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Analytical Columns

For Analytical Chromatography a wide range of Columns are available to enhance the results, effectiveness and efficiency of your work

In this section we introduce you to

 

Comparison of various Analytical Column Parameters

Column1

Relative Flow
Rate
 (ml/min)

Solvent
Reduction
 (%)

Theoretical
Sensitivity
Increase

Recommended

I.D. (mm)

Internal Volume (μl)

Flow Cell
(μl)

Injection Volume (μl)

4.6 

1500

 1.0

-

 -

8.0

 20

4.0 

1133

0.76

24

1.3

 8.0

15

3.2  

725

0.48

 52

2

2.0 - 8.0

  10

2.1  

300

0.20

80

5

1.0 - 2.0

 5

1.0

71

0.047

95

21

 ≤1.0

 1

Capillary & Nano columns

1.0

 

40 - 240(μl/min)

 

21 -

 

 

0.5

 

 10 - 60(μl/min)

 

 84 4

 

 

0.3

 

3 - 18(μl/min)

 

230 11

 

 

0.15

 

0.05 - 5(μl/min)

 

920 44

 

 

0.075

 

 0.02 - 1.5(μl/min)

 

3700 178

 

 

         1)150 mm length

Loadability for proteomics columns

Column ID (Col length 10 & 20 cm)

Column load

9.4 mm

16 mg

4.6 mm

4  mg

2.1 mm

1 mg

1.0 mm

250 ug

 

 

Please do also take flow rate precision into consideration!

Suppose you’re using a 1-mm i.d. column.  The normal flow rate would be 50 l/min.  At the beginning of the gradient, with 99% A/1% B, the flow rate of B would be 0.5 l/min.  At the end of the gradient, with 1% A/99% B, the flow rate of A would be 0.5 l/min.  That means that in order to deliver a gradient with precision at that flow rate, your pump would have to be able to pump 0.5 l/min with precision.  If the specifications of the pump don’t claim that, then don’t bother trying to use it for such columns without a flow splitter. 

 With a 2.1-mm i.d. column the pump must be able to deliver 2 l/min with precision, a more attainable figure (but still not one that all HPLC pumps do attain).  Dead volume is another concern, even if your pumps do have the requisite precision.

 

Standard Analytical Columns  4.6 mm and 4 mm ID

Traditionally columns of either 4.6 or 4.0 mm internal Performance diameter (i.d.) and 150 or 250 mm length have been used as the industry’s standard for analytical applications. Such columns are still the most popular although there is an on-going shift to shorter and narrower i.d. columns.

UHPLC columns are designed to be operated at 1000 bar and are available up to 150 mm length. To stand the high pressure the precission of the column is much higher and so ist the surface roughness and purity. The end-fittings cant be removed with UHPLC columns

Most products are supplied in standart HPLC columns and many are now also available in UHPLC columns. ACE pack under the brand ACE Exel all particle sizes into UHPLC columns.

We also supply Guard Cartridge Columns.  The Hichrom universal guard cartridge system is recommended for use with analytical columns

Optimum Flow

 A flow rate of 0.76 ml/min through a 4.0 mm i.d. column gives the same relative flow rate as 1 ml/min through a 4.6 mm i.d.

Performance

The efficiency of a column depends on the choice of particle size and column length. The mode and quality of manufacture will also affect its performance. Particle size refers to the average diameter of the spherical silica particle. Commonly used silicas have a distribution of diameters. Hence a material of nominal particle size 5μm can typically contain silica particles between 4.0 and 6.5 μm diameter.

Smaller particles give higher efficiencies for constant column length. However at the same time column back pressure increases significantly resulting in an effective maximum column length. The pore size of a silica particle determines the retentivity and capacity characteristics.

MEDIUM BORE (3.2 mm i.d.) COLUMNS

  • Solvent consumption reduced by 50%
  • Sensitivity increased up to x2
  • Uses standard analytical HPLC equipment
  • Significant cost-saving
  • Performance comparable to standard 4.6 mm i.d. columns

Initial interest in the use of analytical columns of narrower bore than the industry standard 4.6 mm arose for scientific reasons. The ability to couple HPLC with techniques capable of providing characterisation data of the solute molecules necessitated the use of lower solvent volumes. Increased assay sensitivity and the need for less sample were added features.

