Rheodyne MX Solutions Guide

User Manual:

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Page Count: 10

MX Series
Modular Automation for
Nano, Micro, and Analytical Scale
HPLC Applications
Solutions Guide
T
Table of Contents
able of Contents
Sample Injection
Two-Column Selection
Alternating Column Regeneration
High Speed Sample Enrichment
High Speed Sample Clean Up and Enrichment
Column Backflushing
MS Solvent Diversion
Multi-Dimensional Proteomic Peptide Separation
2
3
4
5
6
7
8
9
Solutions Guide
MX Automated Valves
2321168B
page 1 of 9
9/04
Sample Injection
The sample and the sample's injection into the system are the most critical
factors in any analytical process. Therefore, the quality, reproducibility, and
flexibility of the sample injection valve are important.
Sample injectors commonly have two positions, (1) LOAD and (2) INJECT.
The LOAD position allows sample loading into the sample loop. The INJECT
position flushes the sample from the sample loop onto a column.
Sample injectors are situated after the pump and before the column. It is
common practice to have a filter before the injector to minimize any pump seal
debris from entering the injector. A filter between the injector and the column
will trap particles that otherwise may block the column frit.
Nano-Scale and Analytical-Scale Injectors
Various MX Valves for sample injection are available:
Nano-Scale MX Four-Port Injection Valve,
Analytical-Scale MX Six-Port Injection Valve, and
Analytical-Scale MX Syringe Loading Injection Valve.
To order, contact your authorized Rheodyne MX dealer. See www.rheodyne.com for a complete list of dealers.
Nano-Scale
MX7984-000 MX Nano Injector,10 nL internal loop (Biocompatible DuraLifeTM II)
Analytical-Scale
MX7900-000 MX Two-Position, Six-Port (Stainless Steel)
MX9900-000 MX Two-Position, Six-Port (Biocompatible)
MX7925-000 MX Syringe Loading Injector (Stainless Steel)
MX9925-000 MX Syringe Loading Injector (Biocompatible )
Sample Injection
Sample Injection
2321168B
page 2 of 9
9/04
Position 2 (INJECT)
1.00
1.00
Waste
Column
Column
Pump
Pump
1
2
34
5
6
1
2
34
5
6
Needle Port
Position 1 (LOAD)
Position 2 (INJECT)
Waste
Waste
Part Number Description
MX Automated Valves
Nano-Scale
Analytical-Scale 2-Position, 6-Port
Analytical-Scale Syringe Loading Injector
1.00
1.00
Pump
Pump
Column
Column
Sample Loop
Needle Port
1
2
3
4
5
6
1
2
3
4
5
6
Position 1 (LOAD)
Position 2 (INJECT)
Waste
1.00 1.00
Nano Pump Nano Pump
Column
Column
Auto
Sampler
Waste
1
23
4
1
23
4
Position 1 (LOAD) Position 2 (INJECT)
Auto
Sampler
Two-Column Selection
Column selection can help separation scientists to develop reproducible, rugged
methods (frequently validated) in the shortest time possible. Automated column
selection can dramatically cut time, save costs, and increase productivity while
improving analytical results. With a variety of columns from which to choose,
any laboratory can optimize selectivity (different phases), resolution (different
particle sizes), time (different lengths), and load/sensitivity (different diameters,
injection volumes, and concentration strategies) for shared users.
Column selection allows multiple users to select methods using the correct
column for their assay and conveniently washout and store columns. Columns
are stored in the solvent of choice and require no manual changes. Different
types of samples can run at night and over weekends using different methods
resulting in better utilization of existing equipment and increased productivity.
In a validated environment, use column selection for column to column
reproducibility, method ruggedness, cross method conformational purity
analysis on columns with different selectivity, sample long-term stability, solute
specificity, column equilibration, and column quality control. The MX Two-
Position, Six-Port Switching Valve, illustrated to the right, is a self-contained,
electrically actuated valve for automated two-column selection.
T
Two-Column Selection
wo-Column Selection
To order, contact your authorized Rheodyne MX dealer. See www.rheodyne.com for a complete list of dealers.
