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Shula Levin's WebSite of HPLC and LC-MS


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אתר הבית של שולה לוין בנושאי כרומטוגרפיה נוזלית

Various Topics in HPLC


Home |  HPLC Topics  |  LC-MS TOPICS  l   HPLC Course  |  Educational  |  Links  |  Courses  | Contact Me   |  on ISRAEL  | Personal


General Introduction

הקדמה כללית

HPLC Method Search & Develop
חיפוש ופיתוח שיטות

Quantitation & Validation  of Method
עבודה כמותית\ולידציה

Qualification of Instrumentation
כיול של מיכשור

Modes of LC סוגי הפרדה

-  SPE -GPC -Ions 
Peptides Proteins  \Clinical
Chiral \ 

Detectors & Detection סוגי גלאים

Prepare the Lab for HPLC הכנת המעבדה לעבודה

Operational & Troubleshootingתיפעול ואיתור תקלות

Ultra Performance Liquid Chromatography

UPLC Materials

Scale up to Preparative Chromatography

כרומטוגרפיה פרפרטיבית



Books, Magazines & Presentations

ספרות מקצועית

Introduction and Overview הקדמה וסקירה כללית

Before you even touch an HPLC system you need to learn about the technique and technology.  The next links bring to you primers and  introductory articles.
Basic Tutorials, videos and animations מדריכים סרטונים ואנימציות:

  A comprehensive HPLC video by Educational Solutions

General Introduction in Hebrew(350k) 
HPLC Basics by Waters
Introduction by Britannica
Basic Theory  of Chromatography by Frey
Excellent startup course by LCResources:: Getting Started    
My own Chromatography Process Movie (4 mb-save first)
  Educational Solutions: video on HPLC (large and comprehensive)
General Introduction to HPLC and  Quantitative approach - handouts (700K)
 Detection in HPLC and Qualification of detectors-handouts ( 673K)
Reversed Phase Chromatography-handouts  (250K)
  Lecture 4: Basic Chromatography: HPLC (RP & IEC) & Capillary Electrophoresis - MAMS 
acr  Fundamentals of HPLC (very basics)  
   HPLC in the pharmaceutical industry - Review

Intorduction to HPLC PPT

Lecture: Introduction to Chromatography 

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Search and/or Development of HPLC Method חיפוש/פיתוח השיטה לעבודה

Where to begin if you need to perform HPLC analysis?

First search in the literature for existing methods, do not re-invent the wheel.  Go to Google and search for your compound plus the two pharases "mobile phase" and "liquid chromatography":
  Waters applications for method development and validation

Understanding and selecting the right HPLC column בחירת הקולונה המתאימה:

Now, you got an application note or a method from the literature.  Chances are (90%) that you got a reversed phase application for your compound.  What is next? There is a huge selection of reversed phase columns, do I need to buy exactly the same column as the literature shows?  Not necessarily, there is a term called: equivalent column.

Optimize and validate the HPLC method אופטימיזציה וולידציה של השיטה:

After you set up your HPLC method, you need to test it on known samples, and you need to validate your method to be sure that it will work properly and accurately from now on.  The next links describe the process and the reason for optimization and validation of HPLC methods.

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Quantitative Work


Validation of HPLC Method for Quantitative Work

 ולידציה של השיטה לעבודה כמותית 

Once you have the method, make sure to assure that it is appropriate for your specific needs, this is done by validation.

FDA reviewer guidance for HPLC method validation
   Quality by Design (QbD) Considerations for analytical methods: FDA perspective
Development and validation of chromatographic method - Summary
  Quality Assurance Guidelines - ICH
   FDA Guidance to the industry on validation of bioanalytical methods 

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Instrumentation Qualification תיקוף מכשירים

