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High-performance liquid chromatography (sometimes referred to as high-pressure liquid chromatography) is a chromatographic technique used to separate a mixture of compounds in analytical chemistry and biochemistry with the purpose of identifying, quantifying and purifying the individual components of the mixture.
Questions related to High-Performance Liquid Chromatography
Mode of sugar analysis depends on sample character and number of samples. Good choice is Aminex carbohydrate column (Pb) or equivalent column and water as mobile phase (temperature required is 80-85 °C). ELSD, RI are good detectors, bud in some cases UV (DAD) at 192-195 nm is possible to use (depend on glucose concentration).
I would like to know what is the best storage condition of a C18 column for a mobile phase of acetonitrile as organic solvent. Is there any other recommended ratio? What's the best storage condition for long term storage and short tern storage(a day or two)?
The best way, to storage a C18 column for short (weekend) or long period, is to wash the column with a mobile phase water and acetonitrile(H2O/ACN HPLC grade) (70:30V/V) for 20 minutes (to remove any salts) and then flush water and acetonitrile(H2O/ACN HPLC grade) (30:70V/V) for 20 minutes (to store the stationary phase in the best conditions). after doing this the column can be capped and stored for the desired time.
I have experienced that when I run on HPLC for several days non-stop (either non-stop loading samples or alternation of loading samples and low-flow stand-by) that the back-pressure increases. I wasn't sure about it, but one colleague confirmed that today. Can it be true? What is the reason for that?
In the previous answers you have many true possible reasons for your system clogging. But I have to mention that the few more frequent are (in order which can change a little depending on the skills of the users):
1.- Purge filter clogged. Check the pressure when pumping without column. Remember to filter your aqueous phases and also the pure organic solvents, ALWAYS!. Its more important for the aqueous phases with buffers dissolved on it, but do it also for pure solvents. We discard brownish filters every day, even with the top brand HPLC solvents and qualities.
2.- Precipitation of the buffer: After preparing your buffer in water people usually check the solution is clear, completely dissolved, and they assume its OK. However, when these aqueous phases are mixed with organic modifiers (with the pump and on the line) the solubilities are changed, and some of the common buffers precipitate. If you have access, check this interesting paper about phosphate: LCGC NORTH AMERICA VOLUME 22 NUMBER 6 PAGE 544 (http://www.chromatographyonline.com/lcgc/data/articlestandard/lcgc/232004/97119/article.pdf). The precipitation normally happens in the mixer, but some particles can go through it and reach the head of the column. Take into account that kinetics of precipitation are not always similar to the kinetics of redissolution. It depends on the buffer. In many cases you are not able to re-dissolve the solids. This is the case also when you have not precipitation but coagulations. If the problem arrive to the point when the column is completely blocked, then it is not reversible because you cannot flow anything through it to redissolve. With modern
3.- Succesive injections of precipitating sample. The concept is the same than the described for buffers. If the sample produce a little of precipitate you need many injections to note the problem, so this can be a silent murder of columns and you have to take care about the method you are using. You can prevent the problem preparing the sample in the same solvent composition of the LC method. Then, you can eliminate the insoluble compounds at the filtration (but you can also lose a part of your analyte here). You cannot use higher organic solvent compositions than the % at the beggining of the run because it can produce tailings. Additionally you can prevent the damage of the column using column guards.
4.- Connectors and tubing. The problem of blocked tubes is much worst in UPLC and narrow tubes compatible with columns of high efficiency and small diameters (narrow bore columns, core shell and similar). PEEK tubing use to clog in the extreme of the tubes because of the cut. In that case you can try cutting some more milimeters of the tube. In the case of stainless steel tubings, you can take out the column bypassing with a dead volume connector, and to flow some seconds of HNO3 30%. Then you have to rinse the acid from the pump with water, leaving it in the tube some minutes (5-10). Finally you have to rinse everything with water and do a solvent rinsing changing with quick gradients (e.g. water->isopropanol->water).
1. The UV/Vis detector can detect single wavelength at one time while the DAD can scan a wavelength range (i.e. 190-800nm). DAD is actually a UV/Vis detector it has an array of numerous photodidoes.
2. DAD will scan all the compounds which can respond in the given wavelength range (say 190-800 nm) there is no need of any adjustment. It will adjust by itself.
3. Both these detectors are equally easy to operate or interpret. But DAD is better than UV/Vis because it can scan whole 190-800 nm region, on the other hand, UV/Vis can scan a single wavelength only. so if you want to scan a wavelength range DAD is better option. But if you know the absorbance wavelength than you can detect that single wavelength by using UV/Vis detector.
In method development, DAD is a better choice because UV/Vis detector is very time-consuming. So for development a new method, I strongly recommend you to use DAD.
I am working with the UV-degradation of bezafiberate in water. Using HPLC- UV detector Bezafiberate has abosrption wavelength of 232 nm and gives peak at 1.7min.
I am expecting that during the UV- degradation process 4-hydroxybenzoic acid is formed, so I changed the wavelength for 4-hydroxybezoic acid to 254 nm. Then again a sharp peak appears at 1.7 min. So, weather this peak is of 4-hydroxybenzoic acid or bezafibrate.
First inject the pure standards of both the compounds 4-hydroxybezoic acid as well as Bezafiberate. If their retention times are same, then you need to modify your mobile phase. What is the phase that you are currently using and the HPLC column? Have you checked the area under curve or height of the Bezafiberate peak before and after UV degradation? Is there an increase in the peak height/area or decrease? Bezafiberate may not be completely degrading into 4-hydroxybezoic acid and therefore you are still getting a peak at 1.74 min.
I am helping a professor determine the D/L amino acid ratio in heated egg shells using HPLC, but before that we need to isolate the amino acids from the matrix without causing unwanted racemization. Any suggestions with supporting literature will be much appreciated.
