Chromatographic Method Development - Science topic

In my opinion try to use Methanol instead of Acetonitrile.....

I have no practical experience, however, I hope the provided link will be very much helpful for you. Please have a look on the following link:

I used HPX-87H column to seperate methyl formate and formic acid, but failed.Does anyone have any advice?

Hi, would you help me to prepare

1,1′-(2,6-Pyridinediyl)bis(3-methylimidazolium) Dibromide, I couldnt find any good paper. Thank you

Hello,

There are a few different ways to approach your problem.

With 8 Cys present, hepcidin-25 should be electrochemically active. It may be that your sample is oxidized and no longer detectable. The easiest way to address this is to treat the samples (standards and biological0, first with a reducing reagent like dithiothreitol (DTT). Selvi shows how to do this with glutathione.

(Selvi, M. Bharath. "Biochemical Mechanisms of Homocysteine and its Role in RETINAL VASCULAR DISEASES." PhD diss., BITS Pilani, 2015.

See also: Sakhi, A.K., Russnes, K.M., Smeland, S., Blomhoff, R. and Gundersen, T.E., 2006. Simultaneous quantification of reduced and oxidized glutathione in plasma using a two-dimensional chromatographic system with parallel porous graphitized carbon columns coupled with fluorescence and coulometric electrochemical detection. Journal of Chromatography A, 1104(1-2), pp.179-189.

Zhang, M. and Pfeiffer, C.M., 2004. Comparing the ESA HPLC total homocysteine assay with electrochemical detection to the CDC in-house HPLC assay with fluorescence detection. Clinica chimica acta, 340(1-2), pp.195-200.}

Standards of glutathione or oxidized glutathione (10 µg/mL) are made in buffer. 50 µL of standard is mixed with 5 µL of 50 mM DTT at room temperature for 10 minutes. 10 µL of the mixture is injected into an HPLC (Buffer: 0.1% trifluoroacetic acid, 2% acetonitrile, 98% MilliQ water; Flow rate: 1 mL/min; Pressure: 400 bar; Temperature: 27.7 °C; ECD with glassy carbon electrode; Range: 1 µA; E cell: 0.90; Voltage: 1.16V; Column: ODS Hypersil C-18, 5 µm particle size, 150 X 4.6 mm).

Note that the moles of disulfide bonds per gram of oxidized GSSG is different than the moles of disulfide bonds (possible) in hepcidin-25 by ~1.75:1. So you might want to make the DTT solution a little stronger (~90 mM). You could also add more 50 mM DTT solution but that will dilute your sample.

Since you are open to other methods, I have attached a chapter on thiol-reactive fluorescent dyes that you can use to label Hepcidin-25. Again, remember the stoichiometry and be sure to first reduce any disulfide bonds in Hepcidin-25 (intra- or inter=molecular) and also use enough thiol-reactive dye to label all 8 Cys.

Best regards,

The method was invalid as shown. The consequences of using instrumentation and techniques without proper training often lead to erroneous data being collected and incorrect conclusions or quantitative results being reported. In your case, there are two reasons for this:

(1) The DAD reference wavelength feature on your Agilent 1260 DAD was turned 'ON'. This must be turned 'OFF', not 'ON'. To learn why, please read the attached article below.

(2) Your signal acquisition wavelength is very close to your Reference Wavelength. This guarantees that your data will be invalid as even the peak of interest will fall under the Reference range you specified (360,10), invalidating the method and data obtained.

Those who use HPLC for the first time and/or who have not had any formal training in liquid chromatography (and especially in the use of a diode array detector (aka: PDA)) often makes these mistakes. Clients often use systems setup just like this one and are unaware of the problems caused until a negative peak shows up. The appearance of the new, negative peak is this hint that all of your previous data may be invalid and the only reason you discovered it now is because this time the system had to subtract-out so much data from your main peak that it ended up being negative!

Added an answer

Goodluck

I have face same problem, but in my situation i have a purified compound using column, that is soluble in methanol. For estimation of its quality using tlc plate which solvent system is best.

Yes you may use salt for several reasons.

See why and how to prepare in the attached HILIC tips

Hi Mel, maybe this will be helpful to you:

Greetings Markus

I'm assuming you are using the same elution for the flash as is being used for the TLC. The Rf shown doesn't  suggest the chromatography will be good. The non-polar compound has an Rf of (essentially) 1, and the product has an Rf of 0.65. Retention on a column is, in column volumes (CV)= 1/Rf. The column volume approximates the void volume on an HPLC. So the impurity comes out at 1 CV and the impurity comes out at ~1.5 CV. The column volume is merely the space taken by the mobile phase in the column and nothing can elute faster than 1 CV.

You would prefer the compound to come out at 2-5 CV, or an Rf of 0.2 through 0.5 and that will be difficult to achieve as you are running 4% EtOAc in pet ether now . I suggest trying a less polar mobile phase such pet ether/THF or pet ether/ether. One can also try pet ether/toluene. Pet ether/dichloromethane (DCM) could work but DCM is nearly as polar as EtOAc. Likewise, pet ether/acetone is similar in polarity to pet ether/EtOAc and so probably won't work well.

For TLC, make sure the chamber has time to saturate with vapor, and make sure vapor can't escape. A watch glass covering a beaker doesn't give the best results because the beaker spout allows vapor to escape. Use a proper TLC chamber or jar that can be completely closed.

