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HPLC Guide
HPLC Guide
HPLC
(High Performance Liquid chromatography) HPLC 1
300 1
1. Fucose 2. Galactosam ine 3. Glucosam ine 4. Galactose 5. Glucose 6. M annose
2 3
m V
4 5
Detector
20.00
Waste
ab cd Pump
)flows 50-5000 L/min
HPLC
HPLC
11 1
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2
Gel Permeation / Size Exclusion Gel Filtration - GPC / SEC/ / GFC
ION EXCHANGE
Cation Exchange
+ + + + +
Anion Exchange
Reversed phase
HPLC Reversed Phase
Reversed Phase
RP-18
(octadecylsilane) ODS
C-18
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(EDTA) UV-VIS (Diode Array) (Liquid LC-MS chromatography- Mass spectrometry) Reversed Phase pH
mole/m2
(Bonding Density)
mole/m2 3
O + OH
:NORMAL PHASE R=H :REVERSED PHASE . R= C18; C8; C4; CN; C1 Etc
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(triethylamine,
(Solvent Strength)
Reversed Phase
pH
pH
pH pH
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(Degradation HPLC
UV-VIS
4
Overlay of Chromatograms
a
AU
min 4.
b
AU
33.80
35.00
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UV-VIS
UV-VIS
20
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HPLC 1
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Ion Chromatography
30 5
94 10 5
2 H+ OH-
pH pH
pH Suppressor Suppressor
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suppression
30 25
2 10 5 10 4
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Narrow standards
M1
Log Mw
Calibration curve
1st Moment = Mn (Number Average) 2nd Moment = Mw (Weight Average) 3rd Moment = Mz (Z Average) 4th Moment = Mz+1 (Z+1 Average) Mw/Mn = Polydispersity
10
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baseline
HPLC
standards (Narrow Unknown) (Broad unknown) GPC Reversed phase Reversed phase GPC Reversed phase GPC Reversed phase 1 45 15 GPC GPC Seal wash
11
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Chiral Separations
UV ref
HPLC GPC
UV-VIS
12
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(recycling)
Specifications
HPLC (Specifications)
HPLC
13
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References
1. 2. 3. 4. 5. 6. 7. 8. 9. U.D. Neue, HPLC Columns, Theory, Technology, and Practice, Wiley-VCH, New York, 1997. V. R. Meyer, Practical High-performance Liquid Chromatography, (2nd Edition) Wiley, New York, 1994. M.C. McMaster, HPLC: A Practical User's Guide, VCH Publishers, Inc., 1994. D. Parriott, A Practical Guide to HPLC Detection, Academic Press, New York, 1993. S. Lindsay, High Performance Liquid Chromatography (Series: Analytical Chemistry by Open Learning), Wiley, Chichester, 1993. B.A. Bidlingmeyer, Practical HPLC Methodology and Applications, Wiley, 1992. R. P. W. Scott, Liquid Chromatography Column Theory, Wiley, New York, 1992. J.W. Dolan, L.R. Snyder, Troubleshooting LC Systems, Humana Press, 1989. L.R. Snyder, J.L. Glajch, J.J. Kirkland, Practical HPLC Method Development, Wiley, 1988. 10. M. T. Gilbert, High Performance Liquid Chromatography, Wright, Bristol, 1987. 11. E. Katz, Quantitatie Analysis Using Chromatographic Techniques, Wiley, Chichester, 1987. 14