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HPLC
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MS
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e-Learning for analytical scientists
Packed with practical information that will help
improve your skills and productivity
Used by over 35,000 chromatographers worldwide, CHROMacademy will
help increase your knowledge, efficiency and productivity in the lab. With
a vast library of high-quality animated and interactive e-learning topics,
webcasts, tutorials and troubleshooting tools we can help you refresh your
chromatography skills or learn something completely new.
CHROMacademy is simply the best training tool for analytical scientists see for yourself!

3. HPLC | GC | MS | Sample Prep
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Each channel is packed with e-learning modules,
webcasts, tutorials and interactive tools

4. High Performance Liquid
Chromatography
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Injection Valve Anatomyy
It is necessary to divert the flow of mobile phase
away from the sampling system (i.e. the syringe)
when aspirating the sample and / or filling the sample loop prior to injection. This is achieved using
the injection valve containing a Rotor Seal.
Isolation Seal
The interface between the HPLC capillaries and the
stator is known as the Stator Face...
Rotor Seal
Stator
Stator Face
e-Learning for Analytical Chemists
THE THEORY OF HPLC
• Introduction to HPLC
• Chromatographic Parameters
• Band Broadening
• Column chemistry
• Reverse phase (partition) chromatography
• Ion-Pair Chromatography
• Normal phase (absorption) chromatography
• Gradient HPLC
• Quantitative and Qualitative HPLC
• Fast HPLC
• HILIC
• Supercritical Fluid Chromatography
• Introduction to Ion Chromatography
INSTRUMENTATION OF HPLC
• Mobile Phase Considerations
• HPLC Solvent Pumping Systems
• Autosamplers
• Detectors
www.chromacademy.com

5. Gas Chromatography
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THEORY AND INSTRUMENTATION OF GC
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e-Learning for Analytical Chemists
Introduction to GC
Chromatographic Parameters
Band Broadening
Gas Supply and Pressure Control
Sampling Techniques
Sample Introduction
GC Columns
GC Temperature Programming
GC Detectors
Supercritical Fluid Chromatography
1. Aims & Objectives
2. Introduction
3. Supercritical Fluids
4. Modes of Chromatography
5. SFC Applications
6. SFC Instrumentation
7. Packed & Capillary SFC
8. The Mobile Phase
9. Exhaust Gases
10. Organic Modifiers
11. Pumping Issues
12. SFC Columns
13. SFC Stationary Phases & Column Selection
14. Detection
15. Pressure Regulators
16. Advantages and Disadvantages of SFC
www.chromacademy.com

6. Mass Spectrometry
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FUNDAMENTAL LC-MS
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Nebulising Gas
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M
M M
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Non-Volatile Analyte
is Charged in Solution
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Corona Electrode Pin
e-Learning for Analytical Chemists
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Introduction
Electro Spray Ionization Theory
Electro Spray Ionization –Instrumentation
Mass Analyzers
Atmospheric Pressure Chemical Ionization
(APCI)
Atmospheric Pressure Photo Ionization (APPI)
Solvents, Buffers and Additives
Vacuum Systems
Flow Rates and Flow Splitting
Orbitrap Mass Analyzers
FUNDAMENTAL GC-MS
• Introduction
• GC Considerations
• GC -MS Interfaces
MS INTERPRETATION
• General Interpretation Strategies
• Fundamentals of MS Proteomics Research
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7. Sample Preparation
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SOLID PHASE EXTRACTION
• Molecular Properties
1. Learning Aims & Objectives
2. Functional groups
3. Molecular Properties
4. Functional group interactions
5. Hydrophobic or Non-Polar Groups
6. Hydrophobic Interactions -Solubility
7. Hydrophobic Interactions -Sorbents
8. Polar Groups
9. Polar Interactions -Solubility
10. Polar Interactions -Sorbents
11. Ionic groups
12. Ionic groups -pH | Ka | pKa | Ionic strength
13. Ionic Interactions -Solubility | Sorbents
14. Ionic Interactions -Chelating Groups
15. Chelating Groups
16. Chelating Interactions
17. Solubility
18. Sorbents
19. Protein Binding
20. Assessment
• SPE Overview
• SPE Mechanisms
• Method Development
• Primary Sample Preparation Techniques
• Liquid / Liquid Extraction Techniques
• Approaches to Automation for SPE
e-Learning for Analytical Chemists

8. Interactive Learning
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e-Learning for Analytical Chemists
CHROMacademy contains a vast library of high-quality
animated and interactive e-learning topics