Use solid load for best results (dry the sample onto silica)- see this app note for a comparison of loading techniques: http://www.teledyneisco.com/en-us/liquidChromatography/Chromatography%20Documents/Application%20Notes/Overview%20of%20Silica%20Column%20Sample%20Loading%20Techniques%20App%20Note.pdf

If you are running a flash column on an instrument with such a solvent system, make sure the equilibration is aet to 5-7 column volumes. The EtOAc adsorbs on the silica, and compounds often don't elute until this adsorption is complete. If packing columns, pack in at least 1-2% ethyl acetate of you are running a step gradient for the same reason. People often dry-pack columns and this may be one case where that can adversely affect the chromatography.

Dear Carlos,

how did you confirm the identity of the two drugs? Dou you analyze standard solutions of both drugs separately? I mean, are you sure the identity of the two peaks obtained?

Regrds.

Have you seen Resteks GC literature? A method outline is here:-

If you are going to use the column in the near future just flow 10X column volumes of Mobile A and B, without the buffer.  It really depends on your sample matrix and the crap that is injected.  Long term storage is given by the manufacturer.

Dear Naziruddin

As said by Mr. Neshat, you can use the same calibration run for each sample. You can add another internal standard inside you sample if you want to see the stability of the sample too. But for the same compound, you can definitely use the same calibration run, with the same method.

No HPLC method information was provided. That means there are hundreds of reasons why it might happen. It is usually something very simple. As Jack suggested, wash the column (properly with a stronger solution, NOT the mobile phase which will do nothing at all) OR replace the column with a NEW one and see what happens.

Additionally, please review this article for ideas?

I have never stored a ion exchange column in anything other than the eluent I was running it with.

Do you work in SIM or SCAN mode? In case you are working in SCAN mode, what is m/z rage you are using? Can you identify (based on MS spectra) the compound giving you the problem?

First off, do you mean a b-DEX column? That is a chiral phase, and has to be handled with great care. 

You claim to be running an analysis in acetonitrile but you show that your maximum temperature is well below the boiling point of the acetonitrile. As is obvious to anyone with any experience in chromatography this would simply not be possible. If this is the actual case then you need to completely change your analysis. If you truly are using ACN as your solvent (a very poor choice for GC, by the way) then you are building up solvent all through your system and you are probably flooding your detector. This is consistent with the chromatographic traces that you have shown.

Secondly, 220C for a FID is WAY too low. You will get all kinds of crazy things happen. Raise your detector temperature up to no less than your injection temperature. 280 C as an injection temperature for a maximum oven temperature of 70 C is WAY, WAY too high!!! Lower your injection temperature.

You are in a university environment. You clearly are in need of some very basic GC training. Surely your university has someone who actually knows something about GC? You should consult them.

This is not unusual!  Even a 'standard' that is 95.0% 'pure' has 5.0% other stuff (crap, process intermediate)'.  Thus, you are likely to have many peaks.  In addition a standard that is a DL racemic mixture would have 50% D and 50% L stereoisomer.

very strange doesn't begin to explain this protein and the protein has no native environment; it is an engineered hodgepodge of several antigens expressed as a single protein.

Great answers given above. I would add one thing:  Can you watch the column pressure in real time?  Is it a gradual change in pressure or erratic? If it's erratic, it could be a seal or air bubble.  Make sure the lines are primed well when you switch mobile phases. 

First, terminology. It is all called "HPLC".  Terms like "UFLC" and "UPLC" are trademarks ("UFLC" https://trademarks.justia.com/772/99/prominence-77299484.html & http://hplctips.blogspot.com/2015/08/terminiology-which-is-it-uplc-uhplc-or.html).

Please refer to this free, linked article to solve your problem, "Diagnosing & Troubleshooting HPLC Pressure Fluctuation Problems (Unstable Baseline) ". I am sure you will find the solution after following the suggestions provided in the article.

More information is needed. Please provide the detailed chromatography conditions (flow rate, exact mobile phase composition and times, injection volume, plus ALL HPLC column dimensions and type).

Hi Dean,

there was a similar question about a year ago on RG, I attached the link FYI. In general, every company will tell you that they are the best. We use Agilent 1200 system. However, we never had a bad experience regarding the customer support. From the instrument reliability, we do not have a comparison, but this also depends on the maintenance work you do. Any nice instrument will break down if the maintenance is bad or not existent at all...

You could also check supercritical LC. Rather expensive, but the solvent is CO2 which simply evaporates and you do not need to freeze-dry your samples. Also available in preparative scale.

Good luck, Marcus

In agricultural chemistry, we often face much more difficult matrices than plasma samples.  The matrix we encounter frequently prevents complete dissolution of the residues of interest.  To circumvent this issue, we typically dissolve samples with vortex mixing in a small amount of organic solvent (50-100 µL of MeOH or ACN) followed by aqueous mobile phase to a final volume of 1 to 2 mL.  This usually allows for analysis of compounds with extremely different polarities in the presence of extracted plant and animal tissues.  By keeping the percent organic at or below 10%, there is usually minimal peak shifting (any peak shifting can be readily accounted for by dissolving standards in the same solvent).  We frequently use this technique to dissolve samples even when the analyses require large injection volumes (900 µL).       

By NMR it seems that there is unbound C18 material (or mineral oil) on the plate. I have seen this especially on older plates; it is either unbound C18 or oils absorbed onto the plate (vacuum pump oil from processing the plate during manufacturing?). Mark the top of the plate and elute the preparative reversed phase plate with chloroform or methylene chloride all the way to the top end of the plate. This will concentrate hydrophobic things like C18 residues at the top of the plate. Dry the plate at room temperature, do not heat or put under vacuum!  Then run your sample in the same direction on the plate. This should give you a cleaner background for the NMR spectrum of your eluted band.

