good ppt

1. Name :- JAY V PATEL
Enrollment No :- 16064561054
Institute :- MEHSANA URBAN INSTITUTE OF SCIENCES
Guide Name :- PROF . HETAL PATEL

2. INTRODUCTION OF CHRTOMATOGRAPHY :
What is chromatography ?
 Chromatography is define as the method of separating a mixture of
components into individual component. The separation uses a column
[stationary phase] and solvent [mobile phase].
Principle of chromatography:-
 Chromatography is an analytical technique . commonly used for separating a
mixture of chemical substance in to it’s individual components. The sample
are subjected flow by mobile liquid on to or through the stable stationary
phase.

3. Type of Chromatography :
1) Liquid chromatography :- In this chromatography used to
analyze metal ions and organic compound.
2) Gas chromatography :- Used to analyze volatile gas.
3) Thin layer chromatography :- This is a simple and rapid
method to check the purity of the organic component.
4) Paper chromatography :- The most common type of
chromatography. The paper is stationary phase and liquid is mobile
phase.

4. HPLC : High Pressure Liquid Chromatography.
What is HPLC ?
 HPLC is an analytical technique, commonly used for
separating a mixture of chemical substance in to it’s
individual components. The sample are subjected flow by
mobile liquid on to or through the stable stationary phase.
 The main principle HPLC is adsorption.

5.  Type of HPLC :
1. Reverse Phase :- Non-polar stationary phase and a polar
mobile phase. Ex.-Silica gel ,C18
2. Normal Phase:- Polar Stationary phase and non polar mobile
phase. EX. -Silica gel
3. Ion Exchange :- Stationary phase contains ionic groups and the
mobile phase is an aqueous buffer.
4. Size Exclusion :- There is interaction between the sample
compound and the column. Large molecules elute first and small
molecules elute later.

6. Instrumentation:-

8. 1. Solvent Reservoir
2. Pump
3. Sample Injector
4. Column
5. Detector
6. Integration
 Main Part of Instruments:-

9.  Advantages of HPLC:-
 The time Required for separation very less for HPLC
than GC.
 High pressure is used in HPLC.
 It is a sensitively.
 Need a small sample with a high accuracy and
precis.
 Disadvantages of HPLC:-
 HPLC has very costly.
 It is a difficulty to clean and devices are very
expensive.
 Solvent consuming
 Need a skill to run the instruments.

10.  Calibration:-
 What is calibration?
 Calibration means to known how accurate Testing and
measuring instrument employed in the quality
measuring system are known as calibration.

11.  HPLC System Calibration:
1. Pump:- Flow rate Accuracy Test:-
 Keep water as a mobile phase in respective reservoir line.
 Connect union in place of column.
 Purge the line and then allow the system to stabilize for about 5 minutes at 1.0 ml/min flow
rate. Check for constant pressure.
 Take a previously dried glass beaker.
Take it’s weight W1 =32.950 gm.
 Allow the effluent from detector waste in the beaker for 5 minute.
Now take beaker + water weight W2 = 37.837 gm.
 Calculate actual volume with help of following equation.
V = D/(W2-W1)
where, D is the density if the water at particular temperature.

12. Flow rate Weight of
empty
beaker (W1)
Empty
beaker +
water (W2)
Difference in
gm.
W =
(W2 - W1)
Conversion
in ml
Acceptance
criteria +or-
2% of
theoretical
volume
1 ml/min 32.950 gm. 37.837 gm. 4.887 gm. 4.9 ml 4.8 ml to 5.2
ml
2 ml/min 33.060 gm 42.985 gm 9.925 gm 9.95 ml 9.8 ml to
10.2 ml
Results are complies as per acceptance criteria.

13. 2. Detector:-
Wavelength Accuracy Test
Chromatographic parameters:-
Column C18, 250 * 4.6mm, 5μ
Mobile Phase Water : Methanol (70:30)
Flow Rate 1 ml/min
Injection Volume 10μl
Detector At different wavelength (269nm, 270nm, 271nm,……..to…….275nm.)
Run Time 4 to 8 minutes (at least 1.2 times of retention time of analyte)
Diluent Mobile Phase

14.  Take 20 mg of caffeine in a 100mL volumetric flask. Add 10 ml of
mobile phase. Sonicate to dissolve. Make up the volume up to 100ml
with mobile phase (200ppm solution).
 Further dilute 5 ml of this solution to 50 ml with mobile phase
(20ppm solution).
 Stabilize the system for constant baseline.
 Inject the sample with 269 nm to 275 nm by the increment of 1nm
wavelength and record the chromatogram. Find the highest response
from the results and consider its respective wavelength as actual
wave length.
 System Suitability Criteria :
Theoretical Plates – Not less than 2000
Tailing factor – Not more than 2

15. Wavelength Response in Height Acceptance criteria 272
+or-2 nm
269nm 97481
270nm 105866
271nm 110025 271
272nm 104145
273nm 56669
274nm 58552
275nm 64955

