HPLC HINTS and TIPS FOR CHROMATOGRAPHERS

Techniques To Enhance Negative Mode ESI in LC/MS

2012-02-21T08:00:00.003-05:00

Many sample types require MS analysis using negative ion electrospray mass spectrometry (ES MS) mode. Sensitivity can be less in this mode, compared to positive mode, as they employ mostly non-polar solvents which do not promote ion formation. In the past we discussed how adduct formation can be employed to enhance ion formation. There are other techniques which can be used as well. Here are a few tips which can be used to improve the quality of the signal obtained under negative mode ES MS conditions.

(1) Negative ion formation and signal response can be improved by choosing the right solution chemistry. One method to improve solvent desolvation and reduce the electrical discharge (noise) is to add isopropyl alcohol (2-Propanol) to the mobile phase. Reported in the literature nearly twenty years ago, as little as a 10% solution has been found to increase the signal level and decrease the noise level under many conditions. The addition of methanol to the mobile phase also can improve the signal, though usually not to the same degree as IPA.

(2) Another technique used to improve ionization involves the pH of the solution or mobile phase used. Higher pH values are often better. Post column addition of a basic solution which adds proton acceptors to the mix, such as ammonium hydroxide (20 or 30 mM), can also improve signal response and stability.

(3) APCI mode: If running nonpolar to semi-polar small molecular weight compounds, especially those which are volatile, this is still the source of choice. *Conventional ESI mode is best for polar to neutral molecules in small to large Mw ranges.

Popular Microsoft Windows® Short Cuts:

2012-01-03T08:00:00.001-05:00

Here is a short list of some of the most popular Windows short cuts. Hopefully, you will spot a few that you do not know about and can use.

Windows Keyboard Logo Symbol + L: Lock the computer (without using CTRL+ALT+DELETE).

ALT+TAB: Easily switch between open programs.
ALT+F4: Quit the program.
ALT+F4: Closes the current window.

SHIFT+DELETE: Delete an item permanently.
SHIFT: Press and hold down the SHIFT key while you insert a CD-ROM to bypass the automatic-run feature.

CTRL+ESC: Open Start menu.
CTRL+C: Copy.
CTRL+X: Cut.
CTRL+V: Paste.
CTRL+Z: Undo.
CTRL+B: Bold Font.
CTRL+U: Underline Font.
CTRL+I: Italic Font.
CTRL+Z: Undo the last command

A Full List of Microsoft Short Cut Commands Can Be Found In This Key Directory (Listed by Application): http://support.microsoft.com/search/default.aspx?query=Microsoft%20shortcut%20commands&catalog=LCID%3D1033

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

2011-12-01T08:00:00.014-05:00

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 prepare the buffers such that the solution is below 3 mM in concentration. The lowest concentration should be used that yields reproducible results. Only use the highest purity 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.

HPLC Solvent Properties Table

2011-11-01T08:00:00.000-04:00

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

HPLC PUMP SOLVENT COMPRESSIBILITY VALUES

2011-10-17T08:00:00.032-04:00

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

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.

UV / VIS HPLC DETECTOR SIGNAL BANDWIDTH (bw):

2011-09-30T08:00:00.000-04:00

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 at 250 nm and continuing through to 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 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 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 and also away from any potential co-eluting peaks. *Be sure and calculate the actual 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.

How Do C18 HPLC Phases Differ ?

2011-09-14T08:00:00.012-04:00

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?

Pressure Drop Across an HPLC / UHPLC Column

2011-08-26T08:00:00.000-04:00

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 the calculations for you.

To create this back pressure table, I had to make a few assumptions. Here they are: Pore Volume = 0.70; Flow Resistivity = 1,000; 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. Naturally, you should optimize column efficiency when developing a method. The higher pressure limits of many UHPLC systems allow the use of higher flow rates with these particles.

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

Conversion Factors microgram, nanogram, ppm, ppb and percent:

2011-08-01T08:00:00.002-04:00

Many years ago I made up this handy sample concentration conversion table . I find that it is still useful when reporting results to clients.

