User Information Plan Template
Form Rev. Feb 25, 1999
Document Information:
Filename
USERINFO.DOC
Current Owner
Deborah Perkins
Product Identifier
Techniques of HPLC. H1186A
Project Identifier
SS980032
Current Lifecycle
Test
Phase
Revision Log:
Version
Date
A.00
99/05/19
A.01
99/11/09
Reason For Update
Initial user information plan.
Preliminary
Final update
Course Description
H1186A
Techniques of HPLC with Instrumentation
H1186A is a 4-day, instructor-led, hands-on course providing the student with knowledge
of techniques of HPLC including details of the separation process, general HPLC
procedures, gradient elution, instrumentation, troubleshooting, quantification, modes of
HPLC, and method development.
To prepare for the course, you will need to provide instruments equipped with
ChemStations, order needed supplies, and tools. The following items are provided by
SSD:
An instructor manual with laboratory and lecture details (as hidden text)
A student manual
Powerpoint slides
Product Delivery Requirements
1. Course Description: H1186A is a 4-day, instructor-led, hands-on course providing the
student with knowledge of techniques of HPLC including details of the separation
process, general HPLC procedures, gradient elution, instrumentation, troubleshooting,
quantification, modes of HPLC, and method development.
2. Course Outline with correlation to file names:
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DAY 1
Module 1
Introduction to HPLC
mod1.doc, mod1.ppt
Module 2
HPLC Instrumentation
mod2.doc, mod2.ppt,
Laboratory: Solvent Delivery Systems
mod2lab1.doc
Laboratory: Autosampler and Detector
mod2lab2.doc
Module 3
Practical HPLC Procedures
mod3.doc, mod3.ppt
Module 4
Chromatographic Resolution and the
Separation
mod4.doc, mod4.ppt
Laboratory: HPLC Parameters
mod4lab.doc
Common Troubleshooting
mod5.doc, mod5.ppt
Module 6
Gradient Elution
Laboratory: Gradient Elution
mod6.doc, mod6.ppt
mod6lab.doc
Module 7
Qualitative and Quantitative Analysis
mod7.doc,mod7.ppt
Module 8
Column Hardware
mod8.doc, mod8.ppt
Laboratoy: Column Hardware
mod8lab.doc
DAY 2
Module 5
DAY 3
(optional, pick 1 of last 3 labs)
DAY 4
Module 9
Reversed- Phase HPLC
Module 10 Reversed-Phase Separations of Ionic
Samples
mod9.doc, mod9.ppt
mod10.doc, mod10.ppt
Laboratory: Separation of Weakly Ionic
Compounds(optional)
mod10lab.doc
Module 11 Ion Exchange HPLC
mod11.doc, mod11.ppt
Module 12 Normal Phase HPLC
mod12.doc, mod12.ppt
Module 13 Size Exclusion Chromatography
mod13.doc, mod13.ppt
Module 14 Method Development
mod14.doc, mod14.ppt
Laboratory: Develop a Quantitative Method
For the Separation of Caffeine in Beverages
(optional)
Module 15 Method Validation(appendix, optional)
Hewlett-Packard Company
mod14lab.doc
mod15.doc, mod15.ppt
For Internal Use Only
Bibliography
biblio.doc
Complete Manual
H1186A.doc
Manual Cover
Cover.doc
PostScript Student Manual
H1186Ast.ps
Student Manual Cover
Stcover.ps
PostScript Instructor Manual
H1186Ains.ps
Instructor Manual Cover
Inscover.ps
3. For marketing purposes of this product see the Product Release Bulletin (if
available):
Product Release Bulletin available? Yes
Product Release Bulletin location: SSD Website
http://cagsso.atl.hp.com/development/main.htm
Find H1186A then the file prodrel.doc.
4. Instructor or Delivery Agent skills and training required to deliver the product:
Care should be taken to ascertain instructor experience and expertise for this
course. This course leans heavily towards the chemistry/methods development side
of HPLC techniques. The following are guidelines:

At least 10 years of HPLC method development experience in an industrial
setting, or

Analytical Chemistry faculty at a college or university whose research area is
separations.

