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אתר הבית של שולה לוין בנושאי כרומטוגרפיה נוזלית

Various Topics in HPLC

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Do you Use HPLC? Please answer this poll:

What LC mode do you use? (answer all that applies)
Mostly Reversed Phase mode
Ion Exchange Chromatography
Mostly Normal Phase mode
Size Exclusion Chtomatography (SEC or GPC)
Chiral chromatography
Affinity Chromatography
Hydropilic Interaction Chromatography (HILIC)
 

Do you Use UPLC? Please answer this poll:

Do you use U-HPLC (UPLC) in your daily work
Yes, I use mostly U-HPLC (UPLC)
Yes, but only here and there
Yes, very rarely, I still prefer to use HPLC
No, but I would like to use it if I have access to
No, I think it is just a hype and have no future
 

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 \  SFC  \ TLC

Detectors & Detection סוגי גלאים

Prepare the Lab for HPLC הכנת המעבדה לעבודה

Operational & Troubleshootingתיפעול ואיתור תקלות

Ultra Performance Liquid Chromatography (UPLC)חומר על UPLC

Scale up to Preparative Chromatographyכרומטוגרפיה פרפרטיבית

Scaling Down to U-HPLC/Fast LC
מיזעור הכרומטוגרפיה

Books, Magazines & Presentationsספרות 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 HebrewHPLC Guide - Hebrew Article(350k) 
HPLC Basics by Waters
Introduction by Britannica
Users' Guide by UKy
Some aspects of pharmaceutical analysis using high performance liquid chromatography Article by Cope and Davidson

Basic Theory  of Chromatography by Frey
Lectures on Chromatography - LC and GC by Dr. Efimov
Excellent startup course by LCResources::  http://www.lcresources.com: Getting Started     
Chromatographic Techniques  by Weib
Glossary of HPLC
Chromatography - Creating a Central Science
My own Chromatography Process Movie (4 mb-save first)
ACDLabs Animations and Simulations
  Educational Solutions: video on HPLC (large and comprehensive)
  Chromatographic separations: Theory and Applet simulating chromatogram
General Introduction to HPLC and  Quantitative approach - handouts (700K)
 Detection in HPLC and Qualification of detectors-handouts ( 673K)
Reversed Phase Chromatography-handouts  (250K)
Determination of caffeine in soft drinks by High Performance Liquid Chromatography -Example of an experiment and procedure by Univ. of Adelaide
Determination of caffeine in beverages - example for HPLC experiment  from Rice University
Determination of diet soft drink components by HPLC-example of experiment - doc  - by Pomona College
Lecture on chromatography  from MSU      HPLC- State of the Art 
  Lecture 4: Basic Chromatography: HPLC (RP & IEC) & Capillary Electrophoresis - MAMS 
  Course on HPLC modes (ppt)
  Lecture on modes of HPLC 
  acr  Fundamentals of HPLC (very basics)
   Basic Chemistry
   http://www.mournetrainingservices.co.uk/hplc_video.html   
   HPLC site: Examples of how to produce results in HPLC  
   HPLC in the pharmaceutical industry - Review 

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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":
 

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.

   Analytical_Method_Development.ppt
RSC chapter on variability of analytical measurements-precision and repeatability 
FDA reviewer guidance for HPLC method validation
HPLC Method Validation
Development and validation of chromatographic method - Summary
FDA Guidance for Industry: Analytical Procedures and Methods Validation 
Lancaster's presentation on analytical method development and validation
Valid Analytical Measurements -Website - See publications
Performance Verification of HPLC
FDA Guideline for submitting samples and analytical data for methods validation
Control of the quality of analytical methods
  Quality Assurance Guidelines - ICH
  Validation of Computerized Liquid Chromatographic Systems -FDA guidance
  Norms and guidelines for  the practical estimation of uncertainty in analytical measurements 
  Precision in HPLC
   Key Elements of Bioanalytical Method Validation for Small Molecules 
   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

    Qualification of Analytical Instruments for Use in the Pharmaceutical Industry  

   Operational and Performance Qualification of HPLC: LC-GC

    Equipment Specifications for Liquid Chromatography -MS/MS (QQQ) with HPLC System

 
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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
Importance of Controlling Mobile Phase pH in Reversed Phase HPLC
Preparing Buffered Mobile Phases for Reversed Phase HPLC by Mac-Mod
Selecting water for HPLC by MilliporeMilliQ system
Solvent Preparation by Millipore
Sample preparation - filtration products 
Buffers in RP HPLC- why TFA?    TFA in the mobile phase - WesternAnalytical
On-line recipes for Buffers - PFG
Troubleshooting in HPLC - Chemistry and instrumentation          
The HPLC Doctor - U of Lisbon
Chromatography Forum - Join the discussion group
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
The Column Companion - mac-mod.com: Maximize your  use of  HPLC columns
HPLC Manual HPLC Manual
Correcting Peak Tailing Problems in Reversed Phase HPLC
Regeneration of Reversed  Phase  Columns  (LC-GC Europe  July  2003)
Isranalytica 2006- Unexpected Peaks in the Chromatogram (by me 2 MB)
A Troubleshooting Guide to Plumbing Problems in HPLC by MAC-MOD
Starting out right HPLC system
  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  
  Guideline for the transfer of analytical test procedures 
   Top 10 HPLC Column Myths  by Ronald E. Majors
   HPLC Troubleshooting (Proteomics page)

   "Ghost" peaks: Background contamination   

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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.


