Phenomenex Gemini C18 Guide

Gemini HPLC/UHPLC Columns
Tips for Care and Use
Phenomenex products are available worldwide.
www.phenomenex.com/mysupport QUALITY
MANAGEMENT SYSTEM
CERTIFIED BY DNV GL
9001:2008
pH Flexible LC
General Information
Each Gemini column manufactured by Phenomenex is individually prepared and tested. Every column is supplied with a
Certificate of Quality Assurance (CQA) which indicates testing conditions, operating parameters, and column details. The
column details, including specifications and performance test results should be entered into your information management
system for easy tracking and reference. Electronic copies of your column’s quality documentation can also be acquired at:
www.phenomenex.com/mysupport.
Inspection
Upon receipt of column, please verify that the column you received is the one you ordered (i.e. dimensions, particle size, media).
Additionally, please check the column for any physical damage potentially caused during shipment. Test the column immediately to
verify performance and record the result of your test in your column information management system.
Typical Flow Rate, Backpressure, Temperature:
Here are some typical values for common dimensions of Gemini HPLC and UHPLC columns. These numbers are not absolute values
and can differ based on LC system, running parameters and sample analytes/matrix. The values below have been created using a
solvent system of Acetonitrile and Water.
Gemini
Phases Shipping Solvent
†
Particle
Sizes
(µm)
Pore
Size
(Å)
Surface
Area
(m
2
/g)
Carbon
Load
(%) pH Stability Reversed Phase Normal Phase HILIC
100%
Aqueous
Stable
C18
Acetonitrile /Water (65:35) 3,5,10 110 375 14 1.0-12.0*
NX-C18 Acetonitrile /Water (75:25) 3,5,10 110 375 14 1.0-12.0*
C6-Phenyl Acetonitrile /Water (65:35) 3,5 110 375 12 1.0-12.0*
†Depending on column dimensions, organic to aqueous solvent ratios may vary slightly.
* pH stability under gradient conditions. pH stability is 1.5-10 under isocratic conditions.
Column Characteristics
Particle
Size (µm)
Internal
Diameter (mm)
Typical Flow
(mL/min)
Typical Pressure (PSI)
50 mm** 150 mm** 250 mm**
3 0.3 0.005 500 1000 NA
3 0.5 0.015 700 1400 NA
3 1.0 0.05 800 1000 NA
3 2.0 0.2 650 1550 NA
3 3.0 0.5 950 1900 2700
3 4.6 1 1000 1700 2600
5 2.0 0.2 400 650 850
5 3.0 0.5 550 1100 1550
5 4.6 1 1400 1500 1950
5 10.0 5 1400 1600 1800
5 21.2 20 500 800 1400
5 30.0 40 500 900 1200
10 4.6 1 NA NA 1400
10 10.0 5 NA NA 1400
10 21.2 20 NA 400 500
10 30.0 40 NA 400 600
10 50.0 50 NA 300 300
** Column Length
Typical Flow Rates (Independent of particle size):
i. 1.0 mL/min for 4.6 mm ID
ii. 0.2-0.6 mL/min for 2.1 mm ID
Maximum Backpressure:
iii. > 5,000 psi (345 bar) may compromise column longevity.
Maximum Temperature:
iv. Recommended max temperature for Gemini LC columns is
60°C, however temperature limits are dependent on your
running parameters. Running at a pH greater than 8 at 60°C will
compromise column lifetime.
v. Continuous use of Gemini columns at the maximum
temperature limit may compromise column longevity.

Reversed Phase
Name: Reversed Phase 2 Test Mix
Part No.: AL0-3045
Contents: Uracil, Acetophenone, Toluene, Naphthalene
Solvent: Acetonitrile/Water (65:35 v/v)
Detection: UV @ 254 nm
Injection Vol.*: Depends on dimensions
* See next page for suggested loading capacities based on ID.