To measure the D to L ratios you need to first hydrolize the proteins into their constituent amino acids. They most common way to do that is to dissolve the eggshell into 7M HCl and then heat the solution at 110 C for at least 6 hrs and up to 22 hrs. You can also vary the temperature, but I think the "industry standard" is 110 C. That hydrolysis process causes racemization. There's no way around it. But, it's a relatively small amount. If you use a standardized method then your results will be comparable to everyone else's. There is a lot of literature on amino acids in eggshell. Authors off the top of my head to look for are Gifford Miller (INSTARR, Colorado) and Collin Murray-Wallace (Univ. of Wollongong?)
I measure dopamine produced by dopaminergic neurons in cell culture experiments by HPLC-ECD. Sample preparation for injection into HPLC is one-step deproteinization by perchloric acid, but the last time, the samples that I received contained an impurity that interfered with the dopamine assay. The impurity is very polar like dopamine and is not removed by dichloromethan or ethylacetate in acidic or alkaline condition. Does anybody knows a better way for sample preparation of dopamine samples to get ride of that impurity?
Adsorption onto alumina or boronate affinity columns at alkaline pH (8.6) followed by acid elution (0.1 M acetic acid) works with plasma samples but for lower concentrations the eluent may need to be concentrated by lyophillization.
I am looking for metabolites to use as biological markers for canine leishmaniasis. My problem is that the concentration of urine samples are quite different. I tried to normalize with creatine but doesn´t work well. Does someone know a statistical/mathematical method to normalize the raw data from HPLC-MS?
Dieterle F, Ross A, Schlotterbeck G, Senn H. Probabilistic quotient normalization as robust method to account for dillution of complex biological mixtures. Application to 1H NMR metabolomics. Analytical Chemistry. 2006;78:4281–4290. doi: 10.1021/ac051632c. PMID: 16808434
If you use Matlab I can send you the code.
I also suggest you read:
Veselkov, K. A., et al, Optimized Preprocessing of Ultra-Performance Liquid Chromatography/Mass Spectrometry Urinary Metabolic Profiles for Improved Information Recovery. Analytical Chemistry, 2011, 83(15): 5864–5872.
State-of-the art data normalization methods improve NMR-based metabolomic analysis.Kohl SM, Klein MS, Hochrein J, Oefner PJ, Spang R, Gronwald W. Metabolomics. 2012 Jun;8(Suppl 1):146-160. Epub 2011 Aug 12. PMID: 22593726
You may also want to have a quick look at Metaboanlayst (online tool): www.metaboanalyst.ca .It has a few normalization methods that you may which to quickly try out.
Xia J, Mandal, R, Sinelnikov IV, Broadhurst D, Wishart DS. MetaboAnalyst 2.0-a comprehensive server for metabolomic data analysis. Nucleic Acids Res. 40(Web Server issue), W127-133 (2012).
PQN has been very popular with NMR data which is well known to be very reproducible (w.r.t. instrument precision), however, I personally have not seen a massive improvement on LC-MS data with this method (most of my work is serum though) However, there are more complex issues that arise with LC-MS data - regarding instrument precision over time. Columns degrade and MS instruments get dirty. This results in signal attenuation which has nothing to do with sample volume (but will effect the ability for PQN to work properly). I am a big proponent of using Quality Control samples to monitor "drift" and then correct for the drift using software.
I have attached a recent review:
Dunn WB , Wilson ID, Nicholls AW Broadhurst D. The importance of experimental design and QC samples in large-scale and MS-driven untargeted metabolomic studies of humans Bioanalysis September 2012, Vol. 4, No. 18, Pages 2249-2264 , DOI 10.4155/bio.12.204
I dont know what about plasma sample type preparation... But for the chromatography I would like to add, that PFP column is useful for rapid determination of methionine, for better separation of amino acids try to use ultra-pure silica column. One advantage - You dont have tyo derivatize methionine :)
I washed it with formic acid and the oil stain disappeared, however I ran a sample on it with HPLC UV detection and found a horrible profile (different to the one I had found before contamination). Has anyone had experience with this type of contamination before? Do you think my column is ruined?
I have tried the following mobile phase solvents and ratios of them.
I am using 1ml/min FR and 298nm WL. Either I do not get the peak, it is too near the solvent peak, peaks not reproducible or they do not increase proportionally to increasing concentration:-
1. Methanol, Acetonitrile, Both combined at 50:50 ratio.
2. KH2PO4, K2HPO4, Orthophosphoric acid solution, Ammonium acetate buffer (at different molarities and pH), water.
Note: UV spec WL detection of the drug was 200, but this would be not appropriate as an HPLC WL choice.
Essential oils- I'm assuming they are volatile so I would run those on GC-MS.
Alkaloids and flavanoids generally run well on HPLC. You _may_ need to switch to negative mode to see flavanoids although I've seen them in positive mode. Please note that I'm speaking generalities since some compounds of each group could be difficult to elute or coelute with other compounds.
I personally like to do initial purification of alkaloids with strong cation exchange columns and initial purification of flavanoids on strong anion exchange columns. I then do any further purification on C18. The initial purification on the ion exchangers removes a lot of compounds that could co-elute.
We would like to buy an HPLC system but I do not have any experience with preparative HPLC. We aim to analyse secondary metabolites from medicinal plants, collecting the compounds and then employing structure elucidation other systems. Do you have any suggestion about preparative ones or both together, analytical and preparative for phytochemical analysis? If so, let me know the system(s)- (brand, model etc)
The use of preparative HPLC mainly depends on the amount of product you want to purify. The amounf of product to product will govern the choice of column size (the internal diameter of the column and not its length), and this will impose the flow rate. With analytical HPLC you can work up to 5 or even 10 mL./min (but keep in mind that it is not very good for the pump to work at the upper limit for a long time). For higher flow rates you must work with preparative pumps and consequently use preparative HPLC.
However, if you do not have an analytical HPLC, i would recommand you to buy one first. And thanks to the automatic injector, what you can do is to inject lower volumes (so less quantity of compound) but repeat the injection a lot of times.
Do not also forget that the softwares dedicated for analytical HPLC are not the same that for preparative ones, espacially for the collection module what is very important for purification !
