1. 1
A Training REPORT
ON
VARIOUS INSTRUMENTS USED IN ANALYTICAL DIVISION OF
PHARMACEUTICAL INDUSTRIES
Submitted in Partial Fulfillment of the Required Credits for the Degree of
BACHELOR OF technology
IN
BIOTECHNOLOGY
SuPERVISEDBY Submitted by
Mr. Sumit Yadav Preeti
Sr. ResearchScientist jv-b/10/2051
Analytical Research& Development Center B.tech-M.tech(Biotechnology)
XII Trimester
Dr. TG.Chandrashekher
Head Global Quality, R&D &RA
Kusum Healthcare Pvt. Ltd.
Faculty of ENGINEERING & technologY
4. 4
CERTIFICATE
The report is hereby approved as a bonafide and creditable training work
“Various instruments used in analytical division of pharmaceutical
industries” carried out and presented by Preeti (jv-b/10/2051) in a manner to
warrant its acceptance in partial fulfillment of the required credits for the degree of
B.tech-M.tech in Biotechnology. However, the undersigned do not necessarily
endorse or take responsibility for any statement or opinion expressed or conclusion
drawn there in, but only approve the report for the purpose for which it is
submitted.
(…………………………) (Dr. Khusbhu Verma)
Supervisor (External) Supervisor (Internal)
Kusum Healthcare Pvt. Ltd. Jayoti Vidyapeeth Women’s University
(Mr. Sumit Yadav)
Coordinator
Department of Analytical Research& Development Center,
Kusum Healthcare Pvt. Ltd.
(Dr. Promod Raghav)
Dean
Faculty of Engineering and Technology,
Jayoti Vidyapeeth Women’s University
5. 5
ACKNOWLEDGEMENT
I express my gratitude to all those who helped me to prepare and complete my
training work entitled “Various instruments used in Analytical division of
pharmaceutical industries”. First of all, I convey my deep gratitude and heart full
thanks to Dr. Rahul Jain and Mr. Shyam S. Shrivastava, Kusum Healthcare
Pvt. Ltd. for his inspiration, cooperation and encouragement for pursuing my
dissertation. His valuable suggestion and guidance helped me a lot to complete my
work in this institution with in a very short period.
I render my sincere respect and heart full gratitude to Gargi Sharma, Food and
Biotechnology Department, Jayoti Vidyapeeth Women’s University, Jaipur. I am
also thankful to all the faculty members, for their valuable suggestion towards
completing the dissertation work. I am also grateful to all my class mates, who
helped me directly or indirectly in completing my dissertation/training/project
work successfully.
Last but not least, I am really ever grateful to my parents, who remained a constant
source of encouragement and inspiration during the completion of this work
successfully in Jayoti Vidyapeeth women’s University, Jaipur.
Preeti
jv-b/10/2051
Food and Biotechnology Department
Jayoti Vidyapeeth Women’s University, Jaipur
6. 6
DECLARATION
"I hereby declare that this submission is my own work and that, to the best of my
knowledge and belief, it contains no material previously published or written by
another person nor materials which have been accepted for the award of any other
degree or diploma of any university or institution of higher learning, except where
due acknowledgment has been made in the text.”
Place.………………… Preeti
Date.…………………. jv-b/10/2051
B.tech-M.tech (Biotechnology)
XII Trimester
7. 7
INDEX
S. No. Topic Page NO.
1.
2.
3.
4.
5.
6.
Introduction of Pharmaceutical industry:
pH Meter:
Introduction
Principle
How does it work
Cleaning
Calibration
Acceptance criteria
Procedure
Precaution
Balance:
Cleaning
Calibration
Acceptance criteria
Procedure
Precaution
Karl-Fischer Auto Titrator:
Introduction
Principle
How does it work
Cleaning
Procedure
KF titration
Determination of water factor
Result
Determination of moisture content
Dissolution Test Apparatus:
Introduction
Types
Example
UV Spectroscopy:
Introduction
Principle
Cleaning
Procedure
6-7
8-12
13-19
20-24
25-28
29-36
9. 9
INTRODUCTION OF PHARMACEUTICAL INDUSTRY WHERE
I HAVE COMPLETED MY TRAINING WORK:
Kusum Healthcare Pvt. Ltd. was founded in 1997, as a 100% export-oriented
unit, with its headquarters in New Delhi.
The company`s first formulation plant was started in Bhiwadi, Rajasthan in
2007.