Hi, I think there are many publications for analysis of oxytetracycline by HPLC, see Tabel 3 of our chapter; unfortunately I do not have soft copy of the published version, I just have the proofs, but I t think you can still read it.

Best regards

No need to concern yourself with "improving" sensitivity at this stage if so far you are having reliability problems. First you must obtain reliable and reproducible results. Since your initial observations saw large variations between two injections from the same vial, you will need to start with the basics first. For example: Are you injecting a volume that is within the range of the injector? What is the injection volume? Is it too small or too large? Is liquid evaporating from the vial? Is the cap on too tight (causing vacuum to form, resulting in less sample being drawn the next time)? Have you tested the system with stds (forget the MS, just use a UV/VIS detector) to verify that you can obtain excellent results and reproducibility before you start looking at the dozens of settings on the MS side. Troubleshooting is a logical, step-wise process. First verify the hardware, column, method and techniques used (i.e. how you prepare samples), then check one thing at a time to find out how to obtain a good quality method. Once you have that, then look at the MS portion.

Regarding sensitivity: With a high quality LC-MS method, next optimize the method used and all of the MS settings to obtain low noise and high signal. Heat, gas flow, capEx voltages and so on. Ask yourself if your LC method is hurting or improving the sensitivity? Don't use chemicals that suppress ionization (e.g. TEA, TFA). Don't use non-volatile additives. Formic acid is a great acid to use in Pos mode. It replaces acetic acid and improves ionization. How about using an adduct to improve the response?

Most importantly of all, get help from your own university's LC/MS expert to assist you in developing a method and running the samples. Learning LC/MS takes several years and I suspect you do not have that extra time available, so enlist some help to get the job done. This is to be expected as learning to run LC-MS methods is not like learning to use an analytical balance. I am sure there are people there to help you.

Additionally, I have attached a web link to a page with some free articles that may assist you. I teach HPLC and LC/MS method development and optimization professionally for a living. The link has many articles and topics that I present in my classes. Some may apply to your work..

VFAs are hard by HPLC. Not a good choice. GC-FID works quite well. Use a wax column or other very polar phase and you won't have to derivatize.

We've run them by both direct injection and headspace. I prefer the headspace method. You would put a sample into a headspace vial (say 5 mL for a 20 mL HS vial) and add 1 mL of an acidified saturated salt solution. You need to get the pH of the sample down below 3 to get them all into the headspace efficiently. Heat the vial to 80C, allow it to equilibrate (say 30 minutes) and then extract a volume of the HS and inject it. We typically inject 1 mL of HS using a 20:1 spilt ratio with a Restek RTX-Wax 20 meter column. You'll get better results with the capillary (you can adapt your packed inlet) but it will certainly work with your packed column and you can run under very low split conditions. 

It can be, but is not the "norm". For HPLC, area is the preferred technique as it does a better job with peak shapes that are not perfectly symmetrical. Peak height can be very accurate if baseline noise is low and you have a gaussian shaped peak (close to perfect, which is often the case in well developed GC methods).

Your best bet is to look through a crystal structure database and generate calculated powder patterns. The peaks in a PXRD spectrum will vary depending on how the Keggin units pack.

Other authors that tried to measure polyhydroxyalkanoate composition by HPLC technique used an ion exchange column. Maybe your problem is you are not using the correct column.

In the following lines you can find the method used by Karr et al. (1983) to determine PHA using a HPLC:

Digestion of PHB and Aminex-HPLC analysis of crotonic acid: Samples ranging from 0.01 to 500 mg of PHB-containing material were digested in 1 ml of concentrated sulfuric acid at 90°C for 30 min. The tubes were cooled on ice, after which, a 4-ml volume of 0.014 N H2SO4 was added with rapid mixing. Before analysis by HPLC, samples were diluted an additional 5- to 100-fold with 0.014 N H2SO4 containing 0.8 mg of adipic acid per ml as an internal standard and filtered through a 0.45-,um HAWP membrane filter (Millipore Corp., Bedford, Mass.) to remove particulate material. The injection volumes ranged from 10 to 50 p.1 or sample concentrations from 0.2 to 560 ,ug/ml. Samples were eluted with 0.014 N H2SO4 at a flow rate of 0.7 ml/min from an Aminex HPX-87H ionexclusion organic acid analysis column (300 by 7.8 mm) (Bio-Rad Laboratories, Richmond, Calif.) preceded by an ion-exclusion guard column of Aminex HPX-85X. HPLC was performed with either a Waters Associates 6000 A solvent delivery system with U6K injector or a series 3 chromatograph (The PerkinElmer Corp., Norwalk, Conn.) with a variable loop injector (Rheodyne, Inc., Berkeley, Calif.). Absorbance of crotonic acid was measured at 214 nm (Waters 441 absorbance detector) or 210 nm (PerkinElmer LC-55 B detector). The amount of crotonic acid produced from PHB was calculated from the regression equation derived from known crotonic acid standards.

I hope this answer help you to solve your problem

Best regards.

Use a chromatography sprayer, see links. 

Thanks Joseph, our green solvent for super-critical extraction is water. Still we need further purification for fine product delivery.

Thank you. Sorry, I should have checked that. I blindly assumed that organic solvent should have melting point below -100C or something which is not true. Thanks again.

