Translator for HPLC HINTS and TIPS for Chromatographers

Thursday, December 1, 2011

Adduct formation in LC-MS Analysis (esp. ESI)

Almost everything you analyze by Electrospray ionization mass spectrometry will create an adduct with something in the system. Normally, hydrogen is the most common adduct formed (M+1), but other chemicals, often in trace amounts may form adducts with your sample too. Sometimes we can take advantage of this fact and introduce our own adduct into the system (post column) to increase signal sensitivity or help us isolate one signal from another (the addition of an adduct can sometimes increase the signal seen for one species, but not the other). 

One of my favorite elements to form an adduct with is sodium (Na+). Two common forms are; sodium citrate and sodium acetate. Both have PKA’s between 3 and 6 so a variety of buffered solutions can be prepared for use. However, it is very important that we keep the concentration of sodium as low as possible so as to not clog the mass detector or suppress ionization completely (and see nothing BUT Sodium for weeks …). My suggestion is to initially prepare the buffers such that the solution is equal to or below 3 mM in concentration. The lowest concentration should be used that yields reproducible results. Ranges from 1 mM to 5 mM are common. Only use the highest purity, volatile buffers (some manufacturer’s use names such as “ultra” to describe them) when preparing these ‘doping’ solutions for post-column addition and be sure and filter them through a 0.2 micron filter before use. A syringe pump can be used to deliver the solution during the run. A low flow rate should be used to infuse the adduct solution into the main inlet of the detector. Make sure you have a simple way of controlling the pump through the system (e.g. ‘On’ / ‘Off’, contact closure) so the flow can be turned off when you are not acquiring data.

Ammonium (NH4) is another popular adduct to add to the system, Often in the form of ammonium acetate. Whichever adduct you use in your system, always start off testing as low a concentration as possible. Monitor the baseline carefully for noise and also to see if the addition of the compound is suppressing or enhancing the signal generated for your compound. Careful use of adducts in your system can provide you with another means to selectively enhance the signal of some compounds without changing the original chromatography method.

I must again emphasize to use the lowest concentration of doping agent. Proper pH control and mode choice are also very important. Use of a syringe pump for infusion, post column can help you to quickly optimize the fragmentor settings in real time.

Tuesday, November 1, 2011

HPLC Solvent Properties Table (e.g. Polarity, Density, Boiling Point...)

Here is a link to a table listing common chromatography solvents and their physical properties (e.g. viscosity, polarity, UV absorption...). 

http://www.hplctools.com/lcsolvent.htm

Monday, October 17, 2011

HPLC PUMP SOLVENT COMPRESSIBILITY VALUES

Have you ever noticed excessive pump ripple (baseline noise) that is not caused by a defective check valve ? The ripple might be due to an incorrect HPLC Pump solvent compressibility setting.

We normally think of liquids as not being compressible in general. Hydraulic systems take advantage of this physical fact and many innovations have been developed using this concept. However, in high pressure liquid chromatography (HPLC) we routinely subject different liquids to very high pressures which can result in measurable liquid compression. The degree of actual compression varies for each liquid (see table). Though the amount of compression is very small, it is enough to change the flow rate of the system. When multiple solvents are mixed together at different proportions, such as is common when running a gradient, the measured flow rate can vary from the set flow rate during the entire run. This flow rate accuracy issue can be compensated for using the built-in solvent compressibility compensation software which is found in most modern HPLC systems. Many of these systems will allow you to manually enter the actual liquid compressibility values for each solvent (pump channel) used. This can result in better baseline stability and less pump noise. I would like to point out that the small improvement gained in performance is best implemented AFTER other major changes have been addressed first (i.e. such as fully degassing your solvents; filtering samples before injecting; selecting the best signal bandwidth and sampling rate values for your detector and insuring that your pumping system has received regular maintenance). 
 
Note how Water has a compressibility value of ~ 46, but a very common solvent such as Methanol has a value of 120. These two are very different. *Most pumps are pre-set with a compressibility value of '100'. A 50/50 mixture of the two run isocratically might benefit from a manually edited compressibility value of 83 [(46 + 120) = 166 / 2 = 83)]. *This is a best guess value as the best compressibility value for a mixture of liquids must be determined through actual experiments. Choose the value which results in the lowest pump pressure ripple and/or noise. 