For structure determination (i suppose by NMR), you do not need high quantity of compound (several mg or several tens of mg). So, indeed, an analytical HPLC may be converted to "semi prep" HPLC with columns of 1 or 2 cm internal diameter.
Otherwise, if you need to isolate sevarl hundreds of mg or several grams, i would recommand you to acquire a preparative HPLC which uses columns with internal diameter of several cm.
In our lab, we have recently acquired a preparative HPLC from Interchim with a 3 detection (ELSD, UV, RI) to work up to 250 mL/min with maximum pressure of 250 bars and to work with columns of 2 cm or more. As for the price, it is around 65 000 €.
Measure calibration standards of known concentrations which are in the expected range of the concentration of your samples. Repeat these measurement for minimum 3 times. Prepare these standard solutions indepently to prevent errors.
After that use your preffered spreadsheet an fit your data to a linear function. Then you can calculate both values.
Use the following equations:
LOD = sx0 * t * sqrt(1/n + 1/m + x²/Qx)
LOQ = k * sx0 * t * sqrt(1/n + 1/m +(k * LOD)²/Qx)
sx0: standard deviation of procedure (sx0 = sy/a where sy is the standard deviation of the slope and a ist the slope)
t: t-value from t-test
n: number of calibration samples
m: number of repetitions per sample
x: mean value of concentrations
Qx: sum (xi - x)² where xi is the corresponding sample
Before going to TLC or HPLC you need to do a simple identification test for your Phytosterols in plants . The test is that , extract your Phytosterols containing extract with chloroform.Take 1ml of this chloroform extract in a test tube and add 0.5ml of acetic anhydride and then add 1-2 of conc H2SO4 and observe the changes. You will observe a brown or red color ring in the test tube and a greenish or bluish coloration over the ring, which indicates the presence of Phytosterols.
I would advise you to use LCMS of GCMS rather than HPLC as the detection limit for a normal HPLC may not be very useful for measuring urine levels of BPA as the levels may sometimes be too low for detection by HPLC. Of course the best way is to use LCMSMS or GCMSMS.
Please have a look at our paper as follows: Urinary Bisphenol A Concentrations and Their Implications for Human Exposure in Several Asian Countries. Environ.Sci. Technol. 2011, 45, 7044–7050.
I've been working on detecting metabolites in human serum using C18 Eclipse-Plus column plus ion-pairing buffer and methanol gradient up to 30%. The low-molecular fraction was extracted by centrifugation with 3 kDa cut-off filters.
Now we're performing experiments with culture medium, containing 15% FBS, growth factors and some other proteins. The 3 kDa technique doesn't work because of great volume losses, which make the amount of low-molecular fraction unsufficient for HPLC method in use.
15% of serum seems to be small enough not to extract it before every tun, so I'm trying to establish the method for column cleaning and regeneration after several analyses so that it isn't 'killed' with proteins from medium too soon.
It is sometimes very hard, if not imposible, to remove proteins from a C18 column. Many proteins are very hydrophobic in nature and can litarally stick to your column. Gradually flushing you column with methanol or acetonitrile might not remove the proteins from the column. Organic solvents can often act as denaturants. In this case the hydrophobicity of the denatured protein usually increases and as a consequence it can stick to the column even stronger.
An elegant solution could maybe be the use of hyperbranched monolithic columns. If I am not mistaken these could be flushed with a NaOH solution, which should remove (hydrolize) any protein from the column.
how to differentiate between IP3 and IP6 on UV-vis detector of HPLC?
best way is by their retention times ... being one much more polar than the other one it shoud not be much difficult to undetstand which is which. the very low UV absorbance will be given mostly by phosphate groups and will be in the same range for both compounds,one having an molar extintion coefficient nearly double than the other one they will have peaks area corresponding with their relative amount ...
if you have a reference the job will be straighforward
probably you will need some secondary detection system to enhance phosphate UV/vis detection ... i am not an expert in this specific issue
I am interested to purify a mixture of proteins using HPLC and LC/MSMS. the analysis of HPLC fractions showed the occurrence of the same protein (but different set of peptides) in many distinct HPLC fractions that present different retention times and molecular mass in sdspage. Any explanations?
It is not surprising that you identify different peptides, from the same protein, in different HPLC fractions. I'd be worried if this wasn't the case. Just because you see different masses on SDS-PAGE, this doesn't mean the proteins are different. As has been said, there are many PTMs that can lead to a range of masses in SDS-PAGE.
Mass spec doesn't lie, it identifies what is in your sample. So you either have issues with the sample itself, ie. carryover, contamination, etc, or you are seeing the same protein (modified in some way) in different mass bands on SDS-PAGE. You just need to work out which case you have.
I'm stumped on an HPLC question and I'd really appreciate any advice/direction from any experts out there!
I have two HPLC chromatograms that came from a sample that underwent two different treatments. The chromatograms are similar in shape but not identical - some peaks differ in height and there are a small number of peaks that are present in one sample but not the other.
Is there a statistical test to determine if the two chromatograms are significantly different?
I'm adding more information about what I've done and what I've found, in case that's helpful.
I have a bacterial isolate that produces a range of interesting extracellular compounds - antibiotics, antifungal compounds, siderophores etc. Preliminary experiments showed that changing growth conditions changes which compounds are produced.
I wanted to compare compound production when the isolate was grown at room temperature vs. 37C. So I cultured the isolate using all the same conditions (culture media, agitation speed, volume, type of cultureware, incubation time etc) with temperature being the only difference.
After a set incubation period, I removed filtrate from each of the cultures and ran them on HPLC with UV detection using the same column, solvent, wavelength, flow rate, everything. I also ran a media-only control so I know which peaks to exclude. Then I analysed each peak individually to identify what compound was produced. That alone told me that there were differences - some compounds were made only by the room temperature isolate, some were made only by the isolate at 37C.
Also, the overall chromatograms just *look* different - similar but definitely not identical. I was worried just looking wasn't 'scientific' enough though which is why I was wondering if there's a formal test / method to compare chromatograms and determine if they are different.
Thank you again for all the suggestions!
