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What are C18 HPLC columns?

The C18 column is probably the most popular type of column for high-performance liquid chromatography (HPLC). Providing robust performance for the analysis of a wide variety of compounds, C18 has been used in pharmaceuticals, materials, food, environmental, manufacturing, and other industries. C18 columns constitute a mainstay in many analytical chemists’ inventory for testing.

This article gives an overview of C18 columns - the chemical properties of the C18 functional group and the various types of C18 HPLC columns available.

 

C18 functional group

C18 is shorthand for the octadecyl alkyl substituent, which is a straight 18-long fully saturated carbon chain. Functionalized to silica gel, it is called octadecylsilyl and commonly referred to as ODS

C18 exhibits the typical features of the alkyl group - non-polar and ionically inert in most common conditions. Retention from a C18 stationary phase comes from weak van der Waals intermolecular force that attracts hydrophobic compounds. C18 together with other alkyl phases such as C8 and C4 constitute the most fundamental type of reverse phase HPLC. C8 and C4 have fewer carbon constituents in their alkyl chains and thus provide less attraction towards hydrophobic compounds. Compounds are commonly tested if they will separate sufficiently on C18, C8, and C4 phases first before moving on to other phases.

 

Types of C18 columns

C18 has been functionalized on many different kinds of silica gels. A wide variety of HPLC columns have been developed for the analysis of many different analytes. Pore design of these columns vary significantly in terms surface area, pore volume, pore diameter, and other parameters. We discuss different types of C18 columns here.

The most common application of C18 phase is the analysis of small particles which have <5,000 Da molecular weight. These columns are called small or narrow pore C18 columns. These columns feature a pore diameter that is usually around 8-12 nm which leads to exclusion of large particles from the pore volume. Column performance is dependent on how well the bonded phase is functionalized to the silica gel - the amount of C18 groups on the phase or carbon load, the bonding type of the C18 group, and how well residual silanols have been end-capped. 

C18 silica InertSustain

InertSustain C18 from GL Sciences is a narrow pore column featuring all the modern technologies for C18 HPLC columns. At a 10 nm pore size with a 14% carbon load, it is especially suited for the analysis of many small particles. A new surface modification method provides InertSustain C18 with a uniformly bonded phase and high degree of end-capping allowing high inertness, high efficiency, and wide pH compatibility for a wide range of analyses.

Samples analyzed using mobile phases with higher aqueous content are usually difficult to separate using a normal C18 column. The usual cause is the C18 groups preventing access of the water molecules into the pore volume in a phenomenon called phase dewetting. Aqueous C18 columns are designed to tackle this problem by having some phase modifications such as a slightly lower carbon load than normal C18 columns.

C18 silica InertSustain

InertSustain AQ-C18 is a modified version of InertSustain C18 designed for water-rich mobile phases. InertSustain AQ-C18 has a different bonding scheme than InertSustain C18 allowing it to provide retentivity for basic, neutral, and acidic compounds even with a 100% water mobile phase.

For faster chromatographic runs, C18 columns with larger pore sizes and lower carbon load are employed. These fast or rapid C18 columns allow for shorter analyte paths through the column for quicker runs. The sacrifice is that peaks are closer in results, making it crucial to have a well-bonded phase in order to preserve comparable peak shapes with non-rapid C18 columns.

C18 silica InertSustain

For faster chromatographic runs, check out InertSustainSwift C18. InertSustainSwift C18 has a larger pore size (20 nm) than InertSustain C18. Using the same modern silica modification technology as InertSustain C18, InertSustainSwift C18 can process runs faster while preserving relative peak distances and peak shapes.

For large molecules such as proteins and peptides, wide pore C18 columns are used. The pore sizes of these columns are even larger than fast columns, usually around 30 nm. Unlike fast columns, however, the large pore size exists not for a quicker run time but for large molecules to be able to access the pore volume. Good wide pore columns will feature good phase definition in order to deliver excellent analyte resolution.

WP300 C18 silica Inertsil

Inertsil WP300 C18 is a 30 nm wide pore column designed for analysis of biomolecules such as proteins and peptides. Inersil WP300 features the same silica technologies such as excellent phase bonding and high-degree of endcapping to deliver the same column performance as narrow pore columns for large molecules. 

There are other C18 phases that exist with modified phases for specialized applications such C18 modified with additional functional groups for enhanced steric selectivity or have interspersed ionizable groups for mixed-mode interaction. For more, check out GL Sciences’ full selection of C18 columns.