The limited general availability of low dispersion volume equipment and the greater difficulties of manufacturing narrow bore HPLC columns have restricted developments. However the cost and environmental issues associated with the purchase and disposal of solvents is becoming increasingly important. Consequently the utility of the intermediate 3.2 mm i.d. column is now being strongly recommended by a number of organisations. The choice represents a compromise between using standard 4.6 mm i.d. columns and microbore columns of 2.1 mm i.d. or less. The 3.2 mm i.d. columns offer a 50% saving in solvent consumption for the same linear dynamic flow without necessarily requiring a change to lower dispersion volume injectors and flow cells. For satisfactory use of 2.1 mm or lower i.d. columns such a change is essential. When a 3.2 mm i.d. column is used with a Rheodyne valve injector model 7125 or 7725 and an 8μl flow cell some loss in performance is observed (Table 1).

The loss decreases with increase in  retention time. In practice chromatograms obtained from 3.2 and 4.6 mm i.d. columns are very similar in appearance (Figure 1).

Analytical Col Fig 1

Guard cartridge columns

The Hichrom universal guard cartridge system is recommended for use with medium bore column

Equipment

A Rheodyne 7125 or 7725 valve injector and an 8μl flow cell can be used with 3.2 mm i.d. columns containing 5μm particles. For critical separations a Rheodyne 8125 valve injector and a micro flow detector cell (<5μl) are recommended.

The loss in efficiency of biphenyl and phenanthrene peaks at various retention times in going from 4.6 to 3.2 mm i.d. columns using the above standard equipment is shown below .

Biphenyl

k'

0.93

 2.47

 7.09

Efficiency loss (%)

39

18

 8

Phenanthrene

k'

1.23

3.32

 10.12

Efficiency loss (%)

32

 14

  7

Optimum flow

A flow rate of 0.48 ml/min through a 3.2 mm i.d. column gives the same relative flow rate as 1 ml/min through a 4.6 mm i.d. column. The optimum flow rate for the medium bore column will be < 0.4 ml/min.

Sample size

Existing methods can be readily adapted for use with 3.2 mm columns at the appropriate flow rate. However as an effective increase in detector sensitivity will be observed, care should be taken not to exceed detector linearity. It may be necessary to dilute samples to lower concentration levels.

MICROBORE (2.1 and 1.0 mm i.d.) COLUMNS

  • Solvent consumption reduced by 80-95%
  • Sensitivity increased x5 to x21
  • Significant cost-saving
  • LC-MS applications

Microbore columns (1.0 and 2.1 mm i.d. traditionally packed) and capillary columns (<1 mm i.d.) offer significant theoretical advantages over conventional 4.6 mm i.d. columns. However, current instrumental constraints and manufacturing difficulties have limited their application, especially the narrower 1mm internal diameter and capillary columns. With this in mind Hichrom currently offer a comprehensive range of 1.0 and 2.1 mm i.d. microbore columns. Hichrom’s microbore columns offer minimal loss of performance compared to the corresponding analytical columns and can be used with optimised conventional equipment.

Column Evaluation

Microbore columns often perform poorly because of excessive  system dead volume. Most HPLC systems in use today are designed for standard bore columns and cannot effectively use columns with internal volumes less than 0.5 ml. The extra column volume measured from the injector through to the detector reduces the achievable efficiency of the column. If the calculated theoretical plate value is less than 90% of that measured by the manufacturer, there is probably an excessive amount of extra column volume in the system for the column being used. Early eluting peaks are less diluted and have smaller volumes than later eluting peaks and will consequently be more susceptible to the detrimental effects of excessive extra column volume.

Column Design

Columns are manufactured from high quality 316 stainless steel tubing. Both column ends are terminated with standard 1/4 - 1/16" female reducing unions. Stainless steel frits inset into a PEEK ring (traditional) or PEEK cap (modular cartridge) are used to retain the packing material.