Parts per million (ppm)	Percent (%)
1	0.0001
10	0.001
100	0.01
1,000	0.1
10,000	1.0
100,000	10.0
1,000,000	100.0

ppm = ug/ml

1,000 ug/ml	=	0.1 %	=	1,000 ppm
100 ug/ml	=	0.01 %	=	100 ppm
10 ug/ml	=	0.001 %	=	10 ppm
1 ug/ml	=	0.0001 %	=	1 ppm
100 ng/ml	=	0.00001 %	=	100 ppb
10 ng/ml	=	0.000001 %	=	10 ppb
1 ng/ml	=	0.0000001 %	=	1 ppb

ESI Charge State Calculator Application

2011-07-15T16:15:00.004-04:00

Here is a neat program, created by Robert Winkler, which allows you to calculate the Mw of proteins using data from your electrospray ionization (ESI) mass spec source.

ESIprot 1.0 [http://www.bioprocess.org/esiprot/]

Reference this journal article: Winkler R. "A universal tool for charge state determination and molecular weight calculation of proteins from electrospray ionization mass spectrometry data." Rapid Commun Mass Spectrom, 24(3), 285-294, 2010.

HPLC to UHPLC Conversion Notes

2011-07-04T08:00:00.011-04:00

The use of ultra-high performance liquid chromatography (UHPLC) columns to reduce analysis times and sometimes improve detection limits is a hot topic. UHPLC presents a number of new issues. The incorporation of smaller 1.9 to 3.0 micron particles and smaller frits will raise backpressures and increase system wear and tear. Smaller diameter lines are often used (I.D. of 0.12mm or less) which can increase blockages and clogs if you do not filter your mobile phase and samples through a 0.45 or 0.2 micron filters. Piston seals and valve rotors can wear out early due to the very high pressures, heating and stress imposed on them. You should monitor your HPLC system carefully over time and consider increasing the frequency of preventative maintenance and inspection services as well. However, the smaller particle sizes can provide better resolution in some applications so they are well worth evaluating.

I must answer twenty or so questions each week in the area of UHPLC. The most common questions deal with selection of an UHPLC column and making adjustments for the changes in: (1) Column Dimensions; (2) Flow Rate and (3) Injection Volume. The good news is that all of these questions can be answered with some basic math.

(1) COLUMN DIMENSIONS: Let's start by making things as simple and brief as possible (this is supposed to be a "hint & tip", not a thirty page article). When initially converting from a convention HPLC column (5 micron) to an UHPLC column (1.9 to 3 micron particle) select a column with the same I.D. and length. This way only the particle size changes. *I like to change one variable at a time. If you would like to change the column length to take advantage of some of the increased efficiency (and decreased pressure!) which results from smaller particles, then please refer to the following equation.

     EQUATION A: 'Lc2' = ('Lc1' * 'p2') / 'p1'

[ 'Lc1' = Length of Column #1 in mm; 'Lc2' = Length of Column #2 in mm; 'p1' = particle size of Column #1 in microns; 'p2' = particle size of Column #2 in microns].

   Example: Column # 1 is a standard HPLC column; 4.6 mm x 250 mm (5u) Column. You want to find out the length of an equivalent column which uses 1.9 micron particles instead of the 5 micron particles.

   'Lc2' = (250 * 1.9) / 5 ; Answer is: 'Lc2' = 95 mm. *So a 10 cm long column would be a good choice here.

(2) FLOW RATE: Flow rate is directly proportional to column diameter and as we saw above in Equation A, the particle size can also affect it too. If you keep the column length and internal diameter the same, then the linear flow will be unchanged with the same particle size. A change to the particle size alone will change the flow rate as follows: 'Fc2' = 'Fc1' x ('p1'/'p2').

A change to a smaller diameter column to compensate for the improved efficiency will require a change to the original flow rate to preserve the linear velocity. Please refer to the following equation.

     EQUATION B:   'Fc2' = ('d2' / 'd1')^2 * 'Fc1'

['Fc1' = Flow Rate of Column #1 in ml/min; 'Fc2' = Flow Rate of Column #2 in ml/min; 'd1' = Column #1 Diameter in mm; 'd2' = Column #2 Diameter in mm].

   Example: Column # 1 is a standard 4.6 mm ID Column. You want to find out what the linear flow rate should be if you use a smaller diameter column (2.1mm in this example).

   'Fc2' = (2.1/4.6)^2 * 1.000 ; Answer is: 'Fc2' = 0.208 ml/min. *A flow rate of 200 ul/min would be fine.

(3) INJECTION VOLUME: A change in the column dimension may require a change to the injection volume. The smaller the internal volume of the column, the smaller the injection volume. To calculate the linear change in volume, please refer to the following equation.