18. Highest Height
at 271 nm

19. 3. Auto Sampler:-
Injector linearity
• Chromatographic Parameters
Column C18, 150 × 4.6mm, 5μ
Mobile Phase Water : Methanol (70:30)
Flow Rate 1.0ml/min
Injection Volume 10,20,30,40,50μl
Detector 272nm
Run Time 4 to 8 minutes (at least 1.2 times of retention time of analyte)
Diluent Mobile Phase

20. • Take 20mg of caffeine in a 100ml volumetric flask. Add
10ml of mobile phase. Sonicate to dissolve. Make up the
volume up to 100ml with mobile phase (200ppm solution).
• Further dilute 5ml of this solution to 50 ml with mobile
phase (20ppm solution).
• Stabilize the system for constant baseline.
• Inject the 10, 20, 30, 40 and 50 microliter of 20ppm
caffeine solution in the system.
• System Suitability Criteria :
Theoretical plates – Not less than 2000
Tailing Factor – Not more than 2

23. Injector
volume
area
10 770283
20 1543972
30 2360696
40 3176094
50 3850811
Injector linearity

24. Assay of sorbic acid
 Chromatographic Parameters:-
Column 250mm * 4.6mm , 5µ , C18
Mobile Phase Water : Methanol : Acetonitrile (22:70:8) +
0.3ml Ortho-Phosphoric Acid
Flow Rate 1 ml/min
Injection Volume 10 μl
Detector 240nm
Diluent Water : Methanol : Acetonitrile(22:70:8)
Run time 6 minutes (at least of retention time of
analyte)

25.  Standard Preparation:-
 Take 35 mg of sorbic acid working standard in a 50ml volumetric flask.
Add 10ml of mobile phase. Sonicate to dissolve it.
 Make up the volume up to 50ml with mobile phase (700ppm
solution).
 Further dilution 5ml of this solution to 50ml with mobile
phase(70ppm solution).
 Sample Preparation:-
 Take 25 mg (25mg) of Sample in a 50ml Volumetric Flask
add 10 ml of mobile phase. Sonicate to dissolve it.
 Make up to 100ml with mobile phase(500ppm solution).
 Further dilution 5ml of this solution to 25ml with mobile
phase(20ppm solution).
 Further dilution 2ml of this solution to 50ml with mobile
phase(20ppm solution).

29. solution
No
of
injec
tions
Diluent 1
Standard
Preparation
3
Sample Preparation 2
Bracketing Standard 1
Injection sequence

30. SORBIC ACID ASSAY.

31.  What is Validation?
 Validation is the establishing documented
evidence which provides a high degree of
assurance that a specific process will
consistently produce a product meeting its
predetermined specifications and quality
attributes.

32.  Parameter of validation:-
(1) Specificity:-
 Main pick is compare to another pick result is less
interfirence, and acceptance area2 >resolution .
(2) Accuracy:-
 Closeness to the hypothetical true value. Acceptable value
is ±2.
(3) Linearity:-
 Linearity is acceptable with a coefficient of
determination(r²) of 0.997
(4) Precision:-
 Closeness to the consiquitive analytical result same.
a. Repeatability:- one and more same sample are use in
repeatability. acceptable critearea±2.

33. b. Reproducibility:- sample check in difference place
with different condition. Acceptable criteria ±2.
c. Intermediate precision:- various parameter like
column beach number different, instrument
different, analyst different etc. are follow
(5) Range:- 100-150ppm range is compulsory for
perfect analysis.
(6) Robustness:- In this process system suitability
parameter are follow. In this process depended on
mobile phase composition, buffer pH, flow rate.
(7) LOD:- (limit of detection)
(8) LOQ:- (limit of quantification)

34.  Standard Preparation:-
 Take 35mg of sorbic acid working in a 50ml volumetric
flask. Add 10ml mobile phase. Sonicate to dissolve it. Make
up to 100ml with mobile phase(700ppm solution).
Further dilute 2ml of this solution to 50ml with mobile
phase.
SPECIFICITY

35. Validation of sorbic acid
 Chromatographic Parameters:-
Column 250mm×4.6μ, 5µ, C18
Mobile Phase Water : Methanol : Acetonitrile (22:70:8) +
0.3ml Ortho-Phosphoric Acid
Flow Rate 1 ml/min
Injection Volume 10 μl
Detector 240nm
Diluent Water : Methanol : Acetonitrile(22:70:8)
Run time 6 minutes (at least of retention time of analyte)

36. Injection sequence
solution no of injection
diluent 1
water 1
acetonitrile 1
methanol 1
standard 1

39.  Application of HPLC:-
 Pharmaceutical/Biopharmaceutical:-
 Pharmaceutical quality control.
 Shelf-life determinations of pharmaceutical
products.
 Identification of counterfeit drug products.
 Complex molecules separation.
 Environmental:-
 Bio-monitoring of Pollutants.
 Water monitoring – Phenol content and toxic
components checking.

40.  Clinical:-
 Analysis of antibiotics and blood substances.
 Detection of endogenous neuropeptides in brain
extracellular fluids.
 Food and Flavor:-
 Sugar analysis in fruit juices.
 Ensuring soft drink consistency and quality.

41. THANK YOU