Water

Solvents:


Glassware

Buffers


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 )

- Introduction to UPLC מאמר היכרות בעברית עם , my article in Hebrew

- Setting up Binary Acquity UPLC system - Step by Step for EMPOWER (Hebrew/English) הכנת מערכת לעבודה   
- Setting up Quaternary H-Class UPLC system - for Empower (Hebrew/English)  הכנת מערכת לעבודה -צעד צעד

- Setting up Binary Acquity system - Step by Step for MassLynx (Hebrew/English) הכנת מערכת לעבודה 

- Quaternary H-Class System Quick Reference Card  הנחיות לעבודה נכונה
- Acquity Binary UPLC System Quick Reference Card כרטיס המלצות לעבודה נכונה 

- Required Information from Original HPLC Method to Transfer to UPLC
- Final UPLC (Binary) Method description: Example   (pdf)  

- Transfer between all UPLC types of systems: calculator description(pdf)
- Example of a Method for Quaternary/Binary UPLC systems

-  Acquity Binary UPLC system: Injection Modes Quick Reference Card
- Solvent saving calculator מחשבון חסכון בממסים
Filtration of Mobile Phase - Article by by Subodh Kulkarni, Jesmi George, and Vivek Joshi  

-  Filtration products: Select the right filter (register first)
Impact of water on UPLC operation by Millipore  
-  What is required from a high quality water for UPLC: MilliQ Water for UPLC (Millipore)  (pdf)

- Type I ultrapure water crucial for HPLC and UHPLC (ELGA) (pdf)  Water for UHPLC by Merck (pdf)

- Presentation to the USP people: "UPLC®, Ultra Performance LC®" (pdf)

 - Stimuli Article in the USP: "Transfer of HPLC Procedures to Suitable Columns of Reduced Dimensions and Particle Sizes"  (pdf)

User Manuals מדריכי שימוש:

  • Controlling Contaminations in UPLC and MS



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Special Topics and Applications נושאים נבחרים

שונות Miscellaneous

 

Separation Modes of HPLC (my html presentation)
System Peaks in HPLC (my Article)
Peak Purity and Spectral Matching with Diode-Array Detectors (my Hebrew article-)
  Handouts-Overview on Photo Diode Array Detectors 
 

 

Chiral Chromatography כרומטוגרפיה כיראלית

                                      Back to Top



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)  Hebrew presentation
Sample Preparation Book(Register first to Waters' site here)
Presentation - Introduction
 Oasis Applications Notebook
Sample preparation SPE search for application protocol
 96 well solid phase extraction  



Scaling Up-Preparative Chromatography גימלון להפרדות פרפרטיביות

(login to website  first:  http://www.waters.com)

Preparative chromatography Considerations- by Waters
Presentation-html
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 



Scaling Down מיזעור לכרומטוגרפיה מהירה או ננו


Ultra Performance Liquid Chromatography (UPLC): An IntroductionUltra Performance Liquid Chromatography - sub 2 micron columns
 Separation sciences redifined




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 
Textbooks
Journals
Special Topics
Abstracts
Wiley's News in Separation Sciences
 
Text Books
Amazon: BOOKS ON CHROMATOGRAPHY
Chromatographic Science Books
Analytical Chemistry in a GMP Environment

My Favorite Electronic Library  הספריה האלקטרונית שלי 

Wiley InterScience


http://www3.interscience.wiley.com/search/allsearch

ScienceDirect
http://www.sciencedirect.com/science/publications/journal/chemistry

Beilstein Abstracts @ ChemWeb.com
http://www.chemweb.com/

Scirus
http://www.scirus.com/

CAS - Chemical Abstracts Service
http://www.cas.org

ACS publications
http://pubs.acs.org/journals/query/nonsubscriberSearch.jsp

ISI - Thomson
http://www.isiwebofknowledge.com/

Kluwer Online
http://www.kluweronline.com/

Taylor & Francis Group
http://taylorandfrancis.metapress.com/app/home/main.asp

Ingenta
http://www.ingenta.com/

InfoTrieve
http://www4.infotrieve.com/users/logon.asp?component=AF&dk=/search/databases/newsearch.asp

Engineering & Industry הנדסה ותעשייה 

Applied Science & Technology Index
http://www.hwwilson.com/

Ei Compendex
http://edina.ac.uk/compendex/
 

Biomedical and Life Sciences מדעי החיים

PubMed
http://www.ncbi.nlm.nih.gov/PubMed

Medscape (from WebMD)
http://www.medscape.com

BioMedNet
http://www.bmn.com/

Cambridge Scientific Abstracts
http://www.csa.com/

 




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
  • Liquid Chromatography
  • HPLC Course - ppt
  • LC-REsources: LC-MS extra slides
  • High Performance Liquid Chromatography (HPLC): Haris chapter 25
  • Analytical vs Preparative
  • New trends in packings, stationary phases and columns in HPLC


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


    Introduction


    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




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    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.