Instructor should have direct, significant experience in at least three modes of
HPLC.
The instructor can test his/her readiness to teach the course by answering the
following questions:
1. Describe the separation process using analogy.
2. Describe the main modes of HPLC and their strengths and weaknesses.
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3. Describe how to maintain an HPLC and prepare samples for injection.
4. Be able to relate resolution to efficiency, selectivity, and capacity.
5. Describe how to perform method development in reversed-phase HPLC based
upon a scouting gradient.
6. Describe how check valves work in an HPLC pumping system.
7. Describe typical HPLC detectors and give advantages of each kind.
8. Describe HPLC detectors which give 3 dimensional data.
9. Troubleshoot the HPLC based on chromatographic data.
10. Know the basis of quantification and when area or height is most suitable.
11. Know the advantages and disadvantages of reduced particle size and narrow
bore columns.
12. Know how to select the right stationary phase for reversed-phase HPLC.
13. Know how to optimize ion-pair HPLC.
14. Describe how to optimize separations on an ion exchange column.
15. Know how to determine the molecular weight from SEC.
16. Know how to optimize the performance of SEC.
17. Know the latest advances in HPLC columns.
18. Lead discussions on method development strategies.
19. Describe rugged method validation procedures.
5. Equipment (Instrumentation and Software) required for product delivery:

This course is designed with laboratories requiring an instrument for every two
customers. The course is designed for the HP 1100 Series Modules. Each Liquid
Chromatograph requires:

Solvent degassing

A binary or quaternary pump

Autosampler

Column Oven

Diode Array Detector

ChemStation
Current HPLC 3D ChemStation Configuration and Software:

A.06, or A.07 G2170AA Single Instrument LC2d ChemStation Software
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
A.0, A.07 G2180AA Spectral Processing Software

Pentium 233 64 Mb RAM

CD-ROM drive

Speakers (to listen to directions from maintenance and repair CD-ROM)

17 inch monitor (at least 600 x800)

Laserjet Printer

NT Windows 4.0

3 ½ inch floppy drive

Classroom Projection System (at least 800 x 600 resolution)

Classroom Computer for slide presentation and software demonstration.

pH meter and associated calibration buffers (If performing optional lab Separation of
Weakly Ionic Compounds by Reversed-Phase).

Balance
6. Consumables required for product delivery:
Purchase the following consumables to complete the entire course.

1/instrument

1per2 instruments 3 um column

1 per 2 instruments narrow bore column

1/instrument XDB column

1 4L methanol

1 4L acetonitrile

1 4L HPLC grade water

Acetate Buffer

Antioxidant sample from Supelco Part Number

Vitamin samples Part Number

Spare Parts

Capillaries

Spare waste tubing
Standard Checkout Column
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
Pump Seals