     EQUATION C:   'V2' = 'V1' * {('d2'^2 * 'L2') / ('d1'^2 * 'L1')}

['V1' = Injection Volume #1 in ul; 'V2' = Injection Volume #2 in ul; 'L1' = Column #1 Length in mm; 'L2' = Column #2 Length in mm; 'd1' = Column #1 Diameter in mm; 'd2' = Column #2 Diameter in mm].

   Example: Current injection volume is 10 ul. Column # 1 is a standard 4.6 mm ID x 250 mm Column. You want to find out what the equivalent injection volume should be for a 2.1 mm ID x 150 mm column.

   'V2' = 10 * (2.1^2 * 150) / (4.6^2 * 250) ; Answer is: 'V2' = 1.25 ul.

Some Stereochemistry Terms (Chirality)

2011-06-19T08:00:00.004-04:00

Some of the common terms used in chiral chromatography often need to be reviewed. Here are some of the most common ones we encounter in the chromatography lab each day. There are many detailed chemistry books on the topic, but I will try and present some simple explanations for each here.

"Structural Isomers": When two compounds have the same chemical formula, but different structures, then they are referred to as structural isomers. AKA "Constitutional isomers".

"Stereoisomers": When two compounds have the same chemical formula and are connected to the same molecules, then they are referred to as stereoisomers.

"Enantiomers": When two compounds have the same chemical formula and are connected to the same molecules (they are stereoisomers), but are also non-superimposable mirror images of each other, then they are referred to as chiral enantiomers. *This is another class of stereoisomer.

"Diastereomer": When a stereoisomer compound has two or more chiral centers which are not mirror images of each other, then the compound is a diastereomer.

"Meso Compounds": When a stereoisomer appears chiral due to the presence of more than two chiral centers (but is in fact not chiral), but is superimposable on its mirror image, then it is a meso compound. These are interesting forms and they are optically inactive because the two opposites cancel each other out (no +/- signal).

Chiral HPLC and SFC Column Screening Strategies:

2011-06-01T07:00:00.001-04:00

We are experts in chiral separations and have learned a great deal about how to efficiently and quickly identify the best conditions to resolve a chiral sample by HPLC and SFC. You should always have a clear strategy in mind when developing an automated HPLC or SFC chiral method development screening system to separate racemic samples. Here are a few key points to focus on.

(1) Make sure the chiral sample is as “chemically” pure as possible before you start. By “chemically” pure I mean it should only contain the two complementary enantiomers and not other impurities, starting materials or chemicals which might interfere with the identification or separation of the racemate. Often, many of the intermediate chemicals used in the synthesis of a compound have similar absorbance or retention characteristics. When this happens, you can be fooled into thinking you have resolved your chiral component apart when in fact you have simply resolved a non-enantiomer apart from the racemate. Chiral columns are very poor at discriminating the racemate from other non-chiral species. As such, you may find that the impurities or other components make it difficult to determine the actual HPLC or SFC purity of your chiral sample. Try and remember this phrase: “The chiral purity of a sample is only as good as the chemical purity”. So start with as clean a sample as possible when developing a chiral method.

(2) Column choices are very important when using a fully automated Chiral Screening System such as the LC Spiderling™ Column Selection System. Here are some popular questions we are asked on this topic.

How do you know how many and which types of columns to include in your chiral screening system?

Keep the goal of creating a "screening system" in mind. Don't lose sight of this basic strategy. You want to select the smallest number of different columns which have the widest specificity for your expected sample types. Often five (5) to nine (9) different types of chiral columns will do the job. Screening more columns than that is often a waste of time. Leave a “test” position in your screening system so you can evaluate new columns all of the time. The key is to identify the best ones first.

Normal or Reverse Phase Columns?

Most chiral screening is performed in the normal phase mode (NP provides easy solvent removal for scale-up and there are a wide range of quality columns available which can be used to generate useful data). You can mix reverse phase and normal phase columns in the same system as long as you incorporate a bypass and flush step between the different methods to wash out the old solvent and bring in the new solvent safely. For this brief discussion, we will assume you have a dedicated normal or reversed phase screening system.

How many different mobile phase systems should I use?

Quick answer is as few as possible. The concept of using a screening system is to quickly identify the column (1^st) and mobile phase (2^nd) which baseline resolves the racemate. Select three or four different mobile phase or modifier systems which span the range of polarity needed to increase your chance of retaining the sample on the column, but for no longer than 30 minutes.