SOLVENT COMPRESSIBILITY VALUES TABLE:

Solvent
Compressibility (10-6 per bar)
Water
46
Acetone
126
Acetonitrile
96
Benzene
95
Carbon Tetrachloride
106
Chloroform
100
Cyclohexane
113
Dichloromethane
99
Ethanol
112
Ethyl Acetate
113
Heptane
144
Hexane
158
Isopropanol
100
Methanol
120
Tetrahydrofuran
97
Toluene
90

Notes: 
(1) The values shown above are approximate and assumed to be accurate. They were recorded at a temperature of 20C (Reference: Handbook of Chemistry and Physics #90). Various grades/purity of solvent may have different compressibility values so please verify the values of your own solvents before use. These should serve as a general guideline only.

(2) The variation in pressure which occurs between the pump piston compression and decompression strokes are sometimes reported by the pump's electronics to aid in troubleshooting. Agilent/HP brand systems refer to it as the pressure "ripple" (should be less than 0.5 %) and Waters brand systems report the calculated ratio, "Compression / Decompression Ratio" value using this guideline [1.0 - 1.4 = Normal; 1.4 -1.8 = Fair; > 1.8 = Possible Bubble]. In all cases, continously degass all liquids and input the correct compressibility values for each mobile phase solution to achieve the most stable flow.

Friday, September 30, 2011

UV / VIS, VWD, DAD, PDA HPLC DETECTOR SIGNAL BANDWIDTH (bw):

Modern chromatography UV/VIS detectors offer the operator a choice of one to several hundred different signal wavelength choices (as is the case for Diode Array Detectors). Besides being able to specify a single wavelength, you often can choose a signal bandwidth (bw) to associate with each wavelength [e.g. for a 280 nm signal with 10 nm bandwidth this is often written as: 280 (10) or 280:10]. Signal Bandwidth is the total number of nanometers across the specific signal value chosen. For example: If you selected a signal wavelength at 280 nm and chose a bandwidth value of 10 nm, then you are actually gathering all signal data from 275 nm to 285 nm (5 nm to the left of the apex and 5 nm to the right for a total of 10 nm). Using a narrow bandwidth has the advantage of increasing the signal selectivity of the detector as you are only collecting data within a tight window. If you were to increase the bandwidth to 60 nm in the same example you would now be collecting data starting from 250 nm through 310 nm. The additional data collected over this wide range can reduce the total noise (by averaging it over a wide range), improve the S/N ratio (increases sensitivity), but it also reduces the selectivity. Large bandwidths also increase the chance you may include peak signal data from other co-eluting components into your signal data. You must select a bandwidth range which is always 'safe' from any other potential peak. As with many things in life balance is important. In this case, the balance between selectivity and sensitivity.

  • When developing new methods we recommend that you choose an initial bandwidth value of 10 nm for each signal. This provides a nice balance between selectivity and sensitivity. It is also a common bandwidth value used on many UV/VIS detectors which have a fixed signal bandwidth (such as many single or variable wavelength detectors).

  • If you have determined the exact signal maximum for your sample and you would like to gain additional sensitivity for your sample (and thus decrease selectivity), re-run the analysis using several different, but increasing signal bandwidth values (e.g. 10, 20, 30, 50 and 100 nm). Choose bw values that are safely within the range of the detector, within the limits of the mobile phase's absorption region and also away from any potential co-eluting peaks. *To confirm which value is best, be sure and calculate the actual measured signal to noise ratio of the peak of interest after each analysis. This is a critical step! Do not be fooled by increases in the peak height or area alone as these changes are not always synonymous with better signal to noise ratios. Only by measuring the actual baseline noise level for each run and comparing it with the actual peak signal obtained will you be able to determine if increasing the bandwidth has provided you with better noise reduction and signal strength.

  • To increase spectral signal selectivity choose a bw value that is very narrow. A value such as 2 or 4 nm would allow the detector to collect only signal data that is at or near the apex of your selected wavelength. This can be very useful when trying to discriminate your signal from nearby signal peaks, especially at low wavelengths such as 210 nm.

  • When reporting your method conditions always include the wavelength AND bandwidth used for each signal. In order to accurately reproduce your method, this information is needed. *It should always be included in your method.

Wednesday, September 14, 2011

How Do C18 HPLC Phases Differ ?