Another formulation plant is expected to come up shortly in Indore, Madhya
Pradesh.
Kusum Healthcare Pvt. Ltd. is a part of kusum group of companies which is
fully integrated, young pharmaceutical group on fast track having two
manufacturing units; one of them is Kusum Healthcare at Bhiwadi,
Rajasthan India and the second is Kusum Pharma at Sumy Ukraine.
Both these manufacturing units are WHO GMP certified and PIC/s
compliant.
Kusum Healthcare products are market in 9 countries namely Ukraine,
Uzbekistan, Kazakstan, Moldova, Tajikistan in CIS with fully integrates
operations with sales, marketing, warehousing, distribution, regulatory,
Pharmacovigilanceaned all supportfunctions.
Apart from this products are marketed in Myanmar, Philippines and Vietnam
in the ASEAN region and has started marketing operations in India in the
year 2012.
All the formulations are developed at own formulation R&D centre which is
recognized by Ministry of Science and Technology Government of India.
10. 10
Analytical Development:
Analytical Development at Kusum Healthcare Pvt. Ltd. Research and
Development Center is fully capable of handling analysis for various types
of dosages form like Solid Oral, Semi Solid etc centre (Formulation and
Analytical).
Analytical Development has the following capabilities:
Analytical method development and their validation (for precision, accuracy,
selectivity or specificity, linearity & range limit of detection, limit of
quantization, ruggedness).
Development / preparation of complete specifications.
Stability study of formulations as per ICH guidelines under variety of
conditions applicable.
Study of effect of solvent, alkalies, acids, excipients on the stability of
formulations.
Preformulations studies.
Kinetics and mechanism of degradation of drugs and formulations.
11. 11
PH METER:-
pH is the negative logarithm of the hydrogen ion activity.
pH meter is an instrument used to measure acidity or alkalinity of a solution.
pH is the unit of measure that describes the degree of acidity or alkalinity.
It is measured on a scale of 0 to 14.
Fig: pH meter
PRINCIPLE:
Glass electrode has a silver based electrical wire suspended in a solution of
potassium chloride, contained inside a thin bulb made from a special glass
containing metal salts.
The other electrode is called the reference electrode and has a potassium
chloride wire suspended in a solution of potassium chloride.
Key parts of a pH meter:
1) Solution being tested
2) Glass electrode, consisting of
12. 12
3) A thin layer of silica glass containing metal salts , inside which there is a
potassium chloride solution
4) Internal electrode
5) Internal electrode made from silver/silver chloride
6) Hydrogen ions formed in the test solution interact with the outer surface of
the glass
7) Hydrogen ions formed in the potassium chloride solution interact with the
inner surface of the glass
8) The meter measure the difference in voltage between the two sides of the
glass and converts the “potential difference” into a pH reading
9) Reference electrode acts as a baseline or reference for the measurement and
complete the circuit.
HOW DOES IT WORK?
The potassium chloride inside the glass electrode is a neutral solution with a
pH of 7, so it contains a certain amount of hydrogen ions [H+].
Supposethe unknown solution we are testing (blue) is much more acidic, so
it contains a lot more hydrogen ions.
13. 13
What the glass electrode does is to measure the difference in pH between the
potassium chloride solution inside the glass and the test solution outside the
glass by measuring the difference in the voltages their hydrogen ions
produce.
Since we know the pH of the potassium chloride solution inside the glass,
we can figure out the pH of the test solution.
CLEANING:
After completion of pH measurement rinse the electrode and temperature
sensor with the purified water, soak the excess water with tissue paper and
insert the pH electrode in the storage solution (3 mol/L potassium chloride
solution).
CALIBRATION:
1. Default calibration parameters are set for the calibration with three buffer
solutions
pH 1.68 Buffer solutions
pH 4.00 Buffer solutions
pH 7.00 Buffer solutions
2. Press CAL key to enter calibration mode starting the calibration with first
buffer solution pH 1.68
Wash the pH electrode with distilled water and wipe off the moisture and
immerse it in the first buffer solution and then confirm with the CAL key.
At the calibration time room temperature is enter manually.
The first buffer solution is measured.
3. Continuing the calibration with second buffer solution pH 4.00.
pH electrode is removed from first buffer solution and wash again.
14. 14
pH electrode is immerse in second buffer solution and continue the
calibration procedurewith the CAL key.
Second buffer solution is measured.