I think this question needs to be broken down a bit. Is it correct to say that what you really want to know is 'are the two samples different'? Or even does sample A have more of compound X than sample B? (Or maybe this second question asked for several compounds?) So, if it were me I'd start by looking at a single peak, which represents a compound which you think might have different concentrations between treatments, then quantify the variation in the amount of that compound using either peak area or height. So in order to know whether two treatments are different I'd need to know how much variation there is between replicates of the same treatment (different flasks, NOT samples from the same flask). This variation contains variation from at least (i) HPLC run (machine error, probably small) (ii) subsample from a single flask (your pippetting error, and heterogeneity in the flask, also probably small) and (iii) actual differences between replicate flasks. If you think the difference between the treatments is large compared to the difference between replicates, then two replicates of each treatment may be enough, so four flasks. Just be certain that you have true replication, and that the only difference between the the two sets of flasks is the 'treatment' in question. Controls are also good - you might want to run another four flasks without any biology in them - so you know whether it is an effect of the treatment alone, or the effects of the biology that you are seeing (this depends on what a 'treatment' is in your case). And, if the differences between treatments are not large, you'll need more than two replicates, which I'd recommend anyway, but this depends on your resources. If you do not replicate, you cannot know where the differences between your chromatograms come from.
Thank you for the remark on my reply. Possibly you have not taken the essence of the comment (although you have repeated the same in the following massage what I stated ). Do you think that there is no science in the performance of of each of our fingers performing thousands of work ? I have spent 45 years of my research on protein purification and reported purification of at least 15 glycosidases. Thank you for your cordial wish to do the best in the domain
Sorry, I couldn't copy the URL, so I paste the procedure here:
The procedure we used has proved applicable to most lipid mixtures before further fractionations by HPLC, another low pressure chromatography or thin-layer chromatography.
Glass columns (inside diameter: 8-10 mm, length: 20 cm) with a stopcock (glass or Teflon) at the bottom to control the solvent flow. Solid phase extraction cartridges (SPE columns) available from various companies (Alltech, Waters, Supelco, Analytichem Int...) may be also used if filled with silica.
Pyrex glass wool.
Glass Pasteur pipettes
Silica gel 60 230-400 mesh (Merck)
Chloroform, methanol, acetone
Prepare a slurry of 300 mg silica gel in 2 or 3 ml chloroform and transfer into the column (equipped with a glass wool plug) with the help of a long Pasteur pipette. Open the stopcock to drain slowly the solvent and rinse the column with a portion of chloroform. Allow the solvent level to drop to the top of the gel.
Transfer the lipid sample in chloroform (about 1 ml) to the top of the gel. The amount of lipid that can be practically applied to this type of column is about 10 mg, for higher amounts increase the column size accordingly. A critical study with details on the optimum ratio between lipid mass and sorbent mass has been published (Pernet F et al., J Chromatogr A 2006, 1137, 127).
1- After complete elution of the solvent, add carefully down the sides of the column 10 ml chloroform. This will elute (at about 1 ml/min) neutral lipids (hydrocarbons, pigments, sterols, triglycerides, waxes, fatty acids..).
2- After draining the first solvent, add on the column 15 ml of acetone/methanol (9/1). This will elute glycolipids (cerebrosides, sulfatides, mono- and digalactosyl diglycerides, sterol glycosides) and ceramides.
3- After draining the second solvent, add on the column 10 ml methanol. This will elute all the phospholipids.
Evaporate the three fractions and dissolve the dry lipids with a small volume of chloroform (for neutral lipids) and/or chloroform/methanol (2/1) for all three.
If a quantitative recovery of the free fatty acids are required, it is recommended to elute the neutral lipid fraction with 10 ml chloroform containing 1% acetic acid instead of pure chloroform. All other neutral lipids are present in this fraction.
I am using Xterra C18 250x4.6, 5 um. After injecting the column with plasma samples, I noticed a high elevation in the column back pressure. My mobile phase is composed of Acetonitril and phosphate buffer. Does anyone have any suggestions?
Jul 16, 2013
Although people have answered your querry, but still I want to suggest that don't inject plasma samples directly to HPLC column. First go through some purification steps (which you can choose based on your analyte of interest) like filtration, centrifugation or extraction process for minimizing matrix effects. Use guard column, which will protect your HPLC column from getting blocked by any contaminant.
If after giving Methanol and ACN wash, column is still not clean, then in that case you can pass 50:50 isopropanol and water mixture through column (keep column temperature 45 or 50 degrees). If still column is not clean and giving high back-pressure, then reverse the column and pass isopropanol:water, MeoH and ACN one by one through column for 2-4 hours.
I have been having serious issues with dead volume in our HPLC system, basicallly because the tubings provided for our FRC-10A system are rather thick. I am considering changing it but it seems to be the "official" one, with a terminal plug adaptor. I am not sure how to change this.
Anyone else here have the same system, so we could exchange experiences with it?
There are several possible reactions of Acid chloride like
1. Reaction with water
2. Reaction with alcohols
3. Reaction with phenols
But as your interest to analyze on hplc, then for HPLC with UV/Visible detector most appropriate reaction is “Reaction with phenols”. The reaction with phenol itself is very interesting; you can make aspirin by a very similar reaction.
And Aspirin can easily analyze on HPLC with UV/Visible detector at 275 nm.
If you reacts phenols with Acid chloride the product will be helpful for you not necessary it will be aspirin. The product can easily analyze on UV/Visible detector. And it’s very good as you mentioned you have diode arrary detector (DAD).
I am using the following method:
NH2 column ACN/water = 75/25, flow 1 ml/min, column temp=20C , UV and ELSD detecter , inject volumn 10 micron for analyzing Glucose 10 and 200 ppm but data is really poor, the peak isn't clear, how can I adjust this method?
It is very difficult to analyze glucose or any other sugars on UV detector. You have to derivatise first then you can go for UV detector as said by Dr Pawel.
