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Prep | Flash | TLC
Prep | Flash | TLC
Preparative Chromatography . . . . . . . . 148183
Introduction. . . . . . . . . . . . . . . . . . . . . . 148151
Columns . . . . . . . . . . . . . . . . . . . . . . . 152171


GraceAlpha Columns 152155


Vydac
®
MS Columns 156157


Vydac
®
TP Columns 158160


Alltech
®
Columns 161
Column Hardware . . . . . . . . . . . . . . . . . . 162171


MODcol
®
Spring Columns 162165


Axial SFC Column Hardware 166169


Empty MODcol
®
Flanged Columns 170


Process Scale Columns (Peak Biotech) 171
Column Packing . . . . . . . . . . . . . . . . . . . 172174


MODcol
®
Multipacker
®
Packing Stations 172173


Column Packing Services 174
Media . . . . . . . . . . . . . . . . . . . . . . . . 175181


Introduction 175


GraceAlpha Media 176


Vydac
®
Media 177


Davisil
®
Media 178181
Accessories . . . . . . . . . . . . . . . . . . . . . 182183


Tubing, Fittings, Pumps, Injectors, and Swtching Valves
Flash Chromatography . . . . . . . . . . . . 184187
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 184
GraceResolv Flash Cartridges . . . . . . . . . . . 185187
Flash Accessories . . . . . . . . . . . . . . . . . . 186187
Flash Media . . . . . . . . . . . . . . . . . . . . . . . . 187
Thin Layer Chromatography (TLC) . . . . . 188198
Introduction. . . . . . . . . . . . . . . . . . . . . . . . . 188
TLC Plates . . . . . . . . . . . . . . . . . . . . . . 189193
TLC Accessories . . . . . . . . . . . . . . . . . . . 194198
42000-05-147-signature 05 2008 catalog Grace proof 3 (mll) july 23, 2007
the henderson company 400 west erie, chicago illinois 60610 (312) 951-8973 f (312) 951-6109 148
www.discoverysciences.com
prep |
columns
prep | flash | tlc
The best Vydac
®
media from the leader in
peptide and protein separations for over
twenty-five years
Vydac
®
MS columns provide unique selectivity and
exceptional protein recovery. Higher recoveries and
yield under overloaded preparative conditions makes
this column the leader in preparative reversed-phase
protein purifications.
Applications: Large molecules and proteins
(>2000 molecular weight)
Differentiated Phases: C18, C8, and C4
Specifications: Spheroidal silica, polymerically and
monomerically bonded, endcapped, 300Å pore size
Formats: Capillary, LC/MS, Expedite, Rocket,
Solvent-Reducer, Analytical, Prep, Bulk Media
Preparative Chromatography Introduction
Grace
®
Key Column Families
Scalable Solutions: Silica and Hardware
Grace, the foremost name in silica material science,
manufactures a range of completely scaleable preparative
chromatography columns. To help select the appropriate
column for your application, we describe key column families
and highlight unique phases within these families. Each
column family highlighted is also scalable to bulk media and
available in ton quantities for process scale applications.
Graces silica based media is packed in proprietary column
hardware that extends media lifetime and ensures high
column efficiencies at the preparative scale. An overview
of preparative column hardware can be found on pages
150151.
Column Packing and Operation
It is generally accepted that the most reliable packing method
for high-performance preparative columns involve axial
compression of the adsorbent slurry in the column by a sliding
piston driven by hydraulic or pneumatic force. However,
reliable performance and maximum column life are obtained
only if axial compression force is maintained continuously on
the column bed during use. The patented MODcol
®
Spring
Column and MultiPacker
®
instrument now make it easy and
convenient for scientists to reproducibly pack any media in a
completely portable dynamic axial compression column.
Adsorbent Particle Sizes
Because small-diameter narrow-particle-range adsorbents
generally are expensive, it is more economical to use less
expensive larger diameter materials in preparative columns.
This is often possible because the objectives of preparative
separations differ from those of analytical separations. Speed
and sensitivity may be less important than product purity in
preparative chromatography. This allows preparative columns
to be operated at lower flow rates with gradient profiles altered
in order to compensate for the less efficient mass transfer of
larger adsorbent particles. To simplify method development
and scale-up, Grace provides a range of adsorbent sizes and
grades with identical bonded-phase chemistry.
2030µm
1520µm
10µm
5µm
0
200
400
600
800
1000
160
140
120
100
80
60
40
20
0
Peak W
idth at Half Height (mm)
Ribonuclease Load (µg)
7265
Column Materials: Vydac
®
214TP, 5µm; Vydac
®
214TP, 10µm;
Vydac
®
214TP, 1520µm;
Vydac
®
214TP, 2030µm
Eluent:

2495% ACN in 0.1% aqueous TFA over 30min
at 1.5mL/min
Protein:
Ribonuclease
Protein Loading Capacity of RP-HPLC
Materials of Different Particle Size
Although peak widths are much narrower with small
particle materials at low sample loads, there is little
difference in peak widths at high loads, where the column
is overloaded, allowing larger particles to be used with
minimal decrease in performance.
A New Silica Generation
GraceAlpha combines increased column efficiencies
and resolution with high loading capacity. A new silica
(patent pending) makes this possible by combining a
high-porosity surface, with a dense core, increasing
mass transfer while yielding a mechanically robust
particle.
Applications: Peptides and small molecules
(<2000 molecular weight)
Differentiated Phases: C18, C8, and Silica
Specifications: High purity spherical silica,
monomerically bonded, endcapped, 120Å pores
Formats: Analytical, Prep, Bulk Media
GraceAlpha
Vydac
®
MS
42000-05-148-signature 05 2008 catalog Grace proof 8 (rjh) october 4, 2007
the henderson company 400 west erie, chicago illinois 60610 (312) 951-8973 f (312) 951-6109 149
www.discoverysciences.com
prep |
columns
prep | flash | tlc
Analytical
(initial)
Prep
(final)
Preparative Scale Up
Step 1: Method Optimization
The analytical method is optimized to increase the alpha and
achieve maximum loadability. The most common ways to
optimize the method are through altering selectivity by mobile
phase manipulation or by altering the stationary phase.
Step 2: Loading Study
Once the analytical method is developed the method
loadability should be tested to determine the capacity of the
stationary phase. The sample load will be determined by the
complexity of the sample mixture as well as the stationary
phase being used.
Step 3: Mass Determination
The total mass that is needed from the purification should
be determined prior to starting the campaign. The total mass
needed can be used to balance the necessary throughput,
purity, and yield required from the separation.
Preparative Chromatography Introduction
related products
For analytical HPLC columns,
see pages 24110.
related products
For flash cartridges,
see pages 184187.
7110
7278
Step 4: The Scale-up Factor
The column size needed for the purification can be calculated
based on the output requirements.
D = diameter L = length
Load
final
= Load
initial
x (D
final
)
2
L
final

(D
initial
)
2
L
initial
The sample load scale-up equation determines the loading
capacity of the larger preparative column from the loading
capacity of the analytical column.
Step 5: Linear Velocity
When the column dimensions are changed to optimize
a separation, or to scale a separation to a preparative or
narrow-bore application, the mobile phase flow rate should be
adjusted proportionally to cross-sectional area of the column
to maintain consistent linear velocity and retention times. See
table below for common flow rate conversions (
Table 1).
Flow Rate
final
= Flo