Wear Retainers

Pistons

Frits

Solvent inlet filters

Rotors

Needles

Needle Seats

lamps
7. Samples and Materials required for each laboratory :
You will need the following materials and samples for each laboratory:
Laboratory 1: Solvent Delivery Systems
Instrument: required as described above
Tools:
¼” wrench
4 mm hexagonal key
Spare Parts:
Optional: pump seals (5062-8576)
Wear retainers (01018-22706)
Samples: None
Columns: No specific column required. Suggest that the columns required for exercise
3 be in place.
Solvents: HPLC grade water in channel A, Acetonitrile in channel B
Make certain that if quaternary system, all channels have some mobile phase.
Special Instructions: The lab allows for the students to replace the pump seals or just
look at the pump seals without replacing them. Tell them which you would like them to
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do before the lab starts. Your supervision will be required while the students locate the
components of the HPLC and describe their function. You may want to do this as a
group. This laboratory is done after the completion of the solvent delivery section of the
Instrumentation module.
Laboratory 2: Autosampler and Detector
Instrument: As described above.
Tools:
¼” wrench
2.5 mm hexagonal key
9/64” hexagonal key
Pozidrive #1 screwdriver
Spare Parts:
Have a few on hand in case someone breaks something.
Needle G1313-87201
Rotor Seal 0100-1853
Deuterium Lamp 2140-0590
Samples: None
Columns: No specific column required. Suggest that the columns required for exercise
3 be in place.
Solvents:
HPLC grade water in channel A
HPLC grade acetonitrile or isopropanol in channel B
If channel C or D, Isopropanol for pressure test: note: water may be substituted for the
pressure test.
Special Instructions: Remind the students not to touch the deuterium lamp with their
fingers. Many students experience problems removing the needle without bending it.
This lab may is done after the completion of the detectors section of the Instrumentation
module.
Laboratory 3: HPLC Parameters
Instrument: As described above.
Tools:
None specifically required.
Spare Parts:
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Same as general selection of spare parts.
Samples:
You will need:
Vials and caps
Crimper
Pasteur pipettes and bulb
Graduated cylinder to measure dilution (don’t need any more accuracy than this)
1 vial isocratic test mix per instrument, part number 01080-68704
Stock Solution of Uracil (uracil may be obtained from a variety of sources including
Preparation:
Dilute each vial of HP isocratic test mix 1:3. Spike with uracil stock solution (the
amount is not critical just as long as a peak can be seen from the baseline).
Uracil Stock: Weigh out 33 mg of uracil. Dissolve in 20 mL of water with sonication.
Dilute with 80 mL of methanol or acetonitrile. Dilute solution 1:50. Add approximately
1 mL for each 1:3 dilution.
Columns:
1 SB-C18, 4.6 x 150 mm, 3.5 micron column, part number 863953-902/instrument.
Solvents:
HPLC grade water in channel A
HPLC grade acetonitrile in channel B
Note: experiment is not greatly affected by use of methanol instead of acetonitrile.
Special Instructions:
You will need to review the lab results carefully. Unfortunately, with this sample, the
efficiency doesn’t decrease with increasing temperature. Point out the shape of the curve
N vs. T where N increases with temperature at first, levels off, then slightly falls.
Make certain that the students return the column oven down before going back to the
classroom.
Laboratory 4: Gradient Elution
Instrument: as described above.
Tools:
None specifically required.
Spare Parts:
Same as general selection of spare parts.
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Samples
Place gradient test mix, part number 01080-68794 in labeled vials.
Columns:
1 SB-C18, 4.6 x 150 mm, 3.5 micron column/instrument part number 863953-902.
Solvents:
HPLC grade water in channel A, HPLC grade acetonitrile in channel B, you may have
other assorted solvents for students to try if desired: methanol, THF, isopropanol.
Special Instructions:
We often make this lab into a contest with a simple prize such as an HPLC textbook or an
Agilent Technologies mug or T-shirt. That often makes this the most popular laboratory.
You may want to postpone this laboratory exercise to the end of the course after method
development as it can apply there as well. Skip the method development and weakly
ionic compound labs. That will make a very relaxed pace course where you can explain
each lecture in a little more detail. In other words, do the two instrument labs, the HPLC
separations lab, the column hardware lab and the gradient lab at the end of the course.
Students can then spend as much time optimizing as they like. Also, if you have an even
more complicated sample, the same instructions can apply, but the lab will be more
challenging.
Laboratory Exercise 5: Column Hardware
Instrument: as described above
Tools:
The students will need wrenches to change the columns.
Spare Parts:
Standard spare parts.
Sample
Use the sample from the HPLC Separations laboratory or just isocratic test mix: 0108068704.
Columns:
1 for every 3 instruments SB-C18, 4.6 x 150 mm, 5 micron column, part number 883975902
