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Restek Liquid Chromatography (LC) Columns — Cleaning Recommendations

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Overview

Restek highly recommends the use of guard columns to protect your reversed phase and normal phase analytical LC columns from both frit blockage and column contamination. However, over time, even with this precaution, your LC column may become blocked or contaminated by strongly adsorbed sample components. If you observe decreased column performance, increased backpressure, or other signs of blockage or column contamination, the following LC column regeneration steps may help restore your column.

The following Restek LC columns are covered by this protocol:

Reversed Phase: Normal Phase:
Biphenyl Amino
C1–C18 Cyano
IBD Silica
PAH  
PFP Propyl  
Quat  

For assistance cleaning Restek LC columns not listed above, contact Technical Service.

LC Column Cleaning & Regeneration

  1. For HPLC columns (3, 4, and 5 µm), first turn off any flow and then disconnect the column from both the injector and the detector. Reverse the column flow direction by connecting the outlet end of the column to the pump eluent. For UHPLC columns (1.9 µm), DO NOT FLIP THE COLUMN. Instead, flush the column according to the direction indicated by the arrow on the column itself.

    Figure 1: The flow direction is indicated on the column's label.

  2. Flush the column using the solvent progression (Table I) and flow rate (Table II) indicated for your specific column.

    Table I: Solvent Progressions for Flushing Restek LC Columns

    NOTE: Flush column with a minimum of 20 column volumes of each solvent, in the order listed. Column volume can be estimated using the formula: V = ∏ r2 L where V = column volume in mL, r = column radius in cm, and L = length of the column in cm.

Reversed Phase Columns Normal Phase Columns
1. Water:Methanol (95:5, v/v) 1. Isopropyl Alcohol
2. Methanol 2. n-Hexane
3. Isopropyl Alcohol 3. Ethanol
4. n-Hexane 4. Return to your original mobile phase
5. Isopropyl Alcohol  
6. Methanol  
7. Water:Methanol (95:5, v/v)  
8. Return to your original mobile phase  
 

Table II: Flow Rates for Flushing Restek LC Columns

Column ID (mm) Flow Rate (μL / min)
1.0 25
2.1 100
3.0 / 3.2 250
4.6 500
 

LC Column Storage

To avoid contamination, store LC columns with end plugs securely fastened and be sure to include information describing the storage solvent.

For short-term storage, all LC columns should be flushed with a solvent identical in composition to the most recently used mobile phase minus any buffered, acidic, or basic components.

For long-term storage, reversed phase LC columns should be stored with 50% water/50% organic solvent (e.g., acetonitrile or methanol). Normal phase LC columns should be stored with a nonpolar solvent (e.g., hexane).

GNAR3266-UNV

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LC Column Cleaning and Regeneration

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