Mobile Phase Compatibility
When using any HPLC/UHPLC column, be sure to only use HPLC grade
solvents and materials while also avoiding immiscible solvent/buffer combina-
tions. Additionally, use of solvent ltration is highly recommended to remove
trace impurities from your mobile phase of choice. Gemini NX-C18 columns are
stable in 100% aqueous conditions, but for all Gemini columns please ensure
that mobile phase pH does not exceed individual stationary phase limits. See
chart in column characteristics section for individual Gemini stationary phase
pH limits.
Column Installation
Initial setup of your LC system is very important to ensure column performance:
Ensure that your LC system is ready:
1. Seals, lines, injector clean
2. Lines primed (no dry lines or bubbles)
3. Steady baseline
4. Consistent pressures
Flush LC system pump and line with mobile phase (HPLC grade and
miscible with solvents that column is shipped in).
Mobile phase starting conditions check list:
1. Ensure that HPLC grade mobile phase is well
mixed, ltered, and degassed prior to use.
2. Ensure that column shipping solvent, remaining
solvent in LC system, and mobile phase solvents
are miscible.
Set ow rate to 0.1 mL/min (for 2.1-4.6 mm ID) and install the column making
sure that the arrow is in the direction of ow. Then increase the ow rate to
0.2 mL/min (2.1mm ID) or 1.0 mL/min (4.6 mm ID) for 5-10 minutes. Collect
solvent in a small beaker.
Stop ow and wipe outlet end of column to remove any particulates before
connecting to detector.
Install tting/tubing into outlet end and run minimum 10 column volumes at
low ow (~0.2 mL/min) while monitoring the backpressure.
1. A steady pressure should indicate a constant ow while
pressure uctuation will indicate air in the system.
2. Wide uctuations in pressure may shock and damage
the column so it’s important to monitor the pressure.
Monitor pressure as well as signal from the detector, when both are steady,
the column is ready for use.
Testing Column Performance
When testing column performance, please use the manufacturer approved
test mix.
Column Cleaning
Reversed Phase:
• Clean with a gradient that is closest to the last solvent system on the
system:
For example, if the last injection ended with Buffer/Acetonitrile
(75:25), it's more appropriate to start with 95:5 Water/
Acetonitrile and then move step by step as needed to increase
organic content (i.e. 75:25 Water/Acetonitrileà50:50 Water/
Acetonitrileà5:95 Water/Acetonitrile).
• For hydrophobic or oily materials, try ushing with Isopropyl Alcohol (IPA),
after the column has been ushed with Acetonitrile. When using IPA,
ensure use of a low ow to prevent higher backpressures due to higher
solvent viscosity.
• For materials that are very hydrophobic, try Tetrahydrofuran (THF)
instead.
Tips:
• When cleaning, set your ow rate lower than that of your method ow rate,
especially when attempting to clean using methanol or IPA.
• Cleaning for a longer period of time is often more benecial than adding
more cycles.
• Working with very high amounts of THF is not recommended especially if
system has PEEK tubing. Cleaning with THF is ne if the tubing are metal.
• Reverse ushing the column (against the direction of the arrow on the
column), but reduce the ow. Here are suggested reverse ush ow rates
based on column ID:
0.1 mL/min (2.1 mm ID)
0.3 mL/min (3.0 mm ID)
0.5 mL/min (4.6 mm ID)
Column Regeneration
Reversed Phase
• Apply the same gradient ush as in the cleaning above, overnight at
low ow.
• Reverse ushing is acceptable.
Column Storage
It is very important to make sure that your column is clean before storage.
This includes removal of buffer, salts, sample, and ion-pairing agents. The
recommended storage conditions are:
• Reversed phase: Acetonitrile/Water (65:35 v/v), Methanol can be used in
place of acetonitrile.

