PE Brownlee Columns/Applications

PE Brownlee Columns/Applications
The Perkin-Elmer Corporation

Pharmaceutical Systems
761 Main Avenue, Norwalk, CT 06859-0200
January, 1999
1-888-PE CHROM or FAX 203-761-5062
203-762-4000 (worldwide)
Internet: http://www.perkin-elmer.com
Copyright © 1998 The Perkin-Elmer Corporation.
All rights reserved. E-mail chrom@perkin-elmer.com
Look to us. And see more.

Table of Contents
1. Sales Information
2. Product Information
3. HPLC Basics/Applications

Introduction
Historical Perspective of PE Brownlee Columns
 1977: Bob Brownlee introduced the first finger-tight

cartridge system and founded Brownlee Laboratories
 1983: PE pioneered Fast LC and 3x3TM columns
 1983: Applied Biosystems (ABI) acquired Brownlee Lab
and used narrowbore columns for its protein sequencers
 1993: Perkin-Elmer merged with ABI
 1996/7: PE Brownlee Columns formed by merging
Peco (PE columns) and Brownlee column lines
- Introduced Validated, ValueLine and Popular Phases

Features of PE Brownlee Columns
 Experience: >20 years of manufacturing experience

 Quality: ISO 9001 certified production
process with stringent quality standards
 Convenience: Reusable MPLC® modular cartridge
design for easy column change or
coupling
 Selection: From microbore to semi-preparative --
selection of > 40 sorbents
Unique products: MPLC cartridge system, NewGuard, Fast LC,

narrowbore, microbore, and Validated
Columns...

MPLC Cartridge System
Features
 Ease of Use
- Finger-tight design (7,000 psi, without wrenches)
- Quick change - no need to disconnect tubing from LC system
- Column cartridge label visible through holder cut outs
 Lower Cost
- Re-usable end fittings and interchangeable holders
- Cartridge columns have lower cost than conventional columns
 Versatility
- Wide selection of cartridge lengths and inner diameters
 Easy scale-up to semiprep or “scale down” to narrowbore columns
- Direct coupling of a cartridge to a NewGuard or another MPLC cartridge
with no dead volume

Quality Control Procedures
Used for each silica or bonded phase lot
in PE Brownlee Products
 Support
- Particle size/distribution, pore volume, and surface area.
 Sorbents (bonded phases)
- Determination of % coverage, %C, trace metals contents
- Techniques used -- GC (reagents), elemental analysis,
TGA, AA...
 Packed Columns
- Most columns are shipped with individual test
chromatograms showing plate count, peak symmetry and
pressure drop.

Who are the Customers?
What is our Strategy?
 Pharmaceutical labs
- Ease of use of MPLC cartridge and wide selection of columns
- Promote Validated Line in Method Development, which passes methods to
QA
- Promote Fast LC
 Bioresearch and biotech labs
- Promote Aquapore line (wide-pore) for proteins and peptides
 Industrial/chemical or universities/government labs
- Promote ValueLine -- low cost, high quality
- Promote Spheri, Pecosphere or Popular Phases
 Environmental and food testing labs
- Promote specialty, Spheri, Pecosphere columns

What’s New in FY ‘97/98?
 ValidatedTM Columns -- extra-tight specifications
and highly consistent columns for validated assays
 ValueLineTM Columns -- batch-tested, reduced cost
columns
 Peco columns in MPLC holders
- includes popular Fast LC Peco columns (3x3 and 3x8)
 Specialty Columns
- PL Gel, PL SAX, etc. .......
 “Popular Phases” packed in MPLC cartridges
- Nucleosil®, LiChrospher®, Hypersil®, Kromasil®

Competition
Silica-based columns and PE Brownlee’s equivalent
Company Column PE Brownlee
Equivalence
Waters -Bondapak C18 Validated, Reduced
Symmetry, Resolve Activity (Red. Act.)
Beckman Ultrasphere Spheri
MacMod Zorbax-Rx Validated, Red. Act.
Supelco Supelcosil Spheri
Phase Sep Spherosorb Spheri, Pecosphere
Shandon or HP Hypersil Spheri
EM Science LiChrospher Validated, Red. Act.
(Merck)
Macherey Nagel Nucleosil Validated, Red. Act.
YMC YMC Validated, Red. Act.
Whatman Partisil Validated
Keystone Deltabond Spheri
Chrompack ChromSpher Spheri