 

Column

Features

USP Code

Particle Size (µm)

Pore Size (nm)

Surface Area (m2/g)

Carbon Loading 

End-Capping 

Inertness

 

Recommended pH range

InertSustain C18

First choice with ultra-high inertness and high durability

L1

2, 3, 5, 10

10

350

14

Yes

★★★★★

1 - 10

InertSustain AQ-C18 

First choice for high polar compounds

L1, L96

1.9, 3, 5

10

350

13

Yes

★★★★★

1 - 10

InertSustainSwift C18 

First analysis with ultra-high inertness and high durability

L1

1.9, 3, 5

20

200

9

Yes

★★★★★

1 - 10

InertSustain AX-C18

Analysis of anionic highly polar compounds

L1, L78

3, 5 

20

200

8

Yes

★★★★

1 - 9

InertSustainBio C18

For peptides and protein with 200Å pore size

L1

1.9, 3

20

200

9

Yes

★★★★★

1-10

InertCore Plus C18

Ideal for analyses requiring a high number of theoretical stages 

L1

2.6

9

200

15

Yes

★★★★

1 - 10

Inertsil ODS-HL

Ultra-high retentivity, High-density bonding of C18 phase

L1

3, 5

10

450

23

Yes

★★★★★

2 - 7.5

Inertsil ODS-4

Ultra-high inertness, High plate count, Medium retentivity

L1

2, 3, 5

10

450

11

Yes

★★★★★

2 - 7.5

Inertsil ODS-4V

Inertsil ODS-4 Validated column

L1

3, 5

10

450

11

Yes

★★★★★

2 - 7.5

Inertsil ODS-3

Strong retentivity, Lower column backpressure, Very inert

L1

2, 3, 4, 5, 10

10

450

15

Yes

★★★★

2 - 7.5

Inertsil ODS-3V

Inertsil ODS-3 Validated column 

L1

3, 5

10

450

15

Yes

★★★★

2 - 7.5

Inertsil ODS-SP

Weak retentivity, for hydrophobic compounds

L1

3, 5

10

450

8.5

Yes

★★★★

2 - 7.5

Inertsil ODS-P 

High steric selectivity

L1

3, 5

10

450

29

None

★★★ 

2 - 7.5

Inertsil ODS-EP 

A polar functional group embedded 

L1

5

10

450

9

None

★★★★

2 - 7.5

Inertsil WP300 C18 

Analysis of high weight molecules 

L1

5

30

150

9

Yes

★★★★

2 - 7.5

Inertsil ODS-2

Ultra-pure silica gel is used

L1

5

15

320

18.5

Yes

★★★★

2 - 7.5

Inertsil ODS

Inertness 1st generation 

L1

5, 10

10

350

14

Yes

★★

2 - 7.5

 

Stereoselectivity vs. Relative Retention of GL Sciences Reverse Phase columns

GL Sciences Reverse Phase chart

C18 HPLC Columns Applications

Here are a few applications for several compounds for C18 columns:

Analysis of Resveratrol 

InertSustain C18 Analysis of Resveratrol

Analysis of β-blocker

InertSustain AQ-C18 Analysis of β-blocker

Analysis of Amino acids

InertSustainSwift C18 Analysis of Amino acids

Fatty acid

Inertsil ODS-3 Fatty acid

Analysis of Tryptic Digest of BSA

Inertsil ODS-4 Analysis of Tryptic Digest of BSA

Analysis of Peptides and Proteins

Inertsil WP300 Analysis of Peptides and Proteins

See the rest of C18 applications in our Applications section.

HPLC Column Technical Guide
Table 1 shows pKa values of frequently used buffers in reversed phase HPLC. Ka stands for an acid dissociation constant and pKa is its negative logarithm. The buffer capacity becomes maximum when the pH of the buffer is its pKa...
How to Select the Right Guard Column
Guard columns are installed between the injector and the analytical column of an HPLC system, to protect the analytical column. Guard columns are widely used and are cost effective to prolong the life of  HPLC columns. ..
Column Cleaning & Storage

Download HPLC Column Cleaning & Storage Guide

Topics

  • General method of washing for Reversed Phase Column
  • General method of washing for Normal Phased Column
  • General method of washing for HILIC Column

Download Column Cleaning & Storage Guide

 

 

 

Comparison Between InertSustain C18 & Commercially Available C18 Columns

Compare Performance

 

Download Comparison Between InertSustain C18 & Commercially Available C18 Columns

 

Recommended Operating Pressure (Maximum Operating Pressure)

Recommended Operating Pressure (Maximum Operating Pressure)

We recommend that you use the HPLC columns that we sell under the following pressures.


