Chromatography Forum

We are planning to buy an HPLC that can perform both standard flowrate and Fast LC analysis. The above three systems are the ones that we have been looking at so far. Anyone among you who have these systems in your lab? Which one is better? What are the advantages and disadvantages? Any problem that you have encountered using them? Which one ives the best precision especially when running at low flowrates, like let's say, 0.3ml/min? We pan to hook it up with our Mass Spec.

I would really appreciate all te comments that you can give me. just want to be sure we ae buying the best one. Thanks alot!

One option could be to check out the Merck Chromolith Fastgradient RP-18 column 2mm. Super low pressure, super fast, makes a HPLC act like a UPLC. Works great in all low dead volume LC instruments. Flow rates from 0.2- 1mL/min make them great for LC/MS.

If for example you have an Agilent 1100 system, you can get an upgrade kit to reduce any dead space volume and then use this column, perhaps in liu of buying a UPLC/UHPLC.

http://www.vwrsp.com/catalog/product/in ... m_search=1

UPLC Waters: PSI max = 15000, system volume = 80 µl
1200 RRS Agilent: PSI max = 8 700, system volume = 183 µl

Concerning these informations, UPLC is better but I reveived these informations in a Waters training so I don't really know if they are right. Can someone confirm?

Another piece of unconfirmed information I have received is the following. Can anyone confirm it to be true?

Concerning UHPLC:

Thermo has had a system on the market for about 2 years, as does SSI. The Waters UPLC system pressure limit of 15000 psi (1034 bar) is good only up to 1 mL/min; at 2 mL/min (max flow), the pressure limit is 9000 psi (620 bar); at flows between 1 and 2 mL/min, the pressure limit varies proportionately between 9000 psi and 15000 psi. The Jasco, on the other hand, has a pressure limit of 15000 psi regardless of flow (2 mL/min max).

best wishes,

Rod

We are planning to buy an HPLC that can perform both standard flowrate and Fast LC analysis

when you say this you mean that you want to use both 4-4.6 mm ID column as well as 3 and 2 mm columns?
if so then you will need to look into several major factors:
1. the gradient delay volume- in particular the mixer chamber size if there is any. in order to use the 4-4.6 columns you will need a greater delay volume, otherwise the baseline will look like the waves on the beaches of Hawaii. TFA aplications will look nicer with a bigger mixer size.
2. the injection volume possible from the system autosampler. you will need an autosampler capable of doing 1-50 ul at least, and that is good at the low end between 1-5 in respect to RSD.
3. the type of detecor you will use and the speed of collection rate possible.

i would not worry too much about the flow rate.
all vendors pump can deal with 0.3 ml/min, and if you wish to use 2 or sub 2 micron particel size columns then you will have to push the flow higher in order to be in the optimal range of flow for the particle size which is more then for 5 micron columns.

the waters UPLC comes ready to use only for their columns which they will try to convince you are the best anyway .
you will find out that they can offer you standard connections to the competion's column.
unless you find away to get the waters connection to their columns then you won't be able to connect them to other vendors system. anyway for most systems you won't be able to use them since they generate so much pressure compare to other vendors systems limit (agilents is 600, shimadzu 660 bars)
also check if those system have a pre-heating system in the column oven. without it if you go higher then 40 degress the peak will start to look ugly.

currently there are several vendors that propose some sort of UHPLC instruments that i know of:
waters
agilent
shimadzu
dionex
hitachi
thermo
jasco.

Thank you Bruce, Kwet, chromatographer1, and unmgvar for your replies.

Apart from what you all said in your replies, may I ask how is the performance of your Waters UPLC if you have it in your lab? Any problems encountered? If you have the UPLC, do you have it attached to an API mass spectrometer? I am interested if the Analyst software can really work smoothly with the Waters UPLC system.

Dear unmgvar,

I plan to use the uplc system for columns with particle size between 1.8 to 5 microns, because we have different users which may have different column particle size.