If excessive extra column volume is a problem, several measures can be undertaken to improve the performance of the system. Use connecting tubing that has an internal diameter of 0.010 inches (0.254 mm) or less. Tubing with an i.d. of 0.007 inches (0.177 mm) or lower is preferable for use with microbore columns

Tubing i.d. (inches)

Volume per 5m length (μl)

0.006 (0.152 mm)

0.9

0.010 (0.254 mm)

2.5

0.020 (0.508 mm)

10.1

0.030 (0.762 mm)

 22.8

 

  • Keep the connecting tubing as short as possible between the injector and the column and the column and the detector. For microbore columns, it is desirable to keep these distances less than 5 cm.
  • Use only ‘low dead volume’ fittings and unions. A fingertight column coupler (throughbore 0.007") HI-081 is recommended to connect guard and microbore columns.
  • Make sure that all fittings are undamaged and correctly made.
  • Use a low volume detector flow cell.
  • Use Rheodyne valve injector model 8125.

Microbore columns normally require detector flow cells with volumes of 2μl or less. The design of the flow cell, however, can be just as important as the cell volume. Some low volume flow cells perform worse than larger volume flow cells because of their inadequate design. Figure 2 provides an example of what can happen when a microbore column is used with a typical HPLC system. The microbore column is unable to achieve the baseline separation (B) provided by the standard bore column (A). However, when the microbore column is used in an ‘optimised’ HPLC system that has less than 10μl of extra column volume, excellent resolution and peak shape is obtained (C).

Figure 2. HPLC systems must be optimised to obtain the best performance from microbore columns

Analytical Col Fig 2

Our microbore columns are evaluated using a Rheodyne model 8125 injector fitted with a 5μl loop manufactured from special 0.020" i.d. tubing. A detector of low dispersion volume fitted with a 1μl flow cell monitors column performance.

Microbore Guard Cartridge Columns

Our universal guard cartridge system is recommended for use with microbore columns

 

CAPILLARY AND NANO (<1.0mm i.d.) COLUMNS

  • High sensitivity
  • Low sample mass and volume applications
  • LC-MS and LC-MS/MS applications
  • Very low solvent consumption

Capillary and nano HPLC are gaining acceptance in applications where limited sample amounts lead to problems in detection sensitivity. High sensitivity and high resolution separations can now be achieved for small sample volumes. This is relevant in the areas of pharmacokinetics, trace analysis and particularly the rapidly expanding field of bioanalytical and proteomic analysis. The on-line coupling with a mass spectrometer has been a major driving force behind the development of capillary chromatography.

Sensitivity

The introduction of capillary and nano columns has made possible high sensitivity and high resolution separations for small sample volumes. The Table “Comparison of Column Parameters” at the top of this page shows the theoretical sensitivity increase of various i.d. capillary columns compared with a 1 mm i.d. microbore column. The use of a 0.075 mm (75 μm) i.d. column, for instance, can decrease detection limits by a factor of >3500 relative to a 4.6 mm i.d. column when the same sample size is used, due to lower chromatographic dilution of the sample.

Instrument Modifications

In order to fully exploit the benefits of using capillary dimensions, the HPLC system must be capable of handling
sample volumes in the sub-microlitre range. The use of columns of <1mm i.d. requires either a specially designed micro-LC instrument or extensive modifications of a standard HPLC instrument. The major principles in the conversion are flow rate reduction and elimination of dead volume. Flow rate reduction can be achieved using a high performance, low μl/minute pump or by incorporating a flow splitting tee between a standard HPLC pump and the injector. The majority of the flow can be split to waste or recycled. To ensure that band spreading is kept to a minimum, low dispersion column hardware must be used throughout the system. Connecting capillaries must be dead volume free and as short as possible. A micro-scale injector allowing injection of sub-microlitre sample volumes should be used. Micro flow cells with appropriate internal volume (<1μl) and path length should be used for UV detection.