Reversed phase HPLC columns which utilize the octadecyl functional group often differ in many ways. Besides the particle size, shape of the stationary phase (irregular or spherical), the coating chemistry and end-capping used. Two other  very important ways that columns can differ from one another are in their available surface area and the extent to which those surfaces are covered with the phase coating (i.e. covalently bonded, non-covalently coated and the total carbon %). When comparing columns for use in validated methods, be sure and consider these factors to minimize the number of changes to your method. Always test several columns of the same type to determine the batch to batch reproducibility and variation. Some manufacturers have mastered the art of preparing and packing columns which achieve high batch to batch reproducibility. After all, what good is a column in your method if the results are not reproducible when you purchase another column some time later?

Friday, August 26, 2011

Pressure Drop Across an HPLC / UHPLC Column

Many of you prefer tables of data over equations that you must work out. So, instead of providing you with another equation, I have done some basic measurements for you to provide a general overview of how particle size (porous) effects backpressure.

For simplicity, let us start with a few parameters. Pore Volume = 0.70; Linear Velocity = 1.44 mm/sec; Solvent Viscosity = 0.89 cP at 25C (Water). 

Pore Volume and Flow Resistivity will vary by column type. Obviously the back pressure will be higher with more viscous solvents (e.g. EtOH is 1.20 cP) and lower with less viscous solvents (e.g. ACN is 0.34cP). A Table of HPLC Solvent Viscosity values can be found here [ http://www.hplctools.com/lcsolvent.htm ]. Linear flow rates have been used for all column I.D.'s to better illustrate the relationship between column dimensions and flow rate. If you double the flow rate, then the pressure will approximately double as well. 

Note that when run at traditional linear velocities, most 2.5u particles are within the maximum pressure limits of most HPLC systems (under 400 bars). Only the newer sub 2.0 micron particles used in long columns exceed the 400 bar limit. The higher maximum pressure limits of many UHPLC systems allow the use of higher flow rates with these particles. Naturally, you should optimize both column efficiency and system dwell volume when developing any UHPLC method. Meeting any/all backpressure requirements to run a method does not translate to success in sample analysis. Successful ultra-fast separations require ultra-low system dwell volumes, higher sampling rates and usually smaller flow cell volumes.

Column I.D. (mm)
Particle Size (u)
Column Length (mm)
Flow Rate (mL/min)
Back Pressure (Bars)
4.6
5
250
1.000
89
4.6
5
150
1.000
54
4.6
5
100
1.000
36
4.6
5
50
1.000
18
4.6
3.5
250
1.000
182
4.6
3.5
150
1.000
109
4.6
3.5
100
1.000
73
4.6
3.5
50
1.000
36
4.6
2.5
250
1.000
357
4.6
2.5
150
1.000
214
4.6
2.5
100
1.000
143
4.6
2.5
50
1.000
71
4.6
1.9
250
1.000
618
4.6
1.9
150
1.000
371
4.6
1.9
100
1.000
247
4.6
1.9
50
1.000
124





3.0
5
250
0.430
90
3.0
5
150
0.430
54
3.0
5
100
0.430
36
3.0
5
50
0.430
18
3.0
3.5
250
0.430
184
3.0
3.5
150
0.430
110
3.0
3.5
100
0.430
74
3.0
3.5
50
0.430
37
3.0
2.5
250
0.430
361
3.0
2.5
150
0.430
217
3.0
2.5
100
0.430
144
3.0
2.5
50
0.430
72
3.0
1.9
250
0.430
625
3.0
1.9
150
0.430
375
3.0
1.9
100
0.430
250
3.0
1.9
50
0.430
125





2.1
5
250
0.210
90
2.1
5
150
0.210
54
2.1
5
100
0.210
36
2.1
5
50
0.210
18
2.1
3.5
250
0.210
184
2.1
3.5
150
0.210
110
2.1
3.5
100
0.210
73
2.1
3.5
50
0.210
37
2.1
2.5
250
0.210
360
2.1
2.5
150
0.210
216
2.1
2.5
100
0.210
144
2.1
2.5
50
0.210
72
2.1
1.9
250
0.210
623
2.1
1.9
150
0.210
374
2.1
1.9
100
0.210
249
2.1
1.9
50
0.210
125