Part Number Description
Column A
Column B
Sample Injector
1
2
34
Position 1 (LOAD)
Position 2 (INJECT)
5
6
Column A
Column B
Sample Injector
1
2
34
5
6
Nano-Scale
MX7980-000 MX Nano-Scale Two-Position, Six-Port Switching Valve (Biocompatible DuraLifeTM II)
Analytical-Scale
MX7900-000 MX Two-Position, Six-Port Switching Valve (Stainless Steel)
MX9900-000 MX Two-Position, Six-Port Switching Valve (Biocompatible)
2321168B
page 3 of 9
9/04
MX Automated Valves
Alternating Column Regeneration
Alternating column regeneration using two matched columns can increase
sample throughput. This timesaving process involves equilibrating one column
while using the other for analysis. A switching valve enhances the convenience
of column regenerating by switching between the analyzing column and the
equilibrating column.
For example, consider an analysis with a total time of 30 minutes: 20 minutes
for the analysis and 10 minutes for equilibration in preparation for the next
sample. Using alternating column regeneration, the 10 minute regeneration time
occurs during the analysis thus reducing the total analysis time to 20 minutes.
In this example, alternating column regeneration reduces the analysis time by
one-third. Time saved will depend on the column equilibration time.
Column regeneration is beneficial for laboratories running large quantities of
samples such as for drug discovery and quality assurance. This application
increases efficiency and saves money for repetitive chromatographic analyses.
The Nano-Scale MX Ten-Port Valves (Stainless Steel MX7986-000 and
Analytical-Scale Ten-Port Valves (Stainless Steel MX7960-000 ) make
unattended operation of this technique possible without the wait of a long
equilibration time.
See High Speed Sample Enrichment (Page 5) and High-Speed Sample Clean
Up and Enrichment (Page 6) for alternating pre-columns.
Alternating Column Regeneration
Alternating Column Regeneration
1.00
1.00
1.00
1.00
Part Number Description
To order, contact your authorized Rheodyne dealer. See www.rheodyne.com for a complete list of dealers.
Nano-Scale
MX7986-000 MX Nano-Scale Two-Position, Ten-Port Switching Valve (Biocompatible DuraLifeTM II)
Analytical-Scale
MX7960-000 MX Two-Position, Ten-Port Switching Valve (Stainless Steel)
2321168B
page 4 of 9
9/04
Column 1
Column 2
Sample
Injector
1
2
3
456
Eluent
Pump
Regeneration
Pump
Detector
Waste
7
8
9
10
Column 1
Column 2
Sample
Injector
1
2
3
456
Eluent
Pump
Regeneration
Pump
Detector
Waste
7
8
9
10
Position 1
Position 2
MX Automated Valves
High Speed Sample Enrichment
The clean up or concentration of sample before injection onto an HPLC
column is often the part of the analysis that requires the most time. The on-line
sample preparation methods described here and on page 6 can, when properly
optimized, significantly reduce that time. Furthermore, using two pre-columns
in alternating parallel operation can increase the speed of these on-line methods
even more.
In parallel operation, the functions of the two pre-columns alternate: one
column accepts sample from the autosampler for clean up or enrichment while
the other column transfers an already purified sample to the analytical column.
Aswitching valve in the system then interchanges the functions of the
pre-columns. This technique is especially effective in increasing overall
analysis speed if the analytical column rapidly performs separations. For
example, short, high-speed columns in a LC-MS system often run analyses
quickly. Therefore, pre-columns operating in parallel significantly increase the
speed of the relatively slow sample preparation step.
A typical high-speed enrichment and clean-up system is illustrated to the right.
This system uses a standard chromatograph, an additional LC pump, two
pre-columns, one analytical column, and a Nano-Scale MX Ten-Port Switching
Valve.
In Position 1, the sample injects onto Pre-Column 1, using mobile phase from
Pump A. Pre-Column 1 enriches the sample and passes interfering sample
components to waste. Meanwhile, the previously injected sample, now
enriched and free of interfering compounds, backflushes out of Pre-Column 2
and onto the analytical column using the mobile phase from Pump B. After
completion of the slowest step (either the enrichment on Pre-Column 1 or the
separation on the analytical column), the valve switches to Position 2.