4. Continuing the calibration with third buffer solution pH 7.00
pH electrode is removed from second buffer solution and wash again.
pH electrode is immerse in third buffer solution and continue the
calibration procedurewith the CAL key.
Third buffer solution is measured.
RESULT:
The result of the calibration is displayed in slope.
Note: The desired pH should be in between the calibration range of buffer. If the
desired pH is not in between the calibrated range, recalibrate the pH meter with the
buffers to get the desired range.
The maximum difference between two calibration buffers values should not be
more than 3.00.
Finish the calibration with the OK key.
15. 15
ACCEPTANCE CRITERIA:
Slope of pH calibration should be between 95.0 to 103.0
PROCEDURE:
1. Switch on the instrument using power on/off switch in the rear of the
instrument. Warm up the instrument for just for 1 minute.
2. Wash the electrode with distilled water and wipe off the moisture.
3. When dipping the electrode into the sample the sensor or glass bulb of
electrode must be completely immersed into the sample.
4. Allow time for the reading to stabilize.
5. Record the observed values of pH.
6. Wash the electrode with distilled water and wipe off the moisture and store
the electrode in storage solution (3 mol/L potassium chloride solution).
PRECAUTIONS:
1. After every pH measurement, leave the electrode in storage solution. (3
mol/L potassium chloride solution).
2. Don’t allow the solution to evaporate & crystallize on electrode.
3. Don’t store in distilled water.
4. If the pH meter is not given the stable reading, recalibrate the pH meter with
fresh buffers.
5. Report any discrepancy observed during operation or calibration monitoring
Affix “UNDER MAINTAINANCE” label on the instrument
16. 16
BALANCE:-
Fig: Analytical balance Fig: Micro balance
Cleaning:
Switch ‘OFF’the balance.
Clean the weighing pan using dry tissue / brush / lint free cloth.
Wipe off the pan using tissue / lint free cloth moistened with water. Wipe off
the excess moisture with dry tissue paper / lint free cloth.
Remove Dust/residual material on weighing pan with tissue / lint free cloth
moistened with Isopropyl Alcohol, if required. Wipe off the excess
IsopropylAlcohol using dry tissue/ lint free.
CALIBRATION:
Daily Verification
1. Internal adjustment:
Internal adjustment is performed for analytical and microbalance on the
daily basis before starting the weighing and in the condition of power
failure.
17. 17
Press the ‘Adjust.int’ key on the display and wait for some time till message
appears as ‘ADJUSTMENT DONE’
2. Balance checks:
The balance checks is performed on the daily basis after the internal
adjustment by using the 100 mg of certified standard weight for the
analytical and microbalance.
ACCEPTANCE CRITERIA:
The observed value should be within ± 0.05 % of the actual value.
Note: Ensure that the standard weight used for balance checks is calibrated and
certified.
2 Periodic Calibration
1. Accuracy:
The accuracy of an analytical procedure expresses the closeness of agreement
between the value, which is accepted either as a conventional true value or an
accepted reference value and the found value.
The accuracy of a balance is satisfactory if its weighing value, when
tested with a suitable weight(s), should be within ± 0.10 % of the test
weight value.
Standard weights shall be placed one by one at the centre of the balance
pan.
For accuracy test of balance calibration, following weights is used:
18. 18
For Microbalance:
For Analytical balance:
S. No. Weight
1 1.0000 mg
2 2.0000 mg
3 5.0000 mg
4 10.0000 mg
5 20.0000 mg
6 50.0000 mg
7 100.0000 mg
8 500.0000 mg
9 1.0000000 g
10 2.0000000 g
S. No. Weight
1 20.0 mg
2 50.0 mg
3 100.0 mg
4 200.0 mg
5 500.0 mg
19. 19
Acceptance Criteria: The observed weight should be within ± 0.10 % of the actual
weight for all the certified weights used above.
2. Eccentricity:
Deviation in the measurement value caused by eccentric loading, in other
words, the asymmetrical placement of the centre of gravity of the load relative
to the load receiver.
The eccentricity is performed for microbalance by placing 500 mg standard
weight at the centre and at the four quadrants locations.
The eccentricity is performed for analytical balance by placing 100 g
standard weight at the centre and at the four quadrants locations.
Acceptance criteria: The standard deviation for the five values observed
above should not be more than 0.05 %.