ANS:- Dear sir,

This method may help you,

The development and validation was carried out by using HPLC (waters) separation 2996 series, variable wavelength photodiode array (PDA) detector module equipped with auto-sampler with injection volume 20 μl, 2693 pump, column used was Inertsil ODS 3V (150 × 4.6 mm, i.d., 5 μm) column,

mobile phase consisting of 0.02 M Phosphate buffer adjusted to pH 2.5 using dilute orthophosphoric acid (solvent A) and acetonitrile (solvent B) was set with gradient programming for 18 min was optimized at a fl ow rate of 1 ml/min, 230 nm wavelength, injection volume of 20 μL and ambient temperature was maintained during the entire process to obtain symmetric peaks of MET and GLI.

2.72 g of potassium dihydrogen orthophosphate (0.02M) dissolved in 1000 ml water, adjusted to pH 2.5 using dilute orthophosphoric acid and fi ltered through 0.45 μm membrane fi lter. Mobile phase B: Acetonitrile Diluent: Water: acetonitrile (50:50).

Table 1 Time programming of gradient elution Time Mobile phase-A (0.02 M potassium dihydrogen orthophosphate buffer) Mobile phase-B (acetonitrile) 0 -80: 20 3- 50 :50 5 -30 :70 12- 30: 70 15- 80: 20 18- 80 :20

I believe you can use multiquant for MS data acquired by flow injection analysis. The problem is that your are using a low resolution triple-quad, many species within the 0.2 amu mass resolution of the instrument can co-elute in the injection slug and you will not be able to tell them apart using the 5500. Using chromatography up front gives you the potential to time resolve many of these compounds that are close in mass and detect  (identify) them separately.

Dear madam,

This chomatograpic conditions will help you,

AMINEX HPX-87C(250x4mm) column is used  for carbohydrates, monosaccharide’s,beverages analysis & amino column with  ELSD detector or RI detector .

eluent:-30% ACN

column Temp:70 ⁰C

Bill is right, more information are needed.

Additionally you may check if your solvents are contaminated (use a fresh bottle), clean everything well prior change anything. Also clean the spray chamber/check for dirt. You may also clean the MS capillary which connects the spray chamber with the internal part of the MS instrument. It yould be a contaminated needle, tubing, whatever had contact to something which hardly dissolves in your solvent system and flushed out only slowly. But this is just guessing!

Can you be more specific about what kind of column you are using? Is your protein soluble, does it have an extreme PI, form quaternary stucture, or some other factor that could lead to unusually high or low binding affinity for the column?

Firstly, it is always a good idea to check and make sure your samples actually contain your recombinant protein in concentrations that you expect. I use IPTG-inducable expression and run an SDS-PAGE gel containing uninduced cells, induced cells, lysate, and a sample from each wash as well as the elutions. You can Western blot this gel to ensure that you are in fact obtaining protein, as poor expression may be the culprit for lower than expected purification yields.

You should also ensure your protein is soluble in the buffer you are using. I use bioinformatics calculations on my raw sequence files to estimate the pI and stability of my protein and design my buffer around that. Tris, sodium phosphate, HEPES, and sodium acetate are some common buffers that operate in physiologically relevant pH ranges. To stabilize proteins, sodium chloride is also often employed in the range of 50-200 mM. Generally speaking, more salt is better for stability but this is a case-by-case basis as every protein is unique. I use a Dynamic Light Scattering (DLS) instrument when I am buffer-optimizing to check for polydispersity and aggregation that can be indicative of poor buffer compatibility. Filtering solutions and/or spinning them and discarding any insolubles helps prevent additional protein loss to aggregation.

If these don't help, IMAC columns are eluted using concentrated imidazole washes (250-500mM). If washes are insufficient, I commonly incubate the column resin in my elution buffer and suspend the beads to maximize competition for the column resin. Alternatively, If you are using the column on LC instrument, try reducing the flow rate. As mentioned above, ion exchange columns are eluted with high concentrations of NaCl and the same principles would help improve your elution efficacy if you are getting low yields from the column.

Lastly, if your protein is for some reason still present in high concentrations on the column, it may require stripping of the column resin to remove. You should refer to the product manual for the specific protocol that is appropriate for your column. This may occur if you attempt to purify multiple His-tagged proteins on a single IMAC column, and so this is not advisable. Note that protein removed from a column in this manner is not usable, but regeneration of the column resin may help with purification yields. Most manufacturers recommend that the column be cleaned every 2 to 10 uses, depending on the nature of its use.

I hope this helps, and good luck!

Here are two good recommendations for novice to intermediate level users.

"Introduction to Modern Liquid Chromatography"; by Snyder, Lloyd R./ Kirkland, Joseph J./ Dolan, John W.   ISBN 0470167548

"Practical HPLC Methodology and Applications"; by Brian A. Bidlingmeyer. ISBN 9780471572466

It depends on what kind of data you are handling... 

BTW you might have a look to MetaMS.

Enjoy

Methanol is a very common choice for the HPLC mobile phase organic solvent component in reversed phase HPLC even though acetonitrile is often superior in several aspects. On the other hand, methanol is often less expensive and less toxic than acetonitrile, so it has advantages as well.

If you wish to simply substitute methanol in place of acetonitrile in a Reversed Phase HPLC method that has already been developed with acetonitrile, you should be aware that methanol is a much weaker solvent than acetonitrile and therefore you will not get the same chromatographic results. For example; if you have a method with 20% acetonitrile and then change it to 20% methanol, you will get longer retention than with the acetonitrile.    

Methanol is more polar than acetonitrile so it will increase retention of hydrophobic compounds in reversed phase and the elution order for some analytes could change as well, especially when a Phenyl Hydride column is used. For these reasons, changing to methanol should be viewed as a method development tool and not as interchangeable with acetonitrile. Also, for many SOP's changing from acetonitrile to methanol requires internal approval.

