High Performance Liquid Chromatography (HPLC)
An analytical separation technique that involves the high-pressure
flow of a liquid through a column that contains the stationary phase.
Stationary phase: Can be a solid (LSC) or a liquid (LLC)
A mixture of compounds injected at one end of the column
separates as the compounds pass through.
Separated compounds are detected electronically as they
elute at the other end of the column
1) Solvent or mobile phase
Usually a mixture of an organic solvent (Ex. methanol, IPA)
Sometimes buffered – keeps solutes in electrically neutral form
Solvent polarity affects the separation process
Mobile phase considerations
Must be filtered (to prevent tiny solids from depositing at the
column head) and degassed
Degassing is done by helium sparging
Bubbles could interfere with detection
Role is to pump the solvent at a high pressure (usually from
1000 to 6000 psi) through the packed column
Sample introduction system
Usually a loop injector – see image below
Introduces the injected sample to the flowing mobile phase
Automated injectors are common
A small metal tube (typically 5 to 30 cm long; 1-5 mm i.d.)
that contains the stationary phase
Role is to separate the components of a mixture
Column – Cont.
Much shorter than columns used in GC — Why?
Highly efficient separations achieved in HPLC due to
interactions of both m.p. and s.p. with the components of
vs. GC, where only the s.p. interacts with
No need for long columns
This allows the separation of individual sample components to be controlled and optimised. Columns come in two types: packed and capillary . Most gas chromatography today is performed using capillary columns, as they offer significant advantages for most samples
Different design from those of GC detectors because the
components are dissolved in a liquid m.p. (vs. gas in GC)
HPLC Column Selection
Dependent on the:
(1) type of mixture being separated, and
(2) type of interaction with the s.p
Type of buffer in Hplc
by maheshbhuva | May 18, 2023 | Hplc
Phosphate buffer Citrate buffer Formate buffer Acetate buffer Tris(hydrixymethyl) -aminomethane Phosphate buffer Pka 2.1 Ph range 1.1 tob3. 1 Pka 7.2 Ph range 6.2 to 8.3 Pk 12.3 Ph range 11.3 to 13.3 Citrate Pka 3.1 Ph range 2.1 to 4.1 Pka 4.7 Ph 3.7 to 5.7 Pka 5.4 Ph...
HPLC Calibration As per IPC Pharmacopeia give guidance
by maheshbhuva | Mar 24, 2023 | Hplc
HPLC Calibration we have perform as earlier as per protocol Company will provided Like example Shimanzu HPLC calibration will perform as per that guidance's But now a day In 16th September 2021 As per IPC Pharmacopeia give guidance(Document ID IPC/GD/05) HPLC...
How can read Protocol analyst
by maheshbhuva | Jan 28, 2023 | Analytical Chemistry, Analytical method development hplc,, Hplc
HPLC Analyst ○ Whenever supervisor give work allocation for testing any product. ○ HPLC analyst first seen Test which is Assay by HPLC or RS By HPLC, Chiral test etc ○ Then careful read specification(SOP) not missing any point which is defied in sop ○ Then Seen Which...
How can read Protocol analyst During HPLC analysis and what Is flow ?
by maheshbhuva | Jan 27, 2023 | Analytical Chemistry, Hplc
• HPLC Analyst ○ Whenever supervisor give work allocation for testing any product. ○ HPLC analyst first seen Test which is Assay by HPLC or RS By HPLC, Chiral test etc ○ Then careful read specification(SOP) not missing any point which is defied in sop ○ Then Seen...
by maheshbhuva | Jan 20, 2023 | Analytical Chemistry, Analytical method development hplc,, Hplc
Why does Acetonitrile use in HPLC Method development?
by maheshbhuva | Dec 28, 2022 | Analytical Chemistry, Analytical method development hplc,, Hplc
it is miscible with water, has good eluotropic strength and also low viscosity alone and mixed with water. Acetonitrile high-sensitivity analysis with UV detection in the short-wavelength region. However , organic solvents which have been processed for HPLC IN...
Accuracy – Analytical method validation, hplc, pharmaceutical
by maheshbhuva | Dec 25, 2022 | Analytical Chemistry, Hplc, Method validation
Accuracy is also part of method validation different place Different terminology used for accuracy like recovery, truth value, biaus etc
Specificity -Analytical Method validation in pharmaceutical company
by maheshbhuva | Dec 25, 2022 | Hplc, Method validation
Specificity -Analytical Method validation
HPLC COLUMN – FACTOR AFFECTS,During HPLC analysis
by maheshbhuva | Dec 22, 2022 | Hplc, Hplc column
1) Partial Blockage of column 2) Adsorption of sample impurities inside column surface 3) Poorly packed stationary phase. 4) Mechanical or thermal shocks during Handling. 5) Chemical attack on the stationary phase. https://www.frontlifesciences.com/qualitycontrol-qc/...
What is Internal standard HPLC / GC- How to USE
by maheshbhuva | Dec 14, 2022 | Gc, Hplc
Internal standard how we have to used? Internal standard 1. Quantatives analylsis 2. Purpose is 3. Selection process 4. Example is Quantatives analylsis main three parts • External standard • Internal standard • Standard addition External standard Here we find...