Notes on Cleaning bound Protein from RP HPLC columns

First, a few comments:

  • ·         Before proceeding with any column regeneration or cleaning procedures, always refer to the specific advice provided by the column manufacturer. Approved maintenance and cleaning instructions can often be found in the product guide which comes with the new column. Their guidelines supersede these!
  • ·         Columns are consumable items. After a suitable amount of use, the time and materials required to regenerate them may cost more than the purchase of a replacement column. Always have a new, spare column on hand.
  • ·         Protect your detector. Before washing or cleaning the column, disconnect the column outlet line and direct the column to waste only.

For RP supports, if buffers have been used, always start by washing the column down with ultra-high purity water and some organic solvent (e.g. Water/MeOH, 95%/5%) to remove all salts.

Many polymeric resins (e.g. PS-DVB) can be effectively cleaned using 0.1 M Sodium Hydroxide solution or a mobile phase solution containing equal parts of isopropanol (IPA) and 3 M Guanidine hydrochloride at ~ 50 °C. Optionally, some success has been reported using other solutions such as: 5M Urea (pH 7) buffer solution; 1 M NaCl (pH 7) and even mixtures containing some methylene chloride solvent.

For RP silica based supports, we often use a series of wash solutions. In most cases, pure water or pure organic solvents such as MeOH or ACN will not remove bound protein. An acid, base or ion pairing reagent is often needed. One of the first general wash solutions to start with is a 1% Acetic acid solution in Methanol (50/50). If desired a stronger acid such as Trifluoroacetic acid (TFA) can be swapped for the acetic acid (where possible, start with a weaker acid). This can be followed with a later solution where IPA replaces the MeOH (50/50). In both cases, adjust the pH to ~ 2.5. 5 M Urea solution has also proven effective in removing bound protein from silica supports too. Another salt solution that has shown some promise is 1 M sodium phosphate solution, pH 7.0. Run the salt solutions for about one hour at a moderate flow rate. Follow up with rinses of water and MeOH (80/20), then 90% MeOH/Water.

HPLC Column | Agilent HC-C18(2) Small Molecule Separations LC Column

Agilent HC-C18(2) columns are an excellent choice for separations of many types of compounds.

Agilent HC-C18(2) is a more retentive C18 with a higher carbon load. An excellent value alternative to other high carbon load columns, it also provides superior peak shape for basic compounds.

Features Of Agilent HC-C18(2) Small Molecule Separations LC Column:

  • Higher carbon load - 17% - provides greater retention for moderately polar and non-polar compounds
  • Ideal for non-polar compounds and separations that start at mid-level % organic (at least greater than 10% organic)
  • Good choice for industrial samples or samples dissolved in organic/mostly organic solvents
  • Stable over a very wide pH range (2-9) for maximum flexibility
  • To compare technical information and specifications for different phases, see our phase specifications chart.

HPLC Column | Agilent Prep Small Molecule Separations LC Column

[caption id="" align="aligncenter" width="475"]Agilent Prep Small Molecule Separations LC Column Agilent Prep Small Molecule Separations LC Column[/caption]

Agilent Prep LC Columns are designed for high loadability to purify milligram to gram quantities of products. Column dimensions are available for most preparative sample types ranging from high throughput samples generated in drug discovery to lab scale prep and up to some pilot scale sample preparation. Preparative sized columns are available in 21.2, 30 and 50 mm internal diameters with lengths ranging from 50–250 mm. Columns are available in 5 and 10 µm particle sizes with very high efficiency in every dimension.