As Bruce suggested, you may add some acid to your mobile phase. 0.1 % TFA or HCOOH is commonly used. Be careful with basic conditions, this may destroy your column material. Some organic buffers may help (e.g. HEPES) adjusted to pH 7-8, this increases the solubility of the protein but does not destroy the stationary phase. At first, wash with 10 column volumes 100% H₂O, then add 5-10 column volumes buffer at low flow rate (0.05-0.1 ml/min), remove buffer with H₂O + 0.1 % acid (20 column volumes), then try the organic phase again (with acid added).

Whether you get a single TLC spot for a mixture of two diastereomers or not depends on how differently the diastereomers interact with the stationary phase and the mobile phase. If you get two spots, you have two diastereomers. If you get one, you have to keep trying. One spot does not mean you have only one diastereomer. Predictions are difficult.

Are you looking for the complex calculation (using the van Deemter plot curve, which can be obtained for free online or from one of the wonderful books on chromatography) or just the main guidelines we use in practice to develop methods? A formal plot and calculation can be made which takes into account the particle size and efficiency to determine the optimum flow range. But there is a better way for everyday purposes.

For practical considerations, to maintain linear velocity we use 1.00 ml/min for 4.6 mm ID columns, 200 ul/min for 2.1 mm ID columns and 50 ul/min for 1 mm ID columns.

Have you tried other reducing agents besides 2-mercaptoethanol to keep your protein from aggregating? (Is it highly prone to intramolecular disulfide bond formation?) It is more usual to employ 1 mM ditiothreitol or TCEP to keep the protein reduced.

Here is a reference that deals with prevention of protein aggregation during purification:

Now I see what the USP is doing.  The "L" is their designation for HPLC columns and this is actually a LiChrosphere resin which is a good SEC media for the separation of smaller protein and peptides by that modality;  better than the soft gel media which have some difficulty in this smaller molecular weight range and utilize highly aqueous solvents.  In this case, to take advantage of the SEC functionality but avoid any other interactions of the resin with protein, the method in "Limit of High Molecular Weight  Proteins" calls for a solvent of acidified aqueous arginine with a 20% acetonitrile  organic modifier to prevent insulin and its oligomers from binding to the resin while the acetic acid rather than TFA acts as the ion pairing agent and acidifier for the usual silica-based media stabilization as well as insulin solubility/stability.  A diol bonded phase is very much less hydrophobic but that doesn't mean the absence of any hydrophobic character due to the bonding chemistry that attaches the diols (Journal of Chromatography A

Volume 915, Issues 1–2, 27 April 2001, Pages 35–42 ).  The arginine acts to prevent any further oligomerization through the effect of the guanido group.  TFA is a stronger acid than acetic acid and more denaturing and is not needed since this is not reverse phase chromatography.  

Indeed, reverse phase resins and solvents have protein denaturing effects but the smaller molecular weight proteins that have multiple disulfide bridges, high ratio of bridges to mass, maintain tighter conformations under these conditions compared to other proteins with relatively larger mass and few disulfides.  Not only insulin but growth factors (protein hormones), cytokines (chemokines, lymphokines), TNF family members, TGF beta family members, etc are examples of relatively small molecular weight disulfide-bridged proteins that have been purified by traditional preparative reverse phase chromatography and, after removing the acetonitrile and TFA as though the proteins were small organic molecules, the proteins are fully active.  You might be interested in the CD data of these proteins in these aqueous acetonitrile solvents.

thanks so much for answering my question. Is there any reference based on your suggestions?

Jaya, probably you wont find a GC method using a non polar column for those analytes since the phase of this column has low polarity and works fine with another kind of semi-volatile samples. I  would recommend you use a polar column, the best for VFAs is HP-FFAP, it works pretty good (C2-C6) , great resolution and sensibity, however you can use a regular polyethylene glycol phase column. 

Other solution could be the alcoholic derivatization of VFAs, not  highly recomnended because the losses in the procces but you can try if you can't chose your column.

Regards.

Alexandra 

1st. Apologies for an earlier typo. Your column void volume is ~ 80 ul (not 8ul as it initially appeared from the system truncating the character). At 1ml/min your Tzero would be ~ 0.08 min (4.8 seconds)!

Saddam: I feel that you would benefit the most if you could find someone at your University with proper chromatography training who could help you in this matter. You appear to be going about this method development process without any understanding of chromatography fundamentals and such an approach will result in poor quality data. You still seem focused on column cleaning and receipt of defective columns. I think your efforts would be better spent understanding the technique used first. Once you have gained an understanding of the technique and used the skills to develop a better method, then you can evaluate columns. As someone new to the field you can not expect to master this technique, the use of the instrumentation or even the software without first having the benefit of months and years of proper training first. - And then, years of practical experience with samples. Many scientists using this technique still do not have a good understanding of the technique and how the many parameters affect the data, even after many years of use. It is a complex analytical technique that takes time to master. Having an experienced professional on hand can greatly help guide you through this process as can reading many of the wonderful books on HPLC principles too. "We" can only provide so much guidance to you via this forum. You need more help with the basics and fundamentals first for our comments to be of value. I hope you have someone there who can provide training to you.

There are too many individual problem items in your method and description which need optimization to list here.  However, I wish to comment on one basic area that I hope you will think about and adjust when you are re-developing this method for the samples.

Solubility: You wrote that all of your compounds do dissolve in pure ACN, but none of them dissolve in your buffer solution. Yet, you start your gradient method with 100% buffer!  The same liquid that none of your samples dissolve in. You also mentioned that when you started the gradient at 95% buffer, you had very poor results so changed it to 100% buffer. This makes no sense at all.