Which types of chiral columns are best?

Well, if you ask the different column suppliers this question, then they will most likely answer that the columns “they sell” are the best. At last count, there are over two hundred different chiral columns advertised on the market today. Most are advertised to be the 'best', but in fact they all can not be… In reality, you should evaluate as many different kinds as possible with your own samples to determine which the “best” are. Do not be fooled by examples of the column separating out very simple compounds such as racemic trans-Stibene oxide as this is one of the easiest compounds to separate even if 90% of the chiral stationary phase is missing! Consider also if the column type can separate compounds without the use of fancy modifiers or complex mobile phase mixtures. Simple is better and usually more easily reproduced too. Some of the most commonly used types of chiral columns are the: cellulose/amylase, Protein, Pirkle type and cyclodextrin based columns. All these columns have different preferred mobile phase choices (Most protein columns are run in reverse phase, while all of the others mentioned have versions which can be run in either normal or reverse phase). You should consult with the appropriate manufacturer about how to best use these columns. Do not necessarily select a column because the column has been “reported in the literature to be used by the largest number of people”. Who cares how many people use the column. We routinely see people who develop methods on a particular column and publish their results with it. That alone does not mean that their method or column choice was a good one. Invest some time learning about and evaluating the different columns with your own samples to find out which are best. Ignore the potentially sales biased recommendations and select a chiral column based on your own scientific evaluation and testing. Start with a "full size" columns to maximize the amount of stationary phase the sample comes in contact with. We do not use or recommend the short "scouting" columns available from some suppliers for screening purposes as they will often cause you to miss identifying a column type that works.

Can you provide some free advice as to which columns are the “best”?

OK, we have tried them all (but not for your samples), but here are two of our favorites (in no order):

The Pirkle based Whelk-O 1 (and/or Whelk-O 2) is the only Pirkle based column we have ever used which can produce a significant quantity of racemic separations using simple mobile phase systems. No other Pirkle based chiral column has ever proven to be as good as this one in real world chiral pharmaceutical drug method development. Every other one tried has disappointed us, but this one has been responsible for a number of successful separations in normal and reversed phase modes.

The coated polysaccharide chiral stationary phase made by Daicel, Chiralcel OD (OD-H) is one of the best chiral columns on the market. Note that this is the non-covalently bound coated version of the column. It has a broad range of selectivity not seen in any other types of columns available. At this time, the covalently bound versions just do not measure up to this one.

Power and Surge Protection for Computers & Analytical Instruments

2011-05-15T08:00:00.005-04:00

(1) When selecting an Uninterruptible Power Supply (UPS) for your computers and analytical instruments, only purchase one which outputs a true sinewave voltage (just like real AC power). Most of the UPS systems sold at the local office and electronic supply stores output either square wave A/C or pseudo-sine wave (which is not the real thing). Some brands will tell you they offer true sinewave outputs right in the model number or package, but always double-check the specifications (e.g. APC's "Smart-UPS" offer sinewave outputs, but NOT the Smart-UPS SC models). Using a UPS that outputs something other than true sinewave power can damage the power supply of what it is attached to and this can lead to premature failure of system you are trying to protect.

(2) Calculate the maximum load of the system being protected in Volt Amps (VA) and purchase a system that can provide power well above that load for the time period you want to protect (*I think 20 or 30 minutes of run time, at load, is a realistic guideline to go by). A 700 VA UPS will usually power most desktop computers and a monitor for that amount of time. Most brownouts last for just seconds, but that is enough time to shut everything down or loose communication. If a power outage is more than five minutes in length, then it may be a while before it is restored. In this case, be sure to safely shut everything down manually before you run out of battery reserve power. Once everything has been turned off, remember to turn OFF the UPS system to stop it from slowly draining the battery (most computers are still turned ON and using power when they are OFF). Doing so will preserve the remaining battery capacity. Just turn the UPS back 'ON' when power has been restored.