Features Of Agilent Prep Small Molecule Separations LC Column:

  • High loadability for maximum sample purification
  • Easy scalability from 4.6 mm id to 50 mm id for rapid method development
  • High Throughput 21.2 mm id cartridges for fast purification
  • Exceptional column stability up to pH 10

HPLC Column | Agilent Load & Lock Small Molecule Separations LC Column

Agilent Load & Lock Small Molecule Separations LC Column

Agilent offers a complete range of Load & Lock column systems for laboratory and process preparative LC. They are designed to enable users to easily and quickly pack their own preparative high efficiency columns. This is the right solution for applications ranging in scale from discovery (milligrams) to production (multi-kilos) of pharmaceutical compounds, peptides, and natural products. Our Load & Lock columns have a unique fluid/sample distribution system to maximize productivity. It is the only system that provides dynamic axial compression (DAC) and static "locked" axial compression (SAC) and is designed for easy operation to deliver greater convenience.

Features Of Agilent Load & Lock Small Molecule Separations LC Column:

  • Mobile packing station supports three different column sizes
  • Runs on compressed air with no need for a power supply
  • Quick and easy packing and unpacking within minutes

HPLC Column | Agilent MicroBore Small Molecule Separations LC Column

MicroBore (1 mm id) columns are a good choice when sample sizes are limited. They will improve detection limits 5 times over 2.1 mm id columns when the same sample mass is used. This increase in sensitivity can be critical. MicroBore columns use low flow rates (typically ~50 µL/min). Therefore, these columns are ideal for use with detectors requiring low flow ratessuch as some mass spectrometers. MicroBore columns perform optimally with HPLC systems purchased or modified for microbore use. Guard columns are now available with an adjustable tube stop depth to provide a perfect zero dead volume connection every time.

Features Of Agilent MicroBore Small Molecule Separations LC Column:

  • High sensitivity for small sample sizes
  • Compatible with LC/MS interfaces
  • Wide variety of bonded phases
  • Guard columns available for MicroBore (1.0mm id) and Capillary columns

HPLC Column | Agilent ChiraDex Small Molecule Separations LC column

Applications: Barbiturates, benzothioadiazines, calcium antagonists, PTH-amino acids

Features Of Agilent ChiraDex Small Molecule Separations LC column:

  • For routine separation of enantiomers
  • Available as ChiraDex cartridge columns
  • Novel manufacturing process bonds ß-cyclodextrin to spherical 5-um silica gel by means of a chemical spacer
  • Enantiomeric separations have been achieved with ChiraDex using simple nonchiral solvent systems

HPLC Column | Agilent ZORBAX Extend-C18 Small Molecule Separations LC Column

Extend-C18 columns incorporate a unique patented bidentate silane, combined with a double-endcapping process that protects the silica from dissolution at high pH - up to pH 11.5. Columns are best applied for separations of compounds that are either (1) basic and have little or no retention at low or intermediate pHs, (2) more stable or more soluble at high pH, or (3) basic and show poor peak shape at low or intermediate pH.

Features Of Agilent ZORBAX Extend-C18 Small Molecule Separations LC Column:

  • High efficiency and long life at high pH - up to pH 11.5
  • Unique bidentate bonding and double endcaping provides high pH stability
  • More efficiency and better peak shape than polymer-based columns
  • Improve retention, resolution and peak shape of basic compounds
  • High sensitivity for LC/MS separations of peptides and proteins
  • To compare technical information and specifications for different phases, see our phase specifications chart.

HPLC Column | Agilent ZORBAX Eclipse XDB-CN Small Molecule Separations LC Column

For normal-phase chromatography, the ZORBAX product line offers a choice of bonded and non-bonded silica packings.

Features:

  • Made from highly pure Rx-SIL
  • Excellent choice for normal-phase applications with basic compounds
  • Equilibrates more rapidly than ZORBAX Rx-Sil and is used for many of the same normal-phase applications
  • To compare technical information and specifications for different phases, see our phase specifications chart.

HPLC Column | Agilent LiChrospher Small Molecule Separations LC Column

Agilent LiChrospher silica columns are made with spherical, 'sil' type porous silica particles. LiChrospher 60 RP-select B starts with 60Å pore size silica optimized for symmetrical peak shapes of basic compounds. LiChrospher 100Å is offered in both C8 and C18 as well as an endcapped C18. These columns are noted for high sample capacity and efficiency. The popular LiChrospher packings are offered in the convenient and economical Agilent cartridge configuration. For normal phase chromatography, Agilent supplies polar modified silica gel phases LiChrospher Si, LiChrospher NH2 , LiChrospher DIOL and LiChrospher CN as convenient cartridge columns.