I will leave you with a link to a free webpage which contains many short articles on a variety of chromatography topics. All are focused on teaching and stress the importance of learning to use good chromatography fundamentals. I hope you find it valuable as you begin to learn this wonderful analytical technique.

Good luck to you.

The choice of an internal standards very much depends on the actual problem you are trying to solve (which analytes in which matrix ...).

Generally speaking isotopically labelled standards are the best choice if you want to account for matrix effects hampering your quantification. Often such labelled substances are not availavble, so one would have to think about a substance which has a chemical structure very close to the actual analyte, but at the same time will not likely be present in the sample matrix.

In case the matrix of your samples does not show a high variability, it might be good enough to use an external (but matrix matched) calibration with a recovery test now and then in your sample series. 

Hi Angela,

I am so pleased that you resolved the situation.

Kind regards,

Ade

I wonder wether or not it is clever to use water to wash of Fe(III) cause it may cause precipitation of iron-oxyhydroxides that form as a consequence of Fe(III) initiated hydrolysis. Is one of you aware of negative effects of this precipate formation? I imagin some sort of coating may form, that prevents an effective desorption.

I think you can slightly increase the pH of the enzyme reaction mixture to enhance the solubility of quercetin glucosides if your enzyme can keep its activity in higher pH (pH 7.5 or 8.0). Besides, you can try to use glycerol solution (25% or 50%) to replace DMSO if your enzyme is not sensitive to glycerol.

Hello Francesca: A few general comments, but in general we can not (and should not) tell you which system would be better for your own applications. Everyone's needs are different and their is no one "perfect" system out there. Other individual's experience may or may not be relevant to your application. Both Agilent and Waters offer good HPLC and single Quad systems. Either of them would be fine. The real (and perhaps more important) difference will be found in the experience of the operator and the methods used, not the instrument. Training in how to properly maintain and run both the HPLC and MS system is far more important, than which system is selected. IMHO: All operator's should know how to perform PM's and logically troubleshoot the flow path and system for problems (critical to daily operation).

One item that I would personally insist on is that the HPLC be equipped with a diode array detector (DAD/PDA). LC/DAD/MS is the most common configuration found and allows for the maximum amount of useful data. Each detector's very different, but complementary detection system allows for a wider range of sample types and they are also very helpful for troubleshooting problems found (both instrument and sample).

Perhaps it is possible for you to demo the systems? If so, you could use the time to "see" and get some hands-on time with the software and hardware Maybe run a few samples too?

If you have not done so already, please obtain professional training in how to best use the instrument you select, after installation. If possible, have the training on-site, for several days of hands-on experience. You should learn on your own system, not someone elses (as it will be different). Make sure that the people responsible for running the system on a day-to-day basis are in attendance. Both the software and hardware need to be considered during training, as the learning curve can be steep if you do not have several years of experience using these types of systems.  *Each LC/MS instrument has specific functions and steps that must be learned properly to operate it well. You will learn these and other important tasks much faster having the training done in this fashion.

Also, if all of the service and support for the instrument are to be provided by the same vendor, then be sure to inquire with others working in the same area as you about what their service experiences have been with the vendor. Most vendors provide great service in some areas, and not so great service in others. This question should be asked well in advance of any purchase.

Dear Juhura

You do not really give much information and there are many parameters that can affect what you are describing.

Taking the difference in the retention time between the two peptides (endogenous verses synthetic) I would suggest that they are not the same peptide. The retention times are way different.

How did you come to the "best peak" conclusion? Is this based on peptide mass? What criteria are you using to assess the different peaks?

How are you monitoring the eluted peptides, UV or mass spectrometry? The elution profile of peptides can be very complex and the question is how did you do your sample preparation? How did you get rid of the other compounds in your matrix like nucleic acids, lipids, carbohydrates, proteins, salts etc

Did you run the synthetic peptide as a standard in a separate injection or was it spiked into the cell lysate and if so at which point of the sample preparation?

How are you processing and enriching your sample? Is the cell lysis by detergent or mechanical means? Do you add protease inhibitors for the different classes of proteases?

Is the peptide prone to disulphide bridge formation, in which case there could be intra peptide bridges forming, and these could change the retention time of the peptide but this can be prevented by acetamidation.

If using mass spectrometry to monitor the eluent, then the mass should differ by the number of heavy isotope atoms added during the synthesis of the peptide as the precursor mass. If running MS/MS the fragmentation should be the same with the same mass offset seen for the precursor up to the labelled amino acid(s) after which the masses should exactly coincide.

If the peptide from the whole cell digest is eluting early it would imply a more polar peptide, which could point to a truncated peptide due to cleavage by a proteolytic enzyme during the lysis and sample preparation. This is an often overlooked aspect of sample preparation and when using cells or tissue can be a major complication.

If you have enough heavy peptide you could try adding it to your sample before your cell lysis and check if it also shifts in retention time, but this is a rather expensive experiment, taking the cost of isotope labelled peptides into consideration.

Fragmentation of longer peptides can be quite good depending on the amino acid sequence and ionisation state during mass spectrometry. 

More information could help trouble shoot your problem.

Good luck.

The method is suitable. The MacLafferty product at m/z 74 - makes spotting FAMEs easy and is always present even if the molecular ion gets lost, which can happen in polyunsaturated FAs.

Thank you Alex, Peter and Santosh!

First, I need to determine retention time of my sample, and compare to alkane.

PEEK is generally safe with chloroform (per the manufacturer). Should not cause ghost peaks on its own.