(3) Unless you want to spend really BIG dollars, just put a high quality UPS system on the computer. A high quality UPS system designed for a single high current draw instrument like a chromatograph may cost thousands of dollars. If the system must be maintained at all times, then a serious back up source of energy should be invested in. High current UPS systems are available from a number of manufacturers so it would be wise to investigate these for your application. For everything else, use a dedicated, high quality power conditioner and surge suppressor on the analytical instrument circuits ($85 and up each, not the $20 models sold at most box stores). The computer system is really what you want to protect first. If the computer gets knocked out you loose communication and control of the analytical system. This usually means it will continue to run on and on without stopping. If the computer is protected by a UPS and the power gets glitched (such as a voltage drop/brown-out) or goes out entirely, then the computer may sense this and abort the run or shut the system down (if it has not been shut down). This is the type of approach we have used in our labs for twenty years and it works great during brown outs and black outs. The computers stay on long enough to properly and safely shut down everything without loosing data (which is the key). The instruments are all connected to individual power conditioners/surge suppressors (Tripp lite brand units in our case) so are protected from brown outs and surges. To protect our equipment from large surges, our electrician installed a high amperage DELTA lightning arrestor (100,000 AMP, 3000 joules per pole, unlimited number of surge capacity) on the breaker panel and individual Delta surge capacitors on each critical 20A circuit that supplies power to laboratory equipment. These extras steps provide protection from direct lightning strikes and surges to the panel and equipment.

(4) The UPS Batteries: The lead acid batteries in your UPS system have a finite life. Mark them with a date and keep records on battery changes. Depending on the quality, temperature and load over time, they may need to be replaced in as little as one year or perhaps last as long as four years. When the batteries loose their ability to hold a charge your on-battery run time will drop dramatically so replace them early rather than late. The battery pack can be (and should be) tested on a regular basis. To test the UPS refer to the Operator's Manual for the correct procedure [*the test often involves selecting a time when the computer is not running any critical applications or providing any networked services (such as right before you would normally shut it off for the day). Once it is safe to do so, go ahead and remove the UPS’s A/C plug from the wall. This will remove the UPS system’s source of input power just as if a real power failure had occurred. Your computer system should behave as if nothing has happened. Monitor how long the system stays on until the available UPS run time shows 15 to 20% remaining time left and you will know how long the system can handle a complete power outage. The system will need overnight to fully recharge after this test and you should repeat this test a few times each year to monitor the status of the system]. *Many of the modern UPS systems provide a software application that continuously monitors the UPS modules which are connected to your computer or entire network. This can provide a record of the systems used, battery change dates and current charge levels right from your desktop.

Determination of HPLC Column Dead Time (T zero):

2011-05-01T21:39:00.006-04:00

Column Dead Volume or Time (AKA: Column Void Volume/Dwell Time) is the column volume divided by the flow rate and is usually expressed in minutes. Determining T0 is necessary to find the Resolution Factor (K1) in a separation. Ideally, it is determined by injecting a sample which is unretained by the column and recording the retention time. It can also be calculated if you know a few physical specifications of the column and media used. Here is short list of typical HPLC column dimensions and their void volumes. At a flow rate of 1.000 ml/min these values would also be the void time in minutes.

COLUMN DIMENSIONS (I.D. x Length (mm)) VOID VOLUME (ml)

                         2.1 x 100 0.24
                         2.1 x 150                                                                 0.37
                         2.1 x 250                                                                 0.61
                         2.1 x 300                                                                 0.73

                         4.6 x 100                                                                 1.16
                         4.6 x 150                                                                 1.75
                         4.6 x 250 2.90
                         4.6 x 300                                                                 3.49

                       10.0 x 100                                                                 5.50
                       10.0 x 150                                                                 8.25
                       10.0 x 250                                                               13.75
                       10.0 x 300                                                               16.49

                       Void Volume (ul) = (d^2 *Pi * L * Pore Volume) / 4 ;
                                  *Column Diameter & Length are in mm.

[Note : Assumes an Average Pore Volume of 0.70 (check with the manufacturer for actual value)]

A more detailed version of this table with other common column sizes and tubing volumes for capillary lines is available at the following link: http://www.lc-ms.com/tablescharts.htm

Microsoft "Undo" and "Redo" Shortcut

2011-04-04T08:30:00.001-04:00

Here is a quick Microsoft Windows tip that I often use. They work with most Microsoft Windows based applications. The undo and redo features.

To "Undo" what you just did or typed, hold down CTRL and Z.
To "Redo" what you just did or typed, hold down CTRL and Y.

That is it. I find this shortcut handy when the program I am using either does not have a menu tool for this feature or it is located deep in the menu. This is quick and simple.

Here are a few more that you probably know, but are used often.

To "COPY" something, hold down CTRL and C.
To "CUT" something, hold down CTRL and X.
To "PASTE" something, hold down CTRL and V.