Competition
BioLC and Polymer-based Columns
vs. PE Brownlee equivalent

Competitive Cost Analysis
A glimpse of PE Brownlee vs Competition
Pro duc t US Lis t
PE Bro wnle e (c artridg e )
Validate d C18 220 x 4.6 mm $ 390.00
S phe ri-5 RP-18 220 x 4.6 mm $ 350.00
Pe c o s phe re 5 C18 150x 4.6 mm $ 200.00
Validate d C18 100 x 4.6 $ 275.00
S phe ri-5 VL C18 100 x 4.6 $ 150.00
Co mpe titio n (Co lumn)
HP Hype rs il BDS C18 5µm, 250 x 4, $ 280.00
Wate rs Re s o lve C18 5µm, 300 x3.9 $ 420.00
Wate rs S ymme try C18 5µm, 150x3.9 $ 420.00
LiChro s phe r 60 RP-s e l B, 5µm, 4 x250 $ 390.00
Allte c h Alltima C18, 5µm, 250 x 4.6, c art $ 295.00
MN Nule o s il AB C18,5µm 250 x 4.6 $ 295.00
S phe ris o rb C18 80Å 5µm 250 x 4.6, $ 270.00
Vydac 201HS C18 5µm 90Å 250 x 4.6, c o l $ 350.00
Hype rs il BDS C18 5µm 120Å, 250x4.6 $ 295.00
YMCbas ic Co lumn 5µm 250 x 4.6 $ 490.00
S upe lc o s il LC-ABZ+ 5µm C18 250 x 4.6 $ 419.00
S upe lc o s il LC-18-DB 250 x 4.6 $ 379.00

Sales Support Materials
PE Brownlee Columns
PE Brownlee Sales Binder, including
 Sales/Product Training (Microsoft PowerPoint)
 PE Brownlee Catalog (1996-97)
- 32-p, PE Order No. L-1928
 Applications Notes (96 sheets)
- Biotech, clinical, environmental, food, industrial, GPC, pharmaceutical
 Product Data Sheets
- Validated Columns #1 (L-1918) & #2 (L-1930)
- ValueLine (L-1917), MPLC Cartridges (L-1931)
- Popular phases (L-1942), Fast LC (L-1950)
5 On-site seminars (PowerPoint) for major accounts (L-1952)

PE Brownlee’s On-site Seminar Program
Topics for FY ‘98, Available from LCBU
1. An Overview of PE Brownlee Columns and

applications
2. Column selection in HPLC method development --
The Fundamentals
3. Selecting HPLC columns to better assay
performance
4. Fast LC -- the quickest way to enhance productivity
in the HPLC lab
5. Use of Validated HPLC Columns for consistent and
reproducible assays

Overview
PE Brownlee Products
PE BrownleeTM MPLC Cartridges Length (cm) i.d. (mm)

Conventional and narrowbore cartridges 3, 10, 22 4.6, 2.1
Validated and ValueLine Columns 3, 10, 15, 22, 25 4.6, (2.1)
Peco column line 3, 8, 15, (25) 4.6
NewGuard 1.5 3.2
Prep-10 cartridges 3, 10, 25 10
Popular Phases 15, 25 4.6
Conventional PE Brownlee Columns
Conventional and semi-prep columns 10, 25 4.6, 7.0
Microbore columns 5, 10, 25 1.0
Dimension in brackets might not be available in all products.

Sorbents
Summary of characteristics
Support Type dp Analyte Functionality Pore Sur.
(m) (Å) area
(m2/g)
Spheri silica 5, 10 small C18, C8, 80 180
mol amino, phenyl,
cyano, Si
Aquapore silica 7, 20 biomol. C18, C8, C4, 300 100
AX, CX
Polypore polym 10 biomol. Phenyl, micro/
. sugars DEAE, SP, macro
H, CA
Validated silica 5 small C18 100 350
mol
Pecosphere silica 3,5 small C18, Si 80 170
mol
Red. Act. silica 3,5 small C18, C8 80 200
mol
All reversed-phase silica-based sorbents are treated to eliminate heavy metals and endcapped.

Selection Guides
PE Brownlee Columns
 General Applications (RP)
- Spheri-5 RP-18, RP-8 (5-m, 10 or 22 cm, 4.6 mm i.d.)
 Protein/peptide applications (RP and IEX)
- Aquapore OD-300, BU-300
- Aquapore AX-300, Polypore DEAE
 Pharmaceutical applications (RP)
- Fast LC : Reduced-activity (3-m, C18 or C8, 3 or 8 cm, 4.6 mm i.d.)
- Validated (5-m, C18, 10, 15, 22 or 25 cm, 4.6 or 2.1 mm i.d.), Popular Phases
 Narrowbore and microbore columns
- Spheri-5 or Aquapore (2.1 and 1.0 mm i.d.)
 Sugars/Organic acids
- Polypore CA or H, Spheri-5 amino
Column Selection Guides, PE Brownlee Column Catalog, L-1928, Perkin-Elmer, p 27, 1996.

MPLC Cartridge Hardware
 Patented MPLC cartridge system for leak-free operation up to
7000 psi, with only finger tightening.
 Changing cartridges is easy without using wrenches or
disconnecting of any tubing from your LC system.
 Standard MPLC holders
- For traditional Brownlee columns in 4.6 and 2.1 mm i.d.
cartridges. Available in 3, 10, and 22 cm.
 New Peco MPLC holders have different holder bodies of 3, 8, 15
and 25 cm lengths - Note: Peco-3 and MPLC-3 holders are different.
 NewGuard holder for NewGuard cartridges.
- Use NewGuard System holder for the direct coupling of
NewGuard to analytical cartridges.
- Use NewGuard holder for external attachment to columns.