But in case of ELSD or RI detector. There is no need to derevatise your samples. But in RI difficult to use gradient solvent system. So at last u you should go with ELSD. Use column temperature at 30C. I got good results with glucose, fructose, sucrose, mannitol and rehmnose. But if you want to analyse other sugars little optimisation required with gas pressure=2SLM, drift tube temp.=70-90, neb temp=60-90C.
I have dissolved Lipid A in chloroform-methanol-water 74:23:3. I tried with solvent A: 95:5 acetonitrile-water and B: 85:15 isopropanol-water, and 1Mm ammonium acetate in both A and B. I used UV light, 206nm. I only see a large top for chloroform. Can anyone tell me what I am doing wrong?
I've use a simple mixture of 22 amino acids from Sigma. Many will ionize without derivatization. Alanine and glycine will be the most difficult for analysis. Same goes for fatty acid analysis. You can purchase a mixture. Negative ion mode works better for the fatty acids. Other than those, you will need some TCA cycle metabolites, glycolysis, creatinine, uric acid metabolites, acylcarnitines. The list could be very large and expensive to get some of them.
I want to analyze a double strand oligonucleotide with LC-MS, but it always melts on the column even at 10oC. The upper strand contains 17 T bases and the lower strand contains 17 A bases, and it is in tris buffer. I have tried several times to anneal the double strand oligo but with no success. The LC conditions I used have been used for analyzing other heterogeneous double strand oligos, so it is not denaturing.
well I don't know much about HPLC, but i know you can improve the stability of the duplex. One is to add salt (NaCl or KCl). I don't know how compatible that is for the conditions you are running. Another is to try tetraethylammonium chloride (TEACL), which at 2.4 M causes nucleic acid duplexes of identical length to have the same Tm, regardless of base content (http://www.ncbi.nlm.nih.gov/pubmed?term=powell%20caccone%20TEACL). But I think this is because it destabilizes G+C pairing, and so wouldn't increase A+T stability. What kind of analysis are you doing? Why not use a native PAGE with one strand 5' radiolabeled? You could water cool it or run in the cold room.
You need to provide more specifics here. What coelution are we talking about?
Are you already running HPLC? if do then it depends on the properties of your coeluants. You might try a different column (ion exchange/reverse phase), different mobile solvent, different temperature and flow rates, pH, etc.
Also your coeluants might be in a dynamic equilibrium.
I have recently acquired my first Shimadzu HPLC system, and I have been trying to put it to use immediately. When recovering a sample using our fraction collector, I have noticed a considerable delay between the obtained signal on screen and the drops coming out of the collector tip. I have determined then (both with a dye and using a bubble) a dead volume of about 560 ul, which gives me a delay of over 1 min at 0.5 ml per minute flux. I am rather disappointed at this delay, as it makes correlating the chromatogram with obtained samples a bit tricky (still learning how to compensate with the collector) and, more importantly, because this means an obtained fraction at the detector will gradually dissolve into this dead volume until it gets to my fraction collector. I am sure this costs me resolution.
1) Do you get as much dead volume as this?
2) Did you ever try to reduce this dead volume?
You are correct - 0.5 ml of dead volume is indeed quite a lot and will significantly affect your resolution if you run your chromatography at 0.5 ml/min.
I recommend to check inner diameter (ID) of installed tubings and - if possible - replace them with smaller ID tubing between your detector and fraction collector. For running at 0.5 ml/min you could go down to 150µm ID - but check if that won´t generate too much backpressure (e.g. if running high-viscosity solvents).
Also, to maximize resolution, all installed tubings should be as short as possible and have the minimal ID´s that are still compatible with your intended flow rate. Additionally, any valves, sensors (pH, conductivity, etc) etc, could also be causing dead volumes - if you don´t need them for your analysis take them out of the flow.
Reversed phase HPLC with UV detection at 210nm has been used to determine constituents in commercial Stevia products, but the methodology is limited in its ability to separate all components of interest, especially when higher order oligosaccharides are present. Another problem is that any single UV wavelength is a detection compromise as the constituents exhibit a wide range of absorbance maxima (193, 204, 236, 238, 284nm). Some are only weakly chromophoric.
The Evaporative Light Scattering Detector (ELSD) is a valuable complement to spectroscopic detectors for HPLC. The ELSD makes a measurement of photons scattered from semi- and non-volatile particles that have been dried of mobile phase through evaporation. Because its response is independent of the light absorbing properties of molecules, it can reveal sample components that UV detectors miss and provide a more accurate profile of relative component abundance than is possible with a spectroscopic detector.
Here i had attach an application note of Shimadzu. Hope it will we helpful to you
I am dealing with the separation of CDT isoforms in HPLC. C(arbohydrate) D(eficient) T(ransferrin) is a protein that bears sialic acid residuals ranging from 0 to 6 units, giving rise to the corresponding isoforms. These can be quite satisfactorily separated with MonoQ type columns and a gradient. Can anyone suggest how to improve this separation by using an organic modifier or an ion pairing agent?
I have been having difficulties with a lag between my fraction collector and the cell detector of my Shimadzu HPLC system. I am trying to adjust this delay time by determining the exact time between the onset of a peak and the appearance of the corresponding compound dripping from the fraction collector. I thought the easiest way would be using some strong dye which I could see coming out.
However I do not want to risk our C18 columns or precolumn cartridges with any dye. I have tried using Sudan III at 1mg/ml and flow 1.0 ml/min, using methanol 97% as vehicle. However, I could not see the dye coming out as it had probably diluted too much. I would like to try some stronger dye.
Please, does anyone know a coloured standard/strong dye which is easy to get and could go in a C18 RPHPLC system without affecting the stationary phase?
There is no problem to open the fitings. The only care you have to take is when you put the tube in the hole and before screwing the nut, to press the tube to make it seat against the deep end. Then you can screw it. On the contrary the tube can be a little far and this introduce an additional dead volume and an additional point where the resolved bands lose resolution. 566ul sems to be a lot. 2300ul?????. I think something is not fine at all in that instrument. Maybe it was intially configured for a huge pareparative column and high flow rates where that extracol volumes were acceptable. But it is not for a normal column of 4.6mm or less.