Want to learn more ? Here is a link to a list of common shortcuts provided by Microsoft.

http://support.microsoft.com/kb/126449

CHIRAL HPLC / SFC METHOD DEVELOPMENT (Alcohols):

2011-04-01T08:00:00.004-04:00

We are experts in chiral HPLC and SFC method development of pharmaceutical samples and have operated a contract separations labs for nearly two decades. We have learned a great deal about developing fast and reliable racemate separation methods, often where other companies have failed. The knowledge we have gained has allowed us to develop over ten thousand new chromatographic methods for our clients. This has made our company the leading expert in the chiral separations field.

I would like to share a tip with you regarding the use of different alcohols in chiral method development. That tip is to experiment with different alcohols during the method development process (*Please make sure the alcohol is compatible with your column!). Many of the normal and reversed phase chiral columns can be used with some unconventional alcohols to achieve excellent separations. These alcohols are often used isocratically at 100% concentration for HPLC methods and at levels ~ 15 to 20% for many SFC methods. We have had a great deal of success using 100% pure Methanol for HPLC methods on normal phase style chiral columns (though 100% Ethanol is still one of the best). For SFC methods, Methanol, Ethanol and Butanol (plus mixtures of these) are still some of our favorite co-solvents.

Here is a list of some popular alcohols (HPLC grade) worth using in your chiral method development:

Methanol; Ethanol;1-Butanol; 2-Propanol; 2-Butanol and Acetonitrile (I know this last one is not an alcohol, but it is often overlooked in chiral method development. It works where other solvent systems fail for both HPLC (100%) and less so for SFC (10% as a co-solvent with Methanol).

Many of the above liquids can be used at 100% concentration, but others require mixing with Hexane or Heptane to yield lower concentrations of alcohol. *Remember to always consult with the column manufacturer first to determine which solvents are safe to use.

A note about acids. Weaker is often better in chiral method development. Examples: TFA at 0.01% and Acetic or Formic Acids at 0.1% concentration are often strong enough to ionize most compounds.

Flow Cell Volume & Path Length:

2011-03-16T20:03:00.000-04:00

Modern UV/VIS detectors offer several different flow cell options. The option(s) you select can make a big difference in the level of signal sensitivity, sample dispersion and response you obtain. If you fail to note which type of HPLC flow cell you use in a particular system, then you may discover some problems when transferring a method to a different instrument. Always record the flow cell volume and path length used as part of your method description.

Flow Cells Usually Differ In Three Ways:

(1) Maximum Rated Back-pressure;

(2) Flow Cell Volume and

(3) Flow Cell Path length.

Let’s take a look at these in more detail.

Maximum Rated Back-pressure: Unless the detector is in series with another detector, column or has a back-pressure regulator on it, the expected back-pressure on a typical flow cell’s outlet is just about one bar as it usually is directed to an open waste line. *This topic will be discussed in more detail in the future as part of another “hint and tip” topic. Today we are more concerned about the remaining two options:

Flow Cell Volume: Analytical flow cells are commonly offered in nl to ul sizes. Depending on your instrument setup, column and sample(s), one flow cell volume may make more sense than another. After you have spent time separating and concentrating the peak of interest into a tiny volume you do not want to elute it off the column and mix it with another peak because the cell volume is too large. Ideal cell volume is a compromise between sample dispersion and sensitivity. The best choice will be determined mostly by the actual peak volume of your separated sample. The general rule is that your flow cell volume should be no larger than 10% of your peak volume, but there are some exceptions to this rule. When in doubt, experiment with different cells and do not forget to consider the total volume of all the connecting tubing and valves in your system as these contribute to many issues when the column volume decreases (such as when using mini or narrow bore columns). Some common analytical cell volumes offered by various manufacturers are 2 ul, 6 ul and 13 ul. For narrow bore columns (~ 2.1mm ID) a smaller cell volume (~ 2 ul) will result in less sample dispersion, while a larger cell volume may increase overall sensitivity (esp. when used with a longer path length). Mid-bore or Mid-Size columns (2.1 to 4.6mm ID) often are best suited to cell volumes around 6 ul to minimize dispersion and still provide good sensitivity. A 13ul flow cell could be used to enhance sensitivity too. Standard 4.6mm ID columns often benefit from a 13ul volume cell to provide maximum sensitivity with less concern for dispersion effects with the larger volumes. Keep in mind that these are general rules (and volumes) only. Most samples contain many peaks of varying width & volume, so you will need to select the cell volume that is best suited to most of the peaks in your sample.