Topics related to the hardware of HPLC systems

Committing to Calibration of Instruments
   Wikipedia: Verification and validation
Operational Qualification of HPLC Instruments:       LCGC North America Vol. 20 No 4 Apr 2002 (Grisanti and Zachowski, Ed: Krull&Swartz)
The procurement, qualification and calibration of lab Instruments: An Overview
       (By Herman Lam, Ph.D.) Laboratory Focus-Gazette Edition, 1999, vol.: 3, no. 3, Page 8  
   Qualification of HPLC (Scribd) 
   Performance Qualification of HPLC Instrumentation in Regulated Laboratories
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Operational Instructions and Troubleshooting הוראות תיפעול ואיתור תקלות 

Now that you have a validated method, you start working daily, now you need to know how to operate the instrument, chemistry and software smoothly and trouble-free.  For instructions see Operational Instructions

The following are topics related to the proper operation of the HPLC system, chemistry, fluidics, software, plumbing, etc.

A guide for a trouble-free  operation of HPLC - Hebrew ( by me 210K)
Guidelines in the operation of an HPLC system
Selecting water for HPLC by MilliporeMilliQ system
Solvent Preparation by Millipore
Sample preparation - filtration products 
On-line recipes for Buffers - PFG
Troubleshooting in HPLC - Chemistry and instrumentation          
Performance Verification of HPLC  By Dr. Herman Lam
QUALIFICATION of HPLC System Components IQ, DQ, OQ from BioQC
SOP for handling and working with analytical standards
Maximizing HPLC Reproducibility in Highly Aqueous Mobile Phases
HPLC Manual HPLC Manual
Regeneration of Reversed  Phase  Columns  (LC-GC Europe  July  2003)
Isranalytica 2006- Unexpected Peaks in the Chromatogram (by Shula Levin 2 MB)
  Non-Linear Calibration
Cleaning and regeneration of HPLC columns - LC-GC 
  The Troubelshooting guide of Waters 
  Maintenance of HPLC system used for concentrated rough samples
  Determination of Carryover and Contamination for Mass Spectrometry Based Chromatographic Assays  
   HPLC Troubleshooting (Proteomics page)

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Preparing the Laboratory for HPLC Work הכנת המעבדה לעבודה

Running HPLC analysis routinely is not a simple task.  See instructions in:   Successful Operation of HPLC System.   The laboratory must be properly prepared for it.  The following links are some pointers to manufacturers of HPLC-compatible chemistry and accessories.





Tubing and Connections

Solvent and Sample handling

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Detection and Detectors in HPLC

General introduction: books, presentations and overviews

Light Spectroscopy Detectors

Aerosol Detectors

Electrochemical & Conductivity Detectors

Mass Spectrometry Detection

*  (User guide of Waters' detector: Register to website first) to get the following information on each detector:

  • Theory of operation
  • Installation procedures
  • User procedures
  • Maintenance and troubleshooting procedur

Now that we know HPLC, it is time to proceed to UPLC: Ultra-Performance Liquid Chromatography

זהו השלב למעבר אל העתיד

Ultra Performance Liquid Chromatography (UPLC)

(login here to WATERS' website first to be able to get these pdf files הרשמה לאתר )

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Special Topics and Applications נושאים נבחרים

שונות Miscellaneous




Chiral Chromatography כרומטוגרפיה כיראלית

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Amino acids Peptides/Proteins חומצות אמיניות, פפטידים וחלבונים

Proein Purification Links קישורים לטיהור חלבונים

Ion Analysis and Capillary Electrophoresis אנליזה של יונים

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Gel Permeation (GPC)  or Size Exclusion Chromatography (SEC) הפרדת מאקרומולקולות :

Sample Preparation Topics נושאים בהכנת דוגמאות

Solid Phase Extraction

Animated Gif

Solid Phase Extraction (SPE) 
Sample Preparation Book(Register first to Waters' site here)
Presentation - Introduction
 Oasis Applications Notebook
 96 well solid phase extraction presentation  

Scaling Up-Preparative Chromatography גימלון להפרדות פרפרטיביות

(login to website  first:

Preparative chromatography Considerations- by Waters
Old Presentation-html
Old Course handouts on  Preparative Chromatography
Scale up of Peptide Separations
Study of Selectivity and Loadability of Pharmaceutical Compounds on RPLC Columns
Preparative Loadability as a Function of pH  Preparative Chromatography at High pH
 The Role of the Column in Preparative HPLC 
 Practical Aspects of Preparative HPLC in Pharmaceutical and Development Production 