These types of detectors are very complicated to use and troubleshoot. Perhaps some of these comments will help you troubleshoot the issue(s)?

CAD and ELSD detectors accumulate sample debris from use. This debris increases the total noise level seen over time. Both types of detectors require periodic cleaning. Be sure to measure the actual noise seen under controlled conditions with a std so you can evaluate any changes over time.

The most common reason for high baseline noise with either an ELSD or CAD detector are through the use of non-optimized detector settings. The settings must be adjusted and optimized for each mobile phase and flow rate. The mobile phase must be 100% volatile. The nebulizer atomization and desolvation temperature must be optimized to the flow rate. This involves adjusting the gas pressure, flow rate and temperature to acheive the best sample S/N ratio or lowest noise S/N ratios. This process can take several hours to perform. Try to optimize the settings, step-by-step, changing one thing at a time to determine the best settings for your application. Failure to 'dial-in' the right settings with these detectors will result in peaks which vary in shape, area and height. IOW: Very poor reproducibility. *A dirty atomizer/nebulizer will also cause similar variability in signal.

Note: When optimizing the detector settings, please do not make changes based only on the observed baseline noise. The noise must always be measured relative to the sample signal.  Higher noise does not always mean lower signal. Lower noise does not always mean better signal. Select a gas flow rate and desolvation temperature (based on the mobile phase's boiling point; see Link provided to table of solvent properties) which removes the mobile phase and results in clean atomization. High noise and/or variable signal will result if the temperature is too high or too low. Same is true with the gas flow rates.

Lastly, make sure the exhaust outlet for your CAD is clean, unobstructed and well vented. Clogs or obstructions here can also induce all kinds of noise and variability.

Dear Aurea, 

Thank you for your helpful replies. 

In your opinion, do you consider HPLC to be a qualitative method so we can rely on it for identifying the compounds or it is only quantitative method for measuring the concentration of known compound based on standard solution?  

Thank you, 

Abdel

Dear Áurea Andrade Eiroa,

My PESTANAL standard is dissolved in MeoH and then diluted in my mobile phase.

Is it possible to be responsible my standard?

Thank you for your answer.

Hi Sabine Harrison,

Thanks for your valuable answer.

But is it possible with any system to set different temperatures at column inlet and column out let, if there is any such HPLC then please let us know.

As per my experience with Liquid chromatography it is not possible as column is constructed with metal (SS) and metal is very good conductor of heat. So it doesn't looks feasible to set different temperatures at column inlet and out let. second problem with setting different temperature is length of column as per USP which is 50 mm only.   

In my opinion this should have been performed by programming column oven temperature i.e. by keeping 50°C at start of analysis and 35°C at end of analysis (continuous decrease in temperature like mobile phase gradient) which is a feature of most of HPLC column ovens.

What you are trying to do is a pretty standard analysis. There is a lot of literature on it using a standard GC-FID with similar columns to yours. About 30 years ago I did some work on long chain alcohols with on older version of your column coupled to a magnetic sector mass spectrometer. After the method was developed on the GC-MS we were able to run it as a routine analysis on a GC-FID. The main difference from your method was that I used trimethylsilyl derivatives, but I am confident that your acetyl derivatization is fine.

OK, some ground rules:-

1) The amount of your internal standard injected on column **must not exceed 100ng**. If you do, the chromatography will be bad - Your peak will front, the geometry and retention time of your internal standard will change (dramatically) with only small variations in solvent composition, polarity and concentration. The 'perfect amount' will be between 10ng and 20ng. Please trust me on this :-) Going over 100ng will not give you any better results, honestly. If it does not work at 10-20ng your method or equipment is badly broken...

2) I am assuming that you will use a splitless injection - If you have a standard split/splitless injector **do not inject more than 1µL of sample** - You can use 2µL if you really, really, have to - But that will only be useful if you are doing trace analysis (you are not).

3) Use an initial column temperature 20C lower than the boiling point of your solvent (heptane b.p. 99C) so your initial temp of 80C should be OK. Ramp the column oven quickly to a temperature of 20C above the solvent boiling point - 30C/min should be fine. Then continue with your normal 10C/min ramp. I would go a higher than 240C (maybe 300C) to make sure than any high boilers elute, otherwise they will stay on the column and come out as random wide peaks in later runs.

4) Set your carrier gas flow velocity to 25cm/sec for helium, or 35cm/sec for hydrogen (1.2 or 1.8mL/min).

4) Do not complicate the method any more than you need to. Avoid Pulsed Electronic Pressure Control. You don't need to use it as you only are injecting 1µL of solvent. The heptane solvent will only produce ~210µL of solvent vapour inside your injection liner (A 2mm ID liner will hold ~250µL, a 4mmID liner will hold >1000µL).

So how do we find out what is going on? If we use simple logical steps it should isolate the problem:-

1) Inject 1µL of pure heptane solvent as a blank. Do you get large peaks coming out in the region of your analyte or IS? If you do they might be retained on the column as dirt or as carry-over from a previous injection.

2) Repeat the solvent blank injection. Have most of the peaks gone? If yes - It is now OK. If not consider cleaning the liner and bake the column off at 300C

3) Now make up a 100µM/L C12Acetate internal standard solution - This is equivalent to 23ng/µL. Run 3 replicates of your 23ng/µL internal. Yes I do mean only 1µL of just the C12 internal standard :-) **Do not add any C14 analytical standard**. Your internal standard defines the analyte quantitation retention times. On your 3 separate runs, your internal standard retention time should not vary by more than a couple of seconds and your peak areas should not vary by more than 15%.