Laboratorio de Separaciones Analíticas
División Química Analítica, Facultad de Ciencias Exactas, UNLP
I´m using a conventional HPLC pump equipped with a flow splitter producing on column flow of 2uL/mim (150mmx0.18mm C18 column). How many micrograms of digested protein is recommended for LC-MALDI protein identification?
If you have "micrograms" of protein, I doubt you'll have many problems identifying your protein, provided it's in the database and your methodology is competent.
Longitudinal diffusion of solvents in stop-flow high performance liquid chromatography (HPLC), what's the influence on gradient separation ?
Sep 10, 2012
I've read that compound diffusion is rather limited in stop-flow HPLC (used in LC-NMR especially), but that solvent diffusion can really be problematic in gradient elutions. But I can't find any reference or data about this. Does one of you have any information or reference about the difference between solvent diffusion and compound diffusion or references about the real influence of stop-flow on quality of elution ?
I want to carry out bioanalysis of a drug by administering it to rat, collecting urine and then analysing it by hplc- uv. But I cannot find any literature on how to extract drug or analyse it on hplc. Can anyone offer advice?
Apr 5, 2012
I appreciate your concern and thank you for the same. I am working on eperisone hydrochloride (myorelaxant). Plz help!
I am trying to show anthocyanin and its metabolites in porcine plasma using UPLC/PDA detector using aglycone and phenolic acid standards. I want any method for extracting anthocyanins/phenolics from plasma.
How could it be possible that two different molecules of different physical and chemical properties are eluted on the same retention time on the same analytical parameters for HPLC? According to my studies this phenomenon is against the principles of HPLC.
Apr 16, 2012
There is absolutely no violation! with millions of different types of charged species, you cannot expect millions of different retention times. What you need to do is adjust the properties of the eluent and it will affect retention times of different species depending on their response to these changes. In IC, we use complexing agents (EDTA,crown agents e.g. 18-crown-6 ether)"to accelerate" the elution times of some species.
In my work I use gel filtration column(TOSOH-TSK g3000) to characterize dimer in proteins,
the column guideline suggest the pH range between 2.5-7.5
the recommended protocol is use phosphate buffer pH 7.4
but when I measure a HPLC water (use in cleaning step --from MERCK) the pH is around 8.39
Do I have to adjust the pH to close to acceptable range in the guideline?
What if i didn't adjust , if there anything worse happen to the column?
If I have to adjust the pH of DISTILLED WATER, which reagent will be the best choice?
I have two columns Atlantis and Symmetry (Waters). Earlier, I used Symmetry and my seperation wasn't great. Afterwards I used Atlantis and I got the seperation much better than symmetry but right now my seperation is disturbed. What is happening ?
Can you right here the Chromatographic conditions Like Mobile Phase, Buffer pH, Diluent etc so that I can help you to suggest equivalent column if possible send me on chromatogram of your analysis with detail Chromatographic conditions.
I used APcI MS in the distant past. The important element in the separation will be your choice of mobile phase additives and pH. Please refer to IUPAC guidelines for single laboratory validation steps - this document is very clear and consise in the approach. Good luck.
the concentration of the analyte can affect the retention time especially if you overload the column. But there are other factors that can affect RT also; Temperature,etc.. to better troubleshoot your problem - can you provide us with a few more details about the chromatography you using. Column type, dimensions, Buffers, type of metabolites.
We know that in TLC we use the simple or native amino acid but in case of HPLC first derivatization should be done by using any derivating agent or chemical. I have both the amino acid and derivating agent but don't know how to prepare reaction mixture and in which proportion. Any advice?
Mobile phase and conditions for HPLC analysis of lactic acid and acetic acid. I'm trying to analyse acetic acid and lactic by HPLC for flavour improvement. I used the following HPLC conditions, but couldn't get distinct peaks:
Waters C18 column, 5 μm, 416mm × 250mm
mobile phase: 0.01mol / L phosphate solution
pH = 2.5 (adjusted with NaH2 PO4)
flow rate: 0.6 mL / min
detection wavelength: 210 nm
injection volume: 20μL
column temperature: room temperature
I need help with the solvents and conditions. Thanks
I am experiencing a problem with HPLC analysis of lactic acid. I am getting two peaks with the standard solution of lactic acid that we bought from sigma aldrich. If anyone has experienced such problem or know any solution please do share! we would really appreciate !
Method for sugar extraction from juice for HPLC analysis.
Mar 1, 2012
NH2 column and RID . I also analyze Fructose , Glucose and Sucrose only . But excellent more if ELSD detector in the same injected volume can analyze much more. and how's the resolution ? acceptable. How many types of sugar can it detected?
The problem of precipitation is mostly found with phosphate buffers or biological buffers (TRIS,HIPS,MOPS, etc) in a high organic solvent eluent. Phosphate buffer systems are great but care should be taken when doing gradient runs and when flushing the column after the analysis. First wash the column with the same ratio of water:organic and thereafter prepare the column for storage as described above. Choosing buffers that are highly soluble in organic solvents (formic, acetic, and their ammonium salts) would eliminate the possibility of precipitation and you do not need to go through an extensive water:organic wash cycle before preparing the column for storage. Never leave a column or HPLC/UPLC system with buffer in the system. A last comment - when experimenting with a new buffer:solvent mixture it is a good idea to premix the mixtures you intend to use and leave these mixtures standing for a while to ensure that the buffer will not crystallize or precipitate out of solution on standing or cooling down.
Because this type of columns are generally used in standard in oficial methods. Methods that were develop several years ago where done with this type of columns. And nobody has taken the time and effort to update them (specially if they work). THere is no problem if you develop and validate your methods with smaller columns ( the smart way) since you will save time, solvents and money. Wich is great if you are working in a cash starved university and you will get home early
Hi I was separating carbohydrates by RP-HPLC. I used to get the solvent peak before the analytes and it was normal. All of a sudden, the solvent peak got inversed. I don't know why? Can anyone help me in this?