6 1.0000 g
7 2.0000 g
8 5.0000 g
9 10.0000 g
10 20.0000 g
11 50.0000 g
12 100.0000 g
13 200.0000 g
20. 20
3. Linearity:
Ability of a balance to follow the linear relationship between a load and the
indicated weighing value.
The linearity is performed using 20 mg, 50 mg, 200 mg, 1g, 10 g, 100 g and
200 g standard weight for analytical balance.
The linearity is performed using 1.0 mg, 5.0 mg, 10.0 mg, 50.0 mg, 100 mg,
500 mg and 2 g of standard weight for micro balance.
The linearity plot is drawn between the actual weight and observed weight.
The correlation coefficient should not be less than 0.99000.
4. Repeatability:
Ability of a weighing balance to display identical measurement values for repeated
weighings of the same objects under the same conditions, e.g., the same
measurement procedure, same operator, same measuring system, same operating
conditions, and same location over a short period of time. Repeatability is usually
expressed as the standard deviation of multiple weighings.
Repeatability is assessed by weighing one test weight not less than 10 times.
The repeatability for analytical balance is performed by weighing standard
weight of 100 mg, 10 times.
The repeatability for micro balance is performed by weighing standard
weight of 10 mg, 10 times.
5. Acceptance criteria:
The repeatability is satisfactory if two times the standard deviation of the
weighed value, divided by the actual value of the weight used should be within
± 0.10%
21. 21
6. Systematic error:
Systematic error is an error that is not determined by chance but is introduced
by an inaccuracy (as of observation or measurement) inherent in the
system. Systematic error may also be an error.
Systematic Error: 2 x Standard deviation X100
Actual Mass value
If standard deviation is less than 0.41d, where d is the scale interval, replace
the standard deviation with 0.41d. In this case repeatability is satisfactory if
two times 0.41d, divided by the actual value of the weight used should not
be more than 0.10 %.
The balance should display the weight within the tolerance limits specified
for each of the standard weight range. In the case of non-conformance, clean
the standard weight with tissue paper or lint free cloth and re-weigh.
Procedure:
Open the glass drift shield, place the required vessel/butter
paper/aluminium foil to be used on weighing pan, close the glass drift
shield, and press the tare/zero key (→T←, →0←). The display will show
the ‘ZERO’ reading. Put the material to be weighed on the vessel/butter
paper /aluminum foil or the material to be weighed can also be directly
placed on the weighing pan. Wait until the stability detector ‘O’
disappears.
The reading shown on the display is the weight of the material and take
the printout wherever printer is attached. (Forpositive weights)
22. 22
For taking actual transfer weight, tare the display weight, remove the
vessel/butter paper/aluminum foil from the weighing pan and transfer the
material/mass from the vessel/butter paper/aluminum foil to the flask or
any desired container. Keep the empty vessel/butter paper/aluminum foil
on the weighing pan. The display shows actual transfer in negative. Take
the printout of the value shown.
PRECAUTIONS:
Ensure that balance is kept on vibration free & even surface.
Ensure that balance is cleaned properly.
Ensure that spirit level is in center position or adjust it using the foot
screw to keep it in center.
Check for calibration status label before use.
Check for daily verification status before use.
Ensure the power supply is on and all the connections are tightened.
23. 23
KARL FISCHER TITRATION:
Karl Fischer Titration is a technique for the determination of moisture
content.
The technique was developed by a chemist Karl Fischer.
Karl Fischer provides for the specific detection of water.
PRINCIPLE:
Water Determination Test (Karl Fischer Method) is designed to determine
water content, utilizing the quantitative reaction of water and iodine and
sulfur dioxide in the presence of a lower alcohol such as methanol and an
organic base suchas pyridine, as shown in the following formula:
H2O + I2 + SO2+ CH3OH + 3RN -> [RNH]SO4CH3 + 2[RNH]I
24. 24
A widely used method for the quantitative analysis of water content in a
variety of the sample.
It uses Coulometric and Volumetric titration to determine trace water
amounts in a sample.
Method Volumetric:
In the volumetric titration method, iodine required for reaction with water is
previously dissolved in water determination and water content is determined
by measuring the amount of iodine consumed as a result of reaction with
water in a sample.
Method Coulometric:
In the Coulometric method, first iodine is produced by electrolysis of the
reagent containing iodide ion, and then the water contain in a sample is
determined by measuring the quantity of electricity which is required for the
electrolysis based on the quantitative reaction of the generated iodine with
water.
KF Method: How does it work?