1 for every instrument (same as used in other laboratories) SB-C18 4.6 x 150 mm, 3.5
micron column part number 863953-902.
1 for every 3 instruments SB-C18 2.1 x 150 mm, 5 micron column, part number 883700922.
Solvents:
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HPLC grade water in channel A, HPLC grade acetonitrile in channel B.
Special Instructions:
To save money, buy only a couple of the 2.1 and 4.6(5 micron) columns. The students
can do the experiments out of order and swap columns. Make certain that they
understand this before they start the lab.
Laboratory 6: Separation of Weakly Ionic Compounds by Reversed Phase
Note: This laboratory is optional. You must have a pH meter. Hypersil columns or any
brand that has more residual silanols are more appropriate than the Zorbax columns.
With the hypersil columns, the lab demonstrates the behavior of weakly ionic species.
Instruments: same as described above.
Tools:
The instructor will need a balance and a pH meter. Several containers for buffers.
Samples:
From stock solution, combine equal amounts of aniline, benzoic acid, and benzaldehyde
into vials. Also, provide vials of standards for aniline and benzoic acid. They can infer
the benzaldehyde peak.
Stock Solutions:
Aniline: mix 5 mL of aniline with 95 mL acetonitrile or methanol. Dilute 1:100 with
65/35 acetonitrile/water.
Benzoic Acid: 9.77 g dissolved in 100 mL acetonitirle or methanol. Dilute 1:100 with
65/35 water/methanol or acetonitrile.
Benzaldehyde: 4.0 mL in 96 mL of methanol or acetonitrile. Dilute 1:500 with 65/35
water /acetonitrile or methanol.
These stock solutions will keep for years.
Buffers:
Make 0.010 M Sodium Acetate Buffers in 500 mL quantities at the following pH’s. Use
Acetic acid to adjust the pH.
pH = 5.5, 4.7, and 4.0
The final buffer solution, make it 0.010 M sodium acetate, add 10 mM triethyamine, then
pH to 4.00 with acetic acid.
The students can do the first three buffer solutions in any order as long as the columns are
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well equilibrated. The TEA buffer must be done last.
Columns:
Select a column with many residual silanols. The Zorbax columns do not work well.
This may be a discussion point about the lack of problem with certain columns. A
hypersil column 4.6 x 100 , 5 micron 79916OD-554 works okay. Non-endcapped sil type
columns work the best, such as Lichrospher.
Solvents:
Above Buffers channel A, acetonitrile channel B.
Special Instructions:
If the students do not spend the appropriate time equilibrating the columns, this lab does
not work well. Have the students look at the changes in the UV spectrum as well. Make
certain that the students flush the columns and instruments at the end of this lab.
Laboratory 7: Develop a Quantitative Method for the Separation of Caffeine in
Beverages.
Instrument: as described above.
Tools:
The students will need wrenches to change the columns.
Spare Parts:
Standard spare parts.
Samples:
You will need the following:
Samples of two or three beverages that contain caffeine. Suggestions: Coca-Cola, Dr.
Pepper, Pepsi, coffee, tea, etc. It is better to filter the samples, but do not dilute. A vial
of each per instrument.
Caffeine standards, 3 levels/ instrument at the following levels:
Weigh out 70 mg into a 100 mL volumetric. Dilute with a 50/50 mixture of
water/acetonitrile to the mark.
Repeat with 20 mg and 10 mg. This range of standards should cover beverages from soft
drinks to brewed coffee.
Columns:
The 4.6 x 150, 3.5 micron SB-C18 column as in previous experiments.
Solvents:
HPLC grade Water in channel A, HPLC grade Acetonitrile in channel B.
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Special Instructions:
The problem with this lab is the difficulty encountered with the ChemStation calibration
tables. If you really don’t want to get into that, use graph paper.
Tools:

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
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





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large column wrenches7/16 x ½ and ½ x 9/16
small column wrenches ¼ x 5/16
pozidrive #1
pozidrive #2
ZDV union
Capillary
Small screw driver
Flat wrenches (HP 1050 and HP 1100) 4mm and 14 mm
Allen wrench set 1.5 mm 2.5 mm 3 mm 4 mm 5 mm
Blanking nut
Small beaker
Safety glasses
Sand paper (small strip)
HP 1100 priming tool
HP 1100 Maintenance and Repair CD-ROM (01100-60002)
Floppy disk
Tweezers
Empty capped vial
9/64” hex key
8. Electronic Files required for product delivery:
None required
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9. Activities required prior to product delivery.
Determine which laboratories you will be conducting. Order supplies. Make any stock
solutions or buffers. Set-up HPLC’s and ChemStations . Check out HPLC’s. Run
standard isocratic test mix on all HPLC’s prior to class. Set-up classroom projector for
slide presentations. Make up required samples.
10. Activities required during product delivery.
Monitor students during laboratory exercises. Dispose of solvent waste as needed.
11. Activities required after product delivery.
Make certain that students fill out the evaluation forms.
Dispose of solvents and samples properly.
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