My laboratory received a donation of Waters HPLC system (Detector 2487, Multi-solvent delivery system, 600E series) from Seeding Labs, Boston last year but the equipment is not in use due to need of the software. Anybody who can can come to our aid?
Water's software is a propriety item. They have a security key system. If any of the water's 600 users (or any other system) wants to install the software, first he will De-install from previous computer system. They have two software Millennium and Empower. Now a days only Empower's various versions are available. Best for you talk to Water's local dealer for getting this software with complete detail or ask for visit first. If you have more than one system pl ask for multiple user version. If you need any help regarding operation of the same. You are most welcome.
I am looking for method of analysis of trimethyl benzene and trioctyl phosphate on HPLC.Is it possible to determine these componets on HPLC,I have the technique to analyse on GC but i am searching for HPLC?
Robin's method is fairly simple. A comparatively complicated and expensive one would be to use an agglutinin conjugated column. I think Shodex has a column specially for glucosamine. These have very high affinity for sugars. You can use a PDA at 210 or 280 nm. The mobile phase is typically a high concentration salt solution.
I am trying to develop a heparin affinity column method using a 100mM to 1M NaCl step elution. My protein of interest actually elutes at a 0.5M NaCl step, but though it elutes in a nice, sharp peak, it is not completely eluted: I get peaks in subsequent blank runs. I am using the lower end of recommended pH for mobile phases so all I can think of to get more complete elution is including a nonionic detergent like NP40 or Tween or CHAPS in my elution buffer. Alternate strategies or detergent suggestions are most welcome.
We also wash the column with 2 M NaCl between uses. The first use of the column frequently provides lower yields than subsequent runs as if there are some very high affinity sites that need to be saturated.
According to best of my knowledge these are alkaloids. These may be present in S-tylophorine was dextrorotatory and the (–)-isomer had the R configuration. You have separate in Chiral HPLC column because these are soluble in chloroform. You have try with normal phase HPLC system.
Check its solubility again. Then I can give you HPLC method. Try...
A good starting point would be to use 0.1% TFA in water with an MeCN (with 0.1%TFA) linear gradient from 0% to 60% over 40 mins, with a 5 min re-equilibration at the end. Monitor by UV at 214nm and at 40DegC. Depending on what you get, you can adjust the gradient parameters accordingly.
According to figure 2 and 3 of the paper your findings are in good agreement with the literature
References about longitudinal diffusion of solvents and compounds in stop-flow LC.
Aug 28, 2012
I'm looking for some informations about longitudinal diffusion of solvents, when using gradients, and diffusion of compounds. Especially their influence on the quality of separation according to the time the flow was stopped.
I can't find anything about solvent longitudinal diffusion, but it has been observed by people doing LC-NMR stop-flow experiments.
My personal experience in doing a lot of stop-flow LC-NMR is that diffusion happens a lot less than you might think. I sometimes doesn't happen even when you want it to. (I can explain this if you contact me.)
Your question is specifically about LC; my answer is specifically about LC-NMR. But from an LC-NMR perspective, you get amazingly little diffusion, even after stopping 24 hours (which horrifies most conventional LC people; even though they haven't tried it). Much of what they ascibe to diffusion is actually degraded separations caused by not maintaining system pressure.
Hi! I am working with method development of cephalosporins. I am using buffer and methanol in the ratio of 20:80. I am also using TEA for adjusting the pH. The method worked well but towards the end of the validation, suddenly both the analyte peak are showing tailing just at the end of the peak. Previously it did not happen. Kindly help me
One question, if you are using phosphate buffer at pH 7-7.2, why are you also employing TEA?? Phophate buffers in this range alone - remove the TEA. The main cause of tailing for basic drugs is the interac tion of the acidic silanols and any residual metal ions in the silica with the drugs. What you describe as a 2nd hump I don't believe is tailing.
Thouroughly clean your column with a 4 solvent wash overnight. The sequence is to run 20 column volumes each of 1.water, then 2. methanol, 3. THF, 4. chloroform or dichloromethane and finally 5. methanol for storage. Keep flow rates about half what you normally run at and allow a slight gradient when moving between water/methanol due to the rise in backpressure.
Having cleaned the column equilibrate with phosphate/organic and reinject your sample. You should always use a guard column - it is cheaper to replace than an analytical column!
Development and validation of rapid ion-pair RPLC method for simultaneous determination of certain B-complex vitamins along with vitamin C.
Patil SS, Srivastava AK.
SourceUniversity of Mumbai, Department of Chemistry, Vidyanagari, Santacruz (East), Mumbai 400098, India.
A rapid, simple, and accurate ion-pair RPLC method has been developed for simultaneous analysis of vitamin C and major B-complex vitamins. An RP C18 column thermostated at 30 degrees C was used with gradient elution of mobile phase comprising 10 mM potassium dihydrogen phosphate buffer (containing 3 mM sodium hexane-1-sulfonate, adjusted to pH 2.80 with o-phosphoric acid) and methanol at a flow rate of 1.0 mL/min to achieve the best possible separation and resolution of all vitamins in about 11.00 min. The detection was performed at 274 nm. The method has been implemented successfully for simultaneous determination of vitamins present in 12 multivitamin/multimineral pharmaceutical preparations, as well as in human urine. Typical validation characteristics were evaluated in accordance with International Conference on Harmonization guidelines. Good linearity over the investigated concentration levels was observed. Intraday repeatability was < or = 2.0%, and interday variation was < or = 2.6%, for all vitamins. The method can be used for assay of these vitamins over a wide concentration range with good precision and accuracy; hence, it would be appropriate for routine QC as well as in clinical analysis.
I currently run my sample in a mobile phase with 5% aqueous acetic acid and methanol [20:80], as per a protocol from the literature. Interestingly, I get some new compound peaks which have not been previously reported. I would like to collect the unknown compound when I run my HPLC sample and to determine the structure using NMR or GC. However, since my mobile phase contains acetic acid, I would like to know how to proceed?
Has someone used HPLC-ECD to analyse catecholamines in blood sample?
a) Do results differ between fresh samples and frozen samples? What is the reason for this?
b) Are there any QC samples we can follow to use?
c) Catecholamines are easily to degraded. How do you avoid this degradation, add antioxidant, EDTA or Cysteine?