Position 2 interchanges the functions of the two pre-columns. Sample back-
flushes out of Pre-column 1 and onto the analytical column. Pre-column 2 now
receives the next sample from the autosampler to carry out the next enrichment
step.
1.00 1.00
1.00 1.00
High Speed Sample Enrichment
High Speed Sample Enrichment
Note some of the similarities that this alternating parallel pre-column method
has to the alternating column regeneration method described on page 4. The
purpose of alternating columns is to reduce the analysis time by allowing one
column to separate sample components while another column re-equilibrates
with the initial mobile phase. Various on-line sample preparation schemes and
column switching schemes use a single system to eliminate more than one rate-
limiting step, producing a robust, automated high-speed analysis system.
To order, contact your authorized Rheodyne MX dealer. See www.rheodyne.com for a complete list of dealers.
Nano-Scale
MX7986-000 MX Nano-Scale Two-Position, Ten-Port Switching Valve (Biocompatible DuraLifeTM Il)
Analytical-Scale
MX7960-000 MX Two-Position, Ten-Port Switching Valve (Stainless Steel)
Part Number Description
2321168B
page 5 of 9
9/04
Pre-Column 1
Pre-Column 2
Auto
Sampler
1
2
3
45
Pump B
Waste
Detector
7
8
9
10
Position 1
6
Pump A
Pre-Column 1
Pre-Column 2
Auto
Sampler
1
2
3
45
Pump B
Waste
Detector
7
8
9
10
Position 2
6
Pump A
MX Automated Valves
column inlet. In the process illustrated, samples are both concentrated and
cleaned up. Enrichment can range from a factor of ten to several thousand
(depending on the volume of sample injected) to significantly improve
detection.
Two popular applications illustrate the versatility of sample enrichment. One is
the LC/MS analysis of drugs and metabolites by direct injection of plasma with
minimal previous work-up. By using a restricted access media (RAM)
pre-column, the tightly bound analytes and plasma proteins that would
otherwise destroy the analytical column pass through the pre-column to waste.
Another popular application is the LC/MS analysis of protein tryptic digests.
A very small pre-column, often called a trap column, allows the injection of
several microliters of digest under conditions where the peptides are highly
retained. The switching valve transfers the small volume of concentrated
sample onto a micro-bore or packed capillary column. Sample volumes are
large enough for convenient handling, yet enrichment allows the actual gradient
elution separation of the peptides to take place on micro-columns using low
flow rates that are optimal for MS instruments.
The system uses a standard chromatograph, an additional LC pump, and an
Analytical- or Nano-Scale MX Two-Position, Six-Port Automated Valve (Model
MX7900-000 or MX 7980-000). In Position 1, the sample injects onto the
enrichment column using mobile phase from Pump A. Meanwhile the analysis
of the previous sample is carried out on the analytical column using the mobile
phase from Pump B. In Position 2, the enrichment column switches to the
reverse direction so Pump B backflushes the cleaned-up and concentrated ana-
lytes onto the analytical column. After the transfer of the analytes onto the ana-
lytical column, the valve switches back to Position 1, so the enrichment col-
umn is conditioned and the next sample can inject onto it. Enrichment and
analysis take place concurrently.
High Speed Sample Clean Up and Enrichment
High Speed Sample Clean Up and Enrichment
To order, contact your authorized Rheodyne MX dealer. See www.rheodyne.com for a complete list of dealers.
Nano-Scale
MX7980-000 MX Nano-Scale Two-Position, Six-Port Switching Valve (Biocompatible DuraLifeTM II)
Analytical-Scale
MX7900-000 MX Two-Position, Six-Port Switching Valve (Stainless Steel)
MX9900-000 MX Two-Position, Six-Port Switching Valve (Biocompatible) 2321168B
page 6 of 9
9/04
Part Number Description
High Speed Sample Clean Up and Enrichment
Complex samples such as biological fluids, food extracts, and waste waters
often must be cleaned up and enriched. Before injection onto an HPLC column,
the analytes of interest and contaminants must be separated so they will not
interfere with the separation or destroy the analytical column. Sample
enrichment will also enhance detection.
