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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

&Control Data Processing

m V

4 5

Detector
20.00

0.00 5.00 M inutes

Waste

ab cd Pump
)flows 50-5000 L/min

Fraction Collector Auto Sampler

HPLC Column in Oven

HPLC

HPLC

HPLC 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

(chromatographic mode) HPLC

columns (spheric, irregular) (60, 100, 150, 300, )

Reversed Phase

(3, 5, 7, 10 m) (C3, C4, C8, C18) pH

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

Reversed Phase - Stationary Phase Surface

O O O O O O O O O O O O O Si O R Si Si Si OH O R Si O R Si O O Si OH Si HO O R O Si O O Si O O Si OH HO O O O AlO Si O R O O Si O R O O

O + OH

:NORMAL PHASE R=H :REVERSED PHASE . R= C18; C8; C4; CN; C1 Etc

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(triethylamine,

(Ion Pairing Reagents) tetrabutylamine

(dodecylsulfonate, heptyl, hexyl or octyl sulfonates)

(Solvent Strength)

Reversed Phase

pH

pKa pKa pH 54 pH 4 8 Reversed Phase pKa 8 8 pH

pH

pH pH

0.1 M 0.01 M (Seal wash)

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1.1 Reversed phase Pharmaceutical Assay

(Degradation HPLC

(Impurity profile) products)

(RSD < 1%) 3b 3a

UV-VIS

4
Overlay of Chromatograms

a
AU

min 4.

b
AU

33.80

34.20 34.60 Minutes

35.00

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(Peak Purity) band-pass UV-VIS

System Suitability HPLC 11 1

1.2 Reversed phase Amino Acids

UV-VIS

UV-VIS

20

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1.3. Reversed phase Peptides and Proteins

0.1% TFA Reversed phase 3

HPLC 1

1.4. Reversed phase Food

HPLC GPC UV-VIS

1.5. Reversed phase - Environmental

Reversed phase (Pesticides and herbicides) (Polycyclic aromatic hydrocarbons)

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Ion Chromatography

30 5

94 10 5

2 H+ OH-

pH pH

pH Suppression Suppressors HPLC

pH Suppressor Suppressor

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suppression

HPLC dedicated HPLC Suppressor HPLC HPLC

Gel Permeation Chromatography (GPC)

30 25

2 10 5 10 4

(Molecular size distribution) Refractive Index (RI) UV

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Creation of a Calibration Curve

K 500 M1 K 250 K 100 Polymer distribution K 25

Narrow standards

M1

Log Mw

K500 K250 K100 K25

Calibration curve

Elution Time or Volume

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

HPLC 1 GPC log MW 10 HPLC (Reference Peak)

10

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Total penetration UV-VIS (RI)

baseline

Noise and drift

GPC Broad Narrow standards

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

Narrow Bore Columns

1 25 10 4.6

12

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Extra-column band broadening

Scaling up preparative chromatography HPLC 50 HPLC 25 54 50 10 10

(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