Handling of HPLC

Unit Operation

Schematic of an HPLC

High-performance liquid chromatography (HPLC) is a form of liquid chromatography to 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

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)

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 = octadecylsilyl as the stationary phase) is called reversed phase

liquid chromatography (RPLC).

Ironically the "normal phase" has fewer applications and RPLC is therefore used considerably more

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

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 penetrateNon-polar interactions between analyte hydrophobic portion and bonded phaseWeak interactions

dipole-dipoledipole-induced dipoleinduced dipole-induced dipole

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.

Column Selection guide

Buffers in the mobile phase preparation

Commonly Used Buffers for Reversed Phase HPLC

Buffer pKa  Buffer Range UV Cutoff (nm)Phosphate pKa1 2.1 1.1-3.1 200

7.2 6.2-8.212.3 11.3-13.3

Formic acid* 3.8 2.8-4.8 210Acetic acid*  4.8 3.8-5.8 210Citrate 3.1 2.1-4.1 230

4.7 3.7-5.7 5.4 4.4-6.4

carbonate, pKa1 6,1 5.1-7.1 < 200carbonate, pKa2 10,3 9.3-11.3 < 200Tris 8.3 7.3-9.3 205Triethylamine* 11 10.0-12.0 200ammonia 9,3 8.3-10.3 200 (10 mM)borate 9,2 8.2-10.2 n/aPyrrolidine 11.3 10.3-12.3 200* Volatile buffers

Ion Pair Reagents in the mobile phase

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

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

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

High surface area generally provides greater retention, capacity and resolution for

separating complex, multi-component samples. Low surface area packings generally

equilibrate quickly, especially important in gradient analyses.

Surface Area-Effect on Chromatography

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

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)17CH3Si

CH3

CH3

(CH2)17CH3XOH +monomeric

bonding

Si

CH3

X

(CH2)17CH3X+polymeric

bonding

OH

OH O

O

Si

CH3

(CH2)17CH3

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

Endcapping reduces peak-tailing of polar solutes that interact excessively with the

otherwise exposed, mostly acidic silanols. Non-endcapped packings provide a different

selectivity than do endcapped packings, especially for such polar samples.

End Capping-Effect on Chromatography

HPLC-Detectors

HPLC-Detectors

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 mL in a sonicator applying vacuum &

degas for 20 minutes for volume 5000 mL in a sonicator applying 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.

Selection of Column

Select the Column as Mentioned in the STP or 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

Preparation of Sample and Standard Solutions

Prepare Standard, Sample and system suitability Solutions as per STP or method

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

Filling of the Vials and Labeling Of the Vials

Discard the First 5 mL of 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

HPLC Chromatograms

0 1 2 3 4 5 6 7

Time (minutes)

Ab

so

rba

nc

e

Area =base x height

2

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 moving

more retained

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 solutionAt least the major analyte of interest and, ideally, a closely eluted component or components that could be found in actual samples at known levelsStandard solution can be used

System suitability

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

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

System suitability

• Resolution (R)

R = 1.0 R = 1.5 R = 2.0

Column Efficiency (N)–only one peak of interest–measure of peak sharpness–detection of trace components–generally not less than 2000 (HPLC)–isocratic/isothermal system

Column efficiencynumber of theoretical plates in a chromatogram

System suitability

Relative Standard Deviation (SR or 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

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

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 2

t - retention time of the analyte

ta - retention time of an unretained peak

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

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%

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 variationis met.

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%

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.

Flow Variation

Temperature Variation

Injector Reproducibility

Injector Linearity

Detector Linearity

Carryover

Gradient performance test

Drift and Noise

Performance verification of HPLC

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