If you can't meet these requirements you equipment or method is broken. Likely problems include: "The large peak that you **think** is your Internal standard isn't" (grease from your fingers. plasticiser, detergents, general lab contamination, dirt in the system and/or contamination in the carrier gas?). Or, (less likely): leaks in the injector; or your column pushed too far into the injector.

Assuming that everything is OK now, you can make up the equivalent of a 100µM/L C14Acetate standard (25.6ng/µL) containing 23ng/µL of your internal standard. Now run this sample 3 times. The retention time window for your internal standard should still be within a couple of seconds. Your integration software will then use this to accurately lock onto your analyte. Your replicates for your analyte calculated from the internal standard should be better than 3%.

Now that you have got this to work, the dynamic range of your system for C15Acetate should go from 0.1µM/L (0.026ng on column) up to the maximum capacity of the system for C14Acetate which will be a maximum of <~120ng on column (or roughly 400µM). Sorry, you REALLY can't go above this - But what you can do is dilute your high concentration standard roughly 6 times with pure heptane. Your chromatography will still work. The software should still be able to find your internal standard at 23/6 = ~4ng (assuming that you do not have any other peaks within 2-4 seconds of the internal standard!). The clever software will do the calculations for you, as it is a simple linear relationship and you will get the correct result.  The bad news is that if you do inject a high concentration C14Acetate sample, the results will be complete rubbish because you have exceeded the design capacity of the system, BUT you can dilute those samples 6:1 with pure heptane and run them again. If the sample contains a number of other peaks near the internal standard and the instrument cannot find the internal standard,, dilute your sample about 1:6 with your internal standard solution and apply  a concentration correction factor.

So in conclusion the maximum dynamic range of your column with a FID is 0.02ng to 100ng injected onto the column. If you want to increase this at the top end, use a larger diameter/thicker film column like, say a 0.32mm ID column with a 1µm film thickness - The maximum sample capacity will be ~500ng on column (or roughly 2000µM/L of C14Acetate).

There is another alternative. Your bottom concentration is 15µM/L so you can cover the dynamic range that you want by running your standards diluted - You know that the top amount that you can inject is <100ng and the bottom detection limit of the system will be ~~0.02ng or roughly a 5000:1 dynamic range. So the have the top end dilution at 100µM/L, and go down down to <0.1µM/L but still keep the internal standard at 23 ng/µL (100µM/L).

Good Luck and Best Regards, Tim

Looks to me like these peaks might be the sodium adducts of polyethylene glycols, try adding 0.1% formic acid to your mobile phases to see if these peaks disappear. Did you use Parafilm on or near the solvents? This could be the source of the polyethylene glycols. Never use sealing tape like Parafilm on or near the reservoir bottles or solvent lines on any LC/MS system because this type of contamination is possible. If you need to cover the reservoirs use aluminum foil with the dull side facing the solvent.

Polar phases work much better. The above reasons given for NP are more or less correct, but flammability should never be an issue in a dry nitrogen atmosphere (common to ESI). Normal phase mobile phases will work just fine IF you adapt them to the type of ionization source and instrument you have. For example: we routinely add adducts or doping agents to the mobile phase (or via infusions) to increase ion formation. Esp common in Negative mode (we run lots of Alcohol/Hexane mobile phases w/o a problem).

In all cases, watch the ESI needle (which should be in a pure N atmosphere) for sparking (bad, as it can damage the needle and result in lots of noise). Super low flow rates are more likely to cause problems so one quick fix is to increase the flow rate to suppress it.  Adding something more polar or conductive to the mobile phase will also cure the problem too.

These forums really are not the best place to go into detail about this, but anyone properly trained in LC/MS should know how to run most NP methods on their instrument w/o any danger or problems. *BTW: I professionally teach LC/MS operation and MD and we routinely run NP methods on most manufacturer's instruments. However, there are millions of possible methods so this is not to say that they all will work. Chromatography and MS are bit more complicated than that.

Any sensibility analysis starts by the determination of the number of degrees of freedom. A continuous distillation column has two degrees of freedom when the pressure is fixed. These two variables can be manipulated to produce desired streams purity e.g. reflux and distillate flow rate. To determine the distillate flow rate influence using rigorous simulation, fixing the feed flow rate and composition, it is necessary to choose the values for the number of stages for the stripping and rectifying sections and for the reflux. If any of these values are smaller than its minimal values, there will not be solution and neither convergence. Infinite reflux and infinite number of stages are the simplifying hypotheses which have inspired the name of the  ∞/∞ analysis. This analysis was firstly proposed by Petlyuk and Avet’yan (1971), but without any significant impact on the scientific community. The feasibility of the stream molar compositions obtained by the mass balance is checked by the existence of a residue curve from distillate to bottom (hypothesis of infinite reflux). Attainable flow rates for each column must be inside the interval from xB = xF (B = F and D = 0) to xD = xF (D = F and B = 0) (Bekiaris et al., 1996). The ∞/∞ analysis allows an easy and fast checking of the interrelation of the system streams in a column sequence without any column design consideration. For more details you can go to the link:

Hello Miguel, if selecting those that do have the difference will do the job for you as well, this might help you: http://fiehnlab.ucdavis.edu/projects/MS2Analyzer/

The software needs mgf files as input, but you can use Proteowizard sofrware to convert raw files (or mzXML files) into the mgf format. Than the Excel based program will select spectra according to your conditions (i.e., containing mass fragments, neutral losses, or mz-differecens (that is the category you want to put your 203 in) - just make sure you use the right ppm accuracies....

Hope this helps,

Justin