Acetone as the Organic Mobile Phase Component – AppNote

“Green” Substitute for Acetonitrile in LCMS Method of 17 Amino Acids Acetone has a high UV absorbance “cutoff” and therefore is generally unsuitable for HPLC with UV Detection. However, this is not a problem when using LCMS or Evaporative Light Scattering Detectors (ELSD) such as the Shimadzu Prominence or the Corona CAD. Acetone can be […]

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Acid Washing Autosampler Vials has Consequences

What are the Advantages and Disadvantages of “Acid Washing” Autosampler Vials? The main reason to “Acid Wash” Autosampler Vials is to reduce basic compound adsorption, due to native silanols onto the glass. Some of the consequences to Acid Washing include time and materials to wash but also you run the risk of Chloride and Sodium […]

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Activity

It is the relative strength of the surface of the stationary phase in adsorption chromatography. In the case of silica gel, the more silanol groups are available on the surface, the more active it is. One can modifty activity by adding water or other polar solvent that will bond to active sites, reducing activity. Type-C […]

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Activity in HPLC. Simple and easy definition.

It is the relative strength of the surface of the stationary phase in adsorption chromatography.  In the case of silica gel, the more silanol groups are available on the surface, the more active it is.  One can modifty activity by adding water or other polar solvent that will bond to active sites, reducing activity.   Cogent TYPE-C HPLC Columns […]

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Add 10 micro molar EDTA to Mobile Phase for Better Peak Shapes.

Metals from glass Bottles and Autosampler Vials or your Instrument can be one cause of poor Peak Shapes for the polyprotic acids and Nucleotides in LCMS. This varies greatly from instrument to instrument so you should identify the source of your poor shapes. One possible solution in this case would be to add 10 micro-molar […]

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Adjusted retention time

where tR is the retention time and tM is the hold-up time. The hold-up time is the time of an analyte (small molecule) which completely penetrates the pores and which is not retained at all by the stationary phase.

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Adjusted retention time in HPLC. A Primer.

Adjusted retention time (tR‘) is the retention time adjusted for the hold-up time: tR‘ = tR – tM where tR is the retention time and tM  is the hold-up time. The hold-up time is the time of an analyte (small molecule) which completely penetrates the pores and which is not retained at all by the stationary phase.

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Amide vs. Amine / Amino HPLC Columns. – Tips

The amide functional group should not be confused with the amine or amino moiety. The presence of the carbonyl group imparts the amide functionality with significant physical and chemical differences compared to an amine. Most notably, amines are ionizable (positively charged when ionized) while amides are not. Amines are more reactive than amides, such as with aldehydes, which can present some […]

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Analysis of Cysteine by LC-MS

Cysteine is difficult to analyze by LC-MS because it is hard to ionize and also very sensitive to any metals in the instrument such as tubing, injectors, seals, joints etc. We do not currently have an application note for cysteine. We do have a method for cystine, which is a disulfide bonded cysteine dimer.

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ANP v. HILIC in HPLC and LCMS Ballistic Gradients.

In comparing ANP vs. HILIC, the former has an advantage in using ballistic gradients because HILIC is generally not suitable for these types of gradients. The reason is the semi-permanent water layer found on the surface of all ordinary HPLC grade silica takes too long to re-generate between runs, compared to the timescale of the […]

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ANP v. HILIC when using Ballistic Gradient HPLC or LCMS.

In comparing ANP vs. HILIC, the former has an advantage in using ballistic gradients because HILIC is generally not suitable for these types of gradients. The reason is the semi-permanent water layer found on the surface of all ordinary HPLC grade silica takes too long to re-generate between runs, compared to the timescale of the sharp gradient. In […]

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Aqueous Normal Phase (ANP) HPLC retention mechanism elucidated.

The mechanism of aqueous normal phase (ANP) retention is an active area of investigation. However, much progress has been made in recent years at elucidating how polar compounds are retained by this mechanism and how it differs from the other techniques used for the analysis of hydrophilic molecules.  A recent study has demonstrated that the water layer […]

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Are RSA-Pro X Vials Recommended for PFAS Analysis – FAQ

RSA-Pro X™ Vials & Inserts are not recommended for PFAS or Per- and Polyfluoroalkyl Substances analysis with LCMS / MS or GCMS. During the conversion from RSA™ to RSA-Pro X™, the vials and inserts are exposed to PFAS. While some of these compounds of interest may not be detectable in HPLC, they may be detected, […]

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Asymmetry factor

Measure the left half of the peak width at 10% peak height (A) and then measure the right half of the peak width at 10% peak height (B) (see figure below). The ratio of B over A is the asymmetry factor. A value of 1.0 is perfect symmetry and 0.9 – 1.2 is acceptable. Value […]

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Autoclaving Glass Inserts with plastic feet is not recommended.

Autoclaving glass or plastic inserts with attached plastic springs is not recommended. While the glass can withstand temperatures up to 1,000°F, the polyetheylene spring will become deformed, may crack and will lose its functionality. You can sterilize these inserts through ethylene oxide gas sterilization.

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Autoclaving Inserts with Plastic Springs is Not Recommended.