MPLC Cartridge Holders
Ordering Info
NewGuard System Holder

(10-cm + NewGuard)
22-cm MPLC Holder
10-cm MPLC Holder
P/N PE Brownlee Cartridge Holder

0715-0013 3-cm MP LC Guard Holder
07150-014 10-cm MP LC Holder
0715-0015 22-cm MP LC Holder
0715-0028 3-cm P eco MP LC Holder
0715-0032 13-cm MP LC S ys tem Holder
0715-0033 25-cm MP LC S ys tem Holder
0715-0018 MP LC Union

PE Brownlee MPLC Cartridges
Analytical cartridges for conventional
and narrowbore HPLC
 3 popular supports (Spheri, Aquapore, Polypore) with wide
selection of bonding chemistries
 Same reusable holder for i.d. cartridges.
 Choice of 3, 10 and 22 cm cartridge lengths and 4.6 or 2.1 mm i.d.
 Cartridges are less expensive than conventional columns
 Reduce solvent consumption with narrowbore or shorter
columns
 Individual test chromatogram with each 10 and 22 cm cartridge.
Features:
MPLC cartridge, reputation of quality, cost, selection, compatible to any
HPLC

Comparison of Separations
Flexibility to optimize column length
3 cm
n= 2,500
10 cm
n= 7,000
22 cm
n= 15,000
n = plate count

Validated™ Columns
 Guaranteed batch-to-batch reproducibility.
 Test Chromatogram and Certification of Compliance shipped with each
column.
 Packed with a 5 m reduced activity monofunctional C18 sorbent.
 Compatibility with acidic, neutral and basic analytes.
 Available in 3, 10, 15, 22 or 25 cm MPLC cartridges (4.6 mm i.d.).
- Select 10 or 15 cm cartridge for routine assays, 22 or 25 cm cartridge
for complex samples.
- Use 2.1 mm i.d. cartridges (10 or 22 cm) to reduce solvent use.
 Method Ruggedness Trial Kit - 3 Columns from different silica lots.
Features:
Batch-to-batch consistency MPLC cartridge, certification, reduced activity
Validated Line Data Sheet, L-1918, Perkin-Elmer, Norwalk, CT, 1996.

Selectivity of Validated Columns
packed with 8 different bonded phase lots showing
showing separation factors with < 1.5% deviation
5
(separation factor)
 (toluene/phenol) = 4.98 +/- 1.5 % RSD

4
1
(toluene/DMA) = 1.22 +/- 1.0% RSD
0
1 2 3 4 5 6 7 8 9
Silica lot No.

Validated Columns
Chlorthalidone assays using columns from 3 different silica lots
Validated C18 (100 x 4.6 mm i.d)

34% MeOH, 65% water, 1% HOAC
1 mL/min, 1650 psi, 30 oC
10 uL of degraded chlorthalidone
Absorbance mAU
at 1 ug/uL
impurity Chlorthalidone CBSA
CCA

Validated Columns
Column Life Time Testing
Validated C18 (100 x 4.6 mm i.d.)

pyridine 80% MeOH, 1 mL/min, 35 oC
5 L of a sample containing t-butyl
2% butyl benzene, 0.03% pyridine benzene
0.01% uracil
n = 9510
As = 1.00
1st Injection p = 900 psi
uracil
n = 8900
550th Injection As = 1.02
p = 940 psi
0 2 4
Time (min)

Validated Columns
Ordering Information
P/N Validated C18 Cartridge Column

402-237 NewGuards , 3/pk
402-239 30 x 4.6 mm, 2/pk
402-240 100 x 2.1 mm
402-241 100 x 4.6 mm
403-058 150 x 4.6 mm
402-242 220 x 2.1 mm
402-243 220 x 4.6 mm
403-059 250 x 4.6 mm
402-788 Trial P ack, 100 x 4.6mm, 3 lots

ValueLine™ Columns
 Batch tested reduced price columns (20-30%

saving).
- Other PE Brownlee columns are individually tested.
 Choice of Spheri-5 VL C8, C18 for small molecules
and Aquapore VL C8 for proteins/peptides
 10 or 22 cm 4.6 mm i.d. MPLC cartridges.
Features:
MPLC cartridge, lower cost, same quality as Spheri-5 or Aquapore,
reputation
Ref: ValueLine Data Sheet, L-1917, Perkin-Elmer, Norwalk, CT, 1996.

Popular Phases
 Four Popular 5 m C18 sorbents packed in MPLC formats
- Nucleosil® (Machery-Nagel, 100Å)
- Hypersil® (Shandon, 120Å)
- LiChrosphere® (E. Merck, 100Å)
- Kromasil® (EKA Nobel, 5 m, 100Å)
 Available in 15 or 25 cm MPLC cartridges (4.6 mm i.d.) and NewGuard
cartridges (15 X 3.2 mm)
Part Numbers
Column 15-cm 25-cm NewGuard
Nucleosil C18 0711-0251 0711-0252 0711-0250
Features:
Hypersil C18 0711-0254 0711-0255 0711-0253
The most popular reversed
LiChrospher C18 phase0711-0257
materials packed in MPLC cartridges.
0711-0258 0711-0256
Popular Phases Data
Kromasil C18Bulletin, L-1942, Perkin-Elmer,
0711-0260 Norwalk,
0711-0261 CT, 1996.
0711-0259