Water and iodine are consumed at 1:1 ratio (in the Bunsen reaction).
Once all the water present is consumed, the presence of excess iodine is
determined volumetrically by the indicator electrode of the titrator.
This signals the endpoint of the titration.
The amount of water present in the sample is calculated based the
concentration of iodine in the KF titrating agent (titer) and the KF reagent
consumed in the titration.
25. 25
CLEANING:
Switch of before cleaning.
Clean the instrument with a clean dry cloth. If required, wipe with a cloth
moistened in a mild detergent solution. Subsequently wipe to dryness with a
tissue paper or a dry cloth.
PROCEDURE:
Switch on the instrument and computer.
Open the tiamo 2.5 software by double clicking the software icon located in
desktop and enters the user name & password as entitled.
Four options will appear on the left side screen bar.
1. Workplace
2. Database
3. Method
4. Configuration.
Go to configuration and check the status. It should be OK.
Click on workplace option. Four windows shall be displayed on screen.
1. Workplace
2. Run
3. Report
4. Live Display 1
Before initiating Titration Check Electrode Input, Dosing Device, & Stirrer
to be used in the method (selected for Titration) by manual control as
displayed at bottom of left side.
Select the appropriate method in workplace from RUN window by click on
METHOD shown in run window.
Weigh the desired sample in a beaker and add the appropriate solvent, place
26. 26
it on the magnetic stirrer to be used & put the magnetic bar into it. Dip
appropriate electrode assembly along with dispensing tip in the beaker just
above the stirrer bar.
Enter Sample Name, Batch No., LNB Ref Analyst Name & Sample size or
appropriate information as required.
Click on START for conditioning and wait till ‘Condition OK’ appears on
the screen. Weigh the sample and fill the required details as appear on the
Screen and Press OK.
After getting titration over, burette gets filled and then titrator goes for
conditioning again. Reportappears in report window.
As the experiment is over, remove the beaker; wash the electrode properly
with appropriate solvent wipe with tissue paper.
To take the Print out select the data then click File – Print – Report – Select
the Report template and click O.K.
KF TITRATION:
The instrument supply should be ON.
Rinse the titration vessel with methanol.
On desktop double click on tiamo 2.5 Icon. Screen shows tiamo 2.5 and then
shows initializing program and devices and follow the step 6.3.2 to 6.3.10.
Fill in the titration vessel with methanol by pressing (↓)button on 803 Ti
stand and for empty the beaker press the (↑)button on 803 Ti stand.
In the Run window, select the appropriate method.
Click START, so that the methanol in the vessel gets conditioned. Live
display 1 / KF Titration show conditioning not ok. Wait till ‘conditioning
ok’ appears and drift should be less than 20µl/min.
27. 27
DETERMINATION OF WATER FACTOR:
Press start. Window of sample data request appears. Add the water drop
(weight should be less than 50 mg) in the titration vessel. Take the weight
and enter the weight of sample in gram in sample size. Press OK to continue.
Titration proceeds. After getting titration over burette gets filled and then
Titrator goes for conditioning again. Reportappears in report window.
Repeat the same procedure two more times. Final report have mean of three
determinations with % RSD.
The % RSD should not be more than 1.0%.
Determine the moisture content by the formula given below:
MeanKF FactorX Volume consumed (mL) X 100
% Moisture = -----------------------------------------------------------------
Sample wt. in g X 1000
RESULT:
The individual % moisture content value and mean value should be
98.0 – 102.0 %.
The %RSDof 3 values should be less than 1.0 %.
DETERMINATION OF MOISTURE CONTENT:
Press start. Window of sample data request appears. Add the sample in the
titration vessel. Enter the weight of sample in gram in sample size. Press OK
to continue.
Titration proceeds. After getting titration over burette gets filled and then
titrator goes for conditioning again. Reportappears in report window.
28. 28
DISSOLUTION TEST APPARATUS:
Dissolution is a pharmaceutical defined as the rate of mass transfer from a
solid surface into the dissolution medium or solvent under standardized
condition of liquid/solid interface, temperature and solvent composition.