Jasmine,
you are heading for a very broad range of column packings. I assume that you are also interested in supporting a broad range of column formats. If this is correct, you should base your decision on the pressure, flow rate, and column support characteristics of the different instruments rather than judging on the pressure alone. The detector data rate is important, too. The higher the data rate, the better you can integrate very narrow peaks. As I am from Dionex, let me only summarise flow, pressure, and DAD data rate characteristics (best comparable between these systems) as well as known column format limitations. You may judge what fits your requirements best.
These systems you mentioned in the topic:
Waters Acquity:
Maximum flow rate: 2 mL/min, Pressure Limit: 1000 bar for flow rates up to 1 mL/min (as stated earlier), linear may pressure drop between 1-2 mL/min to a maximum of 600 bar at 2 mL/min, 80 Hz max DAD data rate, max column length 150 mm.
Thermo Accela:
Maximum flow rate of 1 mL/min, max pressure 1000 bar, 20 Hz max DAD data rate
Agilent 1200:
Maximum flow rate: 5 mL/min, max pressure 600 bar, max data rate 80 Hz
Dionex UltiMate 3000 Rapid Separation System:
Max flow rate: 5 mL/min, max pressure: 800 bar, max data rate: (DAD and VWD) 100 Hz, max column length 300 mm
In addition, the RSLC system has other outstanding specs for speed and flexibilty such as 15 s injection cycle time, up to 100 µL injection volume even at 800 bar, column oven temperatures up to 110 °C, instant results with Chromeleon dynamic data processing and its powerful reporting options, ...

I did not realize earlier that you are particularly interested in the integration of the LC control and Analyst. You might be happy to hear that Dionex offers a plug-in for Analyst that integrates the UltiMate system control. When using DCMSLink, all instruments are controlled and all data is stored, reviewed, quantitated and reported by Analyst. DCMSLink is offered free of charge for Dionex instruments.

Cheers,

Holger

I can confirm the information from Kwet, but I am not sure if it becomes more believable .

The detector cell volume of the UV detector of the UPLC is as little as 500 nL, also unmatched by other instruments.

unmgvar: I do not see why one needs a greater mixing volume for a 2.1 mm column than for a 4.6 mm column. I am sure that it won't hurt, but I also do not see the need. If the mixing works well at 1 mL/min for a 2.1 mm column, there is no reason why it will not work for the 4.6 mm column at 1 mL/min.

The loop size of the autosampler can be adjusted for the column that you want to use. I recommend the smaller sizes for the smaller i.d. columns. This is not so much a question of RSD (an injector designed for 2.1 mm column delivers the expected RSD for such a column), but a question of bandspreading. A larger column is more tolerant to large injection volumes than a smaller i.d. column.

The UPLC connects without difficulties to other vendor's columns.

I agree that the thermal environment and column preheating is an important part of the quality of a method.

Besides the small size of the detector volume, a high sampling rate is important for true rendition of the narrow UPLC peaks. As pointed out above the maximum sampling rate of the UPLC is 80 data points per second.

Thank you unmgvar, Hfranz and Uwe for your replies.

They gave me more info to think about regarding what's needed for buying a fast LC system.

Uwe, may I ask for some information about the possibility of hooking up the Waters UPLC with an API mass spectrometer? How is the acquity software on the UPLC integrated with the Analyst software of the API mass spectrometer? Have you seen any system with this combination working without much problem?

Thank again to all of you.

The loop size of the autosampler can be adjusted for the column that you want to use.

This is a very general statement, but you certainly talk about the Acquity system. Jasmin is interested in conventional column formats which take easily injection volumes of 50 µL and more . It might be interesting for her and us: Can these large volume injections be handled at all by this sampler and at the full range of the system pressure? How long does the injection cycle take with the Acquity sampler? How much sample volume is required?

The major benefit of sub 2um particle LC is the significant increase in resolution produced as a result of the high efficiency of the columns and the low dispersion of the UPLC chromatography system. The increased resolution significantly reduces chromatographic co-elution in complex mixture analysis or when the sample matrix is complecated. The increased resolution reduces ion suppression and this combined with the sharper peaks produced by the LC system results in a 3-9 fold increase in sensitivity. The reduction in ion suppression produces also improves analysis reproducibility.

To realize this increase in performance it is critical to control the peak dispersion in the LC system. Pressure is really a side issue as the ACQUITY UPLC system has been designed and tested to routinely run at these higher pressures.

A good reference is the paper by Houghton and Grace in Chromatography today Feb 2008

Pressure is really a side issue as the ACQUITY UPLC system has been designed and tested to routinely run at these higher pressures.

Well, I guess all systems mentioned in this discussion are designed to work within their defined, high pressure range. Sounds very much as if you work for Waters but you do not tell.
Acquity has been designed for high pressures and small volume columns, but can it be considered an all-purpose system with its flow, mixing, column dimension, and injection capabilities? This is what I wanted to understand better.
Thanks for summarising the benefits of small particles, which is the basis for fast analyses regardless of the manufacturer.

Jasmine,

The UPLC system can be hooked up to the API MS running under Analyst. Talk to your sales rep about the details.

A loop size of 50 microL is available for the UPLC instrument. This does easily accomodate standard injection volumes on a classical HPLC column, which also will not reach UPLC pressures, but only HPLC pressures, even at 2 mL/min. However, the system has been designed primarily for UPLC, and not for preparative chromatography...