Amounts of Sample That Can Be Separated
Column Type ID (mm)
Approx.Dead
Volume (mL)*
Typical Flow
Rate (mL)
Typical and
(Max.) Injection
Masses (mg)
Typical and
(Max.) Injection
Volumes (μL)**
Capillary
(Fused Silica) 0.32 0.0075 0.001 - 0.02 0.001 (0.01) 1 (10)
Microbore 1.0 0.07 0.02 - 0.1 0.01 (0.1) 5 (25)
Analytical 4.6 1.5 0.5 - 2.0 0.1 (2.5) 10 (200)
Semi-Prep 10.0 7.3 5.0 - 20 1.0 (25) 50 (1000)
Preparative 20.0 29.2 10 - 200 5.0 (500) 200 (5000)
*The column Dead Volume (Vo) may be estimated from:
Column Dead Volume (mL) = Vo = 0.487 x d2x L
Where: L = column length (cm); 15 cm (150 mm) used for calculation.
d = column ID (cm, not mm)
**The maximum allowable Sample Injection Volume (Vi) can be estimated as
follows: Maximum Injection Volume
Where: Vr = the retention volume of the rst peak (mL)
N = number of theoretical plates per column
Tips for Extending Column Lifetime
• Utilize sample preparation techniques such as solid phase extraction
(Strata®-X SPE products) or accessories (Phenex™Syringe Filters) to
minimize the injection of unwanted contaminants onto your system
and column.
• Use the correct guard column or guard cartridge system (SecurityGuard™) to
help remove particulates before they foul your column.
• Do not overload your column. Inject suitable sample concentrations and
volumes.
• Work in the appropriate separation mode for the column. Please see
Column Characteristics chart for typical modes each stationary phase
is used for.
• Store your column in appropriate solvent(s).
• Solvent switch correctly by slowly acclimating the phase from one
miscible solvent to the other at a low ow: 0.1 mL/min for 2.1 mm ID and
0.5 mL/min for 4.6 mm ID.
Column Warranties
Phenomenex HPLC columns are warranted to meet the stated performance
and quality and to be free of defects in material and workmanship. If you are
unsatised for any reason, please give your Phenomenex Technical Representa-
tive a call. We’ll do our best to solve the problem to your satisfaction. Should it
become necessary to return the column, a Return Authorization Number must be
obtained from Phenomenex rst.
Disclaimers
New columns should be tested with the manufacturers recommended test mix,
and previously used columns should be tested with the same or a suitable test
mix for the analysis. Remember to re-equilibrate the system when changing
solvents. Never change from one solvent to another which is immiscible, without
going through an intermediate solvent which is miscible with both. This will dam-
age the column. Never change to (or from) a buffer/salt solution where the buffer/
salt is not soluble in the second solvent. Again this will damage the column.
Never attempt to remove the column end ttings. This will void the warranty.
Column Shock
Handle columns with care. Do not drop or create physical shock. Do not start
pump at high ow rates, instead ramp up gradually over a few minutes. Set your
pump pressure limit to protect the column in event of blockage. This can create
voids which will detrimentally affect the column’s performance.
Column Questions and Support
If you have any additional questions, please reach out to our amazing technical
team through:
Email: [email protected]
Live Chat: https://www.phenomenex.com/info/page/2015phenomchat
For more information on Gemini UHPLC, HPLC, and Preparative columns,
please visit www.phenomenex.com/Gemini
Trademarks
Phenomenex, Gemini, and Strata are registered trademarks, Phenex and SecurityGuard are
trademarks of Phenomenex.
Gemini is patented by Phenomenex. U.S. Patent Nos. 7,563,367 and 8,658,038 and foreign
counterparts.
SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362
CAUTION: this patent only applies to the analytical-sized guard cartridge holder, and does not apply to SemiPrep,
PREP or ULTRA holders, or to any cartridges.
Strata-X is patented by Phenomenex. U.S. Patent Nos. 7,119,145
© 2017 Phenomenex, Inc. All rights reserved.
Typical Loading Capacities
FL49380117_W
Vr
2√N
=Vi =