9. powered by
Search for things...
1000’s of eLearning topics, articles and applications
Fix things...
interactive tools for chromatographers
We developed our Troubleshooters with busy chromatographers in mind.
In 3 simple steps you overcome your
instrument, separation and quantitation
issues.
GC Troubleshooter
HPLC Troubleshooter

10. HPLC Troubleshooter
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Select up to 3 Chromatographic
Symptoms
Select up to 2 Instrument
Symptoms
Positive baseline drift
Negative baseline drift
Irregular baseline drift
Cycling Baseline (Short Term)
Cycling Baseline (Long Term)
Erratic baseline
Regular baseline noise
Irregular baseline noise
Baseline Spikes
Not the Chromatogram I was expecting!
a Loss of resolution (some peaks)
Loss of resolution (all peaks)
No Peaks
Additional peaks (ghost peaks)
Negative peaks (One or more peaks)
Normal
Peak broadening (Loss of Efficiency)
Peak fronting (one or more peaks)
Peak shouldering (one or more peaks)
Peak splitting (one or more peaks)
Peak tailing (one or more peaks)
Problem
Rounded peaks
Change in selectivity
Decreasing retention times
Increasing retention times
Too much retention
Low pressure
a High pressure
Continuously decreasing pressure
(no sample injection)
Continuously decreasing pressure
(after sample injection)
Continuously increasing pressure
(no sample injection)
Continuously increasing pressure
(after sample injection)
Pressure fluctuation
Leaks in the solvent reserviour
connective tubing
Leaks at the degasser / fittings
Leaks around the pump head
Leaks around the check valves or fittings
Leaks in the autosampler or
connective tubing
Leaks around the sampling valve
Leaks at the column fittings
Leaks from the detector fiitings
Leaks within the detector
Poor quantititive reproducibility
Poor linearity of response
6
Possible Causes
1. Incorrect / non-optimal mobile phase pH
2. Incorrect column geometry / dimensions
3. Buffer precipitation
4. Incorrect buffer / non-optimal buffer concentration
5. Analyte-stationary phase secondary interaction (e.g.
silanol interactions)
6. Incorrect mobile phase
7. Incorrect mobile phase temperature
6more
Solution
8 Mobile phase pH affects retention of ionisable
analytes and the selectivity of separations in
reversed phase HPLC
8 Some methods require a very precise pH in
order to remain robust
8 Low pH tends to increase retention of acidic analytes and decrease retention of basic analytes
6more
Resources
2
2
2
Buffers and pH Meter
This video explains how to prepare a buffer and
use a pH meter.
Buffer preparation
This useful tool will help you to prepare the correct buffer for your HPLC analysis
How to calibrate pH meter
This video describes how to calibrate your pH
6more

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Scientist “A” needed to understand why her chromatogram contained baseline spikes combined
with a loss of sensitivity. She turns to CHROMacademy’s HPLC Troubleshooter and in 2.3 seconds,
CHROMacademy tells her there are 26 possible causes for this combination of symptoms.
Each possible cause is ranked using our unique ‘expert system’ and includes a comprehensive
explanation and suggested fixes backed by 100’s of resources from LCGC magazine,
CHROMacademy and instrument vendors.
Without a CHROMacademy Premier Membership:
Called her manufactures’ representative.
36 hours before someone called back
Lost time of instrument use: 40 hours
Lost $ = $2,000 (@$95K/annum)
Contacted her Lab Manager
2 hours lost before Lab Manager could be located
e-Learning for Analytical Chemists
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19. Saving Time & Money
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Scientist “B” needed to develop a sensitive GC-MS method for a chlorinated pesticide derivative
present in potable water samples at very low levels. The derivative was new and therefore no
applications existed in manufacturers literature.
Using a CHROMacademy Webcast and Tutorial he was able to develop a GC method from a
generic screening method and realized that Chemical Ionization was the route to attain the required
sensitivity. From the Sample Preparation Channel he noted that solid phase extraction could be
used to selectively extract the target analyte and again was able to develop a method from a
suggested generic starting procedure. He used the ‘ask the expert’ function to enquire of the
CHROMacademy academic team how to create a sensitive MS acquisition method whilst checking
for the presence of co-eluting interferences. Method was developed and was ready for validation
within 1 week.
Without a CHROMacademy Premier Membership:
He wished that the department GC-MS expert hadn’t been laid off…..
Method development now begins using ‘guesstimates’
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