Column Availability

Capillary and nano columns, ranging in i.d. from 0.05 mm to 0.8 mm, are available in the ACE, Inertsil and from a number of other manufactures

 

LC-MS COLUMNS

  • Column diameter ≤ 2.1 mm
  • Rapid analysis
  • Characterisation technique
  • High sensitivity
  • Quantitative analysis
  • Peak deconvolution ability

Introduction

Traditional problems associated with the inherent incompatibility of HPLC (high liquid pressure) and mass spectrometry (low vapour pressure) have been largely overcome. LC-MS has become a leading technique offering characterisation of solute molecules.

Column Dimensions

LC-MS interface techniques such as Atmospheric Pressure Chemical Ionisation, Electrospray, Particle Beam or
Thermospray can typically handle maximum flow rates of 200 μl/min. As microbore columns (2 mm i.d.) utilise such flows they are commonly used in LC-MS applications. A 50 x 2.1 mm i.d. column is used for fast speed analysis applications whilst a 250 x 2.1 mm i.d. column will be the one of choice for more complex separations. Where sensitivity is an issue, as in the analysis of peptides and proteins, 1 mm i.d. columns are available.

Availability

We offers a range of ACE, Hichrom, Hypersil, Inertsil, Kromasil, NUCLEOSIL, and Zorbax LC-MS columns, in lengths from 10 to 250 mm and 1.0 and 2.1 mm i.d. Alternative materials and dimensions can readily be supplied. Please enquire for details. Applications LC-MS has numerous applications. These include:

  • Drug metabolism structure elucidation and quantitative studies
  • Protein and peptide identification and sequencing
  • Combinatorial chemistry
  • Agrochemical identification and quantitative studies

Figure 3 shows the use of single-ion monitoring to analyse a mixture of benzodiazepines. Although the total ion current chromatogram (A) obtained on a 150 x 2.1 mm i.d. column only resolves Oxazepam (D), Lorazepam (G) and Estrazolam (C), the four remaining unresolved benzodiazepines can be analysed using a single-ion monitoring technique (Diazepam m/z 285 (B), Clobazam m/z 301 (E), Alprazolam m/z 309 (F) and Triazolam m/z 344 (H)). Although the assay is complete in under 5 minutes, use of a 50 x 2.1 mm rapid analysis column will reduce the analysis time to under 2.5 minutes.

Figure 3 Single-ion monitoring of benzodiazepines

Analytical Col Fig 3

 

RAPID ANALYSIS (2-10cm LENGTH) COLUMNS

  • Analysis time reduced by over 80%
  • Increase in productivity x5
  • Solvent saving up to 95%
  • 1.5 - 5μm particle size silica
  • Wide range of chemistries
  • Baseline resolution maintained

Rapid Analysis columns are designed to analyse large numbers of samples in as short a time as possible (Figure 4) without major sacrifice of column resolution. Quality Control environments particularly benefit from the use of Rapid Analysis columns giving corresponding increases in productivity

Figure 4. Rapid Analysis Columns Save Time

Analytical Col Fig 4

Typical reduction of column length from 250 to 50 mm will offer an 80% saving in analysis time. Often the efficiencies associated with a 250 mm length column containing 5μm silica particles (>20,000 plates) are not required. In such circumstances a 50 mm length column packed with smaller 3.5 μm particles can still generate an adequate 5000 plates.

Simultaneously less solvent is required (Figure 5) especially with the added reduction of column diameter

Figure 5. Rapid Analysis Columns Save Solvent

Analytical Col Fig 5

The advantage of combining a shorter column length with smaller particle size silica without compromising resolution is clearly shown in Figure 6.  Baseline resolution is maintained and the analysis time reduced by over 90%. More recently, developments in sub 3 μm phases have enabled even faster analyses and higher sample throughput.

