Hplc

1. Handling of HPLC
Unit Operation

2. Schematic of an HPLC
High-performance liquidchromatography(HPLC) is a form of liquidchromatographyto separate compounds that are dissolved in solution. HPLC instruments consist of a reservoir of mobile phase, a pump, an injector, a separation column, and a detector

3. Difference between TLC and HPLC
TLC
HPLC
Type of Analysis
qualitative only
qualitative &
quantitative
Stationary Phase
2-dimensional
thin layer plate
3-dimensional
column
Instrumentation
minimal!
much! with many
adjustable parameters
Sample Application
spotting
(capillary)
injection
(Rheodyne injector)
Mobile Phase Movement
capillary action
(during development)
high pressure
(solvent delivery)
Visualization of Results
UV lightbox
“on-line” detection
(variable UV/Vis)
Form of Results
spots, Rf’s
(retention factors)
peaks, Rt’s
(retention times)

4. Theory
Liquid chromatography (LC) is a separation technique in which the Mobile Phase is a Liquid
Stationary Phase is Solid
In the HPLC technique, the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure
HPLC is historically divided into two different sub-classes based on the polarity of the mobile and stationary phases.
They are 1.Normal Phase 2.Reverse Phase
Normal Phase:Technique in which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase, silica as the stationary phase) is called normal phase liquid chromatography (NPLC)
Reverse Phase: Technique in which the Mobile Phase is more polar than the Stationary phase (e.g. water- methanol mixture as the mobile phase and C18 = octadecylsilylas the stationary phase) is called reversed phase liquid chromatography (RPLC).
Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more

5. Proposed Reverse phase Mechanisms
Hydrophobic Theory
Partition Theory
Adsorption Theory
Hydrophobic Theory
Chromatography of “cavities” in solvent created by hydrophobic portion of analyte molecule
Surface Tension
Interaction of polar functions with solvent
Stationary phase is passive

6. Proposed Reverse phase Mechanisms
Partition Theory
Analyte distributes between aqueous mobile phase and organic stationary phase
Correlation between log P and retention
“organic” phase is attached on one end
Does not explain shape selectivity effects
Adsorption Theory
Analytes “land” on surface -do not penetrate
Non-polar interactions between analyte hydrophobic portion and bonded phase
Weak interactions
dipole-dipole
dipole-induced dipole
induced dipole-induced dipole

7. How The Separation of Analyte in Columns
Separation of compounds is based on the competition of the solute and the mobile phase for binding places on the stationary phase. For instance, if normal phase silica gel is used as the stationary phase it can be considered polar. Given two compounds which differ in polarity, the most polar compound has a stronger interaction with the silica and is therefore more capable to dispel the mobile phase from the binding places. Consequently, the less polar compound eluted first and follows the Next compound.

8. Column Selection guide

9. Buffers in the mobile phase preparation
Commonly Used Buffers for Reversed Phase HPLCBufferpKa Buffer RangeUV Cutoff (nm) Phosphate pKa12.11.1-3.12007.26.2-8.212.311.3-13.3Formic acid*3.82.8-4.8210Acetic acid* 4.83.8-5.8210Citrate3.12.1-4.12304.73.7-5.7 5.44.4-6.4carbonate, pKa16,15.1-7.1< 200carbonate, pKa210,39.3-11.3< 200Tris8.37.3-9.3205Triethylamine*1110.0-12.0200ammonia9,38.3-10.3200 (10 mM) borate9,28.2-10.2n/aPyrrolidine11.310.3-12.3200* Volatile buffers

10. Ion Pair Reagents in the mobile phase

12. Common RP Packings and Its properties
Hydrophobic Surface
Particle Size and Shape
Particle Size Distribution
Porosity, Pore Size and Surface Area
Carbon Loading, End capping

13. Spherical particles offer reduced back pressures and longer column life when using viscous mobile phases like 50:50 MeOH:H2O.
Particle Shape-Effect on Chromatography

14. Smaller particles offer higher efficiency, but also cause higher backpressure. Choose 3μm particles for resolving complex, multi-component samples. Otherwise, choose 5 or 10μm pickings.
Particle Size-Effect on Chromatography

15. High surface area generally provides greater retention, capacity and resolution for separating complex, multi-component samples. Low surface area packingsgenerally equilibrate quickly, especially important in gradient analyses.
Surface Area-Effect on Chromatography