Flow Cell Path Length: The flow cell’s path length affects the intensity of light reaching the detector (Beer-Lambert law). For the same volume of sample, the apparent concentration of the sample will appear to be higher if the path length is longer. There is no established standard for ‘path length’ so it is important that you always known what the path length of each flow cell is in your detector (1 cm is rather common). Manufacturer’s offer different flow cells with varying path lengths. Even identical detectors can use flow cells with identical volumes, but have different path lengths. When comparing the analysis results obtained from two different instruments, always make note of the flow cell dimensions used in each instrument. If the method is to be reproduced on a second system, then the flow cells used should have the same geometry (volume and path length). One way that the difference in path length can be used to enhance sensitivity of an existing method is to use a flow cell with a longer optical path length. For example, if your current flow cell has a path length of 6 mm you could replace it with one having a longer path length of 10 mm. This would increase the sample peak response (as more light would be absorbed) in your method. *This fact can be useful to squeeze out additional sensitivity in a method and often does not require any change of column or conditions.

REFERENCE WAVELENGTHS (as used in HPLC UV/VIS):

2011-03-01T15:55:00.000-05:00

The number one problem that I see as a consultant in chromatography laboratories today revolves around the lack of understanding of how to choose appropriate settings for the modern UV/VIS detectors. One specific example of this relates to the optional use and configuration of a “Reference Wavelength” value in the detector. Most manufacturers of advanced HPLC UV/VIS (esp. DAD/PDA) detectors provide this extra feature in their chromatography software, but its use and function are a mystery to most chromatographer's. Allow me to provide a very brief explanation of it here.

If you are running a gradient analysis, then the change in solvent properties (RI) and temperature over time can cause noticeable baseline drift during the run. This drift up or down relative to the starting baseline can cause a number of quantification problems with the analysis reporting software (as flat baselines are more easily integrated than slopes).

Two clever ways to deal with this problem are:

(Method # 1) run the same method again, but with no sample (a blank) and subtract it or...

(Method #2) collect a second channel of data (2nd wavelength signal) at the same time that is as close to the original wavelength, BUT will not overlap with any of the peaks of interest or other compounds in the sample. As such, it can be used as a blank run for baseline subtraction. You can then subtract the ‘blank’ run from your original run and the resulting chromatogram should have a flatter baseline for quantification purposes.

Using the concept of Method #2 described above, many manufacturers’s added a feature known as a the ‘Reference Wavelength’ to their systems. This feature allowed a chromatographer to input a second wavelength value into the method which would be used to subtract out raw data from the primary wavelength during the analysis. This subtraction occurs in real time and the resulting data reported to the user is in fact the result of the subtraction. You will never know what the original data looked like before the reference wavelength was subtracted. Only the manipulated result is provided. If any sample peak(s) appeared in the region (depending on the wavelength and bandwidth specified) where you selected a reference wavelength, then the resulting data would have been clipped off of your real sample and you would never know or have any record of it ! This brings up a serious validation issue as you are modifying the original data with no way of knowing (or documenting) how you have changed it. It is for this reason alone that we teach chromatographers to always turn this feature 'OFF' by default. If they want to make use of the feature, then we suggest that they simultaneously collect data from a second, separate wavelength channel such that the two raw data streams are preserved for validation purposes. The separate signals can be compared, subtracted or manipulated as needed for reporting purposes, but the original data is left secure. This allows you to monitor for contamination, problems or changes during the run.

Observational Notes: A common reason we come across this problem in the lab is that I am called in to diagnose a strange problem where the area of a known sample peak changes in an unexpected way. That "way" includes: going NEGATIVE, increasing in mass or decreasing in mass. *This is due to the reference wavelength feature being turned 'ON' and another peak absorbing in the region selected.

The reason we see this feature cause so many problems in the field appears to be due to the Reference Wavelength feature being turned 'ON' by DEFAULT in the software for most DAD/ PDA modules (should be 'OFF') and to make matters worse, the default values often supplied by the manufacturers are actually used by most chromatographers (what are the odds that the random values placed in the system are even relevant to your analysis?)! We suggest using a ‘canned’ method template in most laboratories which includes a new default value for this feature... 'OFF' for all analysis methods.