Autoclaving glass or plastic Inserts with Attached Plastic Springs is not recommended. While the glass can withstand temperatures up to 1,000°F, the Polyethylene Spring will become deformed, may crack and will lose its functionality. You can Sterilize these Inserts through Ethylene Oxide Gas Sterilization. Inserts Product Page  

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Blocked column from proteins in the sample matrix can be recovered

It is of crucial importance to always filter your samples prior to introduction to an HPLC instrument. Most samples will contain particulates and other matrix components that can cause blockages in the instrument and the column. In addition, some macromolecules such as proteins may need to be precipitated and removed during sample preparation so that they do not […]

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Buffer concentration can affect ANP retention of acids in HPLC Analyses.

If you are using ammonium acetate or formate in an ANP method, the concentration used may play a significant role in the retention of organic acid analytes. Although buffer concentration may affect a chromatographic environment in numerous ways, one particularly significant effect (in the case of acids) is thought to involve adsorbed acetate or formate on the […]

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Calculating Theoretical Plates N half height

The theoretical plate number N0.5 can be calculated from a chromatogram using the equation below: N0.5 = 5.54 (tR/w0.5)2 Where tR is retention time and w0.5 is the peak width at half its height. Remember to use the same units (minutes or seconds) for both values (tR and w0.5).

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Can ANP chromatography be used to retain steroids with HPLC or LCMS?

It always depends on the individual compounds but generally speaking ANP is best suited to compounds of a more polar nature.  In contrast, steroids as a class of compounds are mostly too hydrophobic for ANP and hence are generally poorly retained under these conditions. Reversed phase may be a more suitable chromatographic approach for retention and […]

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Can I make the PTFE Syringe Filter hydrophilic?

Yes, if you are wiling to introduce Methanol into your method. For a 25mm device, if you use 15-20mls of methanol as a blank filtrate, the membrane will give you less back pressure after that for aqueous samples. This is due to “wetting” the membrane with Methanol allowing more aqueous solvents to pass through it […]

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Can I use acetone as a mobile phase solvent in ANP?

If you are using LCMS, the simple answer is yes but pH is also something to consider. Acetone has a high UV cutoff and therefore can be a problem for UV detection. However, this is not a problem when using LCMS or ELS detectors such as the Corona CAD. You can use acetone to replace acetonitrile with the Cogent TYPE-C™ […]

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Can I Use Acetone as a Mobile Phase Solvent in ANP? – FAQ

If you are using LCMS, the simple answer is yes but pH is also something to consider. Acetone has a high UV cutoff and therefore can be a problem for UV detection. However, this is not a problem when using LCMS or ELS detectors such as the Corona CAD. You can use Acetone to replace Acetonitrile […]

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Can I use primary amines with acetone as the mobile phase component in LCMS?

Yes, you can. However, some labs react acetone with primary amines (+heat) to form acetone adducts (imines) in GC and GCMS. This reaction while using acetone as a mobile phase component in LCMS along with primary amines as part of the sample is something to consider. This reaction, according the University of Liverpool requires a primary amine, a ketone or aldehyde (in this […]

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Can I Use Primary Amines With Acetone as the Mobile Phase in LCMS? – FAQ

Yes, you can. Watch out for imines. However, some labs react acetone with primary amines (+heat) to form acetone adducts (imines) in GC and GCMS. This reaction while using acetone as a mobile phase component in LCMS along with primary amines as part of the sample is something to consider. This reaction, according the University of Liverpool requires a primary amine, a ketone […]

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Can the pre-column filters replace my HPLC guard columns?

No, a guard column is both a particulate filter (non dissolved chemicals) and a chemical filter (removes dissolved chemicals). The pre-column filter is a particulate filter and adds extra protection of your expensive analytical and guard columns Click HERE for Column Protection Ordering Information including Column Pre-Filters

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Changing analyte recovery when using syringe filters

Question: I am doing a sunscreen analysis which requires that the sample solution is filtered. My diluent is 95/5 methanol/DI water and I am using MTC 0.45um nylon syringe filters. One day I observe consistent peak areas but the next day, when using the same solution, the peak areas are higher. What has happened? Answer: The syringe […]

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Choosing The Correct HPLC Column Coupler.

We offer a variety of HPLC Column Couplers. To find the Correct Size, simply match your Tubing Internal Dimension (ID) to the Coupler’s ID. Click HERE for HPLC Unions including Column Coupler Ordering Information The different Coupler Colors correspond with those of the HPLC Tubing Products.

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Cogent Amino HPLC Column Designation for RP or NP.

Even though the Cogent NP™ Amino HPLC Column is an excellent choice for non-Hexane, Normal Phase Chromatography, it is also very useful for highly acidic compounds by using Methanol or Acetonitrile in Reversed Phase.  The Cogent NP Amino Column is also a good choice for separation of Sugars when using RI – Refractive Index Detectors. […]

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Compatibility of Cogent TYPE-C Silica columns with UHPLC instruments

Cogent™ HPLC columns are compatible with ultra high performance liquid chromatography (UHPLC) instrumentation. However, it is important to note the low pressure produced in ANP and the pressure limits of these columns. The Cogent 2.o™ is considered “Near UHPLC” with pressure limits near 9,000 psi. If you are using Aqueous Normal Phase (ANP) HPLC it […]

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Compatibility of Cogent TYPE-C Silica HPLC Columns with UHPLC instruments.

Cogent™ HPLC columns are compatible with ultra high performance liquid chromatography (UHPLC) instrumentation. However, it is important to note the low pressure produced in ANP and the pressure limits of these columns. The Cogent 2.o™ is considered “Near UHPLC” with pressure limits near 9,000 psi. If you are using Aqueous Normal Phase (ANP) HPLC it is unlikely […]

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