VeloSep® Column
 VeloSep columns are 3.2 mm i.d. cartridges packed

with 3 µm reversed-phase sorbents for high
throughput analysis
 Improves sensitivity and speed while cutting
solvent consumption by half
 Packed with 3 µm C8 or C18 sorbents; available in
3.2 mm i.d. and 4 or 10 cm lengths.
Features:
MPLC cartridge, save solvent with 3.2 mm columns

Peco Column Line
Perkin-Elmer’s Peco cartridges with new MPLC holders
 Three cartridge lengths (3, 8 and 15 cm), 4.6 mm i.d.
 Cartridges compatible to “old” style Peco cartridge holders (wrench
tighten) and the new Peco MPLC holders (finger-tight).
 Four Peco silica-based supports are available.
- Pecosphere is a small-pore sorbent for HPLC of small molecules.
- Reduced Activity is a small-pore, base-deactivated sorbent
developed for HPLC of basic analytes and pharmaceuticals
- Pecosil (irregular) and Peco HCODS (wide-pore sorbent)
 Pecosphere, Reduced activity Fast LC columns are most popular
- 3x3TM (3 µm, 33 x 4.6 mm, n > 4,000)
- 3x8 (3 µm, 83 x 4.6 mm, n > 10,000)
Features:
“old” Peco 3x3 holder
MPLC cartridge, Fast LC, economical 3x3, 5/pack
Ref. Fast LC data bulletin, L-1950, Perkin-Elmer, Norwalk, CT, 1996.

Benefits of Fast LC
 Fast analysis.
- Isocratic (1-3 min) or gradient (<10 min)
 Rapid method development and validation.
 Increase mass sensitivity by 5 fold.
 Lower column cost -economical 5/packs for 3x3
 Lower solvent consumption (5 fold).
 Compatible to most existing HPLC systems.
References:
J. R. Gant and M.W. Dong, “Practical benefits of high-speed LC in pharmaceutical analysis,” Pharm.
Tech., 11(10), 44,1987.

Chlorthalidone Purity Assay
Using Fast LC
Column: Reduced activity 3x3 C18
(3-µm, 33 x 4.6 mm i.d.)
Mobile Phase: MeOH:H2O:HOAc

Chlorthalidone
(34:65:1)
Flow Rate: 1.5 mL/min
Detection: UV at 235 nm
CCA
CBSA
PE Brownlee LC Application Note LCPH-15.

Analgesics Tablet
Using Fast LC
1 Column: Pecosphere 3x3 C18

(3-µm, 33 x 4.6 mm i.d.)
Mobile Phase: 15% ACN in 0.1% acetic acid
Flow Rate: 2 mL/min
Peak Identification
1. Acetaminophen
2. Caffeine
3. Salicylamide
4. Acetylsalicylic acid

NewGuard Cartridges
 NewGuard cartridges (15 mm x 3.2 mm i.d.) are packed with 5

or 7 µm sorbents
 Prolong column life by eliminating particulates, contaminants,
and strongly bound sample components.
 Negligible loss of efficiency.
 Available in 3/pack, can be coupled directly to any MPLC
cartridge with a union (0715-0018).
 Finger-tight seal to 7000 psi using NewGuard holders
 Can be used for sample pre-concentration (connected to
sample injection loop)
Features:
MPLC cartridge , reliability, economical 3/packs, size, selection

Prep-10 Cartridges
Scaling up of biological separations is practical and economical
using PE Brownlee’s Prep-10 cartridges.
 10 mm i.d. cartridges, packed with Aquapore 20 µm, 300 Å
sorbents are available in 3, 10, and 25 cm cartridges.
 Design incorporates a moveable inlet plug and filter which
compensates for changes in bed volume with use.
 Changing the spent cartridge is easy using the reusable, finger
tight holder (up to 3000 psi).
 Typical sample capacity of the 25 cm cartridge is 50 mg to 1
gram of material. Typical flow rate is 4 to 10 mL/min.
Features:
MPLC cartridge, 3000 psi, cost-effective, high loading capacity (up to 1 gram).

Conventional PE Brownlee Columns
 Conventional PE Brownlee columns (non-cartridge)
use standard compression end fittings compatible
with all HPLC instrumentation.
 These columns do not require MPLC holders.
 Available in analytical (4.6 mm i.d.) and semi-prep
(7.0 mm i.d.) configurations.
 Each column individually tested.
Features:
No holder required, all conventional columns are compatible to any HPLC

Microbore LC (1.0 mm i.d.) columns
 Increase sensitivity and decrease solvent

consumption by a factor of 20 (compared with 4.6
mm i.d.)
 Reduced sample size for pharmacokinetic assays
 Better compatibility with LC/MS interfaces (requires
low flow rates).
 Aquapore for bioseparations and Spheri-5 for small
molecule analysis.
 Each column individually tested.
Features:
High efficiency, increase mass sensitivity, LC/MS, micropurification of proteins

Comparison of Sensitivity:
Microbore LC (1 mm) Narrowbore (2 mm) and conventional (4.6 mm)
Instrument, sample and injection size were identical
200 Microbore = x 20 Column RP-18 Spheri- 5

Microbore
(250 mm x 1 mm i.d.)
Narrowbore
(220 mm x 2.1 mm i.d.)
Conventional
(220 mm x 4.6 mm i.d.)
Sample Universal test mix 2 L

mAU
Mobile Phase: 85% MeOH/H2O at 35oC
Narrowbore = x 5 Flow Rate 50 L/min (microbore)

200 L/min (narrowbore)
1000 L/min (conventional)
Conventional = x 1
Higher mass sensitivity is
0 achieved due to less sample
dilution from the smaller
Time (Min) column volume.

