HPLC Primer – MicroSolv Technology Corporation

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Calculate the internal volume of tubing – How to

To calculate the internal volume (V), you need to know the length of tubing (L) and the inner diameter (ID). You then use the formula for volume of a cylinder. Convert L and ID into cm first. (1 inch = 2.54 cm). This gives V in cm3. 1 cm3 = 1 mL. You can then […]

Choose an appropriate guard column when using a Cogent TYPE-C HPLC column – How To

Two different guard columns are available: 2.0 and 4.0 mm internal diameter (ID). Both sizes are 20mm in length and are used with the same Guard Column Holder. The 2.0mm ID guard columns are for use with the 2.1mm ID analytical columns and the 4.0mm ID guard columns should be used with the 3.0mm and […]

Determine pore volume of an HPLC column using HPLC injections – How To

The interior of an HPLC column is contains tightly packed spherical particles. There are two types of empty space in the column, i.e. volume not occupied by these particles. The first type is the interstitial space between adjacent particles. This actually comprises a small percentage of the total void volume. The majority of the empty […]

How to back flush and wash an HPLC column for reuse – Tips & Suggestions

1. Reverse the flow direction in the column. Attach the column fitting that is normally at the outlet to the tubing that is normally connected to the inlet. Direct the column outlet to waste, not to the tubing leading to the detector. The reason for this is so any contaminants coming off the column don’t end […]

How to determine concentration of an impurity in HPLC with the relative response factor RRF – How to

The concentration can be calculated once you have values for the following two equations: Response Factor (RF) = Peak Area / Concentration Relative Response Factor (RRF) = RFimpurity / RFAPI You can use RRF and RFAPI to solve for RFimpurity. Then you can use the measured peak area of the impurity to solve for its concentration.

How to tighten screw caps properly – Tips & Suggestions

What is the best way to get an optimal seal from autosampler vial screw caps? The “best practices” for applying autosampler vial, screw caps is not to “tighten them down” as much as possible. In fact, this is contra-indicated as it will cause the septa to “dimple” creating “peaks and valleys” instead of sealing, smoothly […]

Integrate a peak with a shoulder – How to

It is always better to have baseline separation between a main peak and any nearby minor peaks. However, this is not always possible and you may have to deal with integration of a peak with a minor shoulder or peak present. It is less accurate to integrate from the base to the point where the […]

Measure signal to noise ratio in an HPLC chromatogram – How to

The best way is to zoom in on the peak of interest using your data analysis software so that the baseline near the peak is also visible as well. Depending on how sharp or narrow your peak is, you’ll want to zoom in so that the time axis covers a range of about 0.5 –1.0 […]

Octanol Water Partition coefficient – Primer

Basically, this is a measure of the hydrophobicity v. hydrophilicity of a compound. It is extremely useful when combined with the pI of your molecule to predict retention times in HPLC. The Octanol-Water Partition coefficient is a physical property used to describe a chemical’s lipophilic or hydrophobic properties. It is the ratio of the concentration […]

Our glossary of terms used in separation science – Primer

Some of the following parameters must be considered to have a validated HPCE method for use with SOP, GMP, GLP and other pharmaceutical, environmental and forensic applications: Accuracy: How close the observed value is the to true value. This is analogous to an archer hitting the “intended” point each time he shoots his arrow. If the […]

pK of common chemical functional groups in HPLC – FAQ

The dissociation constant (pK) is the pH at which 50% of a compound (functional group) is protonated. Following are some examples of Weak Acid and Weak Base Chemical Functional Groups and their approximate pK’s. Acids pK Sulfonic Acid <1 Phosphate Ester 2 Carboxylic Acid 2.5-5 Uracil 8 Sulfonamide 7-9 Guanine 2.2, 9.4 Phenol 8-10 […]

Teflon variations and uses in plastic laboratory tubing – Primer

TEFLON© comes in many forms an varieties. Each form has different properties and costs. Below is a brief explanation of the different types of TEFLON© which is a registered trademark of E.I. DuPont. PFA PerFlourAlkoxy, has very low friction, excellent purity and excellent chemical resistance. Making it ideally suited for products serving the chromatography and other industries, it […]

Terminology and criteria for chirality – Primer

Chiral compound pairs are two non-superimposable mirror images of each other (see Fig. 1). In their chemical structure, they must have one or more chiral centers (depicted by an up dash or down wedge). These chiral centers are usually but not always at a carbon. Compounds that have more than one chiral center cannot be chiral if they […]

Theoretical plate N estimate for real samples – How to

One can estimate a reasonable plate number for any column as: N ≈ 300 L/dp L is the column length (in millimeters) and dp is the particle diameter (in micrometers). For example for 250 mm x 4.6 mm column packed with 5 micron particles, the predicted value is N ≈ 15,000 If the measured value of N is about […]

What does the specification 9-425mm for screw top autosampler vials mean – FAQ

The first number refers to the “Major Diameter” of the screw thread, (Major Diameter is the OD / outside diameter of the screw top vial (not the OD of the vial) at the thread line, measuring on the outside of the threads, minor diameter is the smaller diameter, measuring OD between the threads). We use […]

What is a pyrogen – FAQ

The definition according to answers.com is “A pyrogen is defined as a fever-producing agent. Pyrogens are substances that cause febrile reactions when sufficient amounts enter the circulatory system. Bacterial endotoxin is the most significant pyrogen because of its potency and ubiquity.”

Category: HPLC Primer