Features Of Agilent LiChrospher Small Molecule Separations LC Column:

  • Available in 5 µm particle size
  • Reversed-phase available in RP-C18, RP-18 Encapped, RP-8, RP-Select B, CN
  • Normal-phase available in CN, Diol, NH2, Si
  • Applications—Pharmaceuticals, aromatics
  • LiChrospher RP-18—Spherical silica for acidic, neutral and basic compounds with great batch-to-batch reproducibility
  • LiChrospher CN—Polar modified silica gel phases > polar and hydrophobic properties; also used in normal-phase chromatography.
 

HPLC Column | Agilent ZORBAX PrepHT Small Molecule Separations LC Column

Agilent ZORBAX PrepHT Small Molecule Separations LC Column

ZORBAX optimized phases are now available in preparative dimensions as PrepHT columns. These columns are 21.2 mm and larger in diameter with 5 µm and 7 µm particles to provide high resolving power. They are also available in various lengths to allow optimum resolving power and speed of analyses. In preparative chromatography, it is important to obtain high purity and high recovery along with high throughput. Agilent ZORBAX PrepHT columns have a broad range of selectivities to achieve these objectives.

Features Of Agilent ZORBAX PrepHT Small Molecule Separations LC Column:

  • Easy scale-up from analytical to preparative scale with ZORBAX phases
  • Fast preparative separations, up to 2000 mg
  • 5 to 7 μm particles for high efficiency and high yield
  • Easy to install finger-tight connections seal up to 5000 psi/350 bar
  • Use to maintain selectivity of the analytical phase in your prep separations

HPLC Column | Agilent ZORBAX Eclipse Amino Acid Analysis (AAA) LC Column

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The ZORBAX Eclipse AAA high efficiency HPLC column rapidly separates amino acids following an updated and improved protocol for use with the Agilent 1100 HPLC. Total analysis from injection to injection can be achieved in as little as 14 min (10 min analysis time) on the short, 7.5 cm column and 24 min (16 min analysis time) on the 15 cm column length. Exceptional sensitivity (5 - 50 pmol with DAD,FLD) and reliability are achieved using both OPA and FMOC derivitization chemistries in one fully automated procedure using the Agilent 1100 HPLC instrument. Eclipse AAA columns are used for high resolution rapid analysis of 24 amino acids. The Eclipse AAA is use tested for amino acid analysis (AAA). The Eclipse AAA uses well known OPA and FMOC derivatization techniques.

Features Of  Agilent ZORBAX Eclipse Amino Acid Analysis (AAA) LC Column:

  • High resolution and rapid analysis of 24 amino acids
  • Tested for amino acid analysis
  • Uses well-known OPA and FMOC precolumn derivatization chemistry
  • Easily automated using a detailed online, derivatization protocol available for use with Agilent 1100/1200 autosampler

HPLC Column | Agilent ZORBAX Carbohydrate Analysis Small Molecule Separations LC Column

ZORBAX Carbohydrate Analysis Columns are reproducible, efficient, and flexible. These columns use ZORBAX porous silica microsphere technology. Silica manufacture, bonding and packing are all performed in our ISO9001 facilities. ZORBAX Carbohydrate Analysis Columns can handle high volume injections as much as 50 µL on a 4.6 x 150 mm column.

Features Of Agilent ZORBAX Carbohydrate Analysis Small Molecule Separations LC Column:

  • Reproducible
  • Efficient – uses ZORBAX porous silica microsphere technology; silica manufacturing, bonding and packing are all performed in Agilent's ISO 9001 facilities
  • Flexible – can handle high volume injections – as much as 50 μL on a 4.6 x 150 mm column
  • Recommended for use with refractive index detectors (RID)