Maximum operating pressure of Analytical HPLC Columns

 

 

Analytical Columns Particle Size Maximum Operating Pressure (MPa)

InertSustain Series

InertSustainSwift Series

Inertsil Series

1.9 µm, 2 µm 80

InertSustain Series

InertSustainSwift Series

Inertsil Series

3 µm HP 50

InertSustain Series

InertSustainSwift Series

Inertsil Series

3 µm - 10 µm 20
Unisil 5 µm, 10 µm 20
Titansphere 5 µm 20
InertSphere Sugar-1 5 µm 15
InertCore C18 2.4 µm 100
Capillary EX Columns 3 µm, 5 µm 20
Capillary EX Nano Columns 3 µm, 5 µm 15

 

 

Maximum operating pressure of Guard Columns

 

 

Guard Columns Recommended Max. Operating Pressure (MPa)
Guard Column for UHPLC 80

Cartridge Guard Column E

Cartridge Guard Column Ei

GL Cart, Pre-clean ORG

PREP Guard Cartridge

20

Packed Guard Columns

Packed Mini Guard Columns

Capillary Micro Guard Columns

Preparative Guard Columns

20
Column Use & Care for Inertsil HPLC Columns

HPLC Column Use & Care (Inertsil)

Basic Operating Instructions and Suggested Care of INERTSIL® HPLC Columns

 

Inertsil® HPLC packings are subject to a rigorous array of QC tests in a ISO 9001 compliant facility, with special emphasis on reagent purity, raw material traceability and consistency in raw materials, and fi nal products. A detailed analysis of all of the physical and chemical properties of Inertsil®, combined with tests for chromatographic selectivity and column packing effi ciency, ensure that each lot of Inertsil® is identical to all previous lots and column-to-column reproducibility is of the highest order. To maintain and maximize peak performance of Inertsil® HPLC columns, and ensure long life and stability of columns please read the following instructions before use.

 

UNPACKING

 

Inspect the column upon arrival and contact GL Sciences (info@inertsil.com or 310-265-4424) immediately if you see signs of damage to product or product packaging that may have occured during shipping.

 

SHIPPING SOLVENTS

 

Inertsil® columns are shipped with the solvent used for the fi nal QC test of the column, as detailed on the test chromatogram delivered with the column. When preparing to introduce your desired mobile phase into a new column, be aware of the miscibility of the solvents being introduced to the column and the solvent inside the column. If the new eluent being introduced contains buffer sales, it is recommended that the column is fl ushed with a highly aqueous eluent (such as 90:10 Water:MeCN) before introducing buffer, to avoid precipitation of salts on the column. For extra precaution, introduce new buffered eluents WITHOUT the buffer component for 5-10 column volumes, and then switch to the fully buffered eluent composition. Precipitation of buffer salts on the columns is essentially irreversible and destroys the column! When switching between solvents with vastly different polarities, it may be necessary to fi rst purge the column with a mutually miscible solvent such as Isopropyl Alcohol or Dioxane at a reduced fl ow rate (approximately 50% of normal). Flushing with a minimum of 5 column volumes is recommended ( e.g. 10 mL for a 150 x 4.6mm I.D. column).

Note: With Inertsil® SIL-100A, it is time consuming to fl ush the buffer or water in the pores of the packing after using reversed-phase eluent. In fact, it is nearly impossible to remove water from the surface of a silica column and once water adsorbs on the surface, the selectivity of the column is subsequently altered. (If a subsequent analysis in the normal phase mode is performed, and the fl ush has not been thoroughly completed, the elution order and retention time may vary signifi cantly). Therefore this type of column should be used solely for one type of analysis (basically, normal phase. If you want to use reverse phase on a polar column, we recommend CN, Diol, or Amino.

 

COLUMN CARE

 

Do not drop or bump columns, as this can fracture the uniformity of the bed and result in tailing or split peaks.

Eluent pH - GL Sciences’ InertSustain® HPLC columns are designed for use between pH 1.5 and 10.0, and have known to provide good stability even at pH approaching 11. Other Inertsil® columns, such as ODS-2, ODS-3, and ODS-4 provide optimum lifetime when used with eluents within the pH range of 2.0 to < 8. At pH above 8, silica gels begin to dissolve; at acidic pH below 2.0 certain bonded phases (particularly CN) become hydrolyzed and gradual loss of bonded phase can occur. While many customers use the columns outside both sides of the pH spectrum with excellent results and good column lifetime, the best lifetimes are usually obtained at intermediate pH conditions. One benefi t of Inertsil’s extreme base deactivation is the ability to analyze highly polar compounds at their pKa without peak tailing.