Windows "Desktop Cleanup" Feature

2011-02-20T18:49:00.000-05:00

OK, this is not directly related to chromatography, but I thought that others out there might enjoy this Microsoft Windows tip. Besides being a scientist, I also have provided business and laboratory computer network services to clients for almost two decades. One of the many "annoying" Windows features is the "Desktop Cleanup" feature which pops up on the lower menu bar every sixty days. This annoying little program asks you to review all of the shortcuts on your desktop space to choose which ones you would like to keep. Well, that was one of those "features" that I could do without and promptly disabled it for good. It turns out that it also annoys a large number of my clients so I would like to pass on how you can disable this feature in Windows XP Pro (other versions of Windows are similar, but I will use XP in this example).

To disable the Windows "Desktop Cleanup" Feature:

(1) Right click your mouse in an empty area of your desktop.

(2) Click on 'Properties' (Display Properties).

(3) Click on the 'Display' tab.

(4) Click on 'Customize Desktop'.

(5) Uncheck the box next to "Run Desktop Cleanup Wizard every 60 days".

(6) Click 'OK' to exit out of the menus. Done.

INLINE HPLC DEGASSING MODULES

2011-02-16T17:00:00.009-05:00

INLINE HPLC DEGASSING MODULES:

Modern electronic vacuum degassing systems are a great convenience. Changing Helium gas cylinders, monitoring gas pressures and levels over time have been replaced in many labs with the sound of small gurgling vacuum pumps. While the convenience gained through the use of these devices can be great, there are some important downsides to this technology. One of them will be mentioned here.

Total Channel Volume: The volume of liquid contained in the tubing and vacuum chamber of EACH vacuum degassing channel can be enormous compared to the tiny volume often found in systems which utilized Helium sparging alone. When the system is turned off, this solvent often sits stagnant over night, allowing air to bleed in, and must be flushed out when the system is re-initialized for use the following day. *If you just switched bottles of mobile phase, then the old mobile phase is still inside the system and needs to be completely flushed out before use or you will have some very strange chromatographic results in the coming weeks! This process takes time and should be performed on every channel in your system, even if you are not going to use them all. How much volume you ask ? Well, let’s look at two common examples of vacuum degassing units we find at client sites (but this applies to any manufacturer’s system, just look up the spec’s or measure it on your own).

Agilent/HP Brand Degassing Module, Model G1322A; Each vacuum channel chamber has a volume of about 12 ml. The interconnecting tubing (solvent frit to bottle head, bottle head to degassing unit and degassing unit to pump inlet) can add another 10 – 15 mls more. In this example, we recommend that you flow at least 30 mls of the appropriate solvent through the line before use. This will insure that the channel is primed with fresh mobile phase. That step is for EACH channel so it is best to flush them one-at-a-time, in sequential order, to keep track of them.
Agilent/HP Brand Micro Degassing Module, Model G1379A or G1379B: Each vacuum channel chamber has a volume of about 1 ml. The interconnecting tubing (solvent frit to bottle head, bottle head to degassing unit and degassing unit to pump inlet) can add another 10 – 15 mls more. In this example, we recommend that you flow at least 20 mls of the appropriate solvent through the line before use. This will insure that the channel is primed with fresh mobile phase. That step is for EACH channel so it is best to flush them one-at-a-time, in sequential order, to keep track of them.

NOTE: Some early models of HPLC degassing systems had individual chamber volumes of greater than 30 mls each ! When you add the total tubing volume to that you end up with a flush volume of 40 to 50 mls per channel. These systems took a long time to flush out.

To save time performing this “flush” step, you should take advantage of the highest available flow rate setting of your pump. With the prime purge valve open (so the solvent goes to waste or if appropriate, to a separate collection container for re-use), set the pump flow rate to 10 ml/min and you will be able to flush out a single channel in two or three minutes. Repeat for each of the other channels and then adjust the flow rate back down to your normal equilibration flow rate before closing the prime purge valve again. Our lab performs this flushing procedure at the start of each day to insure the columns receive fresh solvent. This procedure should also be performed any time a mobile phase bottle is changed out to a different bottle or type of mobile phase too.

One last reminder. Please keep in mind that running liquid at 10 ml/min during the flush phase in most degassing modules will not result in very efficient degassing as degassing efficiency is a function of flow rate (surface area of liquid exposed over time). You will still need to wait a period of time until the new solvent is fully degassed once again, but at least you will know it is free of any older mobile phase. *Remember, everything you do before you start an analysis contributes to the final result.