Figure 6. The separation of a mixture of tricyclic antidepressants on both a traditional length (250 mm)
and Rapid Analysis (50 mm) column

Analytical Col Fig 6

 

COMBINATORIAL CHEMISTRY COLUMNS

  • Matched analytical and preparative columns
  • Easy scale-up for lead compound identification
  • High sample throughput
  • Rapid analysis without efficiency loss
  • Fast re-equilibration

Introduction

Traditionally, potential new drugs have been individually synthesised, purified and their structure confirmed prior to the measurement of structure-activity relationships. A combinatorial chemistry approach, in which mixtures of compounds are synthesised and initially tested, has now often replaced the older methods. Rapid chromatographic analysis of each mixture is required to aid characterisation of active and non-active components.

Columns

Combinatorial chemistry analytical columns are designed to analyse large numbers of complex mixtures in as short a time as possible without major loss of column resolution. They will often need to re-equilibrate as soon as possible following rapid gradient changes in the eluent composition needed to accommodate sample mixtures containing a wide polarity range of molecules. Good column stability is an essential requirement.Columns are typically 50 to 100 mm in length, of 3.2 to 4.6 mm internal diameter and packed with 5μm particle size material.

Availability

We offers a wide range of Combinatorial Chemistry columns, including ACE, Hichrom, Kromasil, and Macherey-Nagel. Typical combinatorial column dimensions are shown in Table 1. Please contact Hichrom for information on matched kits and alternative dimensions and materials.

Scale-up

Once a lead compound has been identified it becomes necessary to isolate it using larger diameter HPLC columns. Preparative and analytical columns need to be matched in terms of length and material content so that a one-step scale-up is easily attained. Figure 7 shows the utility of such columns for scale-up purposes. The progressive steps of analytical screening (Figure A), preparative isolation of Peak 2 using x20 increase in sample size (Figure B) and finally checking the purity of the isolated Peak 2 (Figure C), are shown.

Figure 7. Schematic scale-up and isolation of biologically active molecule.

Analytical Col Fig 7

 

Typical Combinatorial Column Dimensions

Column Dimensions (mm)

50 x 4.6,

50 x 21.2

100 x 4.6

100 x 21.2

 

METHOD DEVELOPMENT KITS

Rapid Method Development Kits

  • Five 50 x 4.6 mm columns
  • Wide range of phases
  • Rapid economic column screening

 The use of Rapid Method Development Kits allows the chromatographer to rapidly evaluate a series of bonded
phases for routine analysis. Once the best partial or total resolution of the sample is obtained, when necessary a longer column packed with the same phase can be used to obtain optimum separation (Figure 8).

Figure 8 Selection of Optimum Bonded Phase and Method Optimisation

Analytical Col Fig 8

Our Partner Hichrom is able to offer >95% of the world’s leading brands of HPLC phases. Hence Hichrom’s Rapid Method Development Kits can contain an extensive range of columns packed with the chromatographer’s selection of phases (silica, chemistry and particle size). Please contact us for guidance on column selection or information on our applications support

Many leading manufacturers also offer competitively priced Method Development Kits including ACE,  Kromasil chiral , NUCLEOSIL chiral and others.

 

Selectivity Kits

  • Three 150 x 4.6 / 250 x 4.6 columns
  • Wide range of phases
  • Optimum resolution
  • Detect minor impurities

Chromatographers may wish to optimise the bonded phase on longer columns. Additionally, the use of all three columns within Hichrom’s Selectivity Kits enables the chromatographer to further check for impurities that may remain undetected when only one column is used. Figure 9 demonstrates the use of a Selectivity Kit containing three Vydac C18-bonded reversed-phase columns of differing selectivity aiding the detection of minor impurities in an antihistamine sample. Selectivity Kits are available in a wide range of phases customised to the user’s needs

Figure 9. Use of a Vydac column Selectivity Kit

Analytical Col Fig 9

 

Validation Kits

  • Three columns
  • One phase, three batches
  • Rapid method validation
  • Confirms assay reproducibility, robustness

Method Validation Kits contain three columns of the same phase and dimensions, but which are packed with different batches of material. The kits are available in all column dimensions and phases. They can be used to assess the batch to batch variability of a chosen bonded phase. By validating a number of batches of material, the chromatographer can obtain greater confidence in the reproducibility and long-term robustness of the method.