On-line sample clean up and enrichment uses a pre-column connected to an
analytical column using a switching valve. Automation has advantages over
off-line techniques. On-line analysis improves analytical precision and reduces
solvent volumes since LC pumps meter the flow rates and the volumes of
mobile phase passed through both columns. This on-line procedure can enhance
productivity by allowing automated, unattended processing of large numbers of
samples using an autosampler and a switching valve connected to the controller
of the chromatograph.
Methodologies
In enrichment, analytes are retained and concentrated on an enrichment column
while unwanted components bypass the analytical column to waste. When the
switching valve is actuated it backflushes analytes out of the enrichment column
onto the analytical column. See illustration below. The enrichment column is
typically a reverse phase column where analytes are injected using a mobile
phase composition that causes them to be strongly retained.
Under these conditions, the injection of a large volume of sample onto the
enrichment column results in a highly concentrated sample at the enrichment
1.00
1.00
1.00
1.00
1
2
34
5
6
1
2
34
5
6
Pump A
Waste
Position 1
Analytical
Column
Autosampler
Enrichment
Column
Autosampler
Pump A
Waste
Enrichment
Column
Analytical
Column
Pump B
Position 2
Detector
Detector
MX Automated Valves
Pump B
Column Backflushing
Column backflushing is an analytical method in which a switching valve
reverses the flow through a column. This method allows automatic clean up of
a column without disconnection in situations where sample mixture is trapped
on the head of a column. Rather than force highly retained compounds through
the full length of the column, the reversed flow permits the elution of
compounds in a shorter time period.
For automated column backflushing, connect a Nano-Scale MX Six-Port
Switching Valve (Stainless Steel MX7980-000) or an Analytical-Scale MX Six-
Port Switching Valve (Stainless Steel MX7900-000 or Biocompatible MX9900-
000) to the system in place of the column between the autosampler (or injec-
tor) and the detector. The illustration to the right shows the column connected
to Ports 6 and 3 and a jumper tube connected to Ports 1 and 5 of a MX Two-
Position, Six-Port Switching Valve to enable flow reversal.
Switching the valve from one position to another only changes the direction of
flow through the column. Choosing the appropriate tubing size is an important
factor in using this application to minimize volume added to the system that
could increase dispersion.
Column Backflushing
Column Backflushing
1.00
1.00
To order, contact your authorized Rheodyne MX dealer. See www.rheodyne.com for a complete list of dealers.
Nano-Scale
MX7980-000 MX Nano-Scale Two-Position, Six-Port Switching Valve (Biocompatible DuraLifeTM II)
Analytical-Scale
MX7900-000 MX Two-Position, Six-Port Switching Valve (Stainless Steel)
MX9900-000 MX Two-Position, Six-Port Switching Valve (Biocompatible)
Part Number Description
2321168B
page 7 of 9
9/04
Jumper Tube
Jumper Tube
Pump
Pump
Auto
Sampler
Auto
Sampler
Column
Column
Detector
Detector
1
2
34
5
6
1
2
34
5
6
Position 2
Position 1
MX Automated Valves
MS Solvent Diversion
MS Solvent Diversion
MS Solvent Diversion
In order to enhance the capability of an analytical method, analysts may
combine two or more traditionally separate analytical techniques. Hyphenated
Techniques is the common term for combining two techniques into a single
system such as liquid chromatography-mass spectrometry (LC-MS).
Combining LC with MS presents an enormous range of possibilities for sample
analysis. LC offers the ability to separate almost any mixture, and MS offers
extremely sensitive detection along with qualitative power for target analyte
identification. An LC-MS creates an efficient analysis technique to eliminate a
two-step method for obtaining comprehensive data on an unknown sample.
For example, further identification of the separated sample from the column is
possible with data from the mass spectrometer.
