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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

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Click here: Page on my own Hebrew articles and presentations here  - מאמרים ומצגות שלי בדף אחד

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General Introduction

 

הקדמה כללית

Basic Tutorials, videos and animations מדריכים סרטונים ואנימציות:

  Understanding and selecting the right HPLC column בחירת הקולונה המתאימה

conc
Principles of quantitative work, method validation
hplc
Qualificatin of HPLC Instruments. Topics related to the hardware of HPLC systems

Special Topics

Various modes of chemistry of the separation in HPLC.

Material on detectors and detection in HPLC

prepare
How to get prepared for HPLC work
A. proper operation of the HPLC system, chemistry, fluidics, software, plumbing, etc

Ultra Performance Liquid Chromatography (UPLC)

 

חומר על UPLC

uplc

Resources on UPLC: collection of links from Waters and operational instruction


Scale up to Preparative Chromatography

כרומטוגרפיה פרפרטיבית

PREP

Information on how to scale up to preparative chromatography

Scaling Down to U-HPLC/Fast LC

מיזעור הכרומטוגרפיה

Scale Down

Scaling down to fast chromatography and narrow bore columns

Books, Magazines
ספרות מקצועית

Presentations
מצגות

books

Electronic literature, web presentations and Books recommendations

Click here to go to my Poll on HPLC columns and Sep-Pak:  Are they analytical components or filtration devices?

spe

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.

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Instrumentation Qualification תיקוף מכשירים

Topics related to the hardware of HPLC systems


 
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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




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-)
utilities, applications and information
Handouts-Overview on Photo Diode Array Detectors 
Ion Pair Reagents by Regis
  Course on HPLC modes (ppt)

 

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)  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 - 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)2004 Dr. Shulamit Levin

    Disclaimer הסתייגויות:

    Only material marked with the icon on this specific page is my own, some of them are revision of Waters Inc.'s educational presentationsThe rest of the material (mostly marked  by ) are just links to other resources.   This means that I do not necessarily fully agree with all the content in these resources or responsible for it in any way.
    Do you have a question about HPLC? Click here to ask by Email

    Copyright (C)1997 Dr. Shulamit Levin
    Medtechnica  Analytical  Department