Fig: Dissolution Instrument
29. 29
TYPES:
Fig: Types of dissolution apparatus
DISSOLUTION METHOD FOR DETERMINATION % DRUG RELEASE IN X
TABLETS (BY UV SPECTROSCOPY):
Product Name: X Tablet
Method For: Determination for % drug release in X Tablets
30. 30
Reagents:
Sodium dihydrogen phosphatedihydrate (HPLC grade)
Water (HPLC/Milli-Q grade)
Purified water
Sodium Laruyl Sulphate (SLS)
Dissolution Parameters:
Dissolution media : pH 7.0 Phosphatebuffer with SLS
Dissolution volume : 900 mL
Apparatus : BP-II (Paddle)
Speed : 100 RPM
Temperature : 37.0°C ± 0.5°C
Sampling Interval : 45 min
Sampling volume : 10 mL
Preparation of dissolution media:
Accurately weigh and transfer 1.380 gm Sodium dihydrogen phosphate
dihydrate and 10.000 gm of SLS mixed into 1.0 L of water.
Mix well and adjusted to pH 7.0 with sodium hydroxide solution.
Preparation of diluent:
Use Dissolution Media as Diluent.
31. 31
Preparation of blank:
Use Diluent as blank
Preparation of standard solution:
Accurately weigh and transfer about 25.0 mg of X working standard into a
100 mL volumetric flask.
Add about 60 mL of diluent and sonicate at least 10 min for dissolve.
Cool and make up the volume with diluent and mix well. Dilute 4 mL of this
solution to 100 mL with diluent.mix well.
Filter this solution through 0.45 µm Nylon filter; discard first 3 mL of filtrate.
Preparation of Sample solution:
Transfer one tablet in to each of the six dissolution vessels containing
900mL of dissolution medium which is previously maintained temperature
of 37°C + 0.5°C and carry out dissolution as per prescribed dissolution
parameters.
PROCEDURE:
Auto zero the instrument using blank at 240 nm and measure the
absorbanceof blank at 240 nm
Measure the absorbance of standard solution in six replicate at 240 nm.
%RSD of six absorbanceof standard solution should be less than 2.0.
Measure the absorbanceof sample solution in single at 240 nm.
32. 32
UV SPECTROSCOPY:
UV spectroscopy is type of absorption spectroscopy in which light of ultra-
violet region (200-400 nm.) is absorbed by the molecule.
It measures the intensity of light passing through the sample (I) and
compares it to the density of light before it pass through the sample (Io).
The ratio I / Io of is called transmittance, and is usually expressed as a
percentage (%T)
The absorbanceA is based on the transmittance.
Fig: UV Spectroscopy
PRINCIPLE:
Ultraviolet (UV) spectroscopy is working based on the principle of Beer
Lambert`s Law.
33. 33
It involves, Absorbance is directly proportional to intensity of the colour and
thickness of the medium.
1. Ultraviolet light: Wavelength between 190 and 400nm.
2. Visible light: Wavelength between 400 and 800nm.
Violet: 400 - 420 nm
Indigo: 420 - 440 nm
Blue: 440 - 490 nm
Green: 490 - 570 nm
Yellow: 570 - 585 nm
Orange: 585 - 620 nm
Red: 620 - 780 nm
CLEANING:
Dedust the instrument with a clean dry cottoncloth.
Clean the outer surface the instrument with Isopropyl alcohol and dry it with
tissue paper.
After the completion of the testing, cuvette from the sample compartment.
34. 34
Remove the solution from the curvette and wash with respective solvent in
which solution is prepared.
Rinse with water and then methanol and wipe with tissue paper.
Air dry and keep it properplace.
Fig: UV Spectroscopy Fig: Cuvette
PROCEDURE:
Connect the power supply to UV Spectrophotometers, Computer& Monitor.
Switch on the instrument and computer and wait till internal initialization is
completed.
Double click on UV probesoftware.
Press PC control (F4) in the instrument operate the instrument from
computer.
Connect the Spectrophotometers by single click on the icon “CONNECT”
on the instrument bar.
Go to the menu and and select appropriate mode mentioned below:
1. Kinetics
35. 35
2. Photometric
3. Spectrum
Click on “M” icon to create data collection method for photometric module.
Set a measurement wavelength as per shown in following figure.
Set up method for calibration cure or single point analysis.
Enter the name and other information for the measurement as shown in the
figure:
It is necessary to the enter the file name before measurement of sample.
36. 36
The page will be opened in the wavelength and add the parameter, select
calibration and enter raw data and close the window and method should be
saved in method in saved folder.
Keep blank in both the compartment and perform AUTOZERO.
Enter the standard/sample information into the boxas shown below:
Measure the absorbanceof blank.
To read the standard/test solution, keep the solution in one compartment and
blank in other compartment.