Microbore Column Efficiency Performance
Spheri-5 RP18 (1x250 mm) 75/25 (MeOH/H2O), 30L/min, Pressure: 920 psi,
n = 21,434 for t-butylbenzene
toluene
n = 15,633
Plate count (n) and tailing factor (Tf )
ethylbenzene calculated using the USP method by
n = 18,243 Turbochrom SST software
100 t-butylbenzene
propylbenzene n = 21,434
n = 20,238 Tf = 1.05 Anthracene
mAU (260 nm)
n = 18,300
0 10 20 30 40
Time (min)

Microbore Analysis of Carbamazepine
400 pg each injected : Limits of detection: 8 pg (CBZE); 13 pg (CBZ)
vs 800 pg of CBZ reported in the literature
CBZE (metabolite)
S/N = 157 Column: Spheri 5 RP-18 (100 x 1 mm i.d.)
Mobile Phase: 40% MeOH/H2O, 100 L/min at 35oC
Pressure: 1800 psi
CBZ
mAU (212 nm)
S/N = 90
CHA (int. std)
S/N = 52
N = 5253
Minutes
Specialty Columns
 Columns from other vendors for specialized
applications
- Gel permeation chromatography (GPC) - PE PLGel
- DNA analysis - PE TSK DEAE
- Bioseparations - PE PL SAX
- Environmental- Pickering Carbamate analysis,
PE ChromSpher-3 PAH
- Sugars - Aminex HPX-87C

USP Listing - LC packings
PE Brownlee offerings are underlined
 L1 - C18 (porous silica)  L13
 L2  L14
 L3 - silica  L15
 L4  L16
 L5  L17 - Polypore H
 L6 -  L18
 L7 - C8 (porous silica)  L19 - Polypore CA
 L8 - amino  L20
 L9  L21
 L10 - cyano  L22 - Polypore SP
 L11 - phenyl  L23
 L12  L24

HPLC Basics/Applications

HPLC Overview
HPLC is
 A physical separation technique in which a sample dissolved
in liquid is injected into a column packed with small particles
and is separated into its constituent components
 The most important and widely used analytical technique for
the quantitative analysis of organics and biomolecules
 Applicable to many sample types
- Most useful for pharmaceuticals, biomolecules, and labile organics;
also some ionic compounds)
- Sales of HPLC equipment and consumables worldwide $2.2 billion
in 1998; >100,000 HPLC's are in use worldwide today

Advantages of HPLC
 Rapid and precise quantitative analysis

- Typical analysis time of 5 - 20 min, Precision < 0.5 - 1% RSD
 Automated analysis
- Using autosampler and data system for unattended analysis and
report generation
 High sensitivity detection
- Detection limits of ng to pg
 Quantitative sample recovery
- Preparative technique from g to kg quantities
 Amenable to diverse samples
- Can handle > 60% of all existing compounds vs. 15% for GC
- Can analyze samples with little or minimal preparation

HPLC Equipment
Components of an HPLC System
Solvent
Waste

Common Detectors for HPLC
Detector Attribute
UV/Vis absorbance Most common detector for compounds with absorbance in
(UV/Vis) UV/Vis, ng sensitivity
Diode array Aids identification of analyte via spectral comparison
Fluorescence (Fl) Specific detector with pg sensitivity
Refractive index Universal, used for compounds without chromophores
(RI) Polymers, sugars, triglycerides, organic acids
Mass spectrometry Excellent sensitivity (fg) and specificity,
(MS) definitive identification of analytes
Electrochemical For electroactive compounds
Conductivity For ionic compounds
Infrared For polymer analysis, solvents is restrictive to normal phase
Light-scattering Competes with RI detector, compatible to gradients
Radioactivity Specific, for detecting radioactive-labeled compounds

Manual Injector
 An injector consists of valve, rotor, sample loop and needle port

 Allows precise sample introduction under high pressure (5000 psi)
 Rheodyne 7125 is the industry-standard HPLC injector
 Samples solution is introduced into the sample loop using a
22-gauge blunt tip syringe in the LOAD position
 Sample is injected into the column by switching the valve to INJECT

Mobile Phase
HPLC Modes Analyte
Stationary Phase
 Normal-phase or Liquid-solid (NP, LSC)
- Separation based on adsorption/desorption of the analyte onto a polar
surface (silica)
 Reversed-phase (RPC)
- Separation based on analytes’ partition coefficients between the mobile
phase and the bonded stationary phase
 Ion-exchange (IEC)
- Separation based on ion-exchanging with the counter-ions and ionic
interaction with the bonded ionic group
 Size-exclusion (SEC or GPC)
- Separation based on analyte’s molecular size and “sieving” action of the
column packing
Note: Ion chromatography is IEC with specialized equipment while ion-pairing

chromatography is a special technique in RPC for ionizable compounds.

