Inertsil HPLC Column Care Instructions

Inertsil HPLC packings are subject to a rigorous array of QC tests in a ISO9001 compliant facility, with special emphasis on reagent purity, raw material traceability, and consistency in raw materials and finished products. A detailed analysis of all Inertsil's physical properties, chemical purity, chromatographic selectivity, and column packing efficiency is used to confirm that each lot of Inertsil is identical to all previous lots.

We know how important column consistency is to your work, so GL Sciences goes to great lengths to make certain that every HPLC column we ship to our customers is of the highest quality possible.

When you receive an HPLC Column from GL Sciences, please take the following simple steps:

(1) Check the column for signs of physical damage which may have occurred during shipping. Contact GL Sciences immediately to report any problems. In the U.S., phone (310)265-4424, or email info@inertsil.com.

(2) Make sure that the column you received is the column that you ordered.

(3) Take note of the solvent contained in the column during shipping. (The solvent used for shipping is the same as that used as the mobile phase on the QC test chromatogram ). Before attempting to change solvents, make certain that the eluent you will be introducing into the column is COMPLETELY miscible with the eluent contained in the column, to avoid precipitation of buffer salts or other mobile phase additives.

(4) Test the column to verify column efficiency and back pressure (using one of the later-eluting components of the QC test sample). Contact GL Sciences immediately to report any problems. In the U.S., phone (310)265-4424, or email info@inertsil.com. This is important because the MOST COMMON complaint about HPLC columns is high back pressure. Given that the columns are all tested during QA/QC, high back pressure is almost ALWAYS the result of particulates introduced by the customer during "equilibration" or the first few sample injections.

There are several important things about your analytical method that will greatly affect column life and column performance, including sample preparation, solvent selection, and solvent preparation.

Sample Preparation
Non-ideal chemical and physical interaction of samples with the column frits and column packings is a primary source of problems. Most columns fail because the frit becomes clogged or the stationary phase at the head of the column becomes contaminated (e.g. "gunked up"). (to top)

Samples should be filtered prior to injection. Even sample solutions which appear to be particulate-free can contain small solids which can clog the pores of the column inlet frit. Samples should be filtered through a 0.45µ or 0.2µ syringe filter before injection. Many customers don't like to filter their samples this way or at all; these customers should expect potential GL Sciences' Chromatodisc filters are designed for sample filtration prior to HPLC analysis. Use common sense; the dirtier the sample injected, the more likely the column will clog.

In addition to filtering samples, Guard cartridges can be used to trap "problems" before they reach the analytical column. Guard cartridges are essentially tiny HPLC columns that are cheaper to replace than the analytical column. Ordering information for Inertsil guard cartridges is found on the same pages as the analytical column ordering information. Many customers don't like using guard columns because of the extra cost and the extra potential "dead volume" introduced. As usual, there is a tradeoff involved here. If the guard columns are plumbed correctly, very little dead volume is introduced and the analytical column lifetime can be extended substantially. Alternatively, if the wrong guard column is used or the guard system is not plumbed correctly, dead volume CAN be introduced and eliminate the potential benefit of using a guard system. Please contact GL Sciences before installing any guard system so we can help you minimize costs and maximize results.

Solvent Selection
Columns last longest when they are used with benign eluents. Using eluents of high pH or low pH can dissolve silica or catalyze hydrolysis of the bonded phase. Try to stay within the pH range of 2 - 9 on Inertsil columns. If you must use a pH outside this range, column life might be reduced.

Solvent Preparation
Use a 0.45µ or 0.2µ filter for solvents as well as samples, even HPLC grade solvents!

COLUMN STORAGE
Column storage conditions can have a profound effect on column lifetime and performance-after-storage. Before extended storage (e.g. greater than 2 days), rinse the column COMPLETELY free of eluents containing buffers, ion-pair reagents, or inorganic solutes, by flushing with 20-50 column volumes of the eluent WITHOUT the dissolved additives. Then flush the column with 5-10 column volumes of water (reverse phase columns only). Then flush the column with 20 volumes of storage solvent (shown above)

In-Situ Column Cleaning
Columns that become fouled over time can sometimes be rejuvenated with an aggressive rinsing sequence, as shown below. In all cases, reverse the column (e.g. attach the outlet end of the column to the pump, and pump the eluent directly into a waste reservoir) and flush the column with 50ml volumes of the indicated solvents in the indicated sequences:

* If mobile phase contains a buffer, flush the column with the mobile phase MINUS the buffer first, to avoid precipitation of the buffer in the pure MeOH remaining in the column.

INERTSIL^® HPLC Column Care Instructions
Phase	Shipping Solvent	Storage Solvent
Alkyl RP - ODS, C8, C4, etc.	MeCN:Water, e.g. 60:40	MeCN or MeOH
Aromatic RP - Phenyl, etc.	MeCN:Water, e.g. 60:40	MeCN or MeOH
Normal Phase - CN, Diol, Silica	n-Hexane:Ethanol, e.g. 98:2	n-Hexane:Ethanol or Heptane:IPA
Polar Reverse Phase, CN, Diol		MeCN
- Dropping or otherwise "shocking" columns can disrupt the column bed and cause peak splitting. - Use of eluents in the pH range of 2 - 7.5 will maximize column life, though higher pH eluents have been used successfully. - The lower the operating pressure, the longer the operating life. Though higher pressures are possible, the lower the operating pressure, typically, the longer the operating life of the column.

Reverse Phase		Normal Phase
(C18, C8, Phenyl, CN (RP*)		(Silica, NH₂, CN, Diol)
H₂O:MeCN 90:10 (up to 55°C)	1.	Hexane/Chloroform
Methanol	2.	Methylene Chloride
Acetonitrile	3.	Isopropanol
THF	4.	Methylene Chloride
Methanol	5.	Mobile Phase
Mobile Phase		*Skip H₂O rinse with CN Phase
*Skip H₂O rinse with CN Phase
Ion Exchange		Protein Removal
(AX, CX, NH₂)		(C18, C4, C8, Phenyl)
Distilled Water (up to 55°C)	1.	H₂O:MeCN 90:10
Methanol	2.	0.1% TFA
Acetonitrile	3.	Isopropanol
Methylene Chloride	4.	Acetonitrile
Methanol	5.	H₂O:MeCN 90:10
Mobile Phase*	6.	Mobile Phase