Enter the name of the sample in sample table and click the READ
UNKNOWN icon at the bottom.
To take printout.
APPLICATIONS:
Detection of impurities
As HPLC(High Performance Liquid Chromatography) detector
Dissolution study
37. 37
In case of Dissolution:
ProjectName:X Tablets Experiment: Dissolution
Analyst Name: Analyst Ref:
[Wavelengths]
Wavelength Name: WL240.0
Wavelength: 240.00 nm
[Calibration Curve]
Cal. Curve Type: Raw Data
[Measurement Parameters (Standard)]
[Measurement Parameters (Sample)]
Data Acquired by: Instrument
Data Sample read: Disable
Repeat: Disable
39. 39
S.No.
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
Sample ID
Blank
Standard_1
Standard_2
Standard_3
Standard_4
Standard_5
Standard_6
X INITIAL 10MIN_1
X INITIAL 10MIN_2
X INITIAL 10MIN_3
X INITIAL 10MIN_4
X INITIAL 10MIN_5
X INITIAL 10MIN_6
X INITIAL 15MIN_1
X INITIAL 15MIN_2
X INITIAL 15MIN_3
X INITIAL 15MIN_4
X INITIAL 15MIN_5
X INITIAL 15MIN_6
X INITIAL 30MIN_1
X INITIAL 30MIN_2
X INITIAL 30MIN_3
X INITIAL 30MIN_4
X INITIAL 30MIN_5
X INITIAL 30MIN_6
X INITIAL 45MIN_1
X INITIAL 45MIN_2
X INITIAL 45MIN_3
X INITIAL 45MIN_4
X INITIAL 45MIN_5
X INITIAL 45MIN_6
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6/14/2016
6/14/2016
6/14/2016
6/14/2016
Time
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WL240
-0.000
0.757
0.755
0.752
0.754
0.753
0.753
0.627
0.588
0.135
0.632
0.421
0.663
0.726
0.678
0.338
0.703
0.529
0.691
0.719
0.715
0.500
0.749
0.639
0.704
0.710
0.731
0.574
0.735
0.674
0.692
Comments
40. 40
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:-
HPLC is high performance liquid chromatography.
It is a chromatographic techniques that can separate a mixture of compounds.
It is used in biological and analytical chemistry to identify, quantify and purify
the individual components of a mixture.
Fig: HPLC
PRINCIPLE:
The mobile phase is pumped through the column by a pump.
The mixture to be separated is injected into the flowing mobile phase by a
injector.
When the mobile phase has passed through the column it enters into the
detector that detects.
41. 41
A signal goes from the detector to a printer that presents the separation
graphically.
1. Eluent (Mobile phase):
The mobile phases in HPLC refer to the solvent being continuously
applied to the column or stationary phase.
The mobile phase acts as a carrier to the sample solution.
A sample solution is injected into the mobile phase of an assay through
the injector port.
As a sample solution flow through a column with the mobile phase, the
components of the solution migrate according to the non-covalent
interaction of the compound with the column.
The chemical interaction of the mobile phase and sample with the
column, determine the separation of components contained in the sample.
The solvents or mobile phase used must be passed through the column at
high pressure at about 1000 to 3000psi, this is because as the particles
size of stationary phase is around 5-10µ,so the resistance to the flow of
solvent is high.
42. 42
2. Pump:
The role of the pump is to force a liquid (called the mobile phase)
through the liquid chromatography at a specify flow rate, expressed in
milliliters per min (ml/min).
The normal flow rate in the HPLC are in the 1-2ml/min range.
Typical pump can reach pressure in the range of 6000-9000 psi (400-600
bar).
During the chromatographic experiment, a pump can deliver a constant
mobile phase composition (isocratic) or an increasing mobile phase
composition (gradient).
Fig: Pump
3. Injector:
The injector serves to introduce the liquid sample into the flow stream of
the mobile phase for analysis.
43. 43
It is equipped with 6 port valves so that a sample can be injected into the
flow path at continuous pressure.
For a manual injector, the knob is manually operated to deliver the
sample to the column.
The knob is set to LOAD position for sample injection using a syringe
the sample is injected into the sample loop, which is separated from the
flow path.
The knob is turned to inject position and the eluent travels through the
loop from the pump and delivers the sample to the column.
Typical samples volumes for manual injector are 5-20microliters (µm).
The injector must also be able to withstand the high pressure of the liquid
system.
