HPLC Tips and Suggestions – MicroSolv Technology Corporation

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Acetone as the Organic Mobile Phase Component – AppNote

“Green” substitute for separation of amino acids Caution: Acetone and Amines can react When working with Acetone as an HPLC solvent it is important to note that primary amines could react with Acetone to form imines. However, to achieve this reaction the mobile phase would most likely need to be free of water and or […]

Acetone as the Organic Mobile Phase Component in LCMS.

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 […]

Acid Washing RSA Autosampler Vials has Consequences.

What are the Advantages and Disadvantages of Acid Washing RSA Vials? There is no reason to Acid Wash RSA Vials. The main reason to Wash ordinary Vials is to reduce adsorption due to native silanols on the glass. Since RSA has virtually no silanols, this is not necessary. Some of the consequences to Acid Washing […]

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 […]

Activity in HPLC. Simple and easy definition.

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 […]

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.

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.

Amide vs. Amine/Amino HPLC columns.

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 […]

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.

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 […]

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

Aqueous Normal phase (ANP) defined & compared to Reversed Phase & Normal Phase in HPLC

Aqueous Normal Phase (ANP) is probably the newest HPLC technique or at least the latest to be investigated well. Often we are asked what is ANP. Below is a link with more explanation than this article can provide. What is Aqueous Normal Phase Normal-Phase Chromatography is defined as a chromatographic phase that will increase the retention of a target […]

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 […]

Assuring retention of polar compounds in an ANP gradient in positive mode LCMS.

When using a Cogent Diamond Hydride™ column prepare your solvents for positive ionization in LC-MS: Solvent A: 50% methanol/50% DI water + 0.1% formic acid and Solvent B: 90% acetonitrile/10% DI water/0.1% formic acid. ANP gradients need to start between 100% – 90% of Solvent B (to retain polar compound) and then reduce the percent of Solvent B […]

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 […]

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.

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

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 […]

Blocked TYPE-C HPLC Column from proteins that were in the sample matrix can be recovered: Tip.

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 […]

Calculating relative response factor (RRF) on different HPLC instruments/detectors

The Relative Response Factor (RRF) value you calculate should be the same, irrespective of the instrument or detector used, as long as linearity has been demonstrated on all the involved instruments/detectors.

Calculating relative response factor (RRF) on different instruments/detectors

The RRF value you calculate should be the same, irrespective of the instrument or detector used, as long as linearity has been demonstrated on all the involved instruments/detectors.

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).

Calculating Theoretical Plates N half height. Simple and easy definition.

Can ANP chromatography be used to retain steroids by 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 […]

Can Cogent TYPE-C HPLC columns be used in supercritical fluid chromatography (SFC)?

As far as compatibility in SFC is concerned, the answer is yes. These columns are fully compatible with the typical conditions used in an SFC application and the packing material and hardware will not be damaged by supercritical fluids used in SFC. We are sorry but at this time, we cannot say whether they will […]

Can Cogent TYPE-C HPLC Columns be used in supercritical fluid chromatography (SFC)?

Can concentration changes effect retention time in HPLC? Is this a difference between HILIC and ANP?

No, it can happen at any mode, so it is not a contrast between HILIC and ANP. Of course retention can change with concentration from two sources. All separation processes follow a “Langmuir model“; when a column is overloaded the retention time will decrease. Also, if the sample comes from a complex matrix as you […]

Can concentration changes effect retention time in HPLC? Is this a difference if I use HILIC or ANP?

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 […]

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™ […]

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

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 […]

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

Can I use the knitted reactor coils (KRC) of the post column photochemical reactor with my HPLC fluorescence detector?

The only restriction on whether or not you can use the knitted reactor coils of the post column photochemical reactor is the inlet tubing of your detector. The absolute minimum ID can be 0.008″. It is preferred that the tubing ID be 0.01″. Anything smaller than 0.008″ is not compatible. Click HERE for HPLC Post […]

Can I use trifluoroacetic acid (TFA) to improve the chromatographic performance in LC-MS analysis?

Trifluoroacetic acid (TFA) is very often used as an additive in HPLC, because of its excellent solvating and ion pairing characteristics. It is a highly volatile acid as well, which could make it an additive for LC-MS analysis. However in many studies it was found that TFA causes spray instability and ion suppression in APCI […]

Can I use trimethylamine with Cogent™ HPLC Columns such as the Diamond Hydride™?

Most of the Cogent HPLC columns have an upper pH use limit of 8. However short exposure and possible use depends on how concentrated the solution is, what the pH of the solution is, and how long the column is exposed to the base. Trimethylamine (pKb 4.2/ pKa conjugate acid 9.81) is generally not compatible and not supported with […]

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

Caution when using the term of robustness for HILIC columns.

It is reported that HILIC columns can be less robust than traditional reversed-phase (RP) columns. Partly this is due to the fact that the surface can be easily contaminated and is hard to clean. In other cases, the bonded group is not very robust and is susceptible to being cleaved from the surface in mobile phases […]

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 […]

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.

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. […]

Cogent TYPE-C HPLC Columns offer faster results compared to Ordinary Silica Based Columns.

It has been well documented in the literature over the years that using Cogent TYPE-C™ HPLC columns offers chromatographers faster results during method development and even with routine use. How? 1. Faster Equilibration. Due to the unique nature of the column surface, these columns will fully equilibrate, after gradient runs with only 3-5 columns compared to […]

Column cleaning and care in the reduced particle sized UHPLC Columns

Despite meticulous care, sometimes non-dissolved chemicals and sample particulates may adsorb onto your UHPLC column, causing high pressure issues. Before cleaning your column, it is suggested to remove the column from the detector and allow your wash solvents to flow through the UHPLC tubing. Following this, it may be pragmatic to reverse the column direction […]

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 […]

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

Contaminants leaching into an analysis, suggestions for troubleshooting.

Have you ever run across a situation where Teflon-like compounds where contaminating an analysis? PFA (PerFlourAlkoxy film, a type of Teflon) is leaching out of something from pipette to HPLC and I have no idea who uses this compound and who doesn’t! This is not easy to answer. In an HPLC system more than one […]