Reversed-phase Chromatography (RPC)
 Separation based on partitioning of the FLOW
analyte into a hydrophobic stationary
phase bonded on silica support
(e.g., C18, C8)
- Uses polar mobile phase such as
mixture of methanol/acetonitrile and
water; polar analytes elute first while
non-polar analytes are retained more.
 Most common HPLC mode used in
> 60% all LC separations
- For polar (water-soluble), medium
polarity, and some non-polar analytes.
- Ionic analytes can be separated using
ion-pairing reagents.

Glossary of HPLC Terms
A Review
 Column - a tube that contains packed sorbents
 Mobile phase - a liquid that carries the sample through
the column
 Retention: the tendency of a solute to be retained by the
stationary phase in the column
 Resolution (Rs) - the degree of separation of 2 peaks
 Efficiency - a measure of the narrowness of peak widths
produced by a column
 Selectivity () - the ratio of retention of 2 adjacent peaks
 Sample capacity - the maximum sample load for the
column

HPLC Column
 Type - standard or cartridge
 Materials - stainless steel, PEEK, glass, titanium
 End-fittings and frits (0.5 m)
 Connection fittings (nuts and ferrules)
 Connection tubing (1/16” o.d. and < 0.007” i.d.)

Column Connection Tubing and Fittings
 Common Connection Fittings (SS-316, titanium, plastics)
- Compression nuts 1/16” and ferrules
 Manufacturers: Rheodyne, Swagelok, Parker, Valco, SSI
 cpi type ferrule is industry standard, 45ocone angle, and 3 mm insertion
depth
 Finger-tight UpChurch plastic fittings are reliable and convenient
 Connection Tubing (SS 316, PEEK)
- 1/16” o.d. and < 0.007” i.d.
- Use 0.005” i.d. for low flow applications using small columns and
0.010”-0.020” i.d. for preparative applications
- Tubing should be square cut and deburred
- PEEK tubing is flexible and easy to use

Column Length (L)
 Column efficiency (n) is proportional to L
- HETP (plate height) = L/n
 Columns can be connected together to produce a
longer column with higher efficiency
 Column back pressure is proportional to L
 Common column length is 10 - 25 cm
 Fast LC columns are short columns (3 - 10 cm)
packed with small-particles (dp = 3 m)

Column Diameter (dc )
 Sample capacity is proportional to (dc )2
 Flow rate is proportional to (dc )2
 Detection limit is inversely proportional to (dc )2
Column Type i.d. Sample Flow rate

(mm) capacity (mg) (mL/min)
Semipreparative 8-20 10-50 5-30
Standard Analytical 4.6 1 1
Narrowbore 2.0 0.2 0.2
Microbore 1.0 0.05 0.05

Column Packing Materials
 Support Type
- Silica (or alumina) or Polymer (cross-linked polystyrene)
 Bonded groups
- C18, C8, C4, amino, cyano, phenyl
- diethylaminoethyl (DEAE), sulfo, quaternary ammonium
 Particle size (dp): 3-, 5-, 7-, 10- or 20 m
- Efficiency is inversely proportional to dp
- Column pressure is inversely proportional to (d p)2
 Pore size (dpore) : 60 - 300 Å
- Wide pore materials (300 Å) are used for biomolecules or polymers
 Surface area: 90 - 400 m2/g
- High surface area maximizes solute interaction with bonded groups

Van Deemter’s Plots
Effect of particle diameter dp and flow rate (F) on efficiency
(HETP is inversely proportional to efficiency)
 Plots show relationship

of HETP vs flow rate
and dp
 Small dp yields lower
HETP
 The optimum flow rate
is higher with smaller
dp
 Small-particle columns
have less efficiency
loss at high flow rates

Characteristics of a “Typical” HPLC Column
 100 - 250 mm long x 4.6 mm i.d. packed with 5-m C18 bonded
phase
 Flow rate range 1 - 2 mL /min using mobile phase of methanol
or acetonitrile in water
 Sample loading: 1 ng to 1 mg
 Operating pressure ranges 500 - 2000 psi
 Has 4,000 to 10,000 theoretical plates
 Costs US $150 - 400
 Typical column life time 500 - 2000 injections or 3 - 24 months

Guidelines in LC Mode Selection
Mode is dictated by structures of analytes
 Macromolecules (MW > 2000)

- Polymers GPC
- Biomolecules (proteins, DNA) SEC, RP, IEC
 Organics (MW < 2000)
- Polar RP, NP
- Medium polarity RP
- Non-polar RP, NP
- Ions, ionizable cpds IEC, RP-ion pair
- Group separation LSC, NP
 Preparative LSC, GPC, RP
PE Brownlee Column Catalog, L-1928, Perkin-Elmer, Norwalk, p 27, 1996.