16. Larger pores allow larger solute molecules to be retained longer through maximum exposure to the surface area of the particles. Choose a pore size of 150Å or less for sample MW 2000. Choose a pore size of 300Å or greater for sample MW > 2000.
Pore Size-Effect on Chromatography

17. Monomeric bonding offers increased mass transfer rates, higher column efficiency, and
faster column equilibration.
Polymeric bonding offers increased column stability, particularly when highly aqueous
mobile phases are used. Polymeric bonding also enables the column to accept higher
sample loading
Bonding Type-Effect on Chromatography
Si
R
R
(CH2)17CH3 Si
CH3
CH3
OH + X (CH2)17CH3
monomeric
bonding
Si
CH3
X
+ X (CH2)17CH3
polymeric
bonding
OH
OH O
O
Si
CH3
(CH2)17CH3

18. Higher carbon loads generally offer greater resolution and longer run times. Low carbon loads shorten run times and many show a different selectivity.
Carbon loading-Effect on Chromatography

19. Endcappingreduces peak-tailing of polar solutes that interact excessively with the otherwise exposed, mostly acidic silanols. Non-endcappedpackingsprovide a different selectivity than do endcappedpackings, especially for such polar samples.
End Capping-Effect on Chromatography

20. HPLC-Detectors

21. HPLC-Detectors

22. Handling
Switch ON Main Power supply to the Pump, auto Sampler, Degasser, Column Compartment and Printer
Preparation of Mobile Phase
1.Prepare buffer solution as per STP & mix with the solvents as mentioned in STP .While preparing the mobile phase, add solvents in a same sequence as mentioned in STP & mix thoroughly in mobile phase bottle.
2. Filter mobile phase through 0.45 micron Nylon membrane filter or as suggested in STP.
3. Degas the mobile phase for 10 minutes for volumes 1000,2000,3000 mLin a sonicatorapplying vacuum & degas for 20 minutes for volume 5000 mLin a sonicatorapplying vacuum. Ensure that proper water level & temperature is maintained during sonication. Avoid over Sonication to avoid polymerization of some of the salts.
4. Limit for pH adjustment for mobile phase & buffers meant for its preparation is + 0.02.

23. Selection of Column
Select the Column as Mentioned in the STPor Method
Ensure that there is no air Bubble in 4 Channels
Flush the Column, prior to use, for at least 30 minutes using HPLC Grade Methanol at a flow rate of about
1 mL/minnute
Saturate the Column with Mobile Phase For at least 30 Minutes
Put Appropriate Needle Wash, Seal wash solutions ,Wash Vials as applicable
Preparation of Sample and Standard Solutions
Prepare Standard, Sample and system suitability Solutions as per STP
Use filter as mentioned in STP. First rinse the syringe with diluents, discard initial volume of diluents solution through a filter holder, collect the required volume & after filtration discard the membrane filter.
Check the System suitability Parameters then only Proceed for Next Step

24. Preparation of Sample and Standard Solutions
Prepare Standard, Sample and system suitability Solutions as per STP ormethod
Use filter as mentioned in STP. First rinse the syringe with diluents, discard initial volume of diluents solution through a filter holder, collect the required volume & after filtration discard the membrane filter
Check the System suitability parameters then only Proceed for Next Step

25. Filling of the Vials and Labeling Of the Vials
Discard the First 5 mLof Solution from the Standard and Sample Solution.
Use Fresh Vials for each Preparation
Rinse the HPLC Vials with Respective solution
Fill the Each Vial with Enough Sample solution for all injection
Crimp the vial properly
Label all the test and standard solutions for all tests with at least details such as A.R.No,Solution Name and appropriate replicate preparation number when ever applicable
Label the HPLC,GC Vials with legible marker pen for as follows
XYY, Where X=Unique Quick set YY-Is position at that is to be placed

26. HPLC Chromatograms
0 1 2 3 4 5 6 7
Time (minutes)
Absorbance Area =base x height2
base
height
Peak A
Peak B
Approximation
of peak area by
triangulation
Rt = 3.0 min.
faster moving
less retained
Rt = 6.0 min. slower movingmore retained