We analyse brain samples which are flash frozen then thawed in PCA and sonicated. These samples can be frozen once at -80 but more than one thaw reduces signal, probably due to oxidation. We also use Sigma salts as standards.
In my method, the mobile phase consists of 70% Buffer and 30% MeOH. Drug standards were prepared in water (deionised). Why I am getting negative peak in first 3 minutes though the major portion of mobile phase if Water?
What type/size column and what is your flow rate? If your samples are in water and your mobile phase is a buffer such as acetate, then depending on your detection wavelength, you will get a water dip toward the front of your chromatogram. In most cases, you should have enough retention on your compouds of interest (k' >= 3) to insure that early eluters do not coelute with peaks of interest.
I agree with much of the above. If the peak is consistent in area and retention under the same conditions, then it is a compound that does not absorb UV to the extent that your buffered mobile phase does. The compound could be from your diluent, especially if you are not using a deionized (or equivalent) feed for your water, or it from the matrix of the original sample. Either way, if it is resolved from your peak(s) of interest, then don't bother investigating, unless your protocols demand an investigation.
If you must investigate, run water blanks to observe whether or not the water is the source. Diluent blanks should always be a part of each sequence, anyway. Then, run incurred sample blanks containing all consituents except for the drug susbtance. If, according to protocol, you must remove the peak from the chromatogram, and the diluent is the source, then try switching your diluent to match the mobile phase composition. You should, in many cases have a diluent that matches the initial conditions of your mobile phase, anyway.
If your diluent is water, I assume, under reversed phase conditions (C18, C8, phenyl, etc. column types) that your drug substances elute early, and may coelute with the negative peak. If this is the case, you should find another set of conditions by which to analyze your compound(s). For instance, aqueous normal phase (polar stationary phase) or HILIC mode separations work well for retention of very polar compounds (e.g., nuceosides, amino acids, small peptides). If reversed phase is giving you retention issues, try running an aqueous buffer:ACN system on a silica column (less expensive) to see if you can retain your compounds and gain selectivity over other components. If you decide to change to an aqueous normal phase, make sure your diluent matches the initial conditions.
I have NH2 columns: Сarbohydrate Analysis 3.9x300mm (Waters), Supelcosil LC-NH2 5um 4,6 mm x 25 cm, 5 mkm and YMC Pack NH2 300x6.0mm S-5 mkm, 12nm. I have to detect oligossaccharides from my glycoprotein after enzymatic digestion by PNGase F.
Can I see monosaccharides on these columns? Is it possible to see oligosaccharides using this columns?
ELSD has a non linear respons, so quantification requires standards at different levels. Moreover, the response of the ELSD decreases strongly when analyte concentrations are low. You should use ELSD only when you need to run a gradient, otherwise use RI detection.
There are two disadvantages of the NH2-column:
1) Reducing sugars react with the amino-group, resulting in partial loss of these sugars and modification of your stationnary phase.
2) Injection volume is severely restricted if the sample is dissolved in water, because water is a strong eluent (sugars display bad solubility in the eluent).
I wonder why the NH2-column is getting so much attention nowadays, because its use was ca. 25 years ago abandonned. The cationic ion-exchange columns using just water and RI-detection were suberb for simple mixtures of mono- and disaccharides and the anionic-exchange columns (Dionex) were the right choice for complicated mixtures and/or high sensitivity.
Dionex has several types of columns suited for (oligo)saccharide analysis, in combination with pulsed amperometric detection (PAD) you get very good separations and high sensitivity (ca. 100x more sensitive than RI or ELSD).
I recommend that you add more topics to your question in order to get more answers. I am sure there will be somebody in "Analytical Chemistry", "Environmental Analytical Chemistry", "Gas Chromatography (GC) and "GC-ECD" with a more specific answer to your question.
We are trying to develop a RP-HPLC method to analyse BSA in aqueous medium. I found the "Validation of Analytical Procedures: Text and Methodology" from ICH but does anyone know how to get the similar guideline from FDA? And where can I get the guideline from FDA's website (their website is so complicated)?
Jun 9, 2012
I attached one file. We follow these guidelines and I hope this will be helpful for u.
Few options to shorten the run time or to make the compounds elute faster
1. Increase the flow rate but sometimes it may not work when u have more than 2 products
2. Increase the strength of the Organic solvent either increasing its percentage or by replacing it with stronger oragnic solvent
3. changing pH [Mostly it may alter many things]
4. Change the column - Use shorter length column with less i.d. It ll make many things better but before using such column You shud take care about the pressure of system. So please let us know ur System and Method conditions.
The plant I'm focussing on is wheat and I want to analyze polyamins in it.
May 27, 2012
U need to Extract polyamins from wheat. Either use Solid Phase extraction method or Liquid Liquid Extraction. Better search for literature for sample preparation/extraction procedure for polyamins and optimize as per ur conditions.
Before extraction, u need to develop HPLC method using standards.
Has anybody got any experience using the Hi-Plex Na column with a ELSD for oligosaccharide detection? The mobile phase is water for the prescribed column if using RI detector according to Agilent's brochure, but I would like to know what are the operating conditions used if I were to use the ELSD HPLC?
Hi, thanks for your answers. I am interested to know if anybody has used the ELSD for analyzing oligosaccharides by a ligand exchange Na column and what are the operating conditions (e.g. nebulization temperature, mobile phase flow rate etc.) that I can use as a starting point?
Serotonin can directly solve in water, but it is not stable in the air and easy to be oxidized. Acidic condition, such as 0.1M HCl, plus some anti-oxidant will be better. See the sample preparation part of my chapter in a book:
Dear Huq, check out the whole tubing starting from Pump to the Column inlet for any air trapped, purge the pump for few minutes. If still you have the same problem, checkout the rest of tubing following the column outlet, and as Robin Joshi suggested it could be also due to blockage in column, so better remove the column and use "tubing joint" and then purge, I am sure the problem will be resolved iA.