+
SO3 Na
Common Bonded Groups

Polymeric
or silica p+
support
SO3 Na
Reversed-phase
C18 Octadecyl Si- CH2- CH2- CH2- CH2- CH2- CH2- CH2- CH2- CH2-
CH2- CH2- CH2- CH2- CH2- CH2- CH2- CH2- CH3
C8 Octyl Si- CH2- CH2- CH2- CH2- CH2- CH2- CH2- CH3
C4 Butyl Si- CH2- CH2- CH2- CH3
Normal Phase
Si Silica S i - OH
CN Cyanopropyl Si- CH2- CH2- CH2- CN
Ion-Exchange
SP Sulfo propyl Si - CH2- CH2- CH2- SO3-
CM Carboxymethyl Si-CH2-CO2-
DEAE Diethylaminoethyl Si - CH2-CH2-NH+ (C2H5)2
SAX Triethylaminopropyl Si-CH2-CH2-CH-N+(C2H5)3
In GPC, bonded group is not needed as the pore structure of the support serves as the separation media.

Guidelines
in Bonded Phase Selection
 C18 - Most common bonded phase, very retentive and stable

- Monofunctional C18 is brush type; polyfunctional is polymeric
- C18 from different vendors can have very different retention
and selectivity characteristics
 C8 - Preferred for low organic mobile phase due to better wettability
 C4 - Often preferred for protein separations (C18 for peptides)
 CN - For both NP or RP; Used for or some applications (e.g.
antidepressants)
 Phenyl - For RP separation of medium polarity basic components,
unique selectivity for aromatics
 Amino - Used to replace silica in NP separations; analysis of sugars
Ref.: PE Brownlee Column Catalog, Perkin-Elmer, L-1928, Norwalk, p 27, 1996.

HPLC Applications
Applications Categories
 Pharmaceutical
 Life Sciences and Biotechnology
 Environmental
 Food applications

Pharmaceutical Applications
 For Research and Development

- New drug discovery (Synthetic organic chemistry)
- Metabolism and pharmacokinetics (LC/MS)
 For Quality Control
- Bulk Drug Analysis (Purity and stability testing)
- Formulation Analysis (Content uniformity, dissolution
and stability testing)

Analysis of Analgesic Tablet
Column : Validated C18 (100 x 4.6 mm i.d.)
Mobile phase: 40% MeOH/0.1% H3PO4, 1.0 mL/min
Pressure: 1900 psi
Sample: 2 L injection of a tablet extract in 500 mL
mobile phase
acetaminophen
mAU (240 nm)
acetylsalicylic
acid
caffeine
0 2 4 6
Time (min)

Separation of Antibiotics
Column: Pecosphere-5 C18

(5 µm,150 x 4.6 mm i.d.)
Mobile Phase: 36/63 v/v MeOH/(0.01 M
Phosphate Buffer Adjusted
to pH 7)
Flow Rate: 1 mL/min
Peak Identification
1. 6-Aminopenicillin Acid
2. Ampicillin
3. Penicillin G
4 Oxacillin
5. Cloxacillin
6. Dicloxacillin

LC/MS QUANTITATION
Of Quinolone antibiotic degradation products
Extracted Ion Chromatogram
Photolyte 2 MS
Lomefloxacin MS
Total Ion Chromatogram
PE Brownlee LC Application Note LCPH-19. Look to us. And see more.

Life Science Applications
 Analysis of proteins, peptides and amino acids

 Protein characterization, peptide mapping
 Separation of DNA, RNA, polynucleotides and
PCR products

Separation of a Complex Peptide Mixture
using a C18 bonded silica support in a narrowbore column
Tryptic Map of Glycoprotein Human IgA
Column: Aquapore RP-300 ODS
(7 µm, 300 A, 220 x 2.1 mm)
Mobile Phase: A: 0.1% TFA in ACN

B: 0.1% TFA
2 - 70% A in 45 min
PE Brownlee LC Application Note LCBIO-21. Look to us. And see more.

Environmental Applications
 Samples are:
- Drinking water, wastewater, solid waste, air samples, etc
 Analytes includes
- Pesticides, herbicides and plant growth regulators
 Carbamates, glyphosate, diquat/paraquat, pyrethrins,
organophosphorous cpds.
- Industrial pollutants
 Dyes, surfactants, amines, PAHs, PCBs, dioxins, phenols,
aldehydes)

Residual Carbamates in Spinach Extract:
Post-column OPA derivatization with fluorescence detection
Column: Pickering Carbamate

(5-m, 250 x 4.6 mm)
Mobile Phase: 25% - 75% MeOH/H2O
in 26 min
Flow Rate: 1 mL/min at 42oC
Detection: Post-column reaction
(NaOH/OPA)
Fluorescence
(330/465nm)
Sample: 20 L of spinach extract
prepared by solid phase
extraction
M.W. Dong, et al, ”Practical Considerations for rugged N-methylcarbamate analysis using
HPLC/post-column derivatization system,” LC.GC 10 (1992) 442.