A MX Switching Valve can help make a working combination of the two tech-
niques. An example of an automated LC-MS system consists of a Nano-Scale
MX Six-Port Switching Valve, or Analytical-Scale MX Six-Port Switching
Valve, in-line with an autosampler, a column (LC System), and the mass spec-
trometer (MS System).
The MX Switching Valve operates as an automatic switching valve to divert
undesired portions of the sample from the LC system to waste before the sam-
ple enters the MS. This flow path diversion removes the unwanted portion of
the sample from the entire system thus keeping any non-volatile salts away
from the mass spectrometer.
Non-volatile salts in the mobile phase can quickly degrade the performance of a
mass spectrometer should they enter the high vacuum region. Even if the
mobile phase is free of non-volatile salts, the sample may contain salts. Since
any salts present in the sample should remain in the unretained portion,
switching the column effluent away from the mass spectrometer as the
unretained portion emerges from the column is a good way to keep mass
spectrometer maintenance to a minimum.
To order, contact your authorized Rheodyne dealer. See www.rheodyne.com for a complete list of dealers.
Part Number Description
Nano-Scale
MX7980-000 MX Nano-Scale Two-Position, Six-Port Switching Valve (Biocompatible DuraLifeTM II)
Analytical-Scale
MX7900-000 MX Two-Position, Six-Port Switching Valve (Stainless Steel)
MX9900-000 MX Two-Position, Six-Port Switching Valve (Biocompatible)
The illustration below shows that the undesired portion of the sample is
diverted to waste in Position 1, and the sample flow travels first through the LC
system and then the MS system in Position 2.
2321168B
page 8 of 9
9/04
MX Automated Valves
1.00
1.00
Column
Column
LC/MS Interface
LC/MS Interface
Pump A
Pump A
Autosampler
Position 1
Position 2
Waste
Autosampler
1
2
34
5
6
Waste
1
2
3
5
6
4
Multi-Dimensional Proteomic Peptide Separation
Reverse phase RP chromatography, a one-dimensional separation, is a common
technique used in proteomic peptide separation. A multi-dimensional separation
system can enhance peptide separations.
An autosampler in-line with a Nano-Scale MX Ten-Port Switching Valve
(Stainless Steel MX7986-000) or an Analytical-Scale MX Ten-Port Switching
Valve (Stainless Steel MX7960-000) creates a two-dimensional chromatography
system.
The first dimension of the analysis is separation of a protein on an ion exchange
(SCX) column. Salt fractions eluted from this column trap on an RP trap col-
umn. Switching the valve from Position 1 to Position 2 elutes the peptides from
the trap column for further resolution using a gradient nano pump. The peptides
are then detected and analyzed by ESI-MS or MS-MS. The peptide eluate can
be spotted onto a MALDI target for further analysis.
In Position 1, effluent from the SCX column traps on the RP Trap Column 1.
While Nano RP Column 1 analyzes the sample, RP Trap Column 2 traps the
next salt fraction. In Position 2, the SCX column is in-line with RP Column 2
while RP Trap Column 1 traps the next salt fraction.
To order, contact your authorized Rheodyne MX dealer. See www.rheodyne.com for a complete list of dealers.
Nano-Scale
MX7986-000 MX Nano-Scale Two-Position, Ten-Port Switching Valve (Biocompatible DuraLifeTM II)
Analytical-Scale
MX7960-000 MX Two-Position, Ten-Port Switching Valve (Stainless Steel)
Part Number Description
Multi-Dimensional
Multi-Dimensional
Pr
Proteomic Peptide Separation
oteomic Peptide Separation
2321168B
page 9 of 9
9/04
MX Automated Valves
1.00
1.00
1.00
1.00
Position 1
Position 2
RP Trap
Column 2
RP Trap
Column 1
Nano RP
Column
SCX
Column
Nano Pump
Gradient Pump
Waste
ESI-MS
Interface
MS
3
4
56
7
8
9
10
1
2
RP Trap
Column 2
RP Trap
Column 1
Nano RP
Column
SCX
Column
Nano Pump
Gradient Pump
Waste
ESI-MS
Interface
MS
3
4
56
7
8
9
10
1
2

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