An auto sampler is the automatic version for when the user has many
samples to analyze or when manual injection is not practical.
It can continuously inject variable volume a of 1µl-1ml.
Fig: Injector
44. 44
4. Column:
The heart of a HPLC system is the column.
It is usually made of stainless steel withstand high pressure caused by the
pump to move the mobile phase through the column packing other
material include glass.
The small particle inside the column is called the packing.
Column packing is usually silica gel because of its particle shape surface
properties, pore structure give us a good separation.
The dimensions of the analytical column are usually:
straight length (5-25cm),
diameter of the column (3-5mm),
diameter of particles (3-5µm).
Fig: Column
5. Detector:
The detector can detect the individual molecules that elute from the
column and convert the data into an electrical signal.
The detector provides an output to a recorder or computer that result in
the liquid chromatogram.
45. 45
APPLICATIONS:
Compound identification
Assay determination
Impurity determination
Dissolution studies
In case of dissolution:
Product Name: Z Tablet
Method For: Determination of dissolution of Z in Z Tablet
Reagents:
Methanol (HPLC grade)
Orthophosphoric Acid (85 % or 88 %, HPLC grade)
Water (HPLC/Milli-Q grade)
Purified water
Hydrochloric Acid
Dissolution Parameters:
Dissolution media : 0.1 N Hcl
Dissolution volume : 900 mL
Apparatus : BP- II (Paddle)
Speed : 50 RPM
46. 46
Temperature : 37.0°C ± 0.5°C
Sampling Interval : 45 min
Sampling volume : 10 mL
Preparation of buffer:
Measure and transfer 1 ml of orthophosphoric acid in 1000 mL of water, mix
well.
Filter the solution through 0.45µm membrane filter.
Preparation of mobile phase:
Prepare a suitable quantity of mobile phase by mixing buffer and methanol
in a ratio of 70:30, degas.
Preparation of dissolution Media:
Measure 85 mL of hydrochloric acid and transfer in to 10.0 liter of water.
Mix well.
Preparation of diluent:
Use Dissolution Media as Diluent.
Preparation of blank:
Use Diluent as blank
47. 47
Preparation of standard solution:
Accurately weigh and transfer about 30.0 mg of Z working standard into a
100 mL volumetric flask.
Add about 60 mL of diluent and sonicate to dissolve.
Cooland make up the volume with diluent and mix well.
Dilute 5 mL of this solution to 50 mL with diluent.mix well.
Filter this solution through 0.45 µm Nylon filter; discard first 3 mL of filtrate.
Preparation of Sample solution:
Transfer one tablet in to each of the six dissolution vessels containing 900
mL of dissolution medium which is previously maintained temperature of
37°C + 0.5°C and carry out dissolution as per prescribed dissolution
parameters.
Chromatographic conditions:
Column : Inertsil ODS-3V, 150 mm x 4.6 mm, 5µ
Flow rate : 1.5 mL/min.
Wavelength : 210 nm
Injection volume : 10 l
Column oven temperature : 30 C
Sample oven temperature : 5 C
Run Time : 12 minutes
48. 48
Procedure:
1. Inject five replicates injections of the standard solution in to the
chromatograph and record the chromatogram, system is suitable only if..
The theoretical plate counts for Z peak in the first injection of standard
solution should be more than 2000.
The tailing factor for Z peak in the first injection of standard solution
should be less than 2.0.
The % relative standard deviation (% RSD) of five replicates injection area
counts of Z peak in standard solution should be less than 2.0 %.
2. Inject sample solution in Single.
Retention time of Z peak is about 7.2 minute. (Retention time is given for
information purposeonly)
49. 49
REFERENCES:
1. Sharma Y.R.Elementary Organic Spectroscopy. 1st Edition, S.Chand
Publisher; 2010 .
2. Chatwal G.R.Instrumental methods of chemical analysis, 1st Edition,
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3. Potdar Manohar A. “Pharmaceutical quality assurance” 2nd Edition, Nirali
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Press,p-199.
5. Biopharmaceutics and pharmacokinitics; D.M. Brahamankar, Sunil B.
Jaiswal, vallabh prakashan, p-20-25.
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p-174-176.
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8. Alfred Martin, James Swarbrick, physical pharmacy, 3rd Edition, Varghese
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50. 50
12.Nash Robert A. “Pharmaceutical Process Validation” 3rd Edition, Marcel
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Heyden and Son; 1976.
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