Pressure - To maximize column life operate at pressures up to 20 MPa (~ 3000 psi) for standard HPLC phases (UHPLC columns can be used at higher pressures, as indicated on the test chromatogram). Column Temperature - To maximize column lifetime, limit column temperature to 60C. Sample Dissolution - Samples should be dissolved in the eluent or solvent weaker than the eluent, which helps avoid sample precipitation at the column head and inconsistent retention values. Filter sample with 0.45μ membrane to remove particulate matter before injection. Solvents - Use HPLC or spectroscopy grade solvents that have been fi ltered through a 0.45μ fi lter. Filter all buffer solutions before use. Avoid introduction of particulates onto the column at all costs.

Column Temperature - To maximize column lifetime, limit column temperature to 60C.

Sample Dissolution - Samples should be dissolved in the eluent or solvent weaker than the eluent, which helps avoid sample precipitation at the column head and inconsistent retention values. Filter sample with 0.45μ membrane to remove particulate matter before injection.

Solvents - Use HPLC or spectroscopy grade solvents that have been fi ltered through a 0.45μ fi lter. Filter all buffer solutions before use. Avoid introduction of particulates onto the column at all costs

 

COLUMN PROTECTION

 

Installation of a GL Sciences’ Preclean ORG guard cartridge between the pump and the injector removes organic contamination from aqueous eluent. Guard columns also help protect more valuable analytical columns by binding non-eluting sample components before the analytical column becomes contaminated. 

 

STORAGE OF COLUMNS

 

After using the column with eluent containing buffer or ion-pair reagent, wash the column completely with a saltfree eluent before storing. When storing the column for a long period, store it in 100% organic solvent. MeCN is the most commonly used solvent, though isopropyl alcohol and MeOH are also commonly used. Seal the column with the plugs provided and store it in a location with a stable temperature. Inertsil® columns are manufactured, inspected, packaged and shipped under strict standards of quality control. Should you fi nd any defect in performance, please contact your GL Sciences’ provider, who will ensure your complete satisfaction. We regret, however, that we cannot guarantee the lifetime of columns, nor can we accept any claim when their performance has deteriorated due to no-compliance with the above operating instructions or as a result of normal aging.

 

COMMON PROBLEMS TO AVOID AT INSTALLATION 

 

If there are tailing peaks early in the elution, the cause is probably dead volume, most commonly caused by a pore fi tting connection between the injector and column. Check that the connecting supply line is properly inserted all the way into the column joint. Piping to the injector and to the detector should be as short and have as small an inside diameter as possible. Sub-optimum connections and plumbing have increasing negative effects as the column diameter is decreased.

Baseline drift and noise can be caused by defective pump operation due to air bubbles, reduction of light intensity when using a UV detector. Bubbles often occur during high temperature analysis or the when there are problems with solvent purity.

The most common cause of column back pressure increases or double peaks is blockage of the inlet frit by sample particulates - either from the analyte/matrix or particles created by aging pump seals - or large quantities of lipophilic compounds adsorbing to the head of the column. Pass the eluent through a 0.45μ membrane fi lter before use. The column can also be protected from contamination by using a guard column / cartridge.

 

 

Type Principal Columns Shipping Solvnet
Normal-Phase CN-3, NH2, Diol, Silica n-hexane:Ethanol
Reverse-Phase ODS, C8, Phenyl, C4, MeCN:Water or MeOH:Water
Ion-Exchange SAX, SCX Methanol (100%)
Column Care & Use Manual for InertSustain/Inertsil Series

Thank you for purchasing an InertSustain & Inertsil series column.

 

InertSustain and Inertsil series packing materials are subjected to a rigourous array of QC test in our ISO9001 compliant facility, with special emphasis on reagent purity, consistency and traceability in raw materials, and final products. A detailed analysis of the physical and chemical properties of InertSustain and Inertsil series columns, combined with tests for chromatogaphic sensitivity and column packing material efficiency, ensure that each lot of InertSustain and Inertsil series columns are identical to all previous lots, and column-to-column reproducibilty is of the highest order.

 

To maintain and maximize peak performance of InertSustain and Inertsil series columns, and to ensure long life and stability of your column please read the following instructions before use.

 

Download InertSustain/ Inertsil Series Care & Use Manual

 

 

USP Chart

Download GL Sciences lates USP Chart

 

USP Chart (PDF)

 

 

HPLC Column Specifications

 

 

 

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