Please contact us to discuss the availability of any type of Method Development Kit custom packed to the individual scientist’s requirements

 

GUARD CARTRIDGES

  • Protection for columns from 1.0 - 30mm i.d.
  • No loss in column performance or selectivity
  • Significantly extends column lifetime
  • Packed with the same high performance
  • Silica used in main column
  • Stand-alone or integral design
  • Tested to ensure consistent high level performance
  • Cartridges individually identified
  • Readily disposable and cost effective

Introduction

Guard cartridges are designed to protect valuable analytical and preparative HPLC columns from contamination with impurity particles and irreversibly adsorbed solutes. By placing a guard cartridge between the column and the injector valve, contaminants which would otherwise damage the column are trapped on the disposable cartridge. This procedure significantly extends column lifetime without affecting performance or selectivity (see Figure 10).

Fig 10 Increased column lifetime

Analytical Col Fig 10

Without column protection, column fouling leads to increased back pressure and peak splitting and/or severe peak tailing.

Silica

It is recommended that guard cartridges are packed with the same silica and bonded phase as used in the HPLC column to be protected (this eliminates the possibility of any loss of performance or selectivity). All cartridges packed by our partners conform to this requirement. We are able to supply cartridges packed with any commercially available silica, virtually all analytical and preparative columns can be suitably protected.

 

Guard Cartridge Holder System from Hichrom

For traditional analytical columns (with compression end fittings) a fingertight column coupler (HI-081) is required to connect the holder (HI-161) to the column (see Figure 2). Similar arrangements are available for semi-preparative (7.75 - 21.2mm i.d.) and preparative (30mm i.d.) columns.

Figure 11. Guard cartridge system for traditional analytical columns

Analytical Col Fig 11

Guard Cartridge

Catalogue No

1 Pack Quantity

Holder

Coupler

Catalogue No. for Starter Kit (see  below)

For 1.0mm i.d. columns 

 X-10CE5

 5

HI-161

HI-081

X-10CE5-SK

For 2.1 mm i.d. columns

X-10CM5

 5

HI-161

HI-081

X-10CM5-SK

3.2 - 4.6 mm i.d. columns

X-10C5

 5

HI-161

HI-081

X-10C5-SK

7.75 - 21.2mm i.d. cols

X-10CP3

 3

HI-150

HI-081

 

For 30mm i.d. columns

X-20CP

1

HI-183

HI-083

 

1 When ordering replace ‘X’ with the appropriate silica code - see column listings or contact Hichrom for details.
Example: For a 5 pack of Hichrom 5 C18 guard cartridges for 3.2 - 4.6mm i.d. columns, Catalogue No. = HI-5C18-10C5

Guard Cartridge Replacement

It is generally recommended that for effective column protection, guard cartridges should be replaced when the
column back pressure increases by 10% or column efficiency or resolution decreases by 10%.

 

Starter Kits from Hichrom

Starter kits (see Figure 12) contain five guard cartridges packed with any chosen silica, a free-standing holder (HI-161) and a fingertight column coupler (HI-081).

Analytical Col Fig 12

 

ColumnSaver Pre-Column Filters

  • Economical column protection
  • Protect columns from particulates
  • Compatible with all HPLC columns
  • Ultra-low dead volume

Placed immediately before the column, pre-column filters trap sample particulates and provide a convenient, lower cost solution than the use of guard cartridges. However, for samples that may irreversibly adsorb onto the column, guard cartridges remain the preferred choice for maximum column protection.

The ColumnSaver pre-column filter is simply hand tightened into the column inlet (no tools are required), and is leak-proof to over 5000psi. Both 2μm and 0.5μm versions are available, for protection of 5μm and 3μm columns respectively.

ColumnSaver pre-column filters are universally compatible with all column manufacturers' end fittings, and may be used with either stainless steel or PEEK tubing and nuts (See Figure 13 ). It is recommended that pre-column filters are replaced when a 10-20% increase in back pressure is detected

Analytical Col Fig 13

 

 

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