn within the apparatus.
 
10.  Q Which capsule is bigger in size - size '0' or size '1'? 
A. '0' size