     LITERATURE ספרות מקצועית:

Free pdf Chromatography books: LC and GC ספרים אלקטרוניים בחינם
BookRags-on-line free books:   HPLC 
i-Papers by SCRIBD מאמרים וכתבים בחינם 

Chromatographic Science Series 
Special Topics
News in Separation Sciences
Text Books
Chromatographic Science Books
Analytical Chemistry in a GMP Environment

My Favorite Electronic Library  הספריה האלקטרונית שלי 

Wiley InterScience


Beilstein Abstracts @

CAS - Chemical Abstracts Service

ACS publications

ISI - Thomson

Kluwer Online

Taylor & Francis Group




Biomedical and Life Sciences מדעי החיים


Medscape (from WebMD)

Cambridge Scientific Abstracts


   Free Acess Presentations on the Net מצגות פתוחות לקהל

  • Lab Tech Guy on HPLC wash solvents
  • Planar Chromatography
  • Proteins Basics you need to know for Proteomics
  • Design And Purification Of Proteins
  • High Performance Liquid Chromatography
  • High Performance Liquid Chromatography HPLC
  • Mass Spectrometry
  • HPLC Columns and Stationary Phases
  • The Basics of Chromatography
  • HPLC?
  • Tips and Tricks: HPLC Troubleshooting
  • Column Troubleshooting
  • HPLC Course - ppt
  • LC-REsources: LC-MS extra slides
  • Analytical vs Preparative
  • Stationary Phases

  • Successful Operation of HPLC System הנחיות לתיפעול נכון של כל מערכת


    Before beginning to work with any HPLC instrument one must understand the basic principles of liquid chromatography at its various modes. 

    The liquid chromatograph is very complex and it consists of sophisticated technology and sometimes delicate and total ignorance in the manner it works might sabotage its parts which are very expensive.  In addition, there are chemical processes of distribution between the two phases, the mobile and stationary, while the analysis goes on, and there is a need for a minimum understanding of their theoretical basis to operate the system correctly.  Also, a minimum understanding of the method, in which the system performs data analysis, is needed so that no false data or results will be obtained in the analysis.


    LISTS OF STEPS NEEDED BEFORE ANY RUN BY HPLC  רשימת צעדים מקדימים לפני הרצה :

    • Filter the solvents with membranes with cutoff of 0.22-0.45 mM
    • Use clean and transparent reservoirs through which precipitates and colloids can be distinguished.
    • Make sure that the solvents will be easily mixed with the previous solvents in the same inlets (A, B, C, D).  For example methanol or water should not be placed instead of hexane directly, or any organic solvent should not be placed directly instead of a buffer reservoir.
    • Degass the solvents and purge all the tubing that lead to the pump.
    • Connect the column carefully according to the flow direction marked on it (do not connect directly to the detector).
    • Flow the appropriate solvents through the column at a low flow-rate (0.1-0.5 ml/min) or reach the composition gradually using the appropriate gradient.
    •  Select the appropriate wavelength (or other type of setting) in the detector and wait for stable baseline
    • Prepare the set of methods in the workstation: Instrument method for the control on the system, Processing method for the data processing and the Report method for the report of final results.
    • When the system and the methods are ready a blank run should be performed to test the system and verify that it is clean from interferences.

     The rest of the article  Click   להמשך המאמר 

    Layout of the rest of the article:

    -Solvent delivery system המשאבה

    -The column הקולונה

    -Detector  הגלאי

    -Injector   המזרק

    -Gradient and isocratic  גרדיינט או איזוקרטי

    -Data Processing  עיבוד נתונים

    Home |  HPLC Topics  |  LC-MS TOPICS  l   HPLC Course  |  Educational  |  Links  |  Courses  | Contact Me |  ISRAEL

    Copyright (C)1997  Dr. Shulamit Levin

    Disclaimer הסתייגויות:

    I do not necessarily fully agree with all the content in links to resources or responsible for it in any way.