Food Applications
 Natural food components
- Sugars, amino acids, triglycerides
- Fatty acids and organic acids
- Vitamins
 Food additives
- Preservatives, colors and dyes
 Contaminants
- Mycotoxins (aflatoxins) , pesticide and drug
residues

Analysis of Sweeteners
and Additives in Soft Drinks
Column: Pecosphere 5 x 15C C18
(5 m, 150 x 4.6 mm i.d.)
Mobile Phase: 45% A in B
A: Methanol
B: Water with 0.7 g/L
hexane sulfonic acid
and H3PO4 to pH 2.1
Sample: Filtered, degassed soft
drink
1. Matrix
2. Saccharin
3. Caffeine
4. Aspartame
5. Vanillin
6. Sodium Benzoate
PE Brownlee LC Application Note LCFD-6.

Organic Acids, Sugars and Ethanol in Wines
ethanol
tartaric
shikimic
malic
ethanol
acetic
lactic
citric
glycerol
tartaric
10 20
Column:
30
Two Polypore H
Time (min) (220 x 4.6 mm, 10-µm)
succinic
malic
acetic
Detection: RI and UV (210 nm)
citric
Mobile Phase: 0.01N H2SO4

fructose
glucose
Flow Rate: 0.2 mL/min at 60oC
tartaric
RI Intensity
Sample: 10 L of beverage sample

fructose
diluted 1:4 in mobile phase
shikimic
malic
ethanol
acetic
lactic
citric
ethanol
10 20 30
lactic
citric
Time (min)
glycerol
tartaric
ethanol
glucose
fructose
succinic
acetic
oxalic
lactic
citric
malic
10 20 30
Time (min)
LC Application Note LCFD-26.

Where to get more information ?
on HPLC techniques and methods?
 Books, journals, electronic sources

 Colleagues, library searches
Useful reference sources include:

 AOAC, USP, EPA, ASTM and other methods
 Journals
- J. Chromatography, J. Chromatogr. Sci., J. Liq.
Chromatogr.,
LC.GC, Chromatographia, Anal. Chem. Applications
Reviews, etc.

Summary
 HPLC is powerful quantitative separation technique
for many sample types
 In this training, we reviewed
- Sales and product information of PE Brownlee Columns
- Basic HPLC
- HPLC applications in pharmaceutical, bioscience,
environmental, food and industrial areas

+
1. L. R. Snyder and J.J. Kirkland, Introduction to Modern Liquid Chromatography, John
Wiley & Son, New York, 1979.
2. V. R. Meyer, Practical HPLC, John Wiley & Sons , Chichester, UK, 1994.
3. R. W. Yost, L.S. Ettre and R. D. Conlon, Practical Liquid Chromatography- An
Introduction, Perkin-Elmer, Norwalk, Connecticut, 1980.
4. Official Methods of Analysis of the Association of Official Analytical Chemists
(AOAC), R. Helrich (ed.), 15th edition, Arlington, VA, 1990.
5. J. R. Gant and M. W. Dong, “ Practical Benefits of High-Speed LC in Pharmaceutical
Analysis,” Pharm. Tech., 11(10) :44-58, 1987.
6. L. Nollet (ed.), Food Analysis by HPLC, Marcel Dekker, New York, 1992.
7. W. Hancock (ed), HPLC in biotechnology, John Wiley & Sons, Chichester, UK, 1990.
8. PE Brownlee HPLC Column Catalog, 1996-97, Perkin-Elmer, L-1928, Norwalk, CT,
1996.

PE Brownlee Columns/Applications

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PE Brownlee Columns/Applications

The Perkin-Elmer Corporation

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 1977: Bob Brownlee introduced the first finger-tight

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 Experience: >20 years of manufacturing experience

Unique products: MPLC cartridge system, NewGuard, Fast LC,

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1. An Overview of PE Brownlee Columns and

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PE BrownleeTM MPLC Cartridges Length (cm) i.d. (mm)

Dimension in brackets might not be available in all products.

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NewGuard System Holder

10-cm MPLC Holder

P/N PE Brownlee Cartridge Holder

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Validated Line Data Sheet, L-1918, Perkin-Elmer, Norwalk, CT, 1996.

 (toluene/phenol) = 4.98 +/- 1.5 % RSD

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Validated C18 (100 x 4.6 mm i.d)

impurity Chlorthalidone CBSA

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Validated C18 (100 x 4.6 mm i.d.)

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P/N Validated C18 Cartridge Column

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 Batch tested reduced price columns (20-30%

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 VeloSep columns are 3.2 mm i.d. cartridges packed

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Mobile Phase: MeOH:H2O:HOAc

PE Brownlee LC Application Note LCPH-15.

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1 Column: Pecosphere 3x3 C18

PE Brownlee LC Application Note LCPH-1.

 NewGuard cartridges (15 mm x 3.2 mm i.d.) are packed with 5

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 Increase sensitivity and decrease solvent

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200 Microbore = x 20 Column RP-18 Spheri- 5

Sample Universal test mix 2 L

Narrowbore = x 5 Flow Rate 50 L/min (microbore)

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