27. USP <1225>
System suitability tests are based on the concept that the equipment, electronics, analytical operations, and samples to be analyzed constitute an integral system that can be evaluated as such. System suitability test parameters to be established for a particular procedure depend on the type of procedure being evaluated. They are especially important in the case of chromatographic procedures...
Provides assurances that the system is working properly at the time of analysis
Ensures that both methodology and instrumentation are performing within expectations prior to the analysis of
the test samples
Should be monitored during run time to verify that the criteria remain realistic and achievable
Determined from the analyte peak
Assessed with any significant change in equipment or in a critical reagent
System suitability solution
At least the major analyte of interest and, ideally, a closely eluted component or components that could be found in actual samples at known levels
Standard solution can be used
System suitability

28. System suitability
Acceptance criteria
balance between theoretical and practical performance
sufficiently tight -data quality is assured
not so restrictive that acceptable systems fail
reflective of minimum acceptable performance to generate reliable result
Parameters for a chromatographic method
Resolution -specificity
Column efficiency -specificity
Relative Standard Deviation – precision
Tailing Factor -accuracy and precision
QL -sensitivity
Capacity factor -specificity
Reference Standard Check -analyst

29. Resolution (R)
–function of column efficiency (N)
–measure of the resolving power of the system
–generally, not less than 2.0
–most closely eluting pair
System suitability

30. System suitability
•Resolution(R) R = 1.0 R = 1.5 R = 2.0

31. Column Efficiency (N)
–only one peak of interest
–measure of peak sharpness
–detection of trace components
–generally not less than 2000 (HPLC)
–isocratic/isothermal systemColumn efficiencynumber of theoretical plates in a chromatogram
System suitability

32. Relative Standard Deviation (SRor RSD)
replicate injections of a Standard preparation
assessment of repeatability of the system
five replicate injections of the analyte if the requirement is 2.0% or less
System suitability

33. Tailing Factor (T)
measure of peak symmetry
equals one for perfectly symmetrical peaks
peak asymmetry increases, accuracy
and precision becomes less reliable
generally not more than 2
System suitability
•Tailing factor

34. System suitability
•Capacity Factor (k’ or k)
–measure of where the peak of interest is located with respect to the void volume, i.e., elution time of the non-retained components
–generally, not less than 2t -retention time of the analyteta-retention time of an unretainedpeak

35. System suitability
•Quantitation Limit (QL)
–a dilution of the analytes are injected at the QL concentration
–S/N > 10 for single injection OR
–RSD <15% for multiple injections

36. System suitability
•Reference Standard Check
–duplicate injection of a separately weighed reference solution
–check accuracy of solutions preparation
–the expected result for the second standard should be:
98.0% reference standard 102.0%

37. Adjustments in System Suitability
pH of the Mobile Phase(HPLC):±0.2 units of the value or range specified for aqueous buffer.
Concn. Of Salts in Buffer (HPLC):Within ±10% provided the permitted pH variationismet.
Wavelength of UV-Vis detector (HPLC):Deviations from the wave lengths specified in the method are not permitted.
Column length(GC,HPLC):±70%
Column Inner Diameter(GC,HPLC):±25% for HPLC and ±50% for GC.
Film Thickness (Capillary GC):-50% to 100%
Particle Size (HPLC):can be reduced by as much as 50%.
Particle Size (GC):If it is same ‘Range Ratio’ of the GC mesh support and chromatography meets the requirements of the system suitability.
Flow Rate (GC,HPLC):±50%
Injection Volume (GC,HPLC): Can be reduced as much as is consistent with accepted precision and detection limits.
Column Temperature (HPLC): ±10deg
Oven Temperature (GC): ±10%

38. Adjustments in System Suitability
Ratio of the Components in Mobile Phase (HPLC): Apply to Minor components of 50% or less.
±30% relative. Change in any component cannot exceed ±10% absolute ( i.e. in relation to the mobile phase). Binary Mixtures: eg. For 50:50 : Thirty percent of 50 is 15% absolute but ±10% is only permitted. That means either 40:60 or 60:40 can be made. Ternary Mixtures: for eg.60:35:5: 30% of 35 is 10.5% absolute but only ±10% is permitted. That means change can be made between 25% and 45%. In all cases , a sufficient quantity of the first component is used to give a total of 100%.There fore, Mixture ranges of 50:45:5 to 70:25:5 or 58.5:35:6.5 to 61.5:35:3.5.

39. Flow Variation
Temperature Variation
Injector Reproducibility
Injector Linearity
DetectorLinearity
Carryover
Gradient